Chronic phase HIV/SIV replication is reduced by as much as 10,000-fold in elite controllers (EC) compared to typical progressors, but sufficient viral replication persists in EC tissues to allow viral sequence evolution and induce excess immune activation. Here, we show that productive SIV infection in rhesus monkey EC is strikingly restricted to follicular helper CD4+ T cells (TFH), suggesting that while the potent SIV-specific CD8+ T cells of these monkeys can effectively clear productive infection from extra-follicular sites, their relative exclusion from B cell follicles limits elimination of infected TFH. Indeed, CD8+ lymphocyte depletion of EC monkeys resulted in a dramatic re-distribution of productive SIV infection to non-TFH, with TFH restriction resuming upon CD8+ T cell recovery. Thus, B cell follicles constitute sanctuaries for persistent SIV replication in the presence of potent anti-viral CD8+ T cell responses, potentially complicating efforts to cure HIV infection with therapeutic vaccination or T cell immunotherapy.

Live attenuated SIV vaccines (LAVs) remain the most efficacious of all vaccines in nonhuman primate (NHP) models of HIV/AIDS, yet the basis of their robust protection remains poorly understood. Here, we demonstrate that the degree of LAV-mediated protection against intravenous wildtype SIVmac239 challenge strongly correlates with the magnitude and function of SIV-specific, effector-differentiated T cells in lymph node, but not with such T cell responses in blood or with other cellular, humoral and innate immune parameters. Maintenance of protective T cell responses was associated with persistent LAV replication in lymph node, which occurred almost exclusively in follicular helper T cells. Thus, effective LAVs maintain lymphoid tissue-based, effector-differentiated, SIV-specific T cells that intercept and suppress early wildtype SIV amplification and, if present in sufficient frequencies, can completely control and perhaps clear infection, an observation that provides rationale for development of safe, persistent vectors that can elicit and maintain such responses.

Prophylactic vaccination of rhesus macaques with rhesus cytomegalovirus (RhCMV) vectors expressing simian immunodeficiency virus (SIV) antigens (RhCMV/SIV) elicits immune responses that stringently control highly pathogenic SIV infection, with subsequent apparent clearance of the infection, in ~50% of vaccinees. In contrast, here, we show that therapeutic RhCMV/SIV vaccination of rhesus macaques previously infected with SIV and given continuous combination antiretroviral therapy (cART) beginning 4-9 d post-SIV infection does not mediate measurable SIV reservoir clearance during over 600 d of follow-up on cART relative to RhCMV/control vaccination. However, none of the six animals started on cART on day four or five, across both RhCMV/SIV- and RhCMV/control-vaccinated groups, those rhesus macaques with SIV reservoirs most closely resembling those of prophylactically RhCMV/SIV-vaccinated and protected animals early in their course, showed post-cART viral rebound with up to nine months of follow-up. Moreover, at necropsy, these rhesus macaques showed little to no evidence of replication-competent SIV. These results suggest that the early SIV reservoir is limited in durability and that effective blockade of viral replication and spread in this critical time window by either pharmacologic or immunologic suppression may result in reduction, and potentially loss, of rebound-competent virus over a period of ~two years.

Although X4 tropic SHIVs have been studied extensively, they show distinct infection phenotypes from those of R5 tropic viruses, which play an important role in HIV-1 transmission and pathogenesis. To augment the variety of R5 tropic SHIVs, we generated a new R5 tropic SHIV from the highly pathogenic X4 tropic SHIV-KS661, a derivative of SHIV-89.6. Based on consensus amino acid alignment analyses of subtype B R5 tropic HIV-1, five amino acid substitutions in the third variable region successfully changed the secondary receptor preference from X4 to R5. Improvements in viral replication were observed in infected rhesus macaques after two passages, and reisolated virus was designated SHIV-MK38. SHIV-MK38 maintained R5 tropism through in vivo passages and showed robust replication in infected monkeys. Our study clearly demonstrates that a minimal number of amino acid substitutions in the V3 region can alter secondary receptor preference and increase the variety of R5 tropic SHIVs.

Simian immunodeficiency virus (SIV) challenge of rhesus macaques (RMs) vaccinated with strain 68–1 Rhesus Cytomegalovirus (RhCMV) vectors expressing SIV proteins (RhCMV/SIV) results in a binary outcome: stringent control and subsequent clearance of highly pathogenic SIV in ~55% of vaccinated RMs with no protection in the remaining 45%. Although previous work indicates that unconventionally restricted, SIV-specific, effector-memory (EM)-biased CD8+ T cell responses are necessary for efficacy, the magnitude of these responses does not predict efficacy, and the basis of protection vs. non-protection in 68–1 RhCMV/SIV vector-vaccinated RMs has not been elucidated. Here, we report that 68–1 RhCMV/SIV vector administration strikingly alters the whole blood transcriptome of vaccinated RMs, with the sustained induction of specific immune-related pathways, including immune cell, toll-like receptor (TLR), inflammasome/cell death, and interleukin-15 (IL-15) signaling, significantly correlating with subsequent vaccine efficacy. Treatment of a separate RM cohort with IL-15 confirmed the central involvement of this cytokine in the protection signature, linking the major innate and adaptive immune gene expression networks that correlate with RhCMV/SIV vaccine efficacy. This change-from-baseline IL-15 response signature was also demonstrated to significantly correlate with vaccine efficacy in an independent validation cohort of vaccinated and challenged RMs. The differential IL-15 gene set response to vaccination strongly correlated with the pre-vaccination activity of this pathway, with reduced baseline expression of IL-15 response genes significantly correlating with higher vaccine-induced induction of IL-15 signaling and subsequent vaccine protection, suggesting that a robust de novo vaccine-induced IL-15 signaling response is needed to program vaccine efficacy. Thus, the RhCMV/SIV vaccine imparts a coordinated and persistent induction of innate and adaptive immune pathways featuring IL-15, a known regulator of CD8+ T cell function, that support the ability of vaccine-elicited unconventionally restricted CD8+ T cells to mediate protection against SIV challenge.

To define the contribution of CD8 + T cell responses to control of SIV reactivation during and following antiretroviral therapy (ART), we determined the effect of long-term CD8 + T cell depletion using a rhesusized anti-CD8β monoclonal antibody (mAb) on barcoded SIVmac239 dynamics on stable ART and after ART cessation in Rhesus Macaques (RMs). Among the RMs with full CD8 + T cell depletion in both blood and tissue, there were no significant differences in the frequency of viral blips in plasma, the number of SIV RNA + cells and the average number of RNA copies/infected cell in tissue, and levels of cell-associated SIV RNA and DNA in blood and tissue relative to control-treated RM during ART. Upon ART cessation, both CD8 + T cell-depleted and control RMs rebounded in <12 days with no difference in the time to viral rebound, or in either the number or growth rate of rebounding SIVmac239M barcode clonotypes. However, effectively CD8 + T cell-depleted RMs showed a stable ~2-log increase in post-ART plasma viremia relative to controls. These results indicate that while potent anti-viral CD8 + T cell responses can develop during ART-suppressed SIV infection, these responses effectively intercept post-ART SIV rebound only after systemic viral replication, too late to limit reactivation frequency or the early spread of reactivating SIV reservoirs.

No abstract

Activation of the NF-κB signaling pathway by Protein Kinase C (PKC) agonists is a potent mechanism for human immunodeficiency virus (HIV) latency disruption in vitro. However, significant toxicity risks and the lack of evidence supporting their activity in vivo have limited further evaluation of PKC agonists as HIV latency-reversing agents (LRA) in cure strategies. Here we evaluated whether GSK445A, a stabilized ingenol-B derivative, can induce HIV/simian immunodeficiency virus (SIV) transcription and virus production in vitro and demonstrate pharmacological activity in nonhuman primates (NHP). CD4+ T cells from people living with HIV and from SIV+ rhesus macaques (RM) on antiretroviral therapy (ART) exposed in vitro to 25 nM of GSK445A produced cell-associated viral transcripts as well as viral particles at levels similar to those induced by PMA/Ionomycin, indicating that GSK445A can potently reverse HIV/SIV latency. Importantly, these concentrations of GSK445A did not impair the proliferation or survival of HIV-specific CD8+ T cells, but instead, increased their numbers and enhanced IFN-γ production in response to HIV peptides. In vivo, GSK445A tolerability was established in SIV-naïve RM at 15 μg/kg although tolerability was reduced in SIV-infected RM on ART. Increases in plasma viremia following GSK445A administration were suggestive of increased SIV transcription in vivo. Collectively, these results indicate that GSK445A is a potent HIV/SIV LRA in vitro and has a tolerable safety profile amenable for further evaluation in vivo in NHP models of HIV cure/remission.