Biology subjects

Bar, K. J.

Publications and source records attributed to Bar, K. J..

11 recordsLinked to original sources

bioRxiv2025

Selective infection and loss of PRDM1+ LN Tfh cells in uncontrolled HIV infection precludes formation of Tfh reservoirs under ART

The multifaceted and long-lived nature of HIV-1 reservoirs has proven to be a formidable obstacle in the development of a therapeutic cure of HIV-1. One of the major dimensions of the HIV-1 reservoir is its prevalence and persistence in tissue environments, including in lymph nodes (LN). Within LN, T follicular helper (Tfh) cells are widely considered as the dominant sub-reservoir in viremic people with HIV (PWH). However, whether Tfh cells survive to establish a reservoir in PWH undergoing suppressive antiretroviral therapy (ART) has remained controversial. To address these issues, we deeply phenotyped over 500,000 cells and identified over 2,000 HIV-1 infected cells by employing single-cell multiomics on LN from PWH during viremia and suppressed on ART. While we detected HIV-1 infected Tfh cells, the majority of infected cells during viremia and ART had non-follicular phenotypes and were instead heterogeneously distributed between various CD4+ T-cell subsets. Within-subset comparisons of HIV+ and HIV- cells revealed heightened activation signatures and altered cell cycle states during viremia, but largely similar features during ART. Furthermore, the comparison between viremia and ART in PWH highlighted a noncanonical Tfh subset - defined by high locus accessibility and transcription of PRDM1 - that is selectively depleted during viremia, recovers after ART, and is highly susceptible to infection in vitro. Our work suggests a revised direction for the HIV-1 cure field where a mandate of any comprehensive strategy will be to address the high proportional burden of non-follicular cells within the HIV reservoir.

immunology↗

bioRxiv2025

Dual Role of Glycosylation in Resistance to CD4-binding Site Broadly Neutralizing Antibodies

Broadly neutralizing antibodies (bNAbs) provide a useful tool for HIV cure strategies because of their ability to target conserved regions on the envelope (Env) protein in the context of both virions and infected cells. One of the most well studied bNAbs is the CD4 binding site (CD4bs) antibody, VRC01 and others in its class. A major obstacle to effective cure strategies with bNAbs is viral immune escape. A deeper understanding of escape pathways from VRC01-class antibodies in genetically diverse samples is needed. Using an in vitro viral escape assay where infected CD4+ T cells were cultured in the presence of increasing VRC01 concentrations, complete resistance to VRC01 was detected in Env 246.F3 by day 42. We determined that resistance was due to a mutation at position N276 that resulted in elimination of the glycan-attachment site. As the loss of a glycan at this site is known to increase virus sensitivity to CD4bs antibodies, we explored this finding further. Specifically, we introduced N276D mutation into the 12-virus global Env panel and measured neutralization susceptibility to a panel of CD4bs bNAbs. This N276D mutation increased resistance or sensitivity depending on the Env and bNAb in question, emphasizing a dual role of glycan N276 in VRC01-class bNAb neutralization. The role of this glycan in escape was demonstrated to be dependent on the context of the Env, the bNAb and the glycosylation complexity of the virus due to producer cells. These findings underscore the complexity of glycosylation in genetically diverse HIV escape from antibodies. ImportanceDespite decades of research, an HIV cure remains elusive, largely due to the viruss immense genetic variability and ability to evade immune clearance. While antiretroviral therapy (ART) suppresses viral replication, it does not eradicate the virus and presents long-term challenges related to toxicity, access, and adherence. Broadly neutralizing antibodies (bNAbs), particularly those targeting the conserved CD4 binding site such as VRC01 and others in its class, offer promise for durable control or cure by targeting both circulating virus and infected cells. However, viral escape from bNAbs remains a critical hurdle. In this study, we demonstrate that glycan-mediated escape from VRC01-class bNAbs is highly context-dependent--shaped by Env, bNAb, and the glycosylation patterns introduced by the producer cell. These findings emphasize the dual role of glycans in affecting antibody sensitivity and underscore the importance of viral and host factors in shaping effective bNAb-based cure strategies across diverse HIV-1 strains.

immunology↗

bioRxiv2025

Env-antibody coevolution identifies B cell priming as the principal bottleneck to HIV-1 V2 apex broadly neutralizing antibody development

Broadly neutralizing antibodies (bNAbs) are rarely elicited during HIV-1 infection. To identify obstacles to bNAb development, we longitudinally studied 122 rhesus macaques infected by one of 16 different simian-human immunodeficiency viruses (SHIVs). We identified V2 apex as the most common bNAb target and a subset of Envs that preferentially elicited these antibodies. In 10 macaques, we delineated Env-antibody coevolution from B cell priming to bNAb development. Antibody phylogenies revealed permissive developmental pathways guided by evolving Envs that contained few mutations in or near the V2 apex C-strand, which were a sensitive indicator of apex-targeted responses. The absence of such mutations reflected a failure in bNAb priming. These results indicate that efficiency of B cell priming, and not complexities in Env-guided affinity maturation, is the primary obstacle to V2 apex bNAb elicitation in SHIV-infected macaques and identify specific HIV-1 Envs to advance as novel vaccine platforms. One sentence summaryB cell priming is the primary bottleneck to HIV-1 V2 apex bNAb elicitation.

immunology↗

bioRxiv2025

Therapeutic CD8+T cell tissue retention and immunomodulation during ART interruption fails to prevent SIV rebound

A primary obstacle for HIV elimination is the long-term viral reservoir in lymphoid tissues (LT) that can cause rebound viremia if therapy is stopped. Cytotoxic CD8+ T cells are critical for control of HIV and SIV viremia; however, CD8+ T cells that migrate to LT are primarily non-cytotoxic, calling into question whether these cells could reduce the viral reservoir on ART or control viral replication when therapy is halted. To determine if CD8+ T cells can inhibit viral replication when retained in lymphoid tissues, we inhibited lymphocyte egress from LTs in antiretroviral therapy (ART)-treated SIV-infected rhesus macaques (RM) during analytic treatment interruption (ATI) using the S1PR modulator FTY720 alone or in combination with anti-PD1 and the IL-15 receptor superagonist N-803 to increase cytolytic function. FTY720 retained migrating CD4+ and CD8+ T cells in LT, whereas cytotoxic CD8+ T cells remained in the vasculature. After ATI and viral rebound, activated SIV-specific CD8+ T cells increased in frequency in LT of FTY720-treated RM but failed to become cytotoxic or control plasma viremia compared to controls, even when combined with anti-PD1 and N-803. These findings indicate that LT-localized CD8+ T cells alone are insufficient to delay or prevent plasma viral rebound during ATI. Significance StatementHIV therapeutic and cure strategies have all operated under the assumption that CD8+ T cells can become cytotoxic in lymph nodes to eliminate infected cells from reservoir repositories. Using FTY720 to prevent lymphocyte egress from lymph nodes, we demonstrated that lymphoid tissue-restricted CD8+ T cells are insufficient to prevent SIV rebound during antiretroviral treatment interruption, even in the presence of immunomodulators such as IL-15 receptor superagonist and PD-1 blockade. These findings call into question the ability of lymph node CD8+ T cells to control or eliminate SIV/HIV infection from lymphoid tissues without substantial immunomodulation, a notion contrary to current efforts to engage CD8+ T cells for therapeutic elimination of HIV viral reservoirs during ART.

immunology↗

bioRxiv2023

Assessing the effect of antibody responses on viral rebound dynamics in postnatally SHIV-infected infant Rhesus macaques

AO_SCPLOWBSTRACTC_SCPLOWWhile the benefits of early antiretroviral therapy (ART) initiation in perinatally infected infants are well documented, early ART initiation is not always possible in postnatal pediatric HIV infections, which account for the majority of pediatric HIV cases worldwide. The timing of onset of ART initiation is likely to affect the size of the latent viral reservoir established, as well as the development of adaptive immune responses, such as the generation of neutralizing antibody responses against the virus. How these parameters impact the ability of infants to control viremia and the time to viral rebound after ART interruption is unclear. To gain insight into the dynamics, we utilized mathematical models to investigate the effect of time of ART initiation via latent reservoir size and autologous virus neutralizing antibody responses in delaying viral rebound when treatment is interrupted. We used an infant nonhuman primate Simian/Human Immunodeficiency Virus (SHIV) infection model that mimics breast milk HIV transmission in human infants. Infant Rhesus macaques (RMs) were orally challenged with SHIV.C.CH505 375H dCT and either given ART at 4-7 days post-infection (early ART condition), at 2 weeks post-infection (intermediate ART condition), or at 8 weeks post-infection (late ART condition). These infants were then monitored for up to 60 months post-infection with serial viral load and immune measurements. We develop a stochastic mathematical model to investigate the joint effect of latent reservoir size, the autologous neutralizing antibody potency, and CD4+ T cell levels on the time to viral rebound and control of post-rebound viral loads. We find that the latent reservoir size is an important determinant in explaining time to viral rebound by affecting the growth rate of the virus. The presence of neutralizing antibodies also can delay rebound, but we find this effect for high potency antibody responses only.

immunology↗

bioRxiv2022

Adaptation of a transmitted/founder simian-human immunodeficiency virus for enhanced replication in rhesus macaques

Transmitted/founder (TF) simian-human immunodeficiency viruses (SHIVs) express HIV-1 envelopes modified at position 375 to efficiently infect rhesus macaques while preserving authentic HIV-1 Env biology. TF SHIV.C.CH505 is an extensively characterized virus shown to recapitulate key features of HIV-1 immunobiology, including CCR5-tropism, a tier 2 neutralization profile, reproducible early viral kinetics, and authentic immune responses. SHIV.C.CH505 is used frequently in nonhuman primate studies of HIV, but viral loads after months of infection are variable and typically lower than those in people living with HIV. We hypothesized that additional mutations besides {Delta}375 might further enhance virus fitness without compromising essential components of CH505 Env biology. From sequence analysis of SHIV.C.CH505-infected macaques across multiple experiments, we identified a signature of envelope mutations associated with higher viremia. We then used short-term in vivo mutational selection and competition to identify a minimally adapted SHIV.C.CH505 with just five amino acid changes that substantially improve virus replication fitness in macaques. Next, we validated the performance of the adapted SHIV in vitro and in vivo and identified the mechanistic contributions of selected mutations. In vitro, the adapted SHIV shows improved virus entry, enhanced replication on primary rhesus cells, and preserved neutralization profiles. In vivo, the minimally adapted virus rapidly outcompetes the parental SHIV with an estimated growth advantage of 0.14 days-1 and persists through suppressive antiretroviral therapy to rebound at treatment interruption. Here, we report the successful generation of a well-characterized, minimally adapted virus, termed SHIV.C.CH505.v2, with enhanced replication fitness and preserved native Env properties that can serve as a new reagent for NHP studies of HIV-1 transmission, pathogenesis, and cure. Author SummaryThe power of the nonhuman primate model of HIV to predict outcomes in people living with HIV (PLWH) depends on authentic virus-host interactions. In pursuit of viruses that generate infection that mirrors the effects of HIV-1 in PLWH, we developed a minimally adapted version of a commonly used virus, SHIV.C.CH505, which has better fitness than the parental virus while retaining important biological properties. First, we studied virus sequences from SHIV.C.CH505-infected rhesus macaques to identify a signature of mutations common to animals with higher viral loads. We then tested viruses containing the various mutations in the lab and in animals to determine the most fit version and to identify the contribution of each mutation. Ultimately, we identified a minimally adapted version of SHIV.C.CH505 with just 5 amino acid substitutions that enhances virus replication and preserves CH505 envelope properties, including sensitivity to clinically relevant broadly neutralizing antibodies. This new virus, called SHIV.C.CH505.v2 replicates well in macaques over time and persists through antiretroviral therapy. SHIV.C.CH505.v2 could be an important component of nonhuman primate studies of HIV prevention, therapy, and cure.

microbiology↗

bioRxiv2022

Extensive heterogeneity of the HIV-1 infected CD4+ T-cell reservoir revealed by single-cell viral ASAPseq

Understanding the complexity of the long-lived HIV reservoir during antiretroviral therapy (ART) remains a major impediment for HIV cure research. To address this, we developed single-cell viral ASAPseq to precisely define the unperturbed peripheral blood HIV-infected memory CD4+ T cell reservoir from antiretroviral treated people living with HIV (ART-PLWH) via the presence of integrated accessible proviral DNA in concert with epigenetic and cell surface protein profiling. We identified profound reservoir heterogeneity within and between ART-PLWH, characterized by novel and known surface markers within total and individual memory CD4+ T cell subsets. We further uncovered novel epigenetic profiles and transcription factor motifs enriched in HIV-infected cells that suggest infected cells with accessible provirus, irrespective of reservoir distribution, are poised for reactivation during ART treatment. Together, our findings reveal the extensive inter- and intrapersonal cellular heterogeneity of the HIV reservoir, and establish an initial multiomic atlas to develop targeted reservoir elimination strategies.

microbiology↗

bioRxiv2021

New SHIVs and Improved Design Strategy for Modeling HIV-1 Transmission, Immunopathogenesis, Prevention and Cure

Simian-human immunodeficiency virus (SHIV) chimeras contain the HIV-1 envelope (env) gene embedded within an SIVmac proviral backbone. Previously, we showed that substitution of Env residue 375-Ser by bulky aromatic residues enhances Env binding to rhesus CD4 and enables primary or transmitted/founder (T/F) HIV-1 Envs to support efficient SHIV replication in rhesus macaques (RMs). Here, we test this design strategy more broadly by constructing and analyzing SHIVs containing ten strategically selected primary or T/F HIV-1 Envs corresponding to subtypes A, B, C, AE and AG, each with six allelic variants at position 375. All ten SHIVs bearing wildtype Env375 residues replicated efficiently in human CD4+ T cells, but only one of these replicated efficiently in rhesus CD4+ T cells. This was a SHIV whose subtype AE Env naturally contained a bulky aromatic His residue at position 375. Replacement of wildtype Env375 residues by Trp, Tyr, Phe or His in the other nine SHIVs uniformly led to efficient replication in rhesus CD4+ T in vitro and in RMs in vivo. Env375-Trp - the residue found most frequently among SIV strains infecting Old World monkeys - was favored for SHIV replication in RMs, although some SHIVs preferred Env375-Tyr, -His or -Phe. Nine SHIVs containing optimized Env375 alleles were grown large scale in primary activated rhesus CD4+ T cells to serve as challenge stocks in preclinical prevention trials. These virus stocks were genetically homogeneous, native-like in Env antigenicity and tier-2 neutralization sensitivity, transmissible by rectal, vaginal, penile, oral or intravenous inoculation routes, and exhibited acute and early replication kinetics that were indistinguishable from HIV-1 infection in humans. Finally, to expedite future SHIV constructions and eliminate short redundant elements in tat1 and env gp41 that were spontaneously deleted in chronically infected monkeys, we engineered a simplified second-generation SHIV design scheme and validated it in RMs. Overall, our findings demonstrate that SHIVs bearing primary or T/F Envs with bulky aromatic amino acid substitutions at position Env375 consistently replicate in RMs, recapitulating many features of HIV-1 infection in humans. We further show that SHIV challenge stocks grown in primary rhesus CD4+ T cells are efficiently transmitted by mucosal routes common to HIV-1 infection and can be used effectively to test for vaccine efficacy in preclinical monkey trials.

microbiology↗

bioRxiv2020

Recapitulation of HIV-1 Env-Antibody Coevolution in Macaques Leading to Neutralization Breadth

Neutralizing antibodies elicited by HIV-1 coevolve with viral Envs in distinctive patterns, in some cases acquiring substantial breadth. Here we show that primary HIV-1 Envs, when expressed by simian-human immunodeficiency viruses in rhesus macaques, elicited patterns of Env-antibody coevolution strikingly similar to those in humans. This included conserved immunogenetic, structural and chemical solutions to epitope recognition and precise Env-amino acid substitutions, insertions and deletions leading to virus persistence. The structure of one rhesus antibody, capable of neutralizing 49% of a 208-strain panel, revealed a V2-apex mode of recognition like that of human bNAbs PGT145/PCT64-35M. Another rhesus antibody bound the CD4-binding site by CD4 mimicry mirroring human bNAbs 8ANC131/CH235/VRC01. Virus-antibody coevolution in macaques can thus recapitulate developmental features of human bNAbs, thereby guiding HIV-1 immunogen design. One sentence summaryVirus-antibody coevolution in rhesus macaques recapitulates developmental features of human antibodies.

immunology↗

bioRxiv2019

Analytical treatment interruption after short-term anti-retroviral therapy in a postnatally SHIV infected infant rhesus macaque model

To achieve long-term viral remission in HIV-infected children, novel strategies beyond early anti-retroviral therapy (ART) will be necessary. Identifying clinical predictors of time to viral rebound upon ART interruption will streamline the development of novel therapeutic strategies and accelerate their evaluation in clinical trials. However, identification of these biomarkers is logistically challenging in infants, due to sampling limitations and potential risks of treatment interruption. To facilitate identification of biomarkers predicting viral rebound, we have developed an infant rhesus macaque (RM) model of oral SHIV.CH505.375H.dCT challenge and analytical treatment interruption (ATI) after short-term ART. We used this model to characterize SHIV replication kinetics and virus-specific immune responses during short-term ART or post-ATI and demonstrated plasma viral rebound in 5 out of 6 (83%) infants. We observed a decline in humoral immune responses and partial dampening of systemic immune activation upon initiation of ART in these infants. Furthermore, we documented that infant and adult macaques have similar SHIV replication and rebound kinetics and equally potent virus-specific humoral immune responses. Finally, we validated our models by confirming a well-established correlate of time to viral rebound, namely pre-ART plasma viral load, as well as identified additional potential humoral immune correlates. Thus, this model of infant ART and viral rebound can be used and further optimized to define biomarkers of viral rebound following long-term ART as well as to pre-clinically assess novel therapies to achieve a pediatric HIV functional cure.\n\nIMPORTANCENovel interventions that do not rely on daily adherence to ART are needed to achieve sustained viral remission for perinatally infected children who currently rely on lifelong ART. Considering the risks and expense associated with ART-interruption trials, identification of biomarkers of viral rebound will prioritize promising therapeutic intervention strategies, including anti-HIV Env protein therapeutics. However, comprehensive studies to identify those biomarkers are logistically challenging in human infants, demanding the need for relevant non-human primate models of HIV rebound. In this study, we developed an infant RM model of oral Simian/Human Immunodeficiency virus infection expressing clade C HIV Env, and short-term ART followed by ATI, longitudinally characterizing immune responses to viral infection during ART and post-ATI. Additionally, we compared this infant RM model to an analogous adult RM rebound model and identified virologic and immunologic correlates of time to viral rebound post-ATI.

immunology↗

bioRxiv2019

SHIV.CH505-infected infant and adult rhesus macaques exhibit similar HIV Env-specific antibody kinetics, despite distinct T-follicular helper (Tfh) and germinal center B cell landscapes

Pediatric HIV infection remains a large global health concern despite the widespread use of antiretroviral therapy (ART). Thus, global elimination of pediatric HIV infections will require the development of novel immune-based approaches, and understanding infant immunity to HIV is critical to guide the rational design of these intervention strategies. Despite their immunological immaturity, HIV-infected children develop broadly neutralizing antibodies (bnAbs) more frequently and earlier than adults do. Furthermore, T-follicular helper (Tfh) cells have been associated with bnAb development in HIV-infected children and adults. To further our understanding of age-related differences in the development of HIV-specific immunity, we evaluated the generation of virus-specific humoral immune responses in infant (n=6) and adult (n=12) rhesus macaques (RMs) infected with a transmitted/founder (T/F) simian-human immunodeficiency virus (SHIV.C.CH505). The plasma HIV envelope-specific IgG antibody kinetics were similar in SHIV-infected infant and adult RMs, with no significant differences in the magnitude or breadth of these responses. Interestingly, autologous tier 2 virus neutralization responses also developed with similar frequency and kinetics in infant and adult RMs, despite infants exhibiting significantly higher Tfh and germinal center B cell frequencies compared to adults. Our results indicate that the humoral immune response to SHIV infection develops with similar kinetics among infant and adult RMs, suggesting that the early life immune system is equipped to respond to HIV-1 and promote the production of neutralizing HIV antibodies. ImportanceThere is a lack of understanding on how the maturation of the infant immune system influences immunity to HIV infection, or how these responses differ from those of adults. Improving our knowledge of infant HIV immunity will help guide antiviral intervention strategies that take advantage of the unique infant immune environment to successfully elicit protective immune responses. We utilized a rhesus macaque model of SHIV infection as a tool to distinguish the differences in HIV humoral immunity in infants versus adults. Here, we demonstrate that the kinetics and quality of the infant humoral immune response to HIV are highly comparable to that of adults during the early phase of infection, despite distinct differences in their Tfh responses, indicating that slightly different mechanisms may drive infant and adult humoral immunity.

immunology↗