Comparison of systemic and mucosal immunization with helper-dependent adenoviruses for vaccination against mucosal challenge with SHIV

Abstract

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.

Figure 1. Schedule of Vaccination and SHIV Challenge.

Figure 2. IFN-γ Secreting Cells from PBMCs.

PBMCs in ELISPOT plates were stimulated with 3 pools of 50 to 70 peptides spanning the gp140 region of SF162 envelope for 36 h and IFN-γ secreting spots were detected and counted. Responses in terms of IFN-γ spot forming units (SFU) for 10⁵ total input cells were determined for individual monkeys after subtracting background values of cells cultured in the medium. A) IFN-γ secreting cells from individual animals. B) Mean IFN-γ secreting cells from each group.

Figure 3. Perforin Secreting Cells from PBMCs.

PBMCs were analyzed by ELISPOT as in Fig. 2, but in this case were stained for perforin production. A) Perforin secreting cells from individual animals. B) Mean perforin secreting cells from each group.

Figure 4. Systemic Cytokines, Peripheral and Mucosal T Cell Responses.

A) Cytokine Responses. Heparinized plasma was collected and analyzed for cytokine levels using a human cytometric bead assay (CBA) with standard curves for each cytokine. Statistical comparisons in A are by one way ANOVA. B) T cell responses from PBMCs and colon biopsies. PBMCs and colon biopsies were collected at week 26 before challenge and CD4 and CD8 Tem and Tcm were analyzed for IL-2 and/or IFN-γ production by flow cytometry (Table 1). Statistical comparisons in B are T test comparisons of the "stack" of total cytokine-expressing cells for a given cell type (i.e. CD8 or CD4, Tcm or Tem).

Figure 5. Antibodies Against HIV-1 Env and Species C Adenoviruses.

A) Anti-env antibodies. Plasma was collected from each group and these were analyzed for env-binding antibodies by ELISA against JRFL env (matching the vaccine). B) Anti-adenovirus neutralizing antibodies. Plasma from week 26 was serially diluted and tested for its ability to neutralize HD-Ad vectors expressing luciferase in vitro.

Figure 6. SHIV Viremia After Rectal Challenge.

A) SHIV viral genomes were quantitated from plasma at the indicated times after challenge for each animal. The reported minimal detection for this assay of 30 meq./ml is indicated by a dashed gray line. B) Comparison of mean SHIV viral loads for controls, i.m. vaccinated, ivag. vaccinated, and the combined mean for all vaccinated animals. Asterisks indicate time points at which p<0.05 for controls as compared to combined vaccinated animals by T test. I.m. and ivag. only groups did not reach this p value.