Immunogenicity of a vaccine regimen composed of simian immunodeficiency virus DNA, rMVA, and viral particles administered to female rhesus macaques via four different mucosal routes

Abstract

A comparative evaluation of the immunity stimulated with a vaccine regimen that includes simian immunodeficiency virus (SIV), interleukin 2 (IL-2), and IL-15 DNAs, recombinant modified vaccinia virus Ankara (rMVA), and inactivated SIVmac239 particles administered into the oral and nasal cavities, small intestine, and vagina was carried out in female rhesus macaques to determine the best route to induce diverse anti-SIV immunity that may be critical to protection from SIV infection and disease. All four immunizations generated mucosal SIV-specific IgA. Oral immunization was as effective as vaginal immunization in inducing SIV-specific IgA in vaginal secretions and generated greater IgA responses in rectal secretions and saliva samples compared to the other immunization routes. All four immunizations stimulated systemic T-cell responses against Gag and Env, albeit to a different extent, with oral immunization providing greater magnitude and nasal immunization providing wider functional heterogeneity. SIV-specific T cells producing gamma interferon (IFN-γ) dominated these responses. Limited levels of SIV-specific IgG antibodies were detected in plasma samples, and no SIV-specific IgG antibodies were detected in secretions. Vaccination also induced CD4(+) and CD8(+) T-cell responses in the rectal and vaginal mucosa with greater functional heterogeneity than in blood samples. Rectal T-cell responses were significantly greater in the orally vaccinated animals than in the other animals. The most balanced, diverse, and higher-magnitude vaginal T-cell responses were observed after intestinal vaccination. Significantly higher CD8(+) granzyme B-positive T-cell responses were observed systemically after intestinal vaccination and in rectal cells after oral immunization. The majority of SIV-specific T cells that produced granzyme B did not produce cytokines. Of the immunization routes tested, oral vaccination provided the most diverse and significant response to the vaccine.

Fig 1

Anti-SIV humoral (IgA) responses in secretions during immunization. (A to C) IgA antibodies to SIV gp140 Env and SIV lysate Gag and Pol proteins were measured by an ELISA performed on rectal secretions (A), vaginal secretions (B), and salivary secretions (C) collected at intervals after immunization by each mucosal route. To compare the magnitude of the mucosal IgA responses generated in animals, the SIV antibody concentration in each secretion was normalized against the total IgA concentration and is expressed as specific activity (nanogram of SIV-specific IgA per microgram of total IgA). Graphs on the left depict the specific activity in individual animals at time points corresponding to 2 and 4 weeks after the third DNA immunization (weeks 26 and 28), 2 weeks after the MVA immunization (week 34), and 2 and 5 weeks after the second viral particle immunization (weeks 52 and 54). The specific activity had to fall outside the shaded portion in the graphs to be considered statistically significant. Symbols in the shaded region may be overlapping, but all symbols above this have been separated to permit visualization of positive responders. Peak IgA responses measured during this phase of the study are illustrated in the Tukey graphs on the right. P values were obtained using the Mann-Whitney rank sum test. Int, intestinal; Nas, nasal; Vag, vaginal. (D) The percentage of animals in each group that were found to have significant IgA responses to both Env and Gag/Pol at the mucosal site is shown. (E) The number of animals that demonstrated a significant Env or Gag/Pol IgA response at all three or fewer mucosal sites is presented for each immunization group. Animal identification (ID) numbers for the four immunization groups were as follows: oral, animals 153, 247, 248, 249, 253, 255, and 256; intestinal, animals 146, 155, 246, 257, 258, 259, and 260; nasal, animals 145, 147, 148, 149, 150, 156, and 250; and vaginal, animals 151, 152, 154, 157, 158, 254, and 265.

Fig 2

Anti-SIV plasma IgG concentrations during the immunization regimen. (A and B) Plasma levels of IgG antibodies against SIV Gag/Pol (A) and gp140 (B) were measured by ELISA on the day of the first DNA immunization (day 1), 2 weeks after the second particle immunization (week 52), and at the end of the immunization regimen (week 57). The concentration, measured in nanogram/milliliter for individual animals at the three time points, is reported. The cutoff was calculated as the preimmune sample mean plus 3 SD, and it is 241 ng/ml for Gag/Pol and 111 ng/ml for Env. The concentration had to fall outside the shaded region and be higher than the value for the preimmune sample to be considered significant. Animal identification (ID) numbers for the four immunization groups were as follows: oral, animals 153, 247, 248, 249, 253, 255, and 256; intestinal, animals 146, 155, 246, 257, 258, 259, and 260; nasal, animals 145, 147, 148, 149, 150, 156, and 250; and vaginal, animals 151, 152, 154, 157, 158, 254, and 265.

Fig 3

Circulating cell-mediated total SIV and Gag- and Env-specific T-cell responses during immunization phase. The geometric mean of each vaccinated group is shown during the immunization phase as a total percentage of CD4⁺ T-cell (A to C) and CD8⁺ T-cell (D to F) responses measured in PBMC as production of IFN-γ, TNF-α, and IL-2 and their combination by ICS and flow cytometry analysis upon stimulation with Gag or Env peptide pools. The graphs in columns A and D show the total SIV-specific T-cell responses (Gag plus Env) for vaccinated groups and for each individual animal of the vaccinated group, while the graphs in columns B and E illustrate the T-cell responses elicited against Gag and the graphs in columns C and F illustrate the T-cell responses elicited against Env during immunization until the end of the immunization phase. The immunization route of the groups are shown as follows: orally vaccinated animals are represented in blue, intestinally vaccinated in red, nasally in orange, and vaginally in light blue. Statistical significance between groups was tested by the one-way ANOVA assay (P < 0.05). (G) Qualitative analysis of systemic SIV Gag- and Env-specific cell-mediated responses for each vaccinated group 2 weeks after the third DNA immunization (week 26), 2 weeks after the boost with rMVA (week 34), 2 weeks after the second boost with inactivated SIVmac239 particles (week 52), and 7 weeks after the last immunization (week 57). Pie graphs representing the diversity of CD4⁺ (left) and CD8⁺ (right) T-cell responses in production of IFN-γ, TNF-α, and IL-2 and the simultaneous production of 2 or more cytokines combined are shown for each of the four time points. The total mean percentage SIV-specific response for each group is shown on the upper left side of each pie. Animal identification (ID) numbers for the four immunization groups were as follows: oral, animals 153, 247, 248, 249, 253, 255, and 256; intestinal, animals 146, 155, 246, 257, 258, 259, and 260; nasal, animals 145, 147, 148, 149, 150, 156, and 250; and vaginal, animals 151, 152, 154, 157, 158, 254, and 265.

Fig 4

Rectal SIV-specific cell-mediated T-cell responses during immunization. (A and B) SIV-specific CD4⁺ (Gag [A] and Env [left graph in panel B]) and CD8⁺ T-cell responses (Gag [A] and Env [right graph in panel B]) were detected in MNC isolated from rectal biopsy specimens 2 weeks after the third DNA dose (week 26), 2 weeks after the boost with rMVA (week 34), and 4 weeks after the boost with inactivated SIVmac239 particles (week 54) and are represented as a bar graph for each animal in all four vaccinated groups. Each bar represents the sum of anti-Gag or anti-Env-specific T cells producing IFN-γ, TNF-α, and IL-2 alone or as a double- or triple-cytokine combination, after stimulation with Gag or Env peptide pools. (C to E) Diversity of rectal SIV-specific cell-mediated T-cell responses during immunization. Pie graphs illustrate the diversity of Gag-specific (C) and Env-specific (D) T cells producing IFN-γ, TNF-α, and IL-2 for each group detected at 3 time points, after the third DNA dose (week 26), 2 weeks after the MVA boost (week 34), and 4 weeks after the boost with inactivated SIVmac239 particles (week 54). The SIV-specific T-cell mean percentage of the total CD4⁺ or CD8⁺ T cells is shown for each group beneath each pie. (E) Total mean percentage of SIV-specific Gag⁺ plus Env⁺ CD4⁺ and CD8⁺ T-cell response detected 6 weeks after the last immunization is shown for each vaccinated group. Statistical significance was evaluated by one-way ANOVA and Bonferroni posttest. A P of <0.05 was considered statistically significant.

Fig 5

Vaginal SIV-specific cell-mediated T-cell responses during immunization. (A to D) SIV-specific CD4⁺ (Gag [A] and Env [B]) and CD8⁺ T-cell responses (Gag [C] and Env [D]) were detected in MNC isolated from cervicovaginal biopsy specimens 2 weeks after the third DNA dose (week 26), 2 weeks after the boost with rMVA (week 34), and 4 weeks after the last boost with AT-2-inactivated SIVmac239 particles (week 54). The T-cell responses are represented as a bar graph for each animal in all four vaccinated groups. Each bar represents the sum of anti-Gag or anti-Env-specific T cells producing IFN-γ, TNF-α, and IL-2 alone or as double- or triple-cytokine combination, after stimulation with Gag or Env peptide pools. (C to E) Diversity of vaginal SIV-specific cell-mediated T-cell responses during immunization. Pie graphs illustrate the diversity of SIV Gag-specific (C) and Env-specific (D) T cells producing IFN-γ, TNF-α, and IL-2 for each group at 3 time points, 2 weeks after the third DNA dose (week 26), 2 weeks after the MVA boost (week 34), and 4 weeks after the boost with inactivated SIVmac239 particles (week 54). (E) Mean percentage of SIV-specific Gag⁺ Env⁺ CD4⁺ and CD8⁺ T-cell response detected 6 weeks after the last immunization is shown for each vaccinated group. Statistical significance was evaluated by one-way ANOVA and Bonferroni posttest. A P value of <0.05 was considered statistically significant. Animal ID numbers for the four immunization groups were as follows: oral, animals 153, 247, 248, 249, 253, 255, and 256; intestinal, animals 146, 155, 246, 257, 258, 259, and 260; nasal, animals 145, 147, 148, 149, 150, 156, and 250; and vaginal, animals 151, 152, 154, 157, 158, 254, and 265.

Fig 6

Analysis of the vaccine-induced SIV-specific cytotoxic T-cell responses in circulating PBMC and rectal and vaginal MNC at the end of the immunization regimen. (A to C) Granzyme B (GB) production in combination with IFN-γ, TNF-α, and IL-2 was measured in circulating PBMC (A) and rectal (B) and vaginal (C) MNC at the end of the immunization regimen. The variety of total SIV-specific responses in CD4⁺ and CD8⁺ T-cell population is represented as pie graphs for each vaccinated group, and the total mean percentage is shown for each group beneath each pie graph. The total SIV-specific CD4⁺ (left graph) or CD8⁺ (right graph) mean production of granzyme B and its combination with the three cytokines analyzed is represented as bar plots for each vaccinated group. Two-way ANOVA and Bonferroni tests were performed to analyze statistical significance. A P value of < 0.05 was considered statistically significant.