A consecutive priming-boosting vaccination of mice with simian immunodeficiency virus (SIV) gag/pol DNA and recombinant vaccinia virus strain DIs elicits effective anti-SIV immunity

To evaluate immunity induced by a novel DNA prime-boost regimen, we constructed a DNA plasmid encoding the gag and pol genes from simian immunodeficiency virus (SIV) (SIVgag/pol DNA), in addition to a replication-deficient vaccinia virus strain DIs recombinant expressing SIV gag and pol genes (rDIsSIVgag/pol). In mice, priming with SIVgag/pol DNA, followed by rDIsSIVgag/pol induced an SIV-specific lymphoproliferative response that was mediated by a CD4+-T-lymphocyte subset. Immunization with either vaccine alone was insufficient to induce high levels of proliferation or Th1 responses in the animals. The prime-boost regimen also induced SIV Gag-specific cellular responses based on gamma interferon secretion, as well as cytotoxic-T-lymphocyte responses. Thus, the regimen of DNA priming and recombinant DIs boosting induced Th1-type cell-mediated immunity, which was associated with resistance to viral challenge with wild-type vaccinia virus expressing SIVgag/pol, suggesting that this new regimen may hold promise as a safe and effective vaccine against human immunodeficiency virus type 1.     

Therapeutic vaccination with simian immunodeficiency virus (SIV)-DNA + IL-12 or IL-15 induces distinct CD8 memory subsets in SIV-infected macaques

DNA vaccination is an invaluable approach for immune therapy in that it lacks vector interference and thus permits repeated vaccination boosts. However, by themselves, DNA-based vaccines are typically poor inducers of Ag-specific immunity in humans and non-human primates. Cytokines, such as IL-12 and IL-15, have been shown to be potent adjuvants for the induction and maintenance of cellular immune responses, in particular during HIV infection. In this study, we examined the ability of therapeutic vaccination with SIV-DNA+IL-12 or IL-15 as molecular adjuvants to improve DNA vaccine potency and to enhance memory immune responses in SIV-infected macaques. Our results demonstrate that incorporating IL-12 into the vaccine induces SIV-specific CD8 effector memory T cell (T(EM)) functional responses and enhances the capacity of IFN-gamma-producing CD8 T(EM) cells to produce TNF. Lower levels of PD-1 were expressed on T cells acquiring dual function upon vaccination as compared with mono-functional CD8 T(EM) cells. Finally, a boost with SIV-DNA+IL-15 triggered most T cell memory subsets in macaques primed with either DNA-SIV or placebo but only CD8 T(EM) in macaques primed with SIV-DNA+IL-12. These results indicate that plasmid IL-12 and IL-15 cytokines represent a significant addition to enhance the ability of therapeutic DNA vaccines to induce better immunity.     

Induction of cellular immune responses to simian immunodeficiency virus gag by two recombinant negative-strand RNA virus vectors

A recombinant Newcastle disease virus (rNDV) expressing simian immunodeficiency virus (SIV) Gag protein (rNDV/SIVgag) was generated. The rNDV/SIVgag virus induced Gag-specific cellular immune responses in mice, leading to a specific anti-Gag antiviral immunity. This was evidenced by the inhibition of growth of recombinant vaccinia virus expressing an identical Gag antigen (rVac/SIVgag) but not of wild-type vaccinia virus in rNDV/SIVgag-immunized mice. Among intravenous, intraperitoneal, or intranasal immunization routes, intranasal administration induced the strongest protective response against challenge with rVac/SIVgag. We further demonstrated that these immune responses were greatly enhanced after booster immunization with recombinant influenza viruses expressing immunogenic portions of SIV Gag. The magnitude of the protective immune response correlated with the levels of cellular immune responses to Gag, which were still evident 9 weeks after immunization. These results suggest that rNDV and influenza virus vectors are suitable candidate vaccines against AIDS as well as against other infectious diseases.     

The adjuvant effects of co-stimulatory molecules on cellular and memory responses to HBsAg DNA vaccination

Because DNA vaccines on their own tend to induce weak immune responses in humans, adjuvant methods are needed in order to improve their efficacy. The co-stimulatory molecules 4-1BBL, OX40L, and CD70 have been shown to induce strong T cell activities; therefore, in this study, we investigated whether they may be used as molecular adjuvants for a hepatitis B surface antigen (HBsAg) DNA vaccine (pcDS2) in eliciting strong cellular and memory responses. Compared to mice immunized with pcDS2 alone, addition of the co-stimulatory molecules increased T cell proliferation and an HBsAg-specific antibody response that was marked with a higher ratio of IgG2a/IgG1. Importantly, pcDS2 plus these co-stimulatory molecules elicited a higher level of IFN-gamma and IL-4 in CD4(+) T cells and a higher level of IFN-gamma in CD8(+) T cells. In addition, a significantly robust antigen-specific cytotoxic T lymphocyte (CTL) response and the production of long-term memory CD8(+) T cells were also observed in the groups immunized with pcDS2 plus 4-1BBL, OX40L, or CD70. Consistently, as late as 100 days after immunization, upregulated expressions of BCL-2, Spi2A, IL-7Ra, and IL-15Ra were still observed in mice immunized with pcDS2 plus these co-stimulatory molecules, suggesting the generation of memory T cells in these groups. Together, these results suggest that the co-stimulatory molecules 4-1BBL, OX40L, or CD70 can enhance the immunogenicity of HBsAg DNA vaccines, resulting in strong humoral, cellular, and memory responses. This approach may lead to an effective therapeutic vaccine for chronic hepatitis B virus (HBV) infection.     

免疫共刺激分子OX40L对乙型肝炎核酸疫苗的免疫佐剂作用

[目的]为了进一步增强HBV DNA疫苗的免疫反应,本研究将共刺激分子OX40L 作为HBV DNA疫苗的分子佐剂免疫小鼠,旨在探讨共刺激分子OX40L对HBV DNA疫苗诱导体液和细胞免疫应答的影响.[方法]我们将HBV DNA疫苗(pcDS2)单独或联合共刺激分子质粒pOX40L免疫C57BL/6小鼠;分别在第0,2,4周进行免疫,在第6周检测抗-HBs IgG、IgG1和IgG2a,T淋巴细胞增殖指数,细胞因子表达水平和体内细胞毒性T淋巴细胞杀伤作用(CTL)等免疫学指标.[结果]pceDS2联合pOX40L免疫组小鼠的抗-HBs水平显著提高,抗-HBs IgG亚类以IgG2a占优;免疫小鼠的T淋巴细胞体外经乙型肝炎表面抗原(HBsAg)刺激后,联合免疫组刺激指数(SI)明显高于pcDS2组;联合免疫组CD4 + T淋巴细胞的IL-4和IFN-γ表达水平及CD8 + T淋巴细胞的IFN-γ表达水平显著升高;DNA疫苗免疫的各组小鼠,HBsAg特异性体内CTL高于对照组,其中联合免疫组小鼠的体内CTL杀伤作用最强.[结论]共刺激分子OX40L不仅能增强HBV DNA疫苗诱导特异性体液免疫应答,还能增强特异性细胞免疫反应,尤其增强体内CTL的杀伤活性,为HBV DNA疫苗的研究奠定了基础.     

Next generation DNA vaccines for HIV-1

We studied the effects of first generation HIV-1 plasmid vaccines in 167 individuals. The vaccines were very well tolerated and induced helper T cell responses in most vaccine recipients. However, the CTL responses were below a 20% response rate. Improvement in vaccine potency is an important goal of this technology and a central focus of our laboratory. To improve on these response rates, we used RNA optimized constructs pGag and pEnv). These vaccines express 20-100 fold better than first generation vectors. However, our studies support that additional enhancements are needed to further boost the immune response. We report that we can significantly enhance the induced CD8 effector cell response by including engineered B7 costimulatory molecules. We observed that B7.2 was more effective at driving cellular immune responses than B7.1 as a plasmid vaccine. We developed gene swaps and deletions between these two molecules. This manipulation resulted in a dramatically enhanced cellular immune response as measured by CTL, or ICC or Elispot. We have also explored the use of cytokines as plasmid vaccine adjuvants. We observed that IL-12 and IL-15 were effective as plasmid vaccine adjuvants. Interestingly, IL-15 appeared to allow T cell expansion in the absence of significant T cell help. Improvement of the immune response induced by plasmid vaccines can be engineered in multiple ways. Our studies show that both costimulation as well as cytokine signals can be harnessed for more potent vaccine development. These results have important implications for the design of vaccines for prophylaxis and therapy.     

NEXT GENERATION DNA VACCINES FOR HIV-1

ABSTRACT

We studied the effects of first generation HIV-1 plasmid vaccines in 167 individuals. The vaccines were very well tolerated and induced helper T cell responses in most vaccine recipients. However, the CTL responses were below a 20% response rate. Improvement in vaccine potency is an important goal of this technology and a central focus of our laboratory. To improve on these response rates, we used RNA optimized constructs pGag and pEnv). These vaccines express 20–100 fold better than first generation vectors. However, our studies support that additional enhancements are needed to further boost the immune response. We report that we can significantly enhance the induced CD8 effector cell response by including engineered B7 costimulatory molecules. We observed that B7.2 was more effective at driving cellular immune responses than B7.1 as a plasmid vaccine. We developed gene swaps and deletions between these two molecules. This manipulation resulted in a dramatically enhanced cellular immune response as measured by CTL, or ICC or Elispot. We have also explored the use of cytokines as plasmid vaccine adjuvants. We observed that IL-12 and IL-15 were effective as plasmid vaccine adjuvants. Interestingly, IL-15 appeared to allow T cell expansion in the absence of significant T cell help. Improvement of the immune response induced by plasmid vaccines can be engineered in multiple ways. Our studies show that both costimulation as well as cytokine signals can be harnessed for more potent vaccine development. These results have important implications for the design of vaccines for prophylaxis and therapy.     

Coimmunization with an optimized IL-15 plasmid results in enhanced function and longevity of CD8 T cells that are partially independent of CD4 T cell help

DNA vaccines are a promising technology for the induction of Ag-specific immune responses, and much recent attention has gone into improving their immune potency. In this study we test the feasibility of delivering a plasmid encoding IL-15 as a DNA vaccine adjuvant for the induction of improved Ag-specific CD8(+) T cellular immune responses. Because native IL-15 is poorly expressed, we used PCR-based strategies to develop an optimized construct that expresses 80-fold higher than the native IL-15 construct. Using a DNA vaccination model, we determined that immunization with optimized IL-15 in combination with HIV-1gag DNA constructs resulted in a significant enhancement of Ag-specific CD8(+) T cell proliferation and IFN-gamma secretion, and strong induction of long-lived CD8(+) T cell responses. In an influenza DNA vaccine model, coimmunization with plasmid expressing influenza A PR8/34 hemagglutinin with the optimized IL-15 plasmid generated improved long term CD8(+) T cellular immunity and protected the mice against a lethal mucosal challenge with influenza virus. Because we observed that IL-15 appeared to mostly adjuvant CD8(+) T cell function, we show that in the partial, but not total, absence of CD4(+) T cell help, plasmid-delivered IL-15 could restore CD8 secondary immune responses to an antigenic DNA plasmid, supporting the idea that the effects of IL-15 on CD8(+) T cell expansion require the presence of low levels of CD4 T cells. These data suggest a role for enhanced plasmid IL-15 as a candidate adjuvant for vaccine or immunotherapeutic studies.     

Induction of AIDS virus-specific CTL activity in fresh, unstimulated peripheral blood lymphocytes from rhesus macaques vaccinated with a DNA prime/modified vaccinia virus Ankara boost regimen

The observed role of CTL in the containment of AIDS virus replication suggests that an effective HIV vaccine will be required to generate strong CTL responses. Because epitope-based vaccines offer several potential advantages for inducing strong, multispecific CTL responses, we tested the ability of an epitope-based DNA prime/modified vaccinia virus Ankara (MVA) boost vaccine to induce CTL responses against a single SIVgag CTL epitope. As assessed using both 51Cr release assays and tetramer staining of in vitro stimulated PBMC, DNA vaccinations administered to the skin with the gene gun induced and progressively increased p11C, C-->M (CTPYDINQM)-specific CD8+ T lymphocyte responses in six of six Mamu-A*01+ rhesus macaques. Tetramer staining of fresh, unstimulated PBMC from two of the DNA-vaccinated animals indicated that as much as 0.4% of all CD3+/CD8alpha+ T lymphocytes were specific for the SIVgag CTL epitope. Administration of MVA expressing the SIVgag CTL epitope further boosted these responses, such that 0.8-20.0% of CD3+/CD8alpha+ T lymphocytes in fresh, unstimulated PBMC were now Ag specific. Enzyme-linked immunospot assays confirmed this high frequency of Ag-specific cells, and intracellular IFN-gamma staining demonstrated that the majority of these cells produced IFN-gamma after peptide stimulation. Moreover, direct ex vivo SIV-specific cytotoxic activity could be detected in PBMC from five of the six DNA/MVA-vaccinated animals, indicating that this epitope-based DNA prime/MVA boost regimen represents a potent method for inducing high levels of functionally active, Ag-specific CD8+ T lymphocytes in non-human primates.     

Induction of positive cellular and humoral immune responses by a prime-boost vaccine encoded with simian immunodeficiency virus gag/pol

It is believed likely that immune responses are responsible for controlling viral load and infection. In this study, when macaques were primed with plasmid DNA encoding SIV gag and pol genes (SIVgag/pol DNA) and then boosted with replication-deficient vaccinia virus DIs recombinant expressing the same genes (rDIsSIVgag/pol), this prime-boost regimen generated higher levels of Gag-specific CD4+ and CD8+ T cell responses than did either SIVgag/pol DNA or rDIsSIVgag/pol alone. When the macaques were i.v. challenged with pathogenic simian/HIV, the prime-boost group maintained high CD4+ T cell counts and reduced plasma viral loads up to 30 wk after viral challenge, whereas the rDIsSIVgag/pol group showed only a partial attenuation of the viral infection, and the group immunized with SIVgag/pol DNA alone showed none at all. The protection levels were better correlated with the levels of virus-specific T cell responses than the levels of neutralization Ab responses. These results demonstrate that a vaccine regimen that primes with DNA and then boosts with a replication-defective vaccinia virus DIs generates anti-SIV immunity, suggesting that it will be a promising vaccine regimen for HIV-1 vaccine development.     

Cytokine,chemokine, and costimulatory molecule modulation to enhance efficacy of HIV vaccines

Understanding key intervention points in developing immune responses may allow the rational inclusion of biological adjuvants into vaccines that could potentiate the immune response both quantitatively and qualitatively and enhance effective memory responses. Cytokine and chemokine combinations can potentially help target antigen to the appropriate antigen presenting cell and initiate maturation of these presenting cells, attract cells expressing different chemokine receptors, steer cellular immune responses toward Th1 and CD8 CTL, and enhance systemic and mucosal IgG and secretory IgA antibodies and determine their isotype balance. Animal protection studies suggest that synergistic combinations of cytokines and immunomodulating molecules may be required to protect from a viral challenge. For example, GM-CSF has been shown to be synergistic with IL-12 or CD40 ligand for induction of CTL and for antiviral protection, and the triple combination of GM-CSF, IL-12, and TNF alpha appears to induce the most effective protection in some mouse models. Chemokine-antigen fusions have also been shown to enhance immunogenicity of the antigen. Combinations of costimulatory molecules have been found to be synergistic when incorporated in a vaccine. Combined use of newer more potent vaccine constructs, containing codon optimized epitopes, relevant CpG motifs, cytokines, costimulatory molecules and chemokines, used in heterologous prime-boost strategies with viral vector vaccines or recombinant proteins, might afford the most potent vaccine approaches yet developed. In this review we will discuss the application and delivery of cytokines, costimulatory molecules, and chemokines toward improving current vaccine strategies.     

Sequential priming with simian immunodeficiency virus (SIV) DNA vaccines, with or without encoded cytokines, and a replicating adenovirus-SIV recombinant followed by protein boosting does not control a pathogenic SIVmac251 mucosal challenge

Previously, combination DNA/nonreplicating adenovirus (Ad)- or poxvirus-vectored vaccines have strongly protected against SHIV(89.6P), DNAs expressing cytokines have modulated immunity elicited by DNA vaccines, and replication-competent Ad-recombinant priming and protein boosting has strongly protected against simian immunodeficiency virus (SIV) challenge. Here we evaluated a vaccine strategy composed of these promising components. Seven rhesus macaques per group were primed twice with multigenic SIV plasmid DNA with or without interleukin-12 (IL-12) DNA or IL-15 DNA. After a multigenic replicating Ad-SIV immunization, all groups received two booster immunizations with SIV gp140 and SIV Nef protein. Four control macaques received control DNA plasmids, empty Ad vector, and adjuvant. All vaccine components were immunogenic, but the cytokine DNAs had little effect. Macaques that received IL-15-DNA exhibited higher peak anti-Nef titers, a more rapid anti-Nef anamnestic response postchallenge, and expanded CD8(CM) T cells 2 weeks postchallenge compared to the DNA-only group. Other immune responses were indistinguishable between groups. Overall, no protection against intrarectal challenge with SIV(mac251) was observed, although immunized non-Mamu-A*01 macaques as a group exhibited a statistically significant 1-log decline in acute viremia compared to non-Mamu-A*01 controls. Possible factors contributing to the poor outcome include administration of cytokine DNAs to sites different from the Ad recombinants (intramuscular and intratracheal, respectively), too few DNA priming immunizations, a suboptimal DNA delivery method, failure to ensure delivery of SIV and cytokine plasmids to the same cell, and instability and short half-life of the IL-15 component. Future experiments should address these issues to determine if this combination approach is able to control a virulent SIV challenge.     

Improved cellular immune response elicited by a ubiquitin-fused DNA vaccine against Mycobacterium tuberculosis

This study evaluated the immune response elicited by a ubiquitin (Ub)-fused MPT64 DNA vaccine against Mycobacterium tuberculosis. BALB/c mice were vaccinated with plasmid DNA encoding MPT64 protein, Ub-fused MPT64 DNA vaccine (UbGR-MPT64), and negative DNA vaccines, respectively. MPT64 DNA vaccine immunization induced a Thl-polarized immune response. The production of Thl-type cytokine (interferon-gamma [IFN-γ]) and proliferative T cell responses were enhanced significantly in mice immunized with UbGR-MPT64 fusion DNA vaccine, compared with nonfusion DNA vaccine. Moreover, this fusion DNA vaccine also resulted in an increased relative ratio of IgG2a to IgGl and the cytotoxicity of T cells. IFN-γ intracellular staining of splenocytes indicated that UbGR-mpt64 fusion DNA vaccine activated CD4+ and CD8+ T cells, particularly CD8+ T cells. Thus, this study demonstrated that the UbGR-MPT64 fusion DNA vaccine inoculation could improve antigen-specific cellular immune responses, which is helpful for protection against TB.     

IL-23 induces stronger sustained CTL and Th1 immune responses than IL-12 in hepatitis C virus envelope protein 2 DNA immunization

IL-23 is a heterodimeric cytokine consisting of p19 and the p40 subunit of IL-12. IL-23 has been shown to possess IL-12-like biological activities, but is different in its capacity to stimulate memory T cells in vitro. In this study, we investigated whether IL-23 could influence envelope protein 2 (E2)-specific cell-mediated immunity induced by immunization of hepatitis C virus E2 DNA. We found that IL-23 induced long-lasting Th1 and CTL immune responses to E2, which are much stronger than IL-12-mediated immune responses. Interestingly, IL-23N220L, an N-glycosylation mutant showing reduced expression of excess p40 without changing the level of IL-23, exhibited a higher ratio of IFN-gamma- to IL-4-producing CD4(+) T cell frequency than did wild-type IL-23, suggesting a negative regulatory effect of p40 on Th1-prone immune response induced by IL-23. These data suggest that IL-23, particularly IL-23N220L, would be an effective adjuvant of DNA vaccine for the induction of durable Ag-specific T cell immunity.     

An mRNA vaccine encoding Chikungunya virus E2-E1 protein elicits robust neutralizing antibody responses and CTL immune responses

Arthropod-borne chikungunya virus (CHIKV) infection can cause a debilitating arthritic disease in human. However, there are no specific antiviral drugs and effective licensed vaccines against CHIKV available for clinical use. Here, we developed an mRNA-lipid nanoparticle (mRNA-LNP) vaccine expressing CHIKV E2-E1 antigen, and compared its immunogenicity with soluble recombinant protein sE2-E1 antigen expressed in S2 cells. For comparison, we first showed that recombinant protein antigens mixed with aluminum adjuvant elicit strong antigen-specific humoral immune response and a moderate cellular immune response in C57BL/6 mice. Moreover, sE2-E1 vaccine stimulated 12-23 folds more neutralizing antibodies than sE1 vaccine and sE2 vaccine. Significantly, when E2-E1 gene was delivered by an mRNA-LNP vaccine, not only the better magnitude of neutralizing antibody responses was induced, but also greater cellular immune responses were generated, especially for CD8⁺ T cell responses. Moreover, E2-E1-LNP induced CD8⁺ T cells can perform cytotoxic effect in vivo. Considering its better immunogenicity and convenience of preparation, we suggest that more attention should be placed to develop CHIKV E2-E1-LNP mRNA vaccine.     

IL-12 gene as a DNA vaccine adjuvant in a herpes mouse model: IL-12 enhances Th1-type CD4+ T cell-mediated protective immunity against herpes simplex virus-2 challenge

IL-12 has been shown to enhance cellular immunity in vitro and in vivo. Recent reports have suggested that combining DNA vaccine approach with immune stimulatory molecules delivered as genes may significantly enhance Ag-specific immune responses in vivo. In particular, IL-12 molecules could constitute an important addition to a herpes vaccine by amplifying specific immune responses. Here we investigate the utility of IL-12 cDNA as an adjuvant for a herpes simplex virus-2 (HSV-2) DNA vaccine in a mouse challenge model. Direct i.m. injection of IL-12 cDNA induced activation of resting immune cells in vivo. Furthermore, coinjection with IL-12 cDNA and gD DNA vaccine inhibited both systemic gD-specific Ab and local Ab levels compared with gD plasmid vaccination alone. In contrast, Th cell proliferative responses and secretion of cytokines (IL-2 and IFN-gamma) and chemokines (RANTES and macrophage inflammatory protein-1alpha) were significantly increased by IL-12 coinjection. However, the production of cytokines (IL-4 and IL-10) and chemokine (MCP-1) was inhibited by IL-12 coinjection. IL-12 coinjection with a gD DNA vaccine showed significantly better protection from lethal HSV-2 challenge compared with gD DNA vaccination alone in both inbred and outbred mice. This enhanced protection appears to be mediated by CD4+ T cells, as determined by in vivo CD4+ T cell deletion. Thus, IL-12 cDNA as a DNA vaccine adjuvant drives Ag-specific Th1 type CD4+ T cell responses that result in reduced HSV-2-derived morbidity as well as mortality.     

Interleukin-23 restores immunity to Mycobacterium tuberculosis infection in IL-12p40-deficient mice and is not required for the development of IL-17-secreting T cell responses

Host control of Mycobacterium tuberculosis is dependent on the activation of CD4+ T cells secreting IFN-gamma and their recruitment to the site of infection. The development of more efficient vaccines against tuberculosis requires detailed understanding of the induction and maintenance of T cell immunity. Cytokines important for the development of cell-mediated immunity include IL-12 and IL-23, which share the p40 subunit and the IL-12Rbeta1 signaling chain. To explore the differential effect of IL-12 and IL-23 during M. tuberculosis infection, we used plasmids expressing IL-23 (p2AIL-23) or IL-12 (p2AIL-12) alone in dendritic cells or macrophages from IL-12p40(-/-) mice. In the absence of the IL-12/IL-23 axis, immunization with a DNA vaccine expressing the M. tuberculosis Ag85B induced a limited Ag-specific T cell response and no control of M. tuberculosis infection. Co-delivery of p2AIL-23 or p2AIL-12 with DNA85B induced strong proliferative and IFN-gamma-secreting T cell responses equivalent to those observed in wild-type mice immunized with DNA85B. This response resulted in partial protection against aerosol M. tuberculosis; however, the protective effect was less than in wild-type mice owing to the requirement for IL-12 or IL-23 for the optimal expansion of IFN-gamma-secreting T cells. Interestingly, bacillus Calmette-Guérin immune T cells generated in the absence of IL-12 or IL-23 were deficient in IFN-gamma production, but exhibited a robust IL-17 secretion associated with a degree of protection against pulmonary infection. Therefore, exogenous IL-23 can complement IL-12 deficiency for the initial expansion of Ag-specific T cells and is not essential for the development of potentially protective IL-17-secreting T cells.     

Macaques co-immunized with SIVgag/pol-HIVenv and IL-12 plasmid have increased cellular responses

BACKGROUND: The cell mediated immune profiles following immunization with a recombinant DNA vaccine was assessed in the simian-human immunodeficiency virus (SHIV) and Macaque model. Earlier work demonstrated increased numbers of antigen specific CD8 and CD4 effector cells able to secrete IFN-gamma. METHOD: The vaccine strategy included co-immunization of a DNA based vaccine alone or in combination with a macaque IL-12 expressing plasmid (pmacIL12). Antigen activated lymphocytes were studied for activation of a set of immunological molecules. RESULTS: The current study demonstrates lymphocytes isolated and activated from the group that was immunized with DNA and pmacIL12 had a higher level of IFN-gamma producing cells. We also observed a different immunological profile when comparing the cells isolated from macaques immunized with DNA as compared to those animals that also received pmacIL12. CONCLUSION: The observed immune profiles are reflective of the co-delivery of pmacIL12 and demonstrates that IL-12 can increase the magnitude and polyfunctionality of the cellular immune response.     

A multivalent combination of experimental antituberculosis DNA vaccines based on Ag85B and regions of difference antigens

Two 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 Th1 cytokines IFN-gamma and IL-12 (p40), and predominant cytotoxic T cell activity. High levels of antigen-specific IgG1 and IgG2a antibodies observed in the sera of immunized mice depicted strong humoral responses generated by DNA vaccine constructs. The multivalent combination of three 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 colony-forming unit 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.     

Electroporation enhances immunogenicity of a DNA vaccine expressing woodchuck hepatitis virus surface antigen in woodchucks

The development of therapeutic vaccines for chronic hepatitis B virus (HBV) infection has been hampered by host immune tolerance and the generally low magnitude and inconsistent immune responses to conventional vaccines and proposed new delivery methods. Electroporation (EP) for plasmid DNA (pDNA) vaccine delivery has demonstrated the enhanced immunogenicity of HBV antigens in various animal models. In the present study, the efficiency of the EP-based delivery of pDNA expressing various reporter genes first was evaluated in normal woodchucks, and then the immunogenicity of an analog woodchuck hepatitis virus (WHV) surface antigen (WHsAg) pDNA vaccine was studied in this model. The expression of reporter genes was greatly increased when the cellular uptake of pDNA was facilitated by EP. The EP of WHsAg-pDNA resulted in enhanced, dose-dependent antibody and T-cell responses to WHsAg compared to those of the conventional hypodermic needle injection of WHsAg-pDNA. Although subunit WHsAg protein vaccine elicited higher antibody titers than the DNA vaccine delivered with EP, T-cell response rates were comparable. However, in WHsAg-stimulated mononuclear cell cultures, the mRNA expression of CD4 and CD8 leukocyte surface markers and Th1 cytokines was more frequent and was skewed following DNA vaccination compared to that of protein immunization. Thus, the EP-based vaccination of normal woodchucks with pDNA-WHsAg induced a skew in the Th1/Th2 balance toward Th1 immune responses, which may be considered more appropriate for approaches involving therapeutic vaccines to treat chronic HBV infection.