Dynamics of latent HIV under clonal expansion

The HIV latent reservoir exhibits slow decay on antiretroviral therapy (ART), impacted by homeostatic proliferation and activation. How these processes contribute to the total dynamic while also producing the observed profile of sampled latent clone sizes is unclear. An agent-based model was developed that tracks individual latent clones, incorporating homeostatic proliferation of cells and activation of clones. The model was calibrated to produce observed latent reservoir dynamics as well as observed clonal size profiles. Simulations were compared to previously published latent HIV integration data from 5 adults and 3 children. The model simulations reproduced reservoir dynamics as well as generating residual plasma viremia levels (pVL) consistent with observations on ART. Over 382 Latin Hypercube Sample simulations, the median latent reservoir grew by only 0.3 log10 over the 10 years prior to ART initiation, after which time it decreased with a half-life of 15 years, despite number of clones decreasing at a faster rate. Activation produced a maximum size of genetically intact clones of around one million cells. The individual simulation that best reproduced the sampled clone profile, produced a reservoir that decayed with a 13.9 year half-life and where pVL, produced mainly from proliferation, decayed with a half-life of 10.8 years. These slow decay rates were achieved with mean cell life-spans of only 14.2 months, due to expansion of the reservoir through proliferation and activation. Although the reservoir decayed on ART, a number of clones increased in size more than 4,000-fold. While small sampled clones may have expanded through proliferation, the large sizes exclusively arose from activation. Simulations where homeostatic proliferation contributed more to pVL than activation, produced pVL that was less variable over time and exhibited fewer viral blips. While homeostatic proliferation adds to the latent reservoir, activation can both add and remove latent cells. Latent activation can produce large clones, where these may have been seeded much earlier than when first sampled. Elimination of the reservoir is complicated by expanding clones whose dynamic differ considerably to that of the entire reservoir.

The Reproductive Transition: Effects on Viral Replication, Immune Activation, and Metabolism in Women with HIV infection

Purpose of Review To describe research advances in the menopausal transition (MT) and its effects on HIV replication, immune activation, and metabolic parameters in women living with HIV (WLWH). Recent Findings. Physiologic changes due to declines in ovarian reserve characterize the MT. Evidence suggests that estrogen depletion influences HIV replication and the latent reservoir. Changes in markers of immune activation, waist circumference, and neurocognition, independent of chronologic age, occur before the final menstrual period (FMP). HIV effects on gut microbial translocation and adipose tissue, as well as health disparities in WLWH may contribute. Improved biomarker sensitivity to predict FMP provides opportunities to study MT in WLWH. Summary Research is needed to determine the effects of MT and HIV on virologic and clinical outcomes, using accurate assessments to predict the FMP and menopausal stages. These findings could inform the timing of interventions to prevent early onset of adverse outcomes in WLWH.

Exacerbated AIDS progression by PD-1 blockade during therapeutic vaccination in chronically SIV-infected rhesus macaques after ART treatment interruption

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

The Evolution of Regulatory Elements in the Emerging Promoter-Variant Strains of HIV-1 Subtype C

In a multicentric, observational, investigator-blinded, and longitudinal clinical study of 764 ART-naïve subjects, we identified nine different promoter variant strains of HIV-1 subtype C (HIV-1C) emerging in the Indian population, with some of these variants being reported for the first time. Unlike several previous studies, our work here focuses on the evolving viral regulatory elements, not the coding sequences. The emerging viral strains contain additional copies of the existing transcription factor binding sites (TFBS), including TCF-1α/LEF-1, RBEIII, AP-1, and NF-κB, created by sequence duplication. The additional TFBS are genetically diverse and may blur the distinction between the modulatory region of the promoter and the viral enhancer. In a follow-up analysis, we found trends, but no significant associations between any specific variant promoter and prognostic markers, probably because the emerging viral strains might not have established mono infections yet. Illumina sequencing of four clinical samples containing a coinfection indicated the domination of one strain over the other and establishing a stable ratio with the second strain at the follow-up time points. Since a single promoter regulates viral gene expression and constitutes the master regulatory circuit with Tat, the acquisition of additional and variant copies of the TFBS may significantly impact viral latency and latent reservoir characteristics. Further studies are urgently warranted to understand how the diverse TFBS profiles of the viral promoter may modulate the characteristics of the latent reservoir, especially following the initiation of antiretroviral therapy.

The gammaherpesvirus 68 viral cyclin facilitates expression of LANA

Gammaherpesviruses establish life-long infections within their host and have been shown to be the causative agents of devastating malignancies. Chronic infection within the host is mediated through cycles of transcriptionally quiescent stages of latency with periods of reactivation into detectable lytic and productive infection. The mechanisms that regulate reactivation from latency remain poorly understood. Previously, we defined a critical role for the viral cyclin in promoting reactivation from latency. Disruption of the viral cyclin had no impact on the frequency of cells containing viral genome during latency, yet it remains unclear whether the viral cyclin influences latently infected cells in a qualitative manner. To define the impact of the viral cyclin on properties of latent infection, we utilized a viral cyclin deficient variant expressing a LANA-beta-lactamase fusion protein (LANA::βla), to enumerate both the cellular distribution and frequency of LANA gene expression. Disruption of the viral cyclin did not affect the cellular distribution of latently infected cells, but did result in a significant decrease in the frequency of cells that expressed LANA::βla across multiple tissues and in both immunocompetent and immunodeficient hosts. Strikingly, whereas the cyclin-deficient virus had a reactivation defect in bulk culture, sort purified cyclin-deficient LANA::βla expressing cells were fully capable of reactivation. These data emphasize that the γHV68 latent reservoir is comprised of at least two distinct stages of infection characterized by differential LANA expression, and that a primary function of the viral cyclin is to promote LANA expression during latency, a state associated with ex vivo reactivation competence.

Optimal Control of an HIV Model with Gene Therapy and Latency Reversing Agents

In this paper, we study the dynamics of HIV under gene therapy and latency reversing agents. While previous works modeled either the use of gene therapy or latency reversing agents, we consider the effects of a combination treatment strategy. For constant treatment controls, we establish global stability of the disease-free equilibrium and endemic equilibrium based on the value of R0. We then consider time-dependent controls and formulate an associated optimal control problem that emphasizes reduction of the latent reservoir. Characterizations for the optimal control profiles are found using Pontryagin’s Maximum Principle. We perform numerical simulations of the optimal control model using the fourth-order Runge–Kutta forward-backward sweep method. We find that a combination treatment of gene therapy with latency reversing agents provides better remission times than gene therapy alone. We conclude with a discussion of our findings and future work.

In Vivo Dynamics of the Latent Reservoir for HIV-1: New Insights and Implications for Cure

Although antiretroviral therapy (ART) can reduce viremia to below the limit of detection and allow persons living with HIV-1 (PLWH) to lead relatively normal lives, viremia rebounds when treatment is interrupted. Rebound reflects viral persistence in a stable latent reservoir in resting CD4+ T cells. This reservoir is now recognized as the major barrier to cure and is the focus of intense international research efforts. Strategies to cure HIV-1 infection include interventions to eliminate this reservoir, to prevent viral rebound from the reservoir, or to enhance immune responses such that viral replication is effectively controlled. Here we consider recent developments in understanding the composition of the reservoir and how it can be measured in clinical studies. We also discuss exciting new insights into the in vivo dynamics of the reservoir and the reasons for its remarkable stability. Finally we discuss recent discoveries on the complex processes that govern viral rebound. Expected final online publication date for the Annual Review of Pathology: Mechanisms of Disease, Volume 17 is January 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Clonal Expansion of Infected CD4+ T Cells in People Living with HIV

HIV infection is not curable with current antiretroviral therapy (ART) because a small fraction of CD4+ T cells infected prior to ART initiation persists. Understanding the nature of this latent reservoir and how it is created is essential to development of potentially curative strategies. The discovery that a large fraction of the persistently infected cells in individuals on suppressive ART are members of large clones greatly changed our view of the reservoir and how it arises. Rather than being the products of infection of resting cells, as was once thought, HIV persistence is largely or entirely a consequence of infection of cells that are either expanding or are destined to expand, primarily due to antigen-driven activation. Although most of the clones carry defective proviruses, some carry intact infectious proviruses; these clones comprise the majority of the reservoir. A large majority of both the defective and the intact infectious proviruses in clones of infected cells are transcriptionally silent; however, a small fraction expresses a few copies of unspliced HIV RNA. A much smaller fraction is responsible for production of low levels of infectious virus, which can rekindle infection when ART is stopped. Further understanding of the reservoir will be needed to clarify the mechanism(s) by which provirus expression is controlled in the clones of cells that constitute the reservoir.

Modest de novo Reactivation of Single HIV-1 Proviruses in Peripheral CD4+ T Cells by Romidepsin

A cure for human immunodeficiency virus (HIV-1) is restricted by the continued presence of a latent reservoir of memory CD4+ T cells with proviruses integrated into their DNA despite suppressive antiretroviral therapy (ART). A predominant strategy currently pursued in HIV-1 cure-related research is the “kick and kill” approach, where latency reversal agents (LRAs) are used to reactivate transcription from integrated proviruses. The premise of this approach is that “kicking” latent virus out of hiding allows the host immune system to recognize and kill infected cells. Clinical trials investigating the efficacy of LRAs, such as romidepsin, have shown that these interventions do induce transient spikes in viral RNA in HIV-1-infected individuals. However, since these trials failed to significantly reduce viral reservoir size or significantly delay time to viral rebound during analytical treatment interruptions, it is questioned how much each individual latent provirus is actually “kicked” to produce viral transcripts and/or proteins by the LRA. Here, we developed sensitive and specific digital droplet PCR-based assays with single-provirus level resolution. Combining these assays allowed us to interrogate the level of viral RNA transcripts from single proviruses in individuals on suppressive ART with or without concomitant romidepsin treatment. Small numbers of proviruses in peripheral blood memory CD4+ T cells were triggered to become marginally transcriptionally active upon romidepsin treatment. These novel assays can be applied retrospectively and prospectively in HIV-1 cure-related clinical trials to gain crucial insights into LRA efficacy at the single provirus level.

MAT2A-Mediated S-Adenosylmethionine Level in CD4+ T Cells Regulates HIV-1 Latent Infection

Antiretroviral drugs effectively halt HIV-1 replication and disease progression, however, due to the presence of a stable viral latent reservoir, the infection cannot be cured by antiretroviral drugs alone. Elucidating the molecular mechanisms underlying HIV-1 latent infection remains a critical hurdle that precludes the development of novel therapeutic strategies aiming for a potential functional cure. Cellular metabolism has been reported to affect HIV-1 replication in CD4+ T cells, but it remains largely unclear whether it is involved in the regulation of HIV-1 latency. Here, we performed a sub-pooled CRISPR library knockout screen targeting 1773 metabolic-related genes in a cell model of HIV-1 latent infection and found that Methionine Adenosyltransferase 2A (MAT2A) contributes to HIV-1 latency. MAT2A knockout enhanced the reactivation of latent HIV-1 while MAT2A overexpression did the opposite. Mechanistically, MAT2A modulates HIV-1 latency through S-Adenosylmethionine (SAM)-mediated one-carbon flux. MAT2A knockout resulted in a significant downregulation of DNA and histone methylation at the HIV-1 5’-LTR. Importantly, we found that the plasma level of SAM is positively correlated with HIV-1 DNA in PBMCs from ART-treated infected individuals, suggesting SAM could serve as a potential biomarker for the latent viral reservoir. Overall, this study reveals an important role of MAT2A-mediated one-carbon metabolism in regulating HIV-1 latency and provides a promising target for the development of new strategies for a functional cure of HIV-1.