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.

Making a Monkey out of Human Immunodeficiency Virus/Simian Immunodeficiency Virus Pathogenesis: Immune Cell Depletion Experiments as a Tool to Understand the Immune Correlates of Protection and Pathogenicity in HIV Infection.

Understanding the underlying mechanisms of HIV pathogenesis is critical for designing successful HIV vaccines and cure strategies. However, achieving this goal is complicated by the virus's direct interactions with immune cells, the induction of persistent reservoirs in the immune system cells, and multiple strategies developed by the virus for immune evasion. Meanwhile, HIV and SIV infections induce a pandysfunction of the immune cell populations, making it difficult to untangle the various concurrent mechanisms of HIV pathogenesis. Over the years, one of the most successful approaches for dissecting the immune correlates of protection in HIV/SIV infection has been the in vivo depletion of various immune cell populations and assessment of the impact of these depletions on the outcome of infection in non-human primate models. Here, we present a detailed analysis of the strategies and results of manipulating SIV pathogenesis through in vivo depletions of key immune cells populations. Although each of these methods has its limitations, they have all contributed to our understanding of key pathogenic pathways in HIV/SIV infection.

Natural killer-like B cells are a distinct but infrequent innate immune cell subset modulated by SIV infection of rhesus macaques.

Natural killer-like B (NKB) cells are unique innate immune cells expressing both natural killer (NK) and B cell receptors. As first responders to infection, they secrete IL-18 to induce a critical cascade of innate and adaptive immune cell infiltration and activation. However, limited research exists on the role of NKB cells in homeostasis and infection, largely due to incomplete and erroneous evaluations. To fill this knowledge gap, we investigated the expression of signaling and trafficking proteins, and the in situ localization and transcriptome of naïve NKB cells compared to conventionally-defined NK and B cells, as well as modulations of these cells in SIV infection. Intracellular signaling proteins and trafficking markers were expressed differentially on naïve NKB cells, with high expression of CD62L and Syk, and low expression of CD69, α4ß7, FcRg, Zap70, and CD3z, findings which were more similar to B cells than NK cells. CD20+NKG2a/c+ NKB cells were identified in spleen, mesenteric lymph nodes (MLN), colon, jejunum, and liver of naïve rhesus macaques (RM) via tissue imaging, with NKB cell counts concentrated in spleen and MLN. For the first time, single cell RNA sequencing (scRNAseq), including B cell receptor (BCR) sequencing, of sorted NKB cells confirmed that NKB cells are unique. Transcriptomic analysis of naïve splenic NKB cells by scRNAseq showed that NKB cells undergo somatic hypermutation and express Ig receptors, similar to B cells. While only 15% of sorted NKB cells showed transcript expression of both KLRC1 (NKG2A) and MS4A1 (CD20) genes, only 5% of cells expressed KLRC1, MS4A1, and IgH/IgL transcripts. We observed expanded NKB frequencies in RM gut and buccal mucosa as early as 14 and 35 days post-SIV infection, respectively. Further, mucosal and peripheral NKB cells were associated with colorectal cytokine milieu and oral microbiome changes, respectively. Our studies indicate that NKB cells gated on CD3-CD14-CD20+NKG2A/C+ cells were inclusive of transcriptomically conventional B and NK cells in addition to true NKB cells, confounding accurate phenotyping and frequency recordings that could only be resolved using genomic techniques. Although NKB cells were clearly elevated during SIV infection and associated with inflammatory changes during infection, further interrogation is necessary to acurately identify the true phenotype and significance of NKB cells in infection and inflammation.

SIV infection and ARV treatment reshape the transcriptional and epigenetic profile of naïve and memory T cells in vivo.

Human and simian immunodeficiency viruses (HIV and SIV) are lentiviruses that reverse transcribe their RNA genome with subsequent integration into the genome of the target cell. How progressive infection and administration of antiretrovirals (ARVs) longitudinally influence the transcriptomic and epigenetic landscape of particular T cell subsets, and how these may influence the genetic location of integration are unclear. Here, we use RNAseq and ATACseq to study the transcriptomics and epigenetic landscape of longitudinally sampled naïve and memory CD4+ and CD8+ T cells in two species of non-human primates prior to SIV infection, during chronic SIV infection, and after administration of ARVs. We find that SIV infection leads to significant alteration to the transcriptomic profile of all T cell subsets that are only partially reversed by administration of ARVs. Epigenetic changes were more apparent in animals with longer periods of untreated SIV infection and correlated well with changes in corresponding gene expression. Known SIV integration sites did not vary due to SIV status but did contain more open chromatin in rhesus macaque memory T cells, and the expression of proteasome-related genes at the pre-SIV timepoint correlated with subsequent viremia.IMPORTANCEChronic inflammation during progressive human and simian immunodeficiency virus (HIV and SIV) infections leads to significant co-morbidities in infected individuals with significant consequences. Antiretroviral (ARV)-treated individuals also manifest increased levels of inflammation which are associated with increased mortalities. These data will help guide rational development of modalities to reduce inflammation observed in people living with HIV and suggest mechanisms underlying lentiviral integration site preferences.

Macrophage- and CD4+ T cell-derived SIV differ in glycosylation, infectivity and neutralization sensitivity.

The human immunodeficiency virus (HIV) envelope protein (Env) mediates viral entry into host cells and is the primary target for the humoral immune response. Env is extensively glycosylated, and these glycans shield underlying epitopes from neutralizing antibodies. The glycosylation of Env is influenced by the type of host cell in which the virus is produced. Thus, HIV is distinctly glycosylated by CD4+ T cells, the major target cells, and macrophages. However, the specific differences in glycosylation between viruses produced in these cell types have not been explored at the molecular level. Moreover, it remains unclear whether the production of HIV in CD4+ T cells or macrophages affects the efficiency of viral spread and resistance to neutralization. To address these questions, we employed the simian immunodeficiency virus (SIV) model. Glycan analysis implied higher relative levels of oligomannose-type N-glycans in SIV from CD4+ T cells (T-SIV) compared to SIV from macrophages (M-SIV), and the complex-type N-glycans profiles seem to differ between the two viruses. Notably, M-SIV demonstrated greater infectivity than T-SIV, even when accounting for Env incorporation, suggesting that host cell-dependent factors influence infectivity. Further, M-SIV was more efficiently disseminated by HIV binding cellular lectins. We also evaluated the influence of cell type-dependent differences on SIV's vulnerability to carbohydrate binding agents (CBAs) and neutralizing antibodies. T-SIV demonstrated greater susceptibility to mannose-specific CBAs, possibly due to its elevated expression of oligomannose-type N-glycans. In contrast, M-SIV exhibited higher susceptibility to neutralizing sera in comparison to T-SIV. These findings underscore the importance of host cell-dependent attributes of SIV, such as glycosylation, in shaping both infectivity and the potential effectiveness of intervention strategies.

Immune restoration by TIGIT blockade is insufficient to control chronic SIV infection.

TIGIT is a negative immune checkpoint receptor associated with T cell exhaustion in cancer and HIV. TIGIT upregulation in virus-specific CD8+ T cells and NK cells during HIV/SIV infection results in dysfunctional effector capabilities. In vitro studies targeting TIGIT on CD8+ T cells suggest TIGIT blockade as a viable strategy to restore SIV-specific T cell responses. Here, we extend these studies in vivo using TIGIT blockage in nonhuman primates in an effort to reverse T cell and NK cell exhaustion in the setting of SIV infection. We demonstrate that in vivo administration of a humanized anti-TIGIT monoclonal antibody (mAb) is well tolerated in both cynomolgus macaques and rhesus macaques. Despite sustained plasma concentrations of anti-TIGIT mAb, we observed no consistent improvement in NK or T cell cytolytic capacity. TIGIT blockade minimally enhanced T cell proliferation and virus-specific T cell responses in both magnitude and breadth though plasma viral loads in treated animals remained stable indicating that anti-TIGIT mAb treatment alone was insufficient to increase anti-SIV CD8+ T cell function. The enhancement of virus-specific T cell proliferative responses observed in vitro with single or dual blockade of TIGIT and/or PD-1 highlights TIGIT as a potential target to reverse T cell dysfunction. Our studies, however, reveal that targeting the TIGIT pathway alone may be insufficient in the setting of viremia and that combining immune checkpoint blockade with other immunotherapeutics may be a future path forward for improved viral control or elimination of HIV.IMPORTANCEUpregulation of the immune checkpoint receptor TIGIT is associated with HIV-mediated T cell dysfunction and correlates with HIV disease progression. Compelling evidence exists for targeting immune checkpoint receptor pathways that would potentially enhance immunity and refocus effector cell efforts toward viral clearance. In this report, we investigate TIGIT blockade as an immunotherapeutic approach to reverse immune exhaustion during chronic SIV/SHIV infection in a nonhuman primate model of HIV infection. We show that interfering with the TIGIT signaling axis alone is insufficient to improve viral control despite modest improvement in T cell immunity. Our data substantiate the use of targeting multiple immune checkpoint receptors to promote synergy and ultimately eliminate HIV-infected cells.

Quantitative and Qualitative Distinctions between HIV-1 and SIV Reservoirs: Implications for HIV-1 Cure-Related Studies.

The persistence of the latent viral reservoir is the main hurdle to curing HIV-1 infection. SIV infection of non-human primates (NHPs), namely Indian-origin rhesus macaques, is the most relevant and widely used animal model to evaluate therapies that seek to eradicate HIV-1. The utility of a model ultimately rests on how accurately it can recapitulate human disease, and while reservoirs in the NHP model behave quantitatively very similar to those of long-term suppressed persons with HIV-1 (PWH) in the most salient aspects, recent studies have uncovered key nuances at the clonotypic level that differentiate the two in qualitative terms. In this review, we will highlight differences relating to proviral intactness, clonotypic structure, and decay rate during ART between HIV-1 and SIV reservoirs and discuss the relevance of these distinctions in the interpretation of HIV-1 cure strategies. While these, to some degree, may reflect a unique biology of the virus or host, distinctions among the proviral landscape in SIV are likely to be shaped significantly by the condensed timeframe of NHP studies. ART is generally initiated earlier in the disease course, and animals are virologically suppressed for shorter periods before receiving interventions. Because these are experimental variables dictated by the investigator, we offer guidance on study design for cure-related studies performed in the NHP model. Finally, we highlight the case of GS-9620 (Vesatolimod), an antiviral TLR7 agonist tested in multiple independent pre-clinical studies in which virological outcomes may have been influenced by study-related variables.

Identification of macaque dendritic cell precursors in blood and tissue reveals their dysregulation in early SIV infection.

Distinct dendritic cell (DC) subsets play important roles in shaping immune responses. Circulating DC precursors (pre-DCs) are more susceptible to HIV infection in vitro, which may explain the inefficiency of immune responses against HIV. However, the interplay between HIV and pre-DC is not defined in vivo. We identify human pre-DC equivalents in the cynomolgus macaque and then analyze their dynamics during simian immunodeficiency virus (SIV) infection to illustrate a sharp decrease of blood pre-DCs in early SIV infection and accumulation in lymph nodes (LNs), where they neglect to upregulate CD83/CD86 or MHC-II. Additionally, SIV infection attenuates the capacity of stimulated LN pre-DCs to produce IL-12p40. Analysis of HIV cohorts provides correlation between costimulatory molecule expression on pre-DCs and T cell activation in spontaneous HIV controllers. These findings pinpoint certain dynamics and functional changes of pre-DCs during SIV infection, providing a deeper understanding of immune dysregulation mechanisms elicited in people living with HIV.

SHIV-C109p5 NHP induces rapid disease progression in elderly macaques with extensive GI viral replication.

CCR5-tropic simian/human immunodeficiency viruses (SHIV) with clade C transmitted/founder envelopes represent a critical tool for the investigation of HIV experimental vaccines and microbicides in nonhuman primates, although many such isolates lead to spontaneous viral control post infection. Here, we generated a high-titer stock of pathogenic SHIV-C109p5 by serial passage in two rhesus macaques (RM) and tested its virulence in aged monkeys. The co-receptor usage was confirmed before infecting five geriatric rhesus macaques (four female and one male). Plasma viral loads were monitored by reverse transcriptase-quantitative PCR (RT-qPCR), cytokines by multiplex analysis, and biomarkers of gastrointestinal damage by enzyme-linked immunosorbent assay. Antibodies and cell-mediated responses were also measured. Viral dissemination into tissues was determined by RNAscope. Intravenous SHIV-C109p5 infection of aged RMs leads to high plasma viremia and rapid disease progression; rapid decrease in CD4+ T cells, CD4+CD8+ T cells, and plasmacytoid dendritic cells; and wasting necessitating euthanasia between 3 and 12 weeks post infection. Virus-specific cellular immune responses were detected only in the two monkeys that survived 4 weeks post infection. These were Gag-specific TNFα+CD8+, MIP1ß+CD4+, Env-specific IFN-γ+CD4+, and CD107a+ T cell responses. Four out of five monkeys had elevated intestinal fatty acid binding protein levels at the viral peak, while regenerating islet-derived protein 3α showed marked increases at later time points in the three animals surviving the longest, suggesting gut antimicrobial peptide production in response to microbial translocation post infection. Plasma levels of monocyte chemoattractant protein-1, interleukin-15, and interleukin-12/23 were also elevated. Viral replication in gut and secondary lymphoid tissues was extensive.IMPORTANCESimian/human immunodeficiency viruses (SHIV) are important reagents to study prevention of virus acquisition in nonhuman primate models of HIV infection, especially those representing transmitted/founder (T/F) viruses. However, many R5-tropic SHIV have limited fitness in vivo leading to many monkeys spontaneously controlling the virus post acute infection. Here, we report the generation of a pathogenic SHIV clade C T/F stock by in vivo passage leading to sustained viral load set points, a necessity to study pathogenicity. Unexpectedly, administration of this SHIV to elderly rhesus macaques led to extensive viral replication and fast disease progression, despite maintenance of a strict R5 tropism. Such age-dependent rapid disease progression had previously been reported for simian immunodeficiency virus but not for R5-tropic SHIV infections.

Extended Postexposure Protection Against Vaginal Simian/Human Immunodeficiency Virus Infection With Tenofovir Alafenamide Fumarate/Elvitegravir Inserts in Macaques.

Vaginal inserts that can be used on demand before or after sex may be a desirable human immunodeficiency virus (HIV) prevention option for women. We recently showed that inserts containing tenofovir alafenamide fumarate (TAF, 20 mg) and elvitegravir (EVG, 16 mg) were highly protective against repeated simian/human immunodeficiency virus (SHIV) vaginal exposures when administered to macaques 4 hours before or after virus exposure (93% and 100%, respectively). Here, we show in the same macaque model that insert application 8 hours or 24 hours after exposure maintains high efficacy (94.4% and 77.2%, respectively). These data extend the protective window by TAF/EVG inserts and inform their clinical development for on-demand prophylaxis in women.

Host immunity associated with spontaneous suppression of viremia in therapy-naïve young rhesus macaques following neonatal SHIV infection.

IMPORTANCE: Despite the advent of highly active anti-retroviral therapy, people are still dying from HIV-related causes, many of whom are children, and a protective vaccine or cure is needed to end the HIV pandemic. Understanding the nature and activation states of immune cell subsets during infection will provide insights into the immunologic milieu associated with viremia suppression that can be harnessed via therapeutic strategies to achieve a functional cure, but these are understudied in pediatric subjects. We evaluated humoral and adaptive host immunity associated with suppression of viremia in rhesus macaques infected soon after birth with a pathogenic SHIV. The results from our study provide insights into the immune cell subsets and functions associated with viremia control in young macaques that may translate to pediatric subjects for the design of future anti-viral strategies in HIV-1-infected infants and children and contribute to an understudied area of HIV-1 pathogenesis in pediatric subjects.

Lymph-Node-Based CD3+ CD20+ Cells Emerge from Membrane Exchange between T Follicular Helper Cells and B Cells and Increase Their Frequency following Simian Immunodeficiency Virus Infection.

CD4+ T follicular helper (TFH) cells are key targets for human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) replication and contribute to the virus reservoir under antiretroviral therapy (ART). Here, we describe a novel CD3+ CD20+ double-positive (DP) lymphocyte subset, resident in secondary lymphoid organs of humans and rhesus macaques (RMs), that appear predominantly after membrane exchange between TFH and B cells. DP lymphocytes are enriched in cells displaying a TFH phenotype (CD4+ PD1hi CXCR5hi), function (interleukin 21 positive [IL-21+]), and gene expression profile. Importantly, expression of CD40L upon brief in vitro mitogen stimulation identifies, by specific gene-expression signatures, DP cells of TFH-cell origin versus those of B-cell origin. Analysis of 56 RMs showed that DP cells (i) significantly increase following SIV infection, (ii) are reduced after 12 months of ART in comparison to pre-ART levels, and (iii) expand to a significantly higher frequency following ART interruption. Quantification of total SIV-gag DNA on sorted DP cells from chronically infected RMs showed that these cells are susceptible to SIV infection. These data reinforce earlier observations that CD20+ T cells are infected and expanded by HIV infection, while suggesting that these cells phenotypically overlap activated CD4+ TFH cells that acquire CD20 expression via trogocytosis and can be targeted as part of therapeutic strategies aimed at HIV remission. IMPORTANCE The HIV reservoir is largely composed of latently infected memory CD4+ T cells that persist during antiretroviral therapy and constitute a major barrier toward HIV eradication. In particular, CD4+ T follicular helper cells have been demonstrated as key targets for viral replication and persistence under ART. In lymph nodes from HIV-infected humans and SIV-infected rhesus macaques, we show that CD3+ CD20+ lymphocytes emerge after membrane exchange between T cells and B cells and are enriched in phenotypic, functional, and gene expression profiles found in T follicular helper cells. Furthermore, in SIV-infected rhesus macaques, these cells expand following experimental infection and after interruption of ART and harbor SIV DNA at levels similar to those found in CD4+ T cells; thus, CD3+ CD20+ lymphocytes are susceptible to SIV infection and can contribute to SIV persistence.

Rapid Loss of CD4 T Cells by Pyroptosis during Acute SIV Infection in Rhesus Macaques.

The mechanisms underlying depletion of CD4 T cells during acute HIV-1 infection are not well understood. Here we show that caspase-1-induced pyroptosis, a highly inflammatory programmed cell death pathway, is the dominant mechanism responsible for the rapid depletion of CD4 T cells in gut-associated lymphatic tissue (GALT), spleen, and lymph nodes during acute simian immunodeficiency virus (SIV) infection in rhesus macaques. Upregulation of interferon-gamma inducible factor 16, a host DNA sensor that triggers pyroptosis, was also observed in tissue-resident CD4 T cells and correlated with viral loads and CD4 T cell loss. In contrast, caspase-3-mediated apoptosis and viral cytotoxicity only accounted for a small fraction of CD4 T cell death. Other programmed cell death mechanisms, including mitochondria-induced caspase-independent cell death, necroptosis, and autophagy, did not significantly contribute to CD4 T cell depletion. These data support a model in which caspase-1-mediated pyroptosis is the principal mechanism that results in CD4 T cell loss in the GALT and lymphoid organs and release of proinflammatory cytokines. These findings contribute to our understanding of the pathogenesis of acute SIV infection and have important implications for the development of therapeutic strategies. IMPORTANCE Different mechanisms for CD4 T cell depletion during acute HIV-1 infection have been proposed. In this study, we demonstrate that in early simian immunodeficiency virus infection, depletion of CD4 T cells is primarily due to pyroptosis. Other mechanisms may also contribute in a minor way to CD4 T cell depletion.

Single-Cell Profiling of Latently SIV-Infected CD4+ T Cells Directly Ex Vivo to Reveal Host Factors Supporting Reservoir Persistence.

HIV-1 cure strategies aiming to eliminate persistent infected cell reservoirs are hampered by a poor understanding of cells harboring viral DNA in vivo. We describe a novel method to identify, enumerate, and characterize in detail individual cells infected in vivo using a combination of single-cell multiplexed assays for integrated proviral DNA, quantitative viral and host gene expression, and quantitative surface protein expression without any in vitro manipulation. Latently infected CD4+ T cells, defined as harboring integrated provirus in the absence of spliced viral mRNA, were identified from macaque lymph nodes during acute, chronic, and combination antiretroviral therapy (cART)-suppressed simian immunodeficiency virus (SIV) infection. Latently infected CD4+ T cells were most abundant during acute SIV (~8% of memory CD4+ T cells) and persisted in chronic and cART-suppressed infection. Productively infected cells actively transcribing viral mRNA, by contrast, were much more labile and declined substantially between acute and chronic or cART-suppressed infection. Expression of most surface proteins and host genes was similar between latently infected cells and uninfected cells. Elevated FLIP mRNA and surface CD3 expression among latently infected cells suggest increased survival potential and capacity to respond to T cell receptor stimulation. These findings point to a large pool of latently infected CD4+ T cells established very early in acute infection and upregulated host factors that may facilitate their persistence in vivo, both of which pose potential challenges to eliminating HIV-1 reservoirs. IMPORTANCE Effective combination antiretroviral therapy controls HIV-1 infection but fails to eliminate latent viral reservoirs that give rise to viremia upon treatment interruption. Strategies to eradicate latently infected cells require a better understanding of their biology and distinguishing features to promote their elimination. Tools for studying these cells from patients are currently limited. Here, we developed a single-cell method to identify cells latently infected in vivo and to characterize these cells for expression of surface proteins and host genes without in vitro manipulation, capturing their in vivo state from SIV-infected macaques. Host factors involved in cell survival and proliferation were upregulated in latently infected cells, which were abundant in the earliest stages of acute infection. These studies provide insight into the basic biology of latently infected cells as well as potential mechanisms underlying the persistence of HIV-1/SIV reservoirs to inform development of novel HIV-1 cure strategies.

In PrEP: Long-acting antivirals for HIV prevention.

Two recent publications (Vidal et al., 2021; Xue et al., 2022) report highly potent inhibitors as candidates for HIV pre-exposure prophylaxis (PrEP). They provide strong evidence that administration of long-acting capsid or fusion inhibitors as PrEP offers robust protection against simian-human immunodeficiency virus in the rhesus macaque model of HIV infection.

Phagocytosis by an HIV antibody is associated with reduced viremia irrespective of enhanced complement lysis.

Increasingly, antibodies are being used to treat and prevent viral infections. In the context of HIV, efficacy is primarily attributed to dose-dependent neutralization potency and to a lesser extent Fc-mediated effector functions. It remains unclear whether augmenting effector functions of broadly neutralizing antibodies (bNAbs) may improve their clinical potential. Here, we use bNAb 10E8v4 targeting the membrane external proximal region (MPER) to examine the role of antibody-mediated effector and complement (C') activity when administered prophylactically against SHIV challenge in rhesus macaques. With sub-protective dosing, we find a 78-88% reduction in post-acute viremia that is associated with 10E8v4-mediated phagocytosis acting at the time of challenge. Neither plasma nor tissue viremic outcomes in vivo is improved with an Fc-modified variant of 10E8v4 enhanced for C' functions as determined in vitro. These results suggest that effector functions inherent to unmodified 10E8v4 contribute to efficacy against SHIVSF162P3 in the absence of plasma neutralizing titers, while C' functions are dispensable in this setting, informing design of bNAb modifications for improving protective efficacy.

Exacerbated AIDS Progression by PD-1 Blockade during Therapeutic Vaccination in Chronically Simian Immunodeficiency Virus-Infected Rhesus Macaques after Interruption of Antiretroviral Therapy.

The persistence of cells latently infected with HIV-1, named the latent reservoir, is the major barrier to HIV-1 eradication, and the formation and maintenance of the latent reservoir might be exacerbated by activation of the immunoinhibitory pathway and dysfunction of CD8+ T cells during HIV-1 infection. Our previous findings demonstrated that prophylactic vaccination combined with PD-1 blockade generated distinct immune response profiles and conferred effective control of highly pathogenic SIVmac239 infection in rhesus macaques. However, to our surprise, herein we found that a therapeutic vaccination in combination with PD-1 blockade resulted in activation of the viral reservoir, faster viral rebound after treatment interruption, accelerated AIDS progression, and, ultimately, death in chronically SIV-infected macaques after antiretroviral therapy (ART) interruption. Our study further demonstrated that the SIV provirus was preferentially enriched in PD-1+CD4+ T cells due to their susceptibility to viral entry, potent proliferative ability, and inability to perform viral transcription. In addition, the viral latency was effectively reactivated upon PD-1 blockade. Together, these results suggest that PD-1 blockade may be a double-edged sword for HIV-1 immunotherapy and provide important insight toward the rational design of immunotherapy strategies for an HIV-1 cure. IMPORTANCE As it is one of the most challenging public health problems, there are no clinically effective cure strategies against HIV-1 infection. We demonstrated that prophylactic vaccination combined with PD-1 blockade generated distinct immune response profiles and conferred better control of highly pathogenic SIVmac239 infection in rhesus macaques. In the present study, to our surprise, PD-1 blockade during therapeutic vaccination accelerated the reactivation of latent reservoir and AIDS progression in chronically SIV-infected macaques after ART interruption. Our study further demonstrated that the latent SIV provirus was preferentially enriched in PD-1+CD4+ T cells because of its susceptibility to viral entry, inhibition of SIV transcription, and potent ability of proliferation, and the viral latency was effectively reactivated by PD-1 blockade. Therefore, PD-1 blockade might be a double-edged sword for AIDS therapy. These findings provoke interest in further exploring novel treatments against HIV-1 infection and other emerging infectious diseases.

Long-acting capsid inhibitor protects macaques from repeat SHIV challenges.

Because no currently available vaccine can prevent HIV infection, pre-exposure prophylaxis (PrEP) with antiretrovirals (ARVs) is an important tool for combating the HIV pandemic1,2. Long-acting ARVs promise to build on the success of current PrEP strategies, which must be taken daily, by reducing the frequency of administration3. GS-CA1 is a small-molecule HIV capsid inhibitor with picomolar antiviral potency against a broad array of HIV strains, including variants resistant to existing ARVs, and has shown long-acting therapeutic potential in a mouse model of HIV infection4. Here we show that a single subcutaneous administration of GS-CA1 provides long-term protection against repeated rectal simian-human immunodeficiency virus (SHIV) challenges in rhesus macaques. Whereas all control animals became infected after 15 weekly challenges, a single 300 mg kg-1 dose of GS-CA1 provided per-exposure infection risk reduction of 97% for 24 weeks. Pharmacokinetic analysis showed a correlation between GS-CA1 plasma concentration and protection from SHIV challenges. GS-CA1 levels greater than twice the rhesus plasma protein-adjusted 95% effective concentration conferred 100% protection in this model. These proof-of-concept data support the development of capsid inhibitors as a novel long-acting PrEP strategy in humans.

Efficient treatment and pre-exposure prophylaxis in rhesus macaques by an HIV fusion-inhibitory lipopeptide.

Two HIV fusion-inhibitory lipopeptides (LP-97 and LP-98) were designed with highly potent, long-acting antiviral activity. Monotherapy using a low dose of LP-98 sharply reduced viral loads and maintained long-term viral suppression in 21 SHIVSF162P3-infected rhesus macaques. We found that five treated monkeys achieved potential posttreatment control (PTC) efficacy and had lower viral DNA in deep lymph nodes, whereas monkeys with a stable viral rebound had higher viral DNA in superficial lymph nodes. The tissues of PTC monkeys exhibited significantly decreased quantitative viral outgrowth and fewer PD-1+ central memory CD4+ T cells, and CD8+ T cells contributed to virologic control efficacy. Moreover, LP-98 administrated as a pre-exposure prophylaxis (PrEP) provided complete protection against SHIVSF162P3 and SIVmac239 infections in 51 monkeys via intrarectal, intravaginal, or intravenous challenge. In conclusion, our lipopeptides exhibit high potential as an efficient HIV treatment or prevention strategy.

Lentiviral Infections Persist in Brain despite Effective Antiretroviral Therapy and Neuroimmune Activation.

HIV infection persists in different tissue reservoirs among people with HIV (PWH) despite effective antiretroviral therapy (ART). In the brain, lentiviruses replicate principally in microglia and trafficking macrophages. The impact of ART on this viral reservoir is unknown. We investigated the activity of contemporary ART in various models of lentivirus brain infection. HIV-1 RNA and total and integrated DNA were detected in cerebral cortex from all PWH (n = 15), regardless of ART duration or concurrent plasma viral quantity and, interestingly, integrated proviral DNA levels in brain were significantly higher in the aviremic ART-treated group (P < 0.005). Most ART drugs tested (dolutegravir, ritonavir, raltegravir, and emtricitabine) displayed significantly lower 50% effective concentration (EC50) values in lymphocytes than in microglia, except tenofovir, which showed 1.5-fold greater activity in microglia (P < 0.05). In SIV-infected Chinese rhesus macaques, despite receiving suppressive (n = 7) or interrupted (n = 8) ART, brain tissues had similar SIV-encoded RNA and total and integrated DNA levels compared to brains from infected animals without ART (n = 3). SIV and HIV-1 capsid antigens were immunodetected in brain, principally in microglia/macrophages, regardless of ART duration and outcome. Antiviral immune responses were comparable in the brains of ART-treated and untreated HIV- and SIV-infected hosts. Both HIV-1 and SIV persist in brain tissues despite contemporary ART, with undetectable virus in blood. ART interruption exerted minimal effect on the SIV brain reservoir and did not alter the neuroimmune response profile. These studies underscore the importance of augmenting ART potency in different tissue compartments. IMPORTANCE Antiretroviral therapy (ART) suppresses HIV-1 in plasma and CSF to undetectable levels. However, the impact of contemporary ART on HIV-1 brain reservoirs remains uncertain. An active viral reservoir in the brain during ART could lead to rebound systemic infection after cessation of therapy, development of drug resistance mutations, and neurological disease. ART's impact, including its interruption, on brain proviral DNA remains unclear. The present studies show that in different experimental platforms, contemporary ART did not suppress viral burden in the brain, regardless of ART component regimen, the duration of therapy, and its interruption. Thus, new strategies for effective HIV-1 suppression in the brain are imperative to achieve sustained HIV suppression.