Helen C. Perry

Comparative Immunogenicity in Rhesus Monkeys of DNA Plasmid, Recombinant Vaccinia Virus, and Replication-Defective Adenovirus Vectors Expressing a Human Immunodeficiency Virus Type 1 gag Gene

ABSTRACT Cellular immune responses, particularly those associated with CD3+ CD8+ cytotoxic T lymphocytes (CTL), play a primary role in controlling viral infection, including persistent infection with human immunodeficiency virus type 1 (HIV-1). Accordingly, recent HIV-1 vaccine research efforts have focused on establishing the optimal means of eliciting such antiviral CTL immune responses. We evaluated several DNA vaccine formulations, a modified vaccinia virus Ankara vector, and a replication-defective adenovirus serotype 5 (Ad5) vector, each expressing the same codon-optimized HIV-1 gag gene for immunogenicity in rhesus monkeys. The DNA vaccines were formulated with and without one of two chemical adjuvants (aluminum phosphate and CRL1005). The Ad5-gag vector was the most effective in eliciting anti-Gag CTL. The vaccine produced both CD4+ and CD8+ T-cell responses, with the latter consistently being the dominant component. To determine the effect of existing antiadenovirus immunity on Ad5-gag-induced immune responses, monkeys were exposed to adenovirus subtype 5 that did not encode antigen prior to immunization with Ad5-gag. The resulting anti-Gag T-cell responses were attenuated but not abolished. Regimens that involved priming with different DNA vaccine formulations followed by boosting with the adenovirus vector were also compared. Of the formulations tested, the DNA-CRL1005 vaccine primed T-cell responses most effectively and provided the best overall immune responses after boosting with Ad5-gag. These results are suggestive of an immunization strategy for humans that are centered on use of the adenovirus vector and in which existing adenovirus immunity may be overcome by combined immunization with adjuvanted DNA and adenovirus vector boosting.

Attenuation of Simian Immunodeficiency Virus SIVmac239 Infection by Prophylactic Immunization with DNA and Recombinant Adenoviral Vaccine Vectors Expressing Gag

ABSTRACT The prophylactic efficacy of DNA and replication-incompetent adenovirus serotype 5 (Ad5) vaccine vectors expressing simian immunodeficiency virus (SIV) Gag was examined in rhesus macaques using an SIVmac239 challenge. Cohorts of either Mamu-A*01(+) or Mamu-A*01(−) macaques were immunized with a DNA prime-Ad5 boost regimen; for comparison, a third cohort consisting of Mamu-A*01(+) monkeys was immunized using the Ad5 vector alone for both prime and boost. All animals, along with unvaccinated control cohorts of Mamu-A*01(+) and Mamu-A*01(−) macaques, were challenged intrarectally with SIVmac239. Viral loads were measured in both peripheral and lymphoid compartments. Only the DNA prime-Ad5-boosted Mamu-A*01(+) cohort exhibited a notable reduction in peak plasma viral load (sevenfold) as well as in early set-point viral burdens in both plasma and lymphoid tissues (10-fold) relative to those observed in the control monkeys sharing the same Mamu-A*01 allele. The degree of control in each animal correlated with the levels of Gag-specific immunity before virus challenge. However, virus control was short-lived, and indications of viral escape were evident as early as 6 months postinfection. The implications of these results in vaccine design and clinical testing are discussed.

Potent CD4+ T Cell Responses Elicited by a Bicistronic HIV-1 DNA Vaccine Expressing gp120 and GM-CSF

Virus-specific CD4+ T cell responses have been shown to play a critical role in controlling HIV-1 replication. Candidate HIV-1 vaccines should therefore elicit potent CD4+ as well as CD8+ T cell responses. In this report we investigate the ability of plasmid GM-CSF to augment CD4+ T cell responses elicited by an HIV-1 gp120 DNA vaccine in mice. Coadministration of a plasmid expressing GM-CSF with the gp120 DNA vaccine led to only a marginal increase in gp120-specific splenocyte CD4+ T cell responses. However, immunization with a bicistronic plasmid that coexpressed gp120 and GM-CSF under control of a single promoter led to a dramatic augmentation of vaccine-elicited CD4+ T cell responses, as measured by both cellular proliferation and ELISPOT assays. This augmentation of CD4+ T cell responses was selective, since vaccine-elicited Ab and CD8+ T cell responses were not significantly changed by the addition of GM-CSF. A 100-fold lower dose of the gp120/GM-CSF bicistronic DNA vaccine was required to elicit detectable gp120-specific splenocyte proliferative responses compared with the monocistronic gp120 DNA vaccine. Consistent with these findings, i.m. injection of the gp120/GM-CSF bicistronic DNA vaccine evoked a more extensive cellular infiltrate at the site of inoculation than the monocistronic gp120 DNA vaccine. These results demonstrate that bicistronic DNA vaccines containing GM-CSF elicit remarkably potent CD4+ T cell responses and suggest that optimal Th cell priming requires the precise temporal and spatial codelivery of Ag and GM-CSF.

Quantitative Adenovirus Neutralization Assays Based on the Secreted Alkaline Phosphatase Reporter Gene: Application in Epidemiologic Studies and in the Design of Adenovector Vaccines

Replication-defective recombinant adenoviruses (rAd) are used as vectors for vaccines as well as for gene therapy. To determine type-specific antibodies to adenovirus (Ad) serotypes 2, 5, 24, 34, and 35, we developed quantitative neutralization assays using recombinant adenoviruses with the secreted alkaline phosphatase (SEAP) reporter gene. Among the standardized parameters, the concentration of infectious and noninfectious adenoviral particles used in the assay is critical for a reliable comparison of data from different studies. The usefulness of this assay was demonstrated in a pilot epidemiologic study of 40 healthy individuals. In this study, the highest prevalence of antiadenovirus antibodies was found for the Ad2 serotype (82.5%), followed by Ad5 (35%). The prevalence of antiadenovirus antibodies for the serotypes 24, 34, and 35 was low (7.5%, 2.5%, and 0%, respectively). In addition, epidemiologic parameters such as gender and age were statistically evaluated. A positive association was found between age and the presence of anti-Ad5 antibodies. The assay was also useful for evaluating the presence of antiadenovirus antibodies in the design of vaccines using a rhesus monkey model. In this animal model, it was possible to determine differential dose and time responses, and the specificity for the detection of neutralizing antibodies was assessed. The evaluation of serotype-specific neutralizing antibodies can be of both clinical and epidemiologic importance as a means of selecting the appropriate serotype adenovector(s).

Cytotoxic T Lymphocyte and Helper T Cell Responses following HIV Polynucleotide Vaccination

Expression vectors encoding either HIV-1 gp160/rev, gp120, or rev alone were used for direct vaccination of mice and nonhuman primates. Each vaccine elicited long-lived (> 7 months) helper T cell responses in mice and monkeys as measured by in vitro proliferation of splenocytes following recombinant antigen treatment. Cytokine assays of the cell supernatants showed that approximately 100-fold more gamma-interferon than IL-4 was secreted during culture indicating that these vaccines elicited TH1-like responses. CD8+ CTL activities were also observed both in mice and rhesus. The gp120 and gp160/rev vaccines elicited antigen-specific antibodies, although these responses were more variable and lower magnitude for gp160/rev, and gp120 DNA-vaccinated African green monkeys had moderate levels of neutralizing antibodies. No antibodies were found against rev (an intracellular protein) with either rev vaccine. Similar antibody titers were obtained for gp120 by either intramuscular or intradermal injection although T cell responses were generally lower by intradermal route. These results indicate that DNA vaccines may provide a powerful means to elicit cellular and humoral immune responses against HIV.

Anti-HIV env immunities elicited by nucleic acid vaccines

Plasmid DNA vaccines encoding HIV-1 env were used to immunize mice and nonhuman primates. Plasmids were prepared that produced either secreted gp120 or full-length gp160. Mice immunized with gp120 DNA developed strong antigen-specific antibody responses, CD8+ cytotoxic T lymphocytes (CTL) (following in vitro restimulation with gp120-derived peptide), and showed in vitro proliferation and Th1-like cytokine secretion [gamma-interferon, interleukin (IL)-2 with little or no IL-4] by lymphocytes obtained from all lymphatic compartments tested (spleen, blood, and inguinal, iliac, and mesenteric lymph nodes). This indicated that systemic anti-gp120 cell-mediated immunity was induced by this DNA vaccine. Although similar antibody responses were observed in mice immunized by either intramuscular or intradermal routes, T cell responses were significantly stronger in mice injected intramuscularly. Rhesus monkeys immunized with both gp120 and gp160 DNAs exhibited significant CD8+ CTL responses, following in vitro restimulation of peripheral blood lymphocytes with antigen. These experiments demonstrate that DNA immunization elicits potent immune responses against HIV env in both a rodent and a nonhuman primate species.

Vaccine-Induced Immune Responses in Rodents and Nonhuman Primates by Use of a Humanized Human Immunodeficiency Virus Type 1 pol Gene

ABSTRACT A synthetic gene consisting of the reverse transcriptase (RT) and integrase (IN) domains of human immunodeficiency virus type 1 (HIV-1) pol was constructed using codons most frequently used in humans. The humanized pol gave dramatically improved levels of Rev-independent, in vitro protein production in mammalian cells and elicited much stronger cellular immunity in rodents than did virus-derived gene. Specifically, BALB/c mice were immunized with plasmids and/or recombinant vaccinia virus constructs expressing the synthetic gene. High frequencies of Pol-specific T lymphocytes were detected in these animals by the gamma interferon enzyme-linked immunospot assay against pools of short overlapping peptides. Characterization of the stimulatory peptides from these pools indicates that the optimized gene constructs are able to effectively activate both CD4+ and CD8+ T cells. Immunization of rhesus macaques with DNA vaccines expressing the humanized pol coupled to a human tissue plasminogen activator leader sequence led to pronounced in vitro cytotoxic T-lymphocyte killing activities and enhanced levels of circulating Pol-specific T cells, comparable to those observed in HIV-1-infected human subjects. Thus, optimizing the immunogenic properties of HIV-1 Pol at the level of the gene sequence validates it as an antigen and provides an important step toward the construction of a potent pol-based HIV-1 vaccine component.