DNA vaccines

combined artificial immunogens mimicking virus particles : the central core of the virus-lile particle (dsRNA) is coated with a layer of the following conjugate : spermidine (arginine, lysine, ..)--polyglucine--enzyme substrate^{ref1,
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bacterial ghosts are non-denatured bacterial cell envelopes that are formed by the expression of the plasmid-encoded lysis gene E of bacterial phage fX174 : they can be filled with DNA or drugs through the lysis hole and used as vectors^{ref1,
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replication-defective Bacteria (e.g. Salmonella typhymurium ) can be used as vectors for DNA vaccines as they free the carried plasmid after cell lysis.

coupled with protein adjuvants (DNA fusion vaccine) :

A and B subunits of cholera toxin (CT) or the heat-labile enterotoxin from E.coli (LT)^ref
fragment C of tetanus toxin^{ref1,
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plant viral genes coding coat protein (PVCP) (self-aggregating) drives T_h1 dominant responses

potato virus X coat protein^ref

transgenic B lymphocyte immunization (TLI) as antigen-presenting cell (APC)-based genetic vaccines : DNA can cross the cell membrane by natural means, but the functional relevance of this phenomenon has not been fully elucidated. Here, we analyzed spontaneous transgenesis of human B cells using plasmid DNA coding for a functional Ig H chain gene under the control of a B-cell-specific promoter. Using PCR, RT-PCR, and flow cytometry in combination, spontaneous transgenesis was documented in Burkitt's lymphoma cell lines, EBV-transformed cell lines, and peripheral blood B lymphocytes of the mature naive phenotype (IgM⁺/IgD⁺/CD27^-). By immunoelectron microscopy, the internalized DNA was seen in the lysosomes/late endosomes and in the cytosol proximal to the nucleus. Importantly, spontaneously transgenic B cells expressed the gene, processed the protein and presented to MHC-restricted T lymphocytes a peptide expressed in the transgenic product^ref. A single i.v. injection of transgenic B lymphocytes induce robust and long-lasting T-cell immunity reproducibly and specifically even at very low cell doses (approximately 3 x 10²). T cell priming can occur in the absence of dendritic cells and results in immunological memory with protective effector functions^ref. These properties reflect on simple characteristics, such as time synchronization and initial localization to secondary lymphoid organs of APCs endowed with protracted synthesis and presentation of antigen to T cells^ref.
xenogenic DNA immunization is a mean to overcome immunological ignorance or tolerance which normally prevents responses to self antigens found in tumors. Xenogeneic DNA immunization leads to rotection from syngeneic tumor challenge, inhibition of growth of established tumors and autoimmune depigmentation of cat in mice or dogs with canine malignant melanoma^ref
fusion with genes coding for cell-penetrating peptides (CPP) (e.g. VP22)
domain I of the filamentous bacteriophage coat protein III gene. Vaccination with a DNA construct encoding the domain I fusion generated antigen-specific T_h1-type cellular immune responses against a non-immunogenic tumor antigen^ref

ability to induce immunological danger signals at sites of injection :

the strong immunogenicity has been attributed to the presence in the plasmid backbone (cloned in bacterial cells, usually E.coli) of particular unmethylated CpG motifs that in humans bind to TLR9 activating plasmacytoid dendritic cells (PDCs) eliciting T_h1 polarization. Methylation of the palindromic CpG sequences of bacterial expression vectors abolished their ability to induce an antibody response to the transgene product (a highly immunogenic viral glycoprotein) in mice. The antibody response could be rescued by concomitant injection of oligonucleotides carrying immunostimulatory sequences. The B cell response to 2 plasmid vectors, both expressing the same viral glycoprotein but containing a different content of the highly stimulatory AACGTT motif, was compared. Comparable B cell responses were induced to the 2 constructs given at an optimal vaccine dose while the vector containing additional palindromic sequences resulted in higher antibody titers at a suboptimal dose. These data indicate that deletion of CpG motifs or methylation of such sequences in plasmid DNA can abrogate the immune response to the vector encoded antigen and might thus enhance their usefulness as gene therapy vehicles^ref.

inherently favor the induction of cytotoxic cellular immunity since Ag can be presented through endogenous processing pathways in the context of class I MHC^ref
ability to directly infect APCs
the host cell provides the post-translational modifications to the antigen, allowing its production with the correct native conformation to live infection
combination therapy :

recognition of self-antigens is emerging as a theme for the immune recognition of human cancer^{ref1,
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ref4}. Melanoma and prostate cancer provide usegul models to explore immunization against differentiation antigens^{ref1,
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ref5}. Interaction between CTLA-4 and B7 on APCs results in down-regulation of activated T cells, attenuating the immune response^ref. Blockade of CTLA-4 results in disruption of the binding of CTLA-4 to the costimulatory molecules B71/2 and enhances the response to antigenic tumors and foreign antigens^{ref1,
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genetic immunization for antibody generation in research animals by intravenous delivery of plasmid DNA : the typical objective of genetic immunization is the isolation of polyclonal antibody containing sera or antibody-producing B cells, which can be used to generate monoclonal antibody (MAb)-producing cell lines (either by immortalization or by fusion with myeloma cells to form hybridomas). The same vector can be used for both genetic immunization and antigen production^ref

drawbacks and risks :

limited to protein antigens (hence excluding carbohydrate antigens) and adjuvants (unless added to DNA mixture)
interspecific codon usage bias may be an obstacle for effective induction of immune responses by gene vaccines, and should be corrected by codon optimization^ref
one issue with DNA vaccine formulation is keeping potential adjuvants and the DNA in proximity. DNA delivery results in protein antigen expression on after several hours and can persist for days, a time period where normally the water soluble resiquimod, could be long gone and unable to play a role in local adjuvanticity^ref.

bupivacaine can modulate immune responses induced by DNA vaccines as they form specific complexes with DNA and this formulation serves to increase transfectant DNA protection as well as local delivery^ref, but stimulated only modest levels of overall antibody production, with relatively low level of IgG_2a
levamisole exhibits strongest T_h1 stimulatory activity whereas Tween 80 showed weakest T_h1 activity, as determined by IgG_2a production, and saline formulation induced weak T cell proliferation and DTH, in animals inoculated with a DNA construct expressing the FMDV capsule protein VP1. Furthermore, co-inoculation of levamisole increases the production of IFN-g by > 100-fold as compared to that by DNA inoculation formulated in saline^ref

potential for atypical processing of bacterial and parasite proteins
development of secondary anti-DNA autoantibodies occurs^ref, but not always^ref and is usually transient
in vaccination with recombinant plasmid :

plasmid DNA purification : current good manufacturing practice (cGMP) conditions for obtaining clinical-grade plasmid DNA
Merck Research Laboratories has developed a highly productive Escherichia coli fermentation process to produce plasmid DNA for use as vaccines. The process consists of a fed-batch fermentation in a chemically defined medium. Initiation of the feed stream precedes a growth-limited phase in which plasmid DNA is amplified. The fermentation is only maximally productive for a small fraction of E. coli transformants designated as high-producers, while the predominant low-producer population does not amplify plasmid DNA. In experiments undertaken to probe this phenomenon, transposition of the 768-bp E. coli insertion sequence IS1 into an HIV DNA vaccine vector was observed in several low-producer clones. IS1 was found to insert in or near the neomycin resistance gene in nearly a dozen unique sites from within a single population of plasmid molecules. The fraction of IS1-containing plasmids within several clones was determined by quantitative polymerase chain reaction and was found to increase with increasing cultivation time in the chemically defined medium. Because transposition into an antibiotic-resistance gene is unlikely to affect plasmid amplification, the genomes of high- and low-producers of three different HIV DNA vaccine vectors were subsequently profiled by restriction fragment length polymorphism analysis. In all 3 cases, IS1 insertional mutations were found in the genomes of the predominant low-producers, while the genomes of the high-producers were indistinguishable from untransformed cells. The insertions reside on similarly sized fragments for two of the low-producer clones, and the fragment size is smaller for the third clone. The third clone also produces much less plasmid DNA than a typical low-producer. The results suggest the presence of an IS1 insertional mutation that affects plasmid replication and amplification, possibly in a position-dependent manner^ref.
reduced immunogenicity of DNA vaccine plasmids in mixtures : given alone, each of 9 DNA vaccine plasmids encoding candidate malaria vaccine antigens induced significant antibody titers and, in the 4 cases for which appropriate assays were available, IFN-g responses. Significant suppression or complete abrogation of responses were seen when the plasmids were pooled in a 9-plasmid cocktail and injected in a single site. Removal of single genes from the mixture frequently reduced the observed suppression. Boosting with recombinant poxvirus increased the antibody response in animals primed with either a single gene or the mixture, but, even after boosting, responses were higher in animals primed with single plasmids than in those primed with the 9-plasmid mixture. Boosting did not overcome the suppressive effect of mixing for IFN-g responses. Interactions between components in a multiplasmid DNA vaccine may limit the ability to use plasmid pools alone to induce responses against multiple targets simultaneously^ref.

in vaccination with recombinant viruses :

presence of virally encoded inhibitors of innate immunity and adaptive immunity
presence of neutralizing antibodies in pre-exposed or prevaccinated individuals, which inhibit the initial round of virus infection and replication, thereby reducing the ability of the virus to immunize
individuals who have never previously exposed to the vaccinating virus generate neutralizing antibody after the first vaccination, thereby precluding homologous prime-boost
the strong viral antigens will dominate, thereby inhibiting responses to the weaker tumour antigen. This concern arises from the ability of immunodominant epitopes in antigens to quench responses to subdominant or cryptic epitopes. However, such a quenching phenomenon is, in fact, rarely observed with recombinant viral vaccines. Heterologous prime-boost would certainly diminish the probability of such a phenomenon, because the recombinant tumour antigen would be the only one being repetitively boosted.

in vaccination with recombinant bacteria :

recombinant bacteria having life cycles that involve both phagolysosomal and cytoplasmic stages (e.g. Listeria monocytogenes ) engineered to secrete antigens will load the MHC class II processing pathway during the phagolysosomal phase of its life cycle and the MHC class I pathway during the cytoplasmic phase.
several recombinant bacteria actively induce infected APCs to secrete proinflammatory cytokines such as IL-12
bacterial vaccines containing plasmids with eukaryotic promoter and enhancer elements that drive the expression of the antigen gene can transfer the plasmid directly into eukaryotic transcriptional compartments within infected APCs and result in potent immunization
the expression plasmid containing the in vivo-inducible promoter pagC (P(pagC)) from Salmonella typhimurium and HGV-NS3 gene was introduced into the attenuated Salmonella typhimurium SL7207 to investigate the function of P(pagC). The expressed HGV-NS3 protein was detectable by SDS-PAGE and Western blotting in the recombinant bacteria in the presence of low concentration of Mg²⁺ (<50 mmol/L). When the concentration of Mg²⁺ reached to 50 mmol/L, the amount of expressed HGV-NS3 was decreased significantly. The recombinant bacteria were multiplied in LB medium containing 50 mmol/L of Mg²⁺ and used as a DNA vaccine to orally inoculate C57 mice for 3 times. The results of serum antibodies, T lymphocyte proliferative response and cytotoxic T lymphocyte response of immunized mice showed that the oral vaccine could induce strong humoral and cellular immune responses in mice, which indicates that the P(pagC) is a strong in vivo-inducible promoter and can be used in attenuated Salmonella typhimurium to construct an effective oral vaccine^ref

antitumor vaccination therapies using attenuated Salmonella typhimurium carrying plasmid DNA encoding tumor-associated antigens are currently under preclinical development. In vivo monitoring of attenuated S. typhimurium uptake is established using a bioluminescent lux gene operon plasmid. Following transformation with the lux gene operon construct, mice were fed with various amounts of attenuated S. typhimurium-lux to monitor in vivo clearance over a period of 24 h. The ingested attenuated S. typhimurium-lux cells were almost cleared out 9 h postfeeding, as judged by a significant decrease in bioluminescence. Attenuated S. typhimurium oral DNA vaccine using attenuated S. typhimurium carrying the mouse a-fetoprotein (AFP) gene was found to promote protective immunity against both cancer line CT26-murine a-feto protein (mAFP) that stably expresses AFP and mouse hepatocellular carcinoma (HCC) Hepa1-6. The oral DNA vaccine significantly increased the life span of tumor-challenged mice in both tumor models. Together, these results suggest that vaccination with the attenuated S. typhimurium oral DNA vaccine that carries the AFP gene could be a promising strategy to prevent HCC development^ref

in vaccination with recombinant yeasts :

unlike intramuscular injection, gene gun delivery of DNA drives a strong T_h2 response. In an effort to counter this, the T_h1 cytokines, IL-12 and IL-23 , were genetically fused to the hemagglutinin (HA) gene from influenza APR/8/34, and delivered these DNA constructs to Balb/c mice. Gene gun delivery of the HA gene was able to induce antibody production by all vaccinated mice. Linking of IL-12 caused almost complete suppression of immune responses whereas mice vaccinated with IL-23HA showed long-lived IgG₁antibody levels. Splenocytes from IL-23HA vaccinated mice also tended to produce more IL-5 and IFN-g after restimulation in vitro than splenocytes from HA vaccinated mice. While codelivery of IL-23 did not change the type of immune response it may increase its longevity following vaccination^ref. A comparative study of domestic pig, minipig and mouse in regard to local tolerance and antigen expression of HIV immunotherapeutic using PMED. Pig/minipig is considered a good model for the safety assessment of DNA vaccines due to the similarity to human skin. Local reactions were evaluated at 10min, 4, 24 and 48h. Histology of administration sites revealed epidermal necrosis with associated dermal inflammation at 10min and 4h, and subsequent regeneration with repair at 24 and 48h. The degree and extent of these changes varied according to species. Domestic pig and minipig showed superficial epidermal necrosis and complete repair, while the mouse showed full-thickness epidermal necrosis and partial repair. Expression of HIV antigen was confirmed using immunohistochemistry in all three species at 4, 24 and 48h. The results showed that PMED is an effective system for DNA vaccine delivery as demonstrated by the antigen expression seen as early as 4h^ref
expensive
need for injection => risk of accidental parenteral infections from contaminated needles
need for skilled medical personnel to deliver jabs
biodistribution : to monitor the distribution and duration of gene expression of a DNA vaccine in living organisms, the naked DNA encoding firefly luciferase (Fluc) has been used as an imaging reporter gene, and in vivo bioluminescent images have been evaluated in a murine model. Bioluminescence was observed at the injection site and at inguinal lymph node from 10h to 24h post-injection when DNA vaccine encoding Fluc (pcDNA3.1-Fluc) was injected into the bilateral posterior flanks in mice. Fluc gene expressions at injection sites and unilateral posterior flank inguinal lymph node were also confirmed by RT-PCR. However, when pcDNA3.1-Fluc was injected into the mid-dorsum bioluminescent signals were observed at the injection site for up to 14 days post-injection, but no bioluminescent signals were detected in inguinal lymph nodes. Concurrent mRNA expressions of Fluc gene at injection sites but not at inguinal lymph nodes were confirmed by RT-PCR. These findings suggest that optical imaging using Fluc could be useful for monitoring the location, intensity and duration of gene expression of naked DNA vaccines in living animals non-invasively and repetitively^ref

Design :

the design of an efficient DNA vaccine, involves choice of a suitable expression vector, ensuring optimal expression by codon optimization, engineering CpG motifs for enhancing immune responses and providing additional sequence signals for efficient translation. DyNAVacS is a web-based tool created for rapid and easy design of DNA vaccines. It follows a step-wise design flow, which guides the user through the various sequential steps in the design of the vaccine. Further, it allows restriction enzyme mapping, design of primers spanning user specified sequences and provides information regarding the vectors currently used for generation of DNA vaccines. The web version uses Apache HTTP server. The interface was written in HTML and utilizes the Common Gateway Interface scripts written in PERL for functionality. DyNAVacS is an integrated tool consisting of user-friendly programs, which require minimal information from the user. The software is available free of cost, as a web based application^ref
generating broad cellular immune responses against a diversity of viral epitopes is a major goal of current vaccine strategies for both HIV-1 and other pathogens. Virus-specific CD8⁺T lymphocyte responses, however, are often highly focused on a very limited number of immunodominant epitopes. For an HIV-1 vaccine, the breadth of CD8⁺ T lymphocyte responses may prove critical as a result of the need to cover a wide diversity of viral isolates in the population and to limit viral escape from dominant epitope-specific T lymphocytes. Epitope modification strategies can alter CD8⁺ T lymphocyte epitope immunodominance hierarchies elicited by a DNA vaccine in mice. Mice immunized with a DNA vaccine expressing SIV Gag lacking the dominant D^b-restricted AL11 epitope generated a marked and durable augmentation of responses specific for the subdominant D^b-restricted KV9 epitope. Moreover, anatomic separation strategies and heterologous prime-boost regimens generated codominant responses against both epitopes. These data demonstrate that dominant epitopes can dramatically suppress the immunogenicity of subdominant epitopes in the context of gene-based vaccines and that epitope modification strategies can be utilized to enhance responses to subdominant epitopes^ref
Broad therapeutic application of nucleic acid micro- and nanoparticles will require bioprocesses capable of achieving high loads of structurally intact and functionality active DNA. Condensation of pDNA into nanoparticles has been achieved by sedimentation through R4 peptide and partitioning at a hydrophobic interface. > or = 90% coating efficiency onto microparticles is achieved via this combined bioprocess with the pDNA retaining 85-90% intact supercoil after bioprocessing. SEM analyses of the microparticles produced therefrom reveals bound pDNA and R4 peptide nanoparticles. HPLC and chemical analyses afford quantification of the particle-associated pDNA and R4 peptide along with lactose, raffinose, or trehalose carbohydrate stabilizer, surface coatings uniformly applied by spray freeze-drying. Administration of these particles by gene gun demonstrates delivery to the nucleus of expressive nanoparticles and into rodents and pigs pronounced immunogenicity even after bioprocessing and accelerated degradation. These data support the discovery of a robust bioprocess platform for preparing macromolecule bound bioparticles with potential relevance beyond simple preparation of bioactive DNA vaccine^ref.
2 different DNA vaccine designs aimed to elicit CD8⁺ T cell responses against a specific peptide-epitope either by direct- or cross-presentation. Each carries sequences from tetanus toxin (TT) to provide essential CD4⁺ T cell help. In the first already proven design, the peptide-epitope is fused to the N-terminal domain of fragment C from TT. This appears to act mainly by cross-presentation. In the second design, the peptide-epitope is encoded by a minigene, with induction of T_h responses mediated by coexpression of a hybrid invariant chain molecule, incorporating a single determinant from TT (p30) in exchange for class II-associated invariant chain peptide (CLIP). This design appears to act mainly via direct presentation from transfected APCs. Both vaccines mediated T_h-dependent priming of CD8⁺ T cells in mice, but the kinetics and level of the responses differed markedly, consistent with engagement of distinct pathways of Ag presentation. Importantly, the vaccines could be combined in an alternating prime-boost regime, in either order, generating substantially expanded memory CD8⁺ T cells, with potent effector function. Taken together, these results demonstrate that vaccination protocols involving different modes of Ag presentation at prime and boost can significantly improve the effectiveness of immunization^ref
2 different DNA vaccine designs aimed to elicit CD8⁺ T cell responses against a specific peptide-epitope either by direct- or cross-presentation. Each carries sequences from tetanus toxin (TT) to provide essential CD4⁺ T cell help. In the first already proven design, the peptide-epitope is fused to the N-terminal domain of fragment C from TT. This appears to act mainly by cross-presentation. In the second design, the peptide-epitope is encoded by a minigene, with induction of T_h responses mediated by coexpression of a hybrid invariant chain molecule, incorporating a single determinant from TT (p30) in exchange for class II-associated invariant chain peptide. This design appears to act mainly via direct presentation from transfected APCs. Both vaccines mediated T_h-dependent priming of CD8⁺ T cells in mice, but the kinetics and level of the responses differed markedly, consistent with engagement of distinct pathways of Ag presentation. Importantly, the vaccines could be combined in an alternating prime-boost regime, in either order, generating substantially expanded memory CD8⁺ T cells, with potent effector function. Taken together, these results demonstrate that vaccination protocols involving different modes of Ag presentation at prime and boost can significantly improve the effectiveness of immunization^ref

Delivery :

HSV-1 amplicon vectors efficiently transduce cultured antigen-presenting cells (APC), including both human and murine dendritic cells as well as primary human chronic lymphocytic leukemia (CLL) B cells. Helper-free amplicons have been shown to be especially well-suited for this purpose, since they do not impair the antigen-presenting functions of these target cells. In vivo, amplicon vectors have been used in preclinical studies aimed at the development of therapeutic cancer vaccines, as well as vaccines for Alzheimer's disease, and selected microbial pathogens. Studies in small animal model systems have shown that ex vivo transduction of irradiated tumor cells with amplicon vectors encoding immunomodulatory cytokines such as IL-2 or GM-CSF can elicit protective responses against a tumor challenge. In an experimental model for cancer immunotherapy, direct transduction of preformed tumors with vectors encoding CD40L resulted in slowed tumor growth or tumor eradication. Other studies have examined the ability of amplicons to elicit immune responses against encoded antigens, and have shown that strong cellular immune responses can be generated against amplicon encoded HIV-1 antigens in mice. Thus, amplicon vectors have shown significant promise as vaccine vectors in a range of settings. These promising initial findings highlight the need to perform additional studies, including experiments to evaluate the immunogenicity of amplicon vectors in additional animal models, possibly including nonhuman primates. Overall, amplicon vectors offer compelling advantages when compared to other vaccine-delivery platforms, which include the capacity to incorporate a very large transgene payload and the potential to efficiently transduce mucosal surfaces. It will be important to design future studies to directly test and exploit these features of the amplicon system. The next few years therefore promise to be an exciting and important period in the development of amplicons as vaccine vectors^ref.
replication-deficient adenoviral vectors (recAd) are attractive candidates for DNA vaccination approaches because they are able to activate the innate and adaptive immune systems. The ability of recAd to transduce and activate subsets of dendritic cells, namely plasmacytoid dendritic cells (pDC) and conventional dendritic cells (cDC), was explored. DC were derived from bone marrow precursors in vitro with the help of FLT3-ligand. Sorted populations of pDC and cDC were infected with recAd at various multiplicities of infection. Transduction efficiency, phenotypic maturation and production of IFN-a as well as IL-6 were assessed. Additionally, activation of DC and induction of CTL were determined in vivo. The role of TLR9 in recAd recognition was investigated as it has previously been shown that DNA viruses are recognized via this receptor. RecAd can efficiently transduce pDC as well as cDC in vitro. Both DC subsets mature and produce IFN-a upon interaction with recAd. In the absence of TLR9, activation and cytokine production was only detected in cDC but not in pDC. Importantly, induction of CD8⁺ CTL following in vivo injection of recAd was similar in TRL9-deficient mice when compared with wildtype controls. RecAd can efficiently transduce and activate both pDC and cDC. pDC required TLR9 to detect the presence of recAd whereas cDC also recognized recAd independently of TLR9. These unique immunostimulatory properties support the future development of recombinant Ad as a vector for DNA vaccine approaches^ref
short-interval intradermal DNA delivery, generates robust T-cell responses within 12 d, of a magnitude sufficient to reject established subcutaneous tumors. Moreover, this vaccination strategy confers protecting humoral immunity against influenza A infection within 2 weeks after the start of vaccination. The strength and speed of this newly developed strategy will be beneficial in situations in which immunity is required in the shortest possible time^ref.
intramuscular needle and syringe, Biojector^® 2000, and Mini-Jecttrade mark was compared in cynomolgus monkeys. The elicited cellular and humoral immune responses were comparable in monkeys immunized using these different delivery techniques, suggesting that the needle-free approaches to vaccine administration do not significantly improve the immunogenicity of the plasmid DNA vaccine used in the study^ref.
Packaging plasmids containing CpGs into fusogenic liposomes (FL) derived from conventional liposomes and Sendai virus-derived active accessory proteins is an attractive method for enhancing the efficacy of a DNA vaccine. These CpG-enhanced plasmids (possessing 16 CpG repeats) that were packaged into FL, enhanced ovalbumin (OVA)-specific T cell proliferation and cytotoxic T cell activity after immunization. In fact, vaccination with CpG enhanced plasmid-loaded FL induced effective prophylactic effects compared with 13 repeats CpG containing plasmid in a tumor challenge experiment^ref.
Depletion or dysfunction of CD4 T lymphocytes profoundly perturbs host defenses and impairs immunogenicity of vaccines. Plasmid DNA vaccination with a cassette encoding antigen (OVA) and a second cassette encoding full-length CD40 ligand (CD40L), a molecule expressed on activated CD4 T lymphocytes and critical for T cell helper function, can elicit significant titers of antigen-specific immunoglobulins in serum and Tc1 CD8 T cell responses in CD4-deficient mice. To investigate whether this approach leads to CD4 T cell-independent vaccine protection against a prototypic AIDS-defining infection, Pneumocystis carinii pneumonia, serum from mice vaccinated with PC-pulsed, CD40L-modifed DCs was used to immunoprecipitate PC antigens. Kexin, a PC antigen identified by this approach, was used in a similar DNA vaccine strategy with or without CD40L. CD4-deficient mice receiving DNA vaccines encoding Kexin and CD40L showed significantly higher anti-PC IgG titers as well as opsonic killing of PC compared with those vaccinated with Kexin alone. Moreover, CD4-depleted, Kexin-vaccinated mice showed a 3-log greater protection in a PC challenge model. Adoptive transfer of CD19 cells or IgG to SCID mice conferred protection against PC challenge, indicating a role of humoral immunity in the protection. The results of these studies show promise for CD4-independent vaccination against HIV-related or other opportunistic pathogens^ref
Effects of plasmid type and insertion sequence on in vitro and in vivo multiplication and invasiveness of attenuated Salmonella typhimurium were examined following transformation of the bacteria with eukaryotic expression plasmids pcDNA3 and pCI with or without heterologous gene (Newcastle disease virus F gene). Exogenous plasmids had negative impacts on the replication or invasiveness of the attenuated S. typhimurium in LB broth and/or HeLa cell monolayers as well as on its survival in live chicks. The plasmid pCI had more significant effects than pcDNA3. Introduction of heterologous gene into the plasmids not only exhibited additional negative influence on the host strain but also on their own stability therein. All these results suggest that full consideration should be given to the types of plasmids and their stability within the host strain as well as to their effects on replication and invasiveness of attenuated bacteria as the DNA vaccine delivery vector for improved immune protection^ref
microorganisms : bacterial delivery of plasmid DNA may provide an efficient and low-cost alternative to plasmid purification and injection. Also, the bacterial vector may exhibit potential as an immune adjuvant in vivo.

the ability of live attenuated Salmonella enterica serovar typhimurium (S. typhimurium) as a carrier of DNA vaccine was evaluated using model plasmid encoding b-galactosidase (b-Gal) and BALB/c mice. pBRCMVb, b-Gal expression apparatus having a replication origin from low copy pBR322, was constructed. Comparison of the plasmid stability showed that pBRCMVb remained stable in Salmonella even after oral administration, while pUC-based pCMVb tended to be lost quickly. However, titers for b-Gal specific IgG in sera did not significantly increase in mice orally administered S. typhimurium harboring pBRCMVb. These data suggest that the stability of plasmid in S. typhimurium is associated with its replication origin. Further studies are required to scientifically establish this methodology^ref. Plasmid stability on oral DNA delivery by the attenuated Salmonella enterica serovar Typhimurium vaccine strain BRD509. A DNA vaccine cassette comprising the C fragment of tetanus toxin under control of the cytomegalovirus (CMV) promoter was ligated into plasmid pcDNA3, pUC18, pBBR122, pACYC184, pRSF1010/CAT, pBR322 and pAT153. In vitro and in vivo stability studies revealed that, with the exception of pcDNA3 and pUC18, the plasmids were retained by BRD509. However, pAT153 was the only plasmid to induce a tetanus toxoid-specific antibody response following oral delivery. Plasmid copy number was found to impact on plasmid stability and the induction of antigen-specific humoral responses^ref.
The use of the food-grade bacterium Lactococcus lactis as a DNA delivery vehicle at the mucosal level is an attractive DNA vaccination strategy. Previous experiments showed that recombinant L. lactis expressing the Listeria monocytogenesis inlA gene can deliver a functional gene into mammalian cells. The potential use of non-invasive L. lactis strains as DNA delivery vehicle was explored. 2 shuttle Escherichia coli-L. lactis plasmids, pLIG:BLG1 and pLIG:BLG2, were constructed containing a eukaryotic expression cassette with the cDNA of bovine b-lactoglobulin (BLG). The greatest BLG expression after transfection of Cos-7 cells was obtained with pLIG:BLG1, which was then used to transform L. lactis MG1363. The resulting L. lactis MG1363(pLIG:BLG1) strain was not able to express BLG. The potential of L. lactis as a DNA delivery vehicle was analyzed by detection of BLG in human colon carcinoma Caco-2 cells after 3 hours co-incubation with: i) purified pLIG:BLG1, ii) MG1363(pLIG:BLG1), iii) a mix of MG1363(pLIG) and purified pLIG:BLG1 and iv) MG1363. Both BLG cDNA and BLG expression were only detected in Caco-2 cells co-incubated with MG1363(pLIG:BLG1). There was a decrease in BLG cDNA in Caco-2 cells between 24 and 48 h after co-incubation. BLG expression by Caco-2 cells started at 24 h and increased between 24 and 72 h. BLG secretion by Caco-2 cells started 48 h after co-incubation with MG1363(pLIG:BLG1). Lactococci can deliver BLG cDNA into mammalian epithelial cells, demonstrating their potential to deliver in vivo a DNA vaccine^ref.
E. coli expressing invasin from Yersinia pseudotuberculosis selectively invade nonphagocytic cells in which b₁-integrin is expressed and accessible. Following internalization the E. coli are degraded in the phagosome. Coexpression of listeriolysin O (LLO) mediates release of the content of the bacteria into the cytosol of the invaded cell. In vitro and in vivo experiments demonstrated that gut epithelial cells failed to be invaded by invasive E. coli, due to a basolateral localization of b₁-integrin. By contrast, selective uptake of invasive bacteria from the intestinal lumen into Peyer's patches was observed ex vivo. Once in this structure, invasive E. coli colocalized with dendritic cells and possibly B cells. Oral administration of invasive E. coli coexpressing the model antigen ovalbumin and LLO from Listeria monocytogenes was able to elicit systemic protection against a lethal challenge of B16 tumor cells expressing ovalbumin. These data demonstrate the selectivity of invasin-mediated invasion to the Peyer's patches and indicate the potential of nonpathogenic, invasive E. coli as an oral vaccine with applications in immunotherapy^ref.
cell-wall-deficient Listeria monocytogenes as a DNA delivery vehicle in vitro. First, the d-alanine-deficient (Ddal-dat) L. monocytogenes strain DP-L3506, which undergoes autolysis inside eukaryotic host cells in the absence of d-alanine, was transformed with a plasmid encoding GFP under control of the CMV promoter (pAM-EGFP). Then COS-7 and MC57G cell lines were infected with the transformed DP-L3506 at various multiplicities of infection (MOI) in the presence or absence of d-alanine. Subsequent GFP expression was observed in both cell lines by 24 h post-infection with DP-L3506(pAM-EGFP). Notably, no GFP⁺ cells were observed when d-alanine was omitted. Although transfection efficiency initially increased as a result of d-alanine supplementation, high concentration or long-term supplementation led to sustained bacterial growth that killed the infected host cells, resulting in fewer GFP-expressing cells. Thus, efficient DNA delivery by transformed bacteria must balance bacterial invasion and survival with target cell health and survival^ref
non-replicating bacterial minicells simultaneously co-delivering the nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) and the corresponding DNA vaccine were tested for the ability to generate protective cellular immune responses in mice. It was found that good protection (89%) was achieved after intramuscular administration, moderate protection (31%) was achieved after intranasal administration, and less protection (7%) was achieved following gastric immunization. These results provide a solid foundation on which to pursue the use of bacterial minicells as a non-replicating vaccine delivery platform^ref.

pDNAVACCultra (source : Nature Technology Corp.) is a family of DNA vaccine vectors, optimized and minimized to comply with FDA guidelines regarding content and elimination of extraneous materials, was constructed. The resulting vectors are much smaller than existing vectors, drive higher levels of target gene expression, facilitate high throughput cloning applications, and allow simultaneous cloning into multiple vectors that feature various intracellular targeting destinations for the protein product. The ability to control expression and trafficking is intended to provide a rapid, rational approach to cancer therapy and emerging infectious diseases^ref
in vivo active targeting of DNA vaccine to DCs with Man-lipoplex can enhance cytotoxic T lymphocyte (CTL) activity through efficient gene delivery to dendritic cells (DCs) by mannose receptor-mediated endocytosis. Ovalbumin (OVA) was selected as a model antigen for vaccination; accordingly, OVA-encoding pDNA (pCMV-OVA) was constructed to evaluate DNA vaccination. Mannosylated cationic liposomes (Man-liposomes) were prepared using cholesten-5-yloxy-N-{4-[(1-imino-2-D-thiomannosylethyl)amino]butyl}formamide (Man-C4-Chol) with cationic lipid. The potency of the mannosylated liposome/pCMV-OVA complex (Man-lipoplex) was evaluated by measuring OVA mRNA in CD11c⁺ cells, CTL activity, and the OVA-specific anti-tumor effect after in vivo administration. An in vitro study using DC2.4 cells demonstrated that Man-liposomes could transfect pCMV-OVA more efficiently than cationic liposomes via mannose receptor-mediated endocytosis. In vivo studies revealed that the Man-lipoplex exhibited higher OVA mRNA expression in CD11c⁺ cells in the spleen and peritoneal cavity and provided a stronger OVA-specific CTL response than intraperitoneal (i.p.) administration of the conventional lipoplex and intramuscular (i.m.) administration of naked pCMV-OVA, the standard protocol for DNA vaccination. Pre-immunization with the Man-lipoplex provided much better OVA-specific anti-tumor effect than naked pCMV-OVA via the i.m. route^ref
Encapsulating DNA within degradable delivery vehicles such as micro- or nanospheres provides an effective way to protect the DNA from the surrounding environment prior to delivery. The ability to target these vehicles directly to the cell type of interest provides a way to enhance the overall efficiency of DNA delivery. One means of highly specific cell targeting is through the addition to the vehicle surface of ligands that bind specifically to receptors on the surface of the targeted cell type. Covalent conjugation of ligands to the surface of degradable delivery vehicles can be difficult, as the most commonly used vehicle formulations use materials selected for their general chemical inertness^ref
inert poly-l-lysine (PLL) coated polystyrene particles greatly enhance DNA immunogenicity. Intradermal injection of plasmid DNA encoding for chicken egg ovalbumin (OVA) complexed with PLL-coated polystyrene nanoparticles induced high levels of CD8 T cells as well as OVA-specific antibodies in C57BL/6 mice and furthermore inhibited tumour growth after challenge with the OVA expressing EG7 tumour cell line. Importantly, vaccine efficacy depended critically on the size of the particles used as well as on the presence of the PLL linker. PLL-coated polystyrene nanoparticles of 0.05 mm but not 0.02 mm or 1.0 mm in diameter are highly effective for the delivery of DNA vaccines^ref.
a first intradermal administration of one DNA vaccine dose combined with the slow-release of a second dose conduct to a priming of the bovine herpesvirus 1-specific immune response similar to the one generated by 2 discrete administrations 4 weeks apart. The first experiment demonstrates the efficacy of the slow-release system with well-characterized Alzet^® osmotic pumps, whereas the second experiment extends the same concept with innovative agarose hydrogel implants. These latter implants are cheaper and more convenient than the osmotic pumps or repeated intradermal administrations since they contribute to an efficient priming of the immune response in a single manipulation of the animals^ref
exploiting the activity of VP22 to enhance the delivery of antigens encoded by DNA vaccine plasmids^ref

Adjuvants

for DNA vaccines :

GM-CSF : to assess GM-CSF immune accessory effects in tumor-bearing mice, an animal tumor model was established by inoculating SP2/0 myeloma cells s.c. into the flank of Balb/c mice and 14 days later, injecting either 400 mg recombinant pcDNA3.1/mGM-CSF or a blank plasmid s.c. or i.m. into the tumor four times. The tumor weight, the activities of CTL and NK, the serum levels of IFN-g, IL-2 and lymphocytes infiltrating in tumor tissue were analysed 8 weeks later with MTT, ELISA and pathological section methods. The tumor lump was reduced in mice injected s.c. (0.880 +/- 0.405 g) or i.m. (0.378 +/- 0.411 g) with pcDNA3.1/mGM-CSF compared with control mice injected s.c. (1.548 +/- 0.221g, P < 0.01)or i.m. (1.554 +/- 0.249g, P < 0.001) with a blank vector. Lymphocyte infiltration in tumor tissues was very apparent in mice injected i.m. with pcDNA3.1/mGM-CSF. In contrast, there was no lymphocyte infiltration in tumor tissues of control mice. In addition, the serum concentrations of IFN-g, IL-2 and the activities of CTL and NK cells were significantly increased in mice injected with pcDNA3.1/mGM-CSF compared with a control mice (P < 0.01)^ref. A novel codon-optimized murine GM-CSF gene significantly increased protein expression levels in all cells tested. Although injection of the wild-type GM-CSF plasmids adjuvanted HIV-1 Gag DNA vaccine induced detectable immune responses, co-administration of plasmids encoding the codon-optimized GM-CSF sequence with the DNA vaccine resulted in a strong antibody and CTL responses and a protective immune response against infection with recombinant vaccinia virus expressing HIV-1 Gag^ref
dual-promoter plasmids that carry an antigen and a TLR adaptor molecule such as the TRIF or MyD88 were constructed and administered to mice to determine if these molecules can act as an adjuvant. A DNA vaccine incorporated with the MyD88 genetic adjuvant enhanced antigen-specific humoral immune responses, whereas that with the TRIF genetic adjuvant enhanced cellular immune responses. Incorporating the TRIF genetic adjuvant in a DNA vaccine targeting the influenza HA antigen or the tumor-associated antigen E7 conferred superior protection^ref.
IL-18 : to determine if the properties of IL-18 could be optimized for use as a DNA vaccine adjuvant, a model of IL-18/IL-18R binding was developed to identify variants of human IL-18 that were predicted to improve receptor interactions and potentially bioactivity. The linkage of mature IL-18 to a secretion signal sequence provided improved protein expression from mammalian cells and signal peptidase cleavage of this protein produced the authentic N-terminus. The IL-18 variant proteins secreted this way were bioactive, as demonstrated by their ability to induce IFN-g expression by human peripheral blood mononuclear cells (PBMCs) and to bind to IL-18R, as demonstrated by BIAcore analysis. The IL-18 variants were inhibited by IL-18 binding protein (IL-18BP), the soluble inhibitor of IL-18, as measured by neutralization of the IFN-g response in PBMCs. One variant, V11I/T63A, demonstrated increases both in bioactivity and mammalian cell expression as compared to native IL-18, indicating that this molecule may be particularly well suited for use as a DNA-encoded vaccine adjuvant^ref.
trichostatin-A (TSA) is a specific inhibitor for histone deacetylase (HDAC) and a gene expression enhancer in in vitro DNA viral infection, including HSV-1 and CMV. A study sought to determine if increasing antigen expression of DNA vaccines through inhibition of HDAC-induced chromatin silencing using TSA would enhance DNA vaccine efficacy in vivo. A luciferase assay was used to detect effects of TSA on different promoters in vitro. The effects of TSA on DNA vaccination of mice were determined by neutralization assays for antibody production and interleukin staining for detection of specific T cell responses. Mice receiving TSA in combination with a DNA vaccine exhibited higher antibody responses to the vaccine than mice not given TSA. Co-administration of TSA also enhanced specific CD8 T cells response. Drugs such as TSA that reduce initial gene silencing by preventing histone deacetylation can increase immune responses to DNA vaccination^ref
seeking to combine the immunogenicity of virus-like particles (VLPs) and the ease of production of plasmid DNA, DNA vaccines expressing VLPs consisting of the MLV Gag and modified MLV Env proteins displaying T cell epitopes have been designed. Such DNA vaccines are remarkably efficient immunogens for inducing cellular immune responses. In contrast to similar plasmids harboring a point mutation preventing VLP formation, they induce protection against a lethal viral challenge in mice. Thus, these "plasmo-retroVLPs" represent a promising second-generation DNA vaccine^ref.
plasmid DNAs encoding cytokines enhance immune responses to vaccination in models of infectious diseases and cancer. DNAs encoding cytokines that affect T cells [IL-2, IL-12, IL-15, IL-18, IL-21, and the chemokine CCL21] and antigen-presenting cells [GM-CSF] were compared in mouse models as adjuvants to enhance CD8⁺ T-cell responses and tumor immunity. A DNA vaccine against a self-antigen, gp100, expressed by melanoma was used in combination with DNA encoding cytokines and cytokines fused to the Fc domain of mouse IgG₁ (Ig). Cytokine DNAs generally increased CD8⁺ T-cell responses against gp100; ligation to Fc domains further enhanced T-cell responses; adjuvant effects were sensitive to timing of DNA injection; (d) the most efficacious individual adjuvants for improving tumor-free survival were IL-12/Ig, IL-15/Ig, IL-21/Ig, GM-CSF/Ig, and CCL21; and (e) combinations of IL-2/Ig + IL-12/Ig, IL-2/Ig + IL-15/Ig, IL-12/Ig + IL-15/Ig, and IL-12/Ig + IL-21/Ig were most active; and (f) increased adjuvanticity of cytokine/Ig fusion DNAs was not related to higher tissue levels or greater stability. These observations support the potential of cytokine DNA adjuvants for immunization against self-antigens expressed by cancer, the importance of timing, and the enhancement of immune responses by Fc domains through mechanisms unrelated to increased half-life^ref
nanoparticle-based DNA vaccine delivery system : the nanoparticles were engineered by cooling pre-formed warm microemulsions comprised of emulsifying wax as the oil phase and hexadecyltrimethyl ammonium bromide (CTAB) as the surfactant. However, the poor aqueous stability of the nanoparticles and the emulsifying wax in the nanoparticles may severely limit the applications of the nanoparticles. In the present study, we used lecithin, a more biocompatible material, instead of emulsifying wax, to prepared lecithin-based cationic nanoparticles. The 50% growth inhibition concentration (IC₅₀) of the lecithin-based nanoparticles was found to be more than 1000-fold higher than that of the emulsifying wax-based nanoparticles. Moreover, the stability of the lecithin nanoparticles was also significantly increased. The size of the nanoparticles did not significantly change during a 6-month storage period at room temperature. Finally, when plasmid DNA was adsorbed on their surface, the lecithin nanoparticles successfully transfected cells in culture. These lecithin-based nanoparticles may hold great potentials as a DNA (vaccine) delivery system^ref.
Den123 : a nontoxic self-assembled dendritic spheroidal nanoparticle made of biodegradable monomers) : footpad DNA immunization in Balb/c mice was done three times using the Bet v 1a gene with or without Den123 with 2-week intervals followed by sensitization with rBetv1 (5 mg) in alum twice in a weekly interval. Different doses of pCMV-Betv1 were used (10 mg and 100 mg). The protective role of different formulations was evaluated by measuring the IgG₁, IgG_2a and IgE antibody production, cytokine release of isolated splenocytes and b-hexosaminidase release from the RBL cells. Higher and increasing ratios of IgG_2a/IgG₁ were seen in mice which received plasmids in combination with Den123. Den123 and DNA vaccine synergistically enhanced the Interferon gamma released from splenocytes. In the presence of Den123, IgE inhibition was independent of the dose and type of the injected DNA. All DNA-pre-immunized mice demonstrated low basophil degranulation. It is therefore concluded that administration of the DNA entrapped in Den123 nanoparticles results in sustained release of plasmids, T_h1/T_h2 balanced immune response with promising IgE inhibition. Also higher amounts of DNA contributed to stronger T_h1 response^ref.
recombinant reovirus type 3 s₁ attachment protein genetically modified with a nuclear localization sequence (sigma1-NLS) as a targeting ligand. Purified sigma1-NLS was covalently conjugated to the polycation polyethyleneimine (PEI) using a carboxyl-reactive cross-linking agent and complexed with plasmid DNA. The benefit of the NLS in enhancement of protein delivery into the nucleus was demonstrated by liposome-mediated loading of cells with sigma1 or sigma1-NLS. In L929 fibroblasts loaded with sigma1-NLS, 69% of the internalized protein was recovered in the nuclear fraction after 6 h compared to just 10% when using unmodified sigma1. Transfection of L929 cells with sigma1-NLS-conjugated PEI complexed with a luciferase expression plasmid resulted in a mean 16-fold increase in luciferase activity over complexes made with unmodified PEI, compared to a mean 3-fold boost obtained using sigma1-conjugated PEI. These results suggest that sigma1-NLS is a useful bifunctional targeting ligand suitable for enhancing DNA delivery and subsequent gene expression for both DNA vaccine applications and nonviral gene therapy^ref.
targeting antigens to FcgRs on DCs has been demonstrated to enhance antigen presentation. CCL21 / secondary lymphoid tissue chemokine (SLC) has been shown to increase immune responses not only by promoting coclustering of T cells and DCs in the lymph nodes and spleen but also by regulating their immunogenic potential for the induction of T cell responses. Using HPV 16 E7 as a model antigen, a chemotactic-antigen plasmid DNA vaccine (pSLC-E7-Fc) was constructed by linking SLC and Fc gene sequences to each end of E7 and evaluated its potency of eliciting specific immune response. Immunization with pSLC-E7-Fc generated much stronger E7-specific lymphocyte proliferative and CTL responses than control DNA. All the mice receiving pSLC-E7-Fc prophylactic vaccination remained tumor free upon subcutaneous inoculation of TC-1 cells, while those given control DNA all developed tumors. These tumor-free mice were also protected against TC-1 rechallenge. Complete tumor regression with long-term survival occurred in 72% of mice given pSLC-E7-Fc as therapeutic vaccination. In experimental lung metastasis model wherein TC-1 cells were intravenously injected, therapeutic vaccination with pSLC-E7-Fc significantly reduced the number of tumor nodules in the lung. In vivo depletion with antibodies against CD4⁺or CD8⁺ T cells both resulted in complete abrogation of the pSLC-E7-Fc-induced immunotherapeutic effect. These data indicate that the DNA vaccine constructed by the fusion of SLC and IgG Fc fragment genes to antigen-coding gene is an effective approach to induce potent anti-tumor immune response via both CD4⁺ and CD8⁺ T cells dependent pathways^ref.
CIA07 : a combination of 0.5-2.0-kb DNA fragments and modified LPS at a ratio of 100:1 exhibited higher immunostimulating activity than each substance alone, and its antitumor activity was significantly higher than that of Bacillus Calmette-Guerin in a mouse bladder cancer model. The use of Escherichia coli DNA or lipopolysaccharide (LPS) as an immunotherapy is often associated with unacceptable toxicity and insufficient therapeutic effects. In this study, we investigated the efficacy of using a combination of bacterial DNA fragments and LPS as an anticancer agent. LPS was isolated from an E. coli strain expressing short-carbohydrate-chain-containing LPS and subjected to alkaline hydrolysis to remove lipid A. The ability to induce TNF-a release in human whole blood cells was significantly lower for the LPS devoid of lipid A than for its parent form. The immunostimulating activity of E. coli DNA fragments of various sizes were tested. Those of 0.2-0.5kb in size exhibited the highest activity in whole blood assays, whereas those of size 0.5-2.0kb exhibited the highest adjuvant activity in mice. An intraperitoneal injection of CIA07 at a dose of 25mg/kg body weight caused no apparent adverse effects in mice and guinea pigs. Taken together, these data demonstrate that CIA07 exhibits potent immunostimulating activity with no apparent toxicity, and therefore warrant the further development of CIA07 as an immunotherapy for cancer treatment^ref.
in vivo electroporation of a DNA vaccine adjuvanted with plasmids encoding different cytokines was investigated in large animals. Sheep were injected intramuscularly with a DNA vaccine encoding an antigen of Haemonchus contortus (pNPA) and plasmids encoding different cytokines followed by in vivo electroporation. Plasmids (pCI) carrying the genes of different cytokines including ovine IL-4(pCI-IL4), IL-10(pCI-IL10), GM-CSF(pCI-GMCSF), and MCP-1a(pCI-MCP1a), and pCI-IL4+pCI-GMCSF were co-delivered with pNPA. The results showed that co-delivery of pCI-GMCSF or pCI-IL4+pCI-GMCSF significantly enhanced both antibody responses and T cell proliferation responses to the antigen after two DNA immunisations compared to co-delivery of pCI. In contrast, antibody responses of the sheep that received pCI-IL10 were decreased significantly. Other cytokine expressing plasmids did not significantly alter the measured immune responses. Furthermore, co-delivery of pCI-GMCSF increased IgG₂ response more than IgG₁responses, suggesting a T_h1 bias. However, the increase in IgG₂ over IgG₁ was less apparent when co-delivery of pCI-IL4 with pCI-GMCSF. Interestingly, the co-delivery of pCI-IL4 alone did not increase the IgG₁ titre, suggesting that both pCI-GMCSF and pCI-IL4 are required for optimal IgG₁ production^ref
geldanamycin (GA), a heat-shock protein (HSP) 90 inhibitor, induces degradation of HSP90 client proteins, which may promote the presentation of degradation peptides with MHC class I on cancer cells. GA may enhance the efficacy of DNA vaccination, and investigated the therapeutic effect of the combination of GA and a DNA vaccine against HSP90 clients p185^neu and Met. The efficacy of various doses of GA combined with an N-terminal neu (N'-neu) DNA vaccine was investigated in a transplanted tumor constitutively overexpressing endogenous p185(neu). Low-dose (2.5 mg) but not high-dose (10 mg) GA enhanced the effect of N'-neu DNA vaccination on the inhibition of murine bladder tumor-2 tumors in syngeneic C3H mice. Anti-p185(neu) antibody titers were similar among all treated groups. Significantly increased infiltrations of CD8⁺ T cells and NK cells were observed at tumor sites. GA sensitized tumor cells to the cytotoxic effects of lymphocytes. Depletion of CD8⁺ T cells eliminated most of the therapeutic efficacy; in contrast, depletion of CD4⁺ T cells enhanced the therapeutic efficacy. A similar enhancing effect was observed for the combination of GA and a DNA vaccine targeting the Met oncogene. Combination of GA and DNA vaccination against GA-targeted proteins^ref
DNA vaccine encoding a chimeric fusion between FasL and a truncated version of HIV gp120 as a model antigen. The fusion DNA was used as a priming vaccine, along with boosting with recombinant gp120 protein. Priming with fusion protein DNA resulted in a powerful enhancement of immune responses to the protein boost, and, in the presence of aluminum phosphate, to a strong enhancement in T_h2 type responses. Fas ligand delivered in a separate plasmid also had an adjuvant effect, although it was weaker than that delivered by the fusion protein^ref
dendriplexes, complexes of dendrons and condensed plasmids containing the gene for protective antigen (PA) of Bacillus anthracis, were encapsulated in poly-lactide-co-glycolide (PLGA) particles using the double emulsion method. The 2 dendrons employed are a dendron with three C(18) chains (C(18) dendron) and one with no attached hydrocarbon chains (the C(0) dendron). 3 types of particles were examined, namely PLGA-C(18) dendriplexes, PLGA-C(0) dendriplexes and the control PLGA-naked DNA system. These were characterised by standard biophysical methods such as photon correlation spectroscopy (PCS) and scanning electron microscopy to select the complexes for in vivo testing. 3 intramuscular immunizations were carried out using 14 mg of DNA per dose at weekly intervals in BALB/c mice. Antibodies against rPA were measured using ELISA. Results indicate that the PLGA-C(18) dendriplex particles produced superior levels of anti-PA IgG antibodies in comparison to animals immunized with the PLGA-C(0) dendriplex particles. The level of antibody production was dependent on the number of immunizations, higher antibody levels being measured after two booster vaccinations. However toxin neutralizing antibodies were absent in all treatment groups, and it is likely that the mice lack protection against lethal toxin and anthrax infection. Further studies are needed to optimize the formulation of DNA vaccines and increase the level of anti-lethal toxin antibodies and enhance their functionality^ref

Vaccines :

anti-infectious diseases vaccines^ref :

anti-prion vaccines :

DNA immunization of wild-type mice can break immune tolerance against the prion protein, resulting in the induction of PrP-specific antibody and T-cell responses. PrP immunogenicity was increased by fusion to the lysosomal targeting signal from LIMPII (lysosomal integral membrane protein type II). Although mice immunized with a PrP-LIMPII DNA vaccine showed a dramatic delay in the onset of early disease signs after intracerebral challenge, immunization against PrP also had some deleterious effects. These results clearly confirm the feasibility of using active immunization to protect against TSEs and, in the absence of effective treatments, indicate a suitable alternative for combating the spread of these diseases^ref

anti-viral vaccines

anti-HIV-1 DNA vaccine (see also protein subunit vaccine and therapeutic vaccines )^ref : the ability of SIVDnef to induce a high frequency virus-specific CD4⁺ T cell response with direct effector function may play a key role in protective immunity produced by vaccination with attenuated SIV strains^ref

enzymatically active reverse transcriptase, but not the inactive mutant^ref
oral or rectal immunization with recombinant attenuated Salmonella typhimurium SL7207 strain producing TBI protein (artificial protein containing HIV-1 B and T cell epitopes)^ref
oral and rectal immunization with recombinant attenuated Salmonella typhimurium SL7207 strain producing gp-160^ref
rectal immunization with recombinant attenuated Salmonella typhimurium SL7207 strain could be perspective for delivery plasmid Env DNA and more effective than oral immunization^ref
multi-epitope T-cell immunogen (TCI), 392 amino acids in length and containing 80 epitopes (both CD8⁺ CTL and CD4⁺ T_h)^ref

rectal immunization with recombinant attenuated Salmonella typhimurium SL7207 strain^ref
i.m. injection of an artificial virus like particle containing eukaryotic expression plasmid pcDNA-TCI encapsulated within a spermidine-polyglucin conjugate^ref

a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope with 3 copies of the murine C3d (mC3d₃) added to the carboxyl terminus^ref
AVX101^® (source : AlphaVax, Inc.) consists of a weakened strain of VEEV , which acts as a vector to carry HIV genetic material into host cells. AVX101 is the first vaccine to be tested that has been designed specifically to target HIV-1 subtype C, which is most prevalent in South Africa, rather than subtype B, which is predominant in the developed world and for which other vaccine trials are underway
tgAAC09 (developed by Seattle-based Targeted Genetics and the Columbus Children's Research Institute in Ohio) uses rAAV encoding HIV gag and pro genes and a portion of the reverse transcriptase gene. Unlike many vaccines currently in clinical trials, tgAAC09 potentially could be a single-shot vaccine, which would be useful in developing countries. In addition, animal trials have shown that the vaccine candidate can stimulate both an antibody and a cell-mediated response to HIV. The vaccine focuses on HIV subtype C, which is the most common strain of the virus in India, so India has begun its first-ever phase I clinical trial of an HIV vaccine, which is being conducted by the Indian Council of Medical Research and the International AIDS Vaccine Initiative, is testing the vaccine candidate called The Indian trial is part of a multicountry Phase I trail of tgAAC09, which already has started in Europe with German and Belgian researchers testing the vaccine in partnership with IAVI. IAVI beginning in March 2001 provided $3 million over 18 months to the Indian government to fund HIV vaccine development efforts. The trial, which is expected to take about 15 months to complete, will test the vaccine on 30 male and female volunteers ages 18 to 45 who are HIV-negative and do not have any other significant illnesses. The trial will take place at the National AIDS Research Institute in Pune, India. ICMR Director N.K. The candidate vaccine might stimulate an immune system response that could protect volunteers from potential HIV infection. The volunteers have been made aware of the risks involved, they have been counseled and their health status will be monitored all the time. The European trial -- conducted at the Centre Hospitalier Universitaire Saint-Pierre in Brussels, Belgium -- is testing the safety and immune response of the vaccine in 50 male and female volunteers. If the vaccine is shown to be safe, it might advance to larger clinical trials, which could involve thousands of people at high risk of contracting HIV, such as commercial sex workers. Targeted Genetics expects to report data from the European trial in the first half of 2005. Web resources : Henry J. Kaiser Family Foundation : kaisernetwork.org.
a DNA vaccine consisting of 7 plasmids encoding 9 HIV-1 proteins. Using a needle-free delivery device, the Biojector, together with recombinant mouse GM-CSF, this vaccine induced strong gp160 Env- and p24 Gag-specific cellular and humoral immune responses in mice. The rGM-CSF was crucial for inducing both antibodies and antigen-specific CD8⁺ T cell responses against both gp160 and p24. A GMP-produced lot of this vaccine, intended for human use, was delivered intradermally or intramuscularly into BALB/c mice at a GLP-accredited animal facility. This vaccine induced strong cellular responses independent of the route of immunization; moreover, no signs of toxicity were detected after histopathological examination of various tissues. This vaccine has been approved by the Swedish Medicinal Products Agency and is currently in a Phase I clinical trial^ref.
current evidence suggests that a strong induced CD8 HIV-1-specific cell mediated immune response may be an important aspect of an HIV vaccine. The response rates and the magnitude of the CTL responses induced by current DNA vaccines in humans need to be improved and cellular immune responses to DNA vaccines can be enhanced in mice by co-delivering DNA plasmids expressing immune modulators. 2 reported to work well in the mouse systems are IL-12 and CD40L . The cDNA for macaque IL-12 and CD40L were cloned into DNA vectors. Groups of cynomolgus macaques were immunized with 2 mg of plasmid expressing SIVgag alone or in combination with either IL-12 or CD40L. CD40L did not appear to enhance the cellular immune response to SIVgag antigen. However, more robust results were observed in animals co-injected with the IL-12 molecular adjuvant. The IL-12 expanded antigen-specific IFN-g⁺ effector cells as well as granzyme B production. The vaccine immune responses contained both a CD8 component as well a CD4 component. The adjuvanted DNA vaccines illustrate that IL-12 enhances a CD8 vaccine immune response, however, different cellular profile^ref. Both 2-trimer and 4-trimer forms of macaque CD40L were active in B cell proliferation assays using macaque and human cells. Using human cells, 4-trimer macaque GITRL costimulated CD4⁺ T cell proliferation, and abrogated the immunosuppressive effects of CD4⁺CD25⁺ regulatory T cells on a MLR. These molecular adjuvants provide new tools for vaccine development in the SIV system and other macaque models^ref
prime-boost regimens :

heterologous plasmid DNA prime, live vector boost vaccination regimen appears especially promising in the nonhuman primate/ simian-human immunodeficiency virus (SHIV) challenge model. A series of intramuscular priming immunizations with a plasmid DNA vaccine expressing SIVgag p39, in combination with plasmid expressed rhesus IL-12, could effectively enhance the immunogenicity and postchallenge efficacy of 2 intranasal doses of recombinant vesicular stomatitis virus (rVSV)-based vectors expressing HIV-1 env 89.6P gp160 and SIVmac239 gag p55 in rhesus macaques. In macaques receiving the combination plasmid DNA prime, rVSV boost vaccination regimen, a significantly increased SIVgag- specific cell-mediated and humoral immune responses and significantly lower viral loads postintravenous SHIV89.6P challenge relative to macaques receiving only the rVSV vectored immunizations were observed. In addition, the plasmid DNA prime, rVSV boost vaccination regimen also tended to increase the preservation of peripheral blood CD4⁺ cells and reduce the morbidity and mortality associated with SHIV89.6P infection. An analysis of immune correlates of protection after SHIV89.6P challenge revealed that the prechallenge SHIV-specific IFN-g ELISpot response elicited by vaccination and the ability of the host to mount a virus-specific neutralizing antibody response postchallenge correlated with postchallenge clinical outcome. The correlation between vaccine-elicited cell-mediated immune responses and an improved clinical outcome after SHIV challenge provides strong justification for the continued development of a cytokine-enhanced plasmid DNA prime, rVSV vector boost immunization regimen for the prevention of HIV infection^ref. A mutant full-sized plasmid DNA vaccine regime in macaques was effective against a homologous challenge^{ref1,
ref2} : to evaluate the DNA vaccination regime against a heterologous challenge, a novel plasmid named pSHIV-ZF1*IL-2 was constructed. 4 monkeys were intramuscularly and intradermally injected 4 times with the pSHIV-ZF1*IL-2. Vaccinated monkeys were intravenously challenged with a highly pathogenic, heterologous SHIV at 11 weeks post vaccination. All the vaccinated monkeys suppressed the challenge virus rapidly under the detectable level by 16 weeks post challenge. One vaccinated monkey was protected from a loss of CD4⁺T cells. These results suggest pSHIV-ZF1*IL-2 alone seems partially effective even against a challenge with a heterologous, pathogenic virus^ref.
gp120/gag DNA priming- gp120 protein boosting : immunization of macaques with multivalent DNA encoding gp120 genes from HIV-1 subtypes A, B, C and E and a gag gene followed by boosting with homologous gp120 proteins elicited strong anti-gp120 antibodies capable of neutralizing homologous and to a lesser degree heterologous HIV-1 isolates. Both Env- and Gag-specific cell mediated immune (CMI) responses were detected in the immunized animals. Following rectal challenge with an SHIV isolate encoding HIV-1(Ba-L)env, plasma viremia in the infected immunized animals was significantly lower than that observed in the naive animals. Further, one of 6 immunized animals was completely protected whereas all six naive animals were infected. These results demonstrate that a vaccine based on priming with a polyvalent DNA vaccine from multiple HIV-1 subtypes followed by boosting with homologous Env proteins elicits anti-HIV-1 immune responses capable of controlling rectal transmission of SHIV(Ba-L)^ref
priming with DNA vaccines expressing primary HIV-1 envelope glycoprotein (Env) followed by recombinant Env protein boosting was successful in generating positive neutralizing antibody responses against a clade B primary HIV-1 isolate, JR-FL, that was not easily neutralized. The DNA priming plus recombinant protein boosting approach delivering a polyvalent primary Env formulation was able to generate neutralizing antibodies against primary HIV-1 viral isolates from various genetic subtypes. New Zealand White rabbits were first immunized with DNA vaccines expressing 1, 3 or 8 primary HIV-1 gp120 antigens delivered by a gene gun followed by recombinant gp120 protein boosting. Neutralizing antibody responses were examined by two independently executed neutralization assays: the first one was a single round infection neutralization assay against a panel of 10 primary HIV-1 isolates of subtypes A, B, C and E and the second one used the PhenoSense assay against a panel of 12 pseudovirues expressing primary HIV-1 Env antigens from subtypes A, B, C, D and E as well as 2 pseudoviruses expressing the Env antigens from MN and NL4-3 viruses. Rabbit sera immunized with the DNA priming plus protein boosting approach, but not DNA vaccine alone or Env protein alone, were capable of neutralizing 7 of 10 viruses in the first assay and 12 of 14 viruses in the second assay. More importantly, sera immunized with the polyvalent Env antigens were able to neutralize a significantly higher percentage of viruses than the sera immunized with the monovalent antigens. DNA priming followed by recombinant Env protein boosting can be used to deliver polyvalent Env-antigen-based HIV-1 vaccines to elicit neutralizing antibody responses against viruses with diverse genetic sequence variations^ref

given the importance of the HIV-specific cell-mediated immune response in the early control and resolution of HIV infection and the observed correlation between pre-challenge vaccine elicited CTL responses and post challenge outcome in SHIV/rhesus macaque experiments, several candidate plasmid DNA (pDNA) vaccine designs capable of eliciting robust and balanced cell-mediated immune responses to multiple HIV-1 derived antigens in mice were identified for further vaccine development. To rationally construct candidate vaccines for immunogenicity testing, the relative immunogenicity of the individual HIV-derived vaccine antigens (env, gag, pol, nef, tat and vif) and the relative strength of various transcriptional control elements (HCMV, SCMV, HSV Lap1) in Balb/c mice were determined. Next, a number of 1-, 2-, 3- and 4-vector pDNA vaccine designs were tested for their ability to elicit HIV-1 antigen-specific CMI responses. For these studies, Balb/c mice were immunized with a fixed total pDNA vaccine dose of 100 mg in combination with 25 mg plasmid-based murine IL-12 and tested for the induction of HIV-1 antigen-specific CMI responses by IFN-g ELISpot analysis. The results of this study indicate that all pDNA vaccine designs were capable of eliciting CMI responses to multiple HIV-1 antigens. As a result of this iterative comparative analysis, we have identified a number of pDNA vaccine candidates capable of eliciting potent, balanced CMI responses to multiple HIV-1 derived antigens. These results have important implications for the design and development of an efficacious vaccine for the prevention of HIV-1 infection^ref
optimization of codon usage, enhancement of viral promoter function and selection of secretory leader sequences can work synergistically to improve the final antigen expression and immunogenicity of HIV-1 Env DNA vaccines, indicating they work through different mechanisms. The best result came from the approach that optimized all 3 components in a DNA vaccine design. The levels of HIV-1 env-specific RNA transcripts in transiently transfected 293T cells were higher from the codon-optimized gene than the wild type counterpart. This finding suggested other mechanism may also contribute to the increased antigen expression and immunogenicity of codon-optimized DNA vaccines in addition to the improved tRNA usage in mammalian cells for codon-optimized viral genes as previously reported^ref
a plasmid DNA vaccine containing a fusion gene consisting of an HIV-1 subtype C gag and a modified subtype C pol was compared to a mixture of gag plus pol or gag plus HIV env plasmids. Plasmid DNA was delivered by intramuscular injection followed by electroporation in vivo. 2 vaccinations were sufficient to induce high levels of Gag- and Pol-specific CD4 and CD8 T cells in peripheral blood. The gag-pol fusion plasmid was as immunogenic as the plasmid mixtures. Thus, DNA vaccination by intramuscular electroporation was an effective means for inducing high levels of Gag- and Pol-specific T cells, and a single gag-pol fusion DNA vaccine was sufficient for eliciting immune responses against both antigens^ref
recombinant DNA vaccine delivered intramuscularly and recombinant Listeria monocytogenes delivered orally induced CD8⁺ and CD4⁺ T cell immune responses in rhesus macaques and this vaccine protocol showed partial protection against an SIV239 challenge. The SIV antigen-specific immune responses at the time of challenge and during the subsequent infection course. The immune status of the animals, as measured by the frequency of antigen-specific IFN-g secreting PBMCs, at the time of challenge correlates more strongly with viral loads at set point than peak viral loads. The correlation between the immune response and viral load was strongest early, as viral set-point was just being established and disintegrates overtime. This study demonstrates the cellular immune response to SIV at the time of challenge of a nonhuman primate is able to impact on viral set-point following SIV239 challenge. Further, this study demonstrates that as virus replicates the T cell immune response to SIV antigens induced by the vaccine is modulated by antigen encountered by immune cells during viral replication^ref
a potent DNA vaccine against HIV, combining a vector that takes advantage of the segregation and compartmentalization effect of bovine papilloma virus E2 protein with MultiHIV insert, expressing a fusion gene coding for the non-structural and structural proteins was developed and tested for immunogenicity in mice and humans^ref
the design and preclinical development of a multigene HIV-1 subtype C DNA vaccine are described, developed as part of the South African AIDS Vaccine Initiative (SAAVI). Genetic variation remains a major obstacle in the development of an HIV-1 vaccine and recent strategies have focused on constructing vaccines based on the subtypes dominant in the developing world, where the epidemic is most severe. The vaccine, SAAVI DNA-C, contains an equimolar mixture of 2 plasmids, pTHr.grttnC and pTHr.gp150CT, which express a polyprotein derived from Gag, reverse transcriptase (RT), Tat and Nef, and a truncated Env, respectively. Genes included in the vaccine were obtained from individuals within 3 months of infection and selection was based on closeness to a South African subtype C consensus sequence. All genes were codon-optimized for increased expression in humans. The genes have been modified for safety, stability and immunogenicity. Tat was inactivated through shuffling of gene fragments, whilst maintaining all potential epitopes; the active site of RT was mutated; 124 aa were removed from the cytoplasmic tail of gp160; and Nef and Gag myristylation sites were inactivated. Following vaccination of BALB/c mice, high levels of cytotoxic T lymphocytes were induced against multiple epitopes and the vaccine stimulated strong CD8⁺ IFN-g responses. In addition, high titres of antibodies to gp120 were induced in guinea pigs. This vaccine is the first component of a prime-boost regimen that is scheduled for clinical trials in humans in the USA and South Africa^ref
The worldwide HIV-1 vaccine research endeavor is focused increasingly on subtype C, which is now the predominant strain of the present HIV/AIDS epidemic. Expression cassettes of HIV-1 subtype C gag, pol and versions of gagpol fusion cassettes were constructed and evaluated for their relative abilities to induce cellular immune responses in mice. Animals were vaccinated with DNA or alphavirus replicon particle-based vaccines and cellular immune responses were measured by flow cytometry. 5 new MHC class I-restricted T cell epitopes in subtype C Gag and Pol were identified. Although two CD8⁺ T cell epitopes within Gag were immunodominant in BALB/c and CB6F1 mice, the overall breadth of the T cell responses in mice immunized with plasmids or recombinant alphavirus replicon particles encoding gagpol fusion genes was improved over single antigen genes (i.e. gag or pol alone). The patterns of epitope dominance were consistent among mice although there were variations observed between different animals in the relative contributions of the various epitopes to the total response. These data are consistent with observations in non-human primates^ref and support a subtype C in-frame gagpol fusion gene vaccine^ref
recombinant vaccinia virus-based vaccine combined with DNA vaccine has produced a protective immune response against HIV infection in non-human primates. A recombinant vaccinia virus (LC16m8 strain) has been used in children without severe side effects. The vaccinia virus expressing an HIV(89.6)env gene (vLC-Env) alone or combined with a DNA vaccine expressing the HIV(89.6)env gene (pCAG-Env) was characterized in BALB/c mice. Vaccination of vLC-Env induced much higher HIV-specific humoral and cell-mediated immune responses than that of pCAG-Env. Priming with pCAG-Env further enhanced vLC-Env induced immune responses, especially cell-mediated immune response. Moreover, efficient expression of Env protein was achieved following infection of bone marrow dendritic cells by vLC-Env in vitro. Administration of vLC-Env-infected dendritic cells to mice generated a high cell-mediated immune response. These results demonstrate that priming with pCAG-Env and boosting with vLC-Env represents a logical candidate for vaccination against HIV infection^ref.
in-frame fusion of TNF-a DNA to DNA encoding a large fragment of HIV gp120. The studies were performed using a DNA prime, protein boost regime and a heterologous boosting protein. Fusion of TNF-a DNA enhanced T_h1 related immune responses against both the priming and the boosting gp120. In-frame fusion of IFN-g-encoding DNA at the 5' end of the chimeric molecule, to create a tripartite fusion, had no additional effect on immunogenicity^ref
immunization of macaques with a reverse transcriptase-deleted SHIV(KU2) (DrtSHIV(KU2)) plasmid that contained HIV-1(HXB2) env and SIV gag-nef induced protection against AIDS caused by challenge virus SHIV89.6P with a heterologous env. We further deleted vif and integrase from DrtSHIV(KU2) and substituted the 3'LTR with SV40 poly A sequences, creating D4SHIV(KU2) (M) and a parallel construct containing gag-nef of HIV-1(SF2), D4SHIV(KU2) (H). 6 macaques received two intramuscular injections of the (M) DNA, and another six received three injections of the (H) DNA. 3 of the latter group received 2 post-challenge boosts with (M) DNA vaccine. 7 virus control macaques were inoculated with SHIV89.6P. All twelve immunized macaques were challenged with SHIV89.6P virus, and CMI responses were measured by ELISPOT assays. Virus control animals all developed progressive infection, whereas vaccinated macaques from both groups controlled virus replication, with plasma viral loads dropping to undetectable levels between weeks 6 and 126 p.i. This DNA vaccine was efficacious even though it encoded Env, Gag, and Nef that were genetically distinct from the proteins in the challenge virus. The DNA vaccine induced broad-based protection without using viral proteins to boost the immunity^ref.
strategies designed to overcome the usual propensity of CTLs to focus recognition on a limited number of dominant epitopes. In studies of rhesus monkeys expressing the Mamu-A*01 MHC class I allele, we show that variously configured multiepitope plasmid DNA vaccine constructs elicit CTL populations that do not evidence skewing of recognition to dominant epitopes. Nevertheless, repeated boosting of these vaccinated monkeys with different live recombinant vaccine vectors uncovers and amplifies the usual CTL epitope dominance hierarchy. Importantly, in vitro peptide stimulation of PBMCs from monkeys that have received only a multiepitope plasmid DNA priming immunization uncovers this dominance hierarchy. Therefore, the dominance hierarchy of the vaccine-elicited epitope-specific CTL populations is inherent in the T lymphocytes of the monkeys after initial exposure to epitope peptides, and the ultimate breadth of epitope recognition cannot be modified thereafter. This finding underscores the enormous challenge associated with increasing the breadth of CTL recognition through vaccination^ref.
controlled release of GM-CSF protein by albumin-heparin microparticles administered via intramuscular vaccination in conjunction with HIV DNA vaccines stimulated HIV Gag-specific immune responses. In the murine model, Gag-specific CTL and T_h responses were significantly enhanced by administration of murine GM-CSF microparticles. This effect was comparable to a GM-CSF encoded plasmid. In three of four rhesus monkeys, enhancement of Gag-specific Ab, T_h, and CTL responses was observed 1 month after the first immunization with coadministration of human GM-CSF microparticles and HIV Gag plasmid. The second, third, and fourth booster immunizations, however, did not increase the Gag-specific immune responses. Subsequent application of Gag protein in complete Freund's adjuvant (CFA) significantly enhanced Ab and T_h, but not CTL. However, Gag-specific CTL response was triggered by cytokine and Gag p55-encapsulated microparticles in all animals. The strategy of priming immune responses by coadministration of cytokine microparticles and DNA vaccines, followed by boosting with cytokine and antigen protein-encapsulated microparticles, may prove effective in improving an HIV DNA vaccine design^ref.
novel biocompatible core-shell cationic nanoparticles, composed of an inner hard core of poly(methylmethacrylate) (PMMA) and a hydrophilic tentacular shell bearing positively charged groups and poly(ethyleneglycol) chains covalently bound to the core, were prepared by emulsion polymerization and characterized in vitro and in vivo for DNA vaccine applications. The nanoparticles reversibly adsorbed large amounts of DNA, mainly through electrostatic interactions, preserved its functional structure, efficiently delivered it intracellularly, and were not toxic in vitro or in mice. Furthermore, 2 intramuscular (i.m.) immunizations (4 weeks apart) with a very low dose (1mug) of the plasmid pCV-tat delivered by these nanoparticles followed by 1 or 2 protein boosts induced significant antigen-specific humoral and cellular responses and greatly increased T_h1-type T cell responses and CTLs against HIV-1 Tat^ref
plasmid-derived IL-21 delivered alone or in combination with the IL-15 gene to regulate immune responses to the HIV-1 envelope (Env) glycoprotein induced by DNA vaccination. Mice were injected with the gp140DeltaCFI(HXB2/89.6) vector expressing a modified Env glycoprotein with C-terminal mutations intended to mimic a fusion intermediate, in which the most divergent region encoding the variable V1, V2, and V3 domains of CXCR4-tropic HxB2 virus was replaced with the dual-tropic 89.6 viral strain. Using a recombinant vaccinia virus expressing 89.6 Env glycoprotein (vBD3) in a mouse challenge model, we observed that IL-21 plasmid produced sustained resistance to viral transmission when injected 5 days after DNA vaccination. Moreover, IL-21 in a synergistic manner with IL-15 expression vector augmented the vaccine-induced recall responses to the vBD3 challenge compared with those elicited by immunization in the presence of either cytokine alone. The synergistic combination of IL-21 and IL-15 plasmids promoted expansion of CD8⁺CD127⁺ memory T cell pools specific for a subdominant HLA-A2-restricted Env_121-129 epitope (KLTPLCVTL). Coimmunization with IL-21 and IL-15 plasmid combination resulted in enhanced CD8⁺ T cell function that was partially independent of CD4⁺ T cell help in mediating protection against vBD3 challenge. Furthermore, the use of IL-21 and IL-15 genes was able to increase Ab-dependent cellular cytotoxicity and complement-dependent lysis of Env-expressing target cells through augmentation of Env-specific IgG Ab levels. These data indicate that the plasmid-delivered IL-21 and IL-15 can increase the magnitude of the response to DNA vaccines^ref
to analyze the immunogenicity of HIV-1 DNA vaccine which expressing the chimeric gene gag-gp120 of Chinese prevalent HIV-1 strain and the immunoregulatory activity of IL-6, DNA vaccine plasmid pVAX1-gag-gp120 and eukaryotic expression plasmid pVAX1-IL6 were constructed and the expression in vitro was detected by RT-PCR and Western blotting, the results showed that the gene of interest expressed in the transfected HeLa cells. To explore the immune response in mice coinoculated with HIV-1 DNA vaccine and IL-6 expression plasmid, BALB/c mice were injected i.m. with eukaryotic expression plasmid pVAX1-IL6 and DNA vaccine plasmid pVAX1-gag-gp120. The specific humoral and cellular immunity in mice could be induced by inoculating separately HIV-1 DNA vaccine plasmid or coinoculating with IL-6 expression plasmid, and the specific killing activities of spleen CTL and the level of serum antibodies in the coinoculation group were significantly higher than those in the separate inoculation group. These results strongly support the use of IL-6 as a cytokine adjuvant in DNA vaccination^ref
a multiHIV fusion gene expressing an antigenic fusion protein composed of regulatory HIV-1 proteins Rev, Nef, and Tat, as well as Gag p17/p24 and a stretch of 11 cytotoxic T lymphocyte (CTL) epitope clusters from Pol and Env, was cloned into a novel DNA vector named the Gene Transport Unit (GTU). A mouse H-2^d-restricted HIV-1 gp120 epitope (RGPGRAFVTI) was cloned into the fusion gene as well. In addition to the HIV- 1 genes the GTU codes for a nuclear anchoring protein (bovine papilloma virus E2), ensuring the long maintenance of the vector and a high expression level of the selected immunogens. BALB/c mice were immunized with the GTU-MultiHIV DNA construct by different routes and regimens of immunization to assess the immunogenicity of the DNA vaccine in vivo. Mice developed strong CD8⁺ CTL responses to HIV-1 Env and Gag measured by an ELISPOT-IFN-gamma assay and chromium release assay. In addition, T cell responses to regulatory proteins Rev, Nef, and Tat were induced. Antibody responses were detected to each of the HIV antigens encoded by the DNA construct. Minimal doses of the GTU-MultiHIV DNA delivered by gene gun were potent in inducing significant HIV-specific CTL responses. The equivalent doses of the conventional plasmid expressing MultiHIV DNA delivered by gene gun failed to do so. An ideal DNA vaccine should yield high expression of the viral antigens for a prolonged period of time, and expression of the multiple viral antigens is probably required for the induction of a broad and protective immune response. The GTU-MultiHIV DNA vaccine described is a good vaccine candidate that meets the above criteria^ref
recombinant plasmid DNA (pEK2P-B) expressing an engineered codon-optimized envelope gp140 gene of primary (nonrecombinant) HIV-1 subtype B isolate 6101. Codon usage and RNA optimization of HIV-1 structural genes has been shown to increase protein expression in vitro as well as in the context of DNA vaccines in vivo. To further increase the expression, a synthetic IgE leader with kozak sequences were fused into the env gene. The cytoplasmic tail of the gene was also truncated to prevent recycling. The expression of env by the recombinant pEK2P-B was evaluated using T7 coupled transcription/translation. The construct demonstrated high expression of the HIV-1 env gene in eukaryotic cells as demonstrated in transfected 293-T and RD cells. Immunogenicity of pEK2P-B was evaluated in mice using IFN-g ELISpot assay, and the construct was found to be highly immunogenic and crossreactive with HIV-1 clade C env peptides. Three immunodominant peptides were also mapped out. Furthermore, by performing a CFSE flow cytometry-based proliferation assay, 2.4 and 1.5% proliferation was observed in CD4⁺, CD8⁺, and CCR⁺ memory T cells, respectively. Therefore, this engineered synthetic optimized env DNA vaccine may be useful in DNA vaccine and other studies of HIV-1 immunogenicity^ref
estimating effective doses of novel HIV vaccines is challenging. Dose-response analyses of DNA and fowlpox virus HIV vaccines showed that 1 mg of DNA vaccine and 5 x 10⁷pfu of fowlpox virus booster was immunogenic in macaques. However, this dose was poorly immunogenic in humans. When adjusted for body surface area, the human dose studied was equivalent to a poorly immunogenic lower dose in monkeys. These data provide a rationale for guiding dosing in future trials of HIV vaccine technologies^ref
bicistronic DNA vaccines that encode for HIV env and caspase-3 mutant (casp 3m) that are expressed via the encephalomyocarditis virus internal ribosomal entry site (IRES) or cytomegalovirus (CMV) promoter-dependent translations. While IRES-casp 3m induced weak apoptosis and caused little reduction in antigen expression, CMV-casp 3m elicited strong apoptosis and led to a marked decrease in the antigen expression. Therefore, IRES-casp 3m augmented HIV-specific immune responses, and IRES-casp 3m induced significant protection against the vaccinia-HIV chimeric virus. These results suggest that the appropriate level of apoptosis is important for DNA vaccine development^ref
Vaccine Research Center (VRC) 004 is the first phase 1 dose-escalation study of a multiclade HIV-1 DNA vaccine. VRC-HIVDNA009-00-VP is a 4-plasmid mixture encoding subtype B Gag-Pol-Nef fusion protein and modified envelope (Env) constructs from subtypes A, B, and C. 50 healthy, uninfected adults were randomized to receive either placebo (n=10) or study vaccine at 2 mg (n=5), 4 mg (n=20), or 8 mg (n=15) by needle-free intramuscular injection. Humoral responses (measured by ELISA, Western blotting, and neutralization assay) and T cell responses (measured by ELISA and intracellular cytokine staining after stimulation with antigen-specific peptide pools) were measured. The vaccine was well tolerated and induced cellular and humoral responses. The maximal CD4⁺ and CD8⁺ T cell responses occurred after 3 injections and were in response to Env peptide pools. The pattern of cytokine expression by vaccine-induced HIV-specific T cells evolved over time, with a diminished frequency of IFN-g-producing T cells and an increased frequency of IL-2-producing T cells at 1 year. DNA vaccination induced antibody to and T cell responses against 3 major HIV-1 subtypes and will be further evaluated as a potential component of a preventive AIDS vaccine regimen^ref
immunization of BALB/c mice with HIV gp120 cDNA + an Ii suppressive construct significantly enhances the activation of both gp120-specific T_h cells and CTL. MHC class II⁺/Ii^- APC present endogenously synthesized vaccine antigens simultaneously by MHC class II and class I molecules, activating both CD4⁺ and CD8⁺ T cells. Activated CD4⁺ T cells locally strengthen the response of CD8⁺ CTL, thus enhancing the potency of a DNA vaccine^ref
a novel subtype B env gene (EY2E1-B) with the goal of increasing vaccine antigen immune potency. The vaccine cassette was designed based on subtype B-specific consensus sequence with several modifications, including codon optimization, RNA optimization, the addition of a Kozak sequence, and a substituted immunoglobulin E leader sequence. The V1 and V2 loops were shortened and the cytoplasmic tail was truncated to prevent envelope recycling. 3 different strains of mice (BALB/c, C57BL/6, and HLA-A2 transgenic mice) were immunized three times with pEY2E1-B or the primary DNA immunogen pEK2P-B alone. The analysis of specific antibody responses suggested that EY2E1-B could induce a moderate subtype B-specific antibody response. Moreover, this construct was up to four times more potent at driving cellular immune responses. Epitope mapping results indicated that there is an increase in the breadth and magnitude of cross-reactive cellular responses induced by the EY2E1-B immunogen^ref.
a DNA plasmid encoding HIV-1 env, nef and tat genes was used in mice in a prime-boost immunization regimen with the corresponding recombinant proteins. The genetic immunogen was delivered with a gene gun and the proteins were injected intramuscularly together with the adjuvant AS02A. Immunizations were followed by experimental challenge with pseudotyped HIV-1 subtype A or B virus. In an initial experiment in which animals were challenged 4 weeks after the final immunization, all single modality and prime-boost vaccinations resulted in a significant level of protection as compared to control animals. There was a trend for DNA-alone immunization yielding the highest protection. In a subsequent study, a late challenge was performed 19 weeks after the final immunization. All groups having received the DNA vaccine, either alone or in combination with adjuvanted protein, exhibited strong protection against HIV replication. The subtype-specific protection against the experimental HIV challenge was significantly stronger than the cross-protection. Cellular and humoral immune responses were assessed during immunization and after challenge, but without clear correlation to protection against HIV replication. The data suggest that either DNA or protein antigens alone provide partial protection against an HIV-1/MuLV challenge and that DNA immunization is essential for achieving very high levels of efficacy in this murine HIV-1 challenge model. While prime-boost combinations were more immunogenic than DNA alone, they did not appear to provide any further enhancement over DNA vaccine mediated efficacy. The DNA immunogen might prime low levels of CD8⁺ T cells responsible for virus clearance or possibly a yet unidentified mechanism of protection^ref
novel codon-optimized GM-CSF gene as an adjuvant to enhance the immunity of a DNA vaccine against HIV-1 gag^ref
modified vaccinia virus Ankara (MVA) and plasmid DNA (pTHr) expressed HIV-1 clade A gag p24 and p17 fused to a string of 25 overlapping CD8⁺ T cell epitopes (HIVA). These studies compared intramuscular, subcutaneous, and intradermal MVA at dosage levels ranging from 5x10⁶-2.5x10⁸ pfu. In Study IAVI-010, DNA vaccine was given as a prime at months 0 and 1, followed by MVA as a boost at months 5 and 8. In Study IAVI-011, MVA alone was given at months 0 and 2. Regular safety monitoring was performed. Immunogenicity was measured by the IFN-g ELISPOT assay on peripheral blood mononuclear cells (PBMC). No serious adverse events were attributed to either vaccine; most adverse events were mild or moderate, although MVA resulted in some severe local reactions. 5 vaccine recipients had at least one positive IFN-g ELISPOT response, but none were sustained^ref
HIV DNA vaccines are potent inducers of cell-mediated immune (CMI) response in mice but elicit poor HIV-specific IFN-g-producing T cells in monkeys and humans. In this study, we performed kinetic analyses on splenocytes of BALB/c mice that were immunized by a single injection with a unique DNA vaccine. The response was detectable for at least 63 wk. ELISPOT detection of IFN-g-producing T cells showed a profile with 2 waves separated by a long period of minimal response. Multiparametric FACS analysis showed 2 populations of CD3⁺CD8⁺ T cells that were specific for all HIV Ags. These cells had similar robust proliferation abilities and contained granzyme B. However, only a few produced IFN-g. Both IFN-g-producing and non-IFN-g-producing HIV-specific CD8⁺ T cells were detected in the early stage (week (W)1 and W2 postimmunization (PI)), in the prolonged intermediate period of minimal response (W4-W26 PI), and in the final late phase of increased response (W30-W63 PI). Longitudinal characterization showed that both subsets of cells underwent expansion, contraction, and memory generation/maintenance phases throughout the lifespan of the animal. Altogether, these findings bring insight to the heterogeneity of the immune T cell response induced by a single immunization with this DNA and strengthen the concept that used of the IFN-g-ELISPOT assay alone may be insufficient to detect critical T cell responses to candidate HIV vaccines^ref.
A novel animal model for testing the immunogenicity and protective immune response induced by HIV-1 DNA vaccines was developed. DBA/2 mice were immunized with GTU^®-MultiHIV DNA encoding multigene for Rev, Nef, Tat, optp17/24 and a stretch of Pol/Env epitopes. A single GTU^®-MultiHIV B-clade specific plasmid or Auxo-GTU^®-MultiHIV(mix) (mixture of four plasmids with A, B, C and FGH clade specific MultiHIV antigens) were administered via gene gun and cell-mediated and humoral immune responses were analysed. The protective efficacy of the immune response was evaluated by challenging the mice with syngeneic tumor cells (P815) stably transfected with the MultiHIV fusion gene. Our results show that the strong MultiHIV-specific immune response generated by the GTU^®-MultiHIV vaccines in DBA/2 mice was able to delay the tumor growth substantially, indicating that the CTL response detected in vitro confers protection in vivo. The model described here is a safe and feasible in vivo assay for assessment of the vaccine potency to induce protective cell-mediated immune responses^ref

anti-HBV DNA vaccine (see also protein subunit vaccine ) :

plasmid expressing HBsAg was formulated with poly(lactide-co-glycolide-acid) (PLGA) and cetyltrimethylammonium bromide (CTAB) to generate highly uniform microparticles. Controlled release of DNA from these microparticles was demonstrated in vitro and in vivo using flow cytometry and confocal laser scanning microscopy with the focus on localization and quantitatively evaluation of APCs involved in the expression of target antigen. Compared to mice vaccinated with naked DNA, mice immunized with PLGA-CTAB-DNA microparticles displayed a much higher percentage of CD11c⁺, HBsAg-expressing APCs in the draining lymph nodes at 24 h and day 14 postinoculation. In addition, a prolonged transcription of plasmid DNA was detected by RT-PCR in mice immunized with the microparticles. A significantly enhanced immunogenicity of PLGA-CTAB-DNA over naked DNA was observed in immunized mice, including higher levels of antibody production, IFN-g secretion and CTL activity. Mice immunized with PLGA-CTAB-DNA microparticles also showed greater efficacy of immunoprotection against challenge of transplanted HBsAg-expressing tumor cells. Controlled release of the PLGA-CTAB-DNA microparticles might involve in the mechanisms of its augmented immunogenicity and enhanced immunoprotection^ref. The vaccine was so efficient that the antibodies and HBsAg formed circulating immune complexes and induced kidney and liver lesions similar to those observed in chronically infected patients^ref. To investigate the specific humoral and cellular immune response induced by prime-boost immunization of HBsAg protein vaccine (P), recombinant vaccinia virus vaccine (V) and DNA vaccine (D) in mice, groups of BALB/c mice were primed by one of the 3 vaccines P, V or D and boosted by another vaccine at 2, 5, 8 and 11 week later, thus 9 immune combinations were made: PP, PV, PD, VP, VV, VD, DP, DV and DD. Serum samples were collected at week 2, 5, 8 and 11 and levels of anti-HBsAg IgG antibodies and their sub-isotypes were determined. 7 days after every boost, spleen cells of vaccinated mice were separated and the specific CTL lysis ratio of P815S cells were determined. Among the three HBsAg vaccines P, V and D, V could induce the quickest humoral immune response. The memory humoral immune response induced by P was the strongest. D induced the weakest antibody titer. The ratio of specific IgG₁/IgG_2a indicated that antibody induced by PP was more polarized to T_h2. The other groups induced balanced T_h1/T_h2 immune response. Among all the groups, VD and DV induced the strongest CTL response, and the specific lysis ratio of P815S cells was 71% and 64%, respectively. The exprimental results suggested that among all the immune combinations, PV, PD, VP and VD can induce better humoral immune response while DV and VD can induce stronger CTL response in BALB/c mice^ref.
a polytope DNA vaccine encoding multiple T cell epitopes induced stronger CTL responses than the vaccine encoding the single antigen in H-2^d and H-2^b mice, although the CTL response to L^d-restricted epitope suppressed the CTLs to other epitopes in H-2^d-restricted mice. Interestingly, HSP70 as an adjuvant not only enhanced CTL response to the viral antigen but also overcame this epitope suppression. Furthermore, the polytope DNA vaccine resulted in a long-term down-regulation of hepatitis B virus surface antigen and inhibition of HBV DNA replication in a HBV transgenic mouse model^ref.
electroporation (EP)-based DNA vaccination to induce multi-specific CTL responses to HBV DNA vaccination in normal mice and marked immune responses to multivalent HBV DNA immunization in larger animal species^ref. In rhesus macaques intramuscular vaccination with electroporation using the plasmid encoding for HBV preS(2)-S antigen and an adjuvant plasmid encoding for hIL-2 and hIFN-g can significantly enhance the immunogenicity of the DNA vaccines, resulting in greatly improved antibody responses as well as peptide-stimulated IFN-g producing T cell responses. In addition, different antibody response behaviors resulted from different electro-pulse parameters^ref
enhancement of immune responses to hepatitis B DNA vaccine by superantigen SEA in mice^ref
HBV small (S) and middle (preS2 +S) envelope proteins. After occurrence of lamivudine breakthrough, 10 HBeAg positive patients with chronic hepatitis B were followed longitudinally before, during and after DNA vaccine therapy. Immunizations were well tolerated and adverse physical events were mild and considered unrelated to the vaccine. Proliferative responses to HBsAg were detected in two patients after DNA injections. Following 3 injections of vaccine, IFN-g-producing T-cells specific for the preS2 or the S antigen were detectable in 50 and 100% of the patients, respectively. Each patient recognized at least one peptide within the envelope domain encoded by the vaccine. Anti-preS2 antibodies and seroconversion to anti-HBe were detected in 2 patients^ref
coadministration of a HBV DNA vaccine with prothymosin a as an adjuvant improves antibody responses to HBV S antigen. Higher seroconversion rates and higher antibody titers were also observed. Prothymosin a appears to increase the number and affinity of HBsAg-specific, IFN-g-secreting T cells and to enhance cellular immune response to the preS2S DNA vaccine. Interestingly, administering the DNA separately from the prothymosin a plasmid abrogated the enhancement of DNA vaccine potency. The results suggest that prothymosin a may be a promising adjuvant for DNA vaccines^ref
comparisons in chimpanzees between a current HBV vaccine that contains recombinant HBsAg adsorbed to alum and 2 novel vaccine strategies that have proven superior to the current vaccine in mice.

The first approach was the use of oligodeoxynucleotides containing CpG motifs (CpG ODN) as an adjuvant to Engerix-B^®, a commercial HBV vaccine. The addition of CpG ODN to Engerix-B greatly improved the kinetics and magnitude of the humoral response, suggesting that CpG ODN might allow induction of protective immunity in humans more quickly and with fewer vaccine doses. All animals receiving either control or CpG-containing subunit vaccines at 0 and 4 weeks attained titers of HBsAg-specific antibody (anti-HBs) considered protective (>/=10 mIU/ml) and were indeed protected from challenge at 8 weeks with 10^3.5 50% chimp infectious doses (CID₅₀) of intravenous HBV.
The second approach was a DNA vaccine with a plasmid vector optimized for content of immunostimulatory CpG motifs. Despite the fact that earlier studies had shown four doses of a similar DNA vaccine (except not optimized for CpG content) to induce strong humoral responses in 1 of 2 chimpanzees, in this study 2 doses of DNA vaccine (at 0 and 4 weeks) did not generate any detectable anti-HBs in either of 2 chimpanzees, although it did protect 1 that rapidly developed anti-HBs during the incubation period, suggesting priming of an antibody response. The poor results may be due to an inadequate number of doses or amount of plasmid DNA in these larger animals, but nevertheless point to the need to improve delivery methods for DNA vaccines for use in larger animals such as primates^ref

Mycobacterium tuberculosis heat shock protein70 (HSP70) has both chaperon and cytokine functions, and has been shown to act as an adjuvant when co-administered with peptide antigens or given as fusion proteins. Of 2 truncated HSP70 molecules, N-terminal domain (HSP70_1-360, amino acids 1-360) and C-terminal domain (HSP70_359-610, amino acids 359-610) of mycobacterial HSP70, only the HSP70_359-610-fused HBV DNA vaccination resulted in a significant increase in HBsAg-specific humoral response, while the HSP70_1-360- or the complete HSP70 molecule-fused vaccine did not. Moreover, HSP70_359-610-fused DNA vaccine did not induce anti-HSP70 antibody. Interestingly, HSP70_359-610 not only enhanced HBsAg-specific CTL responses but also overcame the epitope suppression caused by L^d-restricted epitope. Meanwhile, HSP70_359-610 mediated T_h cell balance towards T_h1 pathway. In a HBV transgenic mouse model, the HSP70_359-610 fusion vaccine facilitated clearance of circulating HBsAg and down-regulation of HBV replication. These results suggested that the truncated mycobacterial HSP70 molecule, HSP70_359-610, might be a superior candidate to deliver the adjuvant function in HBV DNA vaccination instead of the complete HSP70 molecule^ref
a DNA vaccine comprising of most HBV genes plus genetically engineered IL-12 DNA (IL-12N222L) in 12 chronic hepatitis B (CHB) carriers being treated with lamivudine (LAM). When the ex vivo and/or cultured IFN-genzyme-linked immunospot (ELISPOT) assay was performed, the detectable HBV-specific IFN-g secreting T-cell responses were observed at the end of treatment and during a follow-up. These T_h1-cell responses, particularly CD4⁺ memory T-cell responses could be maintained for at least 40 weeks after the therapy and correlated with virological responses, but not with ALT elevation. Moreover, DNA vaccination under LAM treatment appeared to be well-tolerated and showed 50% of virological response rate in CHB carriers. Thus, a combination therapy of the DNA vaccine with chemotherapy may be one of new immunotherapeutic methods for the cure of CHB^ref
a synthetic chimeric gene, TBI-HBS, encoding the immunogenic ENV and GAG epitopes of HIV-1 and HBsAg of HBV, was expressed in tomato plants. Tomato fruits containing the TBI-HBS antigen were fed to experimental mice and, on days 14 and 28 post-feeding, high levels of HIV- and HBV-specific antibodies were present in the serum and feces of the test animals. Intraperitoneal injection of a DNA vaccine directing synthesis of the same TBI-HBsAg antigen boosted the antibody response to HIV in the blood serum; however, it had no effect on the high level of antibodies produced to HBV^ref
S. enteritidis recombinant strain E-23/pKDNA(3.1) with HBc antigen expressed by the eukaryotic promoter (DNA vaccine) in the body of mice after their infection per rectum and in the culture of macrophages was studied. The expression of HBc was shown to lead to the loss of the capacity of salmonellae for persistence in parenteral lymphoid tissue and for inducing the formation of anti-HBc antibodies. The study of the interaction of salmonellae with the macrophage culture revealed that the synthesis of HBc antigen inhibited the intracellular multiplication of salmonellae^ref
To test the feasibility of delivering a plasmid encoding IL-15 as a DNA vaccine adjuvant for improving the immune responses induced by HBV core gene DNA vaccine. RT-PCR based strategies were used to develop IL-15 expression constructs. The gene could be expressed in Escherichia coli due to the poor expression of IL-15. Then the bioactivity of IL-15 plasmid expression product was identified by CTLL-2 proliferation assay. 100 mg of DNA from each of the IL-15 eukaryotic expressed plasmid and the recombinant plasmid harboring DNA encoding the 144 amino acids of the N-terminus of HBV core gene (abbreviated pHBc144) was used to co-immunize C57 BL/6 mice. The titer of anti-HBcIgG was detected by ELISA and the antigen-specific CD8⁺ T cells (CD8⁺IFN-g⁺ T cells) were detected by intracellular cytokine staining at different time points. After co-immunization by pIL-15 and pHBc144 DNA vaccine the antigen-specific CD8⁺ cells of mice increased gradually, the first peak of immune response appeared 14 d later, then the number of antigen-specific CD8⁺ Ts cells decreased gradually and maintained at a steady level in 3 mo. After boosting, the number of antigen-specific CD8⁺ T cells reached the second peak 10 d later with a double of the 1st peak, then the number of antigen-specific CD8⁺ T cells decreased slowly. IL-15 as a gene adjuvant had no significant effect on humoral immune responses induced by HBV core gene DNA vaccine, but increased the memory antigen-specific CD8⁺ T cells induced by hepatitis B virus core gene DNA vaccine. DNA vaccine constructed by HBcAg 1-144 amino acid induces effective cell immunity, and cytokine plasmid-delivered IL-15 enhances the longevity of CD8⁺ T cells^ref
to enhance induction of protective immunity against hepadnavirus, healthy woodchucks were immunized with a bicistronic DNA vaccine carrying WHV core (WHc) and woodchuck IFN-g gene sequences. 3 groups, each 3 animals, were injected once or twice with 0.5 mg, 0.9 mg or 1.5 mg per dose of this vaccine. In addition, 4 animals received twice 0.6 mg or 1 mg WHc DNA alone. All animals were challenged with WHV. 4 of 9 animals injected with the bicistronic vaccine and 1 of 4 immunized with WHc DNA became protected from serologically evident infection and hepatitis. This protection was not linked to induction of WHcAg-specific antibodies or T cell proliferative response, and was not associated with enhanced transcription of T_h1 cytokines or 2',5'-oligoadenylate synthetase. Strikingly, all animals protected from hepatitis became reactive for WHV DNA and carried low levels of replicating virus in hepatic and lymphoid tissues after challenge with WHV. This study shows that the bicistronic DNA vaccine encoding for both hepadnavirus core antigen and IFN-g was significantly more effective in preventing hepatitis than that encoding for virus core alone, but neither of them could mount sterile immunity against the virus nor prevent establishment of occult infection^ref
Sheep immunized with the conventional HBsAg subunit vaccine (Engerix-B) as well as sheep immunized with a HBsAg DNA vaccine, combined with electroporation, generated significant antibody responses that were sustained for 25 weeks after primary immunization. At 25 weeks, all experimental groups received a secondary immunization with the HBsAg subunit vaccine. Sheep that received a primary DNA immunization, in combination with electroporation, mounted an anamnestic response similar to the cohort immunized with the HBsAg subunit vaccine. In contrast, animals immunized with DNA vaccines administered without electroporation elicited no detectable memory response. The presence of immune memory was significantly correlated with the induction of a prolonged primary immune response. Thus, a single DNA vaccination, in combination with electroporation, approached the efficacy of the commercial subunit vaccine in the maintenance of long-term protective serum antibody titres and immune memory^ref
Attenuated strains of bacteria have been developed as potential live vectors to express homologous or heterologous antigens of many pathogens for inducing protective immune responses. The non-pathogenic and rapidly growing Mycobacterium smegmatis can be transformed effectively by genes for pathogenic antigens, and has been used as a valuable vector for the development of live vaccines. However, little is known on whether M. smegmatis could be transformed with the genes for HBV antigens and could express those genes, and whether vaccination with recombinant M. smegmatis could induce humoral and cellular immune responses in vivo. Both the core protein and preS1 peptide of the HBV are immunogenic and can induce cellular and humoral immune responses. This made them ideal platform for the development of new vaccines. In the present study, both recombinant M. smegmatis and DNA vaccines were generated to express the CS1 antigen, a fusion protein that comprises truncated core protein (amino acids 1-155) and preS1 peptide (amino acids 1-55) of HBV. Following vaccination of BALB/c mice with the live recombinant M. smegmatis, the CS1-based DNA vaccine, or controls, antigen-specific humoral and cellular immune responses were characterized. Vaccination with live recombinant M. smegmatis induced a stronger cellular immune response and a longer period of humoral immune response than with the DNA vaccination. These results indicate that the recombinant M. smegmatis can express efficiently immunogenic CS1 antigen of HBV in vivo, and may be used for the prevention of HBV infection^ref
single-chain trimers (SCT) composed of peptide epitope b₂-microglobulin (b₂m) and major histocompatiblity complex (MHC) class I heavy chain covalently linked together in a single molecule have been shown to stimulate efficient CTL responses, we investigated the properties of HLA-A2 SCTs encoding the HBV core antigen (HBcAg) epitopes C(18-27) and C(107-115). Transfection of NIH-3T3 cells with pcDNA3.0-SCT-C(18-27) and SCT-C(107-115) leads to stable presentation of HBcAg epitopes at the cell surface. HLA-A2.1/Kb transgenic mice vaccinated with the SCT constructs, either as a DNA vaccine alone or followed by a boost with recombinant vaccinia virus, were shown to generate HBcAg-specific CTL responses by ELISPOT and in vitro IFN-g release experiments. HBcAg-specific CTLs from vaccinated HLA-A2.1/K^b transgenic mice were able to inhibit HBV surface and e antigen expression as indicated by HepG2.2.15 cells. A DNA vaccine encoding a human HLA-A2 SCT with HBV epitopes can lead to stable, enhanced HBV core antigen presentation, and may be useful for the control of HBV infection in HLA-A2⁺ HBV carriers^ref
N-terminal fragment of glycoprotein 96 (gp96) is able to produce an adjuvant effect for production of CTLs with HBV-specific peptides. A new strategy for HBV DNA vaccine design was reported using a partial gp96 sequence. The N-terminal 1-355aa (N355) of gp96 was linked to HBV genes encoding for structural proteins, the major S and middle S2S envelope proteins and the truncated core HBcAg (1-149aa). ELISPOT, tetramer staining and intracellular IFN-g assay were performed to analyze the induced cellular immune responses of our DNA constructs in BALB/c mice and HLA-A2 transgenic mice. The relative humoral immune responses were analyzed in different IgG isotypes. The fusion genes induced 2- to 6-fold higher HBV-specific CD8⁺ T cells as compared to the antigens alone. There was an approximate 10-fold decrease in the humoral immune responses with fusion genes based on HBV envelope proteins. Interestingly, the decreased humoral immune responses were not observed when antigens and plasmid encoding N355 were co-delivered. However, an approximate 20-fold higher antibody level was induced when linking N355 to a truncated HBcAg. Immunization by intramuscular injection resulted in predominantly IgG_2a antibodies, which indicated that these vaccines preferentially prime T_h1 responses. Highly immunogenic fusions by linking the N-terminal fragment of gp96 to HBV antigens. The N-terminal fragment of gp96 may be used as a molecular adjuvant to enhance the potency of DNA vaccines^ref

anti-HCV DNA vaccine :

immunization with a DNA vaccine encoding the HCV structural antigens Core, E1 and E2 elicits a specific immune response against the capsid and envelope proteins in rabbits and Macaca irus (crab-eating macaque monkeys, an alternative animal model to chimpanzees)^ref
HCV core gene immunization induces potent T-cell^{ref1,
ref2,
ref3}, but only weak B-cell reactivity. Injection of HCV core gene led to a decrease in % of T-cell population^ref, providing that HCV core has immunomodulatory properties^{ref1,
ref2,
ref3}. Depletion could be linked to increased apoptosis sensitivity of core-expressing lymphocytes^ref. Recombinant core proteins enhanced the immune response elicited by a co-delivered DNA vaccine encoding HCV core and envelope proteins. Particularly, the mixture of the plasmid pIDKE2 with Co.173, a protein comprising the first 173 aa of HCV core, induces strong humoral response, including antibodies that recognized peptides representing hypervariable region I from different viral isolates. Moreover, positive lymphoproliferative responses against the HCV structural proteins, encoded by the plasmid, were detected after 2 doses with this mixture. When the HCV core protein used in the mixture with pIDKE2 was Co.120, a protein comprising the first 120 aa of the viral antigen, strong humoral response and a positive lymphoproliferative response were also detected. The effectiveness of this formulation was tested in vivo by measuring the protection against infection with a recombinant vaccinia virus expressing HCV core protein. A 2 log reduction in vaccinia virus titer was observed in mice immunized with the mixture of pIDKE2-Co.120. Humoral and cellular immune responses elicited for the mixture of pIDKE2 with either Co.173 or Co.120 was stronger and more diverse than those generated by individual components. In conclusion, our results indicate that formulations comprising both DNA constructs and protein subunit vaccine candidates are able to elicit strong humoral and cellular immunity against several antigens^ref
nucleocapsid (CAP)^ref
protease and ATPase/helicase of nonstructural 3 protein (NS3)^ref
NS3/4A : T cells primed by transdermal DNA-based vaccination entered the liver and cleared NS3/4A-expressing hepatocytes in transiently transgenic CD8^+/+ mice but not in CD8^-/- mice. Hence, peripherally primed NS3/4A-specific CD8⁺ T cells home to the liver and clear HCV protein-expressing hepatocytes^ref
induction of cytotoxic T cell responses in mice by immunization with multi-CTL epitope-based anti-HCV DNA vaccine^ref
multi-CTL epitope gene which encoded 2 HCV epitopes (H-2^d) was cloned into the eucaryotic expression vector pcDNA3.1 to construct a multi-CTL epitope-based DNA vaccine. BALB/c mice were vaccinated with the DNA vaccine and the specific CTL responses to target cells (P815, H-2^d) pulsed by different CTL epitope peptide were detected. The multi-CTL epitope-based DNA vaccine which directed against 2 HCV CTL epitopes induced specific CTL responses to each of the 2 CTL epitopes independently and enhanced the total specific CTL response. The multi-CTL epitope-based DNA vaccine is constructed by using multi-CTL epitopes linked as encoding sequence through natural flanking amino acid residues. It can not only induce specific CTL responses to each CTL epitope independently but also enhance the total specific CTL response^ref
gene electrotransfer (GET) of a novel candidate DNA vaccine encoding an optimized version of the nonstructural region of HCV (from NS3 to NS5B) induced substantially more potent, broad, and long-lasting CD4⁺ and CD8⁺ cellular immunity than naked DNA injection in mice and in rhesus macaques as measured by a combination of assays, including IFN-gamma ELISPOT, intracellular cytokine staining, and CTL assays. A protocol based on three injections of DNA with GET induced a substantially higher CD4⁺ T cell response than an adenovirus 6-based viral vector encoding the same Ag. To better evaluate the immunological potency and probability of success of this vaccine, 2 chimpanzees were immunized and vaccine-induced cell-mediated immunity were compared to that measured in acute self-limiting infection in humans. GET of the candidate HCV vaccine led to vigorous, multispecific IFN-g⁺CD8⁺ and CD4⁺ T lymphocyte responses in chimpanzees, which were comparable to those measured in five individuals that cleared spontaneously HCV infection. These data support the hypothesis that T cell responses elicited by the present strategy could be beneficial in prophylactic vaccine approaches against HCV^ref
a DNA vaccine expressing a secreted form of HCV E2 protein was constructed and used to vaccinate mice and piglets with or without boosting with a recombinant E2 protein vaccine formulated with CpG ODN and 10% Emulsigen. The immunogenicity of HCV E2 vaccines was analyzed by ELISA for antibody responses, MTT assay for lymphocyte proliferation, ELISPOT for the number of IFN-g secreting cells, and CTç assays. Intradermal injection of E2 DNA vaccine induced strong T_h1-like immune responses in mice. In piglets, E2 DNA vaccine elicited moderate and more balanced immune responses. A DNA vaccine prime and protein boost vaccination strategy induced significantly higher E2-specific antibody levels and shifted the immune response towards T_h2-like ones in piglets. A DNA vaccine expressing a secreted form of HCV E2 protein elicited E2-specific immune responses in mice and piglets. Recombinant E2 protein vaccination following DNA immunization significantly increased the antibody response in piglets. These HCV E2 vaccines may represent promising HCV vaccine candidates for further investigations^ref.
HCV genome is highly variable, particularly in the hypervariable region 1 (HVR1) of its E2 envelope gene. The variability of HCV genome has been a major obstacle for developing HCV vaccines. Due to B-cell HVR1 mimotopes mimicking the antigenicity of natural HVR1 epitopes and some T-cell epitopes from the consensus sequence of HCV genes conserving among the different HCV genotypes, we synthesized an minigene of HCV-derived multi-epitope peptide antigen (CMEP), which contains 9 B-cell HVR1 mimotopes in E2, 2 conserved CTL epitopes in C, 1 conserved CTL epitope in NS3 and 1 conserved Th epitope in NS3. This minigene was cloned into a GST expression vector to generate a fusion protein GST-CMEP. The immunogenic properties of CEMP were characterized by HCV infected patients' sera, and found that the reactivity frequency reached 75%. The cross reactivity of anti-CEMP antibody with different natural HVR1 variants was up to 90%. Meanwhile, we constructed an HCV DNA vaccine candidate, plasmid pVAX1.0-st-CMEP carrying the recombinant gene (st) of a secretion signal peptide and PADRE universal T_h cell epitope sequence in front of the CMEP minigene. Immunization of rabbits with pVAX1.0-st-CMEP resulted in the production of antibody, which was of the same cross reactivity as the fusion protein GST-CMEP. Our findings indicate that the HCV-derived multi-epitope peptide antigen in some degree possessed the characteristics of neutralizing HCV epitopes, and would be of the value as a candidate for the development of HCV vaccines^ref
multiple CTL epitope-based anti-HCV DNA vaccine can induce specific CTL responses to each HCV CTL epitope independently and long-term CD8⁺ T cell memory responses, and to determine the cytokine secretion pattern and subtype of epitope-specific cytotoxic T cells. A multi-CTL epitope gene, which consists of two epitopes of HCV (H-2^d-restricted HCV core(133142) and E1(315322)), was cloned into the eukaryotic expression vector pcDNA3.1. BALB/c mice (H-2^d restricted) were vaccinated intramuscularly with this multi-CTL epitope-based DNA vaccine. The epitope-specific CTLs against target cells (P815,H-2^d restricted) pulsed with various CTL epitope peptides were detected by lactate dehydrogenase release assay, and the precursor frequency of epitope-specific CTLs was determined by limiting dilution analysis. Cytokines (IL-2, IL-4, and IFN-g) in culture supernatants were determined by enzyme-linked immunosorbent assay. The multi-CTL epitope-based DNA vaccine directed against two HCV CTL epitopes could induce specific CTL responses to each of the two CTL epitopes independently and long-term CD8⁺ T cell memory responses. The epitope-specific cytotoxic T cells produced T_h1 cytokines^ref.

anti-HDV DNA vaccine : HDV DNA vaccine can produce T_h1 and CTL immune responses but only a low anti-HDV antibody titer is generated with a large hepatitis D antigen (L-HDAg) construct. In contrast, DNA vaccine expressing small hepatitis D antigen (S-HDAg) can generate a high titer of anti-HDV antibodies. Whether the low humoral immunity of L-HDAg DNA vaccine is due to inadequate dosage or can be ameliorated by other modes of immunization needs further evaluation. Plasmid (p25L) encoding L-HDAg and plasmid (pS/p25L) coexpressing HBsAg and L-HDAg were used in this study. The humoral response generated in mice using different plasmid DNA dosages and modes of immunization, including gene gun and in vivo electroporation (EP), were compared. I.m. injection with a high dose of plasmid DNA (10 mg/kg) produced strong antibodies to HBsAg earlier than the usual dose did, but did not augment the anti-HDV response. Gene gun DNA immunization could not provide a better humoral immune response to HDV. EP DNA immunization had a higher anti-HDV seroconversion rate of 80%, but the anti-HDV antibody responses were generally weak (titer < or = 400:1). The low humoral immunogenicity of DNA vaccine with L-HDAg cannot be ameliorated by different dosage, gene gun immunization, or in vivo EP intramuscular injection. DNA vaccine with a L-HDAg construct may not be a candidate HDV vaccine to generate anti-HDV humoral immunity^ref
anti-HEV DNA vaccine : gene fragments encoding p166 and p179, which contain the neutralization antigenic epitopes of a Chinese strain of HEV genotype IV, were cloned into 2 different eukaryotic expression vectors (pTR421 and pCDNA3.1), respectively. The in vitro expression level of p166 and p179 in HepG2 cells transfected by each of the recombinant plasmids with lipofectamine2000 was examined by means of immunofluorescence and Western blot. Meanwhile, the in vivo expression level in muscles of mice was examined with immunohistochemistry staining. 4 recombinant plasmids, pTR421-166, pTR421-179, pCDNA3.1-166 and pCDNA3.1-179, were constructed successfully and confirmed correct with restriction endonuclease analysis and nucleotide sequencing. The antigen expression was only detected in HepG2 cells transfected by pTR421-179 and in myocytes of the mice injected with pTR421-179. Neither in vitro nor in vivo antigen expression was detected with pTR421-166 although p166 was only 13 amino acids shorter than p179 at N terminus. Neither pCDNA3.1-166 nor pCDNA3.1-179 was expressed in vitro and in vivo^ref. To construct the plasmid pcHEV23 containing fragments of HEV ORF2 and ORF3 chimeric gene and to assess its ability to elicit specific immunologic response in mice, the gene encoding the structural protein of HEV ORF2 fragment and full-length ORF3 was amplified by PCR. The PCR products were cloned into an eucaryotic expression plasmid pcDNA3. The resulting plasmid pcHEV23 was used as a DNA vaccine to inoculate BALB/c mice intramuscularly thrice at a dose of 100 or 200 mug. Mice injected with empty pcDNA3 DNA or saline served as control and then specific immune responses in the mice were detected.RESULTS:After 2-3 times of inoculation, all mice injected with pcHEV23 had anti-HEV IgG seroconversion and specific T lymphocyte proliferation. The lymphocyte stimulation index in the group immunized with pcHEV23 (3.1+/-0.49) was higher than that in the control group (0.787+/-0.12, P<0.01). None in the control group had a detectable level of anti-HEV IgG. DNA vaccine containing HEV ORF2 and ORF3 chimeric gene can successfully induce specific humoral and cellular immune response in mice^ref. The complete ORF2 gene (1-660 amino acids, a.a.) coding for capsid protein of HEV as candidate DNA vaccine induced significant specific humoral and cellular immune responses in mice. Gene gun based DNA administration led to higher seroconversion rates and HEV-specific antibody titers as against needle-injection method. The region (458-607a.a.) within ORF2 protein is reported to harbour the predominant neutralization epitope/s (NE) of HEV. The NE DNA also induced HEV-specific immune responses in mice. NE-based DNA-prime-protein boost approach was observed to be superior to NE DNA based approach. Co-administration of plasmid expressing mouse GM-CSF induced immune response at similar level as that with ORF2/NE plasmid alone. IgG1 was the predominant isotype irrespective of the approach used. HEV-specific antibodies in seroconverted mice sera could bind/neutralize HEV in an in vitro ELISA-based assay. In conclusion, efficacy of ORF2 and NE based DNA/DNA-prime-protein-boost approaches are worth exploring in monkey model^ref
anti-influenzavirus A DNA vaccine (see also protein subunit vaccine , killed vaccines and attenuated vaccine ): intranasal (i.n.) vaccination represents an attractive non-invasive alternative to needle-based injection and provides superior protection at mucosal surfaces. However, new formulations are needed to improve efficacy and reduce the refrigerated storage and distribution requirements associated with standard liquid vaccines. Powder offer improved sterility and stability, as well as ease of storage and transportation, enabling cost effective worldwide vaccine delivery. A dry powder formulation of a reporter gene-encoding plasmid DNA was IN administered in rats and reportr gene expression measured in nasal and lung tissues. The results demonstrate elevated gene expression in the nasal cavities, without spill-over into the lung tissues. Using a model plasmid DNA vaccine for influenza, IN dry powder delivery has been demonstrated to induce potent immune responses. The magnitude of the serum antibody response was stronger than the corresponding titers achieved via intramuscular injection or IN administration of a liquid formulation.

an expression vector that allows mammalian cells to express the TLR5 agonistflagellin at the cell surface. In vitro, cell lines expressing FliC stimulated production of proinflammatory cytokines and the up-regulation of costimulatory molecules on monocytes. Mice given the FliC expression vector intradermally exhibited site-specific inflammation and, in combination with vectors expressing Ags, developed dramatic increases in Ag-specific IgG as well as IgA. Surprisingly, mice also developed strong Ag-specific MHC class I-restricted cellular immunity. To determine whether vaccination using FliC vectors could elicit protective immunity to an infectious agent, mice were given dermal injections of FliC expression vector together with a vector encoding the influenza A virus nucleoprotein. This vaccination strategy elicited protective immunity to lethal influenza A virus infection^ref
a phase I clinical trial was conducted to evaluate a monovalent influenza DNA vaccine containing the HA gene from A/Panama/2007/99 delivered by particle-mediated epidermal delivery (PMED). 3 groups of 12 healthy adult subjects received a single dose on day 0 of either 1, 2 or 4 mg of DNA vaccine, delivered as 1, 2 or 4 PMED administrations. The PMED influenza DNA vaccine elicited serum hemagglutination-inhibition (HAI) antibody responses at all three dose levels, with the highest and most consistent responses in subjects vaccinated with the highest dose level. Antibody responses were greatest at the last time point tested, day 56. Treatment-related reactions were mild to moderate, and included skin reactions at the vaccine site^ref
eukaryotic expressing plasmids encoding HA and HA1 of influenza A virus (A/PR/8/34) and studied their expression in HEK293 cells. HA and HA1 genes were amplified by RT-PCR and cloned into pcDNA3.1⁺ to generate pcDNA3.1⁺/HA and pcDNA3.1⁺/HA1, respectively. After verification of the cloning fidelity by restriction endonuclease digestion, PCR, and sequencing, pcDNA3.1⁺/HA and pcDNA3.1⁺/HA1 were transfected into HEK293 cells using PolyFect Transfection Reagent. Immunofluorescence assay was used to detect the transient expressing cells. Fluorescence microscopy revealed strong expression of target gene in HEK293 cells transiently transfected with either pcDNA3.1⁺/HA or pcDNA3.1⁺/HA1. Therefore, the results confirm the successful construction of eukaryotic expressing plasmids capable of driving the eukaryotic expression of influenza virus antigen HA and HA1, which is likely to provide a basis for both further investigation of the mechanism of influenza viral infection and the development of influenza DNA vaccine^ref
plasmid DNA vaccines encoding the influenza virus NP gene (pNP) alone or in combination with the HSV type 1 protein 22 gene (pVP22). Optimal immune responses were observed in BALB/c mice immunized with the combination of pVP22 plus pNP, as assessed by ELISA, ELISPOT and intracellular cytokine staining (ICCS). These mice also showed maximal resistance following challenge with the A/PR/8/34 (H₁N₁) and A/Udron/72 (H₃N₂) strains of influenza virus. The susceptibility of immunized mice to virus infection was significantly increased following depletion of either CD4⁺ or CD8⁺ T cells. These results indicate that a plasmid DNA vaccine encoding pVP22 plus NP induces a high level of cross-protective immunity against influenza virus subtypes^ref.

^ref

anti-Ebola virus DNA vaccine : the small rodent model for ZEBOV - important for the development of antiviral agents and vaccines, was established in 1998 and the minigenome-based reverse-genetic system was developed inn 1999. Protection of non-human primates against a lethal challenge with ZEBOV was first achieved in 2000 and in 2001 an infectious clone for ZEBOV was created.

gene product	vaccine system	animal model	survivors/challenged
GP	vaccinia virus	guinea pig	3/5
	vaccinia virus	cynomolgus macaque	0/3^ref
	VEEV replicon	guinea pig	8/10^ref
		mouse	18/20^ref
		cynomolgus macaque	0/3^ref
	DNA	guinea pig	13/15^{ref1, ref2}
	DNA	mouse	50-100% (dependent on the dose of DNA administered)^ref
sGP	DNA	guinea pig	8/11^ref
GP and NP	VEEV replicon	cynomolgus macaque	0/3^ref
	DNA	guinea pig	8/8^ref
	DNA and adenovirus	cynomolgus macaque	4/4^ref
	a 3-plasmid DNA vaccine encoding the envelope glycoproteins (GP) from Zaire and Sudan/Gulu species, as well as NP	randomized, placebo-controlled, double-blinded, dose-escalation study. Healthy adults, ages 18-44 years, were randomized to receive three injections of vaccine at 2 mg (n=5), 4 mg (n=8), 8 mg (n=8), or placebo (n=6). The vaccine was well-tolerated, with no significant adverse events or coagulation abnormalities.	Specific antibody response to at least 1 of the 3 antigens encoded by the vaccine as assessed by ELISA and CD4⁺ T cell GP-specific responses as assessed by ICS were detected in 20/20 vaccinees. CD8⁺ T cell GP-specific responses were detected by ICS assay in 6/20 vaccinees^ref
NP	VEEV replicon	mouse	20/20^{ref1, ref2}
	VEEV replicon	cynomolgus macaque	0/3^ref
	DNA	guinea pig	5/8^ref
	DNA	mouse	70-80% (dependent on the dose of DNA administered)^ref
VP24	VEEV replicon	mouse	37/60 (combined data for 2 mouse strains, one strain was better protected by the vaccine than the other)^ref
VP30	VEEV replicon	mouse	30/60 (combined data for 2 mouse strains, one strain was better protected by the vaccine than the other)^ref
VP35	VEEV replicon	mouse	23/59 (combined data for 2 mouse strains, one strain was better protected by the vaccine than the other)^ref
VP40	VEEV replicon	mouse	32/60 (combined data for 2 mouse strains, one strain was better protected by the vaccine than the other)^ref

prime-boost strategies :

plasmid DNA prime followed by human adenovirus (Ad) prime

once a month for 3 months, macaque monkeys were injected with plasmid DNA encoding Ebola virus glycoprotein and nucleoprotein (NP) to prime the immune system. 3 months later (5 months after the first DNA injection), the monkeys received an injection of crippled human adenovirus type 5 containing the glycoprotein gene (GP) from the Zaire Ebola virus subtype to boost the immune response. The team then challenged the vaccinated animals 3 months later with a lethal dose of Ebola virus (at 8 months). Significantly, the vaccine induced both humoral and cellular immune responses in the animals, but fail to protect animals immune to Ad5, suggesting natural Ad5 exposure may limit vaccine efficacy in humans^ref. Although it generated protective cellular and humoral immunity in macaques, > 6 months was required to complete the immunizations, making it impractical to limit an acute epidemic.
first phase I clinical trial (Vical) : a three-plasmid DNA vaccine encoding the envelope glycoproteins (GP) from the Zaire and Sudan/Gulu species as well as the nucleoprotein was evaluated in a randomized, placebo-controlled, double-blinded, dose escalation study. Healthy adults, ages 18 to 44 years, were randomized to receive three injections of vaccine at 2 mg (n = 5), 4 mg (n = 8), or 8 mg (n = 8) or placebo (n = 6). Immunogenicity was assessed by ELISA, immunoprecipitation-Western blotting, ICS, and ELISpot assay. The vaccine was well-tolerated, with no significant adverse events or coagulation abnormalities. Specific antibody responses to at least one of the three antigens encoded by the vaccine as assessed by ELISA and CD4⁺ T-cell GP-specific responses as assessed by ICS were detected in 20/20 vaccinees. CD8⁺ T-cell GP-specific responses were detected by ICS assay in 6/20 vaccinees. This Ebola virus DNA vaccine was safe and immunogenic in humans^ref. Steve Rucker, a registered nurse at the NIH, stepped into medical history by becoming the first person ever injected with an experimental vaccine designed to protect against Ebola

chimpanzee-derived replication-defective prime followed by modified vaccinia Ankara (MVA) boost : in a phase I clinical trial, ChAd3Ebola-Zaire (Okairos, owned by GlaxoSmithKline, in collaboration between the U.S. National Institute of Allergy and Infectious Diseases) generated, for the first time, durable protection against lethal EBOV challenge^ref. Human phase 1 safety trials of both vaccines are due to start within end of 2014 in the USA.

boost alone : a mixture of recombinant adenovirus (Ad) vectors encoding Ebola virus (Zaire and Sudan serotypes) glycoprotein (GP) and nucleoprotein (NP) genes completely protects cynomolgus macaques against Ebola virus challenge 28 days after immunization. Protection was highly effective and correlated with the generation of Ebola-specific CD8⁺ T-cell and antibody responses. Even when animals were immunized once with ADVGP/NP and challenged 28 days later, they remained resistant to challenge with either low or high doses of virus. Even though the boost alone appears to be effective, the prime-boost strategy, which requires 4 shots over 6 months, elicits a stronger immune response, and it may be useful for preventive vaccines intended for hospital workers at high risk of exposure to the virus, for example^ref. Phase I human clinical trials of the adenovirus component alone aree expected in late 2004 or early 2005. Yet the adenovirus vaccine could present some problems because many in the general population have previously been exposed to such viruses : 10% of people would fight the adenovirus-5–based vector itself, clearing it before it can work effectivelyso that some teams are working on another vector based on the adenovirus-35 serotype. The high levels of adenovirus required (currently around 10¹⁰ particles) could potentially render the vaccine toxic in humans : in that case, the team would have to dilute the vaccine, potentially limiting its usefulness.

vesicular stomatitis virus : rVSV-ZEBOV-GP (BPSC1001) (Public Health Agency of Canada (PHAC) , then BioProtection Systems (BPS), a wholly-owned subsidiary of NewLink Genetics). VSV strain Indiana (rVSV), with the glycoprotein of the ZEBOV Kikwit 1995 strain replacing the gene for the VSV envelope glycoprotein. The resultant rVSV construct contains surface ZEBOV glycoprotein that exhibits a narrower host-cell tropism in vitro and considerable attenuation of replication, as compared with wildtype VSV^ref1^,^ref2^,^ref3
under development by Crucell and NIH by using AdVac, an adenovirus (Ad)-based vaccination vector. Vaccine-production will utilise Crucell's human designer cell line PER.C6^ref.
Ebola virus glycoprotein (GP) gene expressed from recombinant alphavirus replicon RNAs derived from an attenuated strain of VEEV (that also encodes the replicase function and is capable of efficient intracellular self-amplification) incorporated into virus-like replicon particles (VLP)^ref

anti-Marburg virus DNA vaccine : attenuated recombinant replication-competent VSV vectors expressing either the EBOV glycoprotein or MARV glycoprotein. A single intramuscular injection of the EBOV or MARV vaccine elicited completely protective immune responses in nonhuman primates against lethal EBOV or MARV challenges. Notably, vaccine vector shedding was not detectable in the monkeys and none of the animals developed fever or other symptoms of illness associated with vaccination. The EBOV vaccine induced humoral and apparent cellular immune responses in all vaccinated monkeys, whereas the MARV vaccine induced a stronger humoral than cellular immune response. No evidence of EBOV or MARV replication was detected in any of the protected animals after challenge. These data suggest that these vaccine candidates are safe and highly efficacious in a relevant animal model. After being rendered harmless through gene shuffling, VSV vectors were covered with distinctive proteins that the human body uses to recognize and marshal defences against either Marburg or Ebola. 6 macaques were given a jab against Marburg disease; 4 were then infected with Marburg virus and 2 were infected with Ebola. The 4 infected with Marburg survived; the others died within 10 days. The same procedure was used for jabs against Ebola disease; again, the 4 that got the right shots survived; the other 2 died. There is concern that VSV will revert to a dangerous form or hop over into cattle. Both teams hope their vaccines will be on the market in three to seven years. The jabs could be used earlier than that, however, if they prove useful in preliminary studies and an outbreak demands their use on compassionate grounds^ref. Replication-competent vaccine based on attenuated recombinant vesicular stomatitis virus (rVSV), as a postexposure treatment for MARV haemorrhagic fever. A rhesus macaque model of MARV haemorrhagic fever that produced 100% lethality was used. rVSV vectors expressing the MARV Musoke strain glycoprotein were administerede to 5 macaques 20–30 min after a high-dose lethal injection of homologous MARV. Three animals were MARV-positive controls and received non-specific rVSV vectors. We tested for viraemia, undertook analyses for haematology and serum biochemistry, and measured humoral and cellular immune responses. All 5 rhesus monkeys that were treated with the rVSV MARV vectors as a postexposure treatment survived a high-dose lethal challenge of MARV for at least 80 days. None of these 5 animals developed clinical symptoms consistent with MARV haemorrhagic fever. All the control animals developed fulminant disease and succumbed to the MARV challenge by day 12. MARV disease in the controls was indicated by: high titres of MARV (103–105 plaque-forming units per mL); development of leucocytosis with concurrent neutrophilia at end-stage disease; and possible damage to the liver, kidney, and pancreas. Postexposure protection against MARV in non-human primates provides a paradigm for the treatment of MARV haemorrhagic fever. Indeed, these data suggest that rVSV-based filoviral vaccines might not only have potential as preventive vaccines, but also could be equally useful for postexposure treatment of filoviral infections^ref
anti-VEEV DNA vaccine : DNA vaccines encoding structural proteins of VEEV can elicit immune responses and protection against VEEV though this protection is incomplete against airborne VEEV. Particle-mediated epidermal delivery of a DNA vaccine encoding the E2 glycoprotein of VEEV can be boosted with a mucosally-delivered Ad-based vaccine encoding the same E2 glycoprotein. This results in an improved T_h2-type IgG response, an increase in neutralising antibody and a significant increase in protection against airborne VEEV. This indicates that prime-boost may be a suitable immunisation regimen for providing protection against airborne VEEV^ref.
anti-Lassa virus DNA vaccine : nucleoprotein or glycoprotein gene expressed from recombinant replicon RNAs derived from an attenuated strain of VEEV (that also encodes the replicase function and is capable of efficient intracellular self-amplification) incorporated into virus-like replicon particles (VLP). A single intramuscular vaccination of a replication-competent vaccine against Lassa virus based on attenuated recombinant vesicular stomatitis virus vectors expressing the Lassa viral glycoprotein elicited a protective immune response in 4 nonhuman primates against a lethal Lassa virus challenge. Vaccine shedding was not detected in the monkeys, and none of the animals developed fever or other symptoms of illness associated with vaccination. The Lassa vaccine induced strong humoral and cellular immune responses in the four vaccinated and challenged monkeys. Despite a transient Lassa viremia in vaccinated animals 7 d after challenge, the vaccinated animals showed no evidence of clinical disease. In contrast, the 2 control animals developed severe symptoms including rashes, facial edema, and elevated liver enzymes, and ultimately succumbed to the Lassa infection^ref
anti-West Nile virus (WNV)DNA vaccines (see also attenuated vaccine ) are currently in preclinical trials :

replication-defective Kunjin virus
hybrid with dengue virus (DV)
hybrid with live attenuated yellow fever virus (YFV) with WNV coat proteins (source : Acambis) is in phase I clinical trial

anti-SARS coronavirus (SARS-CoV) DNA vaccine : NIAID Vaccine Research Center (VRC) produced a DNA vaccine encoding the spike (S) glycoprotein of the SARS-CoV induces CD4 and CD8 T cell and neutralizing antibody responses, as well as protective immunity, in a mouse model. Viral replication was reduced by more than six orders of magnitude in the lungs of mice vaccinated with these S plasmid DNA expression vectors, and protection was mediated by a humoral but not a T-cell-dependent immune mechanism^ref. Clinical trials began in December 2004. A secreted recombinant expression plasmid, pVAX-S1, encoding the partial S protein with a signal peptide, was constructed and used to immunize BALB/c mice through electroporation. It was demonstrated that the eukaryotic expression vector pVAX-S1 was successfully constructed by restriction enzyme and sequence analysis. The expressed protein could be detected specifically by Western blot analysis. The serum IgG level of the vaccine group mice was significantly higher than that of the corresponding control group at day 14 after vaccination (P< 0.05). The vaccine group demonstrated significantly higher S1 protein lymphocyte proliferation index (LPI) than the control groups (P < 0.05). Furthermore, in the experimental group, a decrease in the ratio of CD4⁺ to CD8⁺ T-lymphocytes and an increase level of IFN-g in serum were observed. However, IL-4 was not detectable in 2 groups. These results strongly demonstrated that the pVAX-S1 plasmid could induce humoral and cellular immune responses in mice, and may be a potential candidate for a DNA vaccine against the SARS coronavirus^ref. A single amino acid substitution (R441A) in the full-length S DNA vaccine failed to induce NAbs and abolished viral entry when pseudoviruses were generated. However, another substitution (R453A) abolished viral entry while retaining the capacity for inducing NAbs. The difference between R441A and R453A suggests that the determinants for immunogenicity and viral entry may not be identical^ref. SARS coronavirus nucleocapsid protein^ref. Nucleocapsid protein plays a critical role in SARS-CoV pathogenesis, and high-level anti-nucleocapsid antibodies are detected in the patients infected by SARS-CoV. Several studies have shown that there exists an interaction between nucleocapsid (N) and membrane (M) protein. In this paper, we investigate whether the expression of membrane protein can affect the immune responses induced by nucleocapsid DNA immunization. Two recombinant plasmids containing M and N coding sequence were constructed. Moreover, in order to get the antigen for ELISA and in vitro stimulation assay, N protein were expressed and purified from E. coli bacteria. Injection of 20 mg of the mixture of pVAX1-M and pVAX1-N into the Balb/c mice could elicit the humoral and cellular responses. The ELISA analysis using the N antigen or inactivated SARS-CoV particles as capture antigen showed that co-injection of SARS-M could enhance N-induced antibody production, especially IgG_2a subclass. After lymphocytes were stimulated with 10mug/ml purified N antigen, The CD4⁺ and CD8⁺ T cells of N and M plus N group were increased compared with those of control groups, and the M protein could augment the activation of lymphocytes induced by N DNA vaccine. Cytokine ELISA analysis revealed that co-injection of M could enhance the levels of IFN-g, IL-2 release induced by N antigen. Further experiments in field mouse also support the claim that membrane protein can augment the N-specific immune responses. Virus challenge test was conducted in BSL3 bio safety laboratory with Brandt's vole SARS-CoV model, and the results indicated that co-immunization of M and N antigens could reduce the mortality and pathological changes in lung from the virus infection^ref. It has been reported that after natural infection in humans and immunization in animals with SARS-CoV vaccine, antibody is produced and persistent for a long period of time. Mice were immunized i.m. with SARS-CoV S DNA vaccine, and 3 different methods (ELISA, ELISPOT and FACS) were used to evaluate the immune responses when the cells were stimulated in vitro with a pool of peptides overlapping entire SARS spike protein. Pime-immunization with SARS-CoV S DNA vaccine can induce both CD4⁺ and CD8⁺ T cell responses. Boosting with the same vaccine enhances CD4⁺ and CD8⁺ T cell responses in both lymphoid and nonlymphoid organs and were persistent over 2 months. The SARS-CoV S-specific CD4⁺ and CD8⁺ T cells were CD62L^-, a marker for memory cells, and -30 to 50% of the cells expressed IL-7Ra (CD127), a marker for the capacity of effector cells to develop into memory cells. In addition, immunization with the DNA vaccine elicited high levels of antibody production. Taken together, these data demonstrate that immunization with SARS-CoV S DNA vaccine can generate antigen-specific humoral and cellular immune responses that may contribute to long-term protection^ref. After recombination with an immunostimulatory sequence (ISS), the gene encoding SARS spike protein fragment 1 (S1) was cloned into the plasmid pIRES to produce pIRES-ISS-S1. On confirmation of the expression of S1 protein by indirect immunofluorescence assay (IFA), after the transfection of pIRES-ISS-S1 into BHK-21 cells, the DNA vaccine was repeatedly administrated to BALB/c mice. CD4⁺ and CD8⁺ spleen T lymphocytes were analyzed by flow cytometry to evaluate T cell-mediated immune responses, the antigen-specific responses of T cells were evaluated by CTL assay, and the level of IgG in antisera from immunized mice was determined by enzyme-linked immunosorbent assay. The counts of spleen CD4⁺ and CD8⁺ T lymphocytes were increased, that the T cell-mediated immune responses showed antigen specificity, and that IgG was significantly induced with DNA vaccines pIRES-ISS-S1 and pIRES-S1 at titers of 1:320 and 1:160, respectively. These results are promising for the protective immunization of humans^ref. cDNA constructs encoding the structural antigens; spike (S), membrane (M), envelope (E), or nucleocapsid (N) protein, derived from SARS CoV (strain HKU39849, TW1, or FFM-1). As SARS-CoV is thought to infect the alveolar epithelial cell of the lung,in the present study, a type II alveolar epithelial cell clone, T7, was used to analyze the mechanism of CTL against SARS CoV membrane antigens. Mice vaccinated with SARS CoV (N) DNA or (M) DNA using pcDNA 3.1 (+) plasmid vector showed T-cell immune responses (CTL induction and proliferation) against type II alveolar epithelial cells (T7) transfected with SARS (N) or (M) DNA, respectively. To determine whether these DNA vaccines could induce T-cell immune responses in humans as well as in mice, SCID-PBL/hu mice were immunized with these DNA vaccines. PBL from healthy human volunteers were administered i.p. into IL-2Rg-chain-disrupted NOD-SCID mice [IL-2R^-/- NOD-SCID]. SCID-PBL/hu mice thus constructed can be used to analyze the human immune response in vivo. The SCID-PBL/hu mice were immunized with SARS (N) DNA or (M) DNA and analyzed for a human T-cell immune response. The M DNA vaccine enhanced CTL activity and proliferation in the presence of M peptide in SCID-PBL/hu mice. Furthermore, the SARS N DNA vaccine induced CTL activity (IFN-g production by recombinant N protein or N protein-pulsed autologous B blast cells) and proliferation of spleen cells in SCID-PBL/hu mice. These results, demonstrate that SARS M and N DNA vaccines induced human CTL and human T-cell proliferative responses. On the other hand, we have developed SARS DNA vaccines that induce human neutralizing antibodies and human monoclonal antibodies against SARS CoV. Transgenic mice expressing SARS-CoV receptor (ACE2) are also under development. These vaccines are expected to induce immune responses specific for SARS CoV in human and should provide useful tool for development of protective vaccines^ref. SARS M DNA vaccine and N DNA vaccine induced human CTL specific for SARS CoV antigens in SCID mice reconstituted with human PBLs^ref
anti-tick-borne encephalitis virus (TBEV) DNA vaccine : intranasal immunization^ref. Genetic vaccination against TBE has been successful in an experimental animal model using a plasmid containing a non-structural protein (NS1). Such vaccines would provide inexpensive protection against disease, without raising concerns over inducing ADE on subsequent exposure to infectious virus. Attempts to generate chaemeric plasmids to protect against both TBE and dengue fever were less successful. Although these chaemeric plasmids directed the synthesis and secretion of virus protein normally, no protection was observed against either TBE or dengue fever.
anti-Hantavirus DNA vaccine :

aerosol of 5-10 mm particles ideal for respiratory tract delivery
PCR-generated linear DNA fragments (LDF) with biological half-life enhanced by using PTO-modified primers^ref
DNA vaccine plasmid (pWRG/HA-M) contains both the HTNV and ANDV M gene segments. Rhesus macaques vaccinated with pWRG/HA-M produced antibodies that bound the M gene products (i.e., G1 and G2 glycoproteins), and neutralized both HTNV and ANDV. Neutralizing antibody titers elicited by the dual-immunogen pWRG/HA-M, or single-immunogen plasmids expressing only the HTNV or ANDV glycoproteins, increased rapidly to high levels after a booster vaccination administered 1-2 years after the initial vaccination series. Memory responses elicited by this long-range boost exhibited an increased breadth of cross-neutralizing activity relative to the primary response. This is the first time that hantavirus M gene-based DNA vaccines have been shown to elicit a potent memory response, and to elicit antibody responses that neutralize viruses that cause both HFRS and HPS^ref
heat shock proteins (HSPs) have been shown to act as adjuvants when coadministered with peptide antigens or given as fusion proteins and enhance the vaccination efficiency. To evaluate the enhancement of the potency of Hantaan virus (HTNV) nucleocapsid protein (NP) immunogenicity by heat shock protein 70 (HSP70), we developed a novel chimeric HTNV S-HSP70 DNA vaccine plasmid by genetically linking HSP70 gene to the full-length HTNV S segment DNA (HTNV S DNA). C57BL/6 mice were immunized with this plasmid followed by a subsequent boost with homologous recombinant protein. The levels of HTNV NP-specific antibody and cellular immune response were measured by use of ELISA, FACS analysis, cytotoxicity assay, and IFN-g ELISPOT assay. HTNV S-HSP70 DNA vaccination significantly increased the levels of HTNV NP-specific antibody, IgG_2a/IgG₁ ratio, IFN-g producing CD8⁺ T-cell precursor frequencies, and cytotoxic T lymphocyte (CTL) response when compared with immunization with HTNV S DNA alone or HTNV S DNA physically mixed with HSP70 DNA. By contrast, HSP70 DNA or vector DNA immunization could not induce appreciable levels of specific antibodies and CTL response. Thus, we demonstrate for the first time that HSP70-based HTNV S DNA can induce both humoral and cellular immune response specific for HTNV NP and is a promising candidate DNA vaccine for HTNV infection^ref

anti-respiratory syncytial virus (RSV) DNA vaccine : antiviral immunity induced by SFV replicons encoding the RSV fusion (F) and attachement (G) protein is not associated with enhanced immunopathology^ref
anti-measles virus DNA vaccine : measles remains a major worldwide problem partly because of difficulties with vaccination of young infants. New vaccine strategies need to be safe and to provide sustained protective immunity. Sindbis virus replicon particles that express the measles virus (MV) hemagglutinin (SIN-H) or fusion (SIN-F) proteins have been developed. In mice, SIN-H induced high-titered, dose-dependent, MV-neutralizing antibody after a single vaccination. SIN-F, or SIN-H and SIN-F combined, induced somewhat lower responses. To assess protective efficacy, juvenile macaques were vaccinated with a single dose of 10⁶ or 10⁸ SIN-H particles and infant macaques with 2 doses of 10⁸ particles. A dose of 10⁸ particles induced sustained levels of high-titered, MV-neutralizing antibody and IFN-g-producing memory T cells, and most monkeys were protected from rash when challenged with wild-type MV 18 months later. After challenge, there was a biphasic appearance of H- and F-specific IFN-g-secreting CD4⁺ and CD8⁺ T cells in vaccinated monkeys, with peaks 1 and 3-4 months after challenge. Viremia was cleared within 14 days, but MV RNA was detectable for 4-5 months. These studies suggest that complete clearance of MV after infection is a prolonged, phased, and complex process influenced by prior vaccination^ref. Infants younger than age 9 mo do not respond reliably to the live attenuated measles vaccine due the immaturity of their immune system and the presence of maternal Abs that interfere with successful immunization. The immune responses elicited by Sindbis virus replicon-based DNA vaccines encoding measles virus (MV) hemagglutinin (H, pMSIN-H) or both hemagglutinin and fusion (F, pMSINH-FdU) glycoproteins were evaluated in neonatal mice born to naive and measles-immune mothers. Despite the presence of high levels of maternal Abs, neonatal immunization with pMSIN-H induced long-lasting, high-avidity MV plaque reduction neutralization (PRN) Abs, mainly IgG_2a, that also inhibited syncytium formation in CD150⁺ B95-8 cells. IgG secreting plasma cells were detected in spleen and bone marrow. Newborns vaccinated with pMSINH-FdU elicited PRN titers that surpassed the protective level (200 mIU/ml) but were short-lived, had low syncytium inhibition capacity, and lacked avidity maturation. This vaccine failed to induce significant PRN titers in the presence of placentally transferred Abs. Both pMSIN-H and pMSINH-FdU elicited strong T_h1 type cell-mediated immunity, measured by T cell proliferation and IFN-g production, that was unaffected by maternal Abs. Newborns responded to measles DNA vaccines with similar or even higher PRN titers and cell-mediated immunity than adult mice. This study is the first demonstration that a Sindbis virus-based measles DNA vaccine can elicit robust MV immunity in neonates bypassing maternal Abs. Such a vaccine could be followed by the current live attenuated MV vaccine in a heterologous prime-boost to protect against measles early in life^ref.
anti-dengue virus DNA vaccine :

type 2 : the non-structural protein 1 (NS1) has been identified as a promising antigen for the development of vaccines against dengue virus (DENV). Apparently, it can elicit a protective antibody response with complement-fixing activities. pcTPANS1 plasmid contains the secretory signal sequence derived from human tissue plasminogen activator (t-PA) fused to the full length of the DENV-2 non-structural 1 (NS1) gene. Balb/c mice, intramuscularly inoculated with this plasmid, presented high levels of antibodies, recognizing mainly surface-exposed conformational epitopes present in the NS1 protein expressed by insect cells. Long-term antibody response was observed in animals 56 weeks after the first plasmid inoculation, and a rapid, efficient secondary response was observed after a DNA boost. Vaccinated animals were challenged against DENV-2 in two murine models, based on intracerebral (i.c.) and intraperitoneal (i.p.) virus inoculations, and in both cases, pcTPANS1-immunized mice were protected^{ref1,
ref2}
type 3 : DNA vaccine expressing pre-membrane and envelope genes was tested for immunogenicity and protective efficacy in Aotus monkeys. 5 of 6 vaccinated animals demonstrated moderate DEN-specific antibody responses as measured by ELISA and virus neutralization in vitro. By contrast, none of the 6 control animals developed detectable anti-DEN antibodies. When 5 vaccinated animals were challenged with live DEN-3 virus and viremia determined by PCR amplification of viral RNA in serum samples, 1 animal was completely protected and 2 were partially protected as indicated by a decrease in mean days of viremia. The results demonstrate the ability of the DEN-3 DNA vaccine to elicit a neutralizing antibody response and to partially protect against live virus challenge. These findings support the inclusion of this construct in a tetravalent DNA vaccine^ref
dengue tetravalent DNA vaccine consisting of plasmids expressing premembrane and envelope genes of each of 4 serotypes of dengue viruses. BALB/c mice immunized twice with the tetravalent vaccine at a dose of 100 mg (25 mg for each serotype) using a needle-free jet injector developed neutralizing antibodies against all serotypes. There was no interference among the four components included in this combination vaccine. Tetravalent vaccine-immunized mice showed anamnestic neutralizing antibody responses following challenge with each dengue serotype: responses to challenges from serotypes different to those used for neutralization tests were also induced^ref. Immunogenicity of this tetravalent vaccine is synergistically increased in mice by co-immunization with dengue type 2 virus (DENV2) subviral extracellular particles (D2EPs) or inactivated Japanese encephalitis vaccine (JEVAX). A single immunization with a mixture of 100mug of the tetravalent vaccine and 150ng of D2EPs or a 1/10 dose of JEVAX induced moderate levels of neutralizing antibodies in a 90% plaque reduction assay. Immunized mice were protected from "artificial" viremia created by intravenous injection with DENV2^ref
DNA shuffling and screening technologies were used to produce chimeric DNA constructs expressing antigens that shared epitopes from all four dengue serotypes. 3 shuffled constructs (sA, sB and sC) were evaluated in the rhesus macaque model. Constructs sA and sC expressed pre-membrane and envelope genes, whereas construct sB expressed only the ectodomain of envelope protein. 5 of 6, and four of six animals vaccinated with sA and sC, respectively, developed antibodies that neutralized all 4 dengue serotypes in vitro. 4 of 6 animals vaccinated with construct sB developed neutralizing antibodies against 3 serotypes (den-1, -2 and -3). When challenged with live dengue-1 or dengue-2 virus, partial protection against dengue-1 was observed. These results demonstrate the utility of DNA shuffling as an attractive tool to create tetravalent chimeric dengue DNA vaccine constructs, as well as a need to find ways to improve the immune responses elicited by DNA vaccines in general^ref
3 nonreplicating dengue virus type 2 (DENV-2) vaccines: (i) a DNA vaccine containing the prM-E gene region (D), (ii) a recombinant subunit protein vaccine containing the B domain (i.e., domain III) of the E protein as a fusion with the Escherichia coli maltose-binding protein (R), and (iii) a purified inactivated virus vaccine (P). Groups of 4 rhesus macaques each were primed once and boosted twice using seven different vaccination regimens. After primary vaccination, ELISA antibody levels increased most rapidly for groups inoculated with the P and DP combination, and by 1 month after the second boost, ELISA titers were similar for all groups. The highest plaque reduction neutralization test (PRNT) titers were seen in those groups that received the DR/DR/DR combination (geometric mean titer [GMT], 510), the P/P/P vaccine (GMT, 345), the DP/DP/DP combination (GMT, 287), and the R/R/R vaccine (GMT, 200). The next highest titers were seen in animals that received the D/R/R vaccine (GMT, 186) and the D/P/P vaccine (GMT, 163). Animals that received the D/D/D vaccine had the lowest neutralizing antibody titer (GMT, 49). Both ELISA and PRNT titers declined at variable rates. The only significant protection from viremia was observed in the P-vaccinated animals (mean of 0.5 days), which also showed the highest antibody concentration, including antibodies to NS1, and highest antibody avidity at the time of challenge^ref

anti-variola virus DNA vaccine : priming with DNA vaccines and boosting with live vaccinia virus (VacV). DNA plasmids encoding the A4L, A27L and H5R VacV genes were generated. A considerable increase in antigen-specific IFN-g responses, high proliferative and humoral antigen-specific responses were detected in experimental primed Balb/C mice compared to controls after VacV boost. The VacV-DNA plasmids elicited IFN-g production in HLA-A2.1 transgenic mice in response to predicted HLA-A2.1 restricted peptide epitopes, providing valuable data for further vaccine development^ref. An experimental smallpox DNA vaccine comprised of four vaccinia virus genes (4pox) administered by gene gun elicited protective immunity in mice challenged with vaccinia virus, and in nonhuman primates challenged with monkeypox virus^ref. This 4pox DNA vaccine can be efficiently delivered by a novel method involving skin electroporation using plasmid DNA-coated microneedle arrays. Mice vaccinated with the 4pox DNA vaccine mounted robust antibody responses against the four immunogens-of-interest, including neutralizing antibody titers that were greater than those elicited by the traditional live virus vaccine administered by scarification. Moreover, vaccinated mice were completely protected against a lethal (>10LD₅₀) intranasal challenge with vaccinia virus strain IHD-J. To our knowledge, this is the first demonstration of a protective immune response being elicited by microneedle-mediated skin electroporation^ref
anti-Rift Valley fever virus (RVFV) DNA vaccine : the glycoprotein (GP) and nucleocapsid (NC) genes of Rift Valley fever virus (RVFV) were expressed in different expression systems and were evaluated for their ability to protect mice from virulent challenge using a prime-boost regime. Mice vaccinated with a lumpy skin disease virus-vectored recombinant vaccine (rLSDV-RVFV) expressing the two RVFV glycoproteins (G1 and G2) developed neutralising antibodies and were fully protected when challenged, as were those vaccinated with a crude extract of truncated G2 glycoprotein (tG2). By contrast mice vaccinated with a DNA vaccine expressing G1 and G2 did not sero-convert with only 20% of them surviving challenge. Mice vaccinated with the DNA vaccine and boosted with rLSDV-RVFV also failed to sero-convert but 40% survived challenge. Surprisingly, although none of the mice immunised with the purified NC protein sero-converted, 60% of them survived virulent challenge. The rLSDV-RVFV construct was then further evaluated in sheep for its dual protective abilities against RVFV and sheeppox virus (SPV). Vaccinated sheep sero-converted for both viruses and were protected against RVFV challenge, however, neither the immunised or negative control animals showed any significant reactions to the virulent SPV challenge^ref
anti-Rift Valley fever virus (RVFV), Crimean Congo hemorrhagic fever virus (CCHFV),tick-borne encephalitis virus (TBEV), and Hantaan virus (HTNV) DNA vaccines, were tested in mice alone or in various combinations. The bunyavirus vaccines (RVFV, CCHFV, and HTNV) expressed Gn and Gc genes, and the flavivirus vaccine (TBEV) expressed the preM and E genes. All vaccines were delivered by gene gun. The TBEV DNA vaccine and the RVFV DNA vaccine elicited similar levels of antibodies and protected mice from challenge when delivered alone or in combination with other DNAs. Although in general, the HTNV and CCHFV DNA vaccines were not very immunogenic in mice, there were no major differences in performance when given alone or in combination with the other vaccines^ref.
anti-Japanese encephalitis virus (JEV) DNA vaccine : a single intramuscular immunization of DNA carrying the prM and E coding sequences from JEV or WNV protected mice and horses from virus challenge. The use of multivalent combination DNA vaccines designed to immunize against multiple flaviviruses is an interesting area of development, although the immunogenicity of DNA vaccines in humans first needs to be greatly improved. I.m. immunization with DNA vaccines has been shown to induce a broad range of immune responses and protective immunity in many animal models, but it is less effective in primates. One reason for this may be the low expression of vector-encoded antigen in cells. The use of vaccine vector (pCJ-3) containing 2 regulatory elements, a chimeric intron and a bovine growth hormone (BGH) polyadenylation signal, markedly increased antigen expression both in vitro and in vivo. A positive correlation was seen between the level of expression of JEV envelope proteins and the levels of antibodies in C3H/HeN mice. Immunization of mice with pCJ-3/ME (pCJ-3 containing the entire membrane and envelope protein genes) with or without cardiotoxin pretreatment resulted in higher antibody titers than immunization with vector containing only envelope protein and conferred full protection against infection with JEV. Electron microscopy showed that pCJ-3/ME expression resulted in the production of virus-like particles of JEV in vitro. The particles enhanced the production of higher titers of neutralizing antibodies and thus provided immunity against JEV. Consequently, the efficacy of the newly developed DNA vaccines was validated. This should pave the way to clinical trials in man^ref. A recombinant encoding E protein of the Beijing-1 strain derived from JEV was constructed and the humoral, cellular and protective immunity induced was analyzed. The recombinant pJBE containing E (1,500 bps) gene from the Beijing-1 strain of Japanese encephalitis virus was constructed and then transfected into the HepG2 cell line by liposome fusion. The expression of E (about 53 kD) protein in transfected cells was analyzed by Western blot using a specific anti-JEV-E antibody. BALB/c mice were vaccinated with 3 mug of pJBE by the gene-gun technique. JaGAr-01 and Beijing-1 strains (10⁵ PFU/100 ml) of JEV were given to BALB/c mice by i.p. injection 3 weeks after double DNA immunization with a lethal virus challenge. BALB/c mice were observed for 21 days after challenge. An 80% plaque reduction neutralization test was performed to titrate the neutralization antibody before and after viral challenge. A lactate dehydrogenase activity release test was used to examine CTL activity after double DNA immunization. The expression of about 53 kD protein associated with pJBE was determined in transfected HepG2 cells with specific anti-JEV-E antibody. A higher level of neutralization antibodies and the cytotoxicity effect were induced with pJBE immunization using the gene-gun technique, and were similar to those induced with inactivated vaccine derive from the Beijing-1 strain of JEV. Balb/c mice immunized with pJBE survived the challenge with the different strains of JEV; however, Balb/c mice immunized with inactivated vaccine did not survive the challenge with the JaGAr-01 strain of JEV at all. DNA vaccine containing the E protein gene derived from JEV can provide not only better efficacy including humoral and cellular immunity, but also cross-protection against infection with homologous and heterologous JEV^ref
anti-human papillomavirus (HPV) DNA vaccine :

the gene gun approach has been previously used to test several intracellular targeting and intercellular spreading strategies targeting HPV-16 E7. These strategies include the use of the sorting signal of LAMP-1, Mycobacterium tuberculosis heat shock protein 70 (HSP70), calreticulin (CRT) and HSV-1 VP22 proteins. All of these strategies have been shown to be capable of enhancing E7-DNA vaccine potency. DNA vaccines employing these intracellular targeting or intercellular spreading strategies targeting HPV-16 E6 have been characterized for their ability to generate E6-specific CD8⁺ T cell immune responses and antitumor effects against an E6-expressing tumor cell line, TC-1, in C57BL/6 mice. All the intracellular targeting strategies (CRT, LAMP-1, HSP70) as well as the intercellular spreading strategy (VP22) were able to enhance E6 DNA vaccine potency, although the orientation of HSP70 linked to E6 antigen in the E6 DNA vaccine appears to be important for the HSP70 strategy to work. The enhanced E6-specific CD8⁺ T cell immune response in vaccinated mice also translated into potent antitumor effects against TC-1 tumor cells. All of the intracellular targeting and intercellular spreading strategies that have been shown to enhance E7 DNA vaccine potency were also able to enhance E6 DNA vaccine potency^ref
a carcinogenesis model involving the combination of 9,10-dimethyl-1,2-benzanthracene (DMBA) application with physical wounding of hamster lingual mucosa. The presence of a novel hamster oral papillomavirus (HOPV) was demonstrated and its genome sequenced. In the present study, this HOPV hamster model was used to test whether vaccination with the L1 gene could prevent the development of oral carcinoma. DNA plasmids encoding the L1 gene or the vector alone were injected intramuscularly into 20 vaccinated and 20 control hamsters, respectively. The lingual tips of the hamsters were painted with DMBA for 8 weeks. A portion of the lingual tips was excised, and the tips were then painted daily with DMBA until the animals were killed 13 days later. All control hamsters developed lingual carcinoma, whereas 12 of the L1-vaccinated hamsters showed no lesions. These results suggest that immunization with L1 DNA vaccines may prevent the development of papillomavirus-associated oral cancer^ref
for safety reasons the use of a transforming gene like the HPV-16 E7 for DNA vaccination is not feasible in humans. The artificial ("shuffled") HPV-16 E7-gene (HPV-16 E7SH), containing all putative CTLs epitopes and exhibiting high safety features, induces a strong E7-wildtype (E7WT) directed cellular and humoral immune response including tumor protection and regression after in vivo immunization in the murine system. Moreover, the vaccine candidate demonstrated immunogenicity in humans, demonstrated by priming of antigen-specific T cells in vitro. Importantly, the artificial HPV-gene has completely lost its transforming properties as measured in soft agar transformation assays. These results may be of importance for the development of vaccines based on oncogenes or oncoproteins^ref
enhanced immunization after intranasal coadministration of Escherichia coli heat-labile enterotoxin B subunit and human papillomavirus 16-L1 DNA vaccine^ref
codon optimization of the HPV-16 E7 gene resulted in highly efficient translation of E7 and increased the immunogenicity of E7-specific DNA vaccines. Since vaccines targeting E6 also represent an important strategy for controlling HPV-associated lesions, we developed a codon-optimized HPV-16 E6 DNA vaccine (pNGVL4a-E6/opt) and characterized the E6-specific CD8⁺ T cell immune responses as well as the protective and therapeutic anti-tumor effects in vaccinated C57BL/6 mice. Transfection of human embryonic kidney cells (293 cells) with pNGVL4a-E6/opt resulted in highly efficient translation of E6. In addition, vaccination with pNGVL4a-E6/opt significantly enhanced E6-specific CD8⁺ T cell immune responses in C57BL/6 mice. Mice vaccinated with pNGVL4a-E6/opt are able to generate potent protective and therapeutic antitumor effects against challenge with E6-expressing tumor cell line, TC-1. Thus, DNA vaccines encoding a codon-optimized HPV-16 E6 may be a promising strategy for improving the potency of prophylactic and therapeutic HPV vaccines with potential clinical implications^ref
HPV16-DNA-E6E7, constructed with HPV16 E7 and E6 genes was generated and used to against HPV16-induced tumors. An E7 DNA fragment was inserted into E6 gene to produce a recombinant gene (E6E7-DNA). The E6E7-DNA gene was inserted into a mammalian expression vector, pcDNA 3.1+, to construct the DNA vaccine candidate. Animals (C57BL/6 mice) were immunized with the vaccine candidate with various concentrations (50 mg, 100 mg or 200 mg, respectively), and cytotoxicity measurement and tumor protection assay were carried out to examine the immunological effects of the vaccine candidate. Immunization of with HPV16-E6E7-DNA induced HPV16-specific immune response and also conveyed protection against TC-1 induced tumor in vivo. A survival rate (90%) after 45 days of tumor challenge was observed. The animals injected with a higher dosage of the vaccine (200 mg) exhibited prolonged survival duration of > 55 days. No transforming activity of the vaccine candidate was detected, as determined by focus formation and degradation of endogenous p53^ref
modified E7-HSP70, which has been introduced mutations in 2 zinc-binding motifs of E7, will eliminate its transformation potential and enhance the immunogenicity of fusion protein and repress E7 containing tumors with a low level of MHC-I molecules to lung metastatic in murine model. The transforming properties of mutant E7 oncoprotein were examined by the soft agar colony-formation assays, explored the immunogenicity of modified E7-HSP70 gene by various cellular and humor immune responses and evaluated the effect of treating lung metastatic tumor with a low expressing MHC-I molecules by tumor challenge assay and therapeutic experiment. The mutant E7 oncoprotein has completely lost its transforming properties as measured in the soft agar colony-formation assays. Modified E7-HSP70 gene inducted stronger E7-specific cellular immune response than that induced by unmodified E7-HSP70. More importantly, the new construct significantly reduced the number of B16-HPV16E7 lung metastases. The modified E7-HSP70 gene may be as a powerful and safe DNA vaccine in controlling the hematogenous spread of HPV16E7-associated tumors with low expression of MHC-I molecules. In addition, the B16-HPV16E7 lung metastasis model can be used to test the efficacy of various E7-specific vaccines and immunotherapeutic strategies in settings more relevant to clinical requirements^ref
several HLA-A2.1-transgenic rabbit lines were established to provide a host to study CD8⁺ T cell responses during virus infections. HLA-A2.1 protein expression was detected on cell surfaces within various organ tissues. Continuous cultured cells from these transgenic rabbits were capable of presenting both endogenous and exogenous HLA-A2.1-restricted epitopes to an HLA-A2.1-restricted epitope-specific CTL clone. A DNA vaccine containing an HLA-A2.1-restricted human papillomavirus type 16 E7 epitope (amino acid residues 82-90) stimulated epitope-specific CTLs in both PBLs and spleen cells of transgenic rabbits. In addition, vaccinated transgenic rabbits were protected against infection with a mutant cottontail rabbit papillomavirus DNA containing an embedded human papillomavirus type 16 E7/82-90 epitope. Complete protection was achieved using a multivalent epitope DNA vaccine based on epitope selection from cottontail rabbit papillomavirus E1 using MHC class I epitope prediction software. HLA-A2.1-transgenic rabbits will be an important preclinical animal model system to study virus-host interactions and to assess specific targets for immunotherapy^ref
a DNA vaccine (E7IR) was designed for prophylactic and therapeutic treatment of HPV-16⁺ tumors containing 2 repeats of the E7 gene to inactivate transformation and duplicate available epitopes. Mice were vaccinated then tumor challenged, or challenged and then immunized and monitored for tumor volume and survival. Splenocytes were utilized for in vivo CTL assays. The E7IR vaccine demonstrated decreased tumor volume and enhanced survival in prophylactic and therapeutic experiments and improved CTL-mediated lysis^ref
mice vaccinated with a plasmid encoding for an E7 protein fused to a domain of the MHC class II-associated invariant chain (IiE7) were protected against tumor challenge. Mice immunized against an ubiquitinated form of E7 (Ub(Ala)E7) failed to control tumor growth. Protection induced by IiE7 was correlated with the development of CD8⁺ CTL and required the presence of CD4⁺ cells. In vitro studies confirmed that the IiE7 fusion protein was expressed at high levels in the endosomal compartment of transfected cells, while the natural and the ubiquitin-modified form of E7 were mainly nuclear. The present study suggests that an efficient anti-tumor response can be induced in vivo by DNA constructs encoding for E7 protein forms localizing at the endosomal compartment^ref

anti-HHV-1 / HSV-1 DNA vaccine : the use of morphine has been demonstrated to increase susceptibility to infections. HSV-1 is a highly successful pathogen among immunocompromised individuals. In the present study, due to the importance of HSV vaccination in morphine abusers, the effects of chronic morphine exposure on the host response to a HSV-1 gB DNA-based vaccine have been investigated. The study is addressing an important aspect of vaccine development among the susceptible (immunocompromised) hosts. BALB/c mice were exposed to morphine over 11 days. They were then vaccinated with DNA vaccine or KOS strain as a live vaccine. The findings showed that the morphine-treated animals failed to respond to DNA vaccination evaluated by the anti-HSV gB antibody titer, DTH and lethal HSV-1 challenge. Under the same conditions, the KOS vaccine showed a reduced Ab titer and DTH response in morphine-treated mice, but could protect mice against the lethal challenge and was safe for vaccination of morphine-treated animals^ref. DNA vaccines consisting of vector IRES-gD expressing HSV-1 glycoprotein D (gD) and vector IRES-gD-IL-2 coexpressing HSV-1 gD and interleukin-2 (IL-2), respectively, were constructed. After intramuscular inoculation, both vaccines induced in BALB/c mice antibodies as assayed by ELISA and virus neutralization. However, IRES-gD-IL-2 elicited significantly higher levels of IgG (ELISA) and neutralizing antibodies than IRES-gD. Isotyping of sera from mice injected with IRES-gD-IL-2 revealed predominately IgG_2a antibodies. IRES-gD-IL-2 also elicited a higher delayed-type sensitivity (DTH) reaction. However, there was no difference in the protection against lethal challenge with HSV-1 between the 2 vaccines (P>0.05). The results suggest that the vaccination with IRES-gD-IL-2 can efficiently enhance the immune response of mice to HSV-1, particularly through increased cellular immunity^ref. To compare the immunogenity of the herpes simplex virus 1 (HSV-1/HHV-1) recombinant glycoprotein D (gD1), as a potential protective vaccine, Balb/c mice were immunized with either gD1/313 (the ectodomain of the gD1 fusion protein consisting of 313 amino acid residues), or the plasmid pcDNA3.1-gD (coding for a full length gD1 protein, FLgD1). A live attenuated HSV-1 (deleted in the gE gene), and a HSV-1 (strain HSZP)-infected cell extract served as positive controls, and three non-structural recombinant HSV-1 fusion proteins (ICP27, UL9/OBP and thymidine kinase--TK) were used as presumed non-protective (negative) controls. Protection tests showed that the LD50 value of the challenging infectious virus increased 90-fold in mice immunized with ICP27, but remained unchanged in other control mice immunized with TK and OBP polypeptides. A significant protection (the LD50 value of challenging virus increased 800-fold) was noted following immunization with gD1/313, while immunization with the gE-del virus and/or the gD1 DNA vaccine resulted in a > 4,000-fold increase of the challenging virus dose killing 50% of the animals. Using ELISA, elevated antibody titers were detected following immunizations with gD1/313, gE-del virus, and/or HSV-1-infected-cell extract. In addition, all of the three non-structural proteins elicited a good humoral response (with titres ranging from 1:16,000 to 1:128,000). The lowest IgG response (1:8,000) was noted after immunization with the gD1 DNA vaccine. Peripheral blood leukocytes (PBLs) as well as splenocytes from mice immunized with gD1/313, gE-del virus, and gD1-plasmid responded in lymphocyte transformation test (LTT) to the presence of purified gD1/313 antigen. For PBLs, the most significant stimulation of thymidine incorporation was registered at a gD1/313 concentration of 5 mg/100 ml, while the splenocytes from DNA vaccine-immunized mice responded already at a concentration of 1 mg/100 ml^ref
anti-HHV-2 / HSV-2 DNA vaccine : pcDNA3-gD (pgD) and pcDNA3-MIP-1a / CCL3 (pMIP-1a) were co-injected to examine the modulatory effects of MIP-1a on immune phenotype and protection against lethal challenge with HSV-2. T_h-cell proliferative responses were dramatically enhanced by co-injection of pgD and pMIP-1a compared with injection of pgD alone. The secretion of IL-2 and IFN-g was also significantly increased by pgD and pMIP-1a co-injection; however, the production of cytokines IL-4 and IL-10 was not affected by co-injection. pgD and pMIP-1a co-injection resulted in a moderate enhancement of systemic gD-specific antibody level, but mucosal secretory IgA was markedly enhanced. When BALB/c mice were challenged intravaginally with 100 LD₅₀ of HSV-2 strain Sav, pMIP-1a co-injection with pgD improved their survival rate and significantly reduced both the number of mice with lesions and the lesion severity. Therefore, MIP-1a cDNA as a HSV-2 DNA vaccine adjuvant drives antigen-specific T_h1-type responses, reducing HSV-2-derived morbidity and mortality^ref. The prime-boost immunization strategies were studied using a DNA vaccine carrying the full-length glycoprotein D-1 gene and a baculovirus-derived recombinant glycoprotein D, both expressing HSV glycoprotein D-1 protein. Immunization with recombinant DNAs encoding antigenic proteins could induce cellular and humoral responses by providing antigen expression in vivo. Higher immune response, however, occurred when the recombinant proteins followed DNA inoculation. While all groups of the immunized mice and positive control group could resist virus challenge, a higher virus neutralizing antibody level was detected in the animals receiving recombinant protein following DNA vaccination^ref. A mouse model was employed to evaluate protective cellular immune responses induced by an immediate early antigen of HSV-2. Particle-mediated DNA vaccination of mice with a DNA plasmid-encoding ICP27 resulted in the induction of ICP27-specific IFN-g and TNF-a production in Balb/c mice, but little protection to intranasal challenge with wild type HSV-2. However, when the DNA vaccine was supplemented with as little as 50 ng of a vector encoding the A and B subunits of the Escherichia coli heat labile enterotoxin (LT), animals were profoundly protected from morbidity and mortality. The ICP27+LT-mediated protection was correlated with a large increase in ICP27-specific IFN-gand TNF-a production but cytokine-specific monoclonal antibody treatment at the time of challenge showed that protection was mediated predominantly by IFN-g. Furthermore, depletion of T cell subsets prior to infectious challenge demonstrated that removal of either CD8⁺ or CD4⁺ T cells impaired protection with CD8⁺T cells appearing to play a direct effector role. These data demonstrate that augmented cellular immune responses resulting from LT vector plus antigen vector administration to the skin are biologically significant, leading to enhanced protection against mucosal pathogenic challenge^ref. To construct the recombinant eukaryotic expression plasmid of murine MIP-1a and investigate the effect of MIP-1a as an adjuvant on immune response induced by herpes simplex virus type II glycoprotein D (HSV-II gD) DNA vaccine, using total RNA from RAW264.7 cells stimulated with LPS, the whole code sequence of murine MIP-1a was amplified by RT-PCR and inserted into pcDNA3 at HindIII/XbaI restriction sites. The recombinant eukaryotic expression plasmid Pm was transiently expressed in COS-7 cells and its specificity was demonstrated by RT-PCR and Boyden chemotaxis chamber assay. BALB/c mice were immunized with gD DNA vaccine and/or MIP-1a, and the effect of MIP-1a on gD DNA vaccine was evaluated by detecting anti-HSV-II antibody, antigen-specific lymphoproliferative responses, and examining survival rates after mice were challenged intravaginally with HSV-II. The recombinant eukaryotic expression plasmid of murine MIP-1a was constructed, and it was revealed that immune responses of HSV-II gD DNA vaccine were enhanced by coimmunization with MIP-1a. The murine MIP-1a can be used as an adjuvant of HSV-II gD DNA vaccine^ref
anti-HHV-5 / CMV DNA vaccine :

it is unclear if protective immunity can be conferred by a CMV vaccine encoding a single protein subunit, or if multiple viral genes need to be targeted. Using the guinea pig model of congenital CMV infection, these studies examined the immunogenicity and efficacy of a DNA vaccine based on the guinea pig cytomegalovirus (GPCMV) genome cloned as a non-infectious BAC plasmid, modified by transposon insertion into the homolog of the HCMV tegument protein, UL48. Following vaccination of female Hartley guinea pigs with BAC DNA, adverse GPCMV-related pregnancy outcome were assessed after establishment of pregnancy, followed by GPCMV third-trimester challenge. Animals immunized with recombinant BACmid engendered anti-GPCMV antibodies by ELISA assay. Immunogenicity of BAC plasmid DNA was augmented by inclusion of the lipid adjuvant, DOTMA/DOPE, in the vaccine regimen. Among pups born to 12 control (sham-immunized) dams challenged with GPCMV in the third trimester, mortality was 23/35 (66%). In contrast, among evaluable pregnancy outcomes in pups born to 10 BAC-immunized pregnant dams, preconception immunization resulted in reduced pup mortality, to 10/34 pups (29%; p<0.005 versus control, Fisher's exact test). In addition, vaccinated dams had reduced viral load, compared to controls, as assessed by quantitative, real-time PCR^ref.
to construct eucaryotic expression recombinant vector containing vivo truncated region of UL83 gene of human cytomegalovirus, realize its steady expression in Hep-2 cell, and study sheltered effect of the eucaryotic expression recombinant vector as DNA vaccine. A vivo truncated UL83 gene fragment encoding for truncated HCMV pp65 was obtained by PCR from human CMV AD169 stock genome. By gene recombinant ways, the truncated UL83 gene fragment was cloned into eucaryotic expression vector pEGFP-C1 with reported gene coding GFP to construct recombinant vector pEGFP-C1-UL83. The recombinant vector pEGFP-C1-UL83 was tested by different methods including PCR, restriction digestion and gene sequencing. Test results showed the recombinant vector was constructed successfully. After pEGFP-C1-UL83 was transfected into Hep-2 cell by lipofectin mediation, expression of GFP and truncated pp65 fusion protein in Hep-2 cell was observed at different time points by fluorescence microscope. Quantity of fusion protein expression was the highest at 36h point. Then, Hep-2 cell was cultured selectively by RPMI-1640 containing G418 (200 mg/mL) to obtain a new cell stock of expressing truncated UL83 Gene fragment steadily. RT-PCR and Western blot results showed the truncated fragment of UL83 gene could be expressed steadily in Hep-2 cell. The result showed a new cell stock of expressing Tpp65 was established. This cell stock could be useful in some HCMV research fields, for example, it could be a tool in study of pp65 and HCMV infection, and it could provide a platform for the research into the therapy of HCMV infection. Immune sheltered effect of pEGFP-C1-UL83 as DNA vaccine was studied in vivo of HCMV congenital infection mouse model. The mouse model was immunized solely by pEGFP-C1-UL83, and was immunized jointly by pEGFP-C1-UL83 and its expression product. When the mouse was pregnant and brought to bed, differential antibody of anti-HCMV pp65 was tested by indirect ELISA in mother mouse, the infectious virus was separated with the method of virus separation, and pp65 antigen was checked up by indirect immunofluorescence staining in fetal mouse. Results showed differential antibody of anti-HCMV pp65 was produced in mouse model. Tilter of the antibody was from 1:2.51 to 1:50.79. Results of virus separation and pp65 checkup of fetal mouse brain tissue were negative. So the conclusion can be reached that pEGFP-C1-UL83 as DNA vaccine in vivo has sheltered effect which can prevent HCMV vertical transmission from mother mouse to her fetus^ref
HCMV glycoprotein complex II (gcII) consists of 2 glycoproteins, gM and gN. Although gcII specific IgG purified from HCMV positive patient sera can neutralize HCMV, there has been no report describing the generation of virus-neutralizing antibodies by immunization with individual recombinant gM or gN antigens. In the current study, gM and gN antigens were expressed by the mammalian expression vector pJW4303 and used as DNA vaccines to determine the immunogenicity of these proteins. Sera from mice or rabbits immunized with individual or combinations of gM and gN DNA vaccines contained gM and gN specific antibodies as confirmed by ELISA and Western blot analyses. The combined gM and gN antigens induced the strongest antibody responses that recognized both gM and gcII complex while gM DNA vaccine alone could only elicit antibody specific for gM antigen. When given alone, the gN DNA vaccine did not induce detectable gcII specific antibody even though in vitro gN expression was confirmed by the formation of gM/gN complex in FSK cells using a gN-specific monoclonal antibody 14-16A. The neutralizing antibody titer of anti-gM/gN sera (1:128) was higher than that of anti-gM sera (1:32) against the autologous virus, HCMV AD169. Heterologous HCMV strains including Towne and Davis could also be neutralized by the anti-gM/gN antisera. Our data supported the rationale for the use of the HCMV gM/gN protein complex as protective antigens for subunit based HCMV vaccine development. DNA vaccination is an effective approach to express the gM/gN antigen complex in vivo without the need to express and purify these highly insoluble and structurally complicated antigens^ref
DNA immunization of mice with the MCMV homologs of HCMV DNA polymerase (M54) or helicase (M105) was protective against virus replication in the spleen following systemic challenge, with the protection level elicited by the M54 DNA being comparable to DNA expressing the immunodominant IE1-pp89. Intracellular interferon gamma staining of CD8 T cells from mice immunized with either the M54 or M105 DNAs showed strong primary responses that recalled rapidly after viral challenge. M54- and M105-specific CD8 T cells were detected after primary MCMV infection, but their levels were not consistently above background. The conserved, essential proteins of the CMVs thus represent a novel class of CD8 T cell targets that may contribute to a successful HCMV vaccine strategy^ref

Polyvalent DNA vaccines

anti-HBV + anti-HCV DNA vaccine :

a bicistronic vector pcDNA3.0BA was constructed from pcDNA3.0. HCV PC154 gene and HBV preS2S gene were inserted into this vector to form bicistronic expression construct pcDNA3.0BAPC154S2S and monocistronic expression construct pcDNA3.0BAPC154 or pcDNA3.0BAS2S. These plasmids were transiently expressed in COS-7 cells and injected into muscles of BALB/c mice. RESULTS: pcDNA3.0BA contains two cistronic units, which can co-express 2 kinds of genes, with the first immunogen gene and the second gene serving as additional immunogen or as modulator for the immune responses. HBV surface Ag and HCV core Ag were coexpressed in vitro. The antibody responses and lymphoproliferation to antigens were similar between bicistronic and monocistronic expression construct in mice. pcDNA3.0BA is a novel vector, which can coexpress two proteins and elicit polyvalent immune responses^ref.
HBsA and HCV envelope antigen in BALB/c mice^ref

anti-bacterial vaccines

anti-Bacillus anthracis DNA vaccine : a eucaryotic expression plasmid encoding the exosporium antigen BclA, which is a collagen-like surface protein, pSecTag BclA, carries a secretion signal for the recombinant antigen. Using NMRI mice the effects of immunisation with a combination of PA- and BclA-encoding plasmid DNA were compared to immunisations with either PA- or BclA-encoding pDNA. After 3 immunisations the mice were infected with 25xLD₅₀ of B. anthracis Ames spores. The plasmid pSecTag BclA, induced high BclA-specific antibody responses. Vaccination with a combination of PA- and BclA-encoding pDNA led to significantly better survival than immunisation with only PA- or only BclA-encoding plasmids^ref. The current anthrax vaccine in the U.S., the anthrax vaccine adsorbed, has several serious drawbacks, most notably the very lengthy and complicated dosing schedule. Thus, there is a critical need to develop an alternative anthrax vaccine with a simplified immunization schedule. To address this need, we evaluated the feasibility of topically priming or boosting onto the skin using an anthrax protective antigen (PA) protein-encoding DNA vaccine. To this end, topical immunization of mice onto their skin with a perflubron-based microemulsion incorporated with a PA63-encoding plasmid, pGPA, led to significant PA-specific antibody responses, which have anthrax lethal toxin-neutralization activity. Moreover, topical boosting of mice primed with PA protein with the pGPA-incorporated, perflubron-based microemulsion significantly enhanced the anti-PA antibody responses induced. This topical anthrax DNA vaccine has the potential to be combined with a vaccine, such as the current AVA, to produce a simplified and more convenient dosing schedule^ref.
anti-Legionella pneumophila DNA vaccine : immunogenic protein of Lgeionella pneumophila and to examine its protective efficacy against LP in mice. The eukaryotic expression recombinant plasmid of LP immunogenic protein gene (pcDNA3.1-ip) was constructed and used to immunize BALB/c female mice intramuscularly, and antigen specific antibodies, lymphocyte proliferative response, IFN-g production and cytotoxic T-lymphocyte response of the immunized mice were detected. The BALB/c mice immunized by the pcDNA3.1-ip repeatedly were then challenged with LP (10 LD₅₀), and the number of survivors in each group was quantitated. Then the mice were sacrificed and lung tissue were harvested for quantitative culture of LP and observation of the pathologic changes. The DNA vaccine pcDNA3.1-ip could induce specific humoral and cellular immunity in the immunized mice. The results indicated that the immunogenicity of pcDNA3.1-ip was higher than that of pcDNA3.1(+) (P<0.01). The vaccine was effective in protecting mice against infection after LP (10 LD₅₀) challenge. The DNA vaccine encoding immunogenic protein of LP can be used as a candidate for novel vaccine design^ref
anti-Bordetella pertussis DNA vaccine : a toxic N-terminal 180-amino-acid fragment (C180) of pertussis toxin S1 subunit has the most potent ability to induce protective immunity against pertussis toxin (PT) following DNA-based immunization^ref. For the development of a safer pertussis DNA vaccine, three plasmids encoding mutant C180 (C180-R9K, C180-E129G and C180-R9K/E129G) were constructed and tested for their protective immunogenicity and cytotoxicity. All of the gene gun delivery of the plasmid, performed by inserting the mutant C180 gene into a mammalian expression vector pcDNA3.1, successfully induced anti-PT IgG antibody production without the loss of immunogenicity in mice. The immunizations of mice with the plasmids significantly inhibited leukocytosis-promoting activity by PT. Among stably transfected Chinese hamster ovary (CHO) cells expressing mutant C180, the expression of C180-R9K and C180-R9K/E129G was non-toxic to the transfectants, confirming that these mutant C180s have no cytotoxicity to mammalian cells. These results indicate that C180-R9K and C180-R9K/E129G genes, especially C180-R9K/E129G, are candidates for safe and effective antigen DNAs in the development of pertussis DNA vaccine^ref. A recombinant pertussis DNA vaccine containing the pertussis toxin subunit 1 (PTS1), fragments of the filamentous hemagglutinin (FHA) gene and pertactin (PRN) gene encoding FHA and pertactin were constructed. Balb/c mice were immunized with several DNA vaccines and antigen-specific antibodies anti-PTS1, anti-PRN, anti-FHA, cytokines IL-10, IL-4, IFN-g, TNF-a, and splenocyte-proliferation assay were used to describe immune responses. The recombinant DNA vaccine could elicit similar immune responses in mice as that of separate plasmids encoding the 3 fragments, respectively. Mice immunized with DNA and boosted with the corresponding protein elicited more antibodies than those that received DNA as boost. In particular, when the mice were co-immunized with murine granulocyte-macrophage colony-stimulating factor plasmids and boosted with proteins, all 4 cytokines and the 3 antigen-specific antibodies were significantly increased compared to the pVAX1 group. Anti-PTS1, anti- FHA, IL-4 and TNF-a elicited in the colony stimulating factor (CSF) prime-protein boost group showed significant increase compared to all the other groups. This prime and boost strategy has proven to be very useful in improving the immunogenicity of DNA vaccines against pertussis^ref. 3 immunodominant antigen gene fragments of pertussis, pertussis toxin subunit 1 (pts1), fragments of pertactin (prn) and filamentous hemagglutinin (fha), were recombined as fragment pts1-prn-fha named ppf, and it was cloned to plasmid pVAX1 as pVAX1/ppf. Compared to those injected with pVAX1, the mice injected with pVAX1/ppf significantly elicited more antigen specific antibody anti-PTS1, anti-PRN, anti-FHA and cytokine IL-10, IFN-g. When pGM-CSF was coinjected with pVAX1/ppf, the mice showed significantly increases of the three antibodies and cytokine IL-10, IL-4, IFN-g and TNF-alpha compared to those injected with pVAX1 only. The mice in group pVAX1/ppf & pGM-CSF, in particular; induced much more anti-PTS1, IL-4 and TNF-a than those in group pVAX1/ppf. In the intracerebral mouse protection test, the mice immunized with pVAX1/ppf or pVAX1/ppf & pGM-CSF induced protection to a lethal dose of B. pertussis. The results indicate that recombinant DNA vaccine and pGM-CSF coinjection can induce protective immunity against B. pertussis, demonstrating a valuable method to prevent pertussis^ref.
anti-Pseudomonas aeruginosa DNA vaccine : 3 different plasmids each targeting a fusion of outer membrane proteins (OprF/OprI), a protein regulating type III secretion system (PcrV), or an appendage (PilA) were prepared and mice were immunized with single (monovalent) or a combination of these plasmids (multivalent) via intramuscular electroporation (imEPT) or gene gun. Immunization with multivalent DNA vaccine via imEPT induced the most potent protection against lethal pneumonia. Although the serum levels of IgG binding to whole bacteria cells were comparable between groups, the strongest immune protection was associated with the serum levels of T_h1-dominated multivalent IgG, the bronchoalveolar levels of MIP-2 and IFN-g, and the number of neutrophils and macrophages in the bronchoalveolar lavage following intranasal challenge. These results implied the possible clinical application of multivalent DNA vaccine against P. aeruginosa^ref
anti-Mycobacterium tuberculosis DNA vaccine^ref :

mycolyl-transferase antigen 85A :

recombinant modified vaccinia virus Ankara (MVA) expressing antigen 85A (MVA85A) : in the first phase 1 study of any candidate subunit vaccine against tuberculosis, it was found to induce high levels of antigen-specific IFN-g-secreting T cells when used alone in bacille Calmette-Guérin (BCG)-naive healthy volunteers. In volunteers who had been vaccinated 0.5–38 years previously with BCG, substantially higher levels of antigen-specific IFN-g-secreting T cells were induced, and at 24 weeks after vaccination these levels were 5–30 times greater than in vaccinees administered a single BCG vaccination. Boosting vaccinations with MVA85A could offer a practical and efficient strategy for enhancing and prolonging antimycobacterial immunity in tuberculosis-endemic areas^ref.
BALB/c mice were vaccinated with BCG either following DNA priming or prior to DNA boosting. Ag85A specific CD4⁺ and CD8⁺ mediated IFN-g responses were increased in mice primed with DNA prior to BCG, and in BCG vaccinated mice subsequently boosted with DNA. In the latter immunization protocol, antigenic stimulation also induced significant levels of IL-17. Mice were monitored for cachexia and survival following a low dose intravenous challenge with M. tuberculosis H37Rv. Priming with Ag85A but not control DNA increased significantly the protective efficacy of the BCG vaccine as indicated by reduced cachexia and prolonged survival time: 32 weeks versus 23 weeks in one experiment and 33 weeks versus 26 weeks in another experiment (MST in control, TB infected mice: 17 weeks in both experiments). On the other hand, boosting of BCG by subsequent Ag85A DNA in saline or vaxfectin^TM - or recombinant 85A protein or MVA-85A for that matter - did not augment the efficacy of BCG (MST 19-21 weeks in all vaccinated groups versus 11 weeks in control, TB infected mice). Ag85A DNA priming can increase efficacy of BCG and that boosting protocols of BCG may possibly be hampered by the induction of T_h(IL-17) cells^ref
a recombinant form of BCG Tokyo with an Ag85A gene insert was administered once subcutaneously to guinea pigs and its protective efficacy was compared with that of a DNA vaccine encoding Ag85A from Mycobacterium tuberculosis administered twice to guinea pigs by epidermal gene gun bombardment. Vaccination with either the recombinant BCG Tokyo or Ag85A DNA significantly reduced the severity of pulmonary pathology and the number of pulmonary and splenic CFUs (p<0.001). The recombinant BCG Tokyo was better than Ag85A DNA in terms of protective efficacy against M. tuberculosis. When immunogenic synthetic Ag85A peptide was further used as a booster together with recombinant BCG Tokyo (Ag85A) or Ag85A DNA, lung pathology was improved significantly and the number of pulmonary CFU was reduced significantly. Neither recombinant BCG Tokyo, Ag85A DNA, nor the parental BCG Tokyo protected the guinea pigs from hematogenous spread of tubercle bacilli to the spleen because splenic granulomas without central necrosis were recognized. The spleen tissues from guinea pigs vaccinated with recombinant BCG Tokyo or Ag85A DNA expressed IFN-g and IL-2 mRNA at significantly high levels (p<0.001) as evaluated by reverse transcription polymerase chain reaction. It is concluded that peptide boosting is important for the induction of higher protective efficacy by recombinant BCG Tokyo or a tuberculosis DNA vaccine and both recombinant BCG Tokyo (Ag85A) and Ag85A DNA vaccine induce T_h2 cytokine mRNA expression significantly^ref
plasmid DNA vaccine coding for Ag85A alone or for both Ag85A and GM-CSF and investigated the immune adjuvant effects of electroporation and GM-CSF co-expression, alone or in combination, on CD4 and CD8 T cell IFN-gamma responses, CTL activities and immune protection from pulmonary Mycobacterium tuberculosis challenge in a Balb/c mouse model. We have found that use of electroporation allows a single intramuscular (i.m.) DNA injection to be as effective as repeated i.m. DNA injections in activation of both Ag85A-specific CD4 and CD8 T cells. Co-expression of immune-enhancing cytokine GM-CSF by the same plasmid DNA TB vaccine could further enhance T cell activation including CTL activities on top of electroporation. With regard to immune protection from pulmonary M. tb challenge, use of eletroporation also allows a single i.m. DNA injection to be as effective as repeated i.m. DNA injections. Co-expression of GM-CSF transgene also moderately enhances immune protection and such effect is more evident for systemic protection. However, GM-CSF expression has little added effect on immune protection by electroporation-aided immunization protocols^ref

antigen 85B :

Sindbis virus-based DNA construct expressing the tuberculosis antigen 85B (Sin85B) : the protective efficacy of Sin85B was initially assessed by aerogenically challenging immunized C57BL/6 mice with virulent Mycobacterium tuberculosis. At 1 and 7 months postinfection, the lung bacterial burdens were considerably reduced and the lung pathology was improved in vaccinated mice compared to naive controls. Furthermore, the mean survival period for Sin85B-immunized mice (305 +/- 9 days) after the tuberculous challenge was extended 102 days relative to the naive mice (203 +/- 13 days) and was essentially equivalent to the survival time of Mycobacterium bovis BCG-vaccinated mice (294 +/- 15 days). The essential role of IFN-g in Sin85B-mediated protection was established by showing that significantly increased levels of IFN-gmRNA were present postinfection in lung cells from vaccinated mice relative to control mice and by demonstrating that IFN-g depletion prior to challenge abolished the vaccine-induced protection. The substantial antituberculosis protective responses induced by Sin85B immunization of CD4^-/- mice strongly suggested that CD8 cells partially mediate Sin85B-induced protective immunity. Interestingly, Sin85B vaccination did not protect RNase L^-/- (a key enzyme in the innate antiviral response) mice while significant protection was detected in RNase L^-/- mice immunized with either BCG or a conventional DNA plasmid expressing antigen 85B. These data show that immunization with Sin85B offers protection similar to BCG in a murine model of pulmonary tuberculosis and suggest that Sin85B-induced protection is dependent upon both innate and acquired immune mechanisms^ref.
2 candidate DNA vaccines based on the proteins CFP10 and CFP21 encoded by regions of difference (RDs) of Mycobacterium tuberculosis were evaluated individually and in multivalent combination with the immunodominant protein Ag85B for induction of protective immune responses against experimental tuberculosis. Experimental DNA vaccines induced substantial levels of cell-mediated immune responses as indicated by marked lymphocyte proliferation, significant release of the T_h1 cytokines IFN-gamma and IL-12 (p40), and predominant cytotoxic T cell activity. High levels of antigen-specific IgG₁ and IgG_2a antibodies observed in the sera of immunized mice depicted strong humoral responses generated by DNA vaccine constructs. The multivalent combination of 3 DNA vaccine constructs induced maximal T cell and humoral immune responses. All the experimental vaccines imparted significant protection against challenge with M. tuberculosis H(37)Rv (in terms of CFU reduction in lungs and spleen) as compared to vector controls. The level of protection exhibited by multivalent DNA vaccine formulation was found to be equivalent to that of Mycobacterium bovis BCG observed both at 4 and 8 weeks post-challenge. These results show the protective potential of the multivalent DNA vaccine formulation used in this study^ref

hybrid gene ESAT-6 (9.9 kDa) - Ag85B (34.6 kDa)^ref
Mycobacterium leprae 65-kDa heat shock protein (hsp65) : 100 mg plasmid in 50 ml sterile PBS to quadriceps muscle of mice^ref

researchers led by Youngchul Sung of Pohang University of Science and Technology, Republic of Korea took mice with tuberculosis and gave them standard drug therapy with or without an experimental DNA vaccine containing Mycobacterium leprae hsp65 gene. In mice given drugs alone, bacteria numbers rose after treatment was stopped. Mice given the combined treatment showed no such flare-up. In addition, the combination therapy triggered an immune response that significantly reduced re-infection with tuberculosis bacteria^ref. This is the first study showing that giving vaccination and drugs together can accelerate the disappearance of bacteria as well as protecting against re-infection. Although this study involved mice, the combination treatment may get into the clinic faster than one might think. With the increasing threat of MDR strains, there is a push to get promising therapies into clinical trials
Hsp65 + IL-12 using gene gun bombardment and the hemagglutinating virus of Japan (HVJ)-liposome : a mouse IL-12 expression vector (mIL-12 DNA) encoding single-chain IL-12 proteins comprised of p40 and p35 subunits were constructed. In a mouse model, a single gene gun vaccination with the combination of Hsp65 DNA and mIL-12 DNA provided a remarkably high degree of protection against challenge with virulent Mycobacterium tuberculosis; bacterial numbers were 100-fold lower in the lungs compared to BCG-vaccinated mice. To explore the clinical use of the DNA vaccines, HVJ-liposome encapsulated Hsp65 DNA and mIL-12DNA (Hsp65+mIL-12/HVJ) were evaluated. The HVJ-liposome method improved the protective efficacy of the Hsp65 DNA vaccine compared to gene gun vaccination. Hsp65+mIL-12/HVJ induced CD8⁺ cytotoxic T lymphocyte activity against Hsp65 antigen. Most importantly, Hsp65+mIL-12/HVJ vaccination resulted in a greater degree of protection than that evoked by BCG. This protective efficacy was associated with the emergence of IFN-g-secreting T cells and activation of proliferative T cells and cytokines (IFN-g and IL-2) production upon stimulation with Hsp65 and antigens from M. tuberculosis. These results suggest that Hsp65+IL-12/HVJ could be a promising candidate for a new tuberculosis DNA vaccine, which is superior to BCG vaccine^ref.
the Hsp65 DNA vaccine had no effect on CFU counts when given alone, but augmented the bactericidal activity of moxifloxacin ^ref
plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly(DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy (effector components of protective immunity) and toxicity (effector components of pathology) induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs^ref

DNA vaccines expressing mycobacterial antigens ESAT-6 (Rv3875), a-crystallin (Rv2031c) and superoxide dismutase A (Rv3846) were evaluated for their immune responses in Balb/c mice and protective efficacy in guinea pigs. Immunization of mice with the DNA vaccines expressing superoxide dismutase A and a-crystallin resulted in markedly higher levels of IFN-g as compared to the levels of IL-10. The DNA vaccine expressing ESAT-6 elicited a mixed T_h1/T_h2 response. Immunization of guinea pigs with these DNA vaccines and subsequent challenge of animals with Mycobacterium tuberculosis H(37)Rv, showed that DNA vaccine expressing superoxide dismutase imparted the maximum protection as observed by a 50 and 10 folds reduction in bacillary load in spleens and lungs, respectively, in comparison to immunization with vector control^ref
an approximately 800bp DNA encoding a mycobacterial synthetic gene designated as VacIII (containing ubiquitin gene UbGR and 4 immunogenic mycobacterial epitopes or genes of ESAT-6, Phos1, Hsp 16.3, and Mtb8.4) was sub-cloned into a bacterial expression vector of pRSET-B resulting in a 6xHis-VacIII fusion gene construction. This recombinant clone was over expressed in Escherichia coli BL-21 (DE-3). The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate. The inclusion bodies were solubilized with 8M urea and the recombinant protein was purified by Ni-NTA column and dialyzed by urea gradient dialysis. This method produced a relatively high yield of recombinant VacIII protein and the cloned VacIII gene offers the potential development of other vaccine formats such as DNA vaccine and recombinant vaccine^ref
the early pulmonary cytokine responses elicited by a low dose M. tuberculosis aerogenic challenge were evaluted in mice that had been immunized with either BCG or a TB DNA vaccine cocktail, 2 vaccine preparations that induce long-term protection in the mouse model of pulmonary TB. Using 3 different assays, specific cytokine responses were elevated in the lungs of vaccinated mice (relative to naive controls) during the second week post-challenge. By measuring cytokine levels in the bronchoalveolar lavage fluid (BAL) and cytokine mRNA concentrations in pulmonary cells, the levels of IFN-g, IL-12, and RANTES were shown to be elevated from days 7-14 post-challenge in the lungs. By intracellular cytokine staining (ICS), increased numbers of lung CD4 and CD8 cells expressing IFN-g were also seen at days 10 and 14 after the infection. Moreover, increased post-challenge IFN-gamma levels were detected using the ICS and cytokine mRNA assays in aging BCG-immunized mice that had been effectively boosted with a TB DNA vaccine. Taken together, these data suggest that the post-infection induction of early T_h1 responses correlate with the induction of long-term protective immunity in vaccinated mice^ref.
mycobacterial heat shock protein 65 (HSP65) and interleukin 12 (IL-12) delivered by the hemagglutinating virus of Japan (HVJ)-liposome (HSP65+IL-12/HVJ). This vaccine provided remarkable protective efficacy in mouse and guinea pig models compared to the BCG vaccine, on the basis of an induction of the CTL activity and improvement of the histopathological tuberculosis lesions, respectively. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. This novel vaccine provided a higher level of the protective efficacy than BCG based upon the assessment of mortality, the ESR, body weight, chest X-ray findings and immune responses. Furthermore, the combination of HSP65+IL-12/HVJ and BCG by the priming-booster method showed a synergistic effect in the TB-infected cynomolgus monkey (100% survival). These data indicate that our novel DNA vaccine might be useful against Mycobacterium tuberculosis for human clinical trials^ref

IL-12

IL-23

IL-27

M. tuberculosis

⁺

M. tuberculosis

Mycobacterium bovis

M. tuberculosis

^ref

Mycobacterium tuberculosis

^ref

anti-Mycobacterium avium DNA vaccine : infection of mice with Mycobacterium avium or immunization with a novel PE gene expressed by M. avium (MaPE) showed that a dominant T-cell immune response was elicited. Immunization with an MaPE DNA vaccine protected mice against an aerosol challenge with Mycobacterium tuberculosis, suggesting that mycobacteria express PE antigens with cross-protective T-cell epitopes^ref
anti-Mycobacterium leprae DNA vaccine :
anti-Mycobacterium ulcerans DNA vaccine : in BALB/c mice infected with M. ulcerans subcutaneously, Hsp65 appeared to be an immunodominant antigen eliciting both humoral and cellular responses. However, vaccination of mice with a DNA vector encoding Mycobacterium leprae Hsp65 only poorly limited the progression of M. ulcerans infection. In contrast, a substantial degree of protection was conferred by subcutaneous vaccination with BCG, suggesting that BCG antigens that are conserved in M. ulcerans, such as TB10.4, the 19kDa antigen, PstS3 and Hsp70, may be interesting to consider as subunit vaccines in future prospects^ref.
anti-Streptococcus pneumoniae DNA vaccine

pneumococcal surface protein A (PspA)-based DNA vaccines^{ref1,
ref2,
ref3}. BALB/c mice were immunized with DNA vaccine vectors expressing the N-terminal region of PspA. Animals immunized with a vector expressing secreted PspA developed higher levels of antibody than mice immunized with the vector expressing the antigen in the cytosol. However, both immunogens elicited similar levels of protection against intraperitoneal challenge. Furthermore, immunization with exactly the same fragment in the form of a recombinant protein, with aluminium hydroxide as an adjuvant, elicited even higher antibody levels, but this increased humoral response did not correlate with enhanced protection. These results show that DNA vaccines expressing PspA are able to elicit protection levels comparable to recombinant protein, even though total anti-PspA IgG response is considerably lower^ref.

anti-Streptococcus mutans DNA vaccine :

2 plasmid DNA constructs were obtained by cloning separately into the eukaryotic expression vector pcDNA3.1/V5-His-TOPO the wall-associated protein A (wapA) gene of Streptococcus mutans GS-5 or its truncated derivative antigen A (agA) gene encoding a known candidate antigen for dental caries vaccine. The immunogenicity of the two constructs, designated pcDNA-wapA and pcDNA-agA, was compared by intranasal immunization of two groups of mice using the cationic DMRIE-C (1,2-dimyristyloxypropyl-3-dimethylhydroxy ethyl ammonium bromide-cholesterol) as an adjuvant. Immunization with pcDNA-wapA or pcDNA- agA resulted in specific salivary IgA and systemic IgG antibodies to the target antigens after two doses given at 3-week intervals. Higher salivary IgA level was observed in the mice immunized with the pcDNA-wapA vaccine compared to those immunized with the pcDNA-agA vaccine. Furthermore, anti-WapA antibody inhibited S. mutans sucrose-dependent adherence suggesting a potential protection against S. mutans colonization of the tooth, while anti-AgA had no significant effect. Indeed, prediction and analysis of protein epitopes showed that WapA contains highly promiscuous MHC-II binding motifs in addition to those found in AgA. Immunodot assay confirmed that WapA bound biotin-labeled dextran, whereas AgA did not. These data indicated that full-length WapA is a better candidate vaccine antigen than the soluble AgA, which is truncated in the hydrophobic membrane and wall-spanning region^ref.
A dual-promoter expression plasmid that harbors the target gene encoding SBR of Streptococcus mutans can be applied as DNA vaccine especially suitable for using attenuated Salmonella as delivery vector to elicit effective mucosal immune responses because of its advantage of possessing dual-promoter. Genes encoding SBR and eGFP were amplified by PCR and inserted to the proper sites of vector pCMVnir. Then IRES sequence was inserted between the genes coding for SBR and eGFP. Furthermore, a DNA fragment encoding tPA signal peptide was fused to the 5' end of target gene. Thereby, construction of the dual-promoter expression plasmid pCN-SSIE was completed and then the plasmid was analyzed with DNA sequencing and endonuclearase digestion mapping. The expressions of SBR protein by attenuated Salmonella SL3261 and CHO cell transformed or transfected by the plasmid were tested respectively. Finally, BALB/c mice were immunized through injecting intramuscularly with plasmid pCN-SSIE and anti-SBR specific IgG in serum was tested. Both DNA sequencing and endonuclearase digestion mapping showed that the construction of pCN-SSIE was successful with its open reading frame being correct. The expressions of SBR protein in transformed attenuated Salmonella SL3261 and transfected CHO were detected, and anti-SBR specific IgG levels in serum of immunized mice were markedly higher than the control^ref. Anti-caries DNA vaccines, pGJA-P and pGJA-P/VAX, encoding Streptococcus mutans antigens fused to CTLA4, which binds to B7 molecule expressed on the surfaces of APCs. Rabbits and monkeys were immunized via intranasal or intramuscular routes. The fusion vaccine induced accelerated and increased specific antibody responses in serum and saliva compared with non-fusion DNA vaccine in rabbits. Significant specific serum IgG and salivary IgA levels could be detected in fusion vaccine-immunized monkeys^ref. To investigate and compare the expression pattern and level of targeted anti-caries plasmids encoding different-size antigens in eukaryotic cells. The A-P fragment of PAc (surface protein antigen) was removed from pGJA-P encoding the signal peptide, extracellular domains of human CTLA-4, human Ig hinge, CH2 and CH3 domains, A-P fragment of PAc and GLU (glucan binding domain) region of GTF-I of Streptococcus mutans, to obtain the plasmid pGJGLU. pCI vector skeleton of pGJA-P or pGJGLU was replaced by pVAX1 to construct plasmids pGJA-P/VAX and pGJGLU/VAX. CTLA4-Ig-GLU fragment was removed from pGJGLU and inserted into the vector pEGFP-N1 to obtain the recombinant plasmid pGJGLU/GFP. The CHO cells were transfected with those plasmids by using liposome and the expression of fusion protein was observed with fluorescence microscope. ELISA was used to detect the expression level of fusion proteins in cultured supernatants. Specific vesicles with green fluorescence could be observed in the CHO cells transfected with pGJGLU/GFP. The recombinant fusion protein could be detected in the cultured supernatants of CHO cells transfected with pGJA-P/VAX, pGJGLU/VAX and pGJGLU/GFP, of which the concentration was different. The highest concentration of recombinant fusion protein was observed in the supernatants of CHO cells transfected with pGJGLU/VAX. CTLA-4 targeted fusion protein could be expressed and secreted by eukaryotic cells. The size of antigen may affect the expression level of CTLA-4 targeted anti-caries DNA vaccine^ref. Recently, pGJA-P/VAX, a new targeted anti-caries DNA vaccine for human trials, was constructed by replacing the pCI vector used in the construction of pGJA-P with pVAX1, the only vector authorized by the US Food and Drug Administration in clinical trials. Intranasal vaccination of mice with pGJA-P/VAX induced strong antibody responses that lasted for > 6 months. Furthermore, pGJA-P/VAX could still be detected at both the inoculation site and the draining cervical lymph nodes 6 months after immunization. Thus, the persistent immune responses are likely due to the DNA depot in the host, which acts as a booster immunization^ref
GBR gene of Streptococcus mutans and study the immunogenicity of recombinant GBR protein. The GBR gene was cloned into the expression vector pTriEx-4 through gene cloning techniques by PCR,T-A clone and etc,the recombinant plasmid pTriEx-4-GBR was analyzed by DNA sequencing and endonuclearase digestion mapping. The recombinant GBR(rGBR) protein expression was induced with IPTG in E. coli JM109(DE3) which was transformed with plasmid pTriEx-4-GBR and then the rGBR protein was purified by affinity chromatography. Balb/c mice were randomly divided into 2 groups: mice of experimental group were inoculated subcutaneously with the rGBR protein and the rGBR protein was replaced for NS in the control group, then the anti-serum was evaluated by ELISA.The data were analysed by statistical software PEMS3.0. The DNA sequence and the reading frame of GBR gene in the reconstructed vector pTriEx-4-GBR was in corresponding with the initial design and the rGBR protein was purified. The level of specific anti-rGBR serum IgG in the experimental group was significantly higher than that in the control group (P<0.005). The expressing plasmid carrying the GBR gene was constructed successfully and the purified recombinant GBR protein can elicit specific murine system immune response, which is necessary for further experiments including construction of bivalent anti-caries DNA vaccine and studies both in vitro and in vivo^ref.

anti-Staphylococcus aureus DNA vaccine : blocking the primary stages of Staphylococcus aureus infection, specifically the bacterial adhesion to cell and the colonization of the mucosal surface, may be the most effective strategy for preventing infections. Clumping factor A (ClfA) is considered to be one of the most important adhesions factors of S. aureus to host cells. The present study describes the immune response of dairy cattle to a DNA vaccine against ClfA and evaluates the ability of specific genetic adjuvants, targeting sequences (GM-CSFand CTLA-4) and transporter molecules (chitosan and copolymer) to modify the immune response of cows. The results show that vaccination of cows with fibrinogen-binding region A induced a strong and specific antibody response to ClfA in comparison with a control group injected with the pCI vector alone. Although the co-expression of both genetic adjuvants and the addition copolymer transporter did not augment the overall antibody response, these approaches decreased the number of non-responsive cows. Chitosan was the only factor that did not enhance the immune response. Three months after the last DNA immunization, 3 cows from each of the pGM-CSF, IRES, pCTLA and pCI groups were injected with 200 mg of recombinant ClfA protein in IFA. A strong humoral response was observed in all groups following this protein boost, with the response occurring slightly earlier in DNA-primed protein boost cows. Sera and milk samples taken from cows after the second DNA injection or after the protein boost (sera only) were analyzed for their ability to block adherence and increase phagocytosis. Pre-incubation of S. aureus with sera or milk from vaccinated cows significantly reduced the pathogen's ability to adhere to MAC-T cells relative to the sera and milk samples from the pCI-injected control cows. Similarly, pools of sera and milk from vaccinated cows increased phagocytosis of S. aureus by neutrophils. After the protein boost, sera were more efficient promoters of phagocytosis, reflecting the higher anti-ClfA antibody level of these sera. DNA-prime/protein boost regimes combined with molecular adjuvants appeared to be effective in generating a strong immune response to S. aureus antigens in cattle^ref. Staphylococcus aureus is the most common etiologic agent of mastitis in bovines and a major cause of economic losses in the dairy industry. Since adhesins, that anchor bacteria to the extracellular matrix (ECM), are among the most important S. aureus virulence factors, we used 4 adhesion factors (fibrinogen binding protein, fibronectin binding protein A, clumping factor A and collagen adhesin) as target in a DNA vaccine. Intranasal immunization with a pDNA mixture coding the four adhesins, triggered significant levels of specific serum and mucosal Ig that inhibited S. aureus adhesion to cow mammary gland epithelial cells in vitro. Splenocytes of immunized mice challenged in vitro with S. aureus extracts showed a strong proliferative response. Finally, immunized mice were significantly protected against intramammary challenge with S. aureus^ref. To investigate the strategy of using a multivalent polyprotein DNA vaccine against S. aureus, a series of plasmids was used to immunize mice followed by infectious challenge. The plasmid vaccines expressed Clumping factor A (Clfa), fibronectin binding protein A (FnBPA) and the enzyme Sortase (Srt) as single proteins or combined as a polyprotein. All animals produced a mixed T_h1 and T_h2 response including functional antigen-specific, mostly IgG_2a antibodies, sustained production of IFN-g and a predominantly CD8⁺ T-cell response. Upon challenge with a virulent S. aureus isolate (Sa042), after 21 days, 55% of the multi-gene vaccinated mice survived infection compared to only 15% of the control groups. Vaccinated mice showed no signs of arthritis when challenged with the less virulent "Newman" strain that caused reactive arthritis in the controls. The results suggest that a multi-gene polyprotein-expressing nucleic acid vaccine alone produces a combined T_h1 and T_h2 response that can contribute to protection against the complex pathogenesis of S. aureus^ref. Methicillin-resistant Staphylococcus aureus (MRSA) is the major pathogen involved in nosocomial infections, leading to high rates of morbidity and mortality in hospitals worldwide. The methicillin resistance occurs due to the presence of an additional penicillin-binding protein, PBP2a, which has low affinity for b-lactam antibiotics. In the past few years, vancomycin has been the only antibiotic option for treatment of infections caused by multiresistant MRSA; however, reports of vancomycin-resistant strains have generated great concerns regarding the treatment to overcome these infections. In the present study, we report preliminary results regarding the humoral immune response generated in BALB/c mice by two different doses of naked DNA vaccine containing an internal region, comprising the serine-protease domain, of the PBP2a of MRSA. The immunization procedure consisted of four immunizations given intramuscularly within 15-day intervals. Blood was collect weekly and anti-PBP2a-specific antibodies were screened by ELISA. BALB/c mice immunized with DNA vaccine anti-PBP2a have shown higher antibody titers mainly after the fourth immunization, and intriguingly, no correlation between the humoral immune response and DNA dose was observed. The DNA vaccine anti-PBP2a induced an immune response by production of specific antibodies anti-MRSA in a non-dose-dependent manner, and it could represent a new and valuable approach to produce specific antibodies for passive immunization to overcome MRSA infections^ref
anti-Porphyromonas gingivalis DNA vaccine :

anti-rgpA DNA vaccine induced protective immune responses against P. gingivalis infection in mice^ref. Reduction in lethality against infection by lethal doses of P. gingivalis was observed in the rgpA DNA vaccine-immunized mice. Cytokine levels in the mouse model with nonlethal doses of infection by P. gingivalis were evaluated to analyze the mechanism of protection by immunization with the rgpA DNA vaccine. After nonlethal challenge with invasive P. gingivalis W50, production of IL-2, IL-4, IL-5 and IL-12 was elevated; however, IFN-g was lower in the serum of the DNA vaccine-immunized mice than in the serum of nonimmunized mice. The regulation of IFN-gproduction elicited by immunization with the rgpA DNA vaccine may play a significant role in protection against P. gingivalis infection in mice^ref.
PcDNA3.1+/kgpcd was delivered into Sprague-Dawley (SD) rats by submandibular gland-targeted injection. The anti-KGPcd sIgA in saliva was measured by indirect ELISA method. IHC staining was used to assess the protection in the animal model. The level of specific anti-KGPcd sIgA in saliva of the experimental group was significantly higher than that of control group. HE staining showed that immunization with recombined plasmid pcDNA3.1+/kgpcd could protect or minimize tissue destruction caused by subsequent P. gingivalis challenge in the rat model. pcDNA3.1+/kgpcd was a good candidate for anti-periodontitis gene vaccine and could provide protection against Porphyromonas gingivalis-caused periodontitis in rat lesion model^ref
a eukaryotic co-expression plasmid pIRES-fimA:IL15, which can be used as an immunoreaction-enhancing DNA vaccine against Porphyromonas gingivalis FimA, and investigate its expression in mammalian cells. The eukaryotic co-expression plasmid pIRES-fimA:IL15 was constructed by molecular cloning methods and characterized by restricted endonuclease mapping, PCR and DNA sequencing. The plasmid was transfected into mammalian cell CHO using Lipofectamine 2000. Expression of fimA gene was detected by Western blot and the protein secretion in cultural medium was analyzed by ELISA. Endonuclease mapping showed that the target genes fimA and IL-15 obtained by PCR had the same molecular size as predicted. The DNA sequencing data also indicated that inserted fimA gene and IL-15 gene had correct DNA sequence and orientation. The recombined plasmid could express FimA in mammalian cell CHO transfected. FimA and IL-15 could be secreted into cultural supernatant detected by ELISA^ref
gingipains are potent virulence factors of Porphyromonas gingivalis and are likely to be associated with the development of periodontitis. It is, therefore, suggested that gingipain inhibition by vaccination could be a useful therapy for adult periodontitis. This study investigated the ability of antibodies raised against the catalytic part and the adhesin/haemagglutinin part of HRgpA to prevent haemagglutination and fibronectin degradation caused by P. gingivalis. 2 DNA vaccines, one containing the adhesin part of HRgpA and one with the catalytic part of HRgpA, were constructed. BALB/c mice were immunized intramuscularly with either catalytic-part-encoding plasmids, adhesin-part-encoding plasmids or empty control plasmids. Sera from mice immunized with the catalytic vaccine or the adhesin vaccine each showed inhibition of human fibronectin degradation. A DNA vaccine encoding the adhesin or catalytic part of HRgpA induces responses that inhibit the degradation of molecules important for the structure and function of gingival and bone tissues^ref

anti-Vibrio cholerae DNA vaccine : Salmonella vaccine strains have been previously reported to evoke immune response against heterologous antigen cloned in the flagellin gene. A non-toxic cholera toxin subunit B epitope was selected by using computer-based program and genetically fused in single and double copy in Salmonella typhimurium flagellin gene. The chimeric flagellin functioned normally as demonstrated by motility assay. Cholera toxin B epitope cloned in flagellin was expressed at the flagellar surface. The expression was verified by Western blotting. Mice administered orally and subcutaneously with aroA flagellin-negative strain of S. dublin expressing the chimeric flagellin gene resulted in generation of antibody against cholera toxin. Mice administered intramuscularly and subcutaneously with naked mammalian expression vector containing the same cholera toxin epitope could also evoke the antibody response though it was less than the chimeric flagellin^ref
anti-Helicobacter pylori DNA vaccine : ureB gene^ref. The H. pylori urease subunit B construct or vector alone was administered to mice via the intranasal route. Spleens and stomachs were examined on day 0 and weeks 3, 6, and 12 after immunization. Proliferation of spleen cells was assessed using the carboxyfluorescein diacetate succinimidyl ester-based flow cytometry assay and cytokine secretion from cultured spleen cells was detected by ELISA, after stimulation with the urease subunit B recombinant antigen. Total RNA was isolated from stomach and spleen tissue and the expression of b-defensin and cytokine genes was monitored by RT-PCR. Immunized mice were challenged with H. pylori and bacterial DNA quantified by TaqMan PCR. Results: The urease B subunit DNA vaccine increased INF-g secretion and splenocyte proliferation without inducing adverse effects in the spleen. Increase in gastric b-defensin 1 and marked induction in local IL-10 : IFN-g ratio up to 12 weeks post-immunization suggest a potential role for local innate immune responses in protection at the site of infection. Although significant bacterial reduction in the stomachs of urease B subunit DNA-immunized mice was observed, intermediate reduction was also noted in the vector group. Increased defensin expression and adjuvant effects of the cytosine preceding guanosine motifs may contribute to this phenomenon. CpG motifs, even without coadministration with antigen, can reduce extracellular bacterial load. In this study, a DNA construct encoding the urease B subunit was assessed for its immune profile and its ability to reduce bacterial colonization in the murine stomach. Local innate immune responses may play a greater role than previously supposed in limiting H. pylori colonization in the gastric mucosa^ref. The development of a vaccine against Helicobacter pylori has become a priority to prevent major morbidity and mortality associated with this infection. Our goal was to prepare and evaluate a DNA vaccine based on the urease B gene (ureB). The ureB gene of H. pylori was amplified and cloned into the eukaryotic expression vector pcDNA3.1/TOPO. Plasmid DNA was purified from transformed Escherichia coli cells and used to immunize mice by the intragastric, i.m., intrarectal (40 mg each) and intranasal (16 mg) route, 3 doses every 2 weeks, with CpG oligodeoxynucleotide (ODN) as adjuvant. 4 weeks after the third dose, animals were orally challenged with Helicobacter felis and were sacrificed 6 weeks later. The stomach was stained to detect the presence of infection. Despite in vitro confirmation of successful cloning and functionality of the ureB gene with expression of a protein morphologically and antigenically identical to urease B, the DNA vaccine did not perform well in vivo. Immunization of mice produced a weak immune response. Overall, intrarectal and intranasal administration seemed more immunogenic than other routes. Protection against challenge was modest and nonsignificant, and slightly better on animals immunized by the intramuscular and intranasal route. A DNA vaccine based on H. pylori urease B was poorly immunogenic and nonprotective at the conditions evaluated. Higher doses, better adjuvants or a prime-boost approach may circumvent these limitations^ref. To construct a live attenuated Salmonella typhimurium (S. typhimurium) strain harboring the H pylori neutrophil activating protein (HP-NAP) gene as an oral recombinant DNA vaccine, and to evaluate its immunogenicity. By genetic engineering methods, the genomic DNA of H pylori was extracted as a template. The total length of the HP-NAP gene was amplified by PCR and cloned into pBT vector for sequencing and BLAST analysis, then subcloned into a eukaryotic expression vector pIRES followed by PCR identification and restriction enzyme digestion. The identified recombinant plasmid pIRES-NAP was transfected into COS-7 cells for target fusion protein expression, and its antigenicity was detected by Western blotting. Then the recombinant plasmid was transformed into a live attenuated S. typhimurium strain SL7207 as an oral vaccine strain, and its immunogenicity was evaluated with animal experiments. A 435 bp product was cloned using high homology with HP-NAP gene in GenBank (> 98%). With identification by PCR and restriction enzyme digestion, a recombinant eukaryotic expression plasmid pIRES-NAP containing the HP-NAP gene of H pylori was successfully constructed. The expressed target protein had a specific reaction with H pylori whole cell antibody and showed a single strip result detected by Western blotting. Oral immunization of mice with recombinant DNA vaccine strain SL7207 (pIRES-NAP) also induced a specific immune response. The successful construction of HP-NAP oral DNA vaccine with good immunogenicity may help to further investigate its immunoprotection effects and develop vaccine against H pylori infection^ref
anti-Orientia tsutsugamushi DNA vaccine : the 56-kD outer membrane protein has previously been shown to be the immunodominant antigen in scrub typhus infections. Its gene was cloned into the DNA vaccine vector pVR1012 as a vaccine candidate (pKarp56). The in vitro expression of this 56-kD antigen by pKarp56 was confirmed in tissue culture by an indirect fluorescence assay and Western blot analysis. The initial antibody responses of mice immunized with varied doses of the pKarp56 were barely detected, but increases were observed after each of three subsequent booster immunizations. Although no protection was observed with a single immunization of pKarp56, after 4 immunizations, 60% of the mice survived a 1,000 x 50% lethal dose (LD₅₀) challenge. These results specifically confirm the importance of the 56-kD protein antigen in protective immunity against O. tsutsugamushi and demonstrate the feasibility of DNA vaccines for the prevention of scrub typhus^ref. The DNA vaccine vector pVR1012 carrying the O. tsutsugamushi Karp strain 47-kDa protein gene (p47Kp) consistently provides outbred mice protection against homologous challenge^ref
anti-Treponema pallidum DNA vaccine : to construct the recombinant plasmid of eukaryotic expression containing Gpd gene from Treponema pallidum and study its immunogenicity in New Zealand White rabbits. Gpd gene was amplified from the genomic DNA of T. pallidum and cloned into appropriate site of pcDNA3. 1 ( + ) vector. After verified that the Gpd antigen gene could be expressed in HeLa cells by Western blot and immunocytochemistry, recombinant plasmids pcDNA3.1 ( + )-Gpd, control plasmid pcDNA3. 1 ( + ) or PBS buffer were administered in 3 groups of New Zeal and White rabbits. Booster immunizations were employed at 2-week interval for three times. ELISA was used for the quantitative detection of the specific antibody in the sera of rabbits. The proliferation response of spleen cells was detected by MTT assay. The results of the Western blot and immunocytochemistry showed that Gpd gene constructed in pcDNA3.1 ( + ) vector could express a fusion protein with a calculated molecular mass of 41kD in HeLa cells and react with positive blood serum from syphilis patients. The significant specific antibody IgG titers were observed and the highest titer was 1:1024 in rabbits after three times with pcDNA3.1 ( + )-Gpd. The proliferation response of spleen cells were significantly higher than that of rabbits injected with pcDNA3.1 ( + ) (p < 0.05). All above results establish a solid basis for future studying the biological activities of Gpd and benefit the development of the syphilis DNA vaccine^ref

anti-protozoal vaccines

anti-Leishmania major DNA vaccine : DNA vaccination was used in a susceptible BALB/c high-dose model of infection to screen 100 novel Leishmania major genes as vaccine candidates. In addition to finding novel protective antigens, several antigens that reproducibly exacerbated disease were identified. The immune response to 2 of these antigens, lmd29 and 584C, that were originally identified in an expressed sequence tag cDNA sequencing project, were examined. In addition to exacerbating disease in susceptible BALB/c mice, these antigens retain a propensity to exacerbate disease in resistant C57BL/6 mice. This ability to exacerbate disease was lost when susceptible BALB/c mice were rendered resistant by disruption of the genes encoding IL-4 alone, IL-4/IL-13, or IL-4, IL-5, IL-9, and IL-13. Failure to exacerbate disease was associated with reduced IL-5 and IL-10 production in IL-4 knockout mice. Treatment of lmd29-vaccinated mice with anti-IL-10R antibody prior to challenge infection converted exacerbation in wild-type BALB/c mice into highly significant antigen-specific protection. These studies demonstrate that some highly immunogenic antigens of L. major, while having an intrinsic capacity to exacerbate disease in the context of otherwise T_h1-promoting DNA vaccine delivery, can be rendered protective by the removal of functional IL-10^ref. Heat shock proteins (HSPs) are highly conserved among different organisms. A mycobacterial HSP65 DNA vaccine was previously shown to have prophylactic and immunotherapeutic effects against Mycobacterium tuberculosis infection in mice. BALB/c mice were immunized with mycobacterial DNA-hsp65 or with DNA-hsp65 and trehalose dymicolate (TDM), both carried by biodegradable microspheres (MHSP/TDM), and challenged with Leishmania major. MHSP/TDM conferred protection against L. major infection, as indicated by a significant reduction of edema and parasite loads in infected tissues. Although high levels of interferon-gamma and low levels of IL-4 and IL-10 were detected in mice immunized with DNA-hsp65 or MHSP/TDM, only animals immunized with MHSP/TDM displayed a consistent T_h1 immune response, i.e., significantly higher levels of anti-soluble Leishmania antigen (SLA) IgG_2a and low anti-SLA IgG₁ antibodies. These findings indicate that encapsulated MHSP/TDM is more immunogenic than naked hsp65 DNA, and has great potential to improve vaccine effectiveness against leishmaniasis and tuberculosis^ref
anti-Leishmania donovani chagasi DNA vaccine : acute visceral leishmaniasis is a progressive disease caused by Leishmania chagasi in South America. The acquisition of immunity following infection suggests that vaccination is a feasible approach to protect against this disease. Since Leishmania homologue of receptors for activated C kinase (LACK) antigen is of particular interest as a vaccine candidate because of the prominent role it plays in the pathogenesis of experimental Leishmania major infection, we evaluated the potential of a p36^LACK DNA vaccine in protecting BALB/c mice challenged with L. chagasi. In this study, mice received intramuscular (i.m.) or subcutaneous (s.c.) doses of LACK DNA vaccine. A significant production of IFN-g was detected by splenocytes from i.m. vaccinated mice in response to L. chagasi antigen and to rLACK protein. However, no reduction in parasite load neither in liver nor in the spleen of vaccinated animals was observed. The lack of protection observed may be explained by a significant production of IL-10 induced by the vaccine^ref. Vaccination of dogs, the domestic reservoir of Leishmania chagasi, could not only decrease the burden of canine visceral leishmaniasis (VL), but could also indirectly reduce the incidence of human VL. Intramuscular vaccination of foxhounds with a Leishmania multicomponent (10 antigen) DNA vaccine resulted in antigen-induced lymphoproliferative and IFN-g (but not IL-4) responses. This response was not augmented by co-administration of canine IL-12 or GM-CSF DNA adjuvants. The multicomponent DNA vaccine also induced a delayed type hypersensitivity (DTH) response to viable L. donovani promastigotes and led to a reduction of parasite burden in an in vitro intracellular infection model, and in the draining lymph node of dogs early after cutaneous challenge. Thus, the multicomponent DNA vaccine was effective in priming dogs for a parasite-specific T_h1 cellular immune response, which was able to restrict parasite growth^ref. The nucleoside hydrolase (NH36) of Leishmania (L.) donovani is a vital enzyme which releases purines or pyrimidines of foreign DNA to be used in the synthesis of parasite DNA. As a bivalent DNA vaccine, the VR1012-NH36 was immunoprotective against visceral and cutaneous murine leishmaniasis. In this work we tested the immunotherapy against Leishmania (L.) chagasi infection, using 2 doses of 100 or 20 mg VR1012-NH36 vaccine (i.m. route), and, as a possible immunomodulator, aqueous garlic extract (8 mg/kg/day by the i.p. route), which was effective in immunotherapy of cutaneous murine leishmaniasis. Liver parasitic load was significantly reduced following treatment with 100 mg (91%) and 20 mg (77%) of the DNA vaccine, and by 20 mg DNA vaccine and garlic extract (76%) (p=0.023). Survival was 33% for saline controls, 100% for the 100 mg vaccine, and 83 and 67% for the 20 mg vaccine with and without garlic extract addition, respectively. Garlic treatment alone did not reduce parasite load (p>0.05), but increased survival (100%). The NH36-DNA vaccine was highly effective as a new tool for the therapy and control of visceral leishmaniasis, while the mild protective effect of garlic might be related to an unspecific enhancement of IFN-g secretion^ref
anti-Toxoplasma gondii DNA vaccine :

GRA1^ref : codon-optimized GRA1 DNA vaccine, wild type GRA1 DNA vaccine and an adjuvanted recombinant GRA1 protein vaccine candidate in BALB/c mice against lethal toxoplasmosis. Of the 3 GRA1 vaccines tested, the recombinant GRA1 protein vaccine results reveal significant increase in immune response and prolonged survival against acute toxoplasmosis compared to DNA vaccinations. Immune response and protection conferred by codon-optimized GRA1 DNA vaccine was slightly better than wild type GRA1 DNA vaccine^ref
GRA1, GRA7, and ROP2^ref
To examine the immune response in BALB/c mice induced by oral mixed Toxoplasma gondii DNA vaccine delivered by live attenuated Salmonella typhimurium, gene fragments SAG1 and SAG2 were amplified from the genomic DNA of T. gondii RH strain by PCR and were subcloned into pcDNA3.1(+/-) eukaryotic expression vector. The positive recombinant plasmid was transformed into aroA- and aroD-attenuated Salmonella typhimurium BRD509 (BRD509/pSAG1/SAG2). After screened by PCR, restrictive enzyme digestion and sequencing, recombinant Salmonellastrain was used to immunize BALB/c mice by i.g. route, 3 times at 2-week interval. Humoral and cellular responses were assayed using ELISA for determining antibody, IFN-g and IL-4. MTT assay was used to detect the proliferation activity of T lymphocytes and the activity of NK killers. FCM was also used to sort the lymphocyte subsets of spleen cells All mice were then infected with highly virulent RH tachyzoites of T. gondii intraperitoneally. Significant increase of specific IgG level was observed in immunized mice with a titer of 1:100. Proliferation activity of specific NK cells and T lymphocytes was highly enhanced in BRD509/ pSAG1/SAG2 vaccinated mice: the killing activity of NK cells was 85% +/- 7%, the proliferation SI of T lymphocytes was 2.83, which resulted in a 5 days longer survival time than mice in control group after challenge infection. The oral mixed DNA vaccine delivered by attenuated Salmonella typhimurium shows an immunoprotection against T. gondii in mice^ref.

⁺

Toxoplasma gondii

⁺

Toxoplasma

^ref

anti-Trypanosoma cruzi DNA vaccine : immunity depends on CTLs^{ref1,
ref2}

amastigote surface proteins^ref :

amastigote surface protein-1 (ASP-1)
ASP-2^ref
ASP-2 and trans-sialidase^ref

trypomastigote genes :

trans-sialidase^{ref1,
ref2} :

in a DNA-priming protein-boosting regimen^ref
killed Propionibacterium acnes or a soluble polysaccharide extracted from bacterium cell wall modulated experimental immunization with plasmidial DNA. As previously described, immunization of BALB/c mice with p154/13 plasmid, containing a gene-encoding catalytic domain of Trypanosoma cruzi trans-sialidase, elicited humoral, cell-mediated and protective immune responses against T. cruzi infection. Both P. acnes and its soluble polysaccharide fraction have the ability to modulate the immune response elicited by p154/13. Treatment with these adjuvants enhanced specific trans-sialidase T_h1 immune response, as revealed by a lower IgG₁/IgG_2a ratio and stronger in vitro IFN-g synthesis by CD4⁺ T cells. The most important fact was that treatment with P. acnes or its soluble polysaccharide fraction in the presence of p154/13 significantly reduced the peak of parasitemia observed 7 to 8 days after T. cruzi challenge. These data suggest that P. acnes or its soluble polysaccharide fraction may improve the protective potential of a DNA vaccine against experimental T. cruzi infection^ref.

trypomastigote surface antigen 1 (TSA-1)^ref : vaccine reduced cardiac tissue damage and improved survival in mice when administered during the acute or chronic phases of T. cruzi infection. ICR mice were infected with 500 T. cruzi blood trypomastigotes and treated during the acute or chronic phases with 2 100 mg doses of DNA vaccine. Analysis of stained splenocytes by flow cytometry indicated that the therapeutic vaccine induced a rapid increase in the number of CD4⁺ and CD8⁺ T cells in both the acute and chronic phases. Also, there was a rapid increase in T. cruzi-specific IFN-g-producing CD8⁺ T cells following treatment during the chronic phase. The effects of these changes on the control of infection required longer time periods to be detectable but resulted in a reduction in myocarditis and T. cruzi parasite burden in both phases of the infection, as assessed by histopathologic analysis and semi-quantitative PCR detection of T. cruzi in cardiac tissue. DNA vaccines that induce CD8⁺ T-cells activity and IFN-g production would be good candidates for effective therapeutic vaccination against T. cruzi infection^ref
complement regulatory protein^ref
paraflagella rod proteins (PFR's)^{ref1,
ref2,
ref3,
ref4,
ref5}

exacerbated inflammation disproportional to parasite load characterizes chronic myocardial lesions in chagasic patients. Chemokines and their receptors are expected to account for the renewed inflammatory processes after the inoculation of the parasite, but their potential unique functions are far from being clear. The effect of a DNA vaccine encoding CCL4/MIP-1b, a CC-chemokine, was evaluated in T. cruzi-elicited myocarditis in rats. Holtzman rats were given intramuscularly cardiotoxin and the CCL4/MIP-1b DNA-containing plasmid (100 mg) was delivered in this muscular site four times. 14 days after last immunization, animals were inoculated with a myotropical CL-Brener T. cruzi clone. Peak of parasitism was observed at day 15 after infection, preceding the peak of myocardial inflammation at day 20. Myocarditis was still intense at day 30, but the inflammatory infiltrates showed a more focal distribution. The expression of CCL2/MCP-1 and CCL4/MIP-1b correlated closely with the kinetics of myocardial inflammation. The CCL4/MIP-1b DNA vaccine induced an increase of the levels of the anti-CCL4/MIP-1b observed in T. cruzi-infected animals. This was associated with an exacerbation of myocardial inflammation and fibrosis, although alterations in parasitemia and myocardial parasitism were not observed. CCL4/MIP-1b plays a role in preventing excessive inflammation and pathology rather than in controlling parasite replication^ref

Web resources

Vaccine possibilities for Chagas' disease

anti-Plasmodium falciparum DNA vaccine^ref: the ability to generate potent antigen-specific T cell responses by vaccination has been a major hurdle in vaccinology. Vaccinia virus and avipox viruses have been shown to be capable of expressing antigens in mammalian cells and can induce a protective immune response against several mammalian pathogens. 2 such vaccine constructs, modified vaccinia virus Ankara (MVA) and FP9 (an attenuated fowlpox virus) both expressing the pre-erythrocytic malaria antigen thrombospondin-related adhesion protein and a string of CD8⁺ epitopes (ME-TRAP). In prime-boost combinations in a mouse model MVA and FP9 are highly immunogenic and induce substantial protective efficacy. A series of human clinical trials using the recombinant MVA and FP9 malaria vaccines encoding ME-TRAP, both independently and in prime-boost combinations with or without the DNA vaccine DNA ME-TRAP, has shown them to be both immunogenic for CD8⁺ T cells and capable of inducing protective efficacy. The safety profiles of these viral vectors and show that anti-vector antibody responses induced by the vectors are generally low to moderate : these vectors are safe and show acceptable side effect profiles for prophylactic vaccination^ref. An important consideration in the development of a malaria vaccine for individuals living in areas of endemicity is whether vaccine-elicited immune responses can be boosted by natural infection. To investigate this question, Plasmodium berghei ANKA blood-stage parasites were used for the infection of mice that were previously immunized with a DNA vaccine encoding the P. berghei sexual-stage antigen Pbs48/45. Intramuscular immunization in mice with 1-2 doses of DNA-Pbs48/45 or of empty DNA vaccine as control did not elicit detectable anti-Pbs48/45 antibodies as determined by ELISA. An infection with P. berghei ANKA 6 weeks after DNA vaccination elicited comparable anti-Pbs48/45 antibody levels in mice which had been primed with DNA-Pbs48/45 or with empty DNA vaccine. However, a repeat infection with P. berghei ANKA resulted in significantly higher anti-Pbs48/45 antibody levels in mice which had been primed with the DNA-Pbs48/45 vaccine than the levels in the mock DNA-vaccinated mice. In parallel and as an additional control to distinguish the boosting of Pbs48/45 antibodies exclusively by gametocytes during infection, a separate group of mice primed with DNA-Pbs48/45 received an infection with P. berghei ANKA clone 2.33, which was previously described as a "nongametocyte producer". To our surprise, this parasite clone too elicited antibody levels comparable to those induced by the P. berghei gametocyte producer clone. The nongametocyte producer P. berghei clone is in fact a defective gametocyte producer that expresses Pbs48/45, much like the gametocyte producer clone, and is therefore capable of boosting antibody levels to Pbs48/45. Taken together, these results indicate that vaccine-primed antibodies can be boosted during repeat infections and warrant further investigation with additional malaria antigens^ref. Plasmodium-based DNA vaccine that was targeted to antigen presenting cells by fusion to CTLA4. Fusion proteins comprising the extra-cellular domain of CTLA4, the hinge, C_H2 and C_H3 domains of human IgG₁ and MSP-1 gene fragments were expressed in COS-7 cells. 3 of the secreted proteins containing the mouse homologue of CTLA4 were shown to bind differently to the human B7-1 molecule expressed on THP-1 cells. Competition binding assays for 2 fusion proteins showed that binding was specific. When C57BL/6 mice were immunized with plasmids encoding the fusion proteins, antibodies against two denatured and one non-denatured MSP-1 gene fragments were successfully induced^ref. BALB/c mice first immunized as neonates (7 d) with a Plasmodium yoelii circumsporozoite protein (PyCSP) DNA vaccine mixed with a plasmid expressing murine GM-CSF (DG) and boosted at 28 d with pox virus expressing PyCSP were protected (93%) as well as mice immunized entirely as adults (70%). Like adults, protection was dependent on CD8⁺ T cells and accompanied by excellent anti-PyCSP IFN-g and CTL responses. Mice born of immune mothers (previously exposed to P. yoelii parasites or immunized with the same vaccine given to the neonates) were also protected and had excellent T-cell responses. These data support assessment of this immunization strategy in neonates/young infants in areas where malaria exacts the greatest toll^ref

anti-helminth vaccines :

anti-Clonorchis sinensis DNA vaccine : cysteine proteinases of C. sinensis are important virulence factors that induce pathological changes associated with larval migration and localized biliary epithelial destruction. This study investigated the immunogenicity and protective efficacy of a DNA vaccine encoding Clonorchis sinensis cysteine proteinase (CsCP). The CsCP cDNA sequence displays significant homology to the mammalian or trematode cathepsin L. Plasmid DNA carrying the CsCP gene (pcDNA3.1-CsCP) was injected into Sprague-Dawley (SD) rats intradermally. Animals injected with pcDNA3.1-CsCP developed CsCP-specific antibodies, which exhibited an IgG_2a dominance in sera. In addition, the DNA vaccine elicited the production of IFN-g, but not IL-4 in splenocytes, suggesting the induction of a typical T_h1 dominated immune response in rats. The pcDNA3.1-CsCP induced a significant level of protection (31.5%, p<0.05) in SD rats challenged with C. sinensis metacercariae. These results indicate that pcDNA3.1-CsCP induces both humoral and cellular immune responses. The CsCP gene may be a good candidate for use in future studies of vaccination against clonorchiasis^ref.Clonorchis sinensis, the Chinese liver fluke, resides chronically in the biliary tract, and fatty acid-binding protein (FABP) is known to play an important role in the intracellular transport of long-chain fatty acids obtained from the host. Although FABP has stimulated considerable interest as a vaccine candidate, the nature of C. sinensis FABP (CsFABP) remains unclear. The immunogenicity and protective efficacy of a DNA vaccine encoding CsFABP was investigated. The intradermal injection of plasmid DNA carrying the CsFABP gene (pcDNA3.1-FABP) into Sprague-Dawley (SD) rats induced both humoural and cellular immune responses. Animals injected with pcDNA3.1-FABP developed FABP-specific antibody, which is dominance of IgG_2a in sera. In addition, the DNA vaccine elicited the production of IFN-g, but not the production of IL-4 in spleen cells stimulated with recombinant FABP. Moreover, pcDNA3.1-FABP induced a significant level of protection, decreased worm burden (40.9%, P < 0.05) in SD rats against C. sinensis metacerariae challenge. These results suggest that pcDNA3.1-FABP induces a typical T_h1-dominated immune response and it is a good candidate for use in future clonorchiasis vaccination studies^ref.
anti-Schistosoma japonicum DNA vaccine :

fatty acid binding protein (Sj14FABP) DNA vaccine enhanced by IL-12. he recombinant plasmids pVIVO2-Sj14FABP and pVIVO2-IL12-Sj14FABP were constructed respectively, and were prepared on a large scale after identification. 48 male BALB/c mice were divided into 4 groups randomly. In group A, B, C, and D, each mouse was injected intramuscularly with 100 microl 0.9% NaCl, 100 mg pVIVO2, 100 mg pVIVO2-Sj14FABP and 100 mg pVIVO2-IL12-Sj14FABP respectively. 30 days after immunization each mouse was challenged with 40 +/- 2 cercariae of S. japonicum. On day 45 after challenge, all mice were sacrificed to count the number of recovered adult worms and the hepatic eggs. Sera from mice were used to detect IgG antibody. The production of IL-2, IL-4 and IFN-g in the supernatant of spleen cells was observed by means of sandwich ABC-ELISA. The recombinant plasmids pVIVO2-Sj14FABP and pVIVO2-IL12-Sj14FABP were constructed. The worm reduction rate in group C and D was 24.11% and 39.4%, as well as liver egg reduction rate of 27.2% and 32.8% respectively. The level of IL-2 and IFN-g in group D increased significantly, while IL-4 secretion decreased (P < 0.01). 30 days after immunization, no higher titer of IgG antibody was shown in all groups. Furthermore, no significant difference on the level of IgG was found among the groups (P > 0.05). Sj14FABP DNA vaccine induces partial protective immunity in BALB/c mice. IL-12 drives the immune response toward a T_h1 direction, and enhances the protective immune effect of the vaccine^ref.
triose-phosphate isomerase (TPI) plasmid DNA vaccine : naive pigs were vaccinated with the TPI DNA-plasmid alone, or in conjunction with IL-12 as pcDNA3.1-P35, pcDNA3.1-P40 plasmids via intramuscular injection. Control pigs were immunized with equivalent amounts of pcDNA3.1. Pigs were immunized 3 times at 21-day intervals and challenged 30 days after the final boost. 45 days post-challenge, pigs were sacrificed and perfused to compare adult worm burdens, female worm burdens, liver egg burdens and granuloma size. Pigs vaccinated with SjCTPI DNA alone had adult worm burdens reduced by 48.3% and that a further decrease in adult worm burdens was not seen in the group vaccinated with SjCTPI DNA in conjunction with IL-12 (46.2% reduction). The SjCTPI DNA vaccines had a more pronounced effect on reducing female worm burdens i.e. 53.6% SjCTPI alone and 59.6% for SjCTPI+IL-12. Vaccination with SjCTPI-DNA reduced liver eggs by 49.4% and this response was significantly enhanced by the addition of IL-12 (65.8% reduction in liver eggs). In addition to the dramatic protective effects seen in vaccinated pigs, we also noted that granuloma size was reduced by 42% in both groups. Thus, vaccination of pigs and other large animals in China with SjCTPI DNA vaccine will likely reduce transmission by reducing adult worm burdens and worm egg output and simultaneously reduce hepatic egg-associated pathology^ref
glyceraldehyde-3-phosphate dehydrogenase (GAPDH): to study the immunological characteristics induced in C57BL/6 mice. The gene encoding GAPDH of S. japonicum from screening of cDNA library was amplified using universal primer T3 promoter and T7 promoter, the PCR product was cloned into the T-A vector pT-Adv. GAPDH-pT-Adv and eukaryotic expression vector pcDNA3 were digested by restriction endonucleases HindIII and XhoI, and the nucleic acid vaccine harboring the gene encoding GAPDH was then constructed by ligating the digested products. C57BL/6 mice were immunized using the purified pcDNA3-SjGAPDH. The expression of GAPDH in local muscle in mice was examined by immunofluorescence assay. The specific characteristics induced by pcDNA3-SjGAPDH were analyzed using SDS-PAGE, Western blotting, and ELISA respectively. Light green fluorescence was observed in local muscle cell under the fluoroscope 24 and 48 hours after immunization, which indicated the expression of GAPDH. The recombinant plasmid induced specific anti-GAPDH IgG, predominantly IgG_2a, IgG_2b and IgG₃. Cytokines IFN-g, IL-2 but IL-4 were detected in C57BL/6 mice vaccinated with the vaccine. The DNA vaccine, pcDNA3-SjGAPDH induces T_h1 type immune response in mice^ref.
to enhance the immunogenicity of the recombinant pVIVO2-IL12-Sj23 vaccine of Schistosoma japonicum by using mixed vegetal polysaccharides as adjuvant. The plasmid pVIVO2-IL12-Sj23 was constructed. 3 groups of BALB/C mice were injected intramuscularly with normal saline (Group A), pVIVO2-IL12-Sj23 plasmid DNA (B), and pVIVO2-IL12-Sj23 plus mixed vegetal polysaccharides (C) respectively, and challenged with S. japonicumcercariae on the 4th week after immunization. Mice were killed to calculate the worm reduction rate and egg reduction rate in liver tissue on the 6th week after infection. Before and 4 weeks after immunization blood samples were collected. The worm reduction rate and egg reduction rate were 64.3% and 79.9%, respectively in group C, 45.5% and 58.4%, respectively in group B, showing a remarkable difference hetween them (P < 0.05). ELISA analysis showed a significantly higher level of IgG specific for Sj23 4 weeks after vaccination in groups B and C (P < 0.05). However, there was no significant difference in IgG level between groups C and B (P > 0.05). When mixed vegetal polysaccharides are used as adjuvant, the effect of the vaccine pVIVO2-IL12-Sj23 can he considerably enhanced^ref
plasmids pVIVO2-mcs-SjFABP-Sj23 and pVIVO2-mes-Sj23-SjFABP, co-expressed bivalent DNA vaccines : the presence of bivalent DNA vaccine in the mouse muscle cells was also tested by indirect immunofluorescent antibody tests (IFAT). 70 BALB/c mice were randomly divided into seven groups to be injected with normal saline, pVIVO2-mcs, pVIVO2-mcs-Sj23, pVIVO2-mcs-SjFABP, pVIVO2-mcs-Sj23-SjFABP, pVIVO2-mcs-SjFABP-Sj23 plasmid DNA, and a mixture of pVIVO2-mcs-Sj23-SjFABP plasmid DNA and amylose adjuvant, respectively. At day 45 after challenge the mice were sacrificed. The number of adult worms and hepatic eggs were counted. Successful construction of co-expressed bivalent DNA vaccines were identified by restriction analysis and sequencing. It was confirmed by IFAT that the bivalent DNA vaccine was expressed in the plasma and on the surface of muscle cells from mouse. The worm reduction rate was 41.20-53.85% and the egg reduction rate was 47.02-53.83% bivalent DNA groups. Furthermore, the worm and egg reduction rates in pVIVO2-mcs-Sj23-SjFABP plasmid DNA and amylose adjuvant group were 68.89% and 84.04% respectively, significantly higher than single antigenic DNA vaccine and bivalent DNA vaccine (P < 0.05). The co-expressed bivalent DNA vaccines of Schistosoma japonicum can induce immune protection in mice. The protective immunity of amylose adjuvant group is higher than that of the univalent DNA and bivalent vaccines^ref
C57BU6 mice were vaccinated i.m. with the Sjc97 DNA twice at an interval of 3 weeks, and challenged with 30 +/- 2 cercariae of Schistosoma japonicum 3 weeks after immunization. Mice in blank plasmid vector control and infection control groups were also infected with same number of cercariae. The mice were sacrificed 7 weeks after challenge infection. The size of single egg granulomas in livers was measured with micrometer. The level of hyaluronic acid (HA) and laminin (LN) in sera of the mice was determined by ELISA. PCR-ELISA was used to examine the expression of TGF-b₁ mRNA in liver. The mean hepatic egg granuloma diameters of the three groups, the Sjc97 DNA, blank plasmid vector and infection control, were 183.75 +/- 42.36 mm, 303.12 +/- 37.36 mm and 304.38 +/- 53.23 mm, respectively. The hepatic granuloma was significantly smaller in the Sjc97 DNA group than that in control. The level of HA and LN in sera of Sjc97 DNA vaccinated mice was markedly lower than those in the two control groups (P < 0.01). The amount of TGF-b₁ mRNA isolated from the livers of mice in Sjc97 DNA group decreased significantly. Sjc97 DNA vaccine may act as an effective inhibitor against formation of egg granuloma and reduce immunopathological damage caused by Schistosoma japonicum in the host^ref.
pVIVO(2)SjFABP-23 : 40 BALB/c mice were divided into 4 groups designated pVIVO(2), pVIVO2Sj23, pVIVO(2)SjFABP and pVIVO(2)SjFABP-23. Each mouse was immunized with 100 mg of the corresponding plasmid DNA by i.m. injection. 28 days post-vaccination, the mice were challenged with S. japonicum cercariae, and the worm and egg burdens were determined 42 days post-challenge. Serum samples were collected from all the mice before and after vaccination and at the end of the experiment, and used for antibody detection. The IFN-g and IL-4 levels were quantified in the supernatants of specifically stimulated spleen cells. The number of worms was reduced by 52%, 40% and 42% in mice respectively immunized with pVIVO(2)SjFABP-23, pVIVO(2)Sj23 or pVIVO(2)SjFABP. A respective 61%, 38% and 39% egg reduction was determined relative to those mice that only received the empty pVIVO2 plasmid. pVIVO(2)SjFABP-23 immunization increased IgG levels against SWAP and SEA. Increased IFN-glevels were detected in the supernatant of specific stimulated spleen cells from mice immunized with the 3 different constructs. The multivalent DNA vaccine developed induced higher levels of protection than the 2 monovalent tested vaccines^ref
the fatty-acid-binding protein (Sj14) and the 23kDa transmembrane protein (Sj23) proteins were selected to construct the DNA-based vaccine. It was successful to construct a bivalent DNA vaccine using 3 strategies: the co-expression of 2 genes, a fusion gene expression and 2 kinds of plasmids in combination (cocktail vaccine). The bivalent DNA was proven to express well in vitro and in vivo by indirect immunofluorescence test (IIF) and reverse transcriptase-polymerase chain reaction (RT-PCR). The protective immunity of bivalent DNA vaccine was higher than that of univalent DNA vaccine (p<0.05). There were 4 groups of bivalent vaccine whose protective immunity was > 50%. Granuloma diameter reduction rates were in the range of 18-39%. There was no significant impact on immunity protection exerted by the 4 factors including dosage, inoculated times, inoculated routes and challenge time after the last immunization in 3 levels (p>0.05)^ref
pcDNA3-Sj22.7 and its immunological effect in mice, the recombinant plasmid pcDNA3-Sj22.7 was used to transfect HeLa cells with liposome-mediated method and the expression of Sj22.7 mRNA and protein was examined using RT-PCR, SDS-PAGE and Western blot. Then, the ability of pcDNA3-Sj22.7 to protect against Schistosoma japonicum challenge infections was analyzed according to worm reduction rate and egg reduction rate after vaccination of mice. The serum levels of specific IgG antibody and T lymphocyte proliferation response were also determined. After the challenge infection, Sj22.7-driven IFN-g and IL-4 was also quantified. pcDNA3-Sj22.7 could express Sj22.7 mRNA and protein in vitro. Immunization resulted in a worm reduction rate of 29.70%, egg reduction rate of 47.25% (liver) and 51.73% (intestine), and egg reduction rate of 25.90% (eggs per female), suggesting induction of significant anti-fecundity in the pcDNA3-Sj22.7 group. ELISA and Western blot analysis indicated that immunized mice generated specific IgG against Sj22.7. T lymphocytes from mice immunized with pcDNA3-Sj22.7 showed a significant proliferation response to rSj22.7. The culture of spleen cells showed that secretion of IFN-g increased but IL-4 decreased. The results indicate hat DNA vaccination by pcDNA3-Sj22.7 is sufficient to elicit significant levels of protective immunity against S. japonicum infection. The DNA vaccine could induce significant cellular and humoral immune response, and display predominant T_h1 type immune responses, which contribute to the protective immunity against challenge infection in mice^ref.
eukaryotic co-expression plasmid containing membrane-anchored Sjcl4FABP and Sjc26GST genes and identify their expression in vitro, Sj14 and Sj26 genes were obtained by RT-PCR with total RNA of Schistosoma japonicum adult worms as the template and cloned into eukaryotic expression plasmid pVAC to construct recombinant plasmids pVAC-Sj14 and pVAC-Sj26. Then a 23 amino-acid signal peptide of human IL-2 upstream Sj14 or Sj26 gene and a membrane-anchored sequence containing 32 amino-acids of carboxyl-terminal of human placental alkaline phosphatase (PLAP) downstream were amplified by PCR as the template of plasmid pVAC-Sj14 or pVAC-Sj26 only to get two gene fragments including Sj14 gene and Sj26 gene. The 2 modified genes were altogether cloned into a eukaryotic co-expression plasmid pIRES, resulting in another new recombinant plasmid pIRES-Sj26-Sj14. The expression of Sj14 and Sj26 genes was detected by RT-PCR and indirect immunofluorescent assays (IFA) when the plasmid pIRES-Sj26-Sj14 was transfected into eukaryotic Hela cells. Restriction enzyme analysis, PCR and sequencing results revealed that the recombinant plasmids pVAC-Sj14, pVAC-Sj26 and plRES-Sj26-Sj14 were successfully constructed and the expression of modified Sj14 and Sj26 genes could be detected by RT-PCR and IFA. A bivalent membrane-anchored DNA vaccine encoding Sj14 and Sj26 genes was acquired and expressed proteins were proved to be mostly anchored in cellular membranes^ref

anti-Schistosoma mansoni DNA vaccine :

cDNA clone encoding the Schistosoma mansoni lung-stage Sm22.6 protein, which is 100% and 79% identical with the 22.6kDa adult worm tegument antigen of S. mansoni and S. japonicum, respectively, was isolated. Further, a recombinant (r) Sm22.6 was produced and an Sm22.6 DNA vaccine was constructed. Western blot analysis confirmed the identity of purified MBP-Sm22.6 fusion protein using anti-MBP (maltose binding protein) and anti-rSm22.6 antibodies. Additionally, C57BL/6 mice were immunized and specific anti-Sm22.6 IgG responses were produced when both vaccination strategies were used. Importantly, only rSm22.6 vaccine provided levels of protection against challenge infection (34.5%). Mice immunized with rSm22.6 induced production of IgG₁ and IgG_2a and synthesis of IFN-g and IL-4 in cultured mouse splenocytes. Finally, rSm22.6 vaccination induced a T_h0 type of immune response and protective immunity that suggests Sm22.6 as a potential candidate to compose an anti-schistosome vaccine^ref
Sm14 cDNA alone or in association with a plasmid expressing murine IL-12 to induce protection against challenge infection. Mice were immunized with four doses of the DNA vaccine and the levels of protection were determined by worm burden recovery after challenge infection. Specific antibody production to rSm14 was determined by ELISA, and cytokine production was measured in splenocyte culture supernatants stimulated with rSm14 and in bronchoalveolar lavage of vaccinated mice after challenge infection. DNA immunization with pCI/Sm14 alone induced 40.5% of worm reduction. However, the use of pCI/IL-12 as adjuvant to pCI/Sm14 immunization failed to enhance protection against challenge infection. Protection induced by pCI/Sm14 immunization correlates with specific IgG antibody production against Sm14, T_h1 type of immune response with high levels of IFN-gand low levels of IL-4 in splenocyte culture supernatants and in brochoalveolar lavage after challenge infection. IL-12 co-administration with pCI/Sm14 induced a significant production of nitric oxide in splenocyte culture supernatants and also lymphocyte suppression, with reduced percentage of T cells producing IFN-g and TNF-a^ref

anti-cancer vaccines : see therapeutic subunit cancer vaccines
anti-chronic degenerative disease vaccine :

Alzheimer disease (AD) is a neurodegenerative disorder characterized by neuropathological hallmarks including deposits of the beta-amyloid peptide (AssP). Studies have shown that immunization with Ab₄₂ peptide reduces both the spatial memory impairments and AD-like neuropathologic changes in Alzheimer disease transgenic mice, but can cause side effect of a cell-mediated autoimmune meningoencephalitis. Recently, some studies showed that DNA vaccination could be used to generate an antibody response to Abeta without the adverse cell-mediated immune effect. 4 DNA vaccine plasmids (pV-GE1, pV-GE2, pV-GE3, and pV-GE4) have been generated against AD by separately fusing Ab epitope sequences (coding for EFGH, DAEFGH, EFGH+EFGH, and EFGH+DAEFGH) with IgG heavy chain coding region of mouse. Meanwhile, the full-length gene Abeta encoding plasmid (pV-Ab), empty vector (pVAX) and synthetic AssP were also included as control. The sera of BALB/c mice immunized via intramuscular with plasmids and peptide were tested by indirect ELISA for auto-AssP immunoreactivity. The results showed that all the DNA vaccine plasmids induced AssP-specific antibodies; moreover pV-GE2 and pV-Ab constructs elicited higher antibody titers than other constructs (P < 0.05). To further enhance the immune response, GM-CSF encoding plasmid (pGM-CSF) and purified BCG-DNA were used as molecular adjuvants. BCG-DNA could enhance humoral and cellular immune responses simultaneously and did not alter the phenotype of the immune responses, whereas pGM-CSF showed no obvious effect on immune response. These results suggest that this immunization strategy of using Ab epitope encoding plasmid + BCG-DNA adjuvant may serve as the basis for developing anti-AD vaccines^ref. Safe and effective nonviral Ab DNA vaccines against AD were developed and administered to model (APP23) mice and evaluated Ab burden reduction. Prophylactic treatments started before Ab deposition reduced Ab burden to 15.5% and 38.5% of that found in untreated mice at 7 and 18 months of age, respectively. Therapeutic treatment started after Ab deposition reduced Ab burden to approximately 50% at the age of 18 months. Importantly, this therapy induced neither neuroinflammation nor T cell responses to Ab peptide in both APP23 and wild-type B6 mice, even after long-term vaccination. Although it is reported that other anti-Ab therapies have pharmacological and/or technical difficulties, nonviral DNA vaccines are highly secure and easily controllable and are promising for the treatment of AD^ref.
atherosclerosis : rabbits were intramuscularly immunized with the plasmid pCR-X8-HBc-CETP encoding a B-cell epitope of cholesteryl ester transfer protein (CETP) C-terminal fragment (CETPC) displayed by Hepatitis B virus core (HBc) particle. This plasmid also contained immunostimulatory sequences (ISS) which included eight CpG motifs 5'-GACGTT-3', functioning as immunomodulators. After anti-CETP antibodies were successfully produced, rabbits were fed with a high-cholesterol diet for 15 weeks, and then the antiatherogenic effects of this DNA immunization were evaluated. The fraction of plasma cholesterol in HDL significantly increased and the fraction of plasma cholesterol in LDL decreased in the pCR-X8-HBc-CETP immunized rabbits compared with those in the saline control group and one group treated with the plasmid pCR-X8-HBc containing ISS but lacking CETP epitope. More importantly, DNA immunization with pCR-X8-HBc-CETP markedly reduced the average percentage of aortic lesions in the entire aorta area by 80.60% compared with the saline control (3.78% versus 19.48%) and the average thickness of hyperplastic coronary artery in this group was also significantly less than in the saline control group (146+/-11mum versus 248+/-18mum). CpG DNA alone could be antiatherogenic in this model because the average % of aortic lesions in pCR-X8-HBc immunized rabbits was 16.53% lower than that of the saline control group and the average thickness of hyperplastic coronary artery was also substantially lower than saline control group (155+/-13 mm versus 248+/-18 mm). Thus, plasmid pCR-X8-HBc-CETP could significantly inhibit the progression of atherosclerosis and be potentially developed as a suitable DNA vaccine against atherosclerosis^ref

anti-toxic vaccines : DNA immunisation gives a useful T_h2-biased hmoral response achievable by epidermal delivery of toxin-encoding genes. Since snake venoms typically consist of numerous molecules, only some of which are toxic, antivenoms are antigenically crude preparations whose therapeutic value would theoretically be enhanced by restricting antibody specificity to toxic venom molecules.

scorpion toxins :

Centruroides noxius : the use of synthetic Cn2 fragments is not the best strategy to rely on, since these peptides instead of eliciting a protective response, reveled an unexpected sensitization phenomena of the immunized animals towards Cn2^ref. A chimera consistng on the Cn2 gene fused in frame to the DC-SIGN gene can elicit specific antibody response against Cn2.
Tityus serrulatus : TsNTxP gene expressed as a fusion protein with maltose-binding protein induced neutralizing antibodies against Ts toxins
Androctonus australis : a synthetic nontoxic analog of toxin II

snake toxins : DNA immunisation protocols can be adapted to induce high titre antibody responses with potential to improve the treatment of systemic snake envenoming that kills 20000 people annually in Africa. Envenoming by the saw-scaled vipers and puff adders are responsible for the majority of these deaths. DNA sequences encoding haemorrhagic, pro- and anti-coagulant and other haemostasis-disruptive venom toxins from these vipers showed extensive cross-specific and cross-generic sequence and structural similarities. The predicted antigenic profiles of these toxin sequences are utilised to design DNA immunisation constructs to generate toxin-specific antibodies with potential to polyspecifically neutralise venoms from the most medically-important African vipers^ref

Bothrops jararaca : gene gun immunization with DNA encoding the carboxy-terminal JD9 domain of jararhagin, a haemorrhage-inducing metalloprotease in the venom of South America pit viper stimulated high titre IgG₁ antibodies that substantially reduced (-70%) the hemorrhage induced by whole venom^ref. Simultaneous delivery with a plasmid encoding murine GM-CSF resulted in a log fold increase in titre, without immunoglobulin class switching. Increasing the number of immunisations resulted in a further log fold augmentation of antibody titre^ref.
Echis ocellatus
Bitis arietans
Naja kaouthia : substitutions in the a-cobratoxin (CTX) cDNA were introduced at two residues critical for binding to the nicotinic acetylcholine receptor (Asp27 to Arg, Arg33 to Gly). The mutated CTX expression cassette was delivered in the context of a replication deficient adenovirus vector (AdmCTX). To assess whether expression of the mutated CTX in vivo leads to the development of protective immunity, BALB/c mice were challenged by IV administration of 2 microg of alpha-cobratoxin protein 21 or 63 days after administration of AdmCTX or Ad- Null (as a control; both, 10(9) particle units). Animals receiving AdmCTX but no a-cobratoxin challenge suffered no ill effects, but > or =80% of naive animals or those receiving the AdNull control vector died within 10 min from the a-cobratoxin challenge. In contrast, 100% of animals receiving a single dose of AdmCTX 21 or 63 days prior to a-cobratoxin challenge survived. The data demonstrates that an adenovirus-based vaccine can be developed to protect against lethal challenge with a potent snake venom. The effectiveness of this approach might serve as a basis to consider the development of a global public health program to protect those at risk for death by snakebite^ref

immunocontraceptive DNA vaccines :

partial cDNA sequence coding for Microtus brandti radde (Brandt's vole) testes-specific lactate dehydrogenase (brLDH-C4) was amplified by reverse transcription-polymerase chain reaction (RT-PCR). By inserting the product into the eukaryotic expression vector pCR3.1, pCR3.1-brLDH-C4' was obtained as the prototype of contraceptive DNA vaccine. Immunization with pCR3.1-brLDH-C4' in BALB/c mice generated antibodies specific to purified brLDH-C4' and native mouse LDH-C4 protein. The birth rate of the pCR3.1-brLDH-C4' immunized mice was found to be decreased significantly (80% lower than that of those immunized with pCR3.1). Functions of the elicited antibodies in sera from pCR3.1-brLDH-C4' inoculated mice were further explored. The results indicated that the antibodies from the mice injected with pCR3.1-brLDH-C4' could cause the agglutination of normal sperm suspension, while the ovarian structure and the development of ovarian follicles of these mice were not impaired, which gives a possible explanation for the immunocontraceptive effects of the pCR3.1-brLDH-C4' DNA vaccine^ref
vaccination of female mice with recombinant fertilization antigen (FA-1)causes a long-term reversible contraceptive effect. Also, a DNA vaccine based upon a dodecamer sequence YLP₁₂ present in sperm causes a reduction in fertility. In the present study, the effects of FA-1 DNA vaccine alone, and FA-1 and YLP₁₂ DNA vaccines together were examined. FA-1 495-bp DNA was cloned into pVAX1 vector to prepare the DNA vaccine. 4 groups of female mice were immunized intradermally by using a gene gun with FA-1 DNA, FA-1 DNA + YLP₁₂ DNA, FA-1 DNA + YLP₁₂ DNA mixed with exogenous synthetic CpG oliogodeoxynucleotide (ODN), or vector DNA alone, respectively. Vaccination with all three formulations caused a significant reduction in fertility, with FA-1 DNA + YLP₁₂ DNA mixed with exogenous synthetic CpG ODN showing the highest reduction. Vaccination with all three formulations raised antibody response in both the sera as well as locally in the vaginal tract, with ODN mixed group demonstrating the highest titers. There was no antibody response in the mice injected with the vector alone. In sera, the highest titers were obtained for the IgG class for all vaccine formulations followed by the IgA class. In vaginal washing, the highest titers were obtained by the IgA class followed by the IgG class. Within the IgG class, the titers for the IgG2a subclass were significantly greater than the IgG1 subclass. The immunocontraceptive effects were long-lasting over 1 year of the observation period and increased with time. These novel findings indicate that the intradermal immunization with a sperm-specific FA-1 DNA vaccine causes a long-term circulating and local immune response resulting in immunocontraceptive effects in female mice. The anti-fertility effects were enhanced when FA-1 DNA vaccine was combined with YLP(12) DNA vaccine and injected with ODN^ref
vaccination with a sperm-specific dodecamer peptide sequence, designated as YLP₁₂, causes a long-term reversible immunocontraceptive effect in female mice. In the present study, the effects of YLP₁₂ DNA vaccine were examined. YLP₁₂ 36 bp cDNA was cloned into pVAX1 vector to prepare the DNA vaccine. 2 additional vaccine constructs were made by in frame cloning of one and two CpG repeats in the YLP(12)-cDNA vaccine. Five groups of female mice were immunized intradermally by using gene gun with YLP₁₂-cDNA, YLP₁₂-cDNA-CpG, YLP₁₂-cDNA-CpG-CpG, YLP(12)-cDNA mixed with exogenous synthetic CpG oligodeoxynucleotide (ODN), or vector DNA alone, respectively. Vaccination with all three constructs and the YLP₁₂ vaccine mixed with exogenous ODN raised antibody response both in the sera as well as locally in the vaginal tract. There was no antibody response in the mice injected with the vector alone. In sera, the highest titers were obtained for the IgG class for all constructs and formulation followed by IgA class. In vaginal washings the highest titers were obtained for the IgA class followed by IgG class. Within the IgG class, the titers for the IgG_2a subclass were significantly greater than the IgG₁ subclass. Immunization with all constructs and formulation caused a significant (P < 0.05 to <0.001) reduction (20-43%) in fertility of female mice. The highest reductions were seen in mice immunized with YLP₁₂-cDNA-CpG-CpG (2 repeats) (43% reduction) and with the YLP₁₂ vaccine administered with exogenous CpG ODN (42% reduction). T lymphocytes obtained from DNA-vaccinated mice showed clearly distinguished comparative RT-PCR analysis of cytokine mRNA expression for T_h1 and T_h2 immune responses compared to T lymphocytes obtained from control animals injected with vector DNA. Expression of both T_h1 cytokines (IL-2 and IFN-g) and T_h2 cytokines (IL-4 and IL-10) was enhanced after DNA vaccination as compared to controls, with a bias towards T_h1 response. The immunocontraceptive effects were long-lasting observed up to 1.3 years of the observation period and increased with time. These novel findings indicate that the intradermal immunization with a sperm-specific DNA vaccine causes a long-term circulating and local immune response resulting in immunocontraceptive effects in female mice^ref
to investigate the enhancement of Lagurus lagurus zona pellucida 3 gene (lzp3) immunization induced by molecular adjuvant C3d in comparison with plasmid pcDNA3-LZP3 (pcD-L) in non-C3d fused form, the eukaryotic expression vector pcDNA3-LZP3-C3d3 (pcD-LC) including lzp3 and 3 copies of C3d in fusion form was constructed. In vitro, RT-PCR and Western blot showed that lzp3 mRNA and protein were expressed in HeLa cells transfected with pcD-L and pcD-LC, respectively. In vivo,the result of ELISA showed that NIH female mice vaccined with pcD-LC elicited higher titer, longer lasting anti-LZP3 antibody than non-C3d fused form pcD-L (P<0.05) by intramuscular gene immunization. MTT analysis indicated that the lymphocytes proliferation response of the pcD-LC primed mice was significantly stronger than that of the blank plasmid injected mice. Antifertility experiment showed that pcD-LC had enhanced effect on blocking fertility of mice (P<0.05) with no disruption of follicular development. These results proved that C3d enhanced immune responses induced by lzp3 vaccine and antifertility effect,and provided the basis for the future study of the Lagurus lagurus reproductive controlling^ref
Several examples have shown that C3d3, when fused to a corresponding antigen, had a strong adjuvant effect on certain specific antibody production. However, C3d3 when fused to hCGb inhibited rather than enhanced the antigen-specific immune response. Using hCGb DNA vaccine preparations, C3d3 inhibited the antigen-specific humoral antibody response and several other immune responses, such as the hCGb specific lymphoproliferation. Such inhibitory effects of C3d3 were not related to the expression level of the target protein, the gender of the test mice, or the vector used. Contrastingly, C3d3 fused with the envelope protein of hepatitis B virus (PreS2/S) used as a control system resulted in the enhancement of both humoral and cell-mediated antigen-specific immune responses against HBV-preS2/S, which was consistent with other groups' adjuvant-effect findings. The mechanisms involved in the inhibitory effect of C3d3 might be possible due to impairing the function of antigen presenting B lymphocytes and reducing the expression of transcription factors (T-bet and GATA-3) and cytokine IL-4. Collectively, unlike its usual expected adjuvant function, the fusion of C3d3 with the tumor-associated antigen hCGbeta was found to inhibit both humoral and cell-mediated antigen-specific immune responses. These findings indicate that research concerning tumor immune escapes and vaccine designs require further extensive attention^ref

immunotolerant DNA vaccines :

to re-establish tolerance to autoantigens by modulating costimulation, a new type of DNA vaccine encoding a membrane-bound preproinsulin (mbPPI) and a chimeric gene vector encoding mutant B7.1/CD40L (mB7.1/CD40L) fusion protein was constructed and tested. This mutant B7.1 binds CTLA4 but not CD28. Young NOD mice immunized with mbPPI along with mB7.1/CD40L DNA vectors significantly reduced diabetes incidence while treatment with CTLA4/IgG₁ exacerbated diabetes. In conclusion, the combination of mbPPI and mB7.1/CD40L was able to protect against autoimmunity and diabetes in NOD mice possibly by promoting a more efficient presentation of autoantigen PPI and inducing specific tolerance to PPI by negatively regulating autoreactive T cells^ref. Incorporation of a cDNA coding for the pro-apoptotic protein BAX into plasmid DNA coding for a secreted form of the pancreatic b-cell antigen glutamic acid decarboxylase (GAD) promotes prevention of type 1 diabetes in NOD mice. Injection of the same vaccine at time of early diabetes onset could ameliorate the disease with efficacy, with 42% of mice overtly diabetic by 40 weeks of age compared to 92% in control groups. In addition, immunological analysis revealed that the DNA vaccine induced CD4⁺CD25⁺ T cells cultured from draining lymph nodes that had immunosuppressive function in vitro. The induced T_regs expressed the foxp3 gene and showed cell-contact-dependent as well as TGF-b- and IL-10-independent immunosuppressive activity. Data also revealed that CD4⁺CD25^- T cells from mice immunized with the DNA vaccine yielded a cell population that was foxp3⁺, showed increased expression of CD25 compared to control, and had immunosuppressive function in vitro, indicating that T_regs could have developed from antigen-induced, peripheral T lymphocytes. In contrast, injection of DNA coding for SGAD55 or BAX alone did not induce T_regs. Altogether, our data confirm that pro-apoptotic DNA vaccination can be used as an immunosuppressive strategy and demonstrate its potential for therapy of pathological autoimmunity^ref.
targeting pathogenic T cells with Ag-specific tolerizing DNA vaccines encoding autoantigens is a powerful and feasible therapeutic strategy for T_h1-mediated autoimmune diseases. However, plasmid DNA contains abundant unmethylated CpG motifs, which induce a strong T_h1 immune response. A novel approach to counteract this undesired side effect of plasmid DNA used for vaccination in T_h1-mediated autoimmune diseases has been reported. In chronic relapsing experimental autoimmune encephalomyelitis (EAE), combining a myelin cocktail + IL-4-tolerizing DNA vaccine with a suppressive GpG oligodeoxynucleotide (GpG-ODN) induced a shift of the autoreactive T cell response toward a protective T_h2 cytokine pattern. Myelin microarrays demonstrate that tolerizing DNA vaccination plus GpG-ODN further decreased anti-myelin autoantibody epitope spreading and shifted the autoreactive B cell response to a protective IgG₁ isotype. Moreover, the addition of GpG-ODN to tolerizing DNA vaccination therapy effectively reduced overall mean disease severity in both the chronic relapsing EAE and chronic progressive EAE mouse models. In conclusion, suppressive GpG-ODN effectively counteracted the undesired CpG-induced inflammatory effect of a tolerizing DNA vaccine in a T_h1-mediated autoimmune disease by skewing both the autoaggressive T cell and B cell responses toward a protective T_h2 phenotype. These results demonstrate that suppressive GpG-ODN is a simple and highly effective novel therapeutic adjuvant that will boost the efficacy of Ag-specific tolerizing DNA vaccines used for treating T_h1-mediated autoimmune diseases^ref
DNA vaccine encoding the Mycobacterium leprae 65-kDa heat shock protein (DNA-hsp65) has a prophylactic and therapeutic effect in experimental murine tuberculosis. However, high homology of the vaccine to the corresponding mammalian hsp60, together with the CpG motifs in the plasmidial vector, could trigger or exacerbate an autoimmune disease. DNA-hsp65 vaccination can induce or modulate arthritis in mice genetically selected for acute inflammatory reaction (AIR), either maximal (AIRmax) or minimal (AIRmin). Mice immunized with DNA-hsp65 or injected with the corresponding DNA vector (DNAv) developed no arthritis, whereas pristane injection resulted in arthritis in 62% of AIRmax mice and 7.3% of AIRmin mice. Administered after pristane, DNA-hsp65 downregulated arthritis induction in AIRmax animals. Levels of interleukin (IL)-12 were significantly lower in mice receiving pristane plus DNA-hsp65 or DNAv than in mice receiving pristane alone. However, when mice previously injected with pristane were inoculated with DNA-hsp65 or DNAv, the protective effect was significantly correlated with lower IL-6 and IL-12 levels and higher IL-10 levels. DNA-hsp65 has no arthritogenic potential and is actually protective against experimentally induced arthritis in mice^ref.

immunosuppressive anti-cytokine therapeutic vaccines for autoimmune diseases : introduction of a foreign T_h epitope into CCL2 DNA vaccine could boost its immunogenicity by inducing strong neutralizing autoantibody against the pathogenic chemokine CCL2 sufficiently to be protective in a classically nonimmune model of disease, Adriamycin nephropathy (AN). Modification of the CCL2 DNA vaccine by replacing a surface loop region of CCL2 sequence with tetanus toxoid T_h epitope P30 elicited a strong self-specific CCL2 autoantibody production, as well as an IFN-g-producing T cell cellular response. The increased immunogenicity of modified CCL2 DNA vaccination but not unmodified CCL2 DNA vaccination was protective against functional and structural renal injury in rat AN. The protective effect of the modified CCL2 DNA vaccine was associated with blockade of glomerular and interstitial macrophage recruitment by neutralizing autoantibody against CCL2, which plays a critical role in eliciting renal injury in AN. Therefore, modification with a foreign T_h epitope breaks self-tolerance by inducing a cellular and humoral response against self-protein and provides a strategy to increase the potency of DNA vaccination sufficiently to afford protection in toxin-induced chronic renal disease^ref
anti-type I hypersensitivity DNA vaccines : creation of hypoallergenic DNA vaccines based on deliberate allergen gene fragmentation, the use of mutations and the routine production of hypoallergenic DNA vaccines by forced ubiquitination. Allergen-expressing DNA replicon vaccines enable a drastic reduction of the vaccine dose without loss of anti-allergic efficacy. DNA multi vaccines and fusion vaccines are being developed for protective and therapeutic applications against certain groups of allergens^{ref1,
ref2}. Ubiquitination offers a general approach for the production of hypoallergenic DNA vaccines^ref.

Bet v 1a : immune responses induced by recombinant and plasmid DNA encoding for Bet v 1a, the major allergen of birch pollen in a mouse system. Balb/c mice were injected intraperitoneally with recombinant Bet v 1a and intradermally with plasmid DNA encoding for the gene of Bet v 1a (pCMV-Bet). In addition, the effect of immunostimulatory DNA sequences was investigated by appending CpG motifs to the gene of Bet v 1a, coinjecting CpG-oligodeoxynucleotides together with the pCMV-Bet construct, or both. IgE and IgG antibody responses, as well as IgG subclasses, were measured by ELISA in sera after each immunization. IFN-g and IL-4 levels were also measured by ELISA in sera and supernatants of allergen-stimulated spleen cells. The primary humoral response to a single treatment with pCMV-Bet was very weak, but the reaction could be boosted to higher levels by 2 additional injections. On the other hand, proliferation assays of spleen cells and measurements of cytokine levels already indicated a cellular response after the first injection of plasmid DNA. After 2 immunizations with pCMV-Bet, the ratio of IgG₁ to IgG_2a pointed to a T_h1 subclass profile. IgE was not detectable in any group at any time during the immune reaction. Accordingly, IL-4 levels were markedly reduced in the serum, as well as in the supernatants, of stimulated spleen cells. Animals immunized with pCMV-Bet containing appended CpG motifs at the 3' end of the Bet v 1a gene and/or with the CpG-ODN GCTAGACGTTAGCGT + pCMV-Bet displayed reduced humoral responses against Bet v 1a when compared with animals injected with pCMV-Bet alone. The levels of IFN-g measured after allergen stimulation of isolated spleen cells were significantly higher in animals immunized with pCMV-Bet plus CpG motifs than with pCMV-Bet alone. Immunization with recombinant Bet v 1a protein elicited a strong T_h2 -type response, including IgE production, a high titer of IgG₁, and IL-4 production in both serum and supernatants of proliferation cultures. In contrast to immunization with protein, DNA immunization induces a strong T_h1 -type response against a relevant inhalant allergen^ref. A DNA-based vaccine encoding the major birch pollen allergen Bet v 1 stably linked to ubiquitin was evaluated for its antiallergic potential in a BALB/c mouse model of allergy. Plasmid DNA was applied to mice before (preventive) or after (therapeutic) sensitization with recombinant Bet v 1. In the preventive setting, mice were exposed to aerosolized allergen in addition. Cytokine production was monitored via ELISPOT and Luminex. IgG₁, IgG_2a, and IgE subclass antibody titers were determined by ELISA. In vitro antigen-specific cross-linking of IgE was measured in a degranulation assay. Bronchoalveolar lavages were analyzed for leukocyte subsets as well as for IFN-g and IL-5, and paraffin sections of lungs were examined for mucus production and endothelial damage. Prevaccination with ubiquitinated Bet v 1-stimulated T_h1-biased immune responses with concomitant suppression of functional IgE, reduction of eosinophil counts in bronchoalveolar lavages, and alleviation of lung pathology, and could also suppress an ongoing IgE response in a therapeutic setting. The data clearly demonstrate that hypoallergenic DNA vaccines encoding ubiquitin fusion constructs induce effective antiallergic immune responses. Ubiquitination of allergen gene vaccines eliminates the risk of IgE cross-linking, thereby meeting the safety requirements for clinical applications^ref. Footpad DNA immunization in Balb/c mice was done 3 times using the Bet v 1a gene with or without Den123 (a nontoxic self-assembled dendritic spheroidal nanoparticle made of biodegradable monomers) with 2-week intervals followed by sensitization with rBetv1 (5 microg) in alum twice in a weekly interval. Different doses of pCMV-Betv1 were used (10 mg and 100 mg). The protective role of different formulations was evaluated by measuring the IgG₁, IgG_2a and IgE antibody production, cytokine release of isolated splenocytes and b-hexosaminidase release from the RBL cells. Higher and increasing ratios of IgG_2a/IgG₁ were seen in mice which received plasmids in combination with Den123. Den123 and DNA vaccine synergistically enhanced the IFN-g released from splenocytes. In the presence of Den123, IgE inhibition was independent of the dose and type of the injected DNA. All DNA-pre-immunized mice demonstrated low basophil degranulation. It is therefore concluded that administration of the DNA entrapped in Den123 nanoparticles results in sustained release of plasmids, T_h1/T_h2 balanced immune response with promising IgE inhibition. Also higher amounts of DNA contributed to stronger T_h1 response^ref.
Der p 1 is a major allergen that reacts with IgE in > 70% allergic sera, hence a good candidate for DNA vaccine development. Previous study demonstrated that intramuscular injection of plasmid DNA containing wild type Der p 1 gene induced specific T_h1 skewed immune response. An optimized construct, containing mouse Igk light chain leader sequence followed by codon optimized mature Der p 1 gene, was generated and used to prevent or treat Der p1 induced allergic asthma in an experimental model. This optimized construct was effective in the inhibition of Der p 1 specific IgE levels, attenuation of T_h2 response and reduction of airway hyperresponsiveness in terms of prophylactic efficacy. It also showed therapeutic efficacy in IgE inhibition and up-regulation of IgG_2a and IFN-g^ref
Der p2 : effectively decreased the levels of IL-4 and IL-13 in the asthmatic mice. Histological evidence and Western blot showed that the expression of STAT6 in the DNA treated mice was markedly attenuated. STAT6 binding to specific DNA motif in lung tissue from the gene vaccinated mice was inhibited^ref. BCG immunization also induced immunomodulation, which appeared to be reliant on the properties of the IFN-g that was produced. Mice were immunized with house dust mite extract (HDM). At the 3 week point, we injected BCG subcutaneously into mice on 3 successive weeks. One week after the BCG injection, we immunized mice with the DNA plasmid encoding for murine T-cell epitope on Der p2 thrice weekly. At 9 weeks after immunization, we measured airway responsiveness. 24hours later, we performed BAL and histological examinations. Co-administration of DNA vaccination and BCG resulted in a partial suppression of the overproduction of goblet cells and the thickness of the peribronchial smooth muscle in ongoing allergic responses. In the BALF, the number of total cells and eosinophils was reduced, and regarding the change of cytokines, the concentration of IL-4 was also decreased, but IFN-g was increased in the co-administration group, opposed to the asthma group. These results suggest that co-administration of vaccination with the DNA encoding T-cell epitope and BCG are effective regarding ongoing allergic response and might constitute an ideal method for combating allergic disease in the future^ref. Immune responses induced by 3 DNA constructs encoding different forms of Der p 2 were analyzed for safe and efficacious vaccination against mite allergy. Mice were immunized intramuscularly with DNA constructs encoding Der p 2 without a signal peptide (p2), with a signal peptide (p52), and with a signal peptide + lysosomal-targeting sequence (p52-LA), respectively, followed by T_h2-skewed protein challenge. Antibody and T-cell cytokine responses were assessed by ELISA. Primed dendritic cells (DCs) were adoptively transferred to naive mice and humoral responses were examined after protein challenge. The circulating Der p 2 protein was detected by sandwich ELISA. Mice immunized with p52-LA showed strong and clear-cut T_h1-type response, as evident by high IFN-g production and elevated levels of Der p 2-specific IgG_2a production whereas construct p2 induced only moderate levels of T_h1 response. In contrast, mice immunized with construct p52 showed a mixed T_h1/T_h2 phenotype and produced substantial circulating Der p 2 protein. Mice adoptively transferred with DCs primed by p52 construct, but not by the p2 or p52-LA constructs, were sensitized to produce high levels of Der p 2-specific IgE. Immunization with DNA construct encoding a signal peptide could potentially prime T_h2-skewed responses and IgE production. The additional inclusion of lysosomal-targeting sequences to such construct could improve the safety and efficacy of DNA vaccination against allergy^ref. After DNA vaccination, BALB/c mice were sensitized by intraperitoneal injection (i.p) and challenged by intranasal instillation of rDer p 2. The lung tissues were assessed using hematoxylin and eosin. Mucus-producing goblet cells were identifed using PAS/alcian blue. The total cell number and composition of bronchoalveolar lavage samples were determined. The levels of the cytokines IL-4 and IFN-g, as well as IgE and IgG_2a in the serum were determined by ELISA. Allergen-specific IL-4 and IFN-g production by spleen cells were also measured by ELISA. Expression of STAT6 in splenocytes were determined by Western blot. DNA vaccine encoding Der p 2 allergen inhibited extensive infiltration of inflammatory cells and production of mucin induced by allergen. The influx of eosinophils into the lung interstitium was significantly reduced after administration of DNA vaccine. Significant reductions of IL-4 and increase in levels of IFN-g in BALF were observed. The allergen-specific IgE was markedly decreased in mice receiving DNA vaccination. Allergen could induce higher IFN-g, weaker IL-4 in cultured spleen cells from mice receiving DNA vaccine. DNA vaccination inhibited STAT6 expression of spleen cells induced by allergen^ref. After sensitized and challenged by Der p 2, the BALB/c mice were immunized with DNA vaccine. The degrees of cellular infiltration were scored. IgE levels in serum and IL-4/IL-13 levels in BALF were determined by ELISA. The lung tissues were assessed by histological examinations. Expressions of STAT6 and NF-kB in lung were determined by immunohistochemistry staining. Vaccination of mice with DNA vaccine inhibited the development of airway inflammation and the production of mucin induced by allergen, and reduced the level of Der p 2-specific IgE level. Significant reductions of eosinophil infiltration and levels of IL-4 and IL-13 in BALF were observed after vaccination. Further more, DNA vaccination inhibited STAT6 and NF-kB expression in lung tissue in Der p 2-immunized mice. These results indicated that DNA vaccine encoding Der p 2 allergen could be used for therapy of allergen-induced allergic airway inflammation in this mouse model^ref.
mixed naked DNA plasmids encoding Der p 1, Der p 2, Der p 3, Der f 1, Der f 2, and Der f 3, the major house dust mite allergens on the allergic inflammation to the whole house dust mites (HDM) crude extract. 300 mg of these gene mixtures were injected into muscle of BALB/c mice. Control mice were injected with the pcDNA 3.1 blank vector. After 3 weeks, the mice were actively sensitized and inhaled with the whole house dust mite extract intranasally. The vaccinated mice showed a significantly decreased synthesis of total and HDM-specific IgE compared with controls. Analysis of the cytokine profile of lymphocytes after challenge with HDM crude extract revealed that mRNA expression of IFN-g was higher in the vaccinated mice than in the controls. Reduced infiltration of inflammatory cells and the prominent infiltration of CD8⁺ T cells were observed in histology of lung tissue from the vaccinated mice. Vaccination with DNA encoding the major house dust mite allergens provides a promising approach for treating allergic responses to whole house dust mite allergens^ref
Phl p 5 : a Sindbis virus replicon-based DNA vaccine encoding the major timothy grass pollen allergen was evaluated for its antiallergic potential compared with a conventional DNA vaccine in a BALB/c mouse model of allergy. Mice were intradermally prevaccinated with plasmid DNA, followed by sensitization and intranasal allergen provocation with recombinant Phl p 5. In vitro proliferation and cytokine secretion was measured in splenocyte cultures. Distribution of IgG₁, IgG_2a, and IgE antibody subclasses was determined by means of ELISA. IgE-mediated degranulation was measured with the basophil release assay. BALF was analyzed for eosinophils, IL-4, IL-5, IL-13, and IFN-g. Mucus production, inflammatory infiltrates, and epithelial damage were determined in lung sections. Both vaccines induced T_h1-biased immune responses, resulting in suppression of functional IgE, reduction of eosinophilia in bronchoalveolar lavage fluid, and alleviation of lung pathology. However, immunization with the replicon DNA vaccine elicited these effects at a 100-fold lower dose compared with the conventional DNA vaccine. The increased immunogenicity of replicon-based DNA vaccines allows for application of extremely low doses, thereby eliminating the concerns associated with conventional DNA vaccines, which have to be administered at milligram amounts to induce immune reactions in human subjects. Their high safety profile makes replicon-based DNA vaccines promising candidates for treatment of type I allergies in the clinic^ref
among the 2 major allergens of Japanese cedar pollen, since Cry j 1 is more important than Cry j 2 as an antigen to increase IgE in atopic dogs sensitized to Japanese cedar pollen, Cry j 1 can be a target for immunotherapy. Rfficacy of DNA vaccination with a plasmid containing the gene of a major allergen of Japanese cedar (Cryptomeria japonica, CJ) pollen, Cry j 1, was examined using a dog model experimentally sensitized to CJ pollen allergen. Cry j 1 DNA plasmid and a vector plasmid (pCAGGS) were injected into six dogs and three dogs, respectively, five times with an interval of 1.5 month. After the treatment with Cry j 1 DNA plasmid, production of IgE against Cry j 1 decreased in four of the six dogs in the treatment group, whereas it increased in the three dogs of the control group. The reactivity to the pollen allergen in intradermal testing and provocation testing were obviously reduced in the treatment group, but not in the control group. The number of mast cells in alveolar area of the lung in the treatment group was smaller than that in the control group. Cry j 1 DNA plasmid was also injected into three atopic dogs sensitive to Cry j 1, resulting in improvement of clinical signs in the pollination season. These findings indicated that Cry j 1 DNA plasmid could regulate mast cell-mediated reaction against Cry j 1, which could be an alternative and effective treatment for CJ pollinosis^ref.
Phl p 5b major allergen from timothy grass pollen : pre-treating the mice with Phl p 5b-DNA-construct significantly inhibited the production of specific IgE and IgG1 antibodies and prevented the development of a lung pathology associated with inflammation. Thus, immunoprophylaxis with an expression vector containing the Phl p 5b sequence totally prevented the emergence of characteristics associated with type I allergy and chronic bronchitis^ref.

in vitro

in vivo

^ref

₁

_2a

in vitro

⁺

₁

_2a

⁺

^ref

DNA vaccination in uteroinduces mucosal immunity and immune memory in the neonate : infectious diseases are the primary cause of neonatal morbidity and mortality in people, resulting in millions of deaths every year. Infection of the newborn with some of the pathogens involved, such as HHV-2 / HSV-2

, HHV-5 / HCMV

, HIV-1

, HBV

or group B Streptococcus spp. (GBS)

, usually occurs at the end of pregnancy, during birth or by breast feeding. The vertical transmission of many of these pathogens significantly increases the risk of neonatal infection. Therefore, active immunization of the fetus might represent an effective approach to reduce the high risk of neonatal diseases.

oral DNA vaccination in utero

a single oral exposure in utero to plasmid DNA encoding a truncated form of glycoprotein D of bovine herpesvirus-1 induced detectable immune responses (immune memory and mucosal immunity) in 80% (12 of 15) of newborn lambs. There was no evidence for the induction of immune tolerance in nonresponding lambs. Responding lambs displayed both systemic and mucosal immune responses and reduced virus shedding following intranasal challenge. Furthermore, strong anamnestic responses were evident for at least 3 mo after birth. The efficacy of in utero oral DNA immunization was further demonstrated with the HBsAg, and protective serum Ab titers occurred in 75% of immunized lambs^ref. Fetal immunization was safe and did not affect fetal gestation, neonatal viability, or significantly alter blood leukocyte populations. In utero immunization resulted in the induction of protective mucosal immunity and immune memory in the newborn lamb. Furthermore, there was no evidence that in utero DNA immunization induced immune tolerance. The uptake and expression of the plasmid DNA already occurred within the epithelium of the oral cavity, correlating with previous findings that local immune responses were found exclusively in the retropharyngeal lymph nodes draining the oral cavity^ref.

intramuscular DNA vaccination in utero :

a single intramuscular in-utero anti-HBV DNA immunization at two-thirds of pig gestation produces, at birth, antibody titers considered protective in humans. The boost of antibody titers in every animal following recall at 4 and 10 months demonstrates the establishment of immune memory. The safety of in-utero fetus manipulation is guaranteed by short-term (no fetus loss, lack of local alterations, at-term spontaneous delivery, breastfeeding) and long-term (2 years) monitoring. Treatment of fetuses closer to delivery results in immune ignorance without induction of tolerance^ref

DNA vaccines for farm animals :

anti-viral vaccines :

anti-bovine viral diarrhea virus (BVDV) DNA vaccine : the major protective antigen, the E2 protein, is cell-associated and not expressed on the cell surface. A DNA vaccine encoding various secreted versions of E2 has been evaluated. In vitro analysis demonstrated that deletion of the transmembrane anchor and addition of the signal sequence of bovine herpesvirus-1 (BHV-1) (gDsDE2) resulted in efficient secretion of E2 into the culture medium. In contrast, full-length E2, either without or with gDs (gDsE2), as well as truncated E2 without gDs (DE2), remained entirely cell-associated. Mice immunized with plasmid encoding gDsDeltaE2 developed significantly higher IgG and virus neutralizing antibody titres compared to animals vaccinated with plasmid encoding E2, DeltaE2 or gDsE2. To optimize secretion of E2, the efficiency of gDs was compared with that of the tissue plasminogen activator signal (tPAs) sequence. In addition, the effect of the plasmid backbone was assessed by comparing two vectors. Four plasmids, pMASIA-gDsDeltaE2, pMASIA-tPAsDeltaE2, pSLKIA-gDsDeltaE2 and pSLKIA-tPAsDeltaE2, were constructed and administered intradermally to mice. The mice immunized with pMASIA-tPAsDeltaE2 developed the strongest and most balanced immune responses. Vaccination of cattle confirmed that pMASIA-tPAsDeltaE2 elicited both strong humoral and cellular immune responses and thus could be a candidate DNA vaccine against BVDV^ref. In order to be able to compare DNA, protein and DNA prime-protein boost regimens, a plasmid was constructed encoding a secreted form of the NADL strain E2 protein (pMASIA-tPAsDE2). Furthermore, a pure secreted recombinant DE2 (rDE2) protein was produced. The rDE2 protein was formulated with a combination of Emulsigen and CpG oligodeoxynucleotide. Groups of calves were immunized with pMASIA-tPAsDE2 or with rDE2, or first with pMASIA-tPAsDE2 and then with rDE2. To evaluate the protection against BVDV, calves were challenged with BVDV strain NY-1 after the last immunization. Although all immunized calves developed humoral and cellular immune responses, the antibody responses in the DNA prime-protein boost group were stronger than those elicited by either the DNA vaccine or the protein vaccine. In particular, E2-specific antibody titres were enhanced significantly after boosting the DE2 DNA-primed calves with rDE2 protein. Moreover, protection against BVDV challenge was obtained in the calves treated with the DNA prime-protein boost vaccination regimen, as shown by a significant reduction in weight loss, viral excretion and lymphopenia, compared with the unvaccinated calves and the animals immunized with the DNA or protein only. These results demonstrate the advantage of a DNA prime-protein boost vaccination approach in an outbred species^ref.
anti-porcine reproductive and respiratory syndrome virus (PRRSV) DNA vaccine :

swine IL-2 and IL-4 on the development of immune responses induced by a PRRSV-ORF7 DNA vaccine (phCMV-ORF7). The 2 cytokines were cloned separately in the eukaryotic expression vector phCMV, and delivered via gene gun as adjuvants for the DNA vaccine. Groups of 3-week-old certified PRRSV-free, castrated male, Yorkshire crossbred pigs, were vaccinated with or without the IL-2 or IL-4. The ensuing humoral and cellular immune responses were analyzed by a PRRSV-specific ELISA, and by an in vitro blastogenic response of peripheral blood mononuclear cells (PBMC) stimulated by viral antigen, respectively. The animals were boosted 21 days post-vaccination and challenged 28 days afterward. The virus loads post-challenge were measured by real time PCR. The group of swine receiving the vaccine plus IL-2 had significant virus-specific blastogenic responses 3 weeks after the vaccine-cytokine boost, when compared to those of the experimental pigs that received the vaccine + IL-4, vaccine alone, unvaccinated controls or the pigs vaccinated with the DNA vaccine cloned in the reverse orientation (phCMV-ORF7(Rev)). None of the experimental swine had detectable specific antibodies against the virus during the vaccination phase. The virus load peak in vaccinated animals was delayed by about 72h as compared to that of the control pigs (unvaccinated and vaccinated with the phCMV-ORF7(Rev) construct). Interestingly, animals that received the phCMV-ORF7 vaccine alone consistently had low virus loads throughout the study. These results demonstrate that IL-2 has a positive inductive effect on the activation of vaccine-induced virus-specific cellular immunity, while IL-4 appeared to have a suppressive effect^ref.
immunogenicity of DNA vaccine expressing GP5 of PRRSV fused with VP22 of bovine herpesvirus 1^ref
the 2 major membrane-associated proteins of PRRSV, GP5 and M (encoded by ORF5 and ORF6 genes, respectively), are associated as disulfide-linked heterodimers (GP5/M) in the virus particle. 3 different DNA vaccine constructs, expressing GP5 alone (pCI-ORF5), M alone (pCI-ORF6) or GP5 and M proteins simultaneously (pCI-ORF5/ORF6), were constructed. In vitro, the co-expressed GP5 and M proteins could form heterodimeric complexes in transfected cells and heterodimerization altered the subcellular localization of GP5. The immunogenicities of these DNA vaccine constructs were firstly investigated in a mouse model. Mice inoculated with pCI-ORF5/ORF6 developed PRRSV-specific neutralizing antibodies at 6 and 8 weeks after primary immunization. However, only some mice developed low levels of neutralizing antibodies in groups immunized with pCI-ORF5 or pCI-ORF6. The highest lymphocyte proliferation responses were also observed in mice immunized with pCI-ORF5/ORF6. Interestingly, significantly enhanced GP5-specific ELISA antibody could be detected in mice immunized with pCI-ORF5/ORF6 compared to mice immunized with pCI-ORF5. The immunogenicities of pCI-ORF5/ORF6 were further evaluated in piglets (the natural host) and all immunized piglets developed neutralizing antibodies at 10 weeks after primary immunization, whereas there was no detectable neutralizing antibodies in piglets immunized with pCI-ORF5. The formation of GP5/M heterodimers may be involved in post-translational modification and transport of GP5 and may play an important role in immune responses against PRRSV infection. More importantly, co-expression of GP5 and M protein in heterodimers can significantly improve the potency of DNA vaccination and could be used as a strategy to develop a new generation of vaccines against PRRSV^ref
to construct gene vaccine of PPV and to investigate the effects of IL-2 as an adjuvant on immune responses in mouse, the recombinant expression plasmid of pCIneo-IL2-VP2 was constructed and transfected into PK-15 cells by lipofectamine, the expressed product was detected by immunofluore assay. To study the immune effects of DNA vaccine in vitro and in vivo, mice were used as the animal model. The recombinant plasmid pCIneo-IL2-VP2, the control plasmid pCI-neo and the PPV live vaccine were immunized by intramuscular injection. Anti-PPV antibodies were measured by ELISA, lymphocyte proliferation activity was detected using MTT method, and the specific killing activities of CTL were assayed too. The immunized mice produced PPV antibody after 1 week, and reached to highest after 4 weeks. Compared with the control group, the pCIneo-IL2-VP2 immunized group produced significant differences in the antibody titers, the lymphocyte proliferation activity and the specific killing activities of CTL. The pCIneo-IL2-VP2 induced humoral and cellular immunity responses similarly to that the live vaccine induced. The PPV DNA vaccine successfully induced humoral and cellular immunity response in mice with the IL-2 gene as an adjuvant^ref.

anti-Newcastle disease virus (NDV) DNA vaccine :

VP2 protein of infectious bursal disease virus (IBDV) protects against NDV and IBDV^ref
fusion (F) protein expressed in a fowlpox virus recombinant^ref. A pair of primers were designed and synthesized according to the previously published sequence of fusion protein (F) gene of NDV and used to amplify F gene by RT-PCR from the genomic RNA of a NDV strain JS5 isolated from goose. The PCR product was identified by sequencing. Then recombinant eukaryotic expression vector pVAX1-F was constructed through inserting F gene into MCS of pVAX1. The recombinant plasmid pVAX1-F was transfected in COS-7 cells, and identified for the transient expression of F gene by indirect immunofluorescent assay. Finally, the recombinant plasmid was transformed into attenuated Salmonella typhimurium SL7207, and the recombinant was screened and designated as SL7207 (pVAX1-F). It was verified that SL7207 (pVAX1-F) as the oral NDV DNA vaccine was safe for chickens after oral immunization at dosage of 10¹⁰ CFU or below. 1-day-old commercial ISA brown chickens were immunized orally with SL7207 (pVAX1-F) at two different dosages (10⁹ CFU and 10⁸ CFU) on day 1, 14 and 28. On day 7 after the last immunization, no significant difference was observed in the body weight between these 2 groups (p > 0.05), and also no significant difference between those 2 groups and negative control group (p > 0.05). Since there were maternal antibodies, high ELISA titers of serum antibodies against NDV were detected in the chickens of all groups on day 14. However, the levels of serum antibodies were decreased in the chickens of all groups on day 28, but the anti-NDV antibody response detected in the sera of chickens immunized with SL7207 (pVAX1-F) at the dosage of 10⁹ CFU were increased and significantly higher than the response induced by immunization with SL7207 (pVAX1) on day 35 (p < 0.05). Intestinal mucosal immune response was observed in chickens immunized with SL7207 (pVAX1-F) at the dosage of 10⁹ CFU or 10⁸ CFU. The high ELISA titers of antibodies against NDV in small intestinal mucosal samples from immunized chickens were on day 28 and 35. After challenged intranasally with virulent NDV strain F48E8, the chickens immunized with SL7207 (pVAX1-F) at the dosage of 10⁹ CFU could be protected with the protective rate of 77.27%, significantly higher than those with SL7207 (pVAX1) (p < 0.05). In summary, the DNA vaccine delivered by attenuated Salmonella typhimurium was safe and has good immunogenicity for chickens. A novel mucosal DNA vaccine was developed and could be useful for controlling the infection and epidemic of ND in the poultry^ref. In order to amplify F gene of NDV HeB02 strain, one pair of primers was designed according to the GenBank sequence, and a 1.66 kb F gene fragment was obtained by RT-PCR. Sequence analysis indicated that the homologies of the nucleotide sequence of HeB02 strain to those of F48 E9, La Sota and Clone30 strains were 88.1%, 84.9% and 83.8% respectively. The expression plasmid pSV-F was constructed by inserting the F gene into the pVAX1 vector, and transfected into the cultured COS 7 cell line via liposomes. The specific 5.9 kD protein was detected by SDS-PAGE and the immunogenicity of the expressed F protein was confirmed by Western blot, ELISA and neutralization test. 3 week-old SPF chickens were subcutaneously immunized twice at week 0 and 3 with 50 microg DNA of plasmid pSV-F by electroporration. 5 weeks later, all chickenss were challenged with 100 x EID₅₀ of NDV HeB02 strain, 1 week post challenge all chickenss were sampled by larynx swabbing to isolate virus and the HI level of NDV was measured. The results indicated that the virus isolation was negtive in all vaccinated chickenss and positive in all control chickens. The HI titres reached to 8.3log2 +/- 1.30 and 7.2log2 +/- 1.23 induced by NDV vaccine and positive cells (pSV-F), respectivily, the HI titres induced by Control cells (pVAX1) was not detected. Furthermore, the HI titres reached to 9.8log2 +/- 1.55 and 8.9log2 +/- 1.77 in vaccinated group with NDV vaccine and positive cells (pSV-F), respectivily, were sinificantly higher than that of the control cells (pVAX1) immunized group( HI titers was 3.0 log2 +/- 1.40, P < 0.01) after challenge. These results showed that the plasmid pSV-F could be as a candidate of DNA vaccine to provide protective immune response against NDV infection^ref

anti-infectious bursal disease virus (IBDV) DNA vaccine :

VP2 large segment gene of IBDV standard challenge (STC)^ref. Recombinant plasmids DNA vaccine encoding VP2 gene of very virulent strain of infectious bursal disease virus (vvIBDV) isolated from Pakistan was investigated with or without coadministration of cytocine-phosphate-guanine oligodeoxynucleotide (CpG ODN) to protect the chickens against the disease. VP2 gene of vvIBDV was successfully amplified by reverse transcription-polymerase chain reaction (RT-PCR) and was cloned into eukaryotic expression plasmid vector, which consisted of human cytomegalovirus (HCMV) immediate early enhancer and promoter, adenopartite leader sequences and SV-40 polyadenylation signal, and this was designated as pRc-VP2. 7-day-old maternal antibodies free chickens were intramuscularly injected with 500 mg of pRc-VP2 with or without CpG ODN twice at 1-week interval. At the age of 21 days the broiler chickens were challenged with 10(5) EID₅₀ of homologous strain of IBDV and observed for 14 days post-challenge. Immunization with pRc-VP2 plus CpG ODN conferred protection in 93% of the chickens as evidenced by the absence of clinical signs, atrophy of bursa of Fabricius (BF) and mortality followed by the group vaccinated with attenuated IBD vaccine and boosted with killed oil based IBDV vaccine, which conferred 90% protection. The protection of chickens injected with pRc-VP2 alone was 67% where as only 20% of the chickens in the negative control group were protected. However, enzyme-linked immunosorbent assay (ELISA) antibody titre in the group vaccinated with pRc-VP2 plus CpG ODN were significantly higher (P<0.05) than the group vaccinated with pRc-VP2 alone as well as the group vaccinated with commercial attenuated IBDV vaccine boosted with commercial oil adjuvanted killed IBDV vaccine. Responsiveness to a mitogenic lectin, phytoheamagglutinin-P was significantly reduced in group immunized with conventional vaccines (live boosted with killed) as compared to all the other groups (P<0.05). The results revealed that co-administration of recombinant plasmids with CpG ODN could protect chickens efficiently from IBDV challenge^ref
VP2, VP4, and VP3 genes of the standard challenge (STC) strain^ref
an oral DNA vaccine carrying the IBDV polyprotein antigen delivered by attenuated Salmonella enterica sv. Typhimurium (S. typhimurium). The recombinant plasmid pCI-VP2/4/3 was transformed by electroporation into an attenuated S. typhimurium strain (Dam() PhoP()) (designated hereafter as SV/pCI-VP2/4/3). The IBDV polyprotein gene was expressed in chicken embryo fibroblast (CEF) cells infected with strain SV/pCI-VP2/4/3, as shown by gene-specific RT-PCR and Western blot. Oral immunization of 7-day-old specific-pathogen-free (SPF) chickens with SV/pCI-VP2/4/3 elicited specific humoral responses as measured by ELISA. Vaccination with the strain SV/pCI-VP2/4/3 at 10⁹CFU per chicken offered 11/15 (73.3%) protection of the chickens against virulent IBDV challenge^ref
This study examined the adjuvant effects of dimethyl dioctadecyl ammonium bromide (DDA), CpG oligodeoxynucleotides (CpG-ODN), and chicken IFN-g (ChIFN-g) on a DNA vaccine (pcDNA-VP243) against the infectious bursal disease virus (IBDV). A plasmid encoding chicken IFN-a was constructed. Twice at 2-week intervals, twoweek-old chickens were injected intramuscularly and intraperitoneally with either a DNA vaccine alone or a DNA vaccine together with the respective adjuvants. On week 2 after the second immunization, the chickens were orally challenged with the highly virulent IBDV. The groups that received the DNA vaccines + either DDA or CpG-ODN showed significantly lower survival rates than the group that received the DNA vaccine alone. However, the survival rates for the DNA vaccine alone and for the DNA vaccine plus ChIFN-gamma were similar. The chickens had no detectable antibodies to the IBDV before the challenge but all the surviving chickens in all groups except for the normal control group showed the induction of antibodies to the IBDV at day 10 after the challenge. As judged by the lymphocyte proliferation assays using the a WST-8 solution performed on the peripheral blood and splenic lymphocytes, the stimulation indices (SI) of the peripheral blood lymphocytes in all groups except for the normal control group were similar immediately before the challenge. At 10 days post-challenge, the SI for DNA vaccine plus either CpG-ODN or ChIFN-g was similar to that of the DNA vaccine control group. For splenic lymphocytes, the SI in the DNA vaccine plus CpG-ODN and DNA vaccine plus ChIFN-g groups were higher than for the DNA vaccine control. These results suggest that DDA actually compromises the protection against the IBDV by DNA vaccine, and CpG-ODN and IFN-gamma had no significant effect^ref

anti-maedi-visna virus DNA vaccine : since conventional vaccines have failed in inducing an effective immune response in sheep^{ref1,
ref2,
ref3}, some proposed to develop a DNA vaccine agaist MVV^ref. Plasmid administration by injection into tibialis muscle seems to be more efficient than injection via the quadriceps route, probably because it is smaller and so local plasmid concentration is higher^ref.
anti-classical swine fever virus (CSFV) DNA vaccine : DNA vectors can be used to deliver vaccine antigens that stimulate effective protective immunity in mice, but in larger, outbred animal species, the protective efficacy is lower or large doses of DNA are required. These data demonstrate that porcine IL-3 when delivered to pigs by DNA vector or in low doses as recombinant protein, can enhance antibody responses to classical swine fever virus antigen expressed from co-delivered DNA, and improve the protective efficacy of the DNA vaccine. The effect was further enhanced when IL-3 was expressed as a fusion protein with the potyvirus coat protein. The adjuvant effect of IL-3 was compared to that of GM-CSF and IL-3 was shown to be at least as efficacious as GM-CSF. The response to IL-3 is novel and suggests, that at least in pigs, IL-3 could be used as an adjuvant for DNA vaccines^ref. CSFV E2 protein eukaryotic expression plasmid pVAXE2 was constructed. The plasmid pVAXE2 was transformed into Salmonella choleraesuis C500 (S. C500) attenuated vaccine strain by electroporation to generate Salmonella choleraesuis engineering strain S. C500/pVAXE2. The characterization of S. C500/pVAXE2 in morphology, growth, biochemistry and serology indicated that it retained the same properties as its original strain S. C500 with exception of kanamycin resistance originated from the plasmid pVAXE2. The plasmid stable in the bacteria after 15 passages. Kunming mice and rabbits were vaccinated 3 times at 2 weeks interval with S. C500/pVAXE2 in oral and intramuscular routes at the dosage of 1 x 10⁸ CFU for mice and 2 x 10⁹ CFU for rabbits each time. The specific antibody response against CSFV and Salmonella choleraesuis was detected by ELISA. 2 weeks after the third boost the immunized rabbits were challenged with 20 ID₅₀ of hog cholera lapinized virus (HCLV), followed by a virulent strain of Salmonella choleraesuis 2 week later than HCLV challenge. All immunized mice and rabbits produced significant antibodies against CSFV and Salmonella choleraesuis, and the immunized rabbits demonstrated the effective protection against the challenge of HCLV and virulent Salmonella choleraesuis. These results indicated the potential of developing multiplex swine DNA vaccine by using this bacteria as the vector^ref. Plasmid DNA based on SFV replicon encoding the E2 glycoprotein of CSFV and evaluated its efficacy in rabbits and pigs. The results showed that the animals immunized with the DNA vaccine developed CSFV-specific neutralizing antibodies and were protected from virulent or lethal challenge. This demonstrates that the SFV replicon-derived DNA vaccine can be a potential marker vaccine against CSFV infections^ref
anti-rabies virus (RV) DNA vaccine : DNA-based immunisation is cheaper and more versatile than vaccine prepared with tissue culture^ref. It has been shown recently that dogs can be protected against a challenge with the rabies virus by injecting a DNA vaccine encoding the RV glycoprotein (G)^ref. Production, purification and analysis of an experimental DNA vaccine against rabies has been reported^ref. Rabid dog exposures result in >99% of human rabies deaths worldwide. 98% of these cases occur in developing countries. Thus, the best protection against human rabies would be prevention through adequate vaccination of the reservoir population. The difficulty in re-locating ownerless, freely roaming dogs for booster vaccinations, in addition to poor coverage with inadequate vaccines, suggests that a potentially inexpensive vaccine that elicits long-term protection after a single-dose could improve control of canine rabies in developing countries. One solution could be a DNA vaccine. Dogs vaccinated intradermally (i.d.) in ear pinnae with a rabies DNA vaccine expressing a RV G produce high levels of neutralizing antibody that persist for at least 6 months. All dogs (100%) vaccinated i.d. in each ear pinna with a one-time 50 mg of rabies DNA vaccine, or intramuscular (i.m.) with a commercial canine rabies vaccine survived a lethal dose of rabies virus given 1 year after. In contrast, 100% of dogs vaccinated i.m. with 100 mg of rabies DNA developed rabies, as did 100% of negative control dogs that were vaccinated i.d. in each ear pinna or i.m. with DNA that did not express the rabies virus G. The data suggest that a one-time i.d. rabies DNA vaccination into ear pinnae offers a new approach to facilitate control of endemic canine rabies in developing countries^ref. A recombinant plasmid pTargeT.rabgp containing the G gene of RV was constructed and produced for immunogenicity studies on mice and dogs. The mice immunized twice with 50 mg or 100 mg of pTargeT.rabgp intramuscularly (i.m.), showed a serum antibody titer of 256 or 64, respectively 14 days post immunization (p.i.). When the vaccinated mice were challenged at 14 days p.i. with RV i.c., they were protected in 81.25% or 87.5%, respectively. A positive cell mediated immune response was observed by lymphocyte proliferation assay and macrophage migration inhibition test. These results indicate that the pTargeT.rabgp plasmid can be used as a rabies DNA vaccine^ref. Kinetics of antibody responses and protection against rabies were investigated after injection of a single dose of rabies DNA vaccine and compared to those induced by 1 or 2 injections of cell culture-derived vaccine in dogs issued from the common local breed and reared in experimental conditions. Rabies DNA vaccine administered intradermally by a jet injector in the inner face of the ear was by far more efficient in inducing long lasting high titers of virus neutralizing antibodies compared to cell culture vaccine Rabisin^® administered either subcutaneously or intramuscularly. Four years after vaccine administration of either DNA or cell culture-derived rabies vaccines, full protection against a rabies peripheral challenge was achieved. Vaccine trials targeting dogs living in field conditions in Tunisia further established that rabies DNA-based vaccination induced a stronger induction of virus neutralizing antibodies compared to Rabisin^®. This report shows for the first time that DNA vaccination could be more efficient under experimental or field conditions in large size mammals than the best commercially available cell culture-derived vaccine. This improvement will hopefully allow a better rabies control in developing countries by using a more efficient vaccination with fewer doses and targeting all categories of dogs^ref. Rabies virus (RV) glycoprotein (gp) consists of three domains: cytoplasmic, transmembrane and ectodomain. It occurs in a complete, membrane-bound form within the infected cell, but it is released from them in a deleted, secreted form lacking the transmembrane domain. A study tested the importance of the transmembrane domain for the capability of the RV gp gene, introduced into mice via a recombinant plasmid (DNA vaccine), to induce immune response and protection against challenge. Although the antibody response to the secreted form of gp was higher than that to complete gp, the protective efficacy of the respective DNA vaccine against challenge was not better than that of the DNA vaccine inducing complete gp. This indicates that the transmembrane domain of RV gp is important for generating protection against rabies and should be present in RV DNA vaccines^ref. A DNA vaccine, using a pCl-neo plasmid encoding the glycoprotein gene of a Mexican isolate of rabies virus, was developed to induce long-lasting protective immunity against rabies virus in dogs. The aim of this work was to evaluate the intranasal (IN) vaccination route in mice and dogs. Mice and dogs were immunized via the intramuscular (IM) and IN routes. Mice received 50 mg of DNA vaccine, a booster on day 30, using the same doses and routes, and on day 90 they were challenged. Dogs received 100 mg of DNA vaccine, with a booster on day 180, and immune responses were studied on day 210. Virus-neutralizing antibodies were detected in blood sera (up to 0.5 IU) in animals immunized via the IN route and when the animals were submitted to a booster, the levels of neutralizing antibodies increased. Animals vaccinated via the IM route presented higher neutralizing antibody titres than those vaccinated IN. Control groups lacked anti-rabies antibodies. On day 90, mice were challenged. From these, a 100 % of the IM vaccinated mice, and an 80 % of the IN vaccinated mice survived the challenge. No animals from the control group survived. Dogs revealed significant virus-neutralizing antibody titres (up to 0.5 IU) on day 30 and, after booster, on day 210 neutralizing antibody titre was higher than 1.8 IU. The main advantage of using DNA vaccines over traditional live ones is that there is no contamination with viruses that could be disseminated in the environment and reproduced in susceptible animals. This study demonstrated that pGQH was succesful when administrated via the IN route. IN vaccination seems attractive due to its easy application and mucosal protection. This form of vaccination could also be advantageous in domestic animal vaccination campaigns, for it is less stressful than the parenteral route (no painful shots)^ref. A DNA vaccine is under production by Indian Immunologicals : large-scale trials with dogs are on and six months is the time the company that will manufacture it says it will take to reach the market^ref.
anti-Hirame rhabdovirus (HIRRV) DNA vaccine : genes encoding the nucleocapsid protein (N) and the C-terminal half of the glycoprotein (G) were amplified by RT-PCR and separately cloned into the eukaryotic expression vector pcDNA 3.1. The G protein expressed by transfected cells was detected by western blot analysis. PCR analyses demonstrated the presence of injected plasmids in fish muscle tissue at 14 days post injection. IHC of muscle tissue injected with the plasmid DNA showed expression of the target protein in myofibrils and sarcoplasm. Flounder fry with an average weight of 3 g were injected with 5 mg of plasmid DNA and challenged at 21 days after immunization. Fish injected with vector DNA or PBS showed >95% cumulative mortality by 16 days after inoculation with the virus. In contrast, fish injected with plasmids containing the N gene, G gene, or N+G gene mixture showed 70, 5, and 2.5% cumulative mortality, respectively. These results show that the G gene is effective for the induction of protective immunity against HIRRV infection in injected fish^ref.

anti-infectious hematopoietic necrosis virus (IHNV) DNA vaccine^{ref1,
ref2,
ref3}. Pre-clinical research work reported in the literature has clearly demonstrated that the application of DNA vaccine technology is highly effective for the control of several viral pathogens in fish. However, the transition from proof of concept to commercialization has proved elusive for a variety of reasons, despite the demonstrable purity, potency, safety and efficacy of early DNA vaccine models. Registration is constrained by a worse-case risk assessment approach by regulators and vague, often non-existent regulations for the registration of DNA vaccines and, most importantly, lack of a licensing precedent. Second, costs to manufacture and secure intellectual property rights remain prohibitively high. This is compounded by the low unit value of the target animals, especially where aquaculture is concerned, and uncertainty with respect to required studies to satisfy regulators. Third, where food animals are concerned, there is a risk that inappropriate association with certain recombinant technologies used in agriculture will confuse consumers and create uncertainty in the marketplace. All combined, these factors have challenged the commercialization of DNA vaccine technology for food animal applications, especially in aquaculture. Vaccine plasmid pIHNw-G, containing the gene for the viral glycoprotein, was detected immediately after intramuscular injection in all tissues analyzed, including blood, but at later time points was found primarily in muscle tissue, where it persisted to 90 days. Glycoprotein expression was detected in muscle, kidney, and thymus tissues, with levels peaking at 14 days and becoming undetectable by 28 days. Histologic examination revealed no vaccine-specific pathologic changes at the standard effective dose of 0.1 mg DNA per fish, but at a high dose of 50 mg an increased inflammatory response was evident. Transient damage associated with needle injection was localized in muscle tissue, but by 90 days after vaccination no damage was detected in any tissue, indicating the vaccine to be safe and well tolerated^ref. The DNA vaccine pIHNw-G encodes the glycoprotein of IHNV. Vaccine performance in rainbow trout was measured 3, 6, 13, 24, and 25 months after vaccination. At three months all fish vaccinated with 0.1 mg pIHNw-G had detectable neutralizing antibody (NAb) and they were completely protected from lethal IHNV challenge with a relative percent survival (RPS) of 100% compared to control fish. Viral challenges at 6, 13, 24, and 25 months post-vaccination showed protection with RPS values of 47-69%, while NAb seroprevalence declined to undetectable levels. Passive transfer experiments with sera from fish after 2 years post-vaccination were inconsistent but significant protection was observed in some cases. The long-term duration of protection observed here defined a third temporal phase in the immune response to IHNV DNA vaccination, characterized by reduced but significant levels of protection, and decline or absence of detectable NAb titers. Examination of multiple tissues showed an absence of detectable long-term histopathological damage due to DNA vaccination^ref. The DNA vaccine based on the glycoprotein gene of IHNV induces a non-specific anti-viral immune response and long-term specific immunity against IHNV. This study characterized gene expression responses associated with the early anti-viral response. Homozygous rainbow trout were injected intra-muscularly (I.M.) with vector DNA or the IHNV DNA vaccine. Gene expression in muscle tissue (I.M. site) was evaluated using a 16,008 feature salmon cDNA microarray. Eighty different genes were significantly modulated in the vector DNA group while 910 genes were modulated in the IHNV DNA vaccinate group relative to control group. Quantitative reverse-transcriptase PCR was used to examine expression of selected immune genes at the I.M. site and in other secondary tissues. In the localized response (I.M. site), the magnitudes of gene expression changes were much greater in the vaccinate group relative to the vector DNA group for the majority of genes analyzed. At secondary systemic sites (e.g. gill, kidney and spleen), type I IFN-related genes were up-regulated in only the IHNV DNA vaccinated group. The results presented here suggest that the IHNV DNA vaccine induces up-regulation of the type I IFN system across multiple tissues, which is the functional basis of early anti-viral immunity^ref. A highly efficacious DNA vaccine against IHNV was mutated to introduce two stop codons to prevent glycoprotein translation while maintaining the plasmid DNA integrity and RNA transcription ability. The mutated plasmid vaccine, denoted pIHNw-G2stop, when injected intramuscularly into fish at high doses, lacked detectable glycoprotein expression in the injection site muscle, and did not provide protection against lethal virus challenge 7 days post-vaccination. These results suggest that the G-protein itself is required to stimulate the early protective antiviral response observed after vaccination with the nonmutated parental DNA vaccine^ref

anti-viral hemorrhagic septicemia (VHS) virus (VHSV) DNA vaccine^{ref1,
ref2} : a recombinant glycoprotein vaccine (VHSVg) and a DNA vaccine (pCMV-VHSg) were injected in Japanese flounder (Paralichthys olivaceus). Each fish was injected with 10 mg vaccine dissolved in 50 ml phosphate buffer saline (PBS). One month after the vaccination, the fish were challenged intraperitoneally with either 1x10² or 1x10³ TCID₅₀ of virus. Fish that received the VHSg DNA vaccine were highly protected against virus infection over a 21-day observation period, with cumulative mortalities ranging from 4 to 10%. However, the recombinant protein vaccine group appeared to have low survival rates. Using microarray analysis, humoral defense-related genes such as complement component C3, complement regulatory plasma proteins, IgM, IgD, MHC class II-associated invariant chain and CD20 receptor were observed to be up-regulated by the VHSg recombinant protein vaccine at 1 or 21 days post vaccination. On the other hand, cellular defense-related genes such as CD8a chain, T-cell immune regulator, MIP1-a and apoptosis-associated protein were not detected. Specific antibodies against VHSVg protein were detected in both vaccinated groups at a titer of 1:40 at 28 days post vaccination^ref. Since previous results showed that IL-8 was induced in rainbow trout (Oncorhynchus mykiss) in response to viral hemorrhagic septicemia virus (VHSV) infection, we have cloned IL-8 in an expression vector (pIL8+) and studied its possible adjuvant effect on the early response to a VHSV immunization model, focusing on the early response of several cytokines induced by a vector coding for the glycoprotein of VHSV (pMCV1.4-G) in the spleen and head kidney. First, we demonstrated that the pIL8⁺ successfully transcribed IL-8, by induction of IL-8 transcription in the muscle and blood, and by a massive infiltration of neutrophils at the muscle inoculation site. The effect of pIL8⁺ co-administration was studied on the expression of 2 pro-inflammatory cytokines, such as IL-1b and TNF-a; cytokines that have mainly an inhibitory role, IL-11 and TGF-b; and a T_h1 type cytokine, IL-18. The co-administration of pIL8+ with pMCV1.4-G modulates the cytokine response that is induced, mainly by having its effect increasing pro-inflammatory cytokines (IL-1b and TNF-a), with a greater impact on the spleen, and to a lesser extent in the head kidney. All these data suggest that IL-8 is able to modulate the early cytokine immune response that is produced in response to a DNA vaccine, and therefore, might be a potential immune adjuvant in fish viral vaccination. More work should be done to determine if this modulation has a beneficial effect on protection as seen in other mammal viral models^ref. A plasmid coding for the CXC chemokine interleukin 8 (IL-8, pIL8+) when co-administered with pMCV1.4-G was able to modulate the expression of induced pro-inflammatory cytokines, therefore, we have also studied the expression of these inducible CC chemokines in fish injected with pMCV1.4-G in the presence of pIL8+, study that will also help establish the relations among the different chemokine groups. All chemokines were induced in the head kidney of fish injected with the DNA vaccine, and the co-administration of pIL8+ together with the vaccine modulated the expression of all CC chemokines studied. In this sense, expression of the inducible CC chemokines was also studied in trout head kidney leucocytes treated with supernatants from EPC cells transfected with pIL8+. In this case, all CC chemokines studied except for CK5B were significantly induced by rainbow trout IL-8^ref
anti-influenzavirus H₅N₁ virus DNA vaccine : recently, the Ministry of Agriculture (MOA) of China after very careful evaluation of their safety and efficacy has certified ...

a recombinant H₅N₁ fowlpox vaccine^ref
an inactivated H₅N₁ reverse genetics vaccine developed by scientists at the Key Laboratory of Animal Influenza, affiliated with Harbin Veterinary Research Institute. Since the H₅N₁ inactivated vaccine has proved immunogenic for ducks and geese, the MOA suggests that if necessary, the new vaccine could be used on waterfowl in the high-risk area, which contains many water bodies, namely lakes and rivers. The vaccine is a muscular-injection formula -- thus, it could not be applied by its introduction into "rivers and lakes" [as erroneously stated in the media]. The new vaccine 3 weeks after the flocks are vaccinated protects chickens for 10 months — 4 months longer than existing vaccines — ducks for 10 months, and geese for 3.

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adenovirus-based influenza A virus vaccine directed against the hemagglutinin (HA) protein of the A/Vietnam/1203/2004 (H₅N₁) (VN/1203/04) strain isolated during the lethal human outbreak in Vietnam from 2003 to 2005. Different portions of HA were expressed from a recombinant replication-incompetent adenoviral vector, achieving vaccine production within 36 days of acquiring the virus sequence. BALB/c mice were immunized with a prime-boost vaccine and exposed to a lethal intranasal dose of VN/1203/04 H₅N₁ virus 70 days later. Vaccination induced both HA-specific antibodies and cellular immunity likely to provide heterotypic immunity. Mice vaccinated with full-length HA were fully protected from challenge with VN/1203/04. The efficacy of adenovirus-based vaccination was next evaluated in domestic chickens, given the critical role of fowl species in the spread of HPAI worldwide. A single subcutaneous immunization completely protected chickens from an intranasal challenge 21 days later with VN/1203/04, which proved lethal to all control-vaccinated chickens within 2 days. These data indicate that the rapid production and subsequent administration of recombinant adenovirus-based vaccines to both birds and high-risk individuals in the face of an outbreak may serve to control the pandemic spread of lethal avian influenza^ref
a replication-incompetent, human adenoviral-vector-based, haemagglutinin subtype 5 influenza vaccine (HAd-H₅HA), which induces both humoral and cell-mediated immune responses against avian H₅N₁ influenza viruses isolated from people. Immunisation of mice with HAd-H₅HA provided effective protection from H₅N₁ disease, death, and primary viral replication (p<0.0001) against antigenically distinct strains of H₅N₁ influenza viruses. Unlike the recombinant H₅HA vaccine, which is based on a traditional subunit vaccine approach, HAd-H₅HA vaccine induced a 3- to 8-fold increase in HA-518-epitope-specific IFN-g-secreting CD8 T cells (p=0.01)^ref

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anti-duck hepatitis B virus (DHBV) DNA vaccine : DHBV preS/S DNA vaccine can protect ducks against DHBV infection, DuIFN-g gene as an immune adjuvant enhances its efficacy^ref. Engagement of CD154 on activated T cells with CD40 on antigen-presenting cells (APCs) potentiates adaptive immune responses in mammals. Soluble multimeric forms of CD154 have been used as an adjuvant or in immunotargeting strategies to enhance vaccine responses. The objective of our study was to examine the ability of duck CD154 (DuCD154) to enhance DNA vaccine responses in the duck hepatitis B model. Constructs were generated to express the functional domain of DuCD154 (tCD154), truncated duck hepatitis B virus (DHBV) core antigen (tcore) and chimera of tcore fused to tCD154 (tcore-tCD154). Expression in LMH cells demonstrated that all proteins were secreted and that tCD154 and tcore-tCD154 formed multimers. Ducks immunized with the plasmid ptcore-tCD154 developed accelerated and enhanced core-specific antibody responses compared to ducks immunized with ptcore or ptcore plus ptCD154. Antibody responses were better sustained in both ptcore-tCD154- and ptcore plus ptCD154-immunized ducks. Core-specific proliferative responses of duck peripheral blood mononuclear cells were enhanced in ducks immunized with ptcore-tCD154 or ptcore alone. This study suggests that the role of CD154 in the regulation of adaptive immune responses had already evolved before the divergence of birds and mammals. Thus, targeting of antigens to APCs with CD154 is an effective strategy to enhance DNA vaccine responses not only in mammalian species but also in avian species^ref.
anti-feline immunodeficiency virus (FIV) DNA vaccine : a vif-deleted FIV provirus (FIVDvifATGg) that co-expresses feline IFN-g was tested as a proviral DNA vaccine to extend previous studies showing efficacy with a FIV-pPPRDvif DNA vaccine. Cats were vaccinated with either FIVDvifATGg or FIV-pPPRDvif proviral plasmid DNA, or with both FIV-pPPRDvif DNA and feline IFN-g expression plasmid (pCDNA-IFNg). A higher frequency of FIV-specific T cell proliferation responses was observed in cats immunized with either FIVDvifATGg or FIV-pPPRDvif plus pCDNA-IFNg, whereas virus-specific CTL responses were comparable between vaccine groups. Antiviral antibodies were not observed post-vaccination. Virus-specific cellular and humoral responses were similar between vaccine groups after challenge with biological FIV isolate (FIV-PPR) at 13 weeks post-immunization. All vaccinated and unvaccinated cats were infected after FIV-PPR challenge and exhibited similar plasma virus loads. Accordingly, inclusion of plasmids containing IFN-g did not enhance the efficacy of FIV-pPPRDvif DNA immunization. Interestingly, the lack of protection associated with FIV-pPPRDvif DNA immunization contrasted with findings from a previous study and suggested multiple factors including timing of FIV-pPPRDvif inoculations and challenge, as well as route of challenge virus delivery, may significantly impact vaccine efficacy^ref.
anti-foot-and-mouth disease virus (FMDV) DNA vaccine : It is known that only the minority of plasmid DNAs effect a cure or prevention after intramuscular injection into host. But what is the fate of the majority? And indeed how many of the injected DNAs work? Till now, little is known about it. To answer these questions, two methods including PCR and autoradiography were used in distribution study in mice that had received a single muscular inoculation of plasmid DNA containing antigenic epitopes of FMDV. The results showed that the plasmid DNAs were distributed by blood circulation and degraded soon. The degradation ratio of super coiled plasmid DNA was 20.9% in 10min, 34.1% in 1h, 86.8% in 1 day and 97.8% in 1 week in sera in vivo. And over a half of the whole were output in urine and faeces. The rest resided most in muscles as 'antigen pool', next in immune organs, kidney, liver, heart, lung and little in brain or gonad. About 40% or 0.5% of total plasmid DNAs, inferring to be effective, resided in muscles or immune organs, respectively. Collective results suggested that 'nude' DNA, as water injection, was characterized as quick absorbent, extensive distribution, but low utilization rate. Finally, the immune mechanism for the DNA vaccine was discussed^ref. Various parameters have been examined for the improvement of immune responses induced by immunisation with FMDV DNA vaccine (pcDNA 3.1/P1-2A3C3D, P1) in swine : increasing the amount of P1 DNA plasmids and pGM-CSF adjuvant plasmids induces stronger FMDV specific and neutralising antibody responses, as well as promoting cytokines IL-8 and IFN-g secretion, in immunised pigs via multiple inoculation sites^ref. The fungus Agaricus blazei Murill (ABM) is particularly rich in polysaccharides, which have shown particularly strong results in treating and preventing cancers. The goal of this study was to investigate whether co-administration of the ABM extract with FMDV DNA vaccine could increase the immune responses. Compared with the control mice, which received FMDV DNA vaccine alone, significant increase in not only the FMDV-specific antibody response but also T cell proliferation was observed in mice which received FMDV DNA vaccine plus the ABM extract. Taken together, these results demonstrated that application of the ABM extract might provide a strategy to improve the efficacy of DNA vaccines^ref. Despite FMDV being responsible for one of the most devastating animal diseases, little is known about the cellular immune mechanisms involved in protection against this virus. In this work we have studied the potential of DNA vaccines based on viral minigenes corresponding to three major B and T-cell FMDV epitopes (isolate C-S8c1) originally identified in natural hosts. The BTT epitopes [VP1_133-156-3A_11-40-VP4_20-34] were cloned into the plasmid pCMV, either alone or fused to ubiquitin, the lysosomal targeting signal from LIMPII, a soluble version of CTLA4 or a signal peptide from the human prion protein, to analyze the effect of processing through different antigenic presentation pathways on the immunogenicity of the FMDV epitopes. As a first step in the analysis of modulation exerted by these target signals, a FMDV infection inhibition assay in Swiss outbred mice was developed and used to analyze the protection conferred by the different BTT-expressing plasmids. Only one of the 37 mice immunized with minigene-bearing plasmids developed specific neutralizing antibodies prior to FMDV challenge. As expected, this single mouse that had been immunized with the BTT tandem epitopes fused to a signal peptide (pCMV-spBTT) was protected against FMDV infection. Interestingly, nine more of the animals immunized with BTT-expressing plasmids did not show viremia at 48h post-infection (pi), even in the absence of anti-FMDV antibodies prior to challenge. The highest protection (50%, 6 out of 12 mice) was observed with the plasmid expressing BTT alone, indicating that the targeting strategies used did not result in an improvement of the protection conferred by BTT epitopes. Interestingly, peptide specific CD4⁺ T-cells were detected for some of the BTT-protected mice. Thus, a DNA vaccine based on single FMDV B and T cell epitopes can protect mice, in the absence of specific antibodies at the time of challenge. Further work must be done to elucidate the mechanisms involved in protection and to determine the protective potential of these vaccines in natural FMDV hosts^ref. Mice were immunized via electroporation with 4 cDNA expression vectors that were constructed to express VP1 of FMDV, as cytoplasmic (cVP1), secreted (sVP1), membrane-anchored (mVP1) or capsid precursor protein (P1), respectively, to evaluate whether expression of VP1 in specific subcellular compartment(s) would result in better immune responses. Electroporation enhanced immune responses to vectors expressing cVP1 or P1 and expedited the immune responses to vectors expressing sVP1 or mVP1. Immunization of mice via electroporation with mVP1 cDNA was better than sVP1 or cVP1 cDNA in eliciting neutralizing antibodies and viral clearance protection. Vaccination with P1 cDNA, nonetheless, yielded the best immune responses and protection among all 4 cDNAs tested. The antigenicity of a VP1 DNA vaccine can be significantly enhanced by altering the cellular localization of the VP1 antigen. Electroporation is a useful tool for enhancing the immune responses of vectors expressing VP1 or P1. By mimicking FMDV more closely than that of transgenic VP1 and eliciting immune responses favorably toward T_h2, transgenic P1 may induce more neutralizing antibodies and better protection against FMDV challenge^ref. As an alternative strategy to classical inactivated viral vaccine against FMDV, naked DNA vaccine is attractive because of safety, flexibility and low cost. However DNA vaccination is usually poorly efficient in target species. Indeed naked DNA plasmids encoding for P1-2A3C3D and GM-CSF proteins did not induce any detectable immunity against FMDV in sheep. Interestingly, formulations of DNA on poly(d,l-lactide-co-glycolide) (PLG) or in lipofectin triggered divergent types of immune responses: PLG stimulated a T cell response and could elicit significant neutralising antibody titers, whereas lipofectin generated even higher antibody titers but no significant T cell response. The DNA/PLG regimen used in 5 sheep protected against clinical symptoms and viraemia and prevented the carrier state in 4 of them. Thus formulated DNA can be remarkably efficient against FMDV in a ruminant species that is usually refractory to DNA vaccination^ref. 2 different types of DNA vaccines encoding the capsid precursor polypeptide (P1) of FMDV were constructed and the immunogenicity were investigated and compared in mouse model. The first DNA vaccine, pcDP1, is a conventional plasmid DNA vaccine in which P1 was driven directly by a cytomegalovirus promoter. The second DNA vaccine, pSCAP1, is a Semliki Forest virus (SFV) replicon-based DNA vaccine encoding the same antigen. In vitro expression and characterization indicated that 2 vaccine vectors could correctly produce the P1 antigen. However, pSCAP1 could induce obvious apoptosis of the transfected cells. After immunization in BALB/c mice, the P1-specific ELISA antibodies, neutralizing antibodies, as well as lymphocyte proliferative responses induced by pSCAP1 were significantly higher than those obtained in mice immunized with pcDP1. Notably, mice immunized with the pSCAP1 had the determined ability of clearing virus in their sera after FMDV challenge. These results indicate that the SFV replicon-based DNA vaccine pSCAP1 are more effective than conventional DNA vaccine and it can be considered a promising approach for the development of a safety and efficacious vaccine against FMDV^ref.
anti-pseudorabies virus (PRV) / suid herpes type 1 DNA vaccine : in the natural host, the pig, the efficacy of a DNA vaccine was compared with the efficacy of a conventional modified live vaccine (MLV) and an inactivated vaccine (IV) in maternally immune newborn piglets. The priming of the immune response and the degree of protection against challenge infection was measured for all vaccine types. Piglets with or without maternal immunity were vaccinated twice, at the age of 5 and 9 weeks, and we assessed protection by challenge infection with virulent PRV at the age of 15 weeks. Vaccination with DNA or conventional vaccines induced both humoral and cell-mediated immune responses in maternally immune animals. DNA vaccination seemed not to suffer from suppression by maternal immunity and resulted in similar or stronger immune responses in maternally immune piglets as compared in naive piglets. In contrast, vaccination with conventional vaccines resulted in weaker immune responses in maternally immune piglets than in naive piglets. Moreover, DNA vaccination provided better protection against challenge infection in maternally immune piglets than in naive piglets, whereas vaccination with conventional vaccines did not^ref. A study evaluated the utility of GM-CSF cDNA as a DNA vaccine adjuvant for glycoprotein B (gB) of PrV in a murine model. The co-injection of GM-CSF DNA enhanced the levels of serum PrV-specific IgG with a 1.5-to 2-fold increase. Moreover, GM-CSF co-injection inhibited the production of IgG_2a isotype. However, it enhanced production of an IgG₁ isotype resulting in humoral responses biased to the T_h2-type against PrV antigen. In contrast, the co-administration of GM-CSF DNA enhanced the T cell-mediated immunity biased to the T_h1-type, as judged by the significantly higher level of cytokine IL-2 and IFN-g production but not IL-4. When challenged with a lethal dose of PrV, the GM-CSF co-injection enhanced the resistance against a PrV infection. This suggests that co-inoculation with a vector expressing GM-CSF enhanced the protective immunity against a PrV infection. This immunity was caused by the induction of increased humoral and cellular immunity in response to PrV antigen^ref. Animals were primed with Sindbis virus-derived plasmids that express viral glycoproteins gC and gD (gC- and gD-pSIN) and subsequently booster immunized with Orf virus (ORFV; Parapoxvirus) recombinants expressing gC and gD (D1701-VrVgC and -VrVgD). The prime-boost vaccination induced strong humoral and cellular-like PRV-specific immune responses. All prime-boost vaccinated pigs survived the lethal challenge infection without PRV-specific clinical symptoms and presented excellent body weight loss attenuation. Most notably, nasal shedding of challenge virus was reduced by more than about 3log₁₀, clearly reducing the risk of infection of non-immunized pigs^ref. Glycoprotein B mediates the absorption and penetration of the pseudorabies virus in the form of an immunodominant Ag, and represents a major target for the development of new vaccines. This study evaluated the efficiency of live attenuated Salmonella typhimurium SL7207 for the oral delivery of DNA vaccine encoding the pseudorabies virus glycoprotein B (pCI-PrVgB) in vivo, leading to the generation of both systemic and mucosal immunity against the pseudorabies virus Ag. An oral transgene vaccination of pCI-PrVgB using a Salmonella carrier produced a broad spectrum of immunity at both the systemic and mucosal sites, whereas the intramuscular administration of a naked DNA vaccine elicited no mucosal IgA response. Interestingly, the Salmonella-mediated oral transgene vaccination of the pseudorabies virus glycoprotein B biased the immune responses to the T_h2-type, as determined by the IgG_2a/IgG₁ ratio and the cytokine production profile. However, oral vaccination mediated by Salmonella harbouring pCI-PrVgB showed inferior protection to systemic immunization against virulent pseudorabies virus infection. The expression of transgene delivered by Salmonella bacteria in antigen-presenting cells of both the systemic and mucosal-associated lymphoid tissues was further demonstrated. These results highlight the potential use of live attenuated S. typhimurium for an oral transgene pseudorabies virus glycoprotein B vaccination to induce broad immune responses^ref. A murine model immunized with plasmid DNA vaccine expressing three glycoproteins pCIgB, pCIgC and pCIgD were used to examine the relative potency of major glycoproteins as well as the contribution of immunological parameters in providing protective immunity against the PrV. Among the three glycoprotein-encoded plasmid DNA vaccines, pCIgB produced the strongest response of PrV-specific IgG in the sera. pCIgB and pCIgD also induced a contrast pattern of immunity that was biased to the T_h1 and T_h2 types, respectively. pCIgC showed the potent inducer of CD8⁺ T-cell-mediated CTL activity against PrV. In addition, a cocktail vaccination of all 3 glycoprotein-encoded plasmid DNA vaccines induced the production of both cytokine types, T_h1 and T_h2 with levels that were the same as that of each immunogen. With regard to protective efficacy, pCIgB induced the most effective protection against a virulent virus challenge and a cocktail vaccination appeared to offer complete protection against a 5 LD₅₀ challenge, but not a 10 LD₅₀ one. pCIgD induced protection that was same as pCIgB, but pCIgC offered no effective protection. These results show the relative potency of the three glycoprotein-encoded PrV DNA vaccines in inducing protective immunity against PrV infection. The results in this study support previous results showing the importance of T_h1-type CD4⁺ T cells and their antibodies in conferring protection^ref. The immune responses induced by pseudorabies virus (PrV) gB-encoded plasmid DNA vaccine coadministered with IFN-g-associated cytokines and chemokines. These cytokines and chemokines included IL-12 and IL-18, as potent inducers of IFN-g, and IP-10, the production of which is IFN-g dependent. A coinjection of either IL-12 or IL-18 strongly suppressed the humoral antibody responses but increased the production of the T_h1-type cytokines IFN-g and IL-2 from immune T cells. Such antibody suppression was closely related to the increased susceptibility against a virulent viral challenge. On the other hand, IP-10 exhibited enhanced immune responses in both antibody responses and IFN-g production of immune T cells and facilitated the prolonged survival of infected mice. In contrast, there was no significant change in the immune responses of the mice that received codelivery of IFN-g. Therefore, IFN-g-associated cytokines, as T_h1-type inducers, can generate unexpected and unwanted effects, and their application as a vaccine adjuvant should be carefully evaluated depending on the target antigens^ref. To evaluate the effect of the genetic adjuvant of chemokines on the adaptive immune responses induced by a plasmid DNA vaccine expressing glycorotein B (gB) of the pseudorabies virus (PrV), a PrV DNA vaccine was co-inoculated with plasmid DNA expressing certain chemokines including CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CXCL8 (MIP-2), and CXCL10 (IP-10). A co-injection of the CCL3 plasmid DNA induced immunity that was biased to the T_h2 pattern, as judged by the ratio of IgG isotypes and the production of IL-4 cytokine generated from stimulated immune T cells. However, CCL5 and CXCL10 induced immune responses of the T_h1-type, which rendered the recipients more resistant to a virulent virus infection. CXCL8 also showed enhanced humoral and cell-mediated immunity (mixed-type pattern) providing effective protection against a viral challenge. However, there was no change in the immune responses induced by the PrV DNA vaccine in CCL4 recipients. These results suggest that co-injection of a chemokine, in the form of an adjuvant preparation, causes a rebalancing of the immunity, which subsequently affects the protective efficacy against a virulent virus infection^ref. No PRV-specific neutralizing or IgG₁ type antibodies were detected in sera 5 days after injection of a DNA vaccine against PRV infection in pigs. PRV-stimulated PBMCs produced IFN-g mRNA in vitro. 2 out of 5 pigs recovered from lethal PRV infection without attenuation of nasal viral excretion^ref.
anti-porcine rotavirus A DNA vaccine : the antigenic determinants of Vp6 gene was amplified from infected MA 104 cell by the reverse transcription-polymerase chain reaction (RT-PCR), the product of which was a 1194bp cDNA segment. Using T-A cloning technique, the PCR product was cloned into pGEM-T vector. Cloning plasmid pGEM-T-Vp6 and the prokaryotic shuttle expression vector pW425t between E. coli and Lactobacillus, were digested by SacI and KpnI double enzymes, respectively. The purified Vp6 gene was subcloned into the expression vector pW425t. Thus, the recombinant pW425t-Vp6 was constructed, which then was transformed into the competence thyA gene-mutant E. coli X13. Treated lysates of bacterium were loaded directly onto SDS-PAGE, on which approximately 44.88 kD fusion protein was observed. The protein was further analyzed using Western blot, which indicated that the protein was reactive with the antibody of rotavirus A. The results lay foundation for further studies on the Lactobacillus subunit vaccine and DNA vaccine of Vp6 gene for prevention and control of porcine rotavirus^ref
anti-equine herpes virus (EHV) DNA vaccine : experimental canarypox virus (ALVAC) and plasmid DNA recombinant vaccines expressing the gB, gC and gD glycoproteins of EHV-1 were assessed for their ability to protect conventional ponies against a respiratory challenge with EHV-1. In addition, potential means of enhancing serological responses in horses to ALVAC and DNA vaccination were explored. These included co-administration of the antigen with conventional adjuvants, complexation with DMRIE-DOPE and co-expression of the antigen along with equine GM-CSF. Groups of EHV primed ponies were vaccinated twice intra-muscularly with one dose of the appropriate test vaccine at an interval of 5 weeks. 2-3 weeks after the second vaccination, ponies were infected intra-nasally with the virulent Ab4 strain of EHV-1 after which they were observed clinically and sampled for virological investigations. The results demonstrated that DNA and ALVAC vaccination markedly reduced virus excretion after challenge in terms of duration and magnitude, but failed to protect against cell-associated viremia. Noteworthy was the almost complete absence of virus excretion in the group of ponies vaccinated with ALVAC-EHV in the presence of Carbopol adjuvant or DNA plasmid formulated with aluminium phosphate. The administration of the DNA vaccine in the presence of GM-CSF and formulated in DMRIE-DOPE and of the ALVAC vaccine in the presence of Carbopol adjuvant significantly improved virus neutralising antibody responses to EHV-1. These findings indicate that DNA and ALVAC vaccination is a promising approach for the immunological control of EHV-1 infection, but that more research is needed to identify the immunodominant protective antigens of EHV-1 and their interaction with the equine immune system^ref
anti-bovine herpesvirus 1 (BHV-1) DNA vaccine, which causes respiratory and genital infections in cattle. Previously we demonstrated that a DNA vaccine encoding a truncated, secreted form of BHV-1 glycoprotein B (tgB) induces CTL responses in C3H mice. C3H mice were immunized with a plasmid encoding tgB or with plasmids encoding tgB and murine IL-12. When the plasmid encoding tgB was delivered intramuscularly or epidermally by a gene gun, co-administration with IL-12 plasmid stimulated the synthesis of more IgG_2a, the production of higher levels of IFN-g, and more effective killing by CTLs. In contrast, after intradermal delivery no effect of co-administration of IL-12 encoding plasmid was observed. Further investigation suggested that antigen and IL-12 need to be expressed in the draining lymph nodes, where IL-12 can have a direct effect on T cells^ref
anti-white spot syndrome virus (WSSV) DNA vaccine : WSSV is a major cause of mortality in shrimp and poses a huge threat to aquaculture industry. The protective efficacy of DNA vaccination and tissue distribution of the immunised recombinant plasmid in black tiger shrimp (Penaeus monodon) was investigated. 4 recombinant constructs were generated by inserting four genes encoding the WSSV structural proteins VP15, VP28, VP35 and VP281 individually into DNA vaccine vector pVAX1. Expression of these proteins from the recombinant plasmids was confirmed in vitro in CHO cell lines. For vaccination experiments, shrimp were immunised with these DNA constructs and later challenged with WSSV. A significant level of protection was offered by the plasmids encoding VP28 or VP281 till 7 weeks whereas protein vaccination failed to protect vaccinated shrimp after 3 weeks of first immunisation. In addition, a tissue distribution study revealed the persistence of immunised DNA at least upto 2 months in the injected shrimp muscle^ref
anti-Eimeria tenella DNA vaccine (avian coccidiosis) : a chimeric construct, the Eimeria tenella antigen gene (3-1E) and chicken interferon gamma gene (ChIFN-g) were subcloned into the mammalian expression vector proVAX forming the plasmids proE and prol, and then linked by splicing overlap extension by PCR to construct the chimeric plasmid prolE; the chimeric protein (rlE) was expressed in Escherichia coli harboring the constructed plasmid pGEX/IE. Broilers were administered 2 i.m. injections with the constructed DNA vaccines (50 mg); in the protein booster groups 100 mg of the rlE were given following the proIE prime. After challenge the proIE-vaccinated chickens showed the protective immunity as demonstrated by significantly reduced oocyst shedding compared with chickens immunized with proE, but the prolE vaccine did not have an additive effect of increasing antibody titer and body weight gain. The chickens in the rlE booster groups had significantly higher specific antibody responses than those immunized with prolE, and displayed further decreased oocyst shedding and increased body weight gain. Taken together, ChIFN-g exerts an adjuvant effect coexpressed with 3-1E and provide the first evidence that the DNA prime-protein booster strategy is able to augment the protective efficacy of chimeric DNA vaccine against challenge with Eimeria tenella^ref

anti-bacterial vaccines :

anti-avian Chlamydophilia psittaci serovar A strain DNA vaccine : major outer membrane protein (MOMP) of an (pcDNA1:MOMPA)^{ref1,
ref2,
ref3}. The DNA vaccine confers partial protection against C. psittaci infection in turkeys. Turkeys were injected intramuscularly with the DNA vaccine : a eukaryotic expression vector (pcDNA1::MOMP) expressing the major outer membrane protein (MOMP) of an avian C. psittaci serovar D strain. Over a period of 11 weeks, cellular uptake of the DNA vaccine was examined by PCR, transcription of the insert by reverse transcript-PCR (RT-PCR) and mRNA translation by immunofluorescence staining of muscle biopsies. The results indicate that the DNA vaccine persists in turkey muscle for at least 10 weeks. Moreover, during this period of time MOMP was continuously expressed, as evidenced by the immunofluorescence staining and RT-PCR. Since C. psittaci infections occur at the age of 3 to 6 and 8 to 12 weeks, a vaccine persistence of 10 weeks seems adequate. Therefore, further research should concentrate on improving the elicited immune response, more specifically the cell-mediated immune response, rather than prolonging the lifespan of the plasmid^ref
anti-Mycobacterium tuberculosis DNA vaccine^ref : DNA injection + electroporation ^ref
anti-Mycobacterium avium subsp. paratuberculosis DNA vaccine : currently, paratuberculosis vaccines are comprised of crude whole-cell preparations of Mycobacterium avium subsp. paratuberculosis. Although effective in reducing clinical disease and fecal shedding, these vaccines have severe disadvantages as well, including seroconversion of vaccinated animals and granulomatous lesions at the site of vaccination. DNA vaccines can offer an alternative approach that may be safer and elicit more protective responses. In an effort to identify protective M. avium subsp. paratuberculosis sequences, a genomic DNA expression library was generated and subdivided into pools of clones (approximately 1,500 clones/pool). The clone pools were evaluated to determine DNA vaccine efficacy by immunizing mice via gene gun delivery and challenging them with live, virulent M. avium subsp. paratuberculosis. 4 clone pools resulted in a significant reduction in the amount of M. avium subsp. paratuberculosis recovered from mouse tissues compared to mice immunized with other clone pools and nonvaccinated, infected control mice. One of the protective clone pools was further partitioned into 10 clone arrays of 108 clones each, and 4 clone arrays provided significant protection from both spleen and mesenteric lymph node colonization by M. avium subsp. paratuberculosis. The nucleotide sequence of each clone present in the protective pools was determined, and coding region functions were predicted by computer analysis. Comparison of the protective clone array sequences implicated 26 antigens that may be responsible for protection in mice. This study is the first study to demonstrate protection against M. avium subsp. paratuberculosis infection with expression library immunization^ref
anti-Mycoplasma hyopneumoniae DNA vaccine : ribonucleotide reductase R2 subunit (NrdF) gene fragment was cloned into eukaryotic and prokaryotic expression vectors and its immunogenicity evaluated in mice immunized orally with attenuated Salmonella typhimurium aroA CS332 harboring either of the recombinant expression plasmids. NrdF is highly conserved among M. hyopneumoniae strains. The immunogenicity of NrdF was examined by analyzing antibody responses in sera and lung washes, and the cell-mediated immune (CMI) response was assessed by determining the INF-g level produced by splenocytes upon in vitro stimulation with NrdF antigen. S. typhimurium expressing NrdF encoded by the prokaryotic expression plasmid (pTrcNrdF) failed to elicit an NrdF-specific serum or secretory antibody response, and IFN-g was not produced. Similarly, S. typhimurium carrying the eukaryotic recombinant plasmid encoding NrdF (pcNrdF) did not induce a serum or secretory antibody response, but did elicit significant NrdF-specific IFN-g production, indicating induction of a CMI response. However, analysis of immune responses against the live vector S. typhimurium aroA CS332 showed a serum IgG response but no mucosal IgA response in spite of its efficient invasiveness in vitro. In the present study we show that the DNA vaccine encoding the M. hyopneumoniae antigen delivered orally via a live attenuated S. typhimurium aroA can induce a cell-mediated immune response. Different live bacterial vaccine carriers may have an influence on the type of the immune response induced^ref
anti-Brucella melitensis DNA vaccine : currently available live attenuated vaccines, although relatively efficacious when administered appropriately, remain abortogenic in livestock, fully pathogenic to humans, and indistinguishable from natural infections by the currently adopted diagnostic tests. Candidate genes, identified as homologous to protective antigens and virulence factors in related pathogens include omp25, flagellin, acvB, Cu,Zn superoxide dismutase (SOD)^{ref1,
ref2}, iron-regulated outer membrane protein B (FrpB), lumazine synthase^ref, bacterioferritin or P39 antigen^{ref1,
ref2}, ribosomal L7/L12 gene^ref, recombinant ribosome recycling factor-homologous protein^ref, glyceraldehyde-3-phosphate-dehydrogenase^ref, and invasion proteins A and B^{ref1,
ref2}. The outer membrane proteins (Omps) of Brucella spp. have been extensively characterized as potential immunogenic and protective antigens. Immunization of BALB/c mice with B. melitensis Omp31 gene cloned in the pCI plasmid (pCIOmp31) conferred protection against B. ovis and B. melitensis infection. Mice vaccinated with pCIOmp31 developed a very weak humoral response, and in vitro stimulation of their splenocytes with recombinant Omp31 did not induced the secretion of IFN-g. Splenocytes from Omp31-vaccinated animals induced a specific CTL activity, which leads to the in vitro lysis of Brucella-infected macrophages. pCIOmp31 immunization elicited mainly CD8⁺ T cells, which mediate cytotoxicity via perforins, but also CD4⁺ T cells, which mediate lysis via the Fas-FasL pathway. In vivo depletion of T-cell subsets showed that the pCIOmp31-induced protection against Brucella infection is mediated predominantly by CD8⁺ T cells, although CD4⁺T cells also contribute. These results demonstrate that the Omp31 DNA vaccine induces cytotoxic responses that have the potential to contribute to protection against Brucella infection. The protective response could be related to the induction of CD8⁺ T cells that eliminateBrucella-infected cells via the perforin pathway^ref

the Cu-Zn superoxide dismutase (SOD) antigen of Brucella abortus was previously identified to be a T cell antigen which induces both proliferation of and IFN-g secretion by T cells from infected mice. I.m. injection of mice with a plasmid DNA carrying the gene for SOD leads to the development of significant protection against B. abortus challenge. It has been reported that the antigen-specific immune responses generated by a DNA vaccine can be enhanced by co-delivery of certain cytokine genes. Delivering IL-2 on the efficacy of SOD DNA vaccine by generating a plasmid (pSecTag-SOD-IL-2) that codes for a secretory fusion protein of SOD and IL-2. Another plasmid (pSecTag-SOD) that codes for only SOD as a secretory protein was used for comparison. BALB/c mice injected intramuscularly with pSecTag-SOD or pSecTag-SOD-IL-2, but not the control plasmid pSecTag, developed SOD-specific antibody and T cell immune responses. Upon in vitro stimulation with recombinant SOD (rSOD) antigen, T cells from mice immunized with pSecTag-SOD-IL-2, in comparison with those from mice immunized with pSecTag-SOD, exhibited a lower proliferation response but produced significantly higher concentrations of IFN-g. Both DNA vaccines, however, induced similar levels of SOD-specific antibodies and cytotoxic T cell response. Although mice immunized with pSecTag-SOD-IL2 showed increased resistance to challenge with B. abortus virulent strain 2308, this increase was not statistically significant from that of pSecTag-SOD vaccinated mice. These results suggest that a SOD DNA vaccine fused to IL-2 did not improve protection efficacy^ref
DNA vaccine encoding L7/L12-P39 of Brucella abortus induces protective immunity in BALB/c mice^ref
a divalent fusion DNA vaccine encoding both the Brucella abortusL7/L12 protein (ribosomal protein) and Omp16 protein (outer membrane lipoprotein), designated pcDNA3.1-L7/L12-Omp16. Intramuscular injection of this divalent DNA vaccine into BALB/c mice elicited markedly both humoral and cellular immune responses. The specific antibodies exhibited a dominance of IgG_2a over IgG₁. In addition, the dual-gene DNA vaccine elicited a strong T-cell proliferative response and induced a large amount of gamma interferon-producing T cells upon restimulation in vitro with recombinant fusion protein L7/L12-Omp16, suggesting the induction of a typical T_h1-dominated immune response in vivo. This divalent DNA vaccine could also induce a significant level of protection against challenge with the virulent strain B. abortus 544 in BALB/c mice. Furthermore, the protection level induced by the divalent DNA vaccine was significantly higher than that induced by the univalent DNA vaccines pcDNA3.1-L7/L12 or pcDNA3.1-Omp16. Taken together, the results of this study verify for the first time that the Omp16 gene can be a candidate target for a DNA vaccine against brucellosis. Additionally, a divalent genetic vaccine based on the L7/L12 and Omp16 genes can elicit a stronger cellular immune response and better immunoprotection than the relevant univalent vaccines can^ref
DNA vaccine encoding the omp31 protein of Brucella melitensis 16M to induce cellular and humoral immune responses in mice. Eukaryotic expression vectors called pTargeTomp31, encoding outer membrane protein (omp31) of B. melitensis 16M were constructed. pTargeTomp31 was injected intramuscularly three times, at 3-week intervals in groups of mice 6 weeks of age. pTargeTomp31 induced good antibody response in ELISA . pTargeTomp31 elicited a T-cell-proliferative response and also induced a strong gamma interferon production upon restimulation with either the omp31 antigen or B. melitensis 16M extract. Animals immunized with this plasmid elicited a strong and long-lived memory immune response. Furthermore, pTargeTomp31 elicited a typical T_h1-dominated immune response in mice, as determined by IgG isotype analysis. This vaccine also provided the moderate degree of protection to the mice. This study for the first time focuses on DNA immunization of a gene from B. melitensis. These results may lead to the development of a DNA-based vaccine for the control of brucellosis in goats^ref
5 candidate genes from the Brucella melitensis 16M genome were selected. Eukaryotic expression plasmids encoding these antigens were constructed and expression was verified in vitro from transfected Cos7 cells. Each vaccine was assessed for protective efficacy in a BALB/c mouse brucellosis infection model. From these experiments 2 protective DNA vaccines were identified: p-omp25 and p-ialB. The Omp25 antigen (BMEI1249) has previously been studied in terms of Brucella virulence, serodiagnosis and as a protective antigen. However, this study represents the first report of a significant protective effect achieved against B. melitensis 16M challenge using the Omp25 antigen in a DNA vaccine approach. The other protective vaccine identified in this study was p-ialB. The ialB candidate (BMEI1584) was selected based upon its' putative function as an invasion protein which was assigned due to shared identity with the invasion protein B (ialB) of Bartonella bacilliformis. This candidate has not previously been investigated with regard to Brucella virulence or pathogenesis. This study is the first report to identify the Brucella invasion protein B (BMEI1584) as a novel protective antigen for brucellosis^ref

anti-Ehrlichia ruminantium DNA vaccine^ref : cpg1^ref, GroEL and GroES homologues^ref. 9 genes of E. ruminantium were selected and tested for protection against challenge in a DBA/2 mouse model, in order to identify candidate genes for incorporation into a recombinant vaccine. Of the 9 DNA vaccine constructs tested, four DNA constructs 14HWORF1/VR1012, 14HWORF2/VR1012, 27HWORF1/VR1012, and HSP58/VR1012 were not protective and were excluded from the study. The remaining five DNA constructs-MAP2/ VR1012, 1HWORF3/ VR1012, 4HWORF1/ VR1012, 18HWORF1/ VR1012, and 3GDORF3/ VR1012-offered partial protection against lethal challenge demonstrated by reduced mortalities compared to control groups. Protection was augmented when DNA primed mice were boosted with a respective homologous recombinant protein. Protection in these 5 groups was associated with the induction of cell-mediated or T_h1 type of immune responses characterized by the production of large amounts of IFN-g and IL-2 in in vitro proliferation assays using E. ruminantium antigens for stimulation. These responses were enhanced when the DNA-vaccinated DBA/2 mice were boosted with specific homologous recombinant protein vaccination. In a preliminary follow-up study, protection conferred by DNA vaccination with individual gene constructs was not enhanced when the protective constructs were administered in combination (including the map-1 gene of E. ruminantium). Further evaluation of these and other untested DNA constructs is necessary to optimize their expression in vivo in the presence of molecular adjuvants, such as the IFN-g gene, GM-CSF gene, IL-12 gene, and CpG motifs to fully evaluate their protective value^ref. Each individual 1H12 ORF protected sheep against a virulent needle challenge with the homologous strain, but all the animals vaccinated with the other cocktails succumbed to the challenge^ref.
anti-Salmonella choleraesuis DNA vaccine^ref : prothymosin alpha (ProT) enhances the efficacy of a pseudorabies DNA vaccine delivered by bacterial vectors. Oral Salmonella choleraesuis vaccine carrying the ProT eukaryotic expression plasmid protected mice against virulent S. choleraesuis challenge. The adjuvant effect of porcine ProT on humoral and cellular immune responses enhanced protective efficacy of the vaccine. Furthermore, both humoral and cellular immune responses played roles in the protective immune responses induced by the vaccine. Collectively, our results show that delivery of the ProT gene carried by attenuated S. choleraesuis augmented the immunogenicity of oral S. choleraesuis vaccine.
anti-Mycobacterium bovis DNA vaccine :

calves vaccinated with a combined DNA vaccine encoding Ag85B, MPT- 64, and MPT-83 antigens from the Mycobacterium tuberculosis for the priming and subsequently boosting with BCG prior to experimental challenge with virulent Mycobacterium bovis (M. bovis) resulted in improved immune responses over immunizing. Vaccination with the combined DNA/BCG induced higher levels of antigen-specific IFN-g in whole-blood cultures 4 weeks after final vaccination and the level of antigen-specific IFN-gamma in response to Ag85, MPT-64, and MPT-83 were still higher 4 weeks after challenge when compared to the combined DNA group. There was a significant bias toward induction of CD4⁺ T cells rather than CD8⁺ T cells responses, and the mean percentage of CD4⁺ T cells was increased about 2.6-fold in PBMC cultures in DNA prime-BCG boost vaccination when compared to the nonvaccinated group. In addition, DNA prime-BCG boost vaccination resulted in stronger humoral immune responses, and the levels of the specific antibodies to three antigens were increased 2- to 32- fold when compared to the combined DNA group. Vaccination with the combined DNA/BCG induced a high level of protection against an intratracheal challenge with virulent M. bovis, based on a significant enhancement of 6 pathological and microbiological parameters of protection compared to the nonvaccinated group. Finally, the combined DNA/BCG increased the protective efficacy by more than 10-100-fold as measured by reduced CFU counts in the lungs from calves challenged with M. bovis compared to the combined DNA and BCG groups. These results suggest that use of the prime-boost strategy offers better protection against bovine tuberculosis than does the combined DNA vaccines and BCG^ref.
vaccination against bovine tuberculosis is likely to become an important disease control strategy in developing countries, which cannot afford a test and slaughter control programme, or in countries which have a wildlife reservoir of Mycobacterium bovis infection. In the past decade, considerable progress has been made in the development and evaluation of tuberculosis vaccines for cattle and for a range of wildlife maintenance hosts including possums, badgers, deer and African buffaloes. Experimental challenge systems have been established for the different target species and the resulting disease process has mimicked that seen in the field. In cattle, neonatal vaccination with BCG appeared to be more effective than vaccination of 6-month-old calves and in most situations no other vaccine has been shown to be better than BCG. However, prime-boost strategies involving combinations of BCG with a protein or DNA vaccine, to improve on BCG vaccination alone, have produced very encouraging results. Differential diagnostic tests have been developed using mycobacterial antigens that are only present in virulent M. bovis to differentiate between BCG-vaccinated and M. bovis-infected cattle. BCG vaccine has been shown to reduce the spread of tuberculous lesions in a range of wildlife species and a prototype oral bait delivery system has been developed. Prospects for the development of improved vaccines against bovine tuberculosis are promising and vaccination approaches could become very valuable in the control and eradication of bovine tuberculosis^ref
the gene encoding MPB83 from Mycobacterium bovis Vallee111 chromosomal DNA was amplified by using PCR, and the PCR product was approximately 600bp DNA segment. Using TA cloning technique, the PCR product was cloned into pGEM-T vector and the cloning plasmid pGEM-T-83 was constructed successfully. pGEM-T-83 and pET28a⁺ were digested by BamHI and EcoRI double enzymes. The purified MPB83 gene was subcloned into the expression vector pET28a⁺, and the prokaryotic expression vector pET28a-83 was constructed. Plasmid containing pET28a-83 was transformed into competence Escherichia coli BL21 (DE3). The bacterium was induced by isopropyl-b-D-thiogalactopyranoside (IPTG) and its lysates were loaded directly onto SDS-PAGE, approximately 26 kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed using Western-blotting. The results indicated that the protein was of antigenic activity of M.bovis. The results were expected to lay foundation for further studies on the subunit vaccine and DNA vaccine of MPB83 gene in their prevention against bovine tuberculosis^ref
Bovine tuberculosis is a major cause of economic loss in countries where it is endemic, and in some countries, it may be a significant zoonotic disease problem. Therefore, new strategies for vaccine development are required, and among them, genetic immunization has potential value. The main goal of this study was to test the Mycobacterium bovis Ag85B gene as a DNA vaccine following challenge with an M. bovis virulent strain (ATCC 19274). Groups of BALB/c mice (n = 10) were immunized four times intramuscularly with the pCI-Ag85B construct or the pCI vector alone as the control. High titers of total IgG, IgG₁, and IgG_2a anti-Ag85B were measured in pCI-Ag85B immunized mice when compared to the pCI control group. Regarding cellular immunity, significant levels of IFN-g (1,100 +/- 157 pg/ml) and tumor necrosis factor alpha (650 +/- 42 pg/ml) but not IL-4 were detected in splenocyte culture supernatants of pCI-Ag85B-vaccinated mice following stimulation with recombinant Ag85B. Further, the main source of IFN-g is CD8⁺ T cells, as demonstrated by intracellular cytokine staining. As far as protection, a significant reduction in bacterial load in spleens (P < 0.05) was detected in pCI-Ag85B-immunized mice compared to the pCI vector control group. Use of the Ag85B DNA vaccine is a promising strategy to control M. bovis infection due to its ability to induce a T_h1 type of immune response. However, protective efficacy needs to be improved, since partial protection was achieved in spleens but not in lungs of vaccinated mice^ref

anti-Escherichia coli DNA vaccine : pcDNA1/faeG19 DNA vaccine in pigs by optimizing the FaeG expression plasmid and by co-administration of plasmid vectors encoding the A and B subunit of the E. coli thermo-labile enterotoxin (LT). Insertion of a Kozak sequence and optimization of vector- (cellular localization, expression) and both vector- and codon-usage were all shown to enhance the in vitro FaeG expression compared to pcDNA1/faeG19. Subsequently, pcDNA1/faeG19 and the vector- and vector- + codon-optimized construct were tested for their immunogenicity in pigs. In line with the in vitro results, antibody responses were better induced with increasing expression. The LT vectors additionally enhanced the antibody response, although not significantly, and were necessary to induce an F4-specific cellular response. These vectors were also added since LT has been described to direct the systemic response towards a mucosal IgA response in mice. The intradermal FaeG DNA prime-oral F4 boost immunization mainly resulted in a systemic IgG response, with only a marginal but significant reduction in F4⁺ E. coli faecal excretion when the piglets were primed with pWRGFaeGopt and pWRGFaeGopt with the LT vectors^ref.
anti-Mycobacterium marinum DNA vaccine : the post-challenge immune and histopathologic responses of hybrid striped bass vaccinated with a DNA vaccine encoding the Mycobacterium marinum Ag85A gene and subsequently challenged with M. marinum were investigated. Juvenile hybrid striped bass Morone saxatilis x M. chrysops were injected intramuscularly with 25 or 50 mg DNA plasmid and developed significant specific protective responses to live bacterial challenge 120 d post-vaccination. Both vaccine groups demonstrated increased survival, reduced splenic bacterial counts, and reduced granuloma formation compared to the control groups 14 d after challenge with approximately 8 x 10⁵ cfu M. marinum g(-1) fish body wt. The vaccine groups also developed more rapidly and significantly increased antibody and lymphoproliferative responses post-challenge compared to control groups, and these post-challenge immune responses appear to be vital against M. marinum infection in vaccinated hybrid striped bass. No significant differences in immune responses were recognized between the 25 and 50 mg vaccination groups, and these groups eventually experienced mortalities, splenic bacterial counts, and granuloma formation 28 d post-challenge comparable to those of the control groups at 14 d post-challenge. Therefore, vaccination of hybrid striped bass with a DNA vaccine encoding the M. marinum Ag85A gene provided significant but limited duration of protection against an acute high-dose M. marinum challenge^ref
combined vaccines :

mice immunized with DNA-IL-12⁺ had significantly reduced CFU counts for M. tuberculosis and B. abortus in lung and spleen, respectively (P<0.001), and DNA-IL-12⁺ elicited better protection than the combined DNA vaccine alone (DNA-IL-12(-)) or with the positive control groups after challenge with a virulent M. tuberculosis strain and B. abortus 2308 infection. The DNA-IL-12⁺ group had stronger antigen-specific IFN-gamma ELISPOT activities and higher levels of antigen-specific CD4⁺ and CD8⁺ T cell responses than either the DNA-IL-12^- or positive control groups. Likewise, antigen-specific IgG titers were also much higher than in other immunized groups. Moreover, DNA-IL-12⁺ gave a stronger IgG_2a-skewed response than did DNA-IL-12^-. In addition, its mean concentrations of IFN-gamma and IL-2 were about 2.5- to 4.5-fold higher than those observed in the DNA-IL-12^--treated mice, and were significantly higher than control groups (P<0.01 or P<0.001), whereas IL-4 and IL-10 secretion were lower^ref

anti-protozoal vaccines :

anti-Neospora caninum DNA vaccine : NcMIC1 is a 460 amino acid Neospora caninum microneme protein implicated in host cell adhesion and invasion processes. In this study, we assessed the potential protectivity of NcMIC1-based vaccination against experimental N. caninum infection in mice, employing both recombinant antigen vaccines and DNA vaccines. Recombinant NcMIC1 (recNcMIC1) was expressed in Escherichia coli as gluthatione-S-transferase-fusion protein. The corresponding NcMIC1 cDNA was cloned into the pcDNA3.1 expression plasmid (pcDNA-MIC1), and expression was checked in transfected Vero cells. Mice (10 animals/group) were vaccinated either with recNcMIC1 antigen suspended in Ribi-adjuvant (3 intraperitoneal injections), pcDNA-NcMIC1 (3 intramuscular injections), or pcDNA-NcMIC1 (twice intramuscularly), followed by 1 intraperitoneal recNcMIC1 antigen boost. Control groups included corresponding treatments with adjuvant, pcDNA3.1 without insert, and PBS (= infection control). All vaccinated and control groups were then challenged intraperitoneally with 2 x 10(6) N. caninum tachyzoites. Animals were inspected daily for a period of 3 wk postinfection (PI). At day 21, all animals were killed and assessed for infection. Before day 21 PI, clinical signs such as walking disorders, rounded back, apathy, and paralysis occurred in infection controls (50% of the mice), pcDNA and adjuvant controls (20% each), and the combined pcDNA-NcMIC1/recNcMIC1-treated group (30%). No clinical symptoms were observed in the recNcMIC1 and pcDNA-NcMIC1 vaccinated groups. All mice were positive for cerebral N. caninum infection as assessed by PCR of brain tissue. However, quantitative real-time PCR revealed that the infection intensity was significantly reduced in the group vaccinated with recNcMIC1 antigen. Immunohistochemistry confirmed these findings. In contrast, the infection intensity was highest in the group vaccinated with the pcDNA-NcMIC1/recNcMIC1 combination, indicating that the sequential application of the DNA vaccine and recombinant antigen had a deleterious effect. Serological analysis showed that only recNcMIC1-immunized animals generated detectable antibody levels recognizing native NcMIC1. Thus, of all protocols applied here, only recNcMIC1 vaccination appears to be suited to reduce cerebral infection in mice challenged with N. caninum tachyzoites^ref

anti-helminth vaccines :

anti-Fasciola hepatica DNA vaccine : ovine CTLA-4 mediated targeting enhanced the speed and magnitude of the primary antibody response and effectively primed for a potent memory response to the pro cathepsin B (FhCatB) antigen compared to conventional DNA vaccination alone, which failed to induce a detectable immune response. While the CpG-augmentation of the CTLA-4 targeted construct did not further enhance the magnitude or isotype profile of the CTLA-4 induced antibody titres, it did result in the induction of significant antigen-specific, lymphocyte-proliferative responses that were not observed in any other treatment group, showing for the first time that significant cellular responses can be induced in sheep following DNA vaccination. In contrast, CpG-augmentation in the absence of CTLA-4 mediated targeting failed to induce a detectable immune response. This is the first study to explore the potential adjuvant effects of ruminant specific CpG motifs on DNA vaccine induced immune responses in sheep. The ability of CpG-augmented CTLA-4 mediated targeting to induce both humoral and cellular immune responses in this study suggests that this may be an effective approach for enhancing the efficacy of DNA vaccines in large out-bred animal models^ref
anti-Taenia spp. DNA vaccine : many studies have provided evidence that the HBV core antigen particle is useful as a vaccine carrier for foreign epitopes. Epitopes KETc1, KETc12, and GK-1 are 3 promising candidates for designing a vaccine against Taenia solium cysticercosis. In the present study, epitopes KETc1 and KETc12 were inserted into the immunodominant loop of the truncated HBc149, and epitope GK-1 was fused to its C-terminus. The fused protein DC-3n was expressed and purified successfully. The polymeric character was tested by SDS-PAGE. After inoculation of BALB/c mice with DC-3n, antibody titers were assayed by ELISA, and the antibody specification was analyzed by Western blot. Dot ELISA was performed to verify the protection of the 3 epitopes. Results showed that the purified polymeric protein was formed, high antibody titers were induced in immunized mice and three antibodies different in molecular weight were induced, serum specific antibody recognized the native peptide localized mainly in cyst wall cells, and there was no specific antibody toward the three epitopes in sera of infected pig and humans. All these revealed that the protein DC-3n was a potential candidate for vaccine against cysticercosis. So the DC-3n sequence and the signal peptide sequence of IL-2 were cloned to a vector pVAX3.0 to construct pVAX-S-DC-3n. Pigs were immunized with pVAX-S-DC-3n. 2 weeks after the immunization booster, pigs were introduced to infectious T. solium eggs. The relative protective rate induced in pigs immunized with the DNA vaccine pVAX-S-DC-3n was 83%^ref. A DNA vaccine, pcDNA3-B, was developed by using the nucleotide sequence of Taenia solium B antigen and cloning into pcDNA3.1 plasmid. The growing pigs were vaccinated by 1 i.m. injection of 200 or 1000 mg pcDNA3-B. The immunization with 1000 mg of pcDNA3-B showed 92.6% protection when the pigs were challenged by T. solium eggs and 4 of the 5 pigs vaccinated had no viable cysts. The results provide encouraging information on the use of pcDNA3-B vaccination for the prevention of cysticercosis^ref

anti-tick vaccines :

plasmid pBMC2 encoding antigen Bm86 from a Colombian strain of cattle tickBoophilus microplus, was used for DNA-mediated immunization of BALB/c mice, employing doses of 10 and 50 mg, delivered by intradermic and intramuscular routes. Anti-Bm86 antibody levels were significantly higher compared to control mice treated with PBS. In the evaluation of Ig isotypes, significant levels of IgG_2a and IgG_2b were observed in mice immunized with 50 mg of pBMC2. Measurement of IL levels (IL-4, IL-5, IL-12(p40)) and IFN-g in the sera of mice immunized with pBMC2 indicated high levels of IL-4 and IL-5, although there were also significant levels of IFN-g. Mice immunized with pBMC2 showed antigen-specific stimulation of splenocytes according to the incorporation of bromodeoxyuridine and IFN-g secretion. In all trials, mice injected intramuscularly with 50 mg of pBMC2 presented the highest immune response. Moreover, cattle immunized with this DNA vaccine showed antibody production significantly different to the negative control. In conclusion, these results suggest the potential of DNA immunization with pBMC2 to induce humoral and cellular immune responses against B. microplus^ref
DNA vaccines utilize host cell molecules for gene transcription and translation to proteins, and the interspecific difference of codon usage is one of the major obstacles for effective induction of specific and strong immune response. In an attempt to improve codon usage effects of DNA vaccine on protein expression, a quantitative study was conducted to clarify the relationship of codon usage in the tick gene bm86 and its potential expression in bovine cells. The calculated relative synonymous codon usage (RSCU) and codon adaptation index (CAI) values of bm86 from Boophilus microplus and a set of 14 highly expressed genes from Bos taurus indicated that some codons utilized frequently in bm86 are rarely used in B. taurus genes and vice versa. The different translational efficiencies obtained suggested that after DNA vaccination using the wild bm86 gene, the protein Bm86 would be expressed in bovines, but it would not be the optimum sequence. However, using the codon-optimized bm86 gene to bovines, whose sequence was theoretically designed, would probably improve the level of the immune response generated against ticks^ref

anti-cancer vaccines : canine malignant melanoma (CMM) is an aggressive neoplasm treated with surgery and/or fractionated RT; however, metastatic disease is common and chemoresistant. Xenogeneic DNA vaccination with tyrosinase family members can produce immune responses resulting in tumor rejection or protection and prolongation of survival. Cohorts of 3 dogs each received increasing doses of xenogeneic plasmid DNA encoding either human tyrosinase (huTyr; 100/500/1500mcg), murine GP75 (muGP75; 100/500/1500mcg), murine tyrosinase (muTyr; 5 dogs each at 100/500mcg), muTyr+/-HuGM-CSF (9 dogs at 50mcg muTyr, 3 dogs each at 100/400/800mcg HuGM-CSF, or 3 dogs each at 50mcg muTyr with 100/400/800mcg HuGM-CSF), or 50 mcg MuTyr intramuscularly biweekly for a total of four vaccinations. The Kaplan-Meier median survival time (KM MST) for all stage II-IV dogs treated with huTyr, muGP75 and muTyr are 389, 153 and 224 days, respectively. Preliminarily, the KM MST for stage II-IV dogs treated with 50mcg MuTyr, 100/400/800mcg HuGM-CSF or combination MuTyr/HuGM-CSF are 242, 148 and >402 (median not reached) days, respectively. Thirty-three stage II-III dogs with loco-regionally controlled CMM across the xenogeneic vaccine studies have a KM MST of 569 days. Minimal to mild pain was noted on vaccination and one dog experienced vitiligo. In dogs vaccinated with HuTyr there is 2- to 5-fold increases in circulating antibodies to human tyrosinase. The results of these trials demonstrate that xenogeneic DNA vaccination in CMM: (1) is safe, (2) leads to the development of anti-tyrosinase antibodies, (3) is potentially therapeutic, and (4) is an attractive candidate for further evaluation in an adjuvant, minimal residual disease Phase II setting for CMM^ref.

The immune responses induced by Microtus brandti lactate dehydrogenase C4 DNA vaccine via oral feeding and nasal instillation in mouse were investigated. The number of newborns of the vaccinated mice was statistically significantly reduced compared with the control mice, but the vaccine failed to affect the birthrate, as all vaccinated mice gave birth^ref
Web resources :

DNA vaccines.com
Genetic Vaccines by Scientific American

DNA vaccines conferences :

#1 : 23-25 October 2002, The Royal College of Physicians of Edinburgh, Scotland, UK
#2 : 17-19 November 2004, The Grimaldi Forum, Monte Carlo, Monaco
DNA Vaccines Forum : How to Enhance DNA Vaccines Safety and Potency? 17-18 March 2005: London, United Kingdom

Companies manufacturing DNA vaccines

Institutions and researchers :

Michael A. Egan, PhD, Senior Scientist, Wyeth Vaccines Research
Raf Lemmens, PhD, Project Manager, GE Healthcare
Matthew Watson, Research Biochemist, Biologics Development and Engineering, Merck and Co., Inc.
Dr. Michel Chartrain, Distinguished Senior Investigator, Biocatalysis and Fermentation, Merck and Co., Inc.
Peter Hobart, Executive Director, Molecular Biology, Vical Inc.
Australia :

Biotechnology at Murdoch University
Department of Biotechnology at University of New South Wales

Belgium :

Unité de Génie Biologique at Université Catholique de Louvain
BioTeC - Bioprocess Technology and Control at Katholieke Universiteit Leuven

Brasil :

Departamento de Engenharia Bioquímica at Universidade Federal do Rio de Janeiro
Engenharia de Bioprocessos e Biotecnologia at Universidade Federal do Rio Grande do Sul
Laboratório de Engenharia Bioquímica at Universidade de São Paulo, Escola Politécnica, Departamento de Engenharia Química
Agrupamento de Biotecnologia da Divisão de Química at Instituto de Pesquisas Tecnológicas do Estado de S. Paulo
Universidade Federal de São Paulo Escola Paulista de Medicina - Departamento de Microbiologia, Imunologia e Parasitologia, Brazil

Canada :

Lorne A. Babiuk

Chile :

Escuela de Ingeniería Bioquímica Universidad Católica de Valparaíso

Cuba :

Centro de Ingeniería Genética y Biotecnología (CIGB)

Denmark :

Biotechnological Institute
Center for Process Biotechnology, Lyngbyat Technical University of Denmark
BioCentrum

France :

École Superieure de Biotechnologie de Strasbourg
Daniel Dory, PhD, Scientist, Viral Genetics and Biosafety Unit, French Food Safety Agency
Dr. Marie-Luise Michel, Senior Scientist, Research Director, INSERIM, Pasteur Institute, Paris, France

Germany :

Department of Biochemical Engineering at Aachen University of Technology
Instute of Biotechnologyat Forschungszentrum Jülich
Gesellschaft für Biotechnologische Forschung (GBF), Braunschweig
Institute of Biochemical Engineering (Institut für Bioverfahrenstechnik) at Universität Stuttgart
Hermann Wagner (University of Munich, Germany)
Joachim Schorr (Qiagen, Germany)
Dr. Martin Schleef (Chief Executive Officer, PLASMID FACTORY GMBH & CO. KG., Germany)
Gunther Hartmann (Ludwig-Maximians-University of Munich, Munich, Germany)
Klaus Cichutek, Vice President, Paul Ehrlich Institute, Langen
Dr. Astrid Breul, Business Manager, DNA Production, Qiagen/pAlliance

India :

Ireland :

Department of Agricultural and Food Engineering at University College Dublin

Israel :

Department of Biotechnology Engineering at Ben-Gurion University of Negev

Italy :

International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste

Japan :

Kyushu Institute of Technology, Dept. of Biochemical Engineering and Science
Department of Biotechnology at University of Osaka
International Center for Biotechnology, Osaka

Korea :

Netherlands, The :

Holland Biotechnology
Department of Biotechnology at Technical University of Delft
Menzo Havenga, Executive Director, Vaccine R&D Division, Crucell Holland BV
John Haanen, Medical Oncologist and Head, Immunotherapy Group, Netherlands Cancer Institute

Norway :

Iacob Mathiesen, Chief Executive Officer, Inovio AS
Agnete B. Fredrikson, Research Fellow, Institute of Immunology, University of Oslo

Portugal :

Universidade do Minho, Braga

Centre for Biological and Chemical Engineering at Instituto Superior Técnico, Lisboa
Instituto de Tecnologia Química e Biológica at Universidade Nova de Lisboa
Escola Superior de Biotecnologia, Porto

Russia :

State Virology and Biotechnology Research Center "Vector", Koltsovo, Novosibirsk region, 630559. Located some 30 km south-east of Novosibirsk, NPO Vektor, a Biopreparat subsidiary, links the Scientific Research Institute of Molecular Biology and the Design Institute of Biologically Active substances to industrial production facilities in Siberia. Vector has more than 100 lab and administrative buildings, including a large isolated virology-research campus. In the late 1980s, under the direction of Lev Sandakhchiev, Vector was reportedly devoted to the development and production of virus weapons. This research is said to have included work on loading Marburg virus into special long-range strategic and operational missile warheads, which had until that time been designed to be loaded with smallpox virus, bubonic plague, and anthrax. As recently as 1998, Iran was actively courting Vector's top scientists and officers, proposing cooperative ventures intended to enhance Tehran's biological capabilities. Vector is now ringed by three brand-new fences and the latest Western-made cameras and motion sensors. The Vector State Research Center of Virology and Biotechnology includes experimental laboratories for performance of basic and research virological investigations involving pathogens under P-4 biocontainment level conditions. The work on special-purpose subject ordered by RAO "Biopreparat" were performed using UKSD-25 units, that is the trials of efficacy of various developed vaccines against the specific viral pathogens and principal aerosol characteristics of viruses. Climatic static-dynamic unit UKSD-25 is designed for investigation of the efficacy of vaccinal preparations against viral pathogens under conditions of maximal protection of the personnel and environment. Two such units are available in the SRC VB "Vector", being located in the buildings No 6 and 6a (P-4 biocontainment virological laboratory buildings). Building No 6 is designed for experimental investigation of specific viral pathogens. The works with infectious material are carried out in the isolated P-4 biocontainment laboratories. Arrangement and design of the laboratories allows to perform investigations of any risk group materials. The building is equipped with unique maintenance and biocontainment systems which exclude any possibility of infecting the personnel or penetration of infectious material into the environment. Total floor space of the building - 6,336 m², including: - working floor space of zone 3 (biocontainment level P-4) - 1,440 m² - working floor space of zone 2 (biocontainment level P-3) - 720 m² - working floor space of the clean zone laboratories - 720 m² - working floor space of the technical premises for maintenance and biocontainment systems - 2,448 m² The building is equipped with: - ventilation systems which maintain the negative pressure of 25 mm of water column inside the zone 3 (14 mm of water column in the zone 2), hermetic entrances, double cascade filters for fine purification; system of waste piping capable of collecting up to 32 m³ of waste water and of treating it at a temperature of 135ºC, productivity rate of 16 m³/hour; transfer units (autoclaves AGP1200/2,5 and disinfecting chambers KDF-4) for treatment of waste, suits and instruments at a temperature of 135ºC; dispense systems for disinfecting solutions (6% hydrogen peroxide and 4% sodium hydroxide) providing the treatment of the entire working area; isolating boxes (biocontainment level P-4); 3 independent power supply sources. The Laboratory Animals Farm is designed for the production and maintenance of the pure lines of laboratory animals which are used in the experiments at SRC VB "Vector". The Animal Farm is housed in the 5-storeyed building with a total floor space of 4,225 m². Each storey contains 10 box stalls for animals with 14-fold air exchange. The farm comprises: feed-preparation unit housed in 2-storeyed building with a total floor space of 1,700 m²; storehouse for foodstuffs for animals with a total floor space of 1,200 m²; one-storeyed building for keeping rabbits, goats, sheep, fowl with a total floor space of 600 m²; and two lock entrances for transport. The Laboratory Animals Farm of the SRC VB "Vector" is the only one farm both over the Ural and in European part of Russia (excluding the Sochi department of the Institute of Pathology and Therapy) where monkey-breeding for medical and biological studies occurs. The farm of SRC VB "Vector" is one of the biggest in Western Siberia considering its scale and production volume. The farm production (laboratory animals - outbred and inbred mice and rats, guinea pigs, rabbits, monkeys) provides the experimental investigations of the Center connected with biotrials of medical and prophylactic preparations as well as the basic research in the field of virology, immunology and biotechnology.
Research Center for Toxicology and Hygienic Regulation of Biopreparation, Serpukhov, Russia
Ivanovski Institute for Virology, Moscow, Russia
Lomonosov Moscow State Academy of fine Chemical Technology (LSAFCT / MITHT), Moscow, Russia
Institute of Poliomyelitis and Virus Encephalitis named after M.P. Chumakov (Director - academician of RAMS Drozdov Sergey Grigorievich) 142782, Moscow, Leninskiy borough, p/î Institute of Poliomyelitis ??l.: (095) 439-90-07; fax: 439-93-21
Scientific Research Institute of Vaccines and Serums named after I.I. Mechnikov (Director - academician of RAMS Semionov Boris Fedorovich) 103064, Moscow, Maliy Kazenniy per., 5À ??l.: (095) 917-49-00; fax 917-54-60