CD8+ lymphocytes do not impact SIV reservoir establishment under ART.

Persistence of the human immunodeficiency virus type-1 (HIV-1) latent reservoir in infected individuals remains a problem despite fully suppressive antiretroviral therapy (ART). While reservoir formation begins during acute infection, the mechanisms responsible for its establishment remain unclear. CD8+ T cells are important during the initial control of viral replication. Here we examined the effect of CD8+ T cells on formation of the latent reservoir in simian immunodeficiency virus (SIV)-infected macaques by performing experimental CD8+ depletion either before infection or before early (that is, day 14 post-infection) ART initiation. We found that CD8+ depletion resulted in slower decline of viremia, indicating that CD8+ lymphocytes reduce the average lifespan of productively infected cells during acute infection and early ART, presumably through SIV-specific cytotoxic T lymphocyte (CTL) activity. However, CD8+ depletion did not change the frequency of infected CD4+ T cells in the blood or lymph node as measured by the total cell-associated viral DNA or intact provirus DNA assay. In addition, the size of the persistent reservoir remained the same when measuring the kinetics of virus rebound after ART interruption. These data indicate that during early SIV infection, the viral reservoir that persists under ART is established largely independent of CTL control.

Combination of CD8ß Depletion and Interleukin-15 Superagonist N-803 Induces Virus Reactivation in Simian-Human Immunodeficiency Virus-Infected, Long-Term ART-Treated Rhesus Macaques.

The "shock and kill" strategy predicates that virus reactivation in latently infected cells is required to eliminate the human immunodeficiency virus (HIV) reservoir. In a recent study, we showed robust and persistent induction of plasma viremia in antiretroviral therapy (ART)-treated simian immunodeficiency virus-infected rhesus macaques (RMs) undergoing CD8α depletion and treated with the interleukin-15 (IL-15) superagonist N-803 (J. B. McBrien et al., Nature 578:154-159, 2020, https://doi.org/10.1038/s41586-020-1946-0). Of note, in that study we used an antibody targeting CD8α, thereby depleting NK cells, NKT cells, and γδ T cells, in addition to CD8+ T cells. In the current proof-of-concept study, we tested whether virus reactivation can be induced by administration of N-803 to simian-human chimeric immunodeficiency virus-infected, ART-treated RMs that are selectively depleted of CD8+ T cells via the CD8ß-targeting antibody CD8b255R1. CD8ß depletion was performed in five SHIVSF162P3-infected RMs treated with ART for 12 months and with plasma viremia consistently below 3 copies/ml. All animals received four weekly doses of N-803 starting at the time of CD8b255R1 administration. The induction of detectable plasma viremia was observed in three out of five RMs, with the level of virus reactivation seemingly correlated with the frequency of CD8+ T cells following CD8ß depletion as well as the level of virus reactivation observed when the same animals underwent CD8α depletion and N-803 administration after 24 weeks of ART. These data indicate that CD8ß depletion and N-803 administration can induce virus reactivation in SHIVSF162P3-infected RMs despite suboptimal depletion of CD8+ T cells and profound ART-induced suppression of virus replication, confirming a critical role for these cells in suppressing virus production and/or reactivation in vivo under ART.IMPORTANCE The "shock and kill" HIV cure strategy attempts to reverse and eliminate the latent viral infection that prevents eradication of the virus. Latency-reversing agents tested in clinical trials to date have failed to affect the HIV viral reservoir. IL-15 superagonist N-803, currently involved in a clinical trial for HIV cure, was recently shown by our laboratory to induce robust and persistent induction of plasma viremia during ART in three in vivo animal models of HIV infection. These results suggest a substantial role for CD8+ lymphocytes in suppressing the latency reversal effect of N-803 by promoting the maintenance of viral latency. In this study, we tested whether the use of a CD8ß-targeting antibody, which would specifically deplete CD8+ T cells, would yield similar levels of virus reactivation. We observed the induction of plasma viremia, which correlated with the efficacy of the CD8 depletion strategy.

Bone Marrow-Derived CD4+ T Cells Are Depleted in Simian Immunodeficiency Virus-Infected Macaques and Contribute to the Size of the Replication-Competent Reservoir.

The bone marrow (BM) is the key anatomic site for hematopoiesis and plays a significant role in the homeostasis of mature T cells. However, very little is known on the phenotype of BM-derived CD4+ T cells, their fate during simian immunodeficiency virus (SIV) infection, and their contribution to viral persistence during antiretroviral therapy (ART). In this study, we characterized the immunologic and virologic status of BM-derived CD4+ T cells in rhesus macaques prior to SIV infection, during the early chronic phase of infection, and during ART. We found that BM memory CD4+ T cells are significantly depleted following SIV infection, at levels that are similar to those measured in the peripheral blood (PB). In addition, BM-derived memory CD4+ T cells include a high frequency of cells that express the coinhibitory receptors CTLA-4 and PD-1, two subsets previously shown to be enriched in the viral reservoir; these cells express Ki-67 at levels similar to or higher than the same cells in PB. Finally, when we analyzed SIV-infected RMs in which viral replication was effectively suppressed by 12 months of ART, we found that BM CD4+ T cells harbor SIV DNA and SIV RNA at levels comparable to those of PB CD4+ T cells, including replication-competent SIV. Thus, BM is a largely understudied anatomic site of the latent reservoir which contributes to viral persistence during ART and needs to be further characterized and targeted when designing therapies for a functional or sterilizing cure to HIV.IMPORTANCE The latent viral reservoir is one of the major obstacles in purging the immune system of HIV. It is paramount that we elucidate which anatomic compartments harbor replication-competent virus, which upon ART interruption results in viral rebound and pathogenesis. In this study, using the rhesus macaque model of SIV infection and ART, we examined the immunologic status of the BM and its role as a potential sanctuary for latent virus. We found that the BM compartment undergoes a similar depletion of memory CD4+ T cells as PB, and during ART treatment the BM-derived memory CD4+ T cells contain high levels of cells expressing CTLA-4 and PD-1, as well as amounts of cell-associated SIV DNA, SIV RNA, and replication-competent virus comparable to those in PB. These results enrich our understanding of which anatomic compartments harbor replication virus and suggest that BM-derived CD4+ T cells need to be targeted by therapeutic strategies aimed at achieving an HIV cure.

Antibody-Mediated CD4 Depletion Induces Homeostatic CD4+ T Cell Proliferation without Detectable Virus Reactivation in Antiretroviral Therapy-Treated Simian Immunodeficiency Virus-Infected Macaques.

A major barrier to human immunodeficiency virus (HIV) eradication is the long-term persistence of latently infected CD4+ T cells harboring integrated replication-competent virus. It has been proposed that the homeostatic proliferation of these cells drives long-term reservoir persistence in the absence of virus reactivation, thus avoiding cell death due to either virus-mediated cytopathicity or immune effector mechanisms. Here, we conducted an experimental depletion of CD4+ T cells in eight antiretroviral therapy (ART)-treated, simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs) to determine whether the homeostatically driven CD4+ T-cell proliferation that follows CD4+ T-cell depletion results in reactivation of latent virus and/or expansion of the virus reservoir. After administration of the CD4R1 antibody, we observed a CD4+ T cell depletion of 65 to 89% in peripheral blood and 20 to 50% in lymph nodes, followed by a significant increase in CD4+ T cell proliferation during CD4+ T cell reconstitution. However, this CD4+ T cell proliferation was not associated with detectable increases in viremia, indicating that the homeostatic activation of CD4+ T cells is not sufficient to induce virus reactivation from latently infected cells. Interestingly, the homeostatic reconstitution of the CD4+ T cell pool was not associated with significant changes in the number of circulating cells harboring SIV DNA compared to results for the first postdepletion time point. This study indicates that, in ART-treated SIV-infected RMs, the homeostasis-driven CD4+ T-cell proliferation that follows experimental CD4+ T-cell depletion occurs in the absence of detectable reactivation of latent virus and does not increase the size of the virus reservoir as measured in circulating cells.IMPORTANCE Despite successful suppression of HIV replication with antiretroviral therapy, current treatments are unable to eradicate the latent virus reservoir, and treatment interruption almost invariably results in the reactivation of HIV even after decades of virus suppression. Homeostatic proliferation of latently infected cells is one mechanism that could maintain the latent reservoir. To understand the impact of homeostatic mechanisms on virus reactivation and reservoir size, we experimentally depleted CD4+ T cells in ART-treated SIV-infected rhesus macaques and monitored their homeostatic rebound. We find that depletion-induced proliferation of CD4+ T cells is insufficient to reactivate the viral reservoir in vivo Furthermore, the proportion of SIV DNA+ CD4+ T cells remains unchanged during reconstitution, suggesting that the reservoir is resistant to this mechanism of expansion at least in this experimental system. Understanding how T cell homeostasis impacts latent reservoir longevity could lead to the development of new treatment paradigms aimed at curing HIV infection.