Table of contents :

advantages drawbacks delivery adjuvants anti-infectious diseases vaccines anti-viral DNA vaccines anti-bacterial DNA vaccines anti-protozoal DNA vaccines anti-cancer DNA vaccines anti-chronic degenerative diseases DNA vaccines anti-toxic DNA vaccines

immunocontraceptive DNA vaccines immunotolerant DNA vaccines immunosuppressive anti-cytokine therapeutic vaccines anti-type I hypersensitivity DNA vaccines oral DNA vaccination in utero DNA vaccines for farm animals web resources  companies manufacturing DNA vaccines plasmid DNA purification DNA vaccines conferences institutions and researchers

• advantages^{ref1, ref2} :
• stable at room temperature
• coupled with delivery vectors :
• 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, ref2}
• 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, ref2}
• 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, ref2, ref3, ref4, ref5, ref6, ref7, ref8, ref9}
• 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, ref2, ref3, ref4}. Melanoma and prostate cancer provide usegul models to explore immunization against differentiation antigens^{ref1, ref2, ref3, ref4, 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, ref2, ref3, ref4, ref5, ref6, ref7}.
• 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
• 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 IgG₁ antibody 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

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.
• Induction of immunity after DNA vaccination is generally considered a slow process. DNA delivery to the skin results in a highly transient pulse of antigen expression. A new rapid and potent intradermal DNA vaccination method consisting of 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.
• The immunogenicity of 3 different methods of intramuscular plasmid DNA administration was compared in cynomolgus monkeys: needle and syringe, Biojector^®) 2000, and Mini-Jecttrade mark. 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
• 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.
• 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

• 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
• 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.
• 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 sigma1 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.

• anti-infectious diseases vaccines^ref :
• anti-viral vaccines
• anti-HIV-1 DNA vaccine (see also protein subunit vaccine and therapeutic vaccines):
• 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
• 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
• 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.
• 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
• 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
• 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. We compared the humoral response generated in mice using different plasmid DNA dosages and modes of immunization, including gene gun and in vivo electroporation (EP). 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.
• 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. These results provide a preliminary indication of the safety and immunogenicity of a prototype epidermal DNA vaccine for influenza^ref
Bioprocess engineering issues that would be faced in producing a DNA vaccine at up to 100 m³ fermentation scale for an influenza pandemic^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
		cynomolgus macaque	0/3ref
	VEEV replicon	guinea pig	8/10ref
		mouse	18/20ref
		cynomolgus macaque	0/3ref
	DNA	guinea pig	13/15ref1, ref2
		mouse	50-100% (dependent on the dose of DNA administered)ref
sGP	DNA	guinea pig	8/11ref
GP and NP	VEEV replicon	cynomolgus macaque	0/3ref
	DNA	guinea pig	8/8ref
	DNA and adenovirus	cynomolgus macaque	4/4ref
NP	VEEV replicon	mouse	20/20ref1, ref2
		cynomolgus macaque	0/3ref
	DNA	guinea pig	5/8ref
		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)