Microglia and macrophages alterations in the CNS during acute SIV infection: A single-cell analysis in rhesus macaques.

Human Immunodeficiency Virus (HIV) is widely acknowledged for its profound impact on the immune system. Although HIV primarily affects peripheral CD4 T cells, its influence on the central nervous system (CNS) cannot be overlooked. Within the brain, microglia and CNS-associated macrophages (CAMs) serve as the primary targets for HIV and the simian immunodeficiency virus (SIV) in nonhuman primates. This infection can lead to neurological effects and establish a viral reservoir. Given the gaps in our understanding of how these cells respond in vivo to acute CNS infection, we conducted single-cell RNA sequencing (scRNA-seq) on myeloid cells from the brains of three rhesus macaques 12 days after SIV infection, along with three uninfected controls. Our analysis revealed six distinct microglial clusters including homeostatic microglia, preactivated microglia, and activated microglia expressing high levels of inflammatory and disease-related molecules. In response to acute SIV infection, the homeostatic and preactivated microglia population decreased, while the activated and disease-related microglia increased. All microglial clusters exhibited upregulation of MHC class I molecules and interferon-related genes, indicating their crucial roles in defending against SIV during the acute phase. All microglia clusters also upregulated genes linked to cellular senescence. Additionally, we identified two distinct CAM populations: CD14lowCD16hi and CD14hiCD16low CAMs. Interestingly, during acute SIV infection, the dominant CAM population changed to one with an inflammatory phenotype. Specific upregulated genes within one microglia and one macrophage cluster were associated with neurodegenerative pathways, suggesting potential links to neurocognitive disorders. This research sheds light on the intricate interactions between viral infection, innate immune responses, and the CNS, providing valuable insights for future investigations.

Dual blockade of IL-10 and PD-1 leads to control of SIV viral rebound following analytical treatment interruption.

Human immunodeficiency virus (HIV) persistence during antiretroviral therapy (ART) is associated with heightened plasma interleukin-10 (IL-10) levels and PD-1 expression. We hypothesized that IL-10 and PD-1 blockade would lead to control of viral rebound following analytical treatment interruption (ATI). Twenty-eight ART-treated, simian immunodeficiency virus (SIV)mac239-infected rhesus macaques (RMs) were treated with anti-IL-10, anti-IL-10 plus anti-PD-1 (combo) or vehicle. ART was interrupted 12 weeks after introduction of immunotherapy. Durable control of viral rebound was observed in nine out of ten combo-treated RMs for >24 weeks post-ATI. Induction of inflammatory cytokines, proliferation of effector CD8+ T cells in lymph nodes and reduced expression of BCL-2 in CD4+ T cells pre-ATI predicted control of viral rebound. Twenty-four weeks post-ATI, lower viral load was associated with higher frequencies of memory T cells expressing TCF-1 and of SIV-specific CD4+ and CD8+ T cells in blood and lymph nodes of combo-treated RMs. These results map a path to achieve long-lasting control of HIV and/or SIV following discontinuation of ART.

Impact of alemtuzumab-mediated lymphocyte depletion on SIV reservoir establishment and persistence.

Persistence of the rebound-competent viral reservoir (RCVR) within the CD4+ T cell compartment of people living with HIV remains a major barrier to HIV cure. Here, we determined the effects of the pan-lymphocyte-depleting monoclonal antibody (mAb) alemtuzumab on the RCVR in SIVmac239-infected rhesus macaques (RM) receiving antiretroviral therapy (ART). Alemtuzumab administered during chronic ART or at the time of ART initiation induced >95% depletion of circulating CD4+ T cells in peripheral blood and substantial CD4+ T cell depletion in lymph nodes. However, treatment was followed by proliferation and reconstitution of CD4+ T cells in blood, and despite ongoing ART, levels of cell-associated SIV DNA in blood and lymphoid tissues were not substantially different between alemtuzumab-treated and control RM after immune cell reconstitution, irrespective of the time of alemtuzumab treatment. Upon ART cessation, 19 of 22 alemtuzumab-treated RM and 13 of 13 controls rebounded with no difference in the time to rebound between treatment groups. Time to rebound and reactivation rate was associated with plasma viral loads (pVLs) at time of ART initiation, suggesting lymphocyte depletion had no durable impact on the RCVR. However, 3 alemtuzumab-treated RM that had lowest levels of pre-ART viremia, failed to rebound after ART withdrawal, in contrast to controls with similar levels of SIV replication. These observations suggest that alemtuzumab therapy has little to no ability to reduce well-established RCVRs but may facilitate RCVR destabilization when pre-ART virus levels are particularly low.

Administration of anti-HIV-1 broadly neutralizing monoclonal antibodies with increased affinity to Fcγ receptors during acute SHIVAD8-EO infection.

Anti-HIV-1 broadly neutralizing antibodies (bNAbs) have the dual potential of mediating virus neutralization and antiviral effector functions through their Fab and Fc domains, respectively. So far, bNAbs with enhanced Fc effector functions in vitro have only been tested in NHPs during chronic simian-HIV (SHIV) infection. Here, we investigate the effects of administering in acute SHIVAD8-EO infection either wild-type (WT) bNAbs or bNAbs carrying the S239D/I332E/A330L (DEL) mutation, which increases binding to FcγRs. Emergence of virus in plasma and lymph nodes (LNs) was delayed by bNAb treatment and occurred earlier in monkeys given DEL bNAbs than in those given WT bNAbs, consistent with faster clearance of DEL bNAbs from plasma. DEL bNAb-treated monkeys had higher levels of circulating virus-specific IFNγ single-producing CD8+ CD69+ T cells than the other groups. In LNs, WT bNAbs were evenly distributed between follicular and extrafollicular areas, but DEL bNAbs predominated in the latter. At week 8 post-challenge, LN monocytes and NK cells from DEL bNAb-treated monkeys upregulated proinflammatory signaling pathways and LN T cells downregulated TNF signaling via NF-κB. Overall, bNAbs with increased affinity to FcγRs shape innate and adaptive cellular immunity, which may be important to consider in future strategies of passive bNAb therapy.

Viral escape mutations do not account for non-protection from SIVmac239 challenge in RhCMV/SIV vaccinated rhesus macaques.

Simian immunodeficiency virus (SIV) vaccines based upon 68-1 Rhesus Cytomegalovirus (RhCMV) vectors show remarkable protection against pathogenic SIVmac239 challenge. Across multiple independent rhesus macaque (RM) challenge studies, nearly 60% of vaccinated RM show early, complete arrest of SIVmac239 replication after effective challenge, whereas the remainder show progressive infection similar to controls. Here, we performed viral sequencing to determine whether the failure to control viral replication in non-protected RMs is associated with the acquisition of viral escape mutations. While low level viral mutations accumulated in all animals by 28 days-post-challenge, which is after the establishment of viral control in protected animals, the dominant circulating virus in virtually all unprotected RMs was nearly identical to the challenge stock, and there was no difference in mutation patterns between this cohort and unvaccinated controls. These data definitively demonstrate that viral mutation does not explain lack of viral control in RMs not protected by RhCMV/SIV vaccination. We further demonstrate that during chronic infection RhCMV/SIV vaccinated RMs do not acquire escape mutation in epitopes targeted by RhCMV/SIV, but instead display mutation in canonical MHC-Ia epitopes similar to unvaccinated RMs. This suggests that after the initial failure of viral control, unconventional T cell responses induced by 68-1 RhCMV/SIV vaccination do not exert strong selective pressure on systemically replicating SIV.

Adeno-associated viral delivery of Env-specific antibodies prevents SIV rebound after discontinuing antiretroviral therapy.

An alternative to lifelong antiretroviral therapy (ART) is needed to achieve durable control of HIV-1. Here we show that adeno-associated virus (AAV)-delivery of two rhesus macaque antibodies to the SIV envelope glycoprotein (Env) with potent neutralization and antibody-dependent cellular cytotoxicity can prevent viral rebound in macaques infected with barcoded SIVmac239M after discontinuing suppressive ART. Following AAV administration, sustained antibody expression with minimal anti-drug antibody responses was achieved in all but one animal. After ART withdrawal, SIV replication rebounded within two weeks in all of the control animals but remained below the threshold of detection in plasma (<15 copies/mL) for more than a year in four of the eight animals that received AAV vectors encoding Env-specific antibodies. Viral sequences from animals with delayed rebound exhibited restricted barcode diversity and antibody escape. Thus, sustained expression of antibodies with potent antiviral activity can afford durable, ART-free containment of pathogenic SIV infection.

No evidence for ongoing replication on ART in SIV-infected macaques.

The capacity of HIV-1 to replicate during optimal antiretroviral therapy (ART) is challenging to assess directly. To gain greater sensitivity to detect evolution on ART, we used a nonhuman primate (NHP) model providing precise control over the level of pre-ART evolution and more comprehensive analyses than are possible with clinical samples. We infected 21 rhesus macaques (RMs) with the barcoded virus SIVmac239M and initiated ART early to minimize baseline genetic diversity. RMs were treated for 285-1200 days. We used several tests of molecular evolution to compare 1352 near-full-length (nFL) SIV DNA single genome sequences from PBMCs, lymph nodes, and spleen obtained near the time of ART initiation and those present after long-term ART, none of which showed significant changes to the SIV DNA population during ART in any animal. To investigate the possibility of ongoing replication in unsampled putative tissue sanctuaries during ART, we discontinued treatment in four animals and confirmed that none of the 336 nFL SIV RNA sequences obtained from rebound plasma viremia showed evidence of evolution. The rigorous nature of our analyses reinforced the emerging consensus of a lack of appreciable ongoing replication on effective ART and validates the relevance of this NHP model for cure studies.

Microglia and macrophages alterations in the CNS during acute SIV infection: a single-cell analysis in rhesus macaques.

Human Immunodeficiency Virus (HIV) is widely acknowledged for its profound impact on the immune system. Although HIV primarily affects peripheral CD4 T cells, its influence on the central nervous system (CNS) cannot be overlooked. Within the brain, microglia and CNS-associated macrophages (CAMs) serve as the primary targets for HIV, as well as for the simian immunodeficiency virus (SIV) in nonhuman primates. This infection can lead to neurological effects and the establishment of a viral reservoir. Given the gaps in our understanding of how these cells respond in vivo to acute CNS infection, we conducted single-cell RNA sequencing (scRNA-seq) on myeloid cells from the brains of three rhesus macaques 12-days after SIV infection, along with three uninfected controls. Our analysis revealed six distinct microglial clusters including homeostatic microglia, preactivated microglia, and activated microglia expressing high levels of inflammatory and disease-related molecules. In response to acute SIV infection, the population of homeostatic and preactivated microglia decreased, while the activated and disease-related microglia increased. All microglial clusters exhibited upregulation of MHC class I molecules and interferon-related genes, indicating their crucial roles in defending against SIV during the acute phase. All microglia clusters also upregulated genes linked to cellular senescence. Additionally, we identified two distinct CAM populations: CD14lowCD16hi and CD14hiCD16low CAMs. Interestingly, during acute SIV infection, the dominant CAM population changed to one with an inflammatory phenotype. Notably, specific upregulated genes within one microglia and one macrophage cluster were associated with neurodegenerative pathways, suggesting potential links to neurocognitive disorders. This research sheds light on the intricate interactions between viral infection, innate immune responses, and the CNS, providing valuable insights for future investigations.

Early antiretroviral therapy in SIV-infected rhesus macaques reveals a multiphasic, saturable dynamic accumulation of the rebound competent viral reservoir.

The rebound competent viral reservoir (RCVR)-virus that persists during antiretroviral treatment (ART) and can reignite systemic infection when treatment is stopped-is the primary barrier to eradicating HIV. We used time to initiation of ART during primary infection of rhesus macaques (RMs) after intravenous challenge with barcoded SIVmac239 as a means to elucidate the dynamics of RCVR establishment in groups of RMs by creating a multi-log range of pre-ART viral loads and then assessed viral time-to-rebound and reactivation rates resulting from the discontinuation of ART after one year. RMs started on ART on days 3, 4, 5, 6, 7, 9 or 12 post-infection showed a nearly 10-fold difference in pre-ART viral measurements for successive ART-initiation timepoints. Only 1 of 8 RMs initiating ART on days 3 and 4 rebounded after ART interruption despite measurable pre-ART plasma viremia. Rebounding plasma from the 1 rebounding RM contained only a single barcode lineage detected at day 50 post-ART. All RMs starting ART on days 5 and 6 rebounded between 14- and 50-days post-ART with 1-2 rebounding variants each. RMs starting ART on days 7, 9, and 12 had similar time-to-measurable plasma rebound kinetics despite multiple log differences in pre-ART plasma viral load (pVL), with all RMs rebounding between 7- and 16-days post-ART with 3-28 rebounding lineages. Calculated reactivation rates per pre-ART pVL were highest for RMs starting ART on days 5, 6, and 7 after which the rate of accumulation of the RCVR markedly decreased for RMs treated on days 9 and 12, consistent with multiphasic establishment and near saturation of the RCVR within 2 weeks post infection. Taken together, these data highlight the heterogeneity of the RCVR between RMs, the stochastic establishment of the very early RCVR, and the saturability of the RCVR prior to peak viral infection.

Potent antibody-dependent cellular cytotoxicity of a V2-specific antibody is not sufficient for protection of macaques against SIV challenge.

Fc-mediated antibody effector functions, such as antibody-dependent cellular cytotoxicity (ADCC), can contribute to the containment HIV-1 replication but whether such activities are sufficient for protection is unclear. We previously identified an antibody to the variable 2 (V2) apex of the HIV-1 Env trimer (PGT145) that potently directs the lysis of SIV-infected cells by NK cells but poorly neutralizes SIV infectivity. To determine if ADCC is sufficient for protection, separate groups of six rhesus macaques were treated with PGT145 or a control antibody (DEN3) by intravenous infusion followed five days later by intrarectal challenge with SIVmac239. Despite high concentrations of PGT145 and potent ADCC activity in plasma on the day of challenge, all animals became infected and viral loads did not differ between the PGT145- and DEN3-treated animals. To determine if PGT145 can protect against a neutralization-sensitive virus, two additional groups of six macaques were treated with PGT145 and DEN3 and challenged with an SIVmac239 variant with a single amino acid change in Env (K180S) that increases PGT145 binding and renders the virus susceptible to neutralization by this antibody. Although there was no difference in virus acquisition, peak and chronic phase viral loads were significantly lower and time to peak viremia was significantly delayed in the PGT145-treated animals compared to the DEN3-treated control animals. Env changes were also selected in the PGT145-treated animals that confer resistance to both neutralization and ADCC. These results show that ADCC is not sufficient for protection by this V2-specific antibody. However, protection may be achieved by increasing the affinity of antibody binding to Env above the threshold required for neutralization.

Deep analysis of CD4 T cells in the rhesus CNS during SIV infection.

Virologic suppression with antiretroviral therapy (ART) has significantly improved health outcomes for people living with HIV, yet challenges related to chronic inflammation in the central nervous system (CNS)-known as Neuro-HIV- persist. As primary targets for HIV-1 with the ability to survey and populate the CNS and interact with myeloid cells to co-ordinate neuroinflammation, CD4 T cells are pivotal in Neuro-HIV. Despite their importance, our understanding of CD4 T cell distribution in virus-targeted CNS tissues, their response to infection, and potential recovery following initiation of ART remain limited. To address these gaps, we studied ten SIVmac251-infected rhesus macaques using an ART regimen simulating suboptimal adherence. We evaluated four macaques during the acute phase pre-ART and six during the chronic phase. Our data revealed that HIV target CCR5+ CD4 T cells inhabit both the brain parenchyma and adjacent CNS tissues, encompassing choroid plexus stroma, dura mater, and the skull bone marrow. Aligning with the known susceptibility of CCR5+ CD4 T cells to viral infection and their presence within the CNS, high levels of viral RNA were detected in the brain parenchyma and its border tissues during acute SIV infection. Single-cell RNA sequencing of CD45+ cells from the brain revealed colocalization of viral transcripts within CD4 clusters and significant activation of antiviral molecules and specific effector programs within T cells, indicating CNS CD4 T cell engagement during infection. Acute infection led to marked imbalance in the CNS CD4/CD8 ratio which persisted into the chronic phase. These observations underscore the functional involvement of CD4 T cells within the CNS during SIV infection, enhancing our understanding of their role in establishing CNS viral presence. Our findings offer insights for potential T cell-focused interventions while underscoring the challenges in eradicating HIV from the CNS, particularly in the context of sub-optimal ART.

Lymphoid tissues contribute to plasma viral clonotypes early after antiretroviral therapy interruption in SIV-infected rhesus macaques.

The rebound-competent viral reservoir, composed of a virus that is able to persist during antiretroviral therapy (ART) and mediate reactivation of systemic viral replication and rebound viremia after ART interruption (ATI), remains the biggest obstacle to treating 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 therapeutic strategies for reducing the rebound-competent viral reservoir. In this study, barcoded simian immunodeficiency virus (SIV), SIVmac239M, was used to infect rhesus macaques to enable monitoring of viral barcode clonotypes contributing to virus detectable in plasma after ATI. Blood and tissues from secondary lymphoid organs (spleen, mesenteric lymph nodes, and inguinal lymph nodes) and from the colon, ileum, lung, liver, and brain were analyzed using viral barcode sequencing, intact proviral DNA assay, single-cell RNA sequencing, and combined CODEX and RNAscope in situ hybridization. Four of seven animals had viral barcodes detectable by deep sequencing of plasma at necropsy, although plasma viral RNA remained below 22 copies per milliliter. Among the tissues studied, mesenteric lymph nodes, inguinal lymph nodes, and spleen contained viral barcodes detected in plasma. CD4+ T cells were the main cell type harboring viral RNA after ATI. Furthermore, T cell zones in lymphoid tissues showed higher viral RNA abundance than B cell zones for most animals. These findings are consistent with lymphoid tissues contributing to the virus present in plasma early after ATI.

HIV-1 Env trimers asymmetrically engage CD4 receptors in membranes.

Human immunodeficiency virus 1 (HIV-1) infection is initiated by binding of the viral envelope glycoprotein (Env) to the cell-surface receptor CD41-4. Although high-resolution structures of Env in a complex with the soluble domains of CD4 have been determined, the binding process is less understood in native membranes5-13. Here we used cryo-electron tomography to monitor Env-CD4 interactions at the membrane-membrane interfaces formed between HIV-1 and CD4-presenting virus-like particles. Env-CD4 complexes organized into clusters and rings, bringing the opposing membranes closer together. Env-CD4 clustering was dependent on capsid maturation. Subtomogram averaging and classification revealed that Env bound to one, two and finally three CD4 molecules, after which Env adopted an open state. Our data indicate that asymmetric HIV-1 Env trimers bound to one and two CD4 molecules are detectable intermediates during virus binding to host cell membranes, which probably has consequences for antibody-mediated immune responses and vaccine immunogen design.

AZD5582 plus SIV-specific antibodies reduce lymph node viral reservoirs in antiretroviral therapy-suppressed macaques.

The main barrier to HIV cure is a persistent reservoir of latently infected CD4+ T cells harboring replication-competent provirus that fuels rebound viremia upon antiretroviral therapy (ART) interruption. A leading approach to target this reservoir involves agents that reactivate latent HIV proviruses followed by direct clearance of cells expressing induced viral antigens by immune effector cells and immunotherapeutics. We previously showed that AZD5582, an antagonist of inhibitor of apoptosis proteins and mimetic of the second mitochondrial-derived activator of caspases (IAPi/SMACm), induces systemic reversal of HIV/SIV latency but with no reduction in size of the viral reservoir. In this study, we investigated the effects of AZD5582 in combination with four SIV Env-specific Rhesus monoclonal antibodies (RhmAbs) ± N-803 (an IL-15 superagonist) in SIV-infected, ART-suppressed rhesus macaques. Here we confirm the efficacy of AZD5582 in inducing SIV reactivation, demonstrate enhancement of latency reversal when AZD5582 is used in combination with N-803 and show a reduction in total and replication-competent SIV-DNA in lymph-node-derived CD4+ T cells in macaques treated with AZD5582 + RhmAbs. Further exploration of this therapeutic approach may contribute to the goal of achieving an HIV cure.

CD4 T cell Responses in the CNS during SIV infection.

Virologic suppression with antiretroviral therapy (ART) has significantly improved health outcomes for people living with HIV, yet challenges related to chronic inflammation in the central nervous system (CNS) - known as Neuro-HIV- persist. As primary targets for HIV-1 with the ability to survey and populate the CNS and interact with myeloid cells to co-ordinate neuroinflammation, CD4 T cells are pivotal in Neuro-HIV. Despite their importance, our understanding of CD4 T cell distribution in virus-targeted CNS tissues, their response to infection, and potential recovery following initiation of ART remain limited. To address these gaps, we studied ten SIVmac251-infected rhesus macaques using an ART regimen simulating suboptimal adherence. We evaluated four macaques during the acute phase pre-ART and six during the chronic phase. Our data revealed that HIV target CCR5+ CD4 T cells inhabit both the brain parenchyma and adjacent CNS tissues, encompassing choroid plexus stroma, dura mater, and the skull bone marrow. Aligning with the known susceptibility of CCR5+ CD4 T cells to viral infection and their presence within the CNS, high levels of viral RNA were detected in the brain parenchyma and its border tissues during acute SIV infection. Single-cell RNA sequencing of CD45+ cells from the brain revealed colocalization of viral transcripts within CD4 clusters and significant activation of antiviral molecules and specific effector programs within T cells, indicating CNS CD4 T cell engagement during infection. Despite viral suppression with ART, acute infection led to significant depletion of CNS CD4 T cells, persisting into the chronic phase. These findings underscore the functional involvement of CD4 T cells within the CNS during SIV infection, enhancing our understanding of their role in establishing CNS viral presence. Our results offer insights for potential T cell-focused interventions while also underscoring the challenges in eradicating HIV from the CNS, even with effective ART.

Long-acting lenacapavir protects macaques against intravenous challenge with simian-tropic HIV.

BACKGROUND: Long-acting subcutaneous lenacapavir (LEN), a first-in-class HIV capsid inhibitor approved by the US FDA for the treatment of multidrug-resistant HIV-1 with twice yearly dosing, is under investigation for HIV-1 pre-exposure prophylaxis (PrEP). We previously derived a simian-tropic HIV-1 clone (stHIV-A19) that encodes an HIV-1 capsid and replicates to high titres in pigtail macaques (PTM), resulting in a nonhuman primate model well-suited for evaluating LEN PrEP in vivo. METHODS: Lenacapavir potency against stHIV-A19 in PTM peripheral blood mononuclear cells in vitro was determined and subcutaneous LEN pharmacokinetics were evaluated in naïve PTMs in vivo. To evaluate the protective efficacy of LEN PrEP, naïve PTMs received either a single subcutaneous injection of LEN (25 mg/kg, N = 3) or vehicle (N = 4) 30 days before a high-dose intravenous challenge with stHIV-A19, or 7 daily subcutaneous injections of a 3-drug control PrEP regimen starting 3 days before stHIV-A19 challenge (N = 3). FINDINGS: In vitro, LEN showed potent antiviral activity against stHIV-A19, comparable to its potency against HIV-1. In vivo, subcutaneous LEN displayed sustained plasma drug exposures in PTMs. Following stHIV-A19 challenge, while all vehicle control animals became productively infected, all LEN and 3-drug control PrEP animals were protected from infection. INTERPRETATION: These findings highlight the utility of the stHIV-A19/PTM model and support the clinical development of long-acting LEN for PrEP in humans. FUNDING: Gilead Sciences as part of a Cooperative Research and Development Agreement between Gilead Sciences and Frederick National Lab; federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. 75N91019D00024/HHSN261201500003I; NIH grant R01AI078788.

Correction: Tracking the Luminal Exposure and Lymphatic Drainage Pathways of Intravaginal and Intrarectal Inocula Used in Nonhuman Primate Models of HIV Transmission.

[This corrects the article DOI: 10.1371/journal.pone.0092830.].

Lymphoid tissues contribute to viral clonotypes present in plasma at early post-ATI in SIV-infected rhesus macaques.

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.

SIV clearance from neonatal macaques following transient CCR5 depletion.

Treatment 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 in CCR5 . 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.

Programming cytomegalovirus as an HIV vaccine.

The initial development of cytomegalovirus (CMV) as a vaccine vector for HIV/simian immunodeficiency virus (SIV) was predicated on its potential to pre-position high-frequency, effector-differentiated, CD8+ T cells in tissues for immediate immune interception of nascent primary infection. This goal was achieved and also led to the unexpected discoveries that non-human primate (NHP) CMVs can be programmed to differentially elicit CD8+ T cell responses that recognize viral peptides via classical MHC-Ia, and/or MHC-II, and/or MHC-E, and that MHC-E-restricted CD8+ T cell responses can uniquely mediate stringent arrest and subsequent clearance of highly pathogenic SIV, an unprecedented type of vaccine-mediated protection. These discoveries delineate CMV vector-elicited MHC-E-restricted CD8+ T cells as a functionally distinct T cell response with the potential for superior efficacy against HIV-1, and possibly other infectious agents or cancers.