Immune and Genetic Correlates of Vaccine Protection Against Mucosal Infection by SIV in Monkeys

Abstract

The RV144 vaccine trial in Thailand demonstrated that an HIV vaccine could prevent infection in humans and highlights the importance of understanding protective immunity against HIV. We used a nonhuman primate model to define immune and genetic mechanisms of protection against mucosal infection by the simian immunodeficiency virus (SIV). A plasmid DNA prime/recombinant adenovirus serotype 5 (rAd5) boost vaccine regimen was evaluated for its ability to protect monkeys from infection by SIVmac251 or SIVsmE660 isolates after repeat intrarectal challenges. Although this prime-boost vaccine regimen failed to protect against SIVmac251 infection, 50% of vaccinated monkeys were protected from infection with SIVsmE660. Among SIVsmE660-infected animals, there was about a one-log reduction in peak plasma virus RNA in monkeys expressing the major histocompatibility complex class I allele Mamu-A*01, implicating cytotoxic T lymphocytes in the control of SIV replication once infection is established. Among Mamu-A*01-negative monkeys challenged with SIVsmE660, no CD8(+) T cell response or innate immune response was associated with protection against virus acquisition. However, low levels of neutralizing antibodies and an envelope-specific CD4(+) T cell response were associated with vaccine protection in these monkeys. Moreover, monkeys that expressed two TRIM5 alleles that restrict SIV replication were more likely to be protected from infection than monkeys that expressed at least one permissive TRIM5 allele. This study begins to elucidate the mechanisms of vaccine protection against immunodeficiency viruses and highlights the need to analyze these immune and genetic correlates of protection in future trials of HIV vaccine strategies.

Fig. 1

Protection against SIV acquisition and peak plasma virus RNA concentrations after infection. Low-dose virus challenges involved weekly intrarectal inoculations with one AID₅₀ of virus challenge stock for 12 consecutive weeks. Plasma SIV RNA concentrations were assessed weekly, and monkeys that had detectable plasma virus were excluded from subsequent virus challenges. Shown are Kaplan-Meier curves for SIV acquisition and peak plasma SIV RNA concentrations during primary infection. (A to C) Three parallel studies were performed: (A) a two-arm experiment with Mamu-A*01–negative rhesus monkeys and an SIVmac251 challenge (n = 40); (B) a two-arm experiment with Mamu-A*01–negative rhesus monkeys and an SIVsmE660 challenge (n = 50); and (C) a two-arm experiment with Mamu-A*01–positive rhesus monkeys and an SIVsmE660 challenge (n = 39). The control arm of each of these experiments comprised sham-vaccinated animals. (D) The results of the two SIVsmE660 challenge studies were combined and displayed as a single study. The differences between the two groups of monkeys in acquisition of infection and in peak plasma virus RNA concentrations were analyzed by the log-rank test and by the Wilcoxon rank-sum test, respectively.

Fig. 2

Vaccine-induced pooled peptide ELISpot responses to SIV proteins. Monkey PBMCs were assessed in a pooled peptide ELISpot assay for SIVmac239 Env-, Gag-, and Pol-specific cellular immune responses on the day of challenge. Each plotted data point represents the sum of spot-forming cell (SFC) responses for PBMCs of an individual monkey specific for SIVmac239 Env, Gag, and Pol. Data points are for two studies: the Mamu-A*01–negative monkeys challenged with SIVsmE660 and the Mamu-A*01–positive monkeys challenged with SIVsmE660. In each study, the monkeys were divided into two groups: vaccinated monkeys that became infected and vaccinated monkeys that did not become infected. No statistically significant differences between the magnitudes of the responses in these groups in each study were noted as determined by the Mann-Whitney test.

Fig. 3

Vaccine-induced virus-specific cellular immune responses. (A and B) PBMCs isolated from Mamu-A*01–negative (A) and Mamu-A*01–positive (B) monkeys after the boost immunization were exposed to pools of overlapping peptides spanning the Gag or Env proteins of SIVsmE660, and the fractions of CD4⁺ or CD8⁺ T cells producing IFN-γ (F), TNF-α (T), or IL-2 (2) were determined by intracellular cytokine staining. Data are presented as the frequency of cytokine-producing CD4⁺ or CD8⁺ T cells from the groups of monkeys. The cytokine profiles of these cells were determined by expressing each cytokine response as a proportion of the total antigen-specific cytokine-producing CD4⁺ and CD8⁺ T cell response. Data were analyzed with the SPICE software and are presented in pie charts as the mean values from the experimental groups of monkeys. No statistically significant differences between the magnitudes of the responses in these groups in each study were noted as determined by the Mann-Whitney test.

Fig. 4

Vaccine-induced antibody binding to SIVsmE660. (A) Sera sampled 2 weeks after the rAd5 boost (week 34) and from the day of first SIVsmE660 viral challenge (week 53) were assayed by ELISA for the level of antibody binding to SIVsmE660 gp140. Values are the reciprocal plasma dilution yielding 50% half-maximal binding (ED₅₀). (B) Avidity index values from sera on the day of challenge. Avidity indices were calculated by dividing ED₅₀ values with NaSCN treatment by ED₅₀ values without treatment. P values were calculated with a Mann-Whitney rank-sum test.

Fig. 5

Vaccine-induced antibody-dependent cell-mediated cytotoxicity responses to SIVmac251gp120. CD3⁻CD20⁻CD56⁺ human PBMCs (NK cells) were incubated with SIVmac251gp120-coated CEM-NKr-CCR5 target cells and plasma sampled from the monkeys on the day of challenge. The expression of CD107a, IFNγ, TNF-α, and MIP-1β by the NK cells was evaluated by intracellular cytokine staining. Data are expressed as percentage of NK cells expressing these molecules. No statistically significant differences between the magnitudes of the responses in these groups in each study were noted as determined by the Mann-Whitney test.

Fig. 6

Neutralization of tiers 1 and 2 SIVsmE660 Env pseudoviruses. (A to D) Serum samples were obtained from Mamu-A*01–negative (A and B) or Mamu-A*01–positive (C and D) monkeys 2 weeks after rAd boost immunization or on the day of challenge (DOC) and were tested for neutralizing activity against tier 1 (CP3C-P-A8) or tier 2 (CR54-P-2A5) SIVsmE660 Env pseudoviruses with the TZM-bl assay. Data are presented as serum ID₅₀ titer (A and C) or percent neutralization (B and D) at a 1:10 serum dilution. Immunized monkeys are grouped retrospectively according to their infection status after SIVsmE660 challenge. Serum-neutralizing antibody activity from sham-vaccinated monkeys (control) is shown in (B) and (D). Data points represent responses from individual monkeys, with the median response indicated by the bar. P values were calculated with a Mann-Whitney rank-sum test.

Fig. 7

Neutralization of SIVsmE660 in human PBMCs. CD8⁺ T cell–depleted, concanavalin A–stimulated human PBMCs were infected at a low MOI with SIVsmE660. Infected PBMCs were subsequently cultured in the presence of a 1:50 dilution of plasma collected from the Mamu-A*01–negative and Mamu-A*01–positive study animals at 6 weeks after rAd vaccination (peak). Virus from the infected PBMC-plasma coculture supernatant was quantified with the TZM-bl assay. Luciferase assay results were normalized to a control plasma and plotted as a relative viral replication. P values were calculated with a Mann-Whitney rank-sum test.

Fig. 8

Effect of TRIM5 genotype on SIV mucosal infection in naïve and vaccinated monkeys. The effect of restrictive or permissive TRIM5 alleles on the percentage of monkeys that remained un-infected after each weekly intrarectal SIVsmE660 exposure is shown in Kaplan-Meier curves. (A and B) Naïve (A) and vaccinated (B) monkeys were divided into two groups: one group expressing only restrictive TRIM5 alleles (black) and the other expressing at least one permissive allele (red). In the control group, 16 monkeys expressed restrictive TRIM5 alleles, whereas 27 monkeys expressed permissive alleles. In the vaccine group, 15 monkeys expressed restrictive alleles, whereas 28 monkeys expressed permissive alleles. The statistical comparisons between monkeys with restrictive and permissive TRIM5 genotypes in control and vaccinated arms were determined by the log-rank test.

Fig. 9

Effect of vaccination on SIV mucosal infection. The effect of vaccination on the percentage of monkeys with either restrictive or permissive TRIM5 genotypes that remain uninfected after each weekly intrarectal SIVsmE660 exposure is shown in Kaplan-Meier curves. (A and B) TRIM5 restrictive (A) or permissive (B) monkeys were divided into control (black) and vaccinated (red) groups. The statistical comparisons between monkeys in control and vaccinated arms for TRIM5 restrictive and permissive genotypes were determined by the log-rank test.