The Frequency of Vaccine-Induced T-Cell Responses Does Not Predict the Rate of Acquisition after Repeated Intrarectal SIVmac239 Challenges in Mamu-B*08+ Rhesus Macaques

Abstract

Approximately 50% of rhesus macaques (RMs) expressing the major histocompatibility complex class I (MHC-I) allele Mamu-B*08 spontaneously control chronic-phase viremia after infection with the pathogenic simian immunodeficiency virus mac239 (SIVmac239) clone. CD8⁺ T-cell responses in these animals are focused on immunodominant Mamu-B*08-restricted SIV epitopes in Vif and Nef, and prophylactic vaccination with these epitopes increases the incidence of elite control in SIVmac239-infected Mamu-B*08-positive (Mamu-B*08⁺ ) RMs. Here we evaluated if robust vaccine-elicited CD8⁺ T-cell responses against Vif and Nef can prevent systemic infection in Mamu-B*08⁺ RMs following mucosal SIV challenges. Ten Mamu-B*08⁺ RMs were vaccinated with a heterologous prime/boost/boost regimen encoding Vif and Nef, while six sham-vaccinated MHC-I-matched RMs served as the controls for this experiment. Vaccine-induced CD8⁺ T cells against Mamu-B*08-restricted SIV epitopes reached high frequencies in blood but were present at lower levels in lymph node and gut biopsy specimens. Following repeated intrarectal challenges with SIVmac239, all control RMs became infected by the sixth SIV exposure. By comparison, four vaccinees were still uninfected after six challenges, and three of them remained aviremic after 3 or 4 additional challenges. The rate of SIV acquisition in the vaccinees was numerically lower (albeit not statistically significantly) than that in the controls. However, peak viremia was significantly reduced in infected vaccinees compared to control animals. We found no T-cell markers that distinguished vaccinees that acquired SIV infection from those that did not. Additional studies will be needed to validate these findings and determine if cellular immunity can be harnessed to prevent the establishment of productive immunodeficiency virus infection.IMPORTANCE It is generally accepted that the antiviral effects of vaccine-induced classical CD8⁺ T-cell responses against human immunodeficiency virus (HIV) are limited to partial reductions in viremia after the establishment of productive infection. Here we show that rhesus macaques (RMs) vaccinated with Vif and Nef acquired simian immunodeficiency virus (SIV) infection at a lower (albeit not statistically significant) rate than control RMs following repeated intrarectal challenges with a pathogenic SIV clone. All animals in the present experiment expressed the elite control-associated major histocompatibility complex class I (MHC-I) molecule Mamu-B*08 that binds immunodominant epitopes in Vif and Nef. Though preliminary, these results provide tantalizing evidence that the protective efficacy of vaccine-elicited CD8⁺ T cells may be greater than previously thought. Future studies should examine if vaccine-induced cellular immunity can prevent systemic viral replication in RMs that do not express MHC-I alleles associated with elite control of SIV infection.

Keywords: human immunodeficiency virus; simian immunodeficiency virus; vaccines.

FIG 1

Experimental design. Ten Mamu-B*08⁺ RMs were vaccinated with an rAd5/rVSV/rRRV regimen expressing the immunodominant Mamu-B*08-restricted epitopes Vif RL8, Vif RL9, and Nef RL10. Six Mamu-B*08⁺ RMs were sham vaccinated and served as the controls. At week 41, vaccine efficacy was assessed by subjecting all monkeys to repeated IR challenges with a marginal dose of SIVmac239 (200 TCID₅₀) every 2 weeks. The results for four vaccinees are color coded in blue in this figure and subsequent ones because they resisted greater than six IR challenges with SIVmac239.

FIG 2

Kinetics of vaccine-induced CD8⁺ T-cell responses targeting Mamu-B*08-restricted SIV epitopes. Fluorochrome-labeled Mamu-B*08 tetramers folded with peptides corresponding to SIV epitopes were used to track vaccine-elicited SIV-specific CD8⁺ T cells in PBMC. (A to D) The percentages of live tetramer⁺ CD8⁺ T cells specific for Vif RL8 (aa 172 to 179) (A), Vif RL9 (aa 123 to 131) (B), Nef RL10 (aa 137 to 146) (C), and Nef RL9b (aa 246 to 254) (D) are shown at multiple time points throughout the vaccine phase. The times of each vaccination (black dotted lines) and the day of the first IR SIVmac239 challenge (red solid line) are indicated in each graph. (E) Comparison of the total magnitude of vaccine-induced CD8⁺ T cells against Vif RL8, Vif RL9, and Nef RL10 at the time of the first SIV challenge between vaccinees in the present experiment and those in our previous Mamu-B*08 SIV vaccine trial (36). The P value was determined by Student's t test after log transformation. Lines represent means, and each symbol denotes one vaccinee. The vaccinees in the present experiment that resisted greater than 6 IR SIV challenges (r10038, r10091, r09041, and r08009) are color coded in blue. The remaining six vaccinees that acquired SIV infection and manifested partial or no control of viral replication are indicated by black symbols.

FIG 3

Memory phenotype of vaccine-induced CD8⁺ T cells in PBMC. RM memory T cells can be classified into three subsets based on the differential expression of the cell surface molecules CD28 and CCR7: fully differentiated effector memory (T_EM2; CD28⁻ CCR7⁻), transitional memory (T_EM1; CD28⁺ CCR7⁻), and central memory (T_CM; CD28⁺ CCR7⁺). Based on the expression of these two markers, the memory phenotype of vaccine-induced CD8⁺ T cells against Vif RL8 (A), Vif RL9 (B), and Nef RL10 (C) in PBMC was delineated at week 37 after rAd5 prime. The panels on the left show the percentages of MHC-I tetramer⁺ CD8⁺ T cells that display each of the three memory signatures. This flow cytometric analysis also evaluated the intracellular expression of the cytotoxicity-associated molecule granzyme B (Gzm B) by each MHC-I tetramer⁺ CD8⁺ T-cell population, as shown in the panels on the right. Lines represent means. The P values were determined by repeated-measurement ANOVA. Vaccinees are color coded as described in the legend of Fig. 2. Data from monkey r08038 were not available for this analysis.

FIG 4

Tissue distribution of vaccine-induced CD8⁺ T cells against Mamu-B*08-restricted SIV epitopes. (A to C) The frequencies of vaccine-elicited CD8⁺ T cells against Vif RL8 (A), Vif RL9 (B), and Nef RL10 (C) were quantified in PBMC and disaggregated lymphocyte suspensions obtained from lymph node (LN) and gut (colon plus rectal) biopsy specimens by fluorochrome-labeled MHC-I tetramer staining. (D) The combined frequencies of Vif RL8-, Vif RL9-, and Nef RL10-specific CD8⁺ T cells in each tissue are shown. The samples for this analysis were harvested at week 37 after rAd5 prime. Lines represent means. The P values were determined by repeated-measurement ANOVA. Vaccinees are color coded as described in the legend of Fig. 2.

FIG 5

Total magnitude and functional profile of vaccine-induced SIV-specific T-cell responses at the time of the first SIV challenge. These analyses were carried out in PBMC collected at study week 41. (A and B) CD8⁺ and CD4⁺ T-cell responses were measured by ICS using pools of SIVmac239 peptides (15-mers overlapping by 11 amino acids). The percentages of responding CD8⁺ (A) and CD4⁺ (B) T cells were calculated by adding the frequencies of positive responses producing any combination of three immunological functions (IFN-γ, TNF-α, and CD107a). Each panel shows the magnitude of T-cell responses against Vif, Nef, and both Rev and Tat and the sum of T-cell responses against these proteins. The P values were determined by repeated-measurement ANOVA. (C to E) Functional profile of vaccine-induced CD8⁺ T cells directed against Vif RL8, Vif RL9, and Nef RL10. ICS was used to evaluate the ability of vaccine-induced CD8⁺ T cells to degranulate (based on CD107a upregulation) and/or produce the cytokines IFN-γ and TNF-α upon stimulation. The antigen stimuli for this assay consisted of peptides corresponding to the Mamu-B*08-restricted epitopes Vif RL8 (C), Vif RL9 (D), and Nef RL10 (E). The combinations of functions tested are shown below each panel. The P values were determined by repeated-measurement ANOVA. Lines represent means. Vaccinees are color coded as described in the legend of Fig. 2.

FIG 6

Acquisition of SIV infection in vaccinees and control animals. (A) Challenge scheme. Macaques were exposed to SIV on day 0 and subsequently bled on days 7 and 10. Plasma collected on days 7 and 10 was assayed for the presence of SIV RNA, and a decision was made as to whether or not to challenge the animals on day 14. Macaques that remained aviremic on both days 7 and 10 were rechallenged, whereas monkeys with a positive VL on either of these days were not rechallenged. (B) Kinetics of SIVmac239 acquisition in vaccinated and control macaques. Individual animals in the vaccine and control groups are depicted along with the IR SIVmac239 exposures that caused (filled circles) or did not cause (empty circles) productive infection. Although macaque r08009 likely acquired infection after the 7th SIV exposure, it ended up being challenged 9 times (see the text for details). Macaque r10091 skipped challenge 7 but was rechallenged two additional times (see the text for details). (C) Survival analysis of SIV-infected vaccinated and control animals. The P value was determined by the Cox proportional hazard model (hazard ratio, 0.59; 95% confidence interval, 0.19 to 1.85).

FIG 7

No signs of anamnestic expansion of vaccine-induced T-cell responses or de novo induction of SIV-specific T-cell responses in r10091 after the 6th SIV exposure. (A) SIV-specific CD8⁺ T-cell responses at week 2.4 after challenge 6. The antigen stimuli for this assay consisted of pools of SIV peptides corresponding to vaccine-encoded SIV proteins (Vif, Nef, and Rev-Tat) or antigens that were not included in the vaccine (Gag, gp120, gp41, and Vpr-Vpx). The results are shown as contour plots displaying the percentages of CD8⁺ T cells producing IFN-γ and/or TNF-α in the absence of stimulation (no stimulus) or in response to the aforementioned SIV peptide pools. (B) Baseline (week of the 1st SIV challenge) levels of vaccine-induced CD8⁺ T-cell responses against Vif, Nef, and Rev-Tat are shown as a reference. In all panels, the cells were gated on live CD14⁻ CD16⁻ CD20⁻ CD3⁺ CD4⁻ CD8⁺ lymphocytes.

FIG 8

Plasma virus concentrations after SIVmac239 infection. The viral loads of animals that acquired SIV infection were log transformed and correspond to the number of vRNA copies per milliliter of plasma. (A to H) Viral load traces for animals in the control group (A) or individual vaccinees that became infected (B to H). The dotted and dashed lines are for reference only and indicate VLs of 10³ and 10⁶ vRNA copies/ml of plasma, respectively. The two unintentional SIV challenges delivered to r08009 are indicated by vertical solid red lines in panel B. (I) Comparison of peak viral loads between SIV-infected vaccinees and control animals. The P value was calculated using Student's t test after log transformation. Lines correspond to geometric means, and each symbol denotes an animal. Monkey r08009 is color coded in blue because it manifested stringent control of chronic-phase viremia.

FIG 9

No evidence for de novo induction of SIV-specific CD8⁺ T-cell responses or SIV rebound following CD8α depletion in vaccinees that resisted repeated IR exposures to SIV. (A) Macaques r10038, r10091, and r09041 were bled 4 days prior to the 10th IR SIV challenge, and an ICS assay was carried out in PBMC. The antigen stimuli for this assay consisted of peptides corresponding to SIV antigens that were included in the vaccine (Vif, Nef, and Rev-Tat) as well as proteins that were not included in the vaccine (Gag, Pol, Vpr, Vpx, and Env). The percentages of responding CD8⁺ T cells displayed in the panel were calculated by adding the background-subtracted frequencies of positive responses producing any combination of IFN-γ, TNF-α, and CD107a. Lines represent means, and each symbol corresponds to one vaccinee. (B to D) To evaluate whether r10038 and r09041 harbored replication-competent SIV, these animals were treated with a single i.v. infusion of 50 mg/kg of a CD8α-depleting MAb 39 weeks after the 10th IR SIVmac239 challenge. Monkey r10091 was not subjected to this procedure because it had to be euthanized prior to it. The absolute counts of CD8⁺ T cells (CD3⁺ CD8α⁺) and NK cells (CD3⁻ CD8α⁺ CD16⁺) per microliter of blood after the CD8α depletion are shown in panels B and C, respectively. (D) Viral loads after CD8α depletion. The VLs were log transformed and correspond to the number of vRNA copies per milliliter of plasma. The dash-dot line indicates the limit of detection (LOD) of the VL assay (15 vRNA copies/ml of plasma).