Zimra R. Israel

Effect of Plasmid DNA Vaccine Design and In Vivo Electroporation on the Resulting Vaccine-Specific Immune Responses in Rhesus Macaques

ABSTRACT Since human immunodeficiency virus (HIV)-specific cell-mediated immune (CMI) responses are critical in the early control and resolution of HIV infection and correlate with postchallenge outcomes in rhesus macaque challenge experiments, we sought to identify a plasmid DNA (pDNA) vaccine design capable of eliciting robust and balanced CMI responses to multiple HIV type 1 (HIV-1)-derived antigens for further development. Previously, a number of two-, three-, and four-vector pDNA vaccine designs were identified as capable of eliciting HIV-1 antigen-specific CMI responses in mice (M. A. Egan et al., Vaccine 24:4510-4523, 2006). We then sought to further characterize the relative immunogenicities of these two-, three-, and four-vector pDNA vaccine designs in nonhuman primates and to determine the extent to which in vivo electroporation (EP) could improve the resulting immune responses. The results indicated that a two-vector pDNA vaccine design elicited the most robust and balanced CMI response. In addition, vaccination in combination with in vivo EP led to a more rapid onset and enhanced vaccine-specific immune responses. In macaques immunized in combination with in vivo EP, we observed a 10- to 40-fold increase in HIV-specific enzyme-linked immunospot assay responses compared to those for macaques receiving a 5-fold higher dose of vaccine without in vivo EP. This increase in CMI responses translates to an apparent 50- to 200-fold increase in pDNA vaccine potency. Importantly, in vivo EP enhanced the immune response against the less immunogenic antigens, resulting in a more balanced immune response. In addition, in vivo EP resulted in an approximate 2.5-log10 increase in antibody responses. The results further indicated that in vivo EP was associated with a significant reduction in pDNA persistence and did not result in an increase in pDNA associated with high-molecular-weight DNA relative to macaques receiving the pDNA without EP. Collectively, these results have important implications for the design and development of an efficacious vaccine for the prevention of HIV-1 infection.

Protection by SIV VLP DNA prime/protein boost following mucosal SIV challenge is markedly enhanced by IL-12/GM-CSF co-administration

Abstract:  The ever increasing number of people infected by human immunodeficiency virus (HIV) throughout the world renders the development of effective vaccines an urgent priority. Herein, we report on an attempt to induce and enhance antiviral responses using a deoxyribonucleic acid (DNA) prime/virus‐like particles (VLP) protein boost strategy adjuvanted with interleukin (IL)‐12/GM‐CSF in rhesus macaques challenged with simian immunodeficiency virus (SIV). Thus, groups of monkeys were administered three consecutive doses of pVecB7 a plasmid expressing VLP with or without plasmids expressing IL‐12 and GM‐CSF at weeks 0, 13 and 26. The VLP boost was administered at week 39 with or without IL‐12. All monkeys were challenged intrarectally with SIVsmE660 2 months following the protein boost. All except one immunized monkey became infected. While all immunized monkeys showed a marked reduction of acute viral peaks, reduction of viral load set points was only achieved in groups whose prime‐boost immunizations were supplemented with IL‐12/GM‐CSF (prime) and/or with IL‐12 (boost). Control of viremia correlated with lack of disease progression and survival. Detection of virus in rectal washes at 1 year post‐challenge was only successful in monkeys whose immunizations did not include cytokine adjuvant, but these loads did not correlate with plasma viral loads. In summary, use of IL‐12 and/or GM‐CSF was shown to provide significant differences in the outcome of SIV challenge of prime/boost immunized monkeys.

Long-Lasting Decrease in Viremia in Macaques Chronically Infected with Simian Immunodeficiency Virus SIVmac251 after Therapeutic DNA Immunization

ABSTRACT Rhesus macaques chronically infected with highly pathogenic simian immunodeficiency virus (SIV) SIVmac251 were treated with antiretroviral drugs and vaccinated with combinations of DNA vectors expressing SIV antigens. Vaccination during therapy increased cellular immune responses. After the animals were released from therapy, the virus levels of 12 immunized animals were significantly lower (P = 0.001) compared to those of 11 animals treated with only antiretroviral drugs. Vaccinated animals showed a persistent increase in immune responses, thus indicating both a virological and an immunological benefit following DNA therapeutic vaccination. Several animals show a long-lasting decrease in viremia, suggesting that therapeutic vaccination may provide an additional benefit to antiretroviral therapy.

The use of cytokines and chemokines as genetic adjuvants for plasmid DNA vaccines

Abstract The direct injection of a naked plasmid DNA vaccine encoding a foreign antigen results in plasmid uptake and protein expression leading to the induction of antigen-specific cellular and humoral immune responses. The ability of DNA vaccine-elicited immune responses to protect against viral and bacterial infections, parasites, cancers, and autoimmune diseases has been well documented in numerous animal models. Phase I human clinical trials have shown that experimental DNA vaccines are safe and well tolerated, however, these preliminary studies indicate that measures must be taken to improve vaccine immunogenicity. One approach to improve the immunogenicity of DNA vaccines is through the co-delivery of cytokine expression plasmids as genetic adjuvants. Studies in a variety of animal models clearly demonstrate that plasmid DNA-encoded immunomodulatory cytokines not only alter the magnitude and direction of the DNA vaccine-elicited immune response, but can also improve vaccine efficacy. These studies suggest that the use of immunomodulatory cytokines with plasmid DNA vaccines may allow clinicians to tailor the resulting immune response to more closely resemble the correlates of protection for a given pathogen.

Combined systemic and mucosal immunization with microsphere-encapsulated inactivated simian immunodeficiency virus elicits serum, vaginal, and tracheal antibody responses in female rhesus macaques.

We determined the efficacy of immunization with microsphere-encapsulated whole inactivated simian immunodeficiency virus (SIV) by combined systemic and mucosal administration to protect female rhesus macaques against vaginal challenge with homologous rhesus PBMC-grown SIVmac251. Animals in one group were primed and boosted intramuscularly. Two groups were primed intramuscularly and boosted either intratracheally or orally. A final group was primed by vaccinia/rgp140 scarification and subdivided for either intratracheal or oral boosting. Strong ELISA titers of circulating SIV-specific IgG and modest IgA responses were elicited in the animals primed intramuscularly. Intratracheal boosting in the intramuscularly primed macaques resulted in high bronchial alveolar wash (BAW) IgG and less pronounced IgA. SIV-specific vaginal wash (VW) IgG was also present in the intramuscular/intramuscular and intramuscular/intratracheal groups. Vaccinia/rgp140 priming gave low ELISA titers to whole SIV, and failed to elicit mucosal antibody regardless of the booster route. No animal in any group developed serum neutralizing antibody to homologous SIVmac251. On vaginal challenge none of the immunized groups was infected at a lesser frequency than the unimmunized controls. These data suggest that the use of microspheres in a combined parenteral and mucosal regimen is an effective method of eliciting IgG and IgA antibody at mucosal surfaces.

Modifying the HIV-1 env gp160 gene to improve pDNA vaccine-elicited cell-mediated immune responses

Plasmid DNA (pDNA) vaccines are effective at eliciting immune responses in a wide variety of animal model systems, however, pDNA vaccines have generally been incapable of inducing robust immune responses in clinical trials. Therefore, to identify means to improve pDNA vaccine performance, we compared various post-transcriptional and post-translational genetic modifications for their ability to improve antigen-specific CMI responses. Mice vaccinated using a sub-optimal 100 mcg dose of a pDNA encoding an unmodified primary isolate HIV-1(6101) env gp160 failed to demonstrate measurable env-specific CMI responses. In contrast, significant env-specific CMI responses were seen in mice immunized with pDNA expression vectors encoding env genes modified by RNA optimization or codon optimization. Further modification of the RNA optimized env gp160 gene by the addition of (i) a simian retrovirus type 1 constitutive RNA transport element; (ii) a murine intracisternal A-particle derived RNA transport element; (iii) a tissue plasminogen activator protein signal leader sequences; (iv) a beta-catenin derived ubiquitination target sequence; or (v) a monocyte chemotactic protein-3 derived signal sequence failed to further improve the induction of env-specific CMI responses. Therefore, modification of the env gp160 gene by RNA or codon optimization alone is necessary for high-level rev-independent expression and results in robust env-specific CMI responses in immunized mice. Importantly, further modification(s) of the env gene to alter cellular localization or increase proteolytic processing failed to result in increased env-specific immune responses. These results have important implications for the design and development of an efficacious vaccine for the prevention of HIV-1 infection.

Boosting of SIV-Specific T Cell Responses in Rhesus Macaques That Resist Repeated Intravaginal Challenge with SIVmac251

Despite repeated high-risk exposure to infectious HIV-1, some individuals remain HIV-1 seronegative and apparently uninfected. The use of nonhuman primate model systems to study SIVmac transmission may help to elucidate the factors responsible for protection in exposed, seronegative (ESN) humans. In an earlier vaccination study, three control rhesus macaques that were exposed to three sequential intravaginal challenges with pathogenic SIVmac251 failed to show evidence of infection after 5 years of observation. 51Cr release assay results suggested that these animals had low-level cytotoxic T lymphocyte responses to SIVmac proteins. We hypothesized that these responses might be an important component of protection from mucosal challenge. We performed an additional intravaginal challenge of all three macaques and monitored SIV-specific T cell responses in peripheral blood, using the sensitive enzyme-linked immunospot (ELISpot) assay. After the fourth challenge, one animal became infected; this animal did not mount a strong SIV-specific T cell response. Two other macaques remained uninfected as determined by peripheral blood mononuclear cell (PBMC) coculture, polymerase chain reaction (PCR), and branched DNA (bDNA) analysis of peripheral blood and lymphoid tissues, but demonstrated boosting of SIV-specific T cell responses after challenge. These results support a protective role for SIVmac-specific T cells in repeatedly exposed, persistently seronegative rhesus macaques.

Induction of mucosal antibody responses by microsphere-encapsulated formalin-inactivated simian immunodeficiency virus in a male urethral challenge model

Male rhesus macaques were immunized mucosally with microsphere-encapsulated formalin-inactivated simian immunodeficiency virus (SIV) particles in a test of immunogenicity and protection against mucosal SIV challenge. Tracheal boosting of animals that had been primed intramuscularly resulted in strong serum ELISA titers to SIV, and evidence of local IgA responses in broncho-alveolar washes. The bulk of the antibody response was against non-envelope epitopes. No neutralizing antibody was observed, and intraurethral challenge with cell-free rhesus-grown virus showed no evidence of protection against infection. Microsphere-based immunization efficiently raises local and system responses, but the resulting immunity to SIV is apparently not sufficient to protect against mucosal challenge.