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Melanoma is the most deadly type of skin cancer, constituting annually ∼ 75% of all cutaneous cancer-related deaths due to metastatic spread. Currently, because of metastatic spread, there are no effective treatment options for late-stage metastatic melanoma patients. Studies over the past two decades have provided insight into several complex molecular mechanisms as to how these malignancies evade immunological control, indicating the importance of immune escape or suppression for tumor survival. Thus, it is essential to develop innovative cancer strategies and address immune obstacles with the goal of generating more effective immunotherapies. One important area of study is to further elucidate the role and significance of myeloid-derived suppressor cells (MDSCs) in the maintenance of the tumor microenvironment. These cells possess a remarkable ability to suppress immune responses and, as such, facilitate tumor growth. Thus, MDSCs represent an important new target for preventing tumor progression and escape from immune control. In this study, we investigated the role of MDSCs in immune suppression of T cells in an antigen-specific B16 melanoma murine system utilizing a novel synthetic tyrosinase (Tyr) DNA vaccine therapy in both prophylactic and therapeutic models. This Tyr vaccine induced a robust and broad immune response, including directing CD8 T-cell infiltration into tumor sites. The vaccine also reduced the number of MDSCs in the tumor microenvironment through the downregulation of monocyte chemoattractant protein 1, interleukin-10, CXCL5 and arginase II, factors important for MDSC expansion. This novel synthetic DNA vaccine significantly reduced the melanoma tumor burden and increased survival in vivo, due likely, in part, to the facilitation of a change in the tumor microenvironment through MDSC suppression.

HIV-1 viral protein R (Vpr) is a virion-associated gene product that profoundly affects T cell proliferation, induces apoptosis and can affect cytokine production in part through interfering with NF-kappa B-mediated transcription from host cells. Collectively, these effects support that Vpr could influence immune activation in vivo. However, this effect of Vpr has not been explored previously. Here we examined the effect of Vpr expression in an in vivo model system on the induction of antigen-specific immune responses using a DNA vaccine model. Vpr co-vaccination significantly altered the immune response to co-delivered antigen. Specifically, in the presence of Vpr, inflammation was markedly reduced compared to antigen alone. Vpr reduced antigen-specific CD8-mediated cytotoxic T lymphocyte activity and suppressed T(h)1 immune responses in vivo as evidenced by lower levels of IFN-gamma. In the presence of Vpr, there is a profound shift in isotype towards a T(h)2 response as determined by the IgG2a:IgG1 ratio. The data support that Vpr compromises antigen-specific immune responses and ultimately effector cell function, thus confirming a strong selective advantage to the virus at the expense of the host.

As we explore the potential improvements to the current DNA vaccine strategies, it may be desirable to investigate methods to improve the level of resulting immune responses. One strategy is the use of cytokine cDNA as molecular adjuvants for DNA-based vaccines. Codelivery of these molecular adjuvants consisting of expression plasmid encoding for cytokines with DNA vaccine constructs is an effective method to modulate the magnitude and direction (humoral or cellular) of the immune responses. We have previously reported on the immunomodulatory effects of codelivering cDNA for interleukin-2 (IL-2) and IL-4 as molecular adjuvants for DNA-based vaccines. In this report, we extend these finding and compare the immunomodulatory effects of IL-2 and IL-4 with those of cDNA for prototypical Thl-type cytokine interferon-y (IFN-gamma) and Th2-type cytokine IL-13. We observed that distinct antigen-specific immune modulation can be achieved by the coinjection of IFN-gamma or IL-13 genes with DNA immunogen cassettes. We observed that IFN-gamma is a strong driver of Thl immune responses. Furthermore, in contrast to previous reports on their similarities in biologic activities, IL-13 and IL-4 cDNA coimmunizations modulated vaccine-induced immune responses differently in this model. Overall, these results further support the potential utility of this strategy as an important tool for the development of vaccines and immune therapies.

DNA or nucleic acid immunization has been shown to induce both antigen-specific cellular and humoral immune responses in vivo. Moreover, immune responses induced by DNA immunization can be enhanced and modulated by the use of molecular adjuvants. To engineer the immune response in vivo towards more T-helper (Th)1-type cellular responses, we investigated the co-delivery of inteferon (IFN)-gamma, interleukin (IL)-12, and IL-18 genes along with DNA vaccine constructs. We observed that both antigen-specific humoral and cellular immune responses can be modulated through the use of cytokine adjuvants in mice. Most of this work has been performed in rodent models. There has been little confirmation of this technology in primates. We also evaluated the immunomodulatory effects of this approach in rhesus macaques, since non-human primates represent the most relevant animal models for human immunodeficiency virus (HIV) vaccine studies. As in the murine studies, we also observed that each Th1 cytokine adjuvant distinctively regulated the level of immune responses generated. Co-immunization of IFN-gamma and IL-18 in macaques enhanced the level of antigen-specific antibody responses. Similarly, co-delivery of IL-12 and IL-18 also enhanced the level of antigen-specific Th proliferative responses. These results extend this adjuvant strategy in a more relevant primate model and support the potential utility of these molecular adjuvants in DNA vaccine regimens.

Activation of T cells requires both TCR-specific ligation by direct contact with peptide Ag-MHC complexes and coligation of the B7 family of ligands through CD28/CTLA-4 on the T cell surface. We recently reported that coadministration of CD86 cDNA along with DNA encoding HIV-1 Ags i.m. dramatically increased Ag-specific CTL responses. We investigated whether the bone marrow-derived professional APCs or muscle cells were responsible for the enhancement of CTL responses following CD86 coadministration. Accordingly, we analyzed CTL induction in bone marrow chimeras. These chimeras are capable of generating functional viral-specific CTLs against vaccinia virus and therefore represent a useful model system to study APC/T cell function in vivo. In vaccinated chimeras, we observed that only CD86 + Ag + MHC class I results in 1) detectable CTLs following in vitro restimulation, 2) detectable direct CTLs, 3) enhanced IFN-γ production in an Ag-specific manner, and 4) dramatic tissue invasion of T cells. These results support that CD86 plays a central role in CTL induction in vivo, enabling non-bone marrow-derived cells to prime CTLs, a property previously associated solely with bone marrow-derived APCs.

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-γ) and chemokines (RANTES and macrophage inflammatory protein-1α) 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.

Several vaccines have been investigated experimentally in the herpes simplex virus type 2 (HSV-2) model system. While it is believed that CD4 + -T-cell responses are important for protection in general, the correlates of protection from HSV-2 infection are still under investigation. Recently, the use of molecular adjuvants to drive vaccine responses induced by DNA vaccines has been reported in a number of experimental systems. We sought to take advantage of this immunization model to gain insight into the correlates of immune protection in the HSV-2 mouse model system and to further explore DNA vaccine technology. To investigate whether the Th1- or Th2-type immune responses are more important for protection from HSV-2 infection, we codelivered the DNA expression construct encoding the HSV-2 gD protein with the gene plasmids encoding the Th1-type (interleukin-2 [IL-2], IL-12, IL-15, and IL-18) and Th2-type (IL-4 and IL-10) cytokines in an effort to drive immunity induced by vaccination. We then analyzed the modulatory effects of the vaccine on the resulting immune phenotype and on the mortality and the morbidity of the immunized animals following a lethal challenge with HSV-2. We observed that Th1 cytokine gene coadministration not only enhanced the survival rate but also reduced the frequency and severity of herpetic lesions following intravaginal HSV challenge. On the other hand, coinjection with Th2 cytokine genes increased the rate of mortality and morbidity of the challenged mice. Moreover, of the Th1-type cytokine genes tested, IL-12 was a particularly potent adjuvant for the gD DNA vaccination.

DNA or nucleic acid immunization has been shown to induce both antigen-specific cellular and humoral immune responses in vivo. Moreover, immune responses induced by DNA immunization can be enhanced and modulated by the use of molecular adjuvants. To further engineer the immune response in vivo, we investigated the induction and regulation of immune responses from the codelivery of Thl cytokines (interleukin-2 [IL-2] and IL-12), Th2 cytokines (IL-4 and IL-10), and granulocyte-macrophage colony-stimulating factor (GM-CSF) genes along with a DNA vaccine construct encoding for simian immunodeficiency virus (SIV) gag/pol proteins. We observed that coinjection with IL-2, IL-4, IL-10, and GM-CSF resulted in increased levels of antigen-specific antibodies. In addition, we found that coinjection with cytokine genes drove the immune responses toward a more Thl or Th2 phenotype. We also observed that coadministration of IL-2, IL-12, and GM-CSF genes resulted in a dramatic enhancement of Th proliferation responses. Moreover, coimmunization with IL-12 genes resulted in a dramatic enhancement of antigen-specific cytotoxic T lymphocyte (CTL) responses. These results support the potential utility of molecular adjuvants in DNA vaccine regimens.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) could in theory attract antigen-presenting cells in muscle following intramuscular DNA immunization, resulting in enhanced antigen-specific immune responses. Thus, such adjuvants could constitute an important addition to a herpes vaccine by amplifying specific immune responses. Here we investigate the utility of GM-CSF cDNA as a vaccine adjuvant for herpes simplex virus (HSV)-2 in a mouse challenge model. GM-CSF cDNA co-injection enhanced levels of specific IgG, IgE and IgA against HSV-2 gD protein significantly higher than gD plasmid vaccination alone. Moreover, GM-CSF co-injection induced a dramatic increase in IgG1 levels, as compared to IgG2a levels, suggesting a Th2 bias in the response. T helper cell proliferation and secretion of cytokines (IL-2 and IFN-gamma) were significantly increased by GM-CSF cDNA co-injection. When challenged with a lethal dose of HSV-2, GM-CSF co-injection increased survival rates to 90%, an improvement as compared to gD vaccination alone (60-63%). Furthermore, GM-CSF cDNA co-injection reduced herpetic lesions and resulted in a faster recovery from lesions. These data indicate that GM-CSF cDNA enhances both humoral and cellular immune responses and enhances vaccine efficacy, resulting in reduced HSV-2-derived morbidity as well as mortality.

The potential roles of CD8(+) T-cell-induced chemokines in the expansion of immune responses were examined using DNA immunogen constructs as model antigens. We coimmunized cDNA expression cassettes encoding the alpha-chemokines IL-8 and SDF-1alpha and the beta-chemokines MIP-1alpha, RANTES, and MCP-1 along with DNA immunogens and analyzed the resulting antigen-specific immune responses. In a manner more similar to the traditional immune modulatory role of CD4(+) T cells via the expression of Th1 or Th2 cytokines, CD8(+) T cells appeared to play an important role in immune expansion and effector function by producing chemokines. For instance, IL-8 was a strong inducer of CD4(+) T cells, indicated by strong T helper proliferative responses as well as an enhancement of antibody responses. MIP-1alpha had a dramatic effect on antibody responses and modulated the shift of immune responses to a Th2-type response. RANTES coimmunization enhanced the levels of antigen-specific Th1 and cytotoxic T lymphocyte (CTL) responses. Among the chemokines examined, MCP-1 was the most potent activator of CD8(+) CTL activity. The enhanced CTL results are supported by the increased expression of Th1 cytokines IFN-gamma and TNF-alpha and the reduction of IgG1/IgG2a ratio. Our results support that CD8(+) T cells may expand both humoral and cellular responses in vivo through the elaboration of specific chemokines at the peripheral site of infection during the effector stage of the immune response.

Cytokines are important regulators of the immune response. They influence immune expression, the development of immunologic memory, and regulation of antigen-specific and nonspecific immune activation as well as allergic responses. In a model system in mice, we have studied the effect of plasmids expressing interleukin (IL)-10 or IL-12 on the modulation of antigen-specific responses. Coadministration of IL-12 or IL-10 genes with DNA immunogens directed the antigen-specific immune response toward a T helper (Thl)-type immunity. In addition to the modulation of antigen-specific immune responses, we studied the induction of delayedtype hypersensitivity (DTH) to contact allergens as an in vivo model of the Thl response. We found that IL12 and IL-10 gene-containing plasmids, and not the bacterial plasmid alone, upregulate this response. Our cytokine gene delivery technique demonstrates an important level of control of the magnitude and direction of induced immune responses and could be advantageous in...

Immunization with nucleic acids has been shown to induce both antigen-specific cellular and humoral immune responses in vivo. We hypothesize that immunization with DNA could be enhanced by directing specific immune responses induced by the vaccine based on the differential correlates of protection known for a particular pathogen. Recently we and others reported that specific immune responses generated by DNA vaccine could be modulated by co-delivery of gene expression cassettes encoding for IL-12, granulocyte-macrophage colony-stimulating factor and the co-stimulatory molecule CD86. To further engineer the immune response in vivo, we investigated the induction and regulation of immune responses following the co-delivery of pro-inflammatory cytokine (IL-1 alpha, TNF-alpha, and TNF-beta), Th1 cytokine (IL-2, IL-12, IL-15, and IL-18), and Th2 cytokine (IL-4, IL-5 and IL-10) genes. We observed enhancement of antigen-specific humoral response with the co-delivery of Th2 cytokine genes IL-4, IL-5, and IL-10 as well as those of IL-2 and IL-18. A dramatic increase in antigen-specific T helper cell proliferation was seen with IL-2 and TNF-alpha gene co-injections. In addition, we observed a significant enhancement of the cytotoxic response with the co-administration of TNF-alpha and IL-15 genes with HIV-1 DNA immunogens. These increases in CTL response were both MHC class I restricted and CD8+ T cell dependent. Together with earlier reports on the utility of co-immunizing using immunologically important molecules together with DNA immunogens, we demonstrate the potential of this strategy as an important tool for the development of more rationally designed vaccines.

We have previously reported on the generation of specific functional immune responses after inoculation of animals with expression vectors encoding HIV-1 genes. This article provides the details of the first application of this new technology to induce immune responses against HIV-2. This virus is molecularly and serologically distinct from HIV-1 and is in fact more closely related to the simian immunodeficiency virus (SIV). Anti-HIV-2 and SIV antibodies were induced in mice of three different haplotypes following a single intramuscular inoculation with an HIV-2/ROD envelope glycoprotein expression vector (pcEnv-2). Boosting of animals with pcEnv-2 induced both anti-HIV-2 neutralizing antibodies and T cell-proliferative responses against HIV-2 and SIVmac proteins. We compared the humoral and cellular immune responses of mice injected with pcEnv-2 and then boosted with either the homologous DNA construct or a recombinant Env protein. Animals boosted with pcEnv-2 generated B and T cell immune responses as strong as those of mice boosted with recombinant gp140 protein in adjuvant. Finally, cellular immune responses were significantly increased with the coadministration of pcEnv-2 and a plasmid expressing interleukin 12. We therefore conclude that DNA plasmid inoculation induces cross-reactive anti-HIV-2 and anti-SIVmac immune responses in mice. This technology should be further investigated as a potential vaccine component for this human pathogen.

The human immunodeficiency virus-1 (HIV-1) is a retrovirus which preferentially infects and kills CD4+ T cells and macrophages, ultimately resulting in immune system failure and multi-pathogen infections. Recent breakthroughs in combination therapy using three different anti-retroviral agents have generated optimism regarding the ability to control viral replication in vivo [46]. However, this therapeutic regimen is costly, and it is too early to tell whether this approach can eradicate established infection [26, 46]. The costs and the stringent administration regimen requirements of these pharmaceutical agents make it clear that these drugs will only be effectively utilized in a limited part of the world population. Therefore, to address the worldwide problem of HIV-1 infection, there remains a need for a prophylactic vaccination strategy designed to control the epidemic through mass immunization campaigns [115].

Nucleic acid or DNA immunization represents a novel approach to both vaccine and immune therapeutic development. DNA vaccination induces antigen-specific cellular and humoral immune responses through the delivery of non-replicating transcription units which drive the synthesis of specific foreign proteins within the inoculated host. We have previously reported on the potential use of DNA immunization as a novel vaccine strategy for HIV-1. We found that both antigen-specific cellular and humoral immune responses could be induced in vivo with various DNA vaccine constructs against different antigenic targets within HIV-1. In order to enhance the DNA vaccine's ability to elicit cell-mediated immune responses, we co-delivered plasmids encoding costimulatory molecule B7 and interleukin-12 genes with DNA vaccine for HIV-1. We observed a dramatic increase in both antigen-specific T helper cell proliferation and CTL response. Eventual development of successful vaccines for HIV-1 would likely involve targeting multiple antigenic components of the virus to direct and empower the immune system to protect the host from viral infection. We present here the utility of multicomponent DNA immunization to elicit specific humoral and cell-mediated immune responses against different antigenic targets of HIV-1 as well as the ability of this immunization strategy to achieve significant enhancements of antigen-specific cellular immune responses.

Cancers employ various strategies to escape immune surveillance including the exploitation of immune checkpoint inhibitors. Checkpoint inhibitors are receptors found on immune and stromal cells whose function can impact the duration or potency of an immune response. Tumor cells often upregulate ligands for these receptors to protect themselves from the host immune response. Monoclonal antibody (MAb) therapeutics which block checkpoint inhibitor-ligand interactions restore T cell destruction of cancer cells in vivo. MAbs that target the inhibitory T cell signaling mediated by CTLA-4 and/or PD-1 checkpoint inhibitors have recently gained regulatory approval for the treatment of some cancers based on remarkable clinical outcomes.Here we have focused on a new method to improve MAb delivery through direct engineering of MAb in the form of synthetic DNA plasmids. This technology would improve many aspects of such a therapy by lowering cost, increasing in vivo expression times and allowing for simple combination formulations in the absence of a host anti-vector immune response, possibly extending use of these groundbreaking therapies to disadvantaged patient populations. We report that “enhanced and optimized” DNA plasmid technology can be used to direct in vivo production of immunoglobulin heavy and light chains of established monoclonal antibodies which can target the immune checkpoint inhibitors LAG3 and PD-1 as determined in Flow cytometry, ELISA and Western blot assays. Both antibodies are produced at physiologically relevant levels in blood and other tissues of mice using electroporation-enhanced delivery of DNA plasmids encoding genes for each antibody. We report that serum antibodies from inoculated animals retain the ability to bind to their targets and are bioactive in vivo and exhibit immune stimulatory effects for host T cells. These studies have significant implications for prophylactic and therapeutic strategies for cancer and other important diseases and warrants further attention.

Viral protein R (Vpr) is a 14kD, 96 amino acid accessory protein of the HIV virion that has been demonstrated to have important functions in the viral replication cycle including, among others, the induction of cell cycle arrest and apoptosis in rapidly proliferating cells, which results in immune dysfunction in infected individuals. Several investigators have studied the potential use of the apoptosis inducing and cell cycle arrest effect of Vpr as an anti-tumor therapeutic. In vitro studies have indicated that Vpr is cytotoxic against a large number of different tumor cell types including a number that are p53 independent. Likewise, some in vivo tumor studies using different delivery platforms/methods have indicated an anti-cancer effect mediated by Vpr. Our group has used the aggressive and poorly immunogenic murine melanoma tumor line B16.F10 as a model to deliver, through in vivo electroporation, Vpr expressing plasmids to established tumors and have demonstrated that this treatment regimen can induce growth attenuation and tumor regression in a proportion of the treated mice and appears to be associated with the induction of intratumoral apoptosis. Overall, to date, the data from a number of research groups, including our own, have indicated that Vpr has biological activity against a number of tumors in both in vivo and in vitro models and, as such, may be a potential candidate for testing in human clinical trials. In this report, we summarize the evidence supporting this hypothesis.

It is widely believed that a Th1 type CD4 response is critical for enhancement of CD8 immunity and for controlling HIV-1 infection. Th2 type responses, such as what might be seen in a chronic parasitic infection, would sacrifice cellular immunity and thus benefit the virus at the expense of the host. However, there has been little direct examination of the hypothesis in a primate model system. Accordingly, the simian immunodeficiency virus (SIV) infected rhesus macaque model was used to investigate the impact of immunisation with SIV expressing DNA constructs and co-injection with IL-4 on the SIV specific immunological responses, lymphocyte cell counts, as well as the impact on viral load. IL-4 is a Th2 type cytokine, which enhances antibody production and inhibits a CD4 Th1 phenotype. Rhesus macaques were infected with 10 AID50 of SIVmac239 and treated with 9-[2-(phosphonomethoxy)propyl]adenine (PMPA) 9 weeks post-infection. During PMPA treatment, animals were immunised with plasmids that expressed the SIV proteins, env, rev, gag and pol. In addition, they were immunised with a construct that encoded the gene for IL-4. IL-4 co-immunisation increased the neutralizing antibody titres in this group. Importantly, the viral loads in animals vaccinated with IL-4 expressing plasmid increased during the immunisation regimens despite the higher neutralizing antibody titres. In addition, neutralizing antibodies did not correlate with viral set point prior to PMPA treatment, however, there was a correlation between viral loads and antibody titres following the treatment with PMPA. Antibody titres decreased following the suppression of viral load. Importantly, vaccination in the absence of IL-4 protected CD4 levels without increasing viral load. The data support the hypothesis that inappropriate immune bias toward a Th2 pathway would ultimately enhance disease progression.

Engineering gene therapy vectors to modulate the immune response is an important goal. In this regard, costimulation of T cells is a critical determinant in immune activation. The costimulatory molecule CD40, expressed on antigen-presenting cells, is thought to interact with CD40 ligand (CD40L) expressed on activated CD4(+) or CD8(+) T cells to further drive interleukin-2 receptor (IL-2R) expression and antigen-specific T cell expansion necessary for both class II and class I responses. To compare the specific roles of these two costimulatory molecules in immune induction in a herpes simplex virus (HSV) model, we constructed plasmid DNAs expressing CD40 and CD40L, coimmunized these molecules with a gD plasmid vaccine, and then analyzed immune modulatory effects as well as protection against lethal HSV-2 challenge. We observed that gD-specific IgG production was unaffected by these molecules. However, a higher production of IgG2a isotype was induced by CD40L coinjection, suggesting that CD40L drives immune responses towards a helper T cell type 1 (Th1) phenotype. CD40L also enhanced Th cell proliferative responses and production of Th1-type cytokines (IL-2 and IFN-gamma) and beta-chemokines (RANTES and MIP-1alpha) from splenocytes. In contrast, CD40 showed slightly increasing effects on T cell proliferation responses and cytokine and chemokine production. When animals were challenged with a lethal dose of HSV-2, CD40L-coimmunized animals exhibited a significantly enhanced survival rate, as compared with CD40 coinjection or gD DNA vaccine alone. This enhanced protection appears to be mediated by Th1-type CD4(+) T cells, as determined by in vitro and in vivo T cell subset deletion. CD40L also promoted migration of CD4(+) T cells into the muscle sites. These studies demonstrate that CD40L can play an important role in protective antigen-specific immunity in a gene-based model system through increased expansion of the CD4(+) Th1 T cell subset in vivo.

Chemokines are inflammatory molecules that act primarily as chemoattractants and as activators of leukocytes. Their role in antigen-specific immune responses is of importance, but their role in disease protection is unknown. Recently it has been suggested that chemokines modulate immunity along more classical Th1 and Th2 phenotypes. However, no data currently exist in an infectious challenge model system. We analyzed the modulatory effects of selected chemokines (interleukin-8 [IL-8], gamma interferon-inducible protein 10 [IP-10], RANTES, monocyte chemotactic protein 1 [MCP-1], and macrophage inflammatory protein 1α [MIP-1α]) on immune phenotype and protection against lethal challenge with herpes simplex virus type 2 (HSV-2). We observed that coinjection with IL-8 and RANTES plasmid DNAs dramatically enhanced antigen-specific Th1 type cellular immune responses and protection from lethal HSV-2 challenge. This enhanced protection appears to be mediated by CD4+T cells, as determined by in vitro and in vivo T-cell subset deletion. Thus, IL-8 and RANTES cDNAs used as DNA vaccine adjuvants drive antigen-specific Th1 type CD4+T-cell responses, which result in reduced HSV-2-derived morbidity, as well as reduced mortality. However, coinjection with DNAs expressing MCP-1, IP-10, and MIP-1α increased mortality in the challenged mice. Chemokine DNA coinjection also modulated its own production as well as the production of cytokines. These studies demonstrate that chemokines can dominate and drive immune responses with defined phenotypes, playing an important role in the generation of protective antigen-specific immunity.

Nucleic acid immunization has been shown to induce both antigen-specific cellular and humoral immune responses in vivo. Moreover, immune responses induced by DNA immunization can be enhanced by the use of molecular adjuvants. For example, coadministration of costimulatory molecules (CD80 and CD86), proinflammatory cytokines (interleukin-1alpha [IL-1alpha], tumor necrosis factor-alpha [TNF-alpha, and TNF-beta), Th1 cytokines (interleukin-2 [IL-2], IL-12, IL-15, and IL-18), Th2 cytokines (IL-4, IL-5, and IL-10), and granulocytes-macrophage colony-stimulating factor (GM-CSF) with DNA vaccine constructs leads to modulation of the magnitude and direction (humoral or cellular) of the immune responses. To further engineer the immune response in vivo, we compared the induction and regulation of immune responses from the codelivery of chemokine (IL-8, interferon-gamma-inducible protein-10 [gammaIP-10], macrophage inhibitory protein-1alpha [MIP-1alpha], and RANTES) genes with codelivery of cytokine genes. We found that as in cytokine gene codelivery, coimmunization with chemokine genes along with DNA immunogen constructs can modulate the direction and magnitude of induced immune responses. We observed that coimmunization with IL-8, gammaIP-10, and MIP-1alpha genes increased the antibody response. We also found that coinjection with IL-8, gammaIP-10, and RANTES resulted in a dramatic enhancement of T helper (Th) proliferation response. Furthermore, among all coinjection combinations, we found that RANTES coinjection caused a high level of cytotoxic lymphocyte (CTL) enhancement. This enhancement of CTL responses observed from the coinjection with RANTES was CD8+ T cell dependent. Together with earlier reports on the utility of coimmunizing immunologically important molecules with DNA immunogens, we demonstrate the potential of this strategy as an important tool for the development of more rationally designed vaccines.

An important limitation of DNA immunization in nonhuman primates is the difficulty in generating high levels of antigen-specific antibody responses; strategies to enhance the level of immune responses to DNA immunization may be important in the further development of this vaccine strategy for humans. We approached this issue by testing the ability of molecular adjuvants to enhance the levels of immune responses generated by multicomponent DNA vaccines in rhesus macaques. Rhesus macaques were coimmunized intramuscularly with expression plasmids bearing genes encoding Th1 (interleukin 2 [IL-2] and gamma interferon)- or Th2 (IL-4)-type cytokines and DNA vaccine constructs encoding human immunodeficiency virus Env and Rev and simian immunodeficiency virus Gag and Pol proteins. We observed that the cytokine gene adjuvants (especially IL-2 and IL-4) significantly enhanced antigen-specific humoral immune responses in the rhesus macaque model. These results support the assumption that antigen-specific responses can be engineered to a higher and presumably more desirable level in rhesus macaques by genetic adjuvants.

Studies have indicated that professional APCs in the periphery, such as dendritic cells and macrophages, play an important role in initiating DNA vaccine-specific immune responses. To engineer the immune response induced by DNA vaccines in vivo we investigated the modulatory effects of codelivering growth factor genes for the hematopoietic APCs along with DNA vaccines. Specifically, we examined the effects on the antigen-specific immune responses following the codelivery of the gene expression cassettes for M-CSF, G-CSF, and GM-CSF along with HIV-1 DNA immunogen constructs. We observed that coimmunization with GM-CSF increased the antibody response and resulted in a significant enhancement of lymphoproliferative response. Furthermore, among all coinjection combinations, we found that M-CSF coinjections resulted in a high level of CTL enhancement. This enhancement of CTL responses observed from the coinjection with M-CSF was CD8+ T cell dependent and was associated with the presence of CD11c+ cells at the site of injection and with the antigen-specific induction of the beta-chemokine MIP-1beta, suggesting a role for this chemokine in CTL induction. These results suggest that hematopoietic growth factors should be further studied as potential adjuvants for in vivo modulators of immune responses.

DNA immunization has been investigated as a potential immunization strategy against infectious diseases and cancer. To enhance a DNA vaccine's ability to induce CTL response in vivo, we co-administered CD80 and CD86 expression cassettes along with HIV-1 immunogens. This manipulation resulted in a dramatic increase in MHC class I-restricted and CD8+ T-cell-dependent CTL responses in both mice and chimpanzees. This strategy of engineering vaccine producing cells to be more efficient T-cell activators could be an important tool for optimizing antigen-specific T-cell-mediated immune responses in the pursuit of more rationally designed vaccines and immune therapies.