Single Cell Analysis of the HIV-1 Latent Reservoir

Human immunodeficiency virus type 1 (HIV-1), the virus that causes acquired immune deficiency syndrome (AIDS), is one of the world’s most serious health and development challenges. Worldwide there are approximately 36.7 million people living with HIV, and tens of millions have died of AIDS-related causes since the beginning of the epidemic. Treatment of HIV-1 infection with combinations of antiretroviral drugs has significantly reduced the death rate and improved the quality of life of HIV-1 infected individuals. Despite over thirty years of HIV-1 research, however, both a cure and a vaccine remain elusive. Complete eradication of HIV-1 by antiretroviral drugs is prevented by the persistence of rare, long-lived, latently infected cells. These cells, called the latent reservoir, are thought to resist immune clearance and viral cytopathic effects by harboring a transcriptionally quiescent integrated HIV-1 provirus. As a result, interruption of suppressive therapy almost inevitably results in rapid viral rebound, which originates from these latently infected cells and prevents HIV-1 cure. It is thought that establishing the reservoir requires intact retroviral integration into the host cell genome and subsequent transcriptional silencing of the integrated provirus. These are rare events and these cells have no known distinguishing surface markers, which has made it difficult to define the precise cellular and molecular nature of the reservoir. The long half-life of the latent reservoir has been attributed to a stable pool of long-lived latently infected CD4+ T cells. An alternative explanation, consistent with the frequent occurrence of monotypic viral sequences, is that infected latent cells are maintained in part by cell proliferation. T cell division and productive HIV-1 transcription are mediated by shared metabolic and transcriptional pathways, and productive HIV-1 infection typically leads to CD4+ T cell death. Thus, how infected cells survive while dividing is unknown. I focused my thesis on characterizing this latent reservoir in virally suppressed, HIV-1 infected individuals and examining the mechanisms of HIV-1 latency. In the first part of this thesis, using a novel single-cell, high throughput integration site sequencing method, I demonstrate that HIV-1 infected cells are capable of cell division, but that the great majority of the largest expanded clones contain defective proviruses which cannot contribute to the replication competent rebound virus. In the second part of this thesis, using an assay to qualitatively and quantitatively characterize the latent reservoir, I suggest that the replication competent latent reservoir may, in fact, be maintained in part by rare cell division events. And finally, I developed a novel isolation strategy which allowed single cell characterization of recently reactivated latent cells. I was able to obtain reactivated latent T cells that produced intact, replication competent HIV-1. By sequencing the T cell receptors, I prove that these isolated latent cells are expanded T cell clones. Single cell gene expression analysis revealed that latent cells share a specific gene profile that prominently includes genes implicated in silencing the virus, T cell exhaustion markers, and genes that may aid in identification of specific CD4+ T cell subsets prone to latent infection. Together, the data supports a model for latency whereby infected T cells turn on a gene expression program that suppresses viral replication during cell division thereby preventing activation of the cell death pathways that are normally triggered by HIV-1 infection

Relationship between intact HIV-1 proviruses in circulating CD4+ T cells and rebound viruses emerging during treatment interruption.

Combination antiretroviral therapy controls but does not cure HIV-1 infection because a small fraction of cells harbor latent viruses that can produce rebound viremia when therapy is interrupted. The circulating latent virus reservoir has been documented by a variety of methods, most prominently by viral outgrowth assays (VOAs) in which CD4+ T cells are activated to produce virus in vitro, or more recently by amplifying proviral near full-length (NFL) sequences from DNA. Analysis of samples obtained in clinical studies in which individuals underwent analytical treatment interruption (ATI), showed little if any overlap between circulating latent viruses obtained from outgrowth cultures and rebound viruses from plasma. To determine whether intact proviruses amplified from DNA are more closely related to rebound viruses than those obtained from VOAs, we assayed 12 individuals who underwent ATI after infusion of a combination of two monoclonal anti-HIV-1 antibodies. A total of 435 intact proviruses obtained by NFL sequencing were compared with 650 latent viruses from VOAs and 246 plasma rebound viruses. Although, intact NFL and outgrowth culture sequences showed similar levels of stability and diversity with 39% overlap, the size of the reservoir estimated from NFL sequencing was larger than and did not correlate with VOAs. Finally, intact proviruses documented by NFL sequencing showed no sequence overlap with rebound viruses; however, they appear to contribute to recombinant viruses found in plasma during rebound

Paired quantitative and qualitative assessment of the replication-competent HIV-1 reservoir and comparison with integrated proviral DNA

HIV-1-infected individuals harbor a latent reservoir of infected CD4⁺ T cells that is not eradicated by antiretroviral therapy (ART). This reservoir presents the greatest barrier to an HIV-1 cure and has remained difficult to characterize, in part, because the vast majority of integrated sequences are defective and incapable of reactivation. To characterize the replication-competent reservoir, we have combined two techniques, quantitative viral outgrowth and qualitative sequence analysis of clonal outgrowth viruses. Leukapheresis samples from four fully ART-suppressed, chronically infected individuals were assayed at two time points separated by a 4- to 6-mo interval. Overall, 54% of the viruses emerging from the latent reservoir showed gp160 env sequences that were identical to at least one other virus. Moreover, 43% of the env sequences from viruses emerging from the reservoir were part of identical groups at the two time points. Groups of identical expanded sequences made up 54% of proviral DNA, and, as might be expected, the sequences of replication-competent viruses in the active reservoir showed limited overlap with integrated proviral DNA, most of which is known to represent defective viruses. Finally, there was an inverse correlation between proviral DNA clone size and the probability of reactivation, suggesting that replication-competent viruses are less likely to be found among highly expanded provirus-containing cell clones

Influenza A Virus Infection of Human Primary Dendritic Cells Impairs Their Ability to Cross-Present Antigen to CD8 T Cells

Influenza A virus (IAV) infection is normally controlled by adaptive immune responses initiated by dendritic cells (DCs). We investigated the consequences of IAV infection of human primary DCs on their ability to function as antigen-presenting cells. IAV was internalized by both myeloid DCs (mDCs) and plasmacytoid DCs but only mDCs supported viral replication. Although infected mDCs efficiently presented endogenous IAV antigens on MHC class II, this was not the case for presentation on MHC class I. Indeed, cross-presentation by uninfected cells of minute amounts of endocytosed, exogenous IAV was ∼300-fold more efficient than presentation of IAV antigens synthesized by infected cells and resulted in a statistically significant increase in expansion of IAV-specific CD8 T cells. Furthermore, IAV infection also impaired cross-presentation of other exogenous antigens, indicating that IAV infection broadly attenuates presentation on MHC class I molecules. Our results suggest that cross-presentation by uninfected mDCs is a preferred mechanism of antigen-presentation for the activation and expansion of CD8 T cells during IAV infection

Ending the evidence gap for pregnancy, HIV and co-infections: ethics guidance from the PHASES project.

INTRODUCTION: While pregnant people have been an important focus for HIV research, critical evidence gaps remain regarding prevention, co-infection, and safety and efficacy of new antiretroviral therapies in pregnancy. Such gaps can result in harm: without safety data, drugs used may carry unacceptable risks to the foetus or pregnant person; without pregnancy-specific dosing data, pregnant people face risks of both toxicity and undertreatment; and delays in gathering evidence can limit access to beneficial next-generation drugs. Despite recognition of the need, numerous barriers and ethical complexities have limited progress. We describe the process, ethical foundations, recommendations and applications of guidance for advancing responsible inclusion of pregnant people in HIV/co-infections research. DISCUSSION: The 26-member international and interdisciplinary Pregnancy and HIV/AIDS: Seeking Equitable Study (PHASES) Working Group was convened to develop ethics-centred guidance for advancing timely, responsible HIV/co-infections research with pregnant people. Deliberations over 3 years drew on extensive qualitative research, stakeholder engagement, expert consultation and a series of workshops. The guidance, initially issued in July 2020, highlights conceptual shifts needed in framing research with pregnant people, and articulates three ethical foundations to ground recommendations: equitable protection from drug-related risks, timely access to biomedical advances and equitable respect for pregnant people's health interests. The guidance advances 12 specific recommendations, actionable within the current regulatory environment, addressing multiple stakeholders across drug development and post-approval research, and organized around four themes: building capacity, supporting inclusion, achieving priority research and ensuring respect. The recommendations describe strategies towards ethically redressing the evidence gap for pregnant people around HIV and co-infections. The guidance has informed key efforts of leading organizations working to advance needed research, and identifies further opportunities for impact by a range of stakeholder groups. CONCLUSIONS: There are clear pathways towards ethical inclusion of pregnant people in the biomedical research agenda, and strong agreement across the HIV research community about the need for - and the promise of - advancing them. Those who fund, conduct, oversee and advocate for research can use the PHASES guidance to facilitate more, better and earlier evidence to optimize the health and wellbeing of pregnant people and their children

In Support of a Patient-Driven Initiative and Petition to Lower the High Price of Cancer Drugs

Comment in Lowering the High Cost of Cancer Drugs--III. [Mayo Clin Proc. 2016] Lowering the High Cost of Cancer Drugs--I. [Mayo Clin Proc. 2016] Lowering the High Cost of Cancer Drugs--IV. [Mayo Clin Proc. 2016] In Reply--Lowering the High Cost of Cancer Drugs. [Mayo Clin Proc. 2016] US oncologists call for government regulation to curb drug price rises. [BMJ. 2015

The immune response fails to control HIV early in initial virus spread

Discontinued antiretroviral therapy (ART) results in uncontrolled HIV replication in most cases. How the virus population that persists during ART escapes immune control remains unknown. In this issue of the JCI, Mitchell and authors investigated plasmacytoid dendritic cells (pDCs) from the blood of individuals living with HIV. After ART was discontinued and as the virus began to spread, an apparently functional pDC response emerged. Notably, these pDCs were initially capable of producing high levels of type I IFN, but rapidly lost this capacity, even before the virus became readily detectable in blood. This study suggests that dysfunctional pDCs are a key initial mechanism associated with poor HIV control. These innate immune responses might be targeted in the emerging efforts to cure HIV disease

The Biology of the HIV-1 Latent Reservoir and Implications for Cure Strategies

Antiretroviral therapy (ART) inhibits HIV replication but is not curative. During ART, the integrated HIV genome persists indefinitely within CD4+ T cells and perhaps other cells. Here, we describe the mechanisms thought to contribute to its persistence during treatment and highlight findings from numerous recent studies describing the importance of cell proliferation in that process. Continued progress elucidating the biology will enhance our ability to develop effective curative interventions