High-level, lasting antiviral immunity induced by a bimodal AIDS vaccine and boosted by live-virus exposure: prevention of viremia

Abstract

Objective: To characterize the correlates of protection from systemic infection in a vaccinated rhesus macaque, RAt-9, which had been challenged sequentially with two related clade C simian/human immunodeficiency viruses (SHIV-Cs) yet remained aviremic for more than 5 years despite indirect evidence of cryptic infection.

Design: To measure long-term anti-SHIV-C immunity, host genetics and gene-expression patterns for protective correlates.

Methods: Long-term immune reactivity was evaluated and identification of virus in RAt-9 was attempted by RT-PCR analysis of concentrated plasma and blood transfer to CD8(+) cell-depleted infant macaques. Full MHC genotyping of RAt-9, TRIM5α and KIR3DL allelic expression analysis of PBMC, and microarray gene expression analysis were performed.

Results: All attempts to detect/isolate virus, including blood transfer to CD8(+) cell-depleted infant rhesus macaques, were negative, and the animal maintained normal levels of memory CD4(+) T cells in both peripheral blood and gut tissues. However, RAt-9 maintained high levels of anti-SHIV-C humoral and cellular immunity, including reactivity to nonvaccine neoantigens (Nef and Rev), up to 63 months postinitial challenge, suggesting chronic sub-threshold infection. RAt-9 expressed the Mamu A*001 allele negative for B*008 and B*017, had a B13 serotype, and had increased expression of killer-cell immunoglobulin-like receptors (KIRs) previously linked to favorable outcomes of lentiviral infection. Elements of the gene expression profiling coincided with genotyping results. RAt-9 also displayed CD8 cell noncytotoxic antiviral response (CNAR) activity.

Conclusion: Monkey RAt-9 is the first example of a virus-exposed, persistently aviremic animal that has maintained long-term, high-level cellular and humoral antiviral immunity in the absence of an identifiable cryptic reservoir.

Fig. 1. Lasting virus-specific immune reactivity in RAt-9

(a) Timeline of immunizations, virus challenges and follow-up of RAt-9. (b) Sera from the time points indicated were tested by ELISA for Ab binding against SIV Gag, HIV Tat and HIV_CN54 gp120; for nAb activity by TZM-bl assay against SHIV-1157ipEL-p, SHIV_SF162P4 and Env-pseudotyped MW965.26; for ADCC activity against HIV-C gp120-coated target cells; and for ADCVI activity (the two values shown are % inhibition of virus replication using sera at 1:100 dilution and effector cells from different naïve RM; nt - not tested). (c, d) IFN-γ ELISPOTs after stimulation of PBMC collected at the time points indicated with (c): SIV Gag peptides, the Mamu A*001 immunodominant Gag peptide, p11C, or HIV Tat (p11C data are not available for time 0) and (d) peptides of neoantigens SIV Nef and HIV-1 Rev;. (e) Intracellular cytokine staining 51 months post-initial virus exposure showing the frequency of central memory (CM) and effector memory (EM) T-cell subsets expressing the indicated cytokines singly or in combinations following SIV Gag-peptide exposure. (f) Antigen-specific T-cell proliferation in responses to SIV Gag and neoantigens (SIV Nef and HIV-1 Rev). (g) SIV Gag peptides (15 amino acids in length and overlapping by 11) recognized by IFN-γ ELISPOT assay 68 months post-first SHIV-C challenge are indicated by underlining. Peptides were tested individually.

Fig. 2. In vitro and in vivo cell depletion studies, anti-HIV-1 activity of CD8⁺ cell soluble factors, KIR genotyping and gene expression analysis

(a) SHIV-1157ipEL-p replication in RAt-9 (left) PBMC with or without depletion of CD8⁺ T cells or NK cells. As control, PBMC of REm-6 (RAt-9’s sister, right) were tested in parallel. (b) Mitogen stimulated, HIV-1 pNL4-3 (X4 strain)-exposed human CD4⁺ T cells from a healthy donor were incubated in the lower chamber of a Transwell plate insert (Corning) containing mitogen-stimulated CD8⁺ T cells from RAt-9 (left) or REm-6 (right) in the upper chamber of the plate. HIV-1 replication was monitored by measuring the concentration of HIV-1 p24 protein by ELISA. (c) RAt-9 blood transfer to anti-CD8 mAb (cM-T807)-treated infant RM. Naïve infant RM were treated with anti-CD8 mAb on days -4 and -1, and infused with 10 ml of whole blood from RAt-9 on d 0; anti-CD8 mAb was also given on days 3 and 6. Right Y-axis, open symbols: peripheral blood CD8⁺ T cells per μl blood; left Y-axis, closed symbols: plasma viremia levels. (d) KIR3DL Allelic Discrimination Plot of RAt-9: 3 H/H homozygous controls (ctl) and 3 H/Q controls are shown as well as the expression plot for REm-6, RAt-9’s sibling. (e) Functional pathway analysis of differentially expressed genes using Ingenuity pathway analysis (IPA) software. Dashed horizontal line indicates the significance threshold (P= 0.05).