Innate and adaptive immune correlates of vaccine and adjuvant-induced control of mucosal transmission of SIV in macaques

Abstract

Adjuvant effects on innate as well as adaptive immunity may be critical for inducing protection against mucosal HIV and simian immunodeficiency virus (SIV) exposure. We therefore studied effects of Toll-like receptor agonists and IL-15 as mucosal adjuvants on both innate and adaptive immunity in a peptide/poxvirus HIV/SIV mucosal vaccine in macaques, and made three critical observations regarding both innate and adaptive correlates of protection: (i) adjuvant-alone without vaccine antigen impacted the intrarectal SIVmac251 challenge outcome, correlating with surprisingly long-lived APOBEC3G (A3G)-mediated innate immunity; in addition, even among animals receiving vaccine with adjuvants, viral load correlated inversely with A3G levels; (ii) a surprising threshold-like effect existed for vaccine-induced adaptive immunity control of viral load, and only antigen-specific polyfunctional CD8(+) T cells correlated with protection, not tetramer(+) T cells, demonstrating the importance of T-cell quality; (iii) synergy was observed between Toll-like receptor agonists and IL-15 for driving adaptive responses through the up-regulation of IL-15Ralpha, which can present IL-15 in trans, as well as for driving the innate A3G response. Thus, strategic use of molecular adjuvants can provide better mucosal protection through induction of both innate and adaptive immunity.

Fig. 1.

Protective efficacy of TLR agonists and IL-15 adjuvanted peptide-primed/rMVA-boosted vaccine regimens. (A) Schedule of intracolorectal immunization protocol. (B) Levels of viral RNA in plasma of the individual animals (denoted by single letters), as well as geometric means of the groups with different immunization protocols following intrarectal challenge with 10 ID₅₀ SIVmac251. *, Mamu-B*17⁺ animals. (C) VL in the colon and spleen were measured at the time of necropsy. Comparisons among groups were performed by two-sided Wilcoxon's rank-sum tests. Bars show mean and SEM of Log₁₀.

Fig. 2.

Cellular immune responses before viral challenge. (A) The sum of CD3⁺CD8⁺ Mamu-A*01-tetramer⁺ cell frequencies (for six epitopes) were measured in the colonic lamina propria at week 19. (B) Within the CM9⁺ CD3⁺CD8⁺ cell population, central memory T lymphocyte (TCM: CD28⁺CD95⁺) and effector memory T lymphocyte (TEM: CD28^-CD95⁺) responses were assessed by eight-color intracellular cytokine staining (ICS) assays. (C) Comparison of CD3⁺CD8⁺ tetramer⁺ cell frequency in different compartments was performed by two-tailed Wilcoxon's matched-pairs rank tests. (D and F) Frequencies of CD8⁺ T cell response (triple positive for IFNγ, IL-2, and TNFα) and CD4⁺ T cell response (IFNγ⁺) in PBMCs were measured at week 19 and comparisons among the groups were performed by two-tailed Wilcoxon's rank sum tests. (E) Correlation between prechallenge CD8⁺ triple-functional T-cell response frequency and set-point plasma VL was evaluated by three-decay dose-dependent inhibition curve with R value shown, and the P value was calculated by Fisher's exact test for the 2 × 2 comparison log VL < or > 4 and log % polyfunctional T cells < or > 0.3 (log of 2). The animals in different groups were color-coded: group 1 in red, group 2 in blue, group 3 in green, and group 4 in orange. Data show mean and SEM.

Fig. 3.

A3G expression levels before viral challenge. (A) Relative mRNA expression level of A3G in the MLN at week 19 was measured by quantitative real-time RT-PCR using macaque-specific A3G primers/probe. Comparisons among the groups were performed by two-sided Wilcoxon's rank sum tests. (B) Correlations between set-point plasma VL and the relative A3G mRNA level in the MLN for all of the animals (black line) were evaluated by a two-tailed Spearman's rank test, and for the animals in the adjuvant only group (purple line and purple filled circles) by a linear regression analysis according to the nature of their distributions. The animals in different groups were color-coded: group 1 in red, group 2 in blue, group 3 in green, group 4 in orange, and group 5 in purple. (C) Comparison of relative mRNA expression level of A3G in pre- and postvaccination PBMC samples from animals receiving both TLR agonists and IL-15 with or without Vac (groups 3 and 5) were assessed by Wilcoxon's matched pairs rank test. (D and E) Different A3G⁺ cell populations were measured by ICS staining in the MLNs (D) and colon IEL (E) of representative animals from different immunization groups (at week 19). (F) Correlations between set-point plasma VL and A3G expression level in the Colon IEL were evaluated by a two-tailed Spearman's rank test, Data show mean and SEM.

Fig. 4.

Mechanisms of the synergistic effects of TLR agonists and IL-15 to induce cellular and innate immunity. Healthy macaque PBMCs were treated with TLR agonists or IL-15 for 3 days and A3G⁺ (A) and IL-15Rα⁺ (B) cell populations were measured by eight-color ICS/surface assays. Monoclonal antibody blocking human IFNα/β receptor chain 2 (50 μg/mL) was used to pretreat the cells for 30 min before TLR agonists and IL-15 were added. Results are representative of at least three independent experiments. (C) The proposed synergy model for TLR agonists and IL-15 to induce cellular and innate immunity. Upon the stimulation of TLR agonists, accessory cells were induced to express type I IFN, which then up-regulates A3G and IL-15Rα expression. As a consequence, surface IL-15Rα binds IL-15, which was provided with vaccine, and delivers an IL-15 signal to neighboring T cells/NK cells through transpresentation. In addition, the IL-15/IL-15Rα complex on the cell surface contributes to the long survival of CD8 memory T cells via recycling of the complex. IL-15 might synergize with TLR agonists to induce A3G and IL-15Rα. (D and E) Different IL-15Rα⁺ cell populations (Methods) were measured by surface staining in the MLNs of representative animals from different immunization groups. Data show mean and SEM.