Vaccination against heterologous R5 clade C SHIV: prevention of infection and correlates of protection

Abstract

A safe, efficacious vaccine is required to stop the AIDS pandemic. Disappointing results from the STEP trial implied a need to include humoral anti-HIV-1 responses, a notion supported by RV144 trial data even though correlates of protection are unknown. We vaccinated rhesus macaques with recombinant simian immunodeficiency virus (SIV) Gag-Pol particles, HIV-1 Tat and trimeric clade C (HIV-C) gp160, which induced cross-neutralizing antibodies (nAbs) and robust cellular immune responses. After five low-dose mucosal challenges with a simian-human immunodeficiency virus (SHIV) that encoded a heterologous R5 HIV-C envelope (22.1% divergence from the gp160 immunogen), 94% of controls became viremic, whereas one third of vaccinees remained virus-free. Upon high-dose SHIV rechallenge, all controls became infected, whereas some vaccinees remained aviremic. Peak viremia was inversely correlated with both cellular immunity (p<0.001) and cross-nAb titers (p<0.001). These data simultaneously linked cellular as well as humoral immune responses with the degree of protection for the first time.

Figure 1. Experimental time line and phylogenetic analysis of Env of immunogen and challenge virus.

(A) Experimental design. Groups 1 and 2 were vaccinated with HIV-1 Tat, HIV1084i gp160, and Gag-Pol particles from either SIVmne (Group 1) or SIVmac239 (Group 2; 100 µg of each immunogen i.m. in incomplete Freund's adjuvant (IFA)); Group 3 controls received IFA alone. Maximally 5 weekly low-dose i.r. challenges of SHIV-1157ipEL-p were given (8,000 50% tissue culture infectious doses (TCID_50, measured by TZM-bl assay). SHIV-1157ipEL-p is heterologous to HIV1084i Env and SIVmne Gag-Pol immunogens. Group 4, unvaccinated rhesus monkeys (RM) challenged identically with the same virus stock as part of other studies. All RM with no viremia at week 7 (shown in red) were rechallenged i.r. with a single high-dose of SHIV-1157ipEL-p (1.5×10⁵ TCID₅₀; red arrows). Group 5: naïve controls for the high-dose challenge. *, Mamu A*001-positive RM. (B) Phylogenetic analysis of Env sequences of immunogen and challenge virus. HIV-1 Env sequences of clade C and non-clade C reference strains were obtained from the Los Alamos HIV-1 sequence database. The evolutionary tree was inferred using the Neighbor-Joining method by MEGA4 software . The immunogen and challenge virus sequences were on different branches within cluster of HIV-C Env sequences.

Figure 2. Humoral immunity at the time of initial low-dose SHIV-1157ipEL-p challenge.

(A) Reciprocal serum antibody ELISA titers against SIVmac251 p27 Gag, HIV-1 Tat and heterologous clade C HIV_CN54 gp120. (B) NAb activity SHIV-1157ipEL-p and, SHIV_SF162P4) and Tier 2 (SHIV-1157ipd3N4, SHIV-2873Nip) viruses was measured in human PBMC in the presence of polymyxin B. TZM-bl neutralization assay was also performed against SHIV-1157ipEL-p. IC₅₀ and IC₉₀ titers, reciprocal dilutions of serum giving 50% and 90% neutralization (inhibitory concentrations), respectively, are shown. *, Mamu A*001-positive RM.

Figure 3. Cellular immunity at the time of initial low-dose SHIV-1157ipEL-p challenge.

(A) The frequency of antigen-specific cells measured by IFN-γ ELISPOT assay. No responses were seen in control Group 3. (B) The frequency of SIV Gag and HIV-1 Tat-specific peripheral blood CD4⁺ and CD8⁺ T cells producing intracellular IFN-γ, IL-2 and TNF-α was measured using multiparameter flow cytometry. (C) Proliferation of peripheral blood CD4⁺ and CD8⁺ T cells in response to SIV Gag or HIV-1 Tat proteins. *, Mamu A*001-positive RM.

Figure 4. Plasma viral RNA (vRNA) loads after SHIV-1157ipEL-p challenges and Kaplan-Meier plots.

(A–D) vRNA loads after low-dose viral challenges for Groups 1-4. Black symbols, vRNA loads of Groups 1-4 during the weekly low-dose challenges (blue arrows); red symbols, vRNA loads of animals later given high-dose challenge (red arrows; see also Figure 1 legend). (E) Kaplan-Meier plots depicting the fraction of monkeys remaining aviremic. (F) Kaplan-Meier plots depicting the fraction of animals not yet having reached peak viremia. (G–H) vRNA loads after a single high-dose SHIV-1157ipEL-p challenge of newly recruited naïve controls (Group 5) plus prior control monkey RAk-11 (G), or rechallenge of vaccinees (H). Probability (P) values were determined by 2-sided log-rank analysis. Horizontal dashed lines in A–D, G–H: limit of RT-PCR assay detection (50 vRNA copies/ml; [25]).

Figure 5. Immune parameters associated with protection.

Vaccinees were ranked in ascending order of peak viremia after all virus challenges (animals that received the high-dose challenge are shown in red), and immune parameters measured on the day of 1^st low-dose virus challenge are presented. W, week of peak viremia. (A) Neutralizing antibody titer (IC₅₀) measured by TZM-bl cell-based assay vs. peak plasma vRNA. (B) Neutralizing antibody titer (IC₉₀) measured by PBMC-based assay vs. peak plasma vRNA. (C). SIV Gag- plus HIV-1 Tat-specific IFN-γ ELISPOTs vs. peak plasma vRNA. (D) SIV Gag- plus HIV-1 Tat-specific CD4⁺ and CD8⁺ T cells producing IL-2, IFN-γ and TNF-α vs. peak plasma vRNA. Inverse correlations of peak vRNA with cellular (P<0.001) and nAb (P<0.001) responses were determined by Spearman analyses. The mean peak plasma viremia of all controls is shown by the X mark. All control animals were tested in neutralization (TZM-bl, PBMC) and ELISPOT assays; one control animal was included in the ICS assays.

Figure 6. Immune responses on the day of high-dose virus challenge.

Cellular and nAb responses at week 0 (day of first virus exposure) and week 7 (day of high-dose virus challenge) for: (A) SIV Gag- and HIV-1 Tat-specific IFN-γ ELISPOT responses. (B) SIV Gag and HIV-1 Tat-specific peripheral blood CD4⁺ and CD8⁺ T cells producing intracellular IFN-γ, IL-2 & TNF-α (measured by multiparameter flow cytometry). (C) nAb titers against the challenge virus (IC₅₀ measured by TZM-bl assay).

Figure 7. Degrees of protection.

(A) Parameters to define different degrees of protection. Transient viremia, vRNA blip(s) (<10⁴ vRNA copies/ml, which is not expected to result in seroconversion); viral containment, lowering of peak vRNA by ≥1 log vs. controls. Anti-Nef (non-vaccine component) responses were measured to detect neo-antigen reactivity. The fold increase in ELISPOTs after high-dose virus challenge compared to the day of high-dose virus challenge was determined to identify anamnestic cellular immune responses. (B) Distribution of peak vRNA. Red lines, means; dashed lines: upper, viremia 1 log lower than mean of controls; middle, 1×10⁴ vRNA copies/ml; lower, limit of vRNA detection (50 copies/ml). (C–E) Anamnestic cellular responses and neo-antigen reactivity in the monkeys indicated. Left panels, IFN-γ ELISPOTs against SIV Gag (open bars) and Nef (striped bars, neo-antigen). Right panels, SIV Gag-specific intracellular cytokine staining.