Stephanie J. Buchl

Protection against Mucosal SHIV Challenge by Peptide and Helper-Dependent Adenovirus Vaccines

Groups of rhesus macaques that had previously been immunized with HIV-1 envelope (env) peptides and first generation adenovirus serotype 5 (FG-Ad5) vaccines expressing the same peptides were immunized intramuscularly three times with helper-dependent adenovirus (HD-Ad) vaccines expressing only the HIV-1 envelope from JRFL. No gag, pol, or other SHIV genes were used for vaccination. One group of the FG-Ad5-immune animals was immunized three times with HD-Ad5 expressing env. One group was immunized by serotype-switching with HD-Ad6, HD-Ad1, and HD-Ad2 expressing env. Previous work demonstrated that serum antibody levels against env were significantly higher in the serotype-switched group than in the HD-Ad5 group. In this study, neutralizing antibody and T cell responses were compared between the groups before and after rectal challenge with CCR5-tropic SHIV-SF162P3. When serum samples were assayed for neutralizing antibodies, only weak activity was observed. T cell responses against env epitopes were higher in the serotype-switched group. When these animals were challenged rectally with SHIV-SF162P3, both the Ad5 and serotype-switch groups significantly reduced peak viral loads 2 to 10-fold 2 weeks after infection. Peak viral loads were significantly lower for the serotype-switched group as compared to the HD-Ad5-immunized group. Viral loads declined over 18 weeks after infection with some animals viremia reducing nearly 4 logs from the peak. These data demonstrate significant mucosal vaccine effects after immunization with only env antigens. These data also demonstrate HD-Ad vectors are a robust platform for vaccination.

Comparison of Systemic and Mucosal Immunization with Helper-Dependent Adenoviruses for Vaccination against Mucosal Challenge with SHIV

Most HIV-1 infections are thought to occur at mucosal surfaces during sexual contact. It has been hypothesized that vaccines delivered at mucosal surfaces may mediate better protection against HIV-1 than vaccines that are delivered systemically. To test this, rhesus macaques were vaccinated by intramuscular (i.m.) or intravaginal (ivag.) routes with helper-dependent adenoviral (HD-Ad) vectors expressing HIV-1 envelope. Macaques were first immunized intranasally with species C Ad serotype 5 (Ad5) prior to serotype-switching with species C HD-Ad6, Ad1, Ad5, and Ad2 vectors expressing env followed by rectal challenge with CCR5-tropic SHIV-SF162P3. Vaccination by the systemic route generated stronger systemic CD8 T cell responses in PBMC, but weaker mucosal responses. Conversely, mucosal immunization generated stronger CD4 T cell central memory (Tcm) responses in the colon. Intramuscular immunization generated higher levels of env-binding antibodies, but neither produced neutralizing or cytotoxic antibodies. After mucosal SHIV challenge, both groups controlled SHIV better than control animals. However, more animals in the ivag. group had lower viral set points than in in the i.m. group. These data suggest mucosal vaccination may have improve protection against sexually-transmitted HIV. These data also demonstrate that helper-dependent Ad vaccines can mediate robust vaccine responses in the face of prior immunity to Ad5 and during four rounds of adenovirus vaccination.

Functional Impairment of Central Memory CD4 T Cells Is a Potential Early Prognostic Marker for Changing Viral Load in SHIV-Infected Rhesus Macaques

In HIV infection there is a paucity of literature about the degree of immune dysfunction to potentially correlate and/or predict disease progression relative to CD4+ T cells count or viral load. We assessed functional characteristics of memory T cells subsets as potential prognostic markers for changing viral loads and/or disease progression using the SHIV-infected rhesus macaque model. Relative to long-term non-progressors with low/undetectable viral loads, those with chronic plasma viremia, but clinically healthy, exhibited significantly lower numbers and functional impairment of CD4+ T cells, but not CD8+ T cells, in terms of IL-2 production by central memory subset in response to PMA and ionomycine (PMA+I) stimulation. Highly viremic animals showed impaired cytokine-production by all T cells subsets. These results suggest that functional impairment of CD4+ T cells in general, and of central memory subset in particular, may be a potential indicator/predictor of chronic infection with immune dysfunction, which could be assayed relatively easily using non-specific PMA+I stimulation.

Prime-boost vaccination using chemokine-fused gp120 DNA and HIV envelope peptides activates both immediate and long-term memory cellular responses in rhesus macaques

HIV vaccine candidates with improved immunogenicity and induction of mucosal T-cell immunity are needed. A prime-boost strategy using a novel HIV glycoprotein 120 DNA vaccine was employed to immunize rhesus macaques. The DNA vaccine encoded a chimeric gp120 protein in fusion with monocyte chemoattractant protein-3, which was hypothesized to improve the ability of antigen-presenting cells to capture viral antigen through chemokine receptor-mediated endocytosis. DNA vaccination induced virus-reactive T cells in peripheral blood, detectable by T cell proliferation, INFγ ELISPOT and sustained IL-6 production, without humoral responses. With a peptide-cocktail vaccine containing a set of conserved polypeptides of HIV-1 envelope protein, given by nasogastric administration, primed T-cell immunity was significantly boosted. Surprisingly, long-term and peptide-specific mucosal memory T-cell immunity was detected in both vaccinated macaques after one year. Therefore, data from this investigation offer proof-of-principle for potential effectiveness of the prime-boost strategy with a chemokine-fused gp120 DNA and warrant further testing in the nonhuman primate models for developing as a potential HIV vaccine candidate in humans.

Evaluation of cellular immune responses in rhesus monkeys subjected to adenovirus-mediated gene transfer into the cervix

We reported previously that direct injection of a recombinant adenovirus (rAd), Ad5CMV-β-gal, into the cervix of the rhesus monkey resulted in efficient β-galactosidase expression in the cervix within 3 days. In these studies, we also observed the induction of anti-adenovirus (Ad)-specific immunoglobulin G responses after 22 days. In the continuation of evaluating the anti-Ad-specific immune responses resulting from this approach of gene targeting to the cervix, we measured the cellular immune responses. The introduction of Ad5CMV-β-gal into the cervix by direct injection, but not by the abrasion technique, resulted in the induction of strong proliferative responses against extracts of cells infected with Ad5CMV-β-gal but not against control uninfected cells. These responses were initially detected at 22 days postinjection and coincided with the abrogation of transgene expression. Significant levels of proliferative responses were maintained for ≤83 days. Multiple injections of rAds had no significant enhancing effect on either the level or longevity of the proliferative responses. At 3 days after the injection of Ad5CMV-β-gal, when the transgene expression in the cervix was clearly evident, proliferative responses against the rAd were not detectable. However, the production of low but significant amounts of interleukin-10, a cytokine characteristic of T helper type 2 responses that promote humoral immune responses, was observed at the 3-day point in these animals. These results suggest that significant differences exist between the kinetics of transgene expression and the priming of specific host immune responses, and that these differences may be important for devising alternate strategies to improve techniques for Ad-mediated gene therapy of cervical cancer.

Hydranencephaly in two rhesus monkeys (Macaca mulatta).

Spontaneously occurring hydranencephaly was diagnosed at necropsy and confirmed histologically in two stillborn fetuses that were delivered from young female rhesus macaques (Macaca mulatta).