HIV-1 vaccine-induced immunity in the test-of-concept Step Study: a case-cohort analysis

Abstract

Background: In the Step Study, the MRKAd5 HIV-1 gag/pol/nef vaccine did not reduce plasma viraemia after infection, and HIV-1 incidence was higher in vaccine-treated than in placebo-treated men with pre-existing adenovirus serotype 5 (Ad5) immunity. We assessed vaccine-induced immunity and its potential contributions to infection risk.

Methods: To assess immunogenicity, we characterised HIV-specific T cells ex vivo with validated interferon-gamma ELISPOT and intracellular cytokine staining assays, using a case-cohort design. To establish effects of vaccine and pre-existing Ad5 immunity on infection risk, we undertook flow cytometric studies to measure Ad5-specific T cells and circulating activated (Ki-67+/BcL-2(lo)) CD4+ T cells expressing CCR5.

Findings: We detected interferon-gamma-secreting HIV-specific T cells (range 163/10(6) to 686/10(6) peripheral blood mononuclear cells) ex vivo by ELISPOT in 77% (258/354) of people receiving vaccine; 218 of 354 (62%) recognised two to three HIV proteins. We identified HIV-specific CD4+ T cells by intracellular cytokine staining in 58 of 142 (41%) people. In those with reactive CD4+ T cells, the median percentage of CD4+ T cells expressing interleukin 2 was 88%, and the median co-expression of interferon gamma or tumor necrosis factor alpha (TNFalpha), or both, was 72%. We noted HIV-specific CD8+ T cells (range 0.4-1.0%) in 117 of 160 (73%) participants, expressing predominantly either interferon gamma alone or with TNFalpha. Vaccine-induced HIV-specific immunity, including response rate, magnitude, and cytokine profile, did not differ between vaccinated male cases (before infection) and non-cases. Ad5-specific T cells were lower in cases than in non-cases in several subgroup analyses. The percentage of circulating Ki-67+BcL-2(lo)/CCR5+CD4+ T cells did not differ between cases and non-cases.

Interpretation: Consistent with previous trials, the MRKAd5 HIV-1 gag/pol/nef vaccine was highly immunogenic for inducing HIV-specific CD8+ T cells. Our findings suggest that future candidate vaccines have to elicit responses that either exceed in magnitude or differ in breadth or function from those recorded in this trial.

Conflict of interest statement

Conflict of Interest

SD, LK, MNR, DVM, JWS, and DRC all are employees and shareholders of Merck & Co, Inc, MJM and SPB have served as investigators on Merck-funded research, SCD, ZM, HJ, ODD, DKC, JH, RA, SGS, and LC have no conflicts of interest.

Figures

Figure 1

Ex vivo HIV-specific CD4+ and CD8+ T cells induced by the Merck trivalent Ad5/HIV vaccine. A) Cytokine and activation marker flow cytometric staining profiles in previously cryopreserved PBMC from one vaccine recipient obtained four weeks after the third immunization (week 30). The Ad5 neutralizing titer for this individual is 893. Cells were gated by forward and side scatter, live vs. dead cells, CD3+ T cells, and then CD4+ and CD8+ T cells. HIV-specific CD4+ (left two columns) and CD8+ (right two columns) T cells were stimulated ex vivo with Gag, Pol and Nef peptide pools that span the sequence encoded by the HIV-1 gene insert. The numbers indicate the percent of CD4+ or CD8+ cells expressing cytokine(s) IFN-γ, IL-2 and/or TNF-α. The responses in the negative control were low (<0.01% for all cytokines, not shown). B and C) The percent CD4+ (B) or CD8+ (C) T cells producing IFN-γ and/or IL-2 in response to HIV-1 Gag, Pol and/or Nef (left panels) or HIV-1 Gag only (right panels), four weeks after the second (week 8) or third (week 30) dose in vaccine recipients who remained HIV-uninfected (matched non-cases) or who became HIV-infected (cases). ICS analyses were performed on PBMC obtained prior to infection in cases. The participants are stratified by Ad5 neutralizing antibody titer (≤ 18 and >18). Indicated above each figure are the numbers of positive responders, both as a ratio and as a percentage. The red dots represent positive responders and the blue dots negative responders; triangles identify PBMC from week 8, and circles identify PBMC from week 30.

Figure 2

Vaccine-induced HIV-specific CD4+ and CD8+ T cells producing multiple cytokines. The left graphs show the percentage of the HIV-specific CD4+ (A) or CD8+ (B) T cells that are producing 1, 2 or 3 cytokines in the vaccine recipients. Within each graph, cases are shown in blue and non-cases in red. ICS analyses were performed on PBMC obtained four weeks following the third vaccination (week 30). None of the cases analyzed were infected at this time point. The right graphs depict the percentage of cells producing IFN-γ, IL-2 or TNF-α among those producing one cytokine, and the percentage of cells co-producing cytokine pairs among those producing two cytokines.

Figure 3

Ex vivo Ad5-specific CD4+ and CD8+ T cells. A) Ad5-specific CD4+ (left panels) and CD8+ T cells (right panels) in a representative donor (same as in Figure 1A). PBMC were stimulated overnight with empty Ad5 vector and evaluated (as in Figure 1A) for intracellular cytokine expression. B and C) The percent CD4+ (B) and CD8+ (C) T cells producing IFN-γ and/or IL-2 in response to Ad5 empty vector stimulation in vaccine recipient cases and matched non- cases. PBMC were analyzed from subjects obtained four weeks after the second (week 8) or the third (week 30) Ad5 vaccination. The participants are stratified by Ad5 neutralizing antibody titer (≤18 and >18). The numbers of positive responders are indicated above each figure as a ratio and as a percentage. The red dots represent positive responders, and the blue dots signify the negative responders; triangles identify PBMC from week 8 and circles signify PBMC from week 30.

Figure 4

Activated CD4+ T cells expressing CCR5. A) Activated T cells expressing CCR5 in a representative donor (same as in Figure 1A). The left panels depict the overall percentage of Ki-67+Bcl-2lo activated CD4+ and CD8+ T cells, respectively. The right panels demonstrate the expression of CCR5 for the overall population of CD4+ or CD8+ T cells in grey, as well as the activated cells (red overlay). The numbers indicate the percent of activated CD4+ and CD8+ T cells expressing CCR5. B) Percent activated (Ki-67+/BcL-2lo) CD4+ T cells expressing CCR5 in vaccine recipient and placebo recipient non-cases and cases four weeks following the third Ad5 vaccination (week 30), stratified by Ad5 neutralizing antibody titer (≤200, red dots; >200, blue dots). Only cases infected after week 30 are included. C) Percent activated CD4+ T cells expressing CCR5 in non-cases at six months following the third Ad5 vaccination (week 52), stratified by Ad5 neutralizing antibody titer.