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The rebound-competent viral reservoir (RCVR), comprised of virus that is able to persist during antiretroviral therapy (ART) and mediate reactivation of systemic viral replication and rebound viremia after antiretroviral therapy interruption (ATI), remains the biggest obstacle to the eradication of HIV infection. A better understanding of the cellular and tissue origins and the dynamics of viral populations that initiate rebound upon ATI could help develop targeted therapeutic strategies for reducing the RCVR. In this study, barcoded SIVmac239M was used to infect rhesus macaques to enable monitoring of viral barcode clonotypes contributing to virus detectable in plasma after ATI. Blood, lymphoid tissues (LTs, spleen, mesenteric and inguinal lymph nodes), and non-lymphoid tissues (NLTs, colon, ileum, lung, liver, and brain) were analyzed using viral barcode sequencing, intact proviral DNA assay, single-cell RNA sequencing, and combined CODEX/RNAscope/ in situ hybridization. Four of seven animals had viral barcodes detectable by deep sequencing of plasma at necropsy although plasma viral RNA remained < 22 copies/mL. Among the tissues studied, mesenteric and inguinal lymph nodes, and spleen contained viral barcodes detected in plasma, and trended to have higher cell-associated viral loads, higher intact provirus levels, and greater diversity of viral barcodes. CD4+ T cells were the main cell type harboring viral RNA (vRNA) after ATI. Further, T cell zones in LTs showed higher vRNA levels than B cell zones for most animals. These findings are consistent with LTs contributing to virus present in plasma early after ATI. ONE SENTENCE SUMMARY: The reemerging of SIV clonotypes at early post-ATI are likely from the secondary lymphoid tissues.

SUMMARY PARAGRAPHTreatment of people with HIV (PWH) with antiretroviral therapy (ART) results in sustained suppression of viremia, but HIV persists indefinitely as integrated provirus in CD4-expressing cells. Intact persistent provirus, the “rebound competent viral reservoir” (RCVR), is the primary obstacle to achieving a cure. Most variants of HIV enter CD4+T cells by binding to the chemokine receptor, CCR5. The RCVR has been successfully depleted only in a handful of PWH following cytotoxic chemotherapy and bone marrow transplantation from donors with a mutation inCCR5. Here we show that long-term SIV remission and apparent cure can be achieved for infant macaques via targeted depletion of potential reservoir cells that express CCR5. Neonatal rhesus macaques were infected with virulent SIVmac251, then treated with ART beginning one week after infection, followed by treatment with either a CCR5/CD3-bispecific or a CD4-specific antibody, both of which depleted target cells and increased the rate of plasma viremia decrease. Upon subsequent cessation of ART, three of seven animals treated with CCR5/CD3-bispecific antibody rebounded quickly and two rebounded 3 or 6 months later. Remarkably, the other two animals remained aviremic and efforts to detect replication-competent virus were unsuccessful. Our results show that bispecific antibody treatment can achieve meaningful SIV reservoir depletion and suggest that functional HIV cure might be achievable for recently infected individuals having a restricted reservoir.

The strain 68-1 rhesus cytomegalovirus (RhCMV)–based vaccine for simian immunodeficiency virus (SIV) can stringently protect rhesus macaques (RMs) from SIV challenge by arresting viral replication early in primary infection. This vaccine elicits unconventional SIV-specific CD8 + T cells that recognize epitopes presented by major histocompatibility complex (MHC)–II and MHC-E instead of MHC-Ia. Although RhCMV/SIV vaccines based on strains that only elicit MHC-II– and/or MHC-Ia–restricted CD8 + T cells do not protect against SIV, it remains unclear whether MHC-E–restricted T cells are directly responsible for protection and whether these responses can be separated from the MHC-II–restricted component. Using host microRNA (miR)–mediated vector tropism restriction, we show that the priming of MHC-II and MHC-E epitope–targeted responses depended on vector infection of different nonoverlapping cell types in RMs. Selective inhibition of RhCMV infection in myeloid cells with miR-142–mediated tropism restriction eliminated MHC-E epitope–targeted CD8 + T cell priming, yielding an exclusively MHC-II epitope–targeted response. Inhibition with the endothelial cell–selective miR-126 eliminated MHC-II epitope–targeted CD8 + T cell priming, yielding an exclusively MHC-E epitope–targeted response. Dual miR-142 + miR-126–mediated tropism restriction reverted CD8 + T cell responses back to conventional MHC-Ia epitope targeting. Although the magnitude and differentiation state of these CD8 + T cell responses were generally similar, only the vectors programmed to elicit MHC-E–restricted CD8 + T cell responses provided protection against SIV challenge, directly demonstrating the essential role of these responses in RhCMV/SIV vaccine efficacy.

The primary human immunodeficiency virus (HIV) reservoir is composed of resting memory CD4+ T cells, which often express the immune checkpoint receptors programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4), which limit T cell activation via synergistic mechanisms. Using simian immunodeficiency virus (SIV)-infected, long-term antiretroviral therapy (ART)-treated rhesus macaques, we demonstrate that PD-1, CTLA-4 and dual CTLA-4/PD-1 immune checkpoint blockade using monoclonal antibodies is well tolerated, with evidence of bioactivity in blood and lymph nodes. Dual blockade was remarkably more effective than PD-1 blockade alone in enhancing T cell cycling and differentiation, expanding effector-memory T cells and inducing robust viral reactivation in plasma and peripheral blood mononuclear cells. In lymph nodes, dual CTLA-4/PD-1 blockade, but not PD-1 alone, decreased the total and intact SIV-DNA in CD4+ T cells, and SIV-DNA and SIV-RNA in B cell follicles, a major site of viral persistence during ART. None of the tested interventions enhanced SIV-specific CD8+ T cell responses during ART or viral control after ART interruption. Thus, despite CTLA-4/PD-1 blockade inducing robust latency reversal and reducing total levels of integrated virus, the degree of reservoir clearance was still insufficient to achieve viral control. These results suggest that immune checkpoint blockade regimens targeting PD-1 and/or CTLA-4, if performed in people living with HIV with sustained aviremia, are unlikely to induce HIV remission in the absence of additional interventions.

Structural characterization of the HIV-1 Envelope (Env) glycoprotein has facilitated the development of Env probes to isolate HIV-specific monoclonal antibodies (mAbs). However, preclinical studies have largely evaluated these virus-specific mAbs against chimeric viruses, which do not naturally infect non-human primates, in contrast to the unconstrained simian immunodeficiency virus (SIV)mac239 clone. Given the paucity of native-like reagents for the isolation of SIV-specific B cells, we examined a method to isolate SIVmac239-specific mAbs without using Env probes. We first activated virus-specific B cells by inducing viral replication after the infusion of a CD8β-depleting mAb or withdrawal of antiretroviral therapy in SIVmac239-infected rhesus macaques. Following the rise in viremia, we observed 2- to 4-fold increases in the number of SIVmac239 Env-reactive plasmablasts in circulation. We then sorted these activated B cells and obtained 206 paired Ab sequences. After expressing 122 mAbs, we identified 14 Env-specific mAbs. While these Env-specific mAbs bound to both the SIVmac239 SOSIP.664 trimer and to infected primary rhesus CD4+ T cells, five also neutralized SIVmac316. Unfortunately, none of these mAbs neutralized SIVmac239. Our data show that this method can be used to isolate virus-specific mAbs without antigenic probes by inducing bursts of contemporary replicating viruses in vivo. Keywords: SIV, monoclonal antibodies, rhesus macaques, CD8b depletion, antiretroviral therapy interruption

In people living with HIV (PLWH) on antiretroviral therapy (ART), virus persists in a latent form where there is minimal transcription or protein expression. Latently infected cells are a major barrier to curing HIV. Increasing HIV transcription and viral production in latently infected cells could facilitate immune recognition and reduce the pool of infected cells that persist on ART. Given that programmed cell death protein 1 (PD-1) expressing CD4 + T cells are preferentially infected with HIV in PLWH on ART, we aimed to determine whether administration of antibodies targeting PD-1 would reverse HIV latency in vivo. We therefore evaluated the impact of intravenous administration of pembrolizumab every 3 weeks on HIV latency in 32 PLWH and cancer on ART. After the first infusion of anti–PD-1, we observed a median 1.32-fold increase in unspliced HIV RNA and 1.61-fold increase in unspliced RNA:DNA ratio in sorted blood CD4 + T cells compared to baseline. We also observed a 1.65-fold increase in plasma HIV RNA. The frequency of CD4 + T cells with inducible virus evaluated using the tat/rev limiting dilution assay was higher after 6 cycles compared to baseline. Phylogenetic analyses of HIV env sequences in a participant who developed low concentrations of HIV viremia after 6 cycles of pembrolizumab did not demonstrate clonal expansion of HIV-infected cells. These data are consistent with anti–PD-1 being able to reverse HIV latency in vivo and support the rationale for combining anti–PD-1 with other interventions to reduce the HIV reservoir.

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., Author summary Antiretroviral therapy (ART) is not a definitive cure for HIV infection, in part, because the virus is able to integrate its genetic material in the host cell and remain in a dormant but fully replication-competent form during ART. These latently-infected cells can persist for long periods of time and remain hidden from the host’s immune system. If ART is stopped, the virus can reactivate from this pool of infected cells and resume HIV replication and disease progression. As such, finding and eliminating cells with latent HIV infection is priority for HIV cure research. One approach is to use compounds referred to as latency-reversing agents, that can induce HIV reactivation during ART. The goal of this approach is to facilitate elimination of infected cells by the virus itself once it reactivates or by the host’s immune system, once virus induction renders the cells detectable by the immune system, while also preventing the virus from infecting new cells due to the continued presence of ART. In this study we report on the activity of a novel latency-reversing agent called GSK445A, a potent activator of the enzyme protein kinase C (PKC). We show that GSK445A can induce HIV and simian immunodeficiency virus (SIV) latency reversal in vitro and has a tolerable saftey profile in nonhuman primates that should permit further testing of this PKC-agonist in strategies to cure HIV.

Although IL-15 has been implicated in the pathogenic hyperimmune activation that drives progressive HIV and SIV infection, as well as in the generation of HIV/SIV target cells, it also supports NK and T cell homeostasis and effector activity, potentially benefiting the host. To understand the role of IL-15 in SIV infection and pathogenesis, we treated two cohorts of SIVmac239-infected rhesus macaques (RM; Macaca mulatta), one with chronic infection, the other with primary infection, with a rhesusized, IL-15–neutralizing mAb (versus an IgG isotype control) for up to 10 wk (n = 7–9 RM per group). In both cohorts, anti–IL-15 was highly efficient at blocking IL-15 signaling in vivo, causing 1) profound depletion of NK cells in blood and tissues throughout the treatment period; 2) substantial, albeit transient, depletion of CD8+ effector memory T cells (TEM) (but not the naive and central memory subsets); and 3) CD4+ and CD8+ TEM hyperproliferation. In primary infection, reduced frequencies of SIV-specific effector T cells in an extralymphoid tissue site were also observed. Despite these effects, the kinetics and extent of SIV replication, CD4+ T cell depletion, and the onset of AIDS were comparable between anti–IL-15– and control-treated groups in both cohorts. However, RM treated with anti–IL-15 during primary infection manifested accelerated reactivation of RM rhadinovirus. Thus, IL-15 support of NK cell and TEM homeostasis does not play a demonstrable, nonredundant role in SIV replication or CD4+ T cell deletion dynamics but may contribute to immune control of oncogenic γ-herpesviruses.

SUMMARYSIVmac239 infection of macaques is a favored model of human HIV infection. However, the SIVmac239 envelope (Env) trimer structure, glycan occupancy, and the targets and ability of neutralizing antibodies (nAbs) to protect against SIVmac239 remain unknown. Here, we report the isolation of SIVmac239 nAbs that recognize a glycan hole and the V1/V4 loop. A high-resolution structure of a SIVmac239 Env trimer-nAb complex shows many similarities to HIV and SIVcpz Envs, but with distinct V4 features and an extended V1 loop. Moreover, SIVmac239 Env has a higher glycan shield density than HIV Env that may contribute to poor or delayed nAb responses in SIVmac239-infected macaques. Passive transfer of a nAb protects macaques from repeated low dose intraveneous SIVmac239 challenge at serum titers comparable to those described for protection of humans against HIV infection. Our results provide structural insights for vaccine design and shed light on antibody-mediated protection in the SIV model.

Simian immunodeficiency viruses (SIVs) are lentiviruses that naturally infect non-human primates of African origin and seeded cross-species transmissions of HIV-1 and HIV-2. Here we report prefusion stabilization and cryo-EM structures of soluble envelope (Env) trimers from rhesus macaque SIV (SIV

Primary HIV-1 infection can be classified into six Fiebig stages based on virological and serological laboratory testing, whereas simian-HIV (SHIV) infection in nonhuman primates (NHPs) is defined in time post-infection, making it difficult to extrapolate NHP experiments to the clinics. We identified and extensively characterized the Fiebig-equivalent stages in NHPs challenged intrarectally or intravenously with SHIV(AD8-EO). During the first month post-challenge, intrarectally challenged monkeys were up to 1 week delayed in progression through stages. However, regardless of the challenge route, stages I–II predominated before, and stages V–VI predominated after, peak viremia. Decrease in lymph node (LN) CD4(+) T cell frequency and rise in CD8(+) T cells occurred at stage V. LN virus-specific CD8(+) T cell responses, dominated by degranulation and TNF, were first detected at stage V and increased at stage VI. A similar late elevation in follicular CXCR5(+) CD8(+) T cells occurred, consistent with higher plasma CXCL13 levels at these stages. LN SHIV(AD8-EO) RNA(+) cells were present at stage II, but appeared to decline at stage VI when virions accumulated in follicles. Fiebig-equivalent staging of SHIV(AD8-EO) infection revealed concordance of immunological events between intrarectal and intravenous infection despite different infection progressions, and can inform comparisons of NHP studies with clinical data.

Strain 68-1 rhesus cytomegalovirus expressing simian immunodeficiency virus (SIV) antigens (RhCMV/SIV) primes MHC-E-restricted CD8+ T cells that control SIV replication in 50%-60% of the vaccinated rhesus macaques. Whether this unconventional SIV-specific immunity and protection is unique to rhesus macaques or RhCMV or is intrinsic to CMV remains unknown. Here, using cynomolgus CMV vectors expressing SIV antigens (CyCMV/SIV) and Mauritian cynomolgus macaques, we demonstrate that the induction of MHC-E-restricted CD8+ T cells requires matching CMV to its host species. RhCMV does not elicit MHC-E-restricted CD8+ T cells in cynomolgus macaques. However, cynomolgus macaques vaccinated with species-matched 68-1-like CyCMV/SIV mounted MHC-E-restricted CD8+ T cells, and half of the vaccinees stringently controlled SIV post-challenge. Protected animals manifested a vaccine-induced IL-15 transcriptomic signature that is associated with efficacy in rhesus macaques. These findings demonstrate that the ability of species-matched CMV vectors to elicit MHC-E-restricted CD8+ T cells that are required for anti-SIV efficacy is conserved in nonhuman primates, and these data support the development of HCMV/HIV for a prophylactic HIV vaccine.

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., Author summary SIV insert-expressing vaccine vectors based on strain 68–1 RhCMV elicit robust, highly effector-memory-biased, unconventionally restricted T cell responses that are associated with an unprecedented level of SIV control after challenge (replication arrest leading to clearance) in slightly over half of vaccinated monkeys. Since efficacy among monkeys vaccinated with the effective 68–1 vaccine is not predicted by standard measures of immunogenicity, we used functional genomics analysis of RhCMV/SIV vaccinated monkeys with known challenge outcomes to identify immune correlates of protection. We found that vaccine efficacy significantly correlates with a vaccine-induced response to IL-15 that includes modulation of immune cell, inflammation, TLR signaling, and cell death programming response pathways. These data suggest that RhCMV/SIV efficacy results from a coordinated and sustained vaccine-mediated induction of innate and adaptive immune pathways featuring IL-15, a known regulator of CD8+ effector-memory T cell function, that cooperates with vaccine-elicited CD8+ T cells to mediate efficacy.

A prophylactic vaccine that confers durable protection against human immunodeficiency virus (HIV) would provide a valuable tool to prevent new HIV/AIDS cases. As herpesviruses establish lifelong infections that remain largely subclinical, the use of persistent herpesvirus vectors to deliver HIV antigens may facilitate the induction of long-term anti-HIV immunity. We previously developed recombinant (r) forms of the gamma-herpesvirus rhesus monkey rhadinovirus (rRRV) expressing a replication-incompetent, near-full-length simian immunodeficiency virus (SIVnfl) genome. We recently showed that 8/16 rhesus macaques (RMs) vaccinated with a rDNA/rRRV-SIVnfl regimen were significantly protected against intrarectal (i.r.) challenge with SIVmac239. Here we investigated the longevity of this vaccine-mediated protection. Despite receiving no additional booster immunizations, the protected rDNA/rRRV-SIVnfl vaccinees maintained detectable cellular and humoral anti-SIV immune responses for more than 1.5 years after the rRRV boost. To assess if these responses were still protective, the rDNA/rRRV-SIVnfl vaccinees were subjected to a second round of marginal-dose i.r. SIVmac239 challenges, with eight SIV-naive RMs serving as concurrent controls. After three SIV exposures, 8/8 control animals became infected, compared to 3/8 vaccinees. This difference in SIV acquisition was statistically significant (P = 0.0035). The three vaccinated monkeys that became infected exhibited significantly lower viral loads than those in unvaccinated controls. Collectively, these data illustrate the ability of rDNA/rRRV-SIVnfl vaccination to provide long-term immunity against stringent mucosal challenges with SIVmac239. Future work is needed to identify the critical components of this vaccine-mediated protection and the extent to which it can tolerate sequence mismatches in the challenge virus. IMPORTANCE We report on the long-term follow-up of a group of rhesus macaques (RMs) that received an AIDS vaccine regimen and were subsequently protected against rectal acquisition of simian immunodeficiency virus (SIV) infection. The vaccination regimen employed included a live recombinant herpesvirus vector that establishes persistent infection in RMs. Consistent with the recurrent SIV antigen expression afforded by this herpesvirus vector, vaccinees maintained detectable SIV-specific immune responses for more than 1.5 years after the last vaccination. Importantly, these vaccinated RMs were significantly protected against a second round of rectal SIV exposures performed 1 year after the first SIV challenge phase. These results are relevant for HIV vaccine development because they show the potential of herpesvirus-based vectors to maintain functional antiretroviral immunity without the need for repeated boosting.

Background The PD1/PD-L1 pathway contributes to the pathogenesis of human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection, and blockade of this pathway may have potential to restore immune function and promote viral control or elimination. In this study, we combined a checkpoint inhibitor anti-PD-L1 (Avelumab) and recombinant human interleukin-15 (rhIL-15) in SIV-infected rhesus macaques (RM). Methods The rhIL-15 was administered as continuous infusion in 2 cycles of 10 days in the context of weekly administration of anti-PD-L1 (Avelumab) in SIV-infected RM receiving combination antiretroviral therapy (cART). Safety, immunological parameters, and viral loads were monitored during the study. Results Administration of rhIL-15/anti-PD-L1 was safe and well tolerated. Treatment resulted in transient increases in proliferating (Ki67+) natural killer and CD8 T cells. In addition, treatment expanded a CXCR3+PD1−/low CD8 T-cell subset with the ability to secrete cytokines. Despite these effects, no changes in plasma viremia were observed after cART interruption. Conclusions Expansion of the CXCR3+PD1−/low CD8 T-cell subset with functional capacity and potential to traffic to sites of viral reservoirs in SIV-infected rhesus macaques had no demonstrable effect on plasma viremia after cART interruption.

An antibody is not the antidote An HIV therapeutic that would give long-term remission without sustained antiretroviral therapy (ART) is a long-term goal. Byrareddy et al. [ Science 354 , 197 (2016)] reported that treating simian immunodeficiency virus (SIV)–positive macaques with an antibody against integrin α 4 β 7 during and after ART results in sustained virologic control after stopping all treatment. Three studies in this issue question the reproducibility of that result. Di Mascio et al. sequenced the virus used in the 2016 study and found that it was a variant with a stop codon in the nef gene rather than a wild-type virus. Abbink et al. used the same antibody for α 4 β 7 as before but tested control of a more commonly used pathogenic virus. Iwamato et al. used the same nef -stop virus as in the earlier paper but combined the antibody against the integrin with an antibody against the SIV envelope glycoprotein, which also blocks viral binding of the integrin. None of these three new studies found that treating with the antibody had any effect on virologic control after stopping ART treatment. Science , this issue p. 1025 , p. 1029 , p. 1033

The biological characteristics of HIV pose serious difficulties for the success of a preventive vaccine. Molecularly cloned SIVmac239 is difficult for antibodies to neutralize, and a variety of vaccine approaches have had great difficulty achieving protective immunity against it in rhesus monkey models. Here we report significant protection against i.v. acquisition of SIVmac239 using a long-lasting approach to vaccination. The vaccine regimen includes a replication-competent herpesvirus engineered to contain a near-full-length SIV genome that expresses all nine SIV gene products, assembles noninfectious SIV virion particles, and is capable of eliciting long-lasting effector-memory cellular immune responses to all nine SIV gene products. Vaccinated monkeys were significantly protected against acquisition of SIVmac239 following repeated marginal dose i.v. challenges over a 4-month period. Further work is needed to define the critical components necessary for eliciting this protective immunity, evaluate the breadth of the protection against a variety of strains, and explore how this approach may be extended to human use.

The HIV/SIV envelope glycoprotein (Env) cytoplasmic domain contains a highly conserved Tyr-based trafficking signal that mediates both clathrin-dependent endocytosis and polarized sorting. Despite extensive analysis, the role of these functions in viral infection and pathogenesis is unclear. An SIV molecular clone (SIVmac239) in which this signal is inactivated by deletion of Gly-720 and Tyr-721 (SIVmac239ΔGY), replicates acutely to high levels in pigtail macaques (PTM) but is rapidly controlled. However, we previously reported that rhesus macaques and PTM can progress to AIDS following SIVmac239ΔGY infection in association with novel amino acid changes in the Env cytoplasmic domain. These included an R722G flanking the ΔGY deletion and a nine nucleotide deletion encoding amino acids 734–736 (ΔQTH) that overlaps therevandtatopen reading frames. We show that molecular clones containing these mutations reconstitute signals for both endocytosis and polarized sorting. In one PTM, a novel genotype was selected that generated a new signal for polarized sorting but not endocytosis. This genotype, together with the ΔGY mutation, was conserved in association with high viral loads for several months when introduced into naïve PTMs. For the first time, our findings reveal strong selection pressure for Env endocytosis and particularly for polarized sorting during pathogenic SIV infectionin vivo.

SUMMARYThe HIV/SIV envelope glycoprotein (Env) cytoplasmic domain contains a highly conserved Tyr-dependent trafficking signal that mediates both clathrin-dependent endocytosis and polarized sorting of Env. Despite extensive characterization, the role of these functions in viral infection and pathogenesis is unclear. An SIV molecular clone (SIVmac239) in which the Tyr-based signal is inactivated by deletion of Gly-720 and Tyr-721 (SIVmac239ΔGY) replicates to high levels acutely in pigtail macaques (PTM) but is rapidly controlled. We previously reported that rhesus macaques and PTM can progress to AIDS following SIVmac239ΔGY infection in association with novel amino acid changes in the Env cytoplasmic domain. These included an R722G flanking the ΔGY deletion and a nine nucleotide deletion that encodes amino acids 734-736 (ΔQTH) and overlaps with the rev and tat open reading frames. We show that molecular clones containing these mutations reconstitute signals for both endocytosis and polarized sorting. In one PTM, a novel genotype was selected, which generated a new signal for polarized sorting but not endocytosis. This mutation by itself was sufficient to maintain high viral loads for several months when introduced into naïve PTMs. These findings reveal, for the first time, strong selection pressure for Env endocytosis and, in particular, for polarized sorting during pathogenic SIV infection in vivo.

An ability to activate latent HIV-1 expression could benefit many HIV cure strategies, but the first generation of latency reversing agents (LRAs) has proven disappointing. We evaluated AKT/mTOR activators as a potential new class of LRAs. Two glycogen synthase kinase-3 inhibitors (GSK-3i's), SB-216763 and tideglusib (the latter already in phase II clinical trials) that activate AKT/mTOR signaling were tested. These GSK-3i's reactivated latent HIV-1 present in blood samples from aviremic individuals on antiretroviral therapy (ART) in the absence of T cell activation, release of inflammatory cytokines, cell toxicity, or impaired effector function of cytotoxic T lymphocytes or NK cells. However, when administered in vivo to SIV-infected rhesus macaques on suppressive ART, tideglusib exhibited poor pharmacodynamic properties and resulted in no clear evidence of significant SIV latency reversal. Whether alternative pharmacological formulations or combinations of this drug with other classes of LRAs will lead to an effective in vivo latency-reversing strategy remains to be determined.IMPORTANCE If combined with immune therapeutics, latency reversing agents (LRAs) have the potential to reduce the size of the reservoir sufficiently that an engineered immune response can control the virus in the absence of antiretroviral therapy. We have identified a new class of LRAs that do not induce T-cell activation and that are able to potentiate, rather than inhibit, CD8+ T and NK cell cytotoxic effector functions. This new class of LRAs corresponds to inhibitors of glycogen synthase kinase-3. In this work, we have also studied the effects of one member of this drug class, tideglusib, in SIV-infected rhesus monkeys. When tested in vivo, however, tideglusib showed unfavorable pharmacokinetic properties, which resulted in lack of SIV latency reversal. The disconnect between our ex vivo and in vivo results highlights the importance of developing next generation LRAs with pharmacological properties that allow systemic drug delivery in relevant anatomical compartments harboring latent reservoirs.

Inducing latency reversal to reveal infected cells to the host immune system represents a potential strategy to cure HIV infection. In separate studies, we have previously shown that CD8+ T cells may contribute to the maintenance of viral latency and identified a novel SMAC mimetic/IAP inhibitor (AZD5582) capable of reversing HIV/SIV latency in vivo by activating the non-canonical (nc) NF-κB pathway. Here, we use AZD5582 in combination with antibody-mediated depletion of CD8α+ cells to further evaluate the role of CD8+ T cells in viral latency maintenance. Six rhesus macaques (RM) were infected with SIVmac239 and treated with ART starting at week 8 post-infection. After 84-85 weeks of ART, all animals received a single dose of the anti-CD8α depleting antibody (Ab), MT807R1 (50mg/kg, s.c.), followed by 5 weekly doses of AZD5582 (0.1 mg/kg, i.v.). Following CD8α depletion + AZD5582 combined treatment, 100% of RMs experienced on-ART viremia above 60 copies per ml of plasma. In comparator groups of ART-suppressed SIV-infected RMs treated with AZD5582 only or CD8α depletion only, on-ART viremia was experienced by 56% and 57% of the animals respectively. Furthermore, the frequency of increased viremic episodes during the treatment period was greater in the CD8α depletion + AZD5582 group as compared to other groups. Mathematical modeling of virus reactivation suggested that, in addition to viral dynamics during acute infection, CD8α depletion influenced the response to AZD5582. This work suggests that the latency reversal induced by activation of the ncNF-κB signaling pathway with AZD5582 can be enhanced by CD8α+ cell depletion.

SHIV infection of Indian rhesus macaques is an important animal model for studying HIV-1 transmission, prevention, immunopathogenesis, and cure. Such research is timely, given recent progress with active and passive immunization and novel approaches to HIV-1 cure., Previously, we showed that substitution of HIV-1 envelope (Env) residue 375-Ser by bulky aromatic residues enhances binding to rhesus CD4 and enables primary HIV-1 Envs to support efficient replication as simian-human immunodeficiency virus (SHIV) chimeras in rhesus macaques (RMs). Here, we test this design strategy more broadly by constructing SHIVs containing 10 primary Envs corresponding to HIV-1 subtypes A, B, C, AE, and AG. All 10 SHIVs bearing wild-type Env375 residues replicated efficiently in human CD4+ T cells, but only one replicated efficiently in primary rhesus cells. This was a subtype AE SHIV that naturally contained His at Env375. Replacement of wild-type Env375 residues by Trp, Tyr, Phe, or His in the other nine SHIVs led to efficient replication in rhesus CD4+ T cells in vitro and in vivo. Nine SHIVs containing optimized Env375 alleles were grown large-scale in primary rhesus CD4+ T cells to serve as challenge stocks in preclinical prevention trials. These virus stocks were genetically homogeneous, native-like in Env antigenicity and tier 2 neutralization sensitivity, and transmissible by rectal, vaginal, penile, oral, or intravenous routes. To facilitate future SHIV constructions, we engineered a simplified second-generation design scheme and validated it in RMs. Overall, our findings demonstrate that SHIVs bearing primary Envs with bulky aromatic substitutions at Env375 consistently replicate in RMs, recapitulating many features of HIV-1 infection in humans. Such SHIVs are efficiently transmitted by mucosal routes common to HIV-1 infection and can be used to test vaccine efficacy in preclinical monkey trials. IMPORTANCE SHIV infection of Indian rhesus macaques is an important animal model for studying HIV-1 transmission, prevention, immunopathogenesis, and cure. Such research is timely, given recent progress with active and passive immunization and novel approaches to HIV-1 cure. Given the multifaceted roles of HIV-1 Env in cell tropism and virus entry, and as a target for neutralizing and nonneutralizing antibodies, Envs selected for SHIV construction are of paramount importance. Until recently, it has been impossible to strategically design SHIVs bearing clinically relevant Envs that replicate consistently in monkeys. This changed with the discovery that bulky aromatic substitutions at residue Env375 confer enhanced affinity to rhesus CD4. Here, we show that 10 new SHIVs bearing primary HIV-1 Envs with residue 375 substitutions replicated efficiently in RMs and could be transmitted efficiently across rectal, vaginal, penile, and oral mucosa. These findings suggest an expanded role for SHIVs as a model of HIV-1 infection.

A favored approach to curing HIV infection aims at inducing viral expression using latency-reversing agents (LRAs) to allow the elimination of infected cells. Here, we tested a combination of two recently identified LRAs, the SMAC mimetic/IAP inhibitor AZD5582, which activates the noncanonical NF-κB pathway, and the antibody (Ab) MT807R1, which depletes CD8α+ cells, in SIV-infected rhesus macaques (RMs) on ART., Inducing latency reversal to reveal infected cells to the host immune system represents a potential strategy to cure human immunodeficiency virus (HIV) infection. In separate studies, we have shown previously that CD8+ T cells may contribute to the maintenance of viral latency and have identified a novel SMAC (second mitochondrial activator of caspases) mimetic/IAP (inhibitor of apoptosis protein) inhibitor (AZD5582) capable of reversing HIV/simian immunodeficiency virus (SIV) latency in vivo by activating the noncanonical (nc) NF-κB pathway. Here, we use AZD5582 in combination with antibody (Ab)-mediated depletion of CD8α+ cells to further evaluate the role of CD8+ T cells in the maintenance of viral latency. Six rhesus macaques (RMs) were infected with SIVmac239 and treated with antiretroviral therapy (ART) starting at week 8 postinfection. After 84 to 85 weeks of ART, all animals received a single dose of the anti-CD8α depleting Ab MT807R1 (50 mg/kg of body weight, administered subcutaneously [s.c.]), followed by five weekly doses of AZD5582 (0.1 mg/kg, administered intravenously [i.v.]). Following combined treatment with CD8α depletion and AZD5582, 100% of RMs experienced on-ART viremia levels above 60 copies per ml of plasma. In comparator groups of SIV-infected, ART-suppressed RMs treated with AZD5582 only or CD8α depletion only, on-ART viremia was experienced by 56% and 57% of the animals, respectively. Furthermore, the increased frequency of viremic episodes during the treatment period was greater in the group treated with CD8α depletion plus AZD5582 than in the other groups. Mathematical modeling of virus reactivation suggested that in addition to viral dynamics during acute infection, CD8α depletion influenced the response to AZD5582. This work suggests that the latency reversal induced by activation of the ncNF-κB signaling pathway with AZD5582 can be enhanced by CD8α+ cell depletion. IMPORTANCE A favored approach to curing HIV infection aims at inducing viral expression using latency-reversing agents (LRAs) to allow the elimination of infected cells. Here, we tested a combination of two recently identified LRAs, the SMAC mimetic/IAP inhibitor AZD5582, which activates the noncanonical NF-κB pathway, and the antibody (Ab) MT807R1, which depletes CD8α+ cells, in SIV-infected rhesus macaques (RMs) on ART. Latency reversal, as defined by increased on-ART viremia, was observed in all six SIV-infected, ART-treated RMs that received this combined treatment. Furthermore, comparison of viral reactivation between these animals and groups of SIV-infected, ART-treated RMs treated with AZD5582 only or CD8α+ cell depletion only showed more frequent increases in viremic episodes when the two treatments were combined. This study provides additional evidence that CD8+ T cells may contribute to the maintenance of HIV/SIV latency on ART and potentially inhibit latency reversal during HIV cure approaches.

An ability to activate latent human immunodeficiency virus type 1 (HIV-1) expression could benefit many HIV cure strategies; however, most latency-reversing agents (LRAs) have proven disappointing. We evaluated AKT/mTOR activators as a potential new class of LRAs. Two glycogen synthase kinase-3 inhibitors (GSK-3i’s), SB-216763 and tideglusib (the latter already in phase II clinical trials), that activate AKT/mTOR signaling were tested. These GSK-3i’s reactivated latent HIV-1 present in blood samples from aviremic individuals on antiretroviral therapy (ART) in the absence of T cell activation, release of inflammatory cytokines, cell toxicity, or impaired effector function of cytotoxic T lymphocytes or NK cells. However, when administered in vivo to simian immunodeficiency virus (SIV)-infected rhesus macaques on suppressive ART, tideglusib exhibited poor pharmacodynamic properties and resulted in no clear evidence of significant SIV latency reversal. Whether alternative pharmacological formulations or combinations of this drug with other classes of LRAs will lead to an effective in vivo latency-reversing strategy remains to be determined. IMPORTANCE If combined with immune therapeutics, latency-reversing agents (LRAs) have the potential to reduce the size of the reservoir sufficiently that an engineered immune response can control the virus in the absence of antiretroviral therapy. We have identified a new class of LRAs that do not induce T-cell activation and that are able to potentiate, rather than inhibit, CD8(+) T and NK cell cytotoxic effector functions. This new class of LRAs corresponds to inhibitors of glycogen synthase kinase-3. In this work, we have also studied the effects of one member of this drug class, tideglusib, in SIV-infected rhesus monkeys. When tested in vivo, however, tideglusib showed unfavorable pharmacokinetic properties, which resulted in lack of SIV latency reversal. The disconnect between our ex vivo and in vivo results highlights the importance of developing novel LRAs with pharmacological properties that allow systemic drug delivery in relevant anatomical compartments harboring latent reservoirs.

Simian immunodeficiency virus (SIV) challenge of rhesus macaques (RMs) vaccinated with 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 suggests 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 RhCMV/SIV vector-vaccinated RMs has not been elucidated. Here, we report that RhCMV/SIV vector administration strikingly alters the whole blood transcriptome of vaccinated RMs, with the sustained induction of specific immune-related pathways, including non-canonical T cell receptor (TCR), toll-lie receptor (TLR), inflammasome/cell death, and interleukin-15 (IL-15) signaling, significantly predicting protection. The IL-15 gene expression signature was further evaluated in an independent RM IL-15 treatment cohort, revealing that in whole blood the response to IL-15 is inclusive of innate and adaptive immune gene expression networks that link with RhCMV/SIV vaccine efficacy. We also show that this IL-15 response signature similarly tracks with vaccine protection in an independent RhCMV/SIV vaccination/SIV challenge RM validation cohort. 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 enable vaccine-elicited CD8+ T cells to mediate protection against highly pathogenic SIV challenge.Author SummarySIV insert-expressing vaccine vectors based on strain 68-1 RhCMV elicit robust, highly effector-memory-biased T cell responses that are associated with an unprecedented level of SIV control after challenge (replication arrest leading to clearance) in slightly over half of vaccinated monkeys. Since efficacy is not predicted by standard measures of immunogenicity, we used functional genomics analysis of RhCMV/SIV vaccinated monkeys with known challenge outcomes to identify immune correlates of protection. We found that arrest of viral replication after challenge significantly correlates with a vaccine-induced response to IL-15 that includes modulation of T cell, inflammation, TLR signaling, and cell death programming. These data suggest that RhCMV/SIV efficacy is not based on chance, but rather, results from a coordinated and sustained vaccine-mediated induction of innate and adaptive immune pathways featuring IL-15, a known regulator of CD8+ effector-memory T cell function, that enable vaccine-elicited CD8+ T cells to mediate efficacy.

Broadly neutralizing antibodies (bNAbs) represent an alternative to drug therapy for the treatment of HIV-1 infection. Immunotherapy with single bNAbs often leads to emergence of escape variants, suggesting a potential benefit of combination bNAb therapy. Here a trispecific bNAb is shown to reduce viremia 100- to 1000-fold in viremic SHIV-infected macaques. After treatment discontinuation, viremia rebounded transiently and returned to low levels, through CD8-mediated immune control. These viruses remained sensitive to the trispecific despite loss of sensitivity to one of the parental bNAbs. Similarly, the trispecific bNAb suppressed the emergence of resistance in viruses derived from HIV-1 infected subjects, in contrast to parental bNAbs. Trispecific HIV-1 antibodies therefore mediates potent antiviral activity in vivo that minimizes the potential for immune escape.

Given the complex biology of human immunodeficiency virus (HIV) and its remarkable capacity to evade host immune responses, HIV vaccine efficacy may benefit from the induction of both humoral and cellular immune responses of maximal breadth, potency, and longevity. Guided by this rationale, we set out to develop an immunization protocol aimed at maximizing the induction of anti-Envelope (anti-Env) antibodies and CD8(+) T cells targeting non-Env epitopes in rhesus macaques (RMs). Our approach was to deliver the entire simian immunodeficiency virus (SIV) proteome by serial vaccinations. To that end, 12 RMs were vaccinated over 81 weeks with DNA, modified vaccinia Ankara (MVA), vesicular stomatitis virus (VSV), adenovirus type 5 (Ad5), rhesus monkey rhadinovirus (RRV), and DNA again. Both the RRV and the final DNA boosters delivered a near-full-length SIVmac239 genome capable of assembling noninfectious SIV particles and inducing T-cell responses against all nine SIV proteins. Compared to previous SIV vaccine trials, the present DNA-MVA-VSV-Ad5-RRV-DNA regimen resulted in comparable levels of Env-binding antibodies and SIV-specific CD8(+) T-cells. Interestingly, one vaccinee developed low titers of neutralizing antibodies (NAbs) against SIVmac239, a tier 3 virus. Following repeated intrarectal marginal-dose challenges with SIVmac239, vaccinees were not protected from SIV acquisition but manifested partial control of viremia. Strikingly, the animal with the low-titer vaccine-induced anti-SIVmac239 NAb response acquired infection after the first SIVmac239 exposure. Collectively, these results highlight the difficulties in eliciting protective immunity against immunodeficiency virus infection. IMPORTANCE Our results are relevant to HIV vaccine development efforts because they suggest that increasing the number of booster immunizations or delivering additional viral antigens may not necessarily improve vaccine efficacy against immunodeficiency virus infection.

Strain 68-1 rhesus cytomegalovirus (RhCMV) vectors expressing simian immunodeficiency virus (SIV) antigens elicit CD8+ T cells that recognize peptide epitopes presented by major histocompatibility complex (MHC)-II and MHC-E molecules, instead of MHC-Ia, and are uniquely able to mediate stringent control and subsequent clearance of highly pathogenic SIV in ∼50% of vaccinated rhesus macaques (RMs). We show that the MHC-E ligand VMAPRTLLL (VL9), encoded by the Rh67 gene (or its HCMV UL40 counterpart) is required for recognition of RhCMV-infected fibroblasts by MHC-E-restricted CD8+ T cells via its ability to promote intracellular MHC-E transport. Moreover, deletion of Rh67 from 68-1 RhCMV/SIV vectors, or mutation of its embedded VL9 ligand, abrogated induction of MHC-E-restricted CD8+ T cell responses, leaving responses that exclusively target MHC-II-restricted epitopes. These MHC-II-presented CD8+ T cell responses, though comparable in response magnitude and functional differentiation to responses arising from the efficacious 68-1 vector, did not protect RMs against SIV challenge, indicating that Rh67/UL40-enabled direct priming of MHC-E-targeted CD8+ T cells is a crucial element of RhCMV/SIV vaccine efficacy.One Sentence SummaryA cytomegalovirus protein (Rh67/UL40) that upregulates MHC-E expression on RhCMV/SIV-vector infected cells is required for induction of MHC-E-restricted CD8+ T cells and for protection against SIV.

Simian immunodeficiency virus (SIV) insert-expressing, 68-1 Rhesus Cytomegalovirus (RhCMV/SIV) vectors elicit major histocompatibility complex (MHC)-E- and -II-restricted, SIV-specific CD8+ T cell responses, but the basis of these unconventional responses and their contribution to demonstrated vaccine efficacy against SIV challenge in the rhesus monkeys (RMs) has not been characterized. We demonstrate that these unconventional responses resulted from a chance genetic rearrangement in 68-1 RhCMV that abrogated the function of eight distinct immunomodulatory gene products encoded in two RhCMV genomic regions (Rh157.5/.4 and Rh158-161). Differential repair of these genes with either RhCMV-derived or orthologous human CMV (HCMV)-derived sequences (UL128/130; UL146/147) leads to either of two distinct CD8+ T cell response types – MHC-Ia-restricted-only, or a mix of MHC-II- and MHC-Ia-restricted CD8+ T cells. Despite response magnitude and functional differentiation being similar to RhCMV 68-1, neither alternative response type mediated protection against SIV challenge. These findings implicate MHC-E-restricted CD8+ T cell responses as mediators of anti-SIV efficacy and indicate that translation of RhCMV/SIV vector efficacy to humans will likely require deletion of all the genes that inhibit these responses from the HCMV/HIV vector.One-sentence summaryEight genes in two spatially distinct RhCMV gene regions control induction of unconventionally restricted CD8+ T cell responses and the efficacy of RhCMV/SIV vaccine vectors against SIV challenge.

Unlike HIV infection, which progresses to AIDS absent suppressive anti-retroviral therapy, nonpathogenic infections in natural hosts, such African green monkeys, are characterized by a lack of gut microbial translocation and robust secondary lymphoid natural killer cell responses resulting in an absence of chronic inflammation and limited SIV dissemination in lymph node B-cell follicles. Here we report, using the pathogenic model of antiretroviral therapy-treated, SIV-infected rhesus macaques that sequential interleukin-21 and interferon alpha therapy generate terminally differentiated blood natural killer cells (NKG2a/clowCD16+) with potent human leukocyte antigen-E-restricted activity in response to SIV envelope peptides. This is in contrast to control macaques, where less differentiated, interferon gamma-producing natural killer cells predominate. The frequency and activity of terminally differentiated NKG2a/clowCD16+ natural killer cells correlates with a reduction of replication-competent SIV in lymph node during antiretroviral therapy and time to viral rebound following analytical treatment interruption. These data demonstrate that African green monkey-like natural killer cell differentiation profiles can be rescued in rhesus macaques to promote viral clearance in tissues., Infection of African green monkeys with SIV is associated with reduced pathogenicity. Here the authors explore the requirement of differentiated NK cell populations in a pathogenic Rhesus macaque model of SIV infection and show administration of IL-21 and IFNα rescues terminally differentiated NK cells, similarly to what found in African green monkeys, and limits the SIV reservoir in antiretroviral therapy treated macaques.

Treatment of HIV infection with either antiretroviral (ARV) therapy or neutralizing monoclonal antibodies (NAbs) leads to a reduction in HIV plasma virus. Both ARVs and NAbs prevent new rounds of viral infection, but NAbs may have the additional capacity to accelerate the loss of virus-infected cells through Fc gamma receptor (FcγR)-mediated effector functions, which should affect the kinetics of plasma-virus decline. Here, we formally test the role of effector function in vivo by comparing the rate and timing of plasma-virus clearance in response to a single-dose treatment with either unmodified NAb or those with either reduced or augmented Fc function. When infused into viremic simian HIV (SHIV)-infected rhesus macaques, there was a 21% difference in slope of plasma-virus decline between NAb and NAb with reduced Fc function. NAb engineered to increase FcγRIII binding and improve antibody-dependent cellular cytotoxicity (ADCC) in vitro resulted in arming of effector cells in vivo, yet led to viral-decay kinetics similar to NAbs with reduced Fc function. These studies show that the predominant mechanism of antiviral activity of HIV NAbs is through inhibition of viral entry, but that Fc function can contribute to the overall antiviral activity, making them distinct from standard ARVs.

The effectiveness of virus-specific strategies, including administered HIV-specific mAbs, to target cells that persistently harbor latent, rebound-competent HIV genomes during combination antiretroviral therapy (cART) has been limited by inefficient induction of viral protein expression. To examine antibody-mediated viral reservoir targeting without a need for viral induction, we used an anti-CD4 mAb to deplete both infected and uninfected CD4(+) T cells. Ten rhesus macaques infected with barcoded SIVmac239M received cART for 93 weeks starting 4 days after infection. During cART, 5 animals received 5 to 6 anti-CD4 antibody administrations and CD4(+) T cell populations were then allowed 1 year on cART to recover. Despite profound CD4(+) T cell depletion in blood and lymph nodes, time to viral rebound following cART cessation was not significantly delayed in anti-CD4–treated animals compared with controls. Viral reactivation rates, determined based on rebounding SIVmac239M clonotype proportions, also were not significantly different in CD4-depleted animals. Notably, antibody-mediated depletion was limited in rectal tissue and negligible in lymphoid follicles. These results suggest that, even if robust viral reactivation can be achieved, antibody-mediated viral reservoir depletion may be limited in key tissue sites.