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HIV-1 establishes a persistent proviral reservoir by integrating into the genome of infected host cells. Current antiretroviral treatments do not target this persistent population of proviruses which include latently infected cells that upon treatment interruption can be reactivated to contribute to HIV-1 rebound. Deep sequencing of persistent HIV proviruses has revealed that greater than 90% of integrated HIV genomes are defective and unable to produce infectious virions. We hypothesized that intragenic elements in the HIV genome support transcription of aberrant HIV-1 RNAs from defective proviruses that lack long terminal repeats (LTRs). Using an intact provirus detection assay, we observed that resting CD4+ T cells and monocyte-derived macrophages (MDMs) are biased towards generating defective HIV-1 proviruses. Multiplex reverse transcription droplet digital PCR identified env and nef transcripts which lacked 5' untranslated regions (UTR) in acutely infected CD4+ T cells and MDMs indicating transcripts are generated that do not utilize the promoter within the LTR. 5'UTR-deficient env transcripts were also identified in a cohort of people living with HIV (PLWH) on ART, suggesting that these aberrant RNAs are produced in vivo. Using 5' rapid amplification of cDNA ends (RACE), we mapped the start site of these transcripts within the Env gene. This region bound several cellular transcription factors and functioned as a transcriptional regulatory element that could support transcription and translation of downstream HIV-1 RNAs. These studies provide mechanistic insights into how defective HIV-1 proviruses are persistently expressed to potentially drive inflammation in PLWH. Author summary: People living with HIV establish a persistent reservoir which includes latently infected cells that fuel viral rebound upon treatment interruption. However, the majority of HIV-1 genomes in these persistently infected cells are defective. Whether these defective HIV genomes are expressed and whether they contribute to HIV associated diseases including accelerated aging, neurodegenerative symptoms, and cardiovascular diseases are still outstanding questions. In this paper, we demonstrate that acute infection of macrophages and resting T cells is biased towards generating defective viruses which are expressed by DNA regulatory elements in the HIV genome. These studies describe an alternative mechanism for chronic expression of HIV genomes. [ABSTRACT FROM AUTHOR]

The APOBEC3 (A3) genes encode cytidine deaminase proteins with potent antiviral and anti-retroelement activity. This locus is characterized by duplication, recombination, and deletion events that gave rise to the seven A3s found in primates. These include three single deaminase domain A3s (A3A, A3C, and A3H) and four double deaminase domain A3s (A3B, A3D, A3F, and A3G). The most potent of the A3 proteins against HIV-1 is A3G. However, it is not clear if double deaminase domain A3s have a generalized functional advantage to restrict HIV-1. In order to test whether superior restriction factors could be created by genetically linking single A3 domains into synthetic double domains, we linked A3C and A3H single domains in novel combinations. We found that A3C/A3H double domains acquired enhanced antiviral activity that is at least as potent, if not better than, A3G. Although these synthetic double domain A3s package into budding virions more efficiently than their respective single domains, this does not fully explain their gain of antiviral potency. The antiviral activity is conferred both by cytidine-deaminase dependent and independent mechanisms, with the latter correlating to an increase in RNA binding affinity. T cell lines expressing this A3C-A3H super restriction factor are able to control replicating HIV-1ΔVif infection to similar levels as A3G. Together, these data show that novel combinations of A3 domains are capable of gaining potent antiviral activity to levels similar to the most potent genome-encoded A3s, via a primarily non-catalytic mechanism. Author summary: Antiviral genes are encoded by all organisms to help protect them from viral infections, including proteins encoded by primates to protect them from viruses similar to HIV-1. These antiviral proteins are also called "restriction factors". Some restriction factors are broadly acting, while others are very specific. During the course of evolution, some of these genes have expanded into multiple copies and rearranged in different versions to give them new activities. In this paper, we validated the hypothesis that one particular antiviral gene family, called the APOBEC3 family, has the capability of making novel combinations of antiviral human genes with as great, or greater, potency against HIV-1 as the most potent natural member of this family. By combining parts of the APOBEC3 proteins into novel combinations, we created potent antiviral versions that act through a mechanism distinct from existing APOBEC3 proteins. [ABSTRACT FROM AUTHOR]

Membrane-associated RING-CH (MARCH) 2 is a member of the MARCH protein family of RING-CH finger E3 ubiquitin ligases that play important roles in regulating the levels of proteins found on the cell surface. MARCH1, 2 and 8 inhibit HIV-1 infection by preventing the incorporation of the envelope glycoproteins into nascent virions. However, a better understanding on the mechanism utilized by MARCH proteins to restrict HIV-1 infection is needed. In this report, we identify an amino acid in human MARCH2, absent in mouse MARCH2, critical for its antiretroviral function. Moreover, we map the domains of human MARCH2 critical for restricting as well as binding to the HIV-1 envelope glycoproteins. In addition, we demonstrate that MARCH2 is present inside nascent virions and reduces particle infectivity by blocking virus entry in a RING-CH-independent manner. Finally, we show that MARCH2 acts as an HIV-1 restriction factor only in primary CD4+ T cells and can prevent cell-to-cell transmission of HIV-1. Our findings reveal important new aspects of the antiviral mechanism utilized by human MARCH2 to restrict HIV-1 that have potential implications to all MARCH proteins with antiviral functions and their viral targets. Author summary: Recently, Membrane-associated RING-CH (MARCH) proteins have emerged as important antiviral factors that block virus infection by preventing viral envelope glycoprotein incorporation into newly formed virions in an ill-defined mechanism. Here, we examine in depth the role of MARCH2 during Human Immunodeficiency Virus type 1 (HIV-1) infection and show that it utilizes two mechanisms to inhibit HIV-1: 1) prevents the envelope glycoprotein incorporation into nascent virions in a RING-CH-dependent manner, 2) when incorporated in virions, it blocks virus-cell membrane fusion in a RING-CH independent manner. We also demonstrate that MARCH2 restricts HIV-1 only in CD4+ T cells and can prevent cell-to-cell transmission. In conclusion, our findings reveal new mechanistic aspects regarding the anti-HIV-1 function of MARCH2. [ABSTRACT FROM AUTHOR]

Most people living with HIV-1 experience rapid viral rebound once antiretroviral therapy is interrupted; however, a small fraction remain in viral remission for an extended duration. Understanding the factors that determine whether viral rebound is likely after treatment interruption can enable the development of optimal treatment regimens and therapeutic interventions to potentially achieve a functional cure for HIV-1. We built upon the theoretical framework proposed by Conway and Perelson to construct dynamic models of virus-immune interactions to study factors that influence viral rebound dynamics. We evaluated these models using viral load data from 24 individuals following antiretroviral therapy interruption. The best-performing model accurately captures the heterogeneity of viral dynamics and highlights the importance of the effector cell expansion rate. Our results show that post-treatment controllers and non-controllers can be distinguished based on the effector cell expansion rate in our models. Furthermore, these results demonstrate the potential of using dynamic models incorporating an effector cell response to understand early viral rebound dynamics post-antiretroviral therapy interruption. Author summary: Most people living with HIV-1 experience rapid viral rebound once antiretroviral therapy is interrupted; however, a small fraction remain in viral remission for an extended duration. The factors that determine viral rebound dynamics after treatment interruption are not well understood. In this study, we built upon a previous theoretical framework to construct dynamic models of virus-immune interactions to study factors that influence viral rebound dynamics. We evaluated these models using viral load data from 24 individuals following antiretroviral therapy interruption. The best-performing model accurately captures the heterogeneity of viral dynamics and robustly shows that people who remain in viral remission for an extended duration after treatment interruption have a higher effector cell expansion rate compared to those who rapidly rebound. These results demonstrate the potential of using viral dynamic models incorporating an effector cell response to understand early viral rebound dynamics post-antiretroviral therapy interruption. [ABSTRACT FROM AUTHOR]

A strong genetic predictor of outcome following untreated HIV-1 infection is the carriage of specific alleles of human leukocyte antigens (HLAs) that present viral epitopes to T cells. Residual variation in outcome measures may be attributed, in part, to viral adaptation to HLA-restricted T cell responses. Variants of the endoplasmic reticulum aminopeptidases (ERAPs) influence the repertoire of T cell epitopes presented by HLA alleles as they trim pathogen-derived peptide precursors to optimal lengths for antigen presentation, along with other functions unrelated to antigen presentation. We investigated whether ERAP variants influence HLA-associated HIV-1 adaptation with demonstrable effects on overall HIV-1 disease outcome. Utilizing host and viral data of 249 West Australian individuals with HIV-1 subtype B infection, we identified a novel association between two linked ERAP2 single nucleotide polymorphisms (SNPs; rs2248374 and rs2549782) with plasma HIV RNA concentration (viral load) (P adjusted = 0.0024 for both SNPs). Greater HLA-associated HIV-1 adaptation in the HIV-1 Gag gene correlated significantly with higher viral load, lower CD4+ T cell count and proportion; P = 0.0103, P = 0.0061, P = 0.0061, respectively). When considered together, there was a significant interaction between the two ERAP2 SNPs and HLA-associated HIV-1 adaptation on viral load (P = 0.0111). In a comprehensive multivariate model, addition of ERAP2 haplotypes and HLA associated adaptation as an interaction term to known HLA and CCR5 determinants and demographic factors, increased the explanatory variance of population viral load from 17.67% to 45.1% in this dataset. These effects were not replicated in publicly available datasets with comparably sized cohorts, suggesting that any true global epistasis may be dependent on specific HLA-ERAP allelic combinations. Our data raises the possibility that ERAP2 variants may shape peptide repertoires presented to HLA class I-restricted T cells to modulate the degree of viral adaptation within individuals, in turn contributing to disease variability at the population level. Analyses of other populations and experimental studies, ideally with locally derived ERAP genotyping and HLA-specific viral adaptations are needed to elucidate this further. Author summary: HIV infection outcome is variable between individuals and understanding the factors that impact this variation is important for efforts towards a HIV cure or vaccine. Here, we found that the level of HIV in the blood is affected by whether an individual carries a specific form of ERAP2, a molecule that influences processing and presentation of the virus to the immune system, as well as the degree to which HIV has mutated to adapt to immune responses. We also show that the interaction of ERAP2 and other known genetic factors explains greater variation in infection outcome than these factors alone. These findings expand our knowledge of the potential importance of viral processing and presentation in the immune response to HIV. [ABSTRACT FROM AUTHOR]

AXL+ Siglec-6+ dendritic cells (ASDC) are novel myeloid DCs which can be subdivided into CD11c+ and CD123+ expressing subsets. We showed for the first time that these two ASDC subsets are present in inflamed human anogenital tissues where HIV transmission occurs. Their presence in inflamed tissues was supported by single cell RNA analysis of public databases of such tissues including psoriasis diseased skin and colorectal cancer. Almost all previous studies have examined ASDCs as a combined population. Our data revealed that the two ASDC subsets differ markedly in their functions when compared with each other and to pDCs. Relative to their cell functions, both subsets of blood ASDCs but not pDCs expressed co-stimulatory and maturation markers which were more prevalent on CD11c+ ASDCs, thus inducing more T cell proliferation and activation than their CD123+ counterparts. There was also a significant polarisation of naïve T cells by both ASDC subsets toward Th2, Th9, Th22, Th17 and Treg but less toward a Th1 phenotype. Furthermore, we investigated the expression of chemokine receptors that facilitate ASDCs and pDCs migration from blood to inflamed tissues, their HIV binding receptors, and their interactions with HIV and CD4 T cells. For HIV infection, within 2 hours of HIV exposure, CD11c+ ASDCs showed a trend in more viral transfer to T cells than CD123+ ASDCs and pDCs for first phase transfer. However, for second phase transfer, CD123+ ASDCs showed a trend in transferring more HIV than CD11c+ ASDCs and there was no viral transfer from pDCs. As anogenital inflammation is a prerequisite for HIV transmission, strategies to inhibit ASDC recruitment into inflamed tissues and their ability to transmit HIV to CD4 T cells should be considered. Author summary: This study highlights the significance of AXL+ Siglec-6+ dendritic cells (ASDC) in HIV transmission, particularly in inflamed peripheral tissues such as anogenital tissues, where HIV transmission is prevalent. It reveals that ASDCs are present in inflamed human tissues, including psoriasis affected skin, colorectal cancer and anogenital tissues. However, they are absent in uninflamed tissues. Furthermore, we investigated the expression of chemokine receptors that facilitate ASDC migration from blood to inflamed tissues, and their interactions with HIV and CD4 T cells. Notably, different subsets of ASDCs exhibit different expression levels of HIV binding receptors and showed trends of different phases of HIV transmission to T cells. Understanding ASDC involvement in HIV transmission could provide valuable insights for developing strategies to inhibit their recruitment to inflamed tissues and their ability to transmit the virus to CD4 T cells, potentially offering new avenues for HIV prevention and treatment. [ABSTRACT FROM AUTHOR]

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. Author summary: The human immunodeficiency virus (HIV) is the causative agent of the acquired immunodeficiency syndrome (AIDS), a lethal condition that necessitates life-long antiretroviral therapy for prevention. The envelope protein (Env) of HIV mediates the viral entry into host cells and is the sole target for the immune system on the surface of the virus. Env's extensive modification with sugars, so called N-glycans, shields crucial epitopes from neutralizing antibodies. Interestingly, key HIV producing cell types, such as CD4+ T cells and macrophages, modify Env with distinct N-glycosylation patterns. However, the specific molecular disparities in N-glycosylation between viruses from these cell types are unexplored, and whether the producer cell type impacts the viral spread and resistance to neutralization is incompletely understood. Utilizing the simian immunodeficiency virus (SIV) as a model for HIV, our study revealed differing N-glycan profiles between SIV Env produced in CD4+ T cells (T-SIV) and macrophages (M-SIV), with T-SIV incorporating more N-glycans of the oligomannose-type into Env than M-SIV. M-SIV displayed enhanced infectivity and transmissibility in in vitro studies. Additionally, M-SIV exhibited greater susceptibility to neutralizing sera compared to T-SIV, while T-SIV showed heightened susceptibility to mannose-specific carbohydrate-binding agents. These findings emphasize the critical role of host cell-dependent attributes in shaping N-glycosylation of Env as well as viral infectivity and spread. These findings have important implications for the development of new biomedical interventions against HIV. [ABSTRACT FROM AUTHOR]

The human immunodeficiency virus (HIV) integrates into the host genome forming latent cellular reservoirs that are an obstacle for cure or remission strategies. Viral transcription is the first step in the control of latency and depends upon the hijacking of the host cell RNA polymerase II (Pol II) machinery by the 5' HIV LTR. Consequently, "block and lock" or "shock and kill" strategies for an HIV cure depend upon a full understanding of HIV transcriptional control. The HIV trans-activating protein, Tat, controls HIV latency as part of a positive feed-forward loop that strongly activates HIV transcription. The recognition of the TATA box and adjacent sequences of HIV essential for Tat trans-activation (TASHET) of the core promoter by host cell pre-initiation complexes of HIV (PICH) has been shown to be necessary for Tat trans-activation, yet the protein composition of PICH has remained obscure. Here, DNA-affinity chromatography was employed to identify the mitotic deacetylase complex (MiDAC) as selectively recognizing TASHET. Using biophysical techniques, we show that the MiDAC subunit DNTTIP1 binds directly to TASHET, in part via its CTGC DNA motifs. Using co-immunoprecipitation assays, we show that DNTTIP1 interacts with MiDAC subunits MIDEAS and HDAC1/2. The Tat-interacting protein, NAT10, is also present in HIV-bound MiDAC. Gene silencing revealed a functional role for DNTTIP1, MIDEAS, and NAT10 in HIV expression in cellulo. Furthermore, point mutations in TASHET that prevent DNTTIP1 binding block the reactivation of HIV by latency reversing agents (LRA) that act via the P-TEFb/7SK axis. Our data reveal a key role for MiDAC subunits DNTTIP1, MIDEAS, as well as NAT10, in Tat-activated HIV transcription and latency. DNTTIP1, MIDEAS and NAT10 emerge as cell cycle-regulated host cell transcription factors that can control activated HIV gene expression, and as new drug targets for HIV cure strategies. Author summary: Latent HIV integrated within the host cell genome poses a major problem for viral eradication. The reactivation of latent HIV depends on host cell transcription factors that are hijacked by the 5' long terminal repeat (LTR) region of the HIV genome to produce viral RNA. At the heart of the LTR of HIV lies a particularly crucial DNA region named the core promoter that is specifically required for the reactivation of HIV by a viral protein named Tat. A significant body of work over more than 30 years has established the specific requirement for the HIV core promoter in Tat's control of HIV latency, but the underlying molecular mechanisms have remained elusive. Here, we identify host cell transcription factors that bind selectively to the HIV core promoter to control activated viral gene expression. Our data reveal three human proteins that act in a complex to reactivate latent HIV, including one that directly recognizes the HIV core promoter, one that regulates chromatin, and a third that binds to the HIV Tat protein. Our data fill a significant and long-standing gap in the understanding of latency and identify new potential drug targets for HIV cure strategies. [ABSTRACT FROM AUTHOR]

Persons living with HIV are known to be at increased risk of developing tuberculosis (TB) disease upon infection with Mycobacterium tuberculosis (Mtb). However, it has remained unclear how HIV co-infection affects subsequent Mtb transmission from these patients. Here, we customized a Bayesian phylodynamic framework to estimate the effects of HIV co-infection on the Mtb transmission dynamics from sequence data. We applied our model to four Mtb genomic datasets collected in sub-Saharan African countries with a generalized HIV epidemic. Our results confirm that HIV co-infection is a strong risk factor for developing active TB. Additionally, we demonstrate that HIV co-infection is associated with a reduced effective reproductive number for TB. Stratifying the population by CD4+ T-cell count yielded similar results, suggesting that, in this context, CD4+ T-cell count is not a better predictor of Mtb transmissibility than HIV infection status alone. Together, our genome-based analyses complement observational household contact studies, and more firmly establish the negative association between HIV co-infection and Mtb transmissibility. Author summary: Many sub-Saharan African countries have seen a considerable rise in TB incidence since the introduction of HIV, suggesting a strong interaction between HIV and TB epidemics. HIV infection is recognized as an important risk factor for developing TB, but the contribution of HIV-infected TB cases to further Mtb transmission is poorly understood. In this study, we analyzed four sets of Mtb genomic sequences collected in different countries, comprising sequences from HIV-negative and HIV-positive TB patients. We applied a phylodynamic model to these sequences, aimed at inferring transmission dynamics within and between different host populations. While our findings support that HIV is a strong risk factor for TB, we show that HIV-positive TB cases generate a significantly lower number of secondary TB cases than HIV-negative cases. This suggests that HIV-positive cases mainly act as sinks in Mtb transmission chains, while HIV-negative cases are a major source of transmission. [ABSTRACT FROM AUTHOR]

Engineered T cells hold great promise to become part of an effective HIV cure strategy, but it is currently unclear how best to redirect T cells to target HIV. To gain insight, we generated engineered T cells using lentiviral vectors encoding one of three distinct HIV-specific T cell receptors (TCRs) or a previously optimized HIV-targeting chimeric antigen receptor (CAR) and compared their functional capabilities. All engineered T cells had robust, antigen-specific polyfunctional cytokine profiles when mixed with artificial antigen-presenting cells. However, only the CAR T cells could potently control HIV replication. TCR affinity enhancement did not augment HIV control but did allow TCR T cells to recognize common HIV escape variants. Interestingly, either altering Nef activity or adding additional target epitopes into the HIV genome bolstered TCR T cell anti-HIV activity, but CAR T cells remained superior in their ability to control HIV replication. To better understand why CAR T cells control HIV replication better than TCR T cells, we performed a time course to determine when HIV-specific T cells were first able to activate Caspase 3 in HIV-infected targets. We demonstrated that CAR T cells recognized and killed HIV-infected targets more rapidly than TCR T cells, which correlates with their ability to control HIV replication. These studies suggest that the speed of target recognition and killing is a key determinant of whether engineered T cell therapies will be effective against infectious diseases. Author summary: Infusion of HIV-specific engineered T cells is a promising approach to eliminate HIV infected cells that reverse latency and begin to replicate HIV after ART withdrawal. However, it is unclear whether chimeric antigen receptors (CAR) or TCRs are the best approach to generate HIV-specific T cells for adoptive T cell therapy. We generated CD8 T cells expressing one of three HIV-specific TCRs (TCR-T) or a previously optimized HIV-specific CAR and interrogated which population controlled HIV replication more effectively. HIV-specific CAR T cells were far superior to any of the TCR-T cells. Increasing TCR affinity or interfering with the ability of Nef to downregulate MHC class I had modest effects on the ability TCR-T cells to control HIV. Inserting additional cognate epitopes within the HIV genome did improve the ability of TCR-T cells to control HIV but HIV CAR T cells were still superior. The ability to control HIV replication best correlated with the ability of engineered T cell to rapidly induce cell death in HIV-infected T cells, suggesting that the speed of recognition and initiation of T cell mediated killing is a key determinant on whether engineered T cells can control viral infections. [ABSTRACT FROM AUTHOR]

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. Author summary: Antiretroviral therapy (ART) has improved health outcomes of people living with HIV. However, there are still challenges, especially in the central nervous system (CNS), where ongoing inflammation can lead to neurological disorders. Our study focused on understanding the role of CD4 T cells in the brain during HIV infection and sub-optimal treatment adherence. We used a model with SIV-infected rhesus monkeys to study the AIDS virus in the brain and surrounding tissues. We discovered that a subset of CD4 T cells, which are vulnerable to HIV, are present throughout the CNS. During the early stages of infection, we noticed high levels of the virus in both the brain and nearby tissues. By examining these CD4 T cells at a single-cell level, we found that they actively respond to the virus by initiating specific antiviral effector functions to fight it. Overall, our study helps us understand the role of CD4 T cells within the CNS during both acute and chronic HIV infection. This knowledge could help us develop new ways to target the virus in the CNS and devise treatments for complications related to Neuro-HIV. [ABSTRACT FROM AUTHOR]

Sustainable HIV remission after antiretroviral therapy (ART) withdrawal, or post-treatment control (PTC), remains a top priority for HIV treatment. We observed surprising PTC in an MHC-haplomatched cohort of MHC-M3+ SIVmac239+ Mauritian cynomolgus macaques (MCMs) initiated on ART at two weeks post-infection (wpi). None of the MCMs possessed MHC haplotypes previously associated with SIV control. For six months after ART withdrawal, we observed undetectable or transient viremia in seven of the eight MCMs, despite detecting replication competent SIV using quantitative viral outgrowth assays. In vivo depletion of CD8α+ cells induced rebound in all animals, indicating the observed PTC was mediated, at least in part, by CD8α+ cells. With intact proviral DNA assays, we found that MCMs had significantly smaller viral reservoirs two wpi than a cohort of identically infected rhesus macaques, a population that rarely develops PTC. We found a similarly small viral reservoir among six additional SIV+ MCMs in which ART was initiated at eight wpi, some of whom exhibited viral rebound. These results suggest that an unusually small viral reservoir is a hallmark among SIV+ MCMs. By evaluating immunological differences between MCMs that did and did not rebound, we identified that PTC was associated with a reduced frequency of CD4+ and CD8+ lymphocyte subsets expressing exhaustion markers. Together, these results suggest a combination of small reservoirs and immune-mediated virus suppression contribute to PTC in MCMs. Further, defining the immunologic mechanisms that engender PTC in this model may identify therapeutic targets for inducing durable HIV remission in humans. Author summary: The ultimate goal of human immunodeficiency virus (HIV) therapeutic development is to enable HIV+ individuals to stop taking daily lifelong antiretroviral therapeutics (ART) and remain in virological remission, an outcome called post-treatment control. Post-treatment control is challenging to study because it is very rare in humans. Thus, there are currently no therapeutics that lead to post-treatment control. Simian immunodeficiency virus (SIV) in rhesus macaques is the most widely used preclinical model for HIV. However, post-treatment control is also uncommon in rhesus macaques. The mechanisms that govern post-treatment control remain largely unknown because PTC is so rare. Our lab observed surprising post-treatment control in a cohort of Mauritian cynomolgus macaques that were initiated on ART two weeks post-infection. While Mauritian cynomolgus macaques have been used for SIV research, they are not routinely used to study post-treatment control. We found that small viral reservoirs and immune-mediated virus suppression contribute to post-treatment control in Mauritian cynomolgus macaques. Moving forward, this model can be used to further define the mechanisms that engender post-treatment control as well as identify therapeutic targets in humans for inducing durable HIV remission. [ABSTRACT FROM AUTHOR]

Recent HIV-1 vaccine development has centered on "near native" soluble envelope glycoprotein (Env) trimers that are artificially stabilized laterally (between protomers) and apically (between gp120 and gp41). These mutations have been leveraged for use in membrane-expressed Env mRNA vaccines, although their effects in this context are unclear. To address this question, we used virus-like particle (VLP) produced in 293T cells. Uncleaved (UNC) trimers were laterally unstable upon gentle lysis from membranes. However, gp120/gp41 processing improved lateral stability. Due to inefficient gp120/gp41 processing, UNC is incorporated into VLPs. A linker between gp120 and gp41 neither improved trimer stability nor its antigenic profile. An artificially introduced enterokinase cleavage site allowed post-expression gp120/gp41 processing, concomitantly increasing trimer stability. Gp41 N-helix mutations I559P and NT1-5 imparted lateral trimer stability, but also reduced gp120/gp41 processing and/or impacted V2 apex and interface NAb binding. I559P consistently reduced recognition by HIV+ human plasmas, further supporting antigenic differences. Mutations in the gp120 bridging sheet failed to stabilize membrane trimers in a pre-fusion conformation, and also reduced gp120/gp41 processing and exposed non-neutralizing epitopes. Reduced glycan maturation and increased sequon skipping were common side effects of these mutations. In some cases, this may be due to increased rigidity which limits access to glycan processing enzymes. In contrast, viral gp120 did not show glycan skipping. A second, minor species of high mannose gp160 was unaffected by any mutations and instead bypasses normal folding and glycan maturation. Including the full gp41 cytoplasmic tail led to markedly reduced gp120/gp41 processing and greatly increased the proportion of high mannose gp160. Remarkably, monoclonal antibodies were unable to bind to this high mannose gp160 in native protein gels. Overall, our findings suggest caution in leveraging stabilizing mutations in nucleic acid-based immunogens to ensure they impart valuable membrane trimer phenotypes for vaccine use. Author summary: A vaccine that induces virus-fighting antibodies to block HIV-1 infection remains elusive. To ablate HIV-1 infection, antibodies must bind to authentic envelope (Env) glycoprotein on the virus surface. However, Env can exist in various forms, many of which are relatively easy targets for non-effective antibodies. Therefore, a key challenge of vaccine design is to create pure authentic Env that is unfettered by these other forms of Env. Vaccine research to date has focused largely on stabilizing soluble Env trimers, as this format simplifies purification and translational studies. However, incomplete Env authenticity may blunt the efficacy of this approach. By comparison, the manufacture of particle-based vaccines that express Env in situ on membranes is cumbersome. In an alternative approach, lipid particles can deliver mRNA vaccines encoding membrane trimers, bypassing the manufacturing challenges of previous methods. Stabilizing mutations derived from soluble trimers are now being leveraged for membrane trimers. Here, we evaluated the effects of these mutations. Our results show that some mutations alter Env conformation, and therefore might best be omitted from nucleic acid-based membrane Env vaccines. [ABSTRACT FROM AUTHOR]

Transcriptional silencing of latent HIV-1 proviruses entails complex and overlapping mechanisms that pose a major barrier to in vivo elimination of HIV-1. We developed a new latency CRISPR screening strategy, called Latency HIV-CRISPR which uses the packaging of guideRNA-encoding lentiviral vector genomes into the supernatant of budding virions as a direct readout of factors involved in the maintenance of HIV-1 latency. We developed a custom guideRNA library targeting epigenetic regulatory genes and paired the screen with and without a latency reversal agent–AZD5582, an activator of the non-canonical NFκB pathway–to examine a combination of mechanisms controlling HIV-1 latency. A component of the Nucleosome Acetyltransferase of H4 histone acetylation (NuA4 HAT) complex, ING3, acts in concert with AZD5582 to activate proviruses in J-Lat cell lines and in a primary CD4+ T cell model of HIV-1 latency. We found that the knockout of ING3 reduces acetylation of the H4 histone tail and BRD4 occupancy on the HIV-1 LTR. However, the combination of ING3 knockout accompanied with the activation of the non-canonical NFκB pathway via AZD5582 resulted in a dramatic increase in initiation and elongation of RNA Polymerase II on the HIV-1 provirus in a manner that is nearly unique among all cellular promoters. Author summary: HIV-1 establishes long-lived latent reservoirs that present a barrier for virus eradication and HIV Cure. One approach to reduce the latent reservoir is the use of small molecules called latency reversal agents (LRA) to trigger immune-mediated clearance of virus-producing cells. However, this approach is limited by the inability of current LRAs to reactivate the majority of latent proviruses and a lack of specificity, highlighting the need for a greater understanding of the interplay of mechanisms involved in the maintenance of HIV-1 latency. We developed a modular CRISPR-based screening, called HIV-CRISPR, in a T-lymphocyte (J-Lat) model of latent infection to gain a comprehensive representation of the pathways involved in HIV-1 latency. As the latent state of HIV-1 is controlled by multiple, parallel mechanisms, we used the screen to study the epigenetic factors in the presence of an LRA targeting a transcriptional activation mechanism. The screens identified novel factors controlling HIV-1 latency both in the J-Lat and primary T cell models of HIV-1 latency. [ABSTRACT FROM AUTHOR]

HIV-1 transmission via sexual exposure is an inefficient process. When transmission does occur, newly infected individuals are colonized by the descendants of either a single virion or a very small number of establishing virions. These transmitted founder (TF) viruses are more interferon (IFN)-resistant than chronic control (CC) viruses present 6 months after transmission. To identify the specific molecular defences that make CC viruses more susceptible to the IFN-induced 'antiviral state', we established a single pair of fluorescent TF and CC viruses and used arrayed interferon-stimulated gene (ISG) expression screening to identify candidate antiviral effectors. However, we observed a relatively uniform ISG resistance of transmitted HIV-1, and this directed us to investigate possible underlying mechanisms. Simple simulations, where we varied a single parameter, illustrated that reduced growth rate could possibly underly apparent interferon sensitivity. To examine this possibility, we closely monitored in vitro propagation of a model TF/CC pair (closely matched in replicative fitness) over a targeted range of IFN concentrations. Fitting standard four-parameter logistic growth models, in which experimental variables were regressed against growth rate and carrying capacity, to our in vitro growth curves, further highlighted that small differences in replicative growth rates could recapitulate our in vitro observations. We reasoned that if growth rate underlies apparent interferon resistance, transmitted HIV-1 would be similarly resistant to any growth rate inhibitor. Accordingly, we show that two transmitted founder HIV-1 viruses are relatively resistant to antiretroviral drugs, while their matched chronic control viruses were more sensitive. We propose that, when present, the apparent IFN resistance of transmitted HIV-1 could possibly be explained by enhanced replicative fitness, as opposed to specific resistance to individual IFN-induced defences. However, further work is required to establish how generalisable this mechanism of relative IFN resistance might be. Author summary: HIV-1 sexual transmission is surprisingly inefficient. In the relatively unusual event of successful transmission, there is typically a severe genetic bottleneck, such that infection is established by just a single (or small number) of genetic variants, described as transmitted founder (TF) viruses. Such limited transmission is considered to arise from both physical and immunological barriers that restrict viruses, such as the interferon system. TF viruses have been described as more interferon resistant than viruses present in chronically infected individuals. Here we found that an IFN-resistant transmitted HIV-1 virus is uniformly resistant to multiple interferon-stimulated genes, which led us to investigate the underlying mechanism. Our characterisation of a single TF and chronic virus pair (closely matched in replicative fitness) revealed that small differences in replicative growth rates could possibly explain the broad interferon resistance frequently associated with transmitted HIV-1, although further studies are required to confirm this. Our study provides insight into how relatively small differences in growth rate could still be phenotypically significant, and that a nonspecific mechanism could possibly explain the interferon resistance of transmitted HIV-1. [ABSTRACT FROM AUTHOR]

The authors offer a response on a comment of their article on the development of two novel methods for estimating the rate of reactivation from HIV reactivation from latency directly from existing clinical data. They are critical on the use of simulation by critics to incorporate multiple reactivation events or a distribution of reactivation rates into their model. They also note that critics' primary argument is that there is strong prior evidence of high reactivation rates.

In infectious HIV-1 particles, the capsid protein (CA) forms a cone-shaped shell called the capsid, which encases the viral ribonucleoprotein complex (vRNP). Following cellular entry, the capsid is disassembled through a poorly understood process referred to as uncoating, which is required to release the reverse transcribed HIV-1 genome for integration into host chromatin. Whereas single virus imaging using indirect CA labeling techniques suggested uncoating to occur in the cytoplasm or at the nuclear pore, a recent study using eGFP-tagged CA reported uncoating in the nucleus. To delineate the HIV-1 uncoating site, we investigated the mechanism of eGFP-tagged CA incorporation into capsids and the utility of this fluorescent marker for visualizing HIV-1 uncoating. We find that virion incorporated eGFP-tagged CA is effectively excluded from the capsid shell, and that a subset of the tagged CA is vRNP associated. These results thus imply that eGFP-tagged CA is not a direct marker for capsid uncoating. We further show that native CA co-immunoprecipitates with vRNP components, providing a basis for retention of eGFP-tagged and untagged CA by sub-viral complexes in the nucleus. Moreover, we find that functional viral replication complexes become accessible to integrase-interacting host factors at the nuclear pore, leading to inhibition of infection and demonstrating capsid permeabilization prior to nuclear import. Finally, we find that HIV-1 cores containing a mixture of wild-type and mutant CA interact differently with cytoplasmic versus nuclear pools of the CA-binding host cofactor CPSF6. Our results suggest that capsid remodeling (including a loss of capsid integrity) is the predominant pathway for HIV-1 nuclear entry and provide new insights into the mechanism of CA retention in the nucleus via interaction with vRNP components. Author summary: The timing, location and mechanisms of HIV-1 capsid disassembly which is referred to as uncoating remains unclear. Direct labeling of HIV-1 capsids, by incorporating a few green fluorescent proteins (GFP) tagged capsid protein (CA) into virions allows to image the spatio-temporal loss of HIV-1 CA during virus infection. However, the localization and functions of a few virion incorporated eGFP-tagged CA proteins remain unclear, since <50% of virus packaged CA proteins participate to form the conical capsid shell that protects the HIV-1 genome. Here we developed several approaches to test the localization and function of eGFP-tagged CA proteins in virions. We found that eGFP-tagged CA proteins are excluded from the conical capsid shell and that a subset of these proteins is associated with the viral ribonucleoprotein complex (vRNPs), through direct interactions between CA and vRNP components. eGFP-tagged CA is retained in the nucleus by virtue of vRNP association and is unlikely to report on HIV-1 capsid disassembly. We also found that HIV-1 capsids become permeabilized and are remodeled during their transport into the nucleus. Our study provides new insights into the ability of CA to interact with vRNPs for its retention in the nucleus and highlights capsid remodeling as a preferred pathway for HIV-1 entry into the nucleus. [ABSTRACT FROM AUTHOR]

Both SIV and SHIV are powerful tools for evaluating antibody-mediated prevention and treatment of HIV-1. However, owing to a lack of rhesus-derived SIV broadly neutralizing antibodies (bnAbs), testing of bnAbs for HIV-1 prevention or treatment has thus far been performed exclusively in the SHIV NHP model using bnAbs from HIV-1-infected individuals. Here we describe the isolation and characterization of multiple rhesus-derived SIV bnAbs capable of neutralizing most isolates of SIV. Eight antibodies belonging to two clonal families, ITS102 and ITS103 which target unique epitopes in the CD4bs region, were found to be broadly neutralizing and together neutralized all SIV strains tested. A rare feature of these bnAbs and two additional antibody families, ITS92 and ITS101 which mediate strain-specific neutralizing activity against SIV from sooty mangabeys (SIVsm), was their ability to achieve near complete (i.e. 100%) neutralization of moderately and highly neutralization-resistant SIV. Overall, these newly identified SIV bnAbs highlight the potential for evaluating HIV-1 prophylactic and therapeutic interventions using fully simian, rhesus-derived bnAbs in the SIV NHP model, thereby circumventing issues related to rapid antibody clearance of human-derived antibodies, Fc mismatch and limited genetic diversity of SHIV compared to SIV. Author summary: Antibodies that can bind to a virus and block infection are called neutralizing antibodies. In human immunodeficiency virus (HIV), antibodies that can block multiple strains of HIV circulating globally are known as broadly neutralizing antibodies. These broadly neutralizing antibodies have the potential to be used to treat HIV-infected individuals or to prevent HIV infection altogether. However, before these broadly neutralizing antibodies can safely be used in humans, they must first be tested in monkeys. Simian immunodeficiency virus (SIV), which causes an acquired immunodeficiency syndrome (AIDS)-like illness in Asian macaques that is similar to AIDS in humans, makes it possible to perform pre-clinical testing in monkeys but requires the use of antibodies that are specific to SIV. Another important consideration for testing antibodies in monkeys is that, like HIV, there are many different strains of SIV and some of these are highly resistant to neutralization. In this study we used blood from SIV-infected macaques to isolate several antibodies that can neutralize multiple strains of SIV, including strains that are typically resistant to neutralization. These SIV broadly neutralizing antibodies closely resemble HIV broadly neutralizing antibodies, making it possible now to test antibody-based vaccines or therapies for HIV using the SIV nonhuman primate model of AIDS. [ABSTRACT FROM AUTHOR]

Glycoprotein Env of human immunodeficiency virus type 1 (HIV-1) mediates viral entry through membrane fusion. Composed of gp120 and gp41 subunits arranged as a trimer-of-heterodimers, Env adopts a metastable, highly dynamic conformation on the virion surface. This structural plasticity limits the temporospatial exposure of many highly conserved, neutralizing epitopes, contributing to the difficulty in developing effective HIV-1 vaccines. Here, we employed antibody neutralization of HIV-1 infectivity to investigate how inter- and intra-gp120 interactions mediated by variable loops V1/V2 and V3 at the Env apex regulate accessibility of the gp41 membrane-proximal external region (MPER) at the Env base. Swapping the V3 loop from EnvSF162 into the EnvHXB2 background shifted MPER exposure from the prefusogenic state to a functional intermediate conformation that was distinct from the prehairpin-intermediate state sensitive to gp41-targeted fusion inhibitors. The V3-loop swap had a profound impact on global protein dynamics, biasing the equilibrium to a closed conformation resistant to most anti-gp120 antibodies, stabilizing the protein to both cold- and soluble CD4-induced Env inactivation, and increasing the CD4 requirements for viral entry. Further dissection of the EnvHXB2 V3 loop revealed that residue 306 uniquely modulated epitope exposure and trimer stability. The R306S substitution substantially decreased sensitivity to antibodies targeting the gp41 MPER and, surprisingly, the gp120 V3-loop crown (residues 312–315), but had only modest effects on exposure of intervening gp120 epitopes. Furthermore, the point mutation reduced soluble CD4-induced inactivation, but had no impact on cold inactivation. The residue appeared to exert its effects by electrostatically modifying the strength of intra-subunit interactions between the V1/V2 and V3 loops. The distinct patterns of neutralization and stability pointed to a novel prefusogenic Env conformation along the receptor activation pathway and suggested that apical Env-regulation of gp41 MPER exposure can be decoupled from much of the dynamics of gp120 subunits. Author summary: Surface glycoprotein Env is the main target for neutralizing antibodies elicited by HIV-1 vaccines. Env spontaneously fluctuates among different structures, limiting exposure of many attractive antibody-binding epitopes and, thereby, confounding vaccine development. To characterize these fluctuations, we examined how exposure of the MPER epitope found at the base of Env is regulated by interactions of the V3 loop located in the apex. Starting with an extremely flexible Env with a readily-exposed MPER, we identified two alterations that substantially restricted antibody access to the epitope. The first, a wholesale swap of V3 loops between HIV-1 strains, energetically stabilized Env in a closed structure that restricted access to antibodies throughout the protein. The second, a point mutation that altered V3-loop charge, specifically destabilized the MPER-exposed conformation but had minimal impact on antibody access to Env regions in between the apex and base. The results indicate that MPER exposure is not explicitly tied to the dynamics of Env regions between the apex and base and suggest a new structural fluctuation during Env activation. [ABSTRACT FROM AUTHOR]

Antibodies are principal immune components elicited by vaccines to induce protection from microbial pathogens. In the Thai RV144 HIV-1 vaccine trial, vaccine efficacy was 31% and the sole primary correlate of reduced risk was shown to be vigorous antibody response targeting the V1V2 region of HIV-1 envelope. Antibodies against V3 also were inversely correlated with infection risk in subsets of vaccinees. Antibodies recognizing these regions, however, do not exhibit potent neutralizing activity. Therefore, we examined the antiviral potential of poorly neutralizing monoclonal antibodies (mAbs) against immunodominant V1V2 and V3 sites by passive administration of human mAbs to humanized mice engrafted with CD34+ hematopoietic stem cells, followed by mucosal challenge with an HIV-1 infectious molecular clone expressing the envelope of a tier 2 resistant HIV-1 strain. Treatment with anti-V1V2 mAb 2158 or anti-V3 mAb 2219 did not prevent infection, but V3 mAb 2219 displayed a superior potency compared to V1V2 mAb 2158 in reducing virus burden. While these mAbs had no or weak neutralizing activity and elicited undetectable levels of antibody-dependent cellular cytotoxicity (ADCC), V3 mAb 2219 displayed a greater capacity to bind virus- and cell-associated HIV-1 envelope and to mediate antibody-dependent cellular phagocytosis (ADCP) and C1q complement binding as compared to V1V2 mAb 2158. Mutations in the Fc region of 2219 diminished these effector activities in vitro and lessened virus control in humanized mice. These results demonstrate the importance of Fc functions other than ADCC for antibodies without potent neutralizing activity. Author summary: In the past decade, HIV-1 has infected an estimated 1.5 to 2 million people every year, but vaccines needed to control this pandemic are unavailable. Among vaccines tested in the human efficacy trials, the RV144 vaccine regimen showed a modest efficacy and revealed non-neutralizing antibodies against the virus envelope glycoproteins as a correlate of reduced virus acquisition. To design more efficacious HIV-1 vaccines, a better understanding about antiviral mechanisms of these antibodies is needed. Here non-neutralizing monoclonal antibodies against two immunogenic sites on the virus envelope were evaluated for passive administration to humanized mice that were subsequently challenged with HIV-1. The antibodies did not block mucosal HIV-1 infection but reduced virus burden. The level of virus reduction correlated with the antibody binding potency and the effector functions mediated through their Fc fragments, which included antibody-dependent phagocytosis and complement activation, but not the commonly studied antibody-dependent cellular cytotoxicity. The importance of the Fc functions was further demonstrated by reduced virus control when mutations were introduced to decrease Fc activities. This study provides new evidence for the important contribution of multiple Fc-dependent antibody functions in immune control against HIV-1. [ABSTRACT FROM AUTHOR]

HIV-1 vaccine immunofocusing strategies may be able to induce broadly-reactive neutralizing antibodies (NAbs). Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets—the V2 apex and fusion peptide (FP). Selection criteria included i) high expression and ii) infectious function, so that trimer neutralization sensitivity can be profiled in pseudovirus (PV) assays. Initially, we boosted the expression of 17 candidate trimers by truncating gp41 and introducing a gp120-gp41 SOS disulfide to prevent gp120 shedding. "Repairs" were made to fill glycan holes and eliminate other strain-specific aberrations. A new neutralization assay allowed PV infection when our standard assay was insufficient. Trimers with exposed V3 loops, a target of non-NAbs, were discarded. To try to increase V2-sensitivity, we removed clashing glycans and modified the C-strand. Notably, a D167N mutation improved V2-sensitivity in several cases. Glycopeptide analysis of JR-FL trimers revealed near complete sequon occupation and that filling the N197 glycan hole was well-tolerated. In contrast, sequon optimization and inserting/removing glycans at other positions frequently had global "ripple" effects on glycan maturation and sequon occupation throughout the gp120 outer domain and gp41. V2 MAb CH01 selectively bound to trimers with small high mannose glycans near the base of the V1 loop, thereby avoiding clashes. Knocking in a rare N49 glycan was found to perturb gp41 glycans, increasing FP NAb sensitivity—and sometimes improving expression. Finally, a biophysical analysis of VLPs revealed that i) ~25% of particles bear Env spikes, ii) spontaneous particle budding is high and only increases 4-fold upon Gag transfection, and iii) Env+ particles express ~30–40 spikes. Taken together, we identified 7 diverse trimers with a range of sensitivities to two targets to allow rigorous testing of immunofocusing vaccine concepts. Author summary: Despite almost 40 years of innovation, a vaccine to induce antibodies that block HIV infection remains elusive. Challenges include the unparalleled sequence diversity of HIV's surface spikes and its dense sugar coat that limits antibody access. A growing number of monoclonal antibodies from HIV infected donors provide vaccine blueprints, but have been difficult to induce by vaccination, due to their unusual features. However, two targets, one at the viral spike apex and another at the side of the spikes are more forgiving in their 'demands' for unusual antibodies. Here, we made a diverse panel of HIV spikes vulnerable at these two sites to be used as vaccines to try to focus antibodies on these targets. Our selection criteria for these spikes were: i) that when expressed on particles, they are infectious, allowing us to evaluate immunogens and vaccine sera using particles made with the same trimers, ii) that spikes are easy to produce by cells in quantities sufficient for vaccine use. Ultimately, we selected 7 trimers that will allow us to explore concepts that could bring us closer to an HIV vaccine. [ABSTRACT FROM AUTHOR]

One strategy for a functional cure of HIV-1 is "block and lock", which seeks to permanently suppress the rebound of quiescent HIV-1 by epigenetic silencing. For the bivalent promoter in the HIV LTR, both histone 3 lysine 27 tri-methylation (H3K27me3) and DNA methylation are associated with viral suppression, while H3K4 tri-methylation (H3K4me3) is correlated with viral expression. However, H3K27me3 is readily reversed upon activation of T-cells through the T-cell receptor. In an attempt to suppress latent HIV-1 in a stable fashion, we knocked down the expression or inhibited the activity of UTX/KDM6A, the major H3K27 demethylase, and investigated its impact on latent HIV-1 reactivation in T cells. Inhibition of UTX dramatically enhanced H3K27me3 levels at the HIV LTR and were associated with increased DNA methylation. In latently infected cells from patients, GSK-J4, which is a potent dual inhibitor of the H3K27me3/me2-demethylases JMJD3/KDM6B and UTX/KDM6A, effectively suppressed the reactivation of latent HIV-1 and also induced DNA methylation at specific sites in the 5'LTR of latent HIV-1 by the enhanced recruitment of DNMT3A to HIV-1. Nonetheless, suppression of HIV-1 through epigenetic silencing required the continued treatment with GSK-J4 and was rapidly reversed after removal of the drug. DNA methylation was also rapidly lost after removal of drug, suggesting active and rapid DNA-demethylation of the HIV LTR. Thus, induction of epigenetic silencing by histone and DNA methylation appears to be insufficient to permanently silence HIV-1 proviral transcription. Author summary: The "block and lock" strategy for a functional HIV-1 cure is based on the premise that permanent inactivation of the HIV-1 can be achieved by epigenetic silencing of the proviral DNA. For cellular genes, long-term silencing is achieved during cell differentiation by the induction of specific epigenetic modifications involving histone and DNA methylation. During HIV-1 silencing, histone methylation and DNA methylation are observed, but both sets of modifications can be reversed upon activation of T-cells through the T-cell receptor or potent latency reversing agents. In an attempt to enhance silencing of HIV-1 transcription, we used an inhibitor of H3K27 demethylases to increase H3K27 methylation. This in turn led to enhanced DNA methylation of HIV-1. Unfortunately, although the treatment effectively silenced HIV-1 and prevented viral reactivation, the silencing effects were short-lived and quickly reversed after removal of the drug. [ABSTRACT FROM AUTHOR]

Understanding the molecular mechanisms by which antibodies target and neutralize the HIV-1 envelope glycoprotein (Env) is critical in guiding immunogen design and vaccine development aimed at eliciting cross-reactive neutralizing antibodies (NAbs). Here, we analyzed monoclonal antibodies (mAbs) isolated from non-human primates (NHPs) immunized with variants of a native flexibly linked (NFL) HIV-1 Env stabilized trimer derived from the tier 2 clade C 16055 strain. The antibodies displayed neutralizing activity against the autologous virus with potencies ranging from 0.005 to 3.68 μg/ml (IC50). Structural characterization using negative-stain EM and X-ray crystallography identified the variable region 2 (V2) of the 16055 NFL trimer to be the common epitope for these antibodies. The crystal structures revealed that the V2 segment adopts a β-hairpin motif identical to that observed in the 16055 NFL crystal structure. These results depict how vaccine-induced antibodies derived from different clonal lineages penetrate through the glycan shield to recognize a hypervariable region within V2 (residues 184–186) that is unique to the 16055 strain. They also provide potential explanations for the potent autologous neutralization of these antibodies, confirming the immunodominance of this site and revealing that multiple angles of approach are permissible for affinity/avidity that results in potent neutralizing capacity. The structural analysis reveals that the most negatively charged paratope correlated with the potency of the mAbs. The atomic level information is of interest to both define the means of autologous neutralization elicited by different tier 2-based immunogens and facilitate trimer redesign to better target more conserved regions of V2 to potentially elicit cross-neutralizing HIV-1 antibodies. Author summary: NHPs immunizations with an HIV-1 immunogen (native-like tier 2 clade C 16055 strain) elicit potent HIV-1 tier 2 autologous polyclonal neutralizing antibodies. To understand the basis of the autologous neutralization, we determined structures of antibodies isolated from the vaccinated NHPs in complex with their epitopes. Our structural analysis reveals that the V2 hypervariable region, unique to 16055, is immunodominant and targeted by antibodies from diverse lineages. Additionally, vaccine-elicited V2 NAbs use different binding angles to avoid Env N-glycan shield and the more negatively charged paratope displays potent autologous neutralizing function. In summary, detailed analysis of how vaccine-elicited monoclonal antibodies interact with the target antigen provide valuable information for the design of immunogens aimed to elicit more broadly HIV-neutralizing antibodies. The use of cocktail/prime-boost sequential regimens that include a range of sequence variation combined with the removal/shielding of unwanted immunodominant epitopes will likely be needed to reach this goal. [ABSTRACT FROM AUTHOR]

We have recently demonstrated that the function of T follicular helper (Tfh) cells from lymph nodes (LN) of HIV-infected individuals is impaired. We found that these cells were unable to provide proper help to germinal center (GC)-B cells, as observed by altered and inefficient anti-HIV antibody response and premature death of memory B cells. The underlying molecular mechanisms of this dysfunction remain poorly defined. Herein, we have used a unique transcriptional approach to identify these molecular defects. We consequently determined the transcriptional profiles of LN GC-Tfh cells following their interactions with LN GC-B cells from HIV-infected and HIV-uninfected individuals, rather than analyzing resting ex-vivo GC-Tfh cells. We observed that proliferating GC-Tfh cells from HIV-infected subjects were transcriptionally different than their HIV-uninfected counterparts, and displayed a significant downregulation of immune- and GC-Tfh-associated pathways and genes. Our results strongly demonstrated that MAF (coding for the transcription factor c-Maf) and its upstream signaling pathway mediators (IL6R and STAT3) were significantly downregulated in HIV-infected subjects, which could contribute to the impaired GC-Tfh and GC-B cell functions reported during infection. We further showed that c-Maf function was associated with the adenosine pathway and that the signaling upstream c-Maf could be partially restored by adenosine deaminase -1 (ADA-1) supplementation. Overall, we identified a novel mechanism that contributes to GC-Tfh cell impairment during HIV infection. Understanding how GC-Tfh cell function is altered in HIV is crucial and could provide critical information about the mechanisms leading to the development and maintenance of effective anti-HIV antibodies. Author summary: Human immunodeficiency virus (HIV) remains a worldwide burden despite available treatments. The virus induces dysregulations in major immune cells and organs including lymph nodes. Germinal center T follicular helper (GC-Tfh) cells are immune cells which induce specific anti-HIV antibodies by helping GC-B cells. In chronic HIV, the interaction between these two cell types is defective, leading to modified and inefficient anti-HIV antibody responses. In this study, we examined the underlying mechanisms of this dysfunction. We observed that proliferating GC-Tfh cells from HIV-infected individuals, displayed distinctive gene expression than those from -uninfected subjects, following GC-B cell interaction. Furthermore, GC-Tfh cells from HIV patients showed a reduction in important immune-related pathway and gene expression. A number of essential GC-Tfh cell genes, such as MAF and its associated genes (IL6R and STAT3), were particularly attenuated in HIV, contributing to the impaired cells function. Moreover, we found an association between MAF function and the key enzyme adenosine deaminase-1 (ADA-1), where supplementation with ADA-1 partially restored the dysfunctional signaling in GC-Tfh cells during chronic infection. Understanding how GC-Tfh cells are altered in HIV is critical to elucidate the mechanisms leading to effective anti-HIV antibodies. [ABSTRACT FROM AUTHOR]

A primary goal of HIV-1 vaccine development is the consistent elicitation of protective, neutralizing antibodies. While highly similar neutralizing antibodies (nAbs) have been isolated from multiple HIV-infected individuals, it is unclear whether vaccination can consistently elicit highly similar nAbs in genetically diverse primates. Here, we show in three outbred rhesus macaques that immunization with Env elicits a genotypically and phenotypically conserved nAb response. From these vaccinated macaques, we isolated four antibody lineages that had commonalities in immunoglobulin variable, diversity, and joining gene segment usage. Atomic-level structures of the antigen binding fragments of the two most similar antibodies showed nearly identical paratopes. The Env binding modes of each of the four vaccine-induced nAbs were distinct from previously known monoclonal HIV-1 neutralizing antibodies, but were nearly identical to each other. The similarities of these antibodies show that the immune system in outbred primates can respond to HIV-1 Env vaccination with a similar structural and genotypic solution for recognizing a particular neutralizing epitope. These results support rational vaccine design for HIV-1 that aims to reproducibly elicit, in genetically diverse primates, nAbs with specific paratope structures capable of binding conserved epitopes. Author summary: The primate immune system generates a diverse repertoire of pathogen-binding proteins called antibodies. Primate antibody repertoires must be diverse to respond to infection by many different pathogens. In this study, we characterized the structure and genetic features of HIV-1 inhibitory antibodies from vaccinated monkeys as a model for human vaccination. We found that individual monkeys responded to HIV vaccination by generating highly similar HIV-1 neutralizing antibodies. The antibodies had nearly identical structures and genetic makeups. Thus, the immune systems of different monkeys generate a common solution for inhibiting viral protein function and infectivity. Success of vaccines in multiple recipients may be augmented by the ability of immune systems to reproducibly devise a common antibody solution to attack vulnerable sites on pathogens. [ABSTRACT FROM AUTHOR]

Evaluating the structure-function relationship of viral envelope (Env) evolution and the development of broadly cross-neutralizing antibodies (bnAbs) in natural infection can inform rational immunogen design. In the present study, we examined the magnitude and specificity of autologous neutralizing antibodies induced in rabbits by a novel HIV-1 clade C Env protein (1PGE-THIVC) vis-à-vis those developed in an elite neutralizer from whom the env sequence was obtained that was used to prepare the soluble Env protein. The novel 1PGE-THIVC Env trimer displayed a native like pre-fusion closed conformation in solution as determined by small angle X-ray scattering (SAXS) and negative stain electron microscopy (EM). This closed spike conformation of 1PGE-THIVC Env trimers was correlated with weak or undetectable binding of non-neutralizing monoclonal antibodies (mAbs) compared to neutralizing mAbs. Furthermore, 1PGE-THIVC SOSIP induced potent neutralizing antibodies in rabbits to autologous virus variants. The autologous neutralizing antibody specificity induced in rabbits by 1PGE-THIVC was mapped to the C3/V4 region (T362/P401) of viral Env. This observation agreed with electron microscopy polyclonal epitope mapping (EMPEM) of the Env trimer complexed with IgG Fab prepared from the immunized rabbit sera. Our study demonstrated neutralization of sequence matched and unmatched autologous viruses by serum antibodies induced in rabbits by 1PGE-THIVC and also highlighted a comparable specificity for the 1PGE-THIVC SOSIP trimer with that seen with polyclonal antibodies elicited in the elite neutralizer by negative-stain electron microscopy polyclonal epitope (ns-EMPEM) mapping. Author summary: The interplay between circulating virus variants and broadly cross neutralizing polyclonal antibodies developed in a subset of elite neutralizers is widely believed to provide strategies for rational immunogen design. In the present study, we studied the structural, antigenic and immunogenic properties of a novel soluble trimeric protein with near native pre-fusion conformation prepared using the primary sequence of an HIV-1 clade C env isolated from the broadly cross neutralizing plasma of an elite neutralizer. This novel SOSIP Env trimer demonstrated comparable antigenic, structural and immunogenic properties that favoured several ongoing subunit vaccine design efforts. Interestingly, ns-EMPEM analysis suggested the novel clade C SOSIP induced a comparable neutralizing antibody specificity in rabbits to one that was elicited during the course of natural infection. A better understanding of how vaccine-induced polyclonal neutralizing antibody responses compares to responses that developed in natural infection will improve our knowledge in designing better vaccine design strategies. [ABSTRACT FROM AUTHOR]

HIV persists during antiretroviral therapy (ART) as integrated proviruses in cells descended from a small fraction of the CD4+ T cells infected prior to the initiation of ART. To better understand what controls HIV persistence and the distribution of integration sites (IS), we compared about 15,000 and 54,000 IS from individuals pre-ART and on ART, respectively, with approximately 395,000 IS from PBMC infected in vitro. The distribution of IS in vivo is quite similar to the distribution in PBMC, but modified by selection against proviruses in expressed genes, by selection for proviruses integrated into one of 7 specific genes, and by clonal expansion. Clones in which a provirus integrated in an oncogene contributed to cell survival comprised only a small fraction of the clones persisting in on ART. Mechanisms that do not involve the provirus, or its location in the host genome, are more important in determining which clones expand and persist. Author summary: In HIV-infected individuals, a small fraction of the infected cells persist and divide. This reservoir persists during fully suppressive ART and can rekindle the infection if ART is discontinued. Because the number of possible sites of HIV DNA integration is very large, each infected cell, and all of its descendants, can be identified by the site where the provirus is integrated (IS). To understand the selective forces that determine the fates of infected cells in vivo, we compared the distribution of HIV IS in freshly-infected cells to cells from HIV-infected donors sampled both before and during ART. We found that, as previously reported, integration favors highly-expressed genes. However, over time, the fraction of cells with proviruses integrated in highly-expressed genes decreases, implying that they grow less well. There are exceptions to this broad negative selection. When a provirus is integrated in a specific region in one of seven genes, the proviruses affect the expression of the target gene, promoting growth and/or survival of the cell. Although this effect is striking, it is only a minor component of the forces that promote the growth and survival of the population of infected cells during ART. [ABSTRACT FROM AUTHOR]

Histoplasmosis was first described in Panama in 1906 in a patient presenting miliary tuberculosis-like symptoms. Hence, in Panama, 7.6% of patients had culture-proven histoplasmosis [[9]]; in Venezuela, histoplasmosis was documented in 43 of 200 (21.3%) [[10]]; in Fortaleza, Brazil, of 378 consecutive hospitalized HIV patients, 164 (43.4%) had disseminated histoplasmosis [[11]]; A recent screening study in hospitalized HIV patients in Brazil found high I Histoplasma i antigen prevalence, ranging from 8.8% in Porto Alegre to 44.8% in Natal. [[18]] However, these data originate from histoplasmosis-aware centers, and in most hospitals across Latin America, histoplasmosis is not diagnosed and not treated, often being mistaken for tuberculosis, thereby leading to unnecessary deaths and inflated tuberculosis statistics. [Extracted from the article]

The ALVAC-HIV clade B/AE and equivalent SIV-based/gp120 + Alum vaccines successfully decreased the risk of virus acquisition in humans and macaques. Here, we tested the efficacy of HIV clade B/C ALVAC/gp120 vaccine candidates + MF59 or different doses of Aluminum hydroxide (Alum) against SHIV-Cs of varying neutralization sensitivity in macaques. Low doses of Alum induced higher mucosal V2-specific IgA that increased the risk of Tier 2 SHIV-C acquisition. High Alum dosage, in contrast, elicited serum IgG to V2 that correlated with a decreased risk of Tier 1 SHIV-C acquisition. MF59 induced negligible mucosal antibodies to V2 and an inflammatory profile with blood C-reactive Protein (CRP) levels correlating with neutralizing antibody titers. MF59 decreased the risk of Tier 1 SHIV-C acquisition. The relationship between vaccine efficacy and the neutralization profile of the challenge virus appear to be linked to the different immunological spaces created by MF59 and Alum via CXCL10 and IL-1β, respectively. Author summary: The ALVAC-based clade C /gp120 combination is currently being tested for efficacy and licensures in South Africa in the HVTN702 HIV vaccine trial using the MF59 adjuvant rather than Alum with the intent of inducing higher neutralizing antibodies and T cell responses. Here, we present studies in 71 macaques that evaluated the relative efficacy of identical ALVAC-HIV B/C HIV vaccines with Alum or MF59 following challenge exposure to SHIV-clade C viral stocks that differed in their neutralization profiles. Our results demonstrated that use of the proprietary Novartis Alum at lower doses affects the proportion of mucosal V2-specific IgG and IgA, and that IgAs and IgGs were respectively associated with an increased or decreased risk of virus acquisition. In contrast, animals immunized with the MF59 regimen had no V2 mucosal responses, and a high level of serum neutralizing antibodies (Tier 1) to easy to neutralize viruses that were associated with a decreased risk of Tier 1 SHIV-C acquisition. In vitro studies suggest the hypothesis that the low doses of proprietary Novartis Alum used in our studies may be the underlying reason for the decreased vaccine efficacy, via lower inflammasome activation and IL-1β production. [ABSTRACT FROM AUTHOR]

HIV-1 gene expression is regulated by host and viral factors that interact with viral motifs and is influenced by proviral integration sites. Here, expression variation among integrants was followed for hundreds of individual proviral clones within polyclonal populations throughout successive rounds of virus and cultured cell replication, with limited findings using CD4+ cells from donor blood consistent with observations in immortalized cells. Tracking clonal behavior by proviral “zip codes” indicated that mutational inactivation during reverse transcription was rare, while clonal expansion and proviral expression states varied widely. By sorting for provirus expression using a GFP reporter in the nef open reading frame, distinct clone-specific variation in on/off proportions were observed that spanned three orders of magnitude. Tracking GFP phenotypes over time revealed that as cells divided, their progeny alternated between HIV transcriptional activity and non-activity. Despite these phenotypic oscillations, the overall GFP+ population within each clone was remarkably stable, with clones maintaining clone-specific equilibrium mixtures of GFP+ and GFP- cells. Integration sites were analyzed for correlations between genomic features and the epigenetic phenomena described here. Integrants inserted in the sense orientation of genes were more frequently found to be GFP negative than those in the antisense orientation, and clones with high GFP+ proportions were more distal to repressive H3K9me3 peaks than low GFP+ clones. Clones with low frequencies of GFP positivity appeared to expand more rapidly than clones for which most cells were GFP+, even though the tested proviruses were Vpr-. Thus, much of the increase in the GFP- population in these polyclonal pools over time reflected differential clonal expansion. Together, these results underscore the temporal and quantitative variability in HIV-1 gene expression among proviral clones that are conferred in the absence of metabolic or cell-type dependent variability, and shed light on cell-intrinsic layers of regulation that affect HIV-1 population dynamics. [ABSTRACT FROM AUTHOR]

The CD4 binding site (CD4bs) of the HIV-1 envelope glycoprotein is susceptible to multiple lineages of broadly neutralizing antibodies (bnAbs) that are attractive to elicit with vaccines. The CH235 lineage (VH1-46) of CD4bs bnAbs is particularly attractive because the most mature members neutralize 90% of circulating strains, do not possess long HCDR3 regions, and do not contain insertions and deletions that may be difficult to induce. We used virus neutralization to measure the interaction of CH235 unmutated common ancestor (CH235 UCA) with functional Env trimers on infectious virions to guide immunogen design for this bnAb lineage. Two Env mutations were identified, one in loop D (N279K) and another in V5 (G458Y), that acted synergistically to render autologous CH505 transmitted/founder virus susceptible to neutralization by CH235 UCA. Man5-enriched N-glycans provided additional synergy for neutralization. CH235 UCA bound with nanomolar affinity to corresponding soluble native-like Env trimers as candidate immunogens. A cryo-EM structure of CH235 UCA bound to Man5-enriched CH505.N279K.G458Y.SOSIP.664 revealed interactions of the antibody light chain complementarity determining region 3 (CDR L3) with the engineered Env loops D and V5. These results demonstrate that virus neutralization can directly inform vaccine design and suggest a germline targeting and reverse engineering strategy to initiate and mature the CH235 bnAb lineage. [ABSTRACT FROM AUTHOR]

The causative mutation responsible for limb girdle muscular dystrophy 1F (LGMD1F) is one heterozygous single nucleotide deletion in the stop codon of the nuclear import factor Transportin 3 gene (TNPO3). This mutation causes a carboxy-terminal extension of 15 amino acids, producing a protein of unknown function (TNPO3_mut) that is co-expressed with wild-type TNPO3 (TNPO3_wt). TNPO3 has been involved in the nuclear transport of serine/arginine-rich proteins such as splicing factors and also in HIV-1 infection through interaction with the viral integrase and capsid. We analyzed the effect of TNPO3_mut on HIV-1 infection using PBMCs from patients with LGMD1F infected ex vivo. HIV-1 infection was drastically impaired in these cells and viral integration was reduced 16-fold. No significant effects on viral reverse transcription and episomal 2-LTR circles were observed suggesting that the integration of HIV-1 genome was restricted. This is the second genetic defect described after CCR5Δ32 that shows strong resistance against HIV-1 infection. [ABSTRACT FROM AUTHOR]

Chronic infection with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) affects an estimated 35 million and 75 million individuals worldwide, respectively. These viruses induce persistent inflammation which often drives the development or progression of organ-specific diseases and even cancer including Hepatocellular Carcinoma (HCC). In this study, we sought to examine inflammatory responses following HIV or HCV stimulation of macrophages or Kupffer cells (KCs), that may contribute to virus mediated inflammation and subsequent liver disease. KCs are liver-resident macrophages and reports have provided evidence that HIV can stimulate and infect them. In order to characterize HIV-intrinsic innate immune responses that may occur in the liver, we performed microarray analyses on KCs following HIV stimulation. Our data demonstrate that KCs upregulate several innate immune signaling pathways involved in inflammation, myeloid cell maturation, stellate cell activation, and Triggering Receptor Expressed on Myeloid cells 1 (TREM1) signaling. TREM1 is a member of the immunoglobulin superfamily of receptors and it is reported to be involved in systemic inflammatory responses due to its ability to amplify activation of host defense signaling pathways. Our data demonstrate that stimulation of KCs with HIV or HCV induces the upregulation of TREM1. Additionally, HIV viral proteins can upregulate expression of TREM1 mRNA through NF-кB signaling. Furthermore, activation of the TREM1 signaling pathway, with a targeted agonist, increased HIV or HCV-mediated inflammatory responses in macrophages due to enhanced activation of the ERK1/2 signaling cascade. Silencing TREM1 dampened inflammatory immune responses elicited by HIV or HCV stimulation. Finally, HIV and HCV infected patients exhibit higher expression and frequency of TREM1 and CD68 positive cells. Taken together, TREM1 induction by HIV contributes to chronic inflammation in the liver and targeting TREM1 signaling may be a therapeutic option to minimize HIV induced chronic inflammation. [ABSTRACT FROM AUTHOR]

BLyS/BAFF is recognized for its role in B-cell ontogenesis, as well as cell fate decision towards the first-line/innate marginal zone (MZ) B-cell pool. Excess BLyS/BAFF is associated with hyperglobulinemia and increased frequencies of activated precursor-like MZ B-cells. Herein, we show that HIV highly-exposed seronegative (HESN) commercial sex workers (CSWs) had lower soluble BLyS/BAFF levels and relative frequencies of BLyS/BAFF expressing cells in their genital mucosa when compared to those from HIV-infected CSWs and HIV-uninfected non-CSWs. Furthermore, we identified genital innate and/or marginal zone (MZ)-like CD1c+ B-cells that naturally bind to fully glycosylated gp120, which frequencies were lower in HESNs when compared to HIV-infected CSWs and HIV-uninfected non-CSWs. Although genital levels of total IgA were similar between groups, HESNs had lower levels of total IgG1 and IgG3. Interestingly, HIV-gp41 reactive IgG1 were found in some HESNs. Low genital levels of BLyS/BAFF observed in HESNs may allow for controlled first-line responses, contributing to natural immunity to HIV. [ABSTRACT FROM AUTHOR]

A major barrier to curing HIV-1 is the long-lived latent reservoir that supports re-emergence of HIV-1 upon treatment interruption. Targeting this reservoir will require mechanistic insights into the establishment and maintenance of HIV-1 latency. Whether T cell signaling at the time of HIV-1 infection influences productive replication or latency is not fully understood. We used a panel of chimeric antigen receptors (CARs) with different ligand binding affinities to induce a range of signaling strengths to model differential T cell receptor signaling at the time of HIV-1 infection. Stimulation of T cell lines or primary CD4+ T cells expressing chimeric antigen receptors supported HIV-1 infection regardless of affinity for ligand; however, only signaling by the highest affinity receptor facilitated HIV-1 expression. Activation of chimeric antigen receptors that had intermediate and low binding affinities did not support provirus transcription, suggesting that a minimal signal is required for optimal HIV-1 expression. In addition, strong signaling at the time of infection produced a latent population that was readily inducible, whereas latent cells generated in response to weaker signals were not easily reversed. Chromatin immunoprecipitation showed HIV-1 transcription was limited by transcriptional elongation and that robust signaling decreased the presence of negative elongation factor, a pausing factor, by more than 80%. These studies demonstrate that T cell signaling influences HIV-1 infection and the establishment of different subsets of latently infected cells, which may have implications for targeting the HIV-1 reservoir. [ABSTRACT FROM AUTHOR]

HIV-1 is dependent on the host cell for providing the metabolic resources for completion of its viral replication cycle. Thus, HIV-1 replicates efficiently only in activated CD4+ T cells. Barriers preventing HIV-1 replication in resting CD4+ T cells include a block that limits reverse transcription and also the lack of activity of several inducible transcription factors, such as NF-κB and NFAT. Because FOXO1 is a master regulator of T cell functions, we studied the effect of its inhibition on T cell/HIV-1 interactions. By using AS1842856, a FOXO1 pharmacologic inhibitor, we observe that FOXO1 inhibition induces a metabolic activation of T cells with a G0/G1 transition in the absence of any stimulatory signal. One parallel outcome of this change is the inhibition of the activity of the HIV restriction factor SAMHD1 and the activation of the NFAT pathway. FOXO1 inhibition by AS1842856 makes resting T cells permissive to HIV-1 infection. In addition, we found that FOXO1 inhibition by either AS1842856 treatment or upon FOXO1 knockdown induces the reactivation of HIV-1 latent proviruses in T cells. We conclude that FOXO1 has a central role in the HIV-1/T cell interaction and that inhibiting FOXO1 with drugs such as AS1842856 may be a new therapeutic shock-and-kill strategy to eliminate the HIV-1 reservoir in human T cells. [ABSTRACT FROM AUTHOR]

CCR5 plays immune functions and is the coreceptor for R5 HIV-1 strains. It exists in diverse conformations and oligomerization states. We interrogated the significance of the CCR5 structural diversity on HIV-1 infection. We show that envelope glycoproteins (gp120s) from different HIV-1 strains exhibit divergent binding levels to CCR5 on cell lines and primary cells, but not to CD4 or the CD4i monoclonal antibody E51. This owed to differential binding of the gp120s to different CCR5 populations, which exist in varying quantities at the cell surface and are differentially expressed between different cell types. Some, but not all, of these populations are antigenically distinct conformations of the coreceptor. The different binding levels of gp120s also correspond to differences in their capacity to bind CCR5 dimers/oligomers. Mutating the CCR5 dimerization interface changed conformation of the CCR5 homodimers and modulated differentially the binding of distinct gp120s. Env-pseudotyped viruses also use particular CCR5 conformations for entry, which may differ between different viruses and represent a subset of those binding gp120s. In particular, even if gp120s can bind both CCR5 monomers and oligomers, impairment of CCR5 oligomerization improved viral entry, suggesting that HIV-1 prefers monomers for entry. From a functional standpoint, we illustrate that the nature of the CCR5 molecules to which gp120/HIV-1 binds shapes sensitivity to inhibition by CCR5 ligands and cellular tropism. Differences exist in the CCR5 populations between T-cells and macrophages, and this is associated with differential capacity to bind gp120s and to support viral entry. In macrophages, CCR5 structural plasticity is critical for entry of blood-derived R5 isolates, which, in contrast to prototypical M-tropic strains from brain tissues, cannot benefit from enhanced affinity for CD4. Collectively, our results support a role for CCR5 heterogeneity in diversifying the phenotypic properties of HIV-1 isolates and provide new clues for development of CCR5-targeting drugs. [ABSTRACT FROM AUTHOR]

A correction to the article "HIV Reactivation from Latency after Treatment Interruption Occurs on Average Every 5-8 Days-Implications for HIV Remission" by Mykola Pinkevych and colleagues in the 2016 issue is presented.

Drug resistant HIV is a major threat to the long-term efficacy of antiretroviral treatment. Around 10% of ART-naïve patients in Europe are infected with drug-resistant HIV type 1. Hence it is important to understand the dynamics of transmitted drug resistance evolution. Thanks to routinely performed drug resistance tests, HIV sequence data is increasingly available and can be used to reconstruct the phylogenetic relationship among viral lineages. In this study we employ a phylodynamic approach to quantify the fitness costs of major resistance mutations in the Swiss HIV cohort. The viral phylogeny reflects the transmission tree, which we model using stochastic birth–death-sampling processes with two types: hosts infected by a sensitive or resistant strain. This allows quantification of fitness cost as the ratio between transmission rates of hosts infected by drug resistant strains and transmission rates of hosts infected by drug sensitive strains. The resistance mutations 41L, 67N, 70R, 184V, 210W, 215D, 215S and 219Q (nRTI-related) and 103N, 108I, 138A, 181C, 190A (NNRTI-related) in the reverse trancriptase and the 90M mutation in the protease gene are included in this study. Among the considered resistance mutations, only the 90M mutation in the protease gene was found to have significantly higher fitness than the drug sensitive strains. The following mutations associated with resistance to reverse transcriptase inhibitors were found to be less fit than the sensitive strains: 67N, 70R, 184V, 219Q. The highest posterior density intervals of the transmission ratios for the remaining resistance mutations included in this study all included 1, suggesting that these mutations do not have a significant effect on viral transmissibility within the Swiss HIV cohort. These patterns are consistent with alternative measures of the fitness cost of resistance mutations. Overall, we have developed and validated a novel phylodynamic approach to estimate the transmission fitness cost of drug resistance mutations. [ABSTRACT FROM AUTHOR]

HIV-1 envelope glycoprotein (Env) mediates virus attachment and entry into the host cells. Like other membrane-bound and secreted proteins, HIV-1 Env contains at its N terminus a signal peptide (SP) that directs the nascent Env to the endoplasmic reticulum (ER) where Env synthesis and post-translational modifications take place. SP is cleaved during Env biosynthesis but potentially influences the phenotypic traits of the Env protein. The Env SP sequences of HIV-1 isolates display high sequence variability, and the significance of such variability is unclear. We postulate that changes in the Env SP influence Env transport through the ER-Golgi secretory pathway and Env folding and/or glycosylation that impact on Env incorporation into virions, receptor binding and antibody recognition. We first evaluated the consequences of mutating the charged residues in the Env SP in the context of infectious molecular clone HIV-1 REJO.c/2864. Results show that three different mutations affecting histidine at position 12 affected Env incorporation into virions that correlated with reduction of virus infectivity and DC-SIGN-mediated virus capture and transmission. Mutations at positions 8, 12, and 15 also rendered the virus more resistant to neutralization by monoclonal antibodies against the Env V1V2 region. These mutations affected the oligosaccharide composition of N-glycans as shown by changes in Env reactivity with specific lectins and by mass spectrometry. Increased neutralization resistance and N-glycan composition changes were also observed when analogous mutations were introduced to another HIV-1 strain, JRFL. To the best of our knowledge, this is the first study showing that certain residues in the HIV-1 Env SP can affect virus neutralization sensitivity by modulating oligosaccharide moieties on the Env N-glycans. The HIV-1 Env SP sequences thus may be under selective pressure to balance virus infectiousness with virus resistance to the host antibody responses. (289 words) [ABSTRACT FROM AUTHOR]

HIV is adept at avoiding naturally generated T cell responses; therefore, there is a need to develop HIV-specific T cells with greater potency for use in HIV cure strategies. Starting with a CD4-based chimeric antigen receptor (CAR) that was previously used without toxicity in clinical trials, we optimized the vector backbone, promoter, HIV targeting moiety, and transmembrane and signaling domains to determine which components augmented the ability of T cells to control HIV replication. This re-engineered CAR was at least 50-fold more potent in vitro at controlling HIV replication than the original CD4 CAR, or a TCR-based approach, and substantially better than broadly neutralizing antibody-based CARs. A humanized mouse model of HIV infection demonstrated that T cells expressing optimized CARs were superior at expanding in response to antigen, protecting CD4 T cells from infection, and reducing viral loads compared to T cells expressing the original, clinical trial CAR. Moreover, in a humanized mouse model of HIV treatment, CD4 CAR T cells containing the 4-1BB costimulatory domain controlled HIV spread after ART removal better than analogous CAR T cells containing the CD28 costimulatory domain. Together, these data indicate that potent HIV-specific T cells can be generated using improved CAR design and that CAR T cells could be important components of an HIV cure strategy. [ABSTRACT FROM AUTHOR]

In order to inform the rational design of HIV-1 preventive and cure interventions it is critical to understand the events occurring during acute HIV-1 infection (AHI). Using viral deep sequencing on six participants from the early capture acute infection RV217 cohort, we have studied HIV-1 evolution in plasma collected twice weekly during the first weeks following the advent of viremia. The analysis of infections established by multiple transmitted/founder (T/F) viruses revealed novel viral profiles that included: a) the low-level persistence of minor T/F variants, b) the rapid replacement of the major T/F by a minor T/F, and c) an initial expansion of the minor T/F followed by a quick collapse of the same minor T/F to low frequency. In most participants, cytotoxic T-lymphocyte (CTL) escape was first detected at the end of peak viremia downslope, proceeded at higher rates than previously measured in HIV-1 infection, and usually occurred through the exploration of multiple mutational pathways within an epitope. The rapid emergence of CTL escape variants suggests a strong and early CTL response. Minor T/F viral strains can contribute to rapid and varied profiles of HIV-1 quasispecies evolution during AHI. Overall, our results demonstrate that early, deep, and frequent sampling is needed to investigate viral/host interaction during AHI, which could help identify prerequisites for prevention and cure of HIV-1 infection. [ABSTRACT FROM AUTHOR]

The process by which drug-resistant HIV-1 arises and spreads spatially within an infected individual is poorly understood. Studies have found variable results relating how HIV-1 in the blood differs from virus sampled in tissues, offering conflicting findings about whether HIV-1 throughout the body is homogeneously distributed. However, most of these studies sample only two compartments and few have data from multiple time points. To directly measure how drug resistance spreads within a host and to assess how spatial structure impacts its emergence, we examined serial sequences from four macaques infected with RT-SHIVmne027, a simian immunodeficiency virus encoding HIV-1 reverse transcriptase (RT), and treated with RT inhibitors. Both viral DNA and RNA (vDNA and vRNA) were isolated from the blood (including plasma and peripheral blood mononuclear cells), lymph nodes, gut, and vagina at a median of four time points and RT was characterized via single-genome sequencing. The resulting sequences reveal a dynamic system in which vRNA rapidly acquires drug resistance concomitantly across compartments through multiple independent mutations. Fast migration results in the same viral genotypes present across compartments, but not so fast as to equilibrate their frequencies immediately. The blood and lymph nodes were found to be compartmentalized rarely, while both the blood and lymph node were more frequently different from mucosal tissues. This study suggests that even oft-sampled blood does not fully capture the viral dynamics in other parts of the body, especially the gut where vRNA turnover was faster than the plasma and vDNA retained fewer wild-type viruses than other sampled compartments. Our findings of transient compartmentalization across multiple tissues may help explain the varied results of previous compartmentalization studies in HIV-1. [ABSTRACT FROM AUTHOR]

Although invasive cytomegalovirus (CMV) disease is uncommon in the era of antiretroviral therapy (ART), asymptomatic CMV coinfection is nearly ubiquitous in HIV infected individuals. While microbial translocation and gut epithelial barrier dysfunction may promote persistent immune activation in treated HIV infection, potentially contributing to morbidity and mortality, it has been unclear whether CMV replication in individuals with no symptoms of CMV disease might play a role in this process. We hypothesized that persistent CMV replication in the intestinal epithelium of HIV/CMV-coinfected individuals impairs gut epithelial barrier function. Using a combination of state-of-the-art in situ hybridization technology (RNAscope) and immunohistochemistry, we detected CMV DNA and proteins and evidence of intestinal damage in rectosigmoid samples from CMV-positive individuals with both untreated and ART-suppressed HIV infection. Two different model systems, primary human intestinal cells differentiated in vitro to form polarized monolayers and a humanized mouse model of human gut, together demonstrated that intestinal epithelial cells are fully permissive to CMV replication. Independent of HIV, CMV disrupted tight junctions of polarized intestinal cells, significantly reducing transepithelial electrical resistance, a measure of monolayer integrity, and enhancing transepithelial permeability. The effect of CMV infection on the intestinal epithelium is mediated, at least in part, by the CMV-induced proinflammatory cytokine IL-6. Furthermore, letermovir, a novel anti-CMV drug, dampened the effects of CMV on the epithelium. Together, our data strongly suggest that CMV can disrupt epithelial junctions, leading to bacterial translocation and chronic inflammation in the gut and that CMV could serve as a target for therapeutic intervention to prevent or treat gut epithelial barrier dysfunction during HIV infection. [ABSTRACT FROM AUTHOR]

Oropharyngeal mucosal epithelia of fetuses/neonates/infants and the genital epithelia of adults play a critical role in HIV-1 mother-to-child transmission and sexual transmission of virus, respectively. To study the mechanisms of HIV-1 transmission through mucosal epithelium, we established polarized tonsil, cervical and foreskin epithelial cells. Analysis of HIV-1 transmission through epithelial cells showed that approximately 0.05% of initially inoculated virions transmigrated via epithelium. More than 90% of internalized virions were sequestered in the endosomes of epithelial cells, including multivesicular bodies (MVBs) and vacuoles. Intraepithelial HIV-1 remained infectious for 9 days without viral release. Release of sequestered intraepithelial HIV-1 was induced by the calcium ionophore ionomycin and by cytochalasin D, which increase intracellular calcium and disrupt the cortical actin of epithelial cells, respectively. Cocultivation of epithelial cells containing HIV-1 with activated peripheral blood mononuclear cells and CD4+ T lymphocytes led to the disruption of epithelial cortical actin and spread of virus from epithelial cells to lymphocytes. Treatment of epithelial cells with proinflammatory cytokines tumor necrosis factor-alpha and interferon gamma also induced reorganization of cortical actin and release of virus. Inhibition of MVB formation by small interfering RNA (siRNA)-mediated silencing of its critical protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) expression reduced viral sequestration in epithelial cells and its transmission from epithelial cells to lymphocytes by ~60–70%. Furthermore, inhibition of vacuole formation of epithelial cells by siRNA-inactivated rabankyrin-5 expression also significantly reduced HIV-1 sequestration in epithelial cells and spread of virus from epithelial cells to lymphocytes. Interaction of the intercellular adhesion molecule-1 of epithelial cells with the function-associated antigen-1 of lymphocytes was important for inducing the release of sequestered HIV-1 from epithelial cells and facilitating cell-to-cell spread of virus from epithelial cells to lymphocytes. This mechanism may serve as a pathway of HIV-1 mucosal transmission. [ABSTRACT FROM AUTHOR]

The interactions that occur during HIV Pr55Gag oligomerization and genomic RNA packaging are essential elements that facilitate HIV assembly. However, mechanistic details of these interactions are not clearly defined. Here, we overcome previous limitations in producing large quantities of full-length recombinant Pr55Gag that is required for isothermal titration calorimetry (ITC) studies, and we have revealed the thermodynamic properties of HIV assembly for the first time. Thermodynamic analysis showed that the binding between RNA and HIV Pr55Gag is an energetically favourable reaction (ΔG<0) that is further enhanced by the oligomerization of Pr55Gag. The change in enthalpy (ΔH) widens sequentially from: (1) Pr55Gag-Psi RNA binding during HIV genome selection; to (2) Pr55Gag-Guanosine Uridine (GU)-containing RNA binding in cytoplasm/plasma membrane; and then to (3) Pr55Gag-Adenosine(A)-containing RNA binding in immature HIV. These data imply the stepwise increments of heat being released during HIV biogenesis may help to facilitate the process of viral assembly. By mimicking the interactions between A-containing RNA and oligomeric Pr55Gag in immature HIV, it was noted that a p6 domain truncated Pr50Gag Δp6 is less efficient than full-length Pr55Gag in this thermodynamic process. These data suggest a potential unknown role of p6 in Pr55Gag-Pr55Gag oligomerization and/or Pr55Gag-RNA interaction during HIV assembly. Our data provide direct evidence on how nucleic acid sequences and the oligomeric state of Pr55Gag regulate HIV assembly. [ABSTRACT FROM AUTHOR]

HIV-1 infection is associated with an early and profound depletion of mucosal memory CD4+ T cells, a population that plays an indispensable role in the regulation of isotype switching and transepithelial transport of antibodies. In this study, we addressed whether the depletion of CD4+ T cell in HIV-1-infected individuals results in altered humoral responses specific to antigens encountered at mucosal surfaces. Comprehensive protein microarray of systemic humoral responses to intestinal microbiota demonstrated reduced IgG responses to antigens derived from Proteobacteria and Firmicutes but not Bacteroidetes. Importantly, intestinal secretions of antiretroviral therapy-treated HIV-1-infected individuals exhibited a significant elevation of IgM levels and decreased IgA/IgM and IgG/IgM ratios of antibodies specific to a variety of microbial and food antigens. The presented findings indicate reduced competence of mucosal B cells for class switch recombination from IgM to other isotypes limiting their capacity to react to changing antigenic variety in the gut lumen. Decreased availability of microbiota-specific IgA and IgG may be an important factor contributing to the translocation of microbial antigens across the intestinal mucosal barrier and their systemic dissemination that drives chronic inflammation in HIV-1-infected individuals. [ABSTRACT FROM AUTHOR]

Computational neutralization fingerprinting, NFP, is an efficient and accurate method for predicting the epitope specificities of polyclonal antibody responses to HIV-1 infection. Here, we present next-generation NFP algorithms that substantially improve prediction accuracy for individual donors and enable serologic analysis for entire cohorts. Specifically, we developed algorithms for: (a) selection of optimized virus neutralization panels for NFP analysis, (b) estimation of NFP prediction confidence for each serum sample, and (c) identification of sera with potentially novel epitope specificities. At the individual donor level, the next-generation NFP algorithms particularly improved the ability to detect multiple epitope specificities in a sample, as confirmed both for computationally simulated polyclonal sera and for samples from HIV-infected donors. Specifically, the next-generation NFP algorithms detected multiple specificities in twice as many samples of simulated sera. Further, unlike the first-generation NFP, the new algorithms were able to detect both of the previously confirmed antibody specificities, VRC01-like and PG9-like, in donor CHAVI 0219. At the cohort level, analysis of ~150 broadly neutralizing HIV-infected donor samples suggested a potential connection between clade of infection and types of elicited epitope specificities. Most notably, while 10E8-like antibodies were observed in infections from different clades, an enrichment of such antibodies was predicted for clade B samples. Ultimately, such large-scale analyses of antibody responses to HIV-1 infection can help guide the design of epitope-specific vaccines that are tailored to take into account the prevalence of infecting clades within a specific geographic region. Overall, the next-generation NFP technology will be an important tool for the analysis of broadly neutralizing polyclonal antibody responses against HIV-1. [ABSTRACT FROM AUTHOR]

Structural rearrangements of HIV-1 glycoprotein Env promote viral entry through membrane fusion. Env is a symmetric homotrimer with each protomer composed of surface subunit gp120 and transmembrane subunit gp41. Cellular CD4- and chemokine receptor-binding to gp120 coordinate conformational changes in gp41, first to an extended prehairpin intermediate (PHI) and, ultimately, into a fusogenic trimer-of-hairpins (TOH). HIV-1 fusion inhibitors target gp41 in the PHI and block TOH formation. To characterize structural transformations into and through the PHI, we employed asymmetric Env trimers containing both high and low affinity binding sites for individual fusion inhibitors. Asymmetry was achieved using engineered Env heterotrimers composed of protomers deficient in either CD4- or chemokine receptor-binding. Linking receptor engagement to inhibitor affinity allowed us to assess conformational changes of individual Env protomers in the context of a functioning trimer. We found that the transition into the PHI could occur symmetrically or asymmetrically depending on the stoichiometry of CD4 binding. Sequential engagement of multiple CD4s promoted progressive exposure of individual fusion inhibitor binding sites in a CD4-dependent fashion. By contrast, engagement of only a single CD4 molecule led to a delayed, but symmetric, exposure of the gp41 trimer. This complex coupling between Env-CD4 interaction and gp41 exposure explained the multiphasic fusion-inhibitor titration observed for a mutant Env homotrimer with a naturally asymmetric gp41. Our results suggest that the spatial and temporal exposure of gp41 can proceed in nonconcerted, asymmetric manner depending on the number of CD4s that engage the Env trimer. The findings have important implications for the mechanism of viral membrane fusion and the development of vaccine candidates designed to elicit neutralizing antibodies targeting gp41 in the PHI. [ABSTRACT FROM AUTHOR]