Probes for HIV

HIV-associated neurological disorders (HAND) affect up to 50% of people living with HIV (PLWH), even in the era of combination antiretroviral therapy (cART). HIV-DNA can be detected in the cerebral spinal fluid (CSF) of approximately half of aviremic ART-suppressed PLWH and its presence is associated with poorer neurocognitive performance. HIV DNA + and HIV RNA + cells have also been observed in postmortem brain tissue of individuals with sustained cART suppression. In this review, we provide an overview of how HIV invades the brain and HIV infection of resident brain glial cells (astrocytes and microglia). We also discuss the role of resident glial cells in persistent neuroinflammation and HAND in PLWH and their potential contribution to the HIV reservoir. HIV eradication strategies that target persistently infected glia cells will likely be needed to achieve HIV cure.

This review describes how advances in CyTOF and high-dimensional analysis methods have furthered our understanding of HIV transmission, pathogenesis, persistence, and immunity.CyTOF has generated important insight on several aspects of HIV biology: (1) the differences between cells permissive to productive vs. latent HIV infection, and the HIV-induced remodeling of infected cells; (2) factors that contribute to the persistence of the long-term HIV reservoir, in both blood and tissues; and (3) the impact of HIV on the immune system, in the context of both uncontrolled and controlled infection. CyTOF and high-dimensional analysis tools have enabled in-depth assessment of specific host antigens remodeled by HIV, and have revealed insights into the features of HIV-infected cells enabling them to survive and persist, and of the immune cells that can respond to and potentially control HIV replication. CyTOF and other related high-dimensional phenotyping approaches remain powerful tools for translational research, and applied HIV to cohort studies can inform on mechanisms of HIV pathogenesis and persistence, and potentially identify biomarkers for viral eradication or control.

Background: HIV-specific chimeric antigen receptor T (CAR T) cells are being developed as a potential approach towards curing HIV infection. During infection, HIV replication is concentrated in B cell follicles, and viral reservoirs such as B cell follicles are a significant barrier to an HIV cure. We developed HIV-specific CAR T cells expressing the follicular homing receptor CXCR5 (CAR/CXCR5 T cells) to target follicular HIV reservoirs. We hypothesized after infusion of CAR/CXCR5 T cells in humanized HIV-infected DRAGA mice, CAR/CXCR5 T cells would accumulate in lymphoid follicles, make direct contact with HIV+ cells, lead to reductions in HIV viral loads, and preserve human CD4 T cells. Methods: Fourteen female humanized DRAGA mice were included in this study. Twelve mice were infected with 10 000 TCID50 of HIV-1 BaL. Levels of HIV-1 plasma viral loads and CD4 T cells were monitored using qRT-PCR and flow cytometry. Two spleens from uninfected mice were used to produce transduced CAR/CXCR5 T cells and transduced cell products (2×105 cells/gram) were infused in six HIV-infected mice. RNAscope combined with immunohistochemistry was used to visualize locations and quantities of CAR/CXCR5 T cells and HIV vRNA+ cells in lymphoid tissues. Results: All mice were HIV-1 detectable nbefore infusion of CAR/CXCR5 T cells. High levels of CAR/CXCR5 T cells and HIV vRNA+ cells were detected at 6 days post-infusion in lymphoid tissues. Many CAR/CXCR5 T cells were found in direct contact with HIV vRNA+ cells. However, many CAR/CXCR5 T cells, presumably CD4+ cells, were HIV vRNA+ and likely spreading infection. No differences in HIV plasma viral loads or CD4 T cell counts were observed between control and treated animals. Conclusions: These studies support the use of the HIV-infected DRAGA mouse model for HIV cure research studies. Using this model, we showed CAR/CXCR5 T cells accumulate in follicle-like structures with HIV vRNA+ cells and come in contact with vRNA+ cells. The simultaneous detection of CAR T cells with high levels of HIV vRNA+ cells indicates the need for HIV-resistant CAR T cells. These preliminary findings demonstrate the HIV-infected DRAGA mouse model is extremely valuable for evaluating HIV cure approaches.

Since the first HIV-cured person was reported in 2009, a strong interest in developing highly sensitive HIV and SIV reservoir assays has emerged. In particular, the question arose about the comparative value of state-of-the-art assays to measure and characterize the HIV reservoir, and how these assays can be applied to accurately detect changes in the reservoir during efforts to develop a cure for HIV infection. Secondly, it is important to consider the impact on the outcome of clinical trials if these relatively new HIV reservoir assays are incorporated into clinical trial endpoints and/or used for clinical decision-making. To understand the advantages and limitations and the regulatory implications of HIV reservoir assays, the National Institute of Allergy and Infectious Diseases (NIAID) sponsored and convened a meeting on September 16, 2022, to discuss the state of knowledge concerning these questions and best practices for selecting HIV reservoir assays for a particular research question or clinical trial protocol.

Background: HIV infection persists predominantly within follicular helper CD4+ T cell-rich B cell follicles of lymphoid tissues. Cytotoxic CD8+ T cells, which are associated with natural control of HIV infection in peripheral blood, are relatively excluded from this niche, representing a potential barrier to cellular immunity and HIV cure. To better understand the mechanisms of HIV control within lymph nodes (LN), we investigated functionality, clonotypic compartmentalization, spatial localization, phenotypic characteristics and transcriptional profiles of LN-resident virus-specific and CXCR5-expressing follicular CD8+ T cells (fCD8) in persons who control HIV without medications. Methods: We obtained paired excisional inguinal LN biopsies and peripheral blood (PB) from 19 spontaneous HIV controllers and 17 HIV+ individuals on long-term ART. HIV-specific CD8+ T cell responses were identified by IFN-γ ELISpot and functional response to antigenic stimulation was measured by flow cytometry and CFSE-based proliferation assay. Clonotypic compartmentalization and transcriptional signatures associated with localization of HIV-specific CD8+ T cells were assessed via TCR and RNA-sequencing. Spatial relationships between ongoing viral replication and fCD8 cytotoxic effector potential in GCs were measured by HIV gagpol RNAscope and immunofluorescence on fixed LN sections. Results: Antigen-induced HIV-specific CD8+ T cell proliferation and cytolytic effector upregulation consistently distinguished spontaneous controllers from noncontrollers in PB (p=0.03) and LN (p=0.04). HIV-specific CD8+ T cells from both compartments shared TCR clonotypic composition (Morisita-Horn Similarity Index 0.8-1.0), consistent with ongoing infiltration from circulation. Migration into LNs was associated with gene signatures of inflammatory chemotaxis and antigen-induced effector function. The cytolytic effectors perforin and granzyme B were elevated among virus-specific CXCR5 + fCD8 s (p

Combination antiretroviral therapy (ART) can suppress plasma HIV-RNA to < 50 copies/mL, decrease HIV transmission, reduce mortality, and improve quality of life for people living with HIV. ART cannot, however, eliminate HIV from an infected individual. The primary barrier to cure HIV infection is the multiple reservoir sites, including adipose tissue, bone marrow, central nervous system, liver, lungs, male and female reproductive system, secondary lymph nodes, and gut-associated lymphoid tissue, established 1 to 2 weeks after acquisition of HIV. Additional challenges include understanding the mechanism(s) by which HIV is maintained at low or undetectable levels and developing treatments that will eradicate or produce a sustained suppression of virus without ART. To date, the most extensive clinical investigations of cure strategies have been the shock-and-kill approach using histone deacetylase inhibitors (HDACis) to induce reactivation of latent HIV. Despite evidence for HIV latency reversal, HDACis alone have not decreased the size of the latent reservoir. Clinical pharmacologic explanations for these results include a low inhibitory quotient (i.e., low potency) within the reservoir sites and intrinsic (e.g., sex differences and reservoir size) and extrinsic (physiochemical and pharmacokinetic drug characteristics) factors. We offer an outline of desired clinical pharmacologic attributes for therapeutics intended for clinical HIV cure research and call for research teams to have early and ongoing involvement of clinical pharmacologists. We believe such a collective effort will provide a solid scientific basis and hope for reaching the goal of a cure for HIV infection.

Background: Early after primary infection, HIV reservoirs are established within multiple tissues, including the brain. As these viral reservoirs are not targeted by antiretroviral therapy (cART), we require robust methods of detection, quantification, and characterization of these viral reservoirs in human tissues. Our recent work developed a multi-component imaging methodology that characterizes and quantifies viral reservoirs within the brain. Methods: The imaging methodology demonstrated utilizes the simultaneous staining of brain tissue from HIV-infected donors using DNAscope, RNAscope, and antibodies for HIV-DNA, HIV-mRNA, and either viral or host proteins, respectively. The panel of patients included in these analyses varied in cART regimen, viral load, years living with HIV, and neurocognitive status, all contrasted to age-matched tissues from uninfected patients. Results: Our group demonstrated that cART is sufficient to reduce the size of the viral reservoirs within the brains of HIV patients. We also found that about half of the cells positive for HIV-DNA expressed HIV-mRNA, and only about one-third expressed viral proteins. HIV proteins varied in expression and bystander uptake by uninfected cells but could provide insight into bystander toxicity. Conclusions: The results found were present irrespective of cART regimen and systemic viral replication but suggested that these viral reservoirs are a major barrier to curing HIV and treating associated neurocognitive disorders.

The ability of antiretroviral drugs to penetrate and suppress viral replication in tissue reservoir sites is critical for HIV remission. We evaluated antiretroviral concentrations in lymph nodes and their impact on HIV transcription.Participants of the RV254/SEARCH010 Acute HIV Infection Cohort in Thailand were enrolled. Group 1 (n  = 6) initiated and continued antiretrovirals with two nucleoside reverse transcriptase inhibitors (NRTIs), dolutegravir (DTG) and mar- aviroc (MVC). Group 2 (n = 12) initiated antiretrovirals with two NRTIs as well as efavirenz and were switched to two NRTIs as well as DTG. Antiretroviral concentrations were measured by mass spectroscopy. HIV RNA+ and DNA+ cells were measured by in-situ hybridization.All participants were MSM. At lymph node biopsy, all had plasma HIV RNA less than 20 copies/ml. Group 2 had longer durations of antiretroviral and DTG use (medians of 135 and 63 weeks, respectively) compared with Group 1 (median 44 weeks for both). TFV-DP, 3TC-TP, DTG and MVC were quantifiable in all lymph node samples from participants receiving those drugs versus carbovir-triphosphate (CBV-TP) in four out of 14. Median ratios of lymph node to peripheral blood concentrations were DTG, 0.014; MVC, 6.9; CBV-TP, 0.38; 3TC-TP, 0.32; and TFV-DP, 3.78. Median inhibitory quotients [ratios of lymph node concentrations to in-vitro inhibitory levels (IC50-or-90)] were DTG, 0.8; MVC, 38.8; CBV-TP, 0.5; 3TC- TP, 4.1; and TFV-DP, 1.8. Ongoing viral transcription was detected in lymph node of all participants. Median lymph node RNA+ cells were 71 350 versus 99 750 cells/g for Groups 1 and 2, respectively (P = 0.111).MVC has enhanced lymph node penetration and thereby may contribute to more complete viral suppression in the lymph node.

To discuss the role of CD4+ T cells with active Human immunodeficiency virus (HIV), meaning infected cells with transcriptional and/or translational viral activity during antiretroviral therapy (ART), focusing on new technologies for its detection, potential cell markers for its characterization, and evidences on the contribution of the active HIV reservoir to long-term viral persistence.HIV-infected cells expressing viral ribonucleic acid are systematically detected in subjects on long-term ART. In recent years, powerful new tools have provided significant insights into the nature, quantification, and identification of cells with active HIV, including the identification of new cell markers, and the presence of viral activity in specific cell populations located in different cellular and anatomical compartments. Moreover, studies on viral sequence integrity have identified cell clones with intact viral genomes and active viral transcription that could potentially persist for years. Together, new investigations support the notion that the active reservoir could represent a relevant fraction of long-term infected cells, and therefore, the study of its cell sources and mechanisms of maintenance could represent a significant advance in our understanding of viral persistence and the development of new curative strategies.The presence of HIV-infected cells with viral expression during ART has been traditionally overlooked for years. Based on recent investigations, this active viral reservoir could play an important role in HIV persistence.

Emerging literature suggests that experiences of discrimination negatively influence health and well-being. It is unfortunately common for people living with HIV (PLWH) to be stigmatized and discriminated against because of their HIV status and other marginalized identities (e.g., ethnicity/race, sexual identity and orientation). To date, little research has specifically examined discrimination in PWLH and its associations with pain and other pain-relevant factors such as mood and sleep. The purpose of this ongoing study was to preliminarily analyze associations among daily experiences of discrimination, pain severity and interference, depressive symptoms, and sleep in PLWH. Participants included 24 PLWH recruited from a local HIV treatment center. Participants completed The Everyday Discrimination Scale (TEDS) followed by the Brief Pain Inventory - Short Form (BPI-SF), the Insomnia Severity Index (ISI), and the Center for Epidemiologic Studies - Depression Scale (CES-D). Initial findings tentatively suggest that more frequent daily experiences of discrimination may be significantly associated with greater pain interference on the BPI-SF (p = .030) and greater severity of insomnia symptoms on the ISI (p = .059). However, it appears that daily experiences of discrimination may not be meaningfully associated with pain severity on the BPI-SF (p = .401) or depressive symptoms on the CES-D (p = .235). Our findings highlight the potentially deleterious effects of daily discrimination experiences on pain and sleep in in PLWH. As this ongoing study recruits a larger sample of PLWH, data will need to be reanalyzed to better determine the durability of these preliminary findings. However, there is potential that findings from this study may assist in elucidating causal pathways linking discrimination to pain and pain relevant health behaviors like sleep in PLWH. Grant support from The Impact of Insomnia on Pain, Physical Function, and Inflammation in HIV (3R01HL147603-03S1).

Currently, there are approximately 38.4 million individuals living with the Human Immunodeficiency Virus (HIV), of which 36.7 million adults, 1.7 million children (

Human Immunodeficiency virus (HIV)-associated neurocognitive disorders are a major burden for people living with HIV whose viremia is stably suppressed with antiretroviral therapy. The pathogenesis of disease is likely multifaceted, with contributions from viral reservoirs including the brain, chronic and systemic inflammation, and traditional risk factors including drug use. Elucidating the effects of each element on disease pathogenesis is near impossible in human clinical or ex vivo studies, facilitating the need for robust and accurate non-human primate models. In this review, we describe the major non-human primate models of neuroHIV infection, their use to study the acute, chronic, and virally suppressed infection of the brain, and novel therapies targeting brain reservoirs and inflammation.