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Abstract To understand the roles of acute-phase viral dynamics and host immune responses in post-acute sequelae of SARS-CoV-2 infection (PASC), we enrolled 136 participants within 5 days of their first positive SARS-CoV-2 real-time PCR test. Participants self-collected up to 21 nasal specimens within the first 28 days post-symptom onset; interviewer-administered questionnaires and blood samples were collected at enrollment, days 9, 14, 21, 28, and month 4 and 8 post-symptom onset. Defining PASC as the presence of any COVID-associated symptom at their 4-month visit, we compared viral markers (quantity and duration of nasal viral RNA load, infectious viral load, and plasma N-antigen level) and host immune markers (IL-6, IL-10, TNF-α, IFN-α, IFN-γ, MCP, IP-10, and Spike IgG) over the acute period. Compared to those who fully recovered, those reporting PASC demonstrated significantly higher maximum levels of SARS-CoV-2 RNA and N-antigen, burden of RNA and infectious viral shedding, and lower Spike-specific IgG levels within 9 days post-illness onset. No significant differences were identified among a panel of host immune markers. Our results suggest early viral dynamics and the associated host immune responses play a role in the pathogenesis of PASC, highlighting the importance of understanding early biological markers in the natural history of PASC.

Abstract Persistence of HIV in people living with HIV (PWH) on suppressive antiretroviral therapy (ART) has been linked to physiological mechanisms of CD4+ T cells. Here, in the same 37 male PWH on ART we measure longitudinal kinetics of HIV DNA and cell turnover rates in five CD4 cell subsets: naïve (TN), stem-cell- (TSCM), central- (TCM), transitional- (TTM), and effector-memory (TEM). HIV decreases in TTM and TEM but not in less-differentiated subsets. Cell turnover is ~10 times faster than HIV clearance in memory subsets, implying that cellular proliferation consistently creates HIV DNA. The optimal mathematical model for these integrated data sets posits HIV DNA also passages between CD4 cell subsets via cellular differentiation. Estimates are heterogeneous, but in an average participant’s year ~10 (in TN and TSCM) and ~104 (in TCM, TTM, TEM) proviruses are generated by proliferation while ~103 proviruses passage via cell differentiation (per million CD4). In simulations, therapies blocking proliferation and/or enhancing differentiation could reduce HIV DNA by 1-2 logs over 3 years. In summary, HIV exploits cellular proliferation and differentiation to persist during ART but clears faster in more proliferative/differentiated CD4 cell subsets and the same physiological mechanisms sustaining HIV might be temporarily modified to reduce it.

The major barrier to an HIV cure is the HIV reservoir: latently-infected cells that persist despite effective antiretroviral therapy (ART). There have been few cohort-based studies evaluating host genomic or transcriptomic predictors of the HIV reservoir. We performed host RNA sequencing and HIV reservoir quantification (total DNA [tDNA], unspliced RNA [usRNA], intact DNA) from peripheral CD4+ T cells from 191 ART-suppressed people with HIV (PWH). After adjusting for nadir CD4+ count, timing of ART initiation, and genetic ancestry, we identified two host genes for which higher expression was significantly associated with smaller total DNA viral reservoir size, P3H3 and NBL1, both known tumor suppressor genes. We then identified 17 host genes for which lower expression was associated with higher residual transcription (HIV usRNA). These included novel associations with membrane channel (KCNJ2, GJB2), inflammasome (IL1A, CSF3, TNFAIP5, TNFAIP6, TNFAIP9, CXCL3, CXCL10), and innate immunity (TLR7) genes (FDR-adjusted q

Background Postacute sequelae of COVID‐19 (PASC) and HIV are both associated with reduced exercise capacity, but whether SARS‐CoV‐2 or PASC are associated with exercise capacity among people with HIV (PWH) is unknown. We hypothesized that PWH with PASC would have reduced exercise capacity from chronotropic incompetence. Methods and Results We conducted cross‐sectional cardiopulmonary exercise testing within a COVID recovery cohort that included PWH with and without prior SARS‐CoV‐2 infection and people without HIV with prior SARS‐CoV‐2 infection (controls). We evaluated associations of HIV, SARS‐CoV‐2, and PASC with exercise capacity (peak oxygen consumption) and chronotropy (adjusted heart rate reserve). We included 83 participants (median age, 54 years; 35% women; 37 PWH): 23 out of 37 (62%) PWH and all 46 controls had prior SARS‐CoV‐2 infection, and 11 out of 23 (48%) PWH and 28 out of 46 (61%) without HIV had PASC. Peak oxygen consumption was reduced among PWH versus controls (80% predicted versus 99%, P=0.005), a difference of 5.5 mL/kg per minute (95% CI, 2.7–8.2; P

HIV-1 persists in a latent reservoir in resting CD4+ T cells despite antiretroviral therapy (ART). The reservoir decays slowly over the first 7 years of ART (t1/2 = 44 months). However, whether decay continues with long-term ART is unclear. Recent integration site studies indicate gradual selection against inducible, intact proviruses, raising speculation that decades of ART might allow treatment interruption without viral rebound. Therefore, we measured the reservoir in 42 people on long-term ART (mean 22 years) using a quantitative viral outgrowth assay. After 7 years of ART, there was no long-term decrease in the frequency of inducible, replication-competent proviruses but rather an increase with an estimated doubling time of 23 years. Another reservoir assay, the intact proviral DNA assay, confirmed that reservoir decay with t1/2 of 44 months did not continue with long-term ART. The lack of decay reflected proliferation of infected cells. Most inducible, replication-competent viruses (79.8%) had env sequences identical to those of other isolates from the same sample. Thus, although integration site analysis indicates changes in reservoir composition, the proliferation of CD4+ T cells counteracts decay, maintaining the frequency of inducible, replication-competent proviruses at roughly constant levels over the long term. These results reinforce the need for lifelong ART.

Some individuals do not return to baseline health following SARS-CoV-2 infection, leading to a condition known as long COVID. The underlying pathophysiology of long COVID remains unknown. Given that autoantibodies have been found to play a role in severity of SARS-CoV-2 infection and certain other post-COVID sequelae, their potential role in long COVID is important to investigate. Here, we apply a well-established, unbiased, proteome-wide autoantibody detection technology (T7 phage-display assay with immunoprecipitation and next-generation sequencing, PhIP-Seq) to a robustly phenotyped cohort of 121 individuals with long COVID, 64 individuals with prior COVID-19 who reported full recovery, and 57 pre-COVID controls. While a distinct autoreactive signature was detected that separated individuals with prior SARS-CoV-2 infection from those never exposed to SARS-CoV-2, we did not detect patterns of autoreactivity that separated individuals with long COVID from individuals fully recovered from COVID-19. These data suggest that there are robust alterations in autoreactive antibody profiles due to infection; however, no association of autoreactive antibodies and long COVID was apparent by this assay.

Here, the authors apply positron emission tomography (PET) imaging to visualize HIV tissue-wide burden in infected individuals using a radiolabeled broadly neutralizing antibody, 89Zr-VRC01, and show that PET tracer lymph node uptake positively correlates with HIV protein levels measured directly from cells obtained from these tissues. This strategy may allow non-invasive characterization of residual HIV infection in the setting of therapeutic interventions.

BACKGROUND The presence and reactivation of chronic viral infections, such as EBV, CMV, and HIV, have been proposed as potential contributors to long COVID (LC), but studies in well-characterized postacute cohorts of individuals with COVID-19 over a longer time course consistent with current case definitions of LC are limited.METHODS In a cohort of 280 adults with prior SARS-CoV-2 infection, we assessed the presence and types of LC symptoms and prior medical history (including COVID-19 history and HIV status) and performed serological testing for EBV and CMV using a commercial laboratory. We used covariate-adjusted binary logistic regression models to identify independent associations between variables and LC symptoms.RESULTS We observed that LC symptoms, such as fatigue and neurocognitive dysfunction, at a median of 4 months following initial diagnosis were independently associated with serological evidence suggesting recent EBV reactivation (early antigen–diffuse IgG positivity) or high nuclear antigen (EBNA) IgG levels but not with ongoing EBV viremia. Serological evidence suggesting recent EBV reactivation (early antigen–diffuse IgG positivity) was most strongly associated with fatigue (OR = 2.12). Underlying HIV infection was also independently associated with neurocognitive LC (OR = 2.5). Interestingly, participants who had serologic evidence of prior CMV infection were less likely to develop neurocognitive LC (OR = 0.52).CONCLUSION Overall, these findings suggest differential effects of chronic viral coinfections on the likelihood of developing LC and association with distinct syndromic patterns. Further assessment during the acute phase of COVID-19 is warranted.TRIAL REGISTRATION Long-term Impact of Infection with Novel Coronavirus; ClinicalTrials.gov NCT04362150.FUNDING This work was supported by NIH/National Institute of Allergy and Infectious Diseases grants (3R01AI141003-03S1, R01AI158013, and K24AI145806); the Zuckerberg San Francisco General Hospital Department of Medicine and Division of HIV, Infectious Diseases, and Global Medicine; and the UCSF-Bay Area Center for AIDS Research (P30-AI027763).

IntroductionHIV-1 persists in resting CD4+ T-cells despite antiretroviral therapy (ART). Determining the cell surface markers that enrich for genetically-intact HIV-1 genomes is vital in developing targeted curative strategies. Previous studies have found that HIV-1 proviral DNA is enriched in CD4+ T-cells expressing the immune checkpoint markers programmed cell death protein-1 (PD-1) or cytotoxic T-lymphocyte associated protein-4 (CTLA-4). There has also been some success in blocking these markers in an effort to reverse HIV-1 latency. However, it remains unclear whether cells expressing PD-1 and/or CTLA-4 are enriched for genetically-intact, and potentially replication-competent, HIV-1 genomes. MethodsWe obtained peripheral blood from 16 HIV-1-infected participants, and paired lymph node from four of these participants, during effective ART. Memory CD4+ T-cells from either site were sorted into four populations: PD-1-CTLA-4- (double negative, DN), PD-1+CTLA-4- (PD-1+), PD-1-CTLA-4+ (CTLA-4+) and PD-1+CTLA-4+ (double positive, DP). We performed an exploratory study using the full-length individual proviral sequencing (FLIPS) assay to identify genetically-intact and defective genomes from each subset, as well as HIV-1 genomes with specific intact open reading frames (ORFs). Results and DiscussionIn peripheral blood, we observed that proviruses found within PD-1+ cells are more likely to have intact ORFs for genes such as tat, rev and nef compared to DN, CTLA-4+ and DP cells, all of which may contribute to HIV-1 persistence. Conversely, we observed that CTLA-4 expression is a marker for cells harbouring HIV-1 provirus that is more likely to be defective, containing low levels of these intact ORFs. In the lymph node, we found evidence that CTLA-4+ cells contain lower levels of HIV-1 provirus compared to the other cell subsets. Importantly, however, we observed significant participant variation in the enrichment of HIV-1 proviruses with intact genomes or specific intact ORFs across these memory CD4+ T-cell subsets, and therefore consideration of additional cellular markers will likely be needed to consistently identify cells harbouring latent, and potentially replication-competent, HIV-1.

In most people living with HIV (PLWH) on effective antiretroviral therapy (ART), cell-associated viral transcripts are readily detectable in CD4+ T cells despite the absence of viremia. Quantification of HIV RNA species provides insights into the transcriptional activity of proviruses that persist in cells and tissues throughout the body during ART (‘HIV reservoir’). One such technique for HIV RNA quantitation, ‘HIV transcription profiling’, developed in the Yukl laboratory, measures a series of HIV RNA species using droplet digital PCR. To take advantage of advances in digital (d)PCR, we adapted the ‘HIV transcription profiling’ technique to Qiagen’s dPCR platform (QIAcuity) and compared its performance to droplet digital (dd)PCR (Bio-Rad QX200 system). Using RNA standards, the two technologies were tested in parallel and assessed for multiple parameters including sensitivity, specificity, linearity, and intra- and inter-assay variability. The newly validated dPCR assays were then applied to samples from PLWH to determine HIV transcriptional activity relative to HIV reservoir size. We report that HIV transcriptional profiling was readily adapted to dPCR and assays performed similarly to ddPCR, with no differences in assay characteristics. We applied these assays in a cohort of 23 PLWH and found that HIV reservoir size, based on genetically intact proviral DNA, does not predict HIV transcriptional activity. In contrast, levels of total DNA correlated with levels of most HIV transcripts (initiated, proximally and distally elongated, unspliced, and completed, but not multiply spliced), suggesting that a considerable proportion of HIV transcripts likely originate from defective proviruses. These findings may have implications for measuring and assessing curative strategies and clinical trial outcomes.

A multiplexed enzyme-linked immunosorbent assay (ELISA) that simultaneously measures antibody binding to multiple antigens can extend the impact of serosurveillance studies, particularly if the assay approaches the simplicity, robustness, and accuracy of a conventional single-antigen ELISA. Here, we report on the development of multiSero, an open-source multiplex ELISA platform for measuring antibody responses to viral infection. Our assay consists of three parts: (1) an ELISA against an array of proteins in a 96-well format; (2) automated imaging of each well of the ELISA array using an open-source plate reader; and (3) automated measurement of optical densities for each protein within the array using an open-source analysis pipeline. We validated the platform by comparing antibody binding to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antigens in 217 human sera samples, showing high sensitivity (0.978), specificity (0.977), positive predictive value (0.978), and negative predictive value (0.977) for classifying seropositivity, a high correlation of multiSero determined antibody titers with commercially available SARS-CoV-2 antibody tests, and antigen-specific changes in antibody titer dynamics upon vaccination. The open-source format and accessibility of our multiSero platform can contribute to the adoption of multiplexed ELISA arrays for serosurveillance studies, for SARS-CoV-2 and other pathogens of significance.

Shortness of breath, chest pain, and palpitations occur as postacute sequelae of COVID-19, but whether symptoms are associated with echocardiographic abnormalities, cardiac biomarkers, or markers of systemic inflammation remains unknown. In a cross-sectional analysis, we assessed symptoms, performed echocardiograms, and measured biomarkers among adults more than 8 weeks after confirmed SARS-CoV-2 infection. We modeled associations between symptoms and baseline characteristics, echocardiographic findings, and biomarkers using logistic regression. We enrolled 102 participants at a median of 7.2 months following COVID-19 onset; 47 individuals reported dyspnea, chest pain, or palpitations. Median age was 52 years, and 41% of participants were women. Female sex, hospitalization, IgG antibody against SARS-CoV-2 receptor binding domain, and C-reactive protein were associated with symptoms. Regarding echocardiographic findings, 4 of 47 participants (9%) with symptoms had pericardial effusions compared with 0 of 55 participants without symptoms; those with effusions had a median of 4 symptoms compared with a median of 1 symptom in those without effusions. There was no strong evidence for a relationship between symptoms and echocardiographic functional parameters or other biomarkers. Among adults more than 8 weeks after SARS-CoV-2 infection, SARS-CoV-2 RBD antibodies, markers of inflammation, and, possibly, pericardial effusions are associated with cardiopulmonary symptoms. Investigation into inflammation as a mechanism underlying postacute sequelae of COVID-19 is warranted.

Despite long-term antiretroviral therapy (ART), HIV-1 persists within a reservoir of CD4+ T cells that contribute to viral rebound if treatment is interrupted. Identifying the cellular populations that contribute to the HIV-1 reservoir and understanding the mechanisms of viral persistence are necessary to achieve an effective cure. In this regard, through Full-Length Individual Proviral Sequencing, we observed that the HIV-1 proviral landscape was different and changed with time on ART across naive and memory CD4+ T cell subsets isolated from 24 participants. We found that the proportion of genetically intact HIV-1 proviruses was higher and persisted over time in effector memory CD4+ T cells when compared with naive, central, and transitional memory CD4+ T cells. Interestingly, we found that escape mutations remained stable over time within effector memory T cells during therapy. Finally, we provided evidence that Nef plays a role in the persistence of genetically intact HIV-1. These findings posit effector memory T cells as a key component of the HIV-1 reservoir and suggest Nef as an attractive therapeutic target.

Analytic treatment interruptions (ATIs) are currently the standard for assessing the impact of experimental interventions aimed at inducing sustained antiretroviral therapy (ART)-free remission in trials related to HIV cure. ATIs are associated with substantial risk to both study participants and their sexual partner(s). Two documented HIV transmissions occurring in the context of ATIs have been recently reported, but recommendations for mitigating the risk of such events during ATIs are limited. We outline a practical approach to risk mitigation during ATI studies and describe strategies we are utilising in an upcoming clinical trial that may be applicable to other centres.

T and natural killer (NK) cells are effector cells with key roles in anti-HIV immunity, including in lymphoid tissues, the major site of HIV persistence. However, little is known about the features of these effector cells from people living with HIV (PLWH), particularly from those who initiated antiretroviral therapy (ART) during acute infection. Our study design was to use 42-parameter CyTOF to conduct deep phenotyping of paired blood- and lymph node (LN)-derived T and NK cells from three groups of HIV+ aviremic individuals: elite controllers (N = 5), and ART-suppressed individuals who had started therapy during chronic (N = 6) vs. acute infection (N = 8), the latter of which is associated with better outcomes. We found that acute-treated individuals are enriched for specific subsets of T and NK cells, including blood-derived CD56-CD16+ NK cells previously associated with HIV control, and LN-derived CD4+ T follicular helper cells with heightened expansion potential. An in-depth comparison of the features of the cells from blood vs. LNs of individuals from our cohort revealed that T cells from blood were more activated than those from LNs. By contrast, LNs were enriched for follicle-homing CXCR5+ CD8+ T cells, which expressed increased levels of inhibitory receptors and markers of survival and proliferation as compared to their CXCR5- counterparts. In addition, a subset of memory-like CD56brightTCF1+ NK cells was enriched in LNs relative to blood. These results together suggest unique T and NK cell features in acute-treated individuals, and highlight the importance of examining effector cells not only in blood but also the lymphoid tissue compartment, where the reservoir mostly persists, and where these cells take on distinct phenotypic features.

The design of future HIV-1 curative therapies requires a more thorough understanding of the distribution of genetically intact HIV-1 within T-cell subsets as well as the cellular mechanisms that maintain this reservoir. These genetically intact and presumably replication-competent proviruses make up the latent HIV-1 reservoir.

Summary: We describe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses, soluble markers of inflammation, and antibody levels and neutralization capacity longitudinally in 70 individuals with PCR-confirmed SARS-CoV-2 infection. Participants represent a spectrum of illness and recovery, including some with persistent viral shedding in saliva and many experiencing post-acute sequelae of SARS-CoV-2 infection (PASC). T cell responses remain stable for up to 9 months. Whereas the magnitude of early CD4+ T cell immune responses correlates with severity of initial infection, pre-existing lung disease is independently associated with higher long-term SARS-CoV-2-specific CD8+ T cell responses. Among participants with PASC 4 months following coronavirus disease 2019 (COVID-19) symptom onset, we observe a lower frequency of CD8+ T cells expressing CD107a, a marker of degranulation, in response to Nucleocapsid (N) peptide pool stimulation, and a more rapid decline in the frequency of N-specific interferon-γ-producing CD8+ T cells. Neutralizing antibody levels strongly correlate with SARS-CoV-2-specific CD4+ T cell responses.

The immune checkpoint molecule PD-1 is expressed on a fraction of CD4+ T cells latently infected with HIV, but whether PD-1 plays a functional role in reservoir persistence remains unknown. Here, Fromentin et al. show that PD-1 blockade potentiates latency reversal ex vivo in CD4+ T cells from ART suppressed individuals.

Although many HIV cure strategies seek to expand HIV-specific CD8+ T cells to control the virus, all are likely to fail if cellular exhaustion is not prevented. A loss in stem-like memory properties (i.e., the ability to proliferate and generate secondary effector cells) is a key feature of exhaustion; little is known, however, about how these properties are regulated in human virus–specific CD8+ T cells. We found that virus-specific CD8+ T cells from humans and nonhuman primates naturally controlling HIV/SIV infection express more of the transcription factor TCF-1 than noncontrollers. HIV-specific CD8+ T cell TCF-1 expression correlated with memory marker expression and expansion capacity and declined with antigenic stimulation. CRISPR-Cas9 editing of TCF-1 in human primary T cells demonstrated a direct role in regulating expansion capacity. Collectively, these data suggest that TCF-1 contributes to the regulation of the stem-like memory property of secondary expansion capacity of HIV-specific CD8+ T cells, and they provide a rationale for exploring the enhancement of this pathway in T cell–based therapeutic strategies for HIV.

The precise role of CD4 T cell turnover in maintaining HIV persistence during antiretroviral therapy (ART) has not yet been well characterized. In resting CD4 T cell subpopulations from 24 HIV-infected ART-suppressed and 6 HIV-uninfected individuals, we directly measured cellular turnover by heavy water labeling, HIV reservoir size by integrated HIV-DNA (intDNA) and cell-associated HIV-RNA (caRNA), and HIV reservoir clonality by proviral integration site sequencing. Compared to HIV-negatives, ART-suppressed individuals had similar fractional replacement rates in all subpopulations, but lower absolute proliferation rates of all subpopulations other than effector memory (TEM) cells, and lower plasma IL-7 levels (p = 0.0004). Median CD4 T cell half-lives decreased with cell differentiation from naïve to TEM cells (3 years to 3 months, p

The latent reservoir is a major barrier to HIV cure. As latently infected cells cannot be phenotyped directly, the features of the in vivo reservoir have remained elusive. Here, we describe a method that leverages high-dimensional phenotyping using CyTOF to trace latently infected cells reactivated ex vivo to their original pre-activation states. Our results suggest that, contrary to common assumptions, the reservoir is not randomly distributed among cell subsets, and is remarkably conserved between individuals. However, reservoir composition differs between tissues and blood, as do cells successfully reactivated by different latency reversing agents. By selecting 8–10 of our 39 original CyTOF markers, we were able to isolate highly purified populations of unstimulated in vivo latent cells. These purified populations were highly enriched for replication-competent and intact provirus, transcribed HIV, and displayed clonal expansion. The ability to isolate unstimulated latent cells from infected individuals enables previously impossible studies on HIV persistence.

The primary reservoir for HIV is within memory CD4+ T cells residing within tissues, yet the features that make some of these cells more susceptible than others to infection by HIV is not well understood. Recent studies demonstrated that CCR5-tropic HIV-1 efficiently enters tissue-derived memory CD4+ T cells expressing CD127, the alpha chain of the IL7 receptor, but rarely completes the replication cycle. We now demonstrate that the inability of HIV to replicate in these CD127-expressing cells is not due to post-entry restriction by SAMHD1. Rather, relative to other memory T cell subsets, these cells are highly prone to undergoing latent infection with HIV, as revealed by the high levels of integrated HIV DNA in these cells. Host gene expression profiling revealed that CD127-expressing memory CD4+ T cells are phenotypically distinct from other tissue memory CD4+ T cells, and are defined by a quiescent state with diminished NFκB, NFAT, and Ox40 signaling. However, latently-infected CD127+ cells harbored unspliced HIV transcripts and stimulation of these cells with anti-CD3/CD28 reversed latency. These findings identify a novel subset of memory CD4+ T cells found in tissue and not in blood that are preferentially targeted for latent infection by HIV, and may serve as an important reservoir to target for HIV eradication efforts.

Latent replication-competent HIV-1 persists in individuals on long-term antiretroviral therapy (ART). We developed the Full-Length Individual Proviral Sequencing (FLIPS) assay to determine the distribution of latent replication-competent HIV-1 within memory CD4+ T cell subsets in six individuals on long-term ART. FLIPS is an efficient, high-throughput assay that amplifies and sequences near full-length (∼9 kb) HIV-1 proviral genomes and determines potential replication competency through genetic characterization. FLIPS provides a genome-scale perspective that addresses the limitations of other methods that also genetically characterize the latent reservoir. Using FLIPS, we identified 5% of proviruses as intact and potentially replication competent. Intact proviruses were unequally distributed between T cell subsets, with effector memory cells containing the largest proportion of genetically intact HIV-1 proviruses. We identified multiple identical intact proviruses, suggesting a role for cellular proliferation in the maintenance of the latent HIV-1 reservoir.

Background: In HIV infection, lymphoid tissue is disrupted by fibrosis. Angiotensin converting enzyme inhibitors have anti-fibrotic properties. We completed a pilot study to assess whether the addition of lisinopril to antiretroviral therapy (ART) reverses fibrosis of gut tissue, and whether this leads to reduction of HIV RNA and DNA levels. Methods: Thirty HIV-infected individuals on ART were randomized to lisinopril at 20mg daily or matching placebo for 24 weeks. All participants underwent rectal biopsies prior to starting the study drug and at 22 weeks, and there were regular blood draws. The primary end point was the change in HIV RNA and DNA levels in rectal tissue. Secondary outcomes included the change in 1) HIV levels in blood; 2) Gag-specific T-cell responses; 3) levels of T-cell activation; and 4) collagen deposition. Results: The addition of lisinopril did not have a significant effect on the levels of HIV RNA or DNA in gut tissue or blood, Gag-specific responses, or levels of T-cell activation. Lisinopril also did not have a significant impact on lymphoid fibrosis in the rectum, as assessed by quantitative histology or heavy water labeling. Conclusions: Treatment with lisinopril for 24 weeks in HIV-infected adults did not have an effect on lymphoid fibrosis, immune activation, or gut tissue viral reservoirs. Further study is needed to see if other anti-fibrotic agents may be useful in reversing lymphoid fibrosis and reducing HIV levels. Clinical Trials Registration: NCT01535235

Studies have demonstrated that intensive ART alone is not capable of eradicating HIV-1, as the virus rebounds within a few weeks upon treatment interruption. Viral rebound may be induced from several cellular subsets; however, the majority of proviral DNA has been found in antigen experienced resting CD4+ T cells. To achieve a cure for HIV-1, eradication strategies depend upon both understanding mechanisms that drive HIV-1 persistence as well as sensitive assays to measure the frequency of infected cells after therapeutic interventions. Assays such as the quantitative viral outgrowth assay (QVOA) measure HIV-1 persistence during ART by ex vivo activation of resting CD4+ T cells to induce latency reversal; however, recent studies have shown that only a fraction of replication-competent viruses are inducible by primary mitogen stimulation. Previous studies have shown a correlation between the acquisition of effector memory phenotype and HIV-1 latency reversal in quiescent CD4+ T cell subsets that harbor the reservoir. Here, we apply our mechanistic understanding that differentiation into effector memory CD4+ T cells more effectively promotes HIV-1 latency reversal to significantly improve proviral measurements in the QVOA, termed differentiation QVOA (dQVOA), which reveals a significantly higher frequency of the inducible HIV-1 replication-competent reservoir in resting CD4+ T cells.

To date, most assays for measuring the human immunodeficiency virus (HIV-1) reservoir do not include memory CD4+ T-cells expressing the activation marker, human leukocyte antigen-antigen D related (HLA-DR). However, little is known concerning the role these cells play in maintaining persistent HIV-1 during effective antiretroviral therapy (ART). To address this issue, we examined, cellular activation/exhaustion markers (Ki67, CCR5, PD-1, Lag-3 and Tim-3) and viral gag-pol DNA sequences within HLA-DR− and HLA-DR+ memory CD4+ T-cell subsets longitudinally from the peripheral blood of six participants over 3 to ≥15 years of effective therapy. HLA-DR expression was readily detected during the study period in all participants. The average expression levels of CCR5, PD-1 and Tim-3 were higher on the HLA-DR+ T-cell subset whereas the average of LAG-3 expression was higher on their HLA-DR− counterpart. The proportion of HIV-infected cells increased within the HLA-DR+ subset by an average of 18% per year of ART whereas the frequency of infected HLA-DR− T-cells slightly decreased over time (5% per year). We observed that 20–33% of HIV-DNA sequences from the early time points were genetically identical to viral sequences from the last time point within the same cell subset during ART. This indicates that a fraction of proviruses persists within HLA-DR+ and HLA-DR− T-cell subsets during prolonged ART. Our HIV-DNA sequence analyses also revealed that cells transitioned between the HLA-DR+ and HLA-DR− phenotypes. The Ki67 expression, a marker for cellular proliferation, and the combined markers of Ki67/PD-1 averaged 19-fold and 22-fold higher on the HLA-DR+ T-cell subset compared to their HLA-DR− counterpart. Moreover, cellular proliferation, as reflected by the proportion of genetically identical HIV-DNA sequences, increased within both T-cell subsets over the study period; however, this increase was greater within the HLA-DR+ T-cells. Our research revealed that cellular transition and proliferation contribute to the persistence of HIV in HLA-DR+ and HLA-DR− T-cell subsets during prolonged therapy. As such, the HIV reservoir expands during effective ART when both the HLA-DR+ and HLA-DR− cell subsets are included, and therapeutic interventions aimed at reducing the HIV-1 reservoir should target HLA-DR+ and HLA-DR− T-cells.

Quantitative viral outgrowth assays (QVOA) use limiting dilutions of CD4+ T cells to measure the size of the latent HIV-1 reservoir, a major obstacle to curing HIV-1. Efforts to reduce the reservoir require assays that can reliably quantify its size in blood and tissues. Although QVOA is regarded as a "gold standard" for reservoir measurement, little is known about its accuracy and precision or about how cell storage conditions or laboratory-specific practices affect results. Owing to this lack of knowledge, confidence intervals around reservoir size estimates-as well as judgments of the ability of therapeutic interventions to alter the size of the replication-competent but transcriptionally inactive latent reservoir-rely on theoretical statistical assumptions about dilution assays. To address this gap, we have carried out a Bayesian statistical analysis of QVOA reliability on 75 split samples of peripheral blood mononuclear cells (PBMC) from 5 antiretroviral therapy (ART)-suppressed participants, measured using four different QVOAs at separate labs, estimating assay precision and the effect of frozen cell storage on estimated reservoir size. We found that typical assay results are expected to differ from the true value by a factor of 1.6 to 1.9 up or down. Systematic assay differences comprised a 24-fold range between the assays with highest and lowest scales, likely reflecting differences in viral outgrowth readout and input cell stimulation protocols. We also found that controlled-rate freezing and storage of samples did not cause substantial differences in QVOA compared to use of fresh cells (95% probability of < 2-fold change), supporting continued use of frozen storage to allow transport and batched analysis of samples. Finally, we simulated an early-phase clinical trial to demonstrate that batched analysis of pre- and post-therapy samples may increase power to detect a three-fold reservoir reduction by 15 to 24 percentage points.

The establishment of HIV-1 latency gives rise to persistent chronic infection that requires life-long treatment. To reverse latency for viral eradiation, the HIV-1 Tat protein and its associated ELL2-containing Super Elongation Complexes (ELL2-SECs) are essential to activate HIV-1 transcription. Despite efforts to identify effective latency-reversing agents (LRA), avenues for exposing latent HIV-1 remain inadequate, prompting the need to identify novel LRA targets. Here, by conducting a CRISPR interference-based screen to reiteratively enrich loss-of-function genotypes that increase HIV-1 transcription in latently infected CD4+ T cells, we have discovered a key role of the proteasome in maintaining viral latency. Downregulating or inhibiting the proteasome promotes Tat-transactivation in cell line models. Furthermore, the FDA-approved proteasome inhibitors bortezomib and carfilzomib strongly synergize with existing LRAs to reactivate HIV-1 in CD4+ T cells from antiretroviral therapy-suppressed individuals without inducing cell activation or proliferation. Mechanistically, downregulating/inhibiting the proteasome elevates the levels of ELL2 and ELL2-SECs to enable Tat-transactivation, indicating the proteasome-ELL2 axis as a key regulator of HIV-1 latency and promising target for therapeutic intervention.

The latent HIV-1 reservoir is comprised of stably integrated and intact proviruses with limited to no viral transcription. It has been proposed that latent infection may be maintained by methylation of pro-viral DNA. Here, for the first time, we investigate the cytosine methylation of a replication competent provirus (AMBI-1) found in a T cell clone in a donor on antiretroviral therapy (ART). Methylation profiles of the AMBI-1 provirus were compared to other proviruses in the same donor and in samples from three other individuals on ART, including proviruses isolated from lymph node mononuclear cells (LNMCs) and peripheral blood mononuclear cells (PBMCs). We also evaluated the apparent methylation of cytosines outside of CpG (i.e., CpH) motifs. We found no evidence for methylation in AMBI-1 or any other provirus tested within the 5′ LTR promoter. In contrast, CpG methylation was observed in the env-tat-rev overlapping reading frame. In addition, we found evidence for differential provirus methylation in cells isolated from LNMCs vs. PBMCs in some individuals, possibly from the expansion of infected cell clones. Finally, we determined that apparent low-level methylation of CpH cytosines is consistent with occasional bisulfite reaction failures. In conclusion, our data do not support the proposition that latent HIV infection is associated with methylation of the HIV 5′ LTR promoter.

Background: Quantifying latently infected cells is critical to evaluate the efficacy of therapeutic strategies aimed at reducing the size of the long-lived viral reservoir, but the low frequency of these cells makes this very challenging. Methods: We developed TILDA (Tat/rev Induced Limiting Dilution Assay) to measure the frequency of cells with inducible multiply-spliced HIV RNA, as these transcripts are usually absent in latently infected cells but induced upon viral reactivation. TILDA requires less than a million cells, does not require RNA extraction and can be completed in two days. Findings: In suppressed individuals on ART, we found the median frequency of latently infected CD4+ T cells as estimated by TILDA to be 24 cells/million, which was 48 times more than the frequency measured by the quantitative viral outgrowth assay, and 6–27 times less than the frequencies of cells harbouring viral DNA measured by PCR-based assays. TILDA measurements strongly correlated with most HIV DNA assays. The size of the latent reservoir measured by TILDA was lower in subjects who initiated ART during the early compared to late stage of infection (p = 0.011). In untreated HIV disease, the frequency of CD4+ cells carrying latent but inducible HIV largely exceeded the frequency of actively producing cells, demonstrating that the majority of infected cells are transcriptionally silent even in the absence of ART. Interpretations: Our results suggest that TILDA is a reproducible and sensitive approach to measure the frequency of productively and latently infected cells in clinical settings. We demonstrate that the latent reservoir represents a substantial fraction of all infected cells prior to ART initiation. Research in context: In this manuscript, we describe the development of a novel assay that measures the magnitude of the latent HIV reservoir, the main barrier to HIV eradication. This novel assay, termed TILDA for Tat/rev Induced Limiting Dilution Assay, requires only 10 ml of blood, does not necessitate extraction of viral nucleic acids, is highly reproducible, covers a wide dynamic range of reservoir sizes and can be completed in two days. As such, TILDA may represent an alternative to existing assays used to evaluate the efficacy of therapeutic strategies aimed at reducing the size of the latent HIV reservoir.

BackgroundIt is unknown if extremely early initiation of antiretroviral therapy (ART) may lead to long-term ART-free HIV remission or cure. As a result, we studied 2 individuals recruited from a pre-exposure prophylaxis (PrEP) program who started prophylactic ART an estimated 10 days (Participant A; 54-year-old male) and 12 days (Participant B; 31-year-old male) after infection with peak plasma HIV RNA of 220 copies/mL and 3,343 copies/mL, respectively. Extensive testing of blood and tissue for HIV persistence was performed, and PrEP Participant A underwent analytical treatment interruption (ATI) following 32 weeks of continuous ART.Methods and findingsColorectal and lymph node tissues, bone marrow, cerebral spinal fluid (CSF), plasma, and very large numbers of peripheral blood mononuclear cells (PBMCs) were obtained longitudinally from both participants and were studied for HIV persistence in several laboratories using molecular and culture-based detection methods, including a murine viral outgrowth assay (mVOA). Both participants initiated PrEP with tenofovir/emtricitabine during very early Fiebig stage I (detectable plasma HIV-1 RNA, antibody negative) followed by 4-drug ART intensification. Following peak viral loads, both participants experienced full suppression of HIV-1 plasma viremia. Over the following 2 years, no further HIV could be detected in blood or tissue from PrEP Participant A despite extensive sampling from ileum, rectum, lymph nodes, bone marrow, CSF, circulating CD4+ T cell subsets, and plasma. No HIV was detected from tissues obtained from PrEP Participant B, but low-level HIV RNA or DNA was intermittently detected from various CD4+ T cell subsets. Over 500 million CD4+ T cells were assayed from both participants in a humanized mouse outgrowth assay. Three of 8 mice infused with CD4+ T cells from PrEP Participant B developed viremia (50 million input cells/surviving mouse), but only 1 of 10 mice infused with CD4+ T cells from PrEP Participant A (53 million input cells/mouse) experienced very low level viremia (201 copies/mL); sequence confirmation was unsuccessful. PrEP Participant A stopped ART and remained aviremic for 7.4 months, rebounding with HIV RNA of 36 copies/mL that rose to 59,805 copies/mL 6 days later. ART was restarted promptly. Rebound plasma HIV sequences were identical to those obtained during acute infection by single-genome sequencing. Mathematical modeling predicted that the latent reservoir size was approximately 200 cells prior to ATI and that only around 1% of individuals with a similar HIV burden may achieve lifelong ART-free remission. Furthermore, we observed that lymphocytes expressing the tumor marker CD30 increased in frequency weeks to months prior to detectable HIV-1 RNA in plasma. This study was limited by the small sample size, which was a result of the rarity of individuals presenting during hyperacute infection.ConclusionsWe report HIV relapse despite initiation of ART at one of the earliest stages of acute HIV infection possible. Near complete or complete loss of detectable HIV in blood and tissues did not lead to indefinite ART-free HIV remission. However, the small numbers of latently infected cells in individuals treated during hyperacute infection may be associated with prolonged ART-free remission.

HIV persists in a small pool of latently infected cells despite antiretroviral therapy (ART). Identifying cellular markers expressed at the surface of these cells may lead to novel therapeutic strategies to reduce the size of the HIV reservoir. We hypothesized that CD4+ T cells expressing immune checkpoint molecules would be enriched in HIV-infected cells in individuals receiving suppressive ART. Expression levels of 7 immune checkpoint molecules (PD-1, CTLA-4, LAG-3, TIGIT, TIM-3, CD160 and 2B4) as well as 4 markers of HIV persistence (integrated and total HIV DNA, 2-LTR circles and cell-associated unspliced HIV RNA) were measured in PBMCs from 48 virally suppressed individuals. Using negative binomial regression models, we identified PD-1, TIGIT and LAG-3 as immune checkpoint molecules positively associated with the frequency of CD4+ T cells harboring integrated HIV DNA. The frequency of CD4+ T cells co-expressing PD-1, TIGIT and LAG-3 independently predicted the frequency of cells harboring integrated HIV DNA. Quantification of HIV genomes in highly purified cell subsets from blood further revealed that expressions of PD-1, TIGIT and LAG-3 were associated with HIV-infected cells in distinct memory CD4+ T cell subsets. CD4+ T cells co-expressing the three markers were highly enriched for integrated viral genomes (median of 8.2 fold compared to total CD4+ T cells). Importantly, most cells carrying inducible HIV genomes expressed at least one of these markers (median contribution of cells expressing LAG-3, PD-1 or TIGIT to the inducible reservoir = 76%). Our data provide evidence that CD4+ T cells expressing PD-1, TIGIT and LAG-3 alone or in combination are enriched for persistent HIV during ART and suggest that immune checkpoint blockers directed against these receptors may represent valuable tools to target latently infected cells in virally suppressed individuals.

Identifying host immune determinants governing HIV transcription, latency and infectivity in vivo is critical to developing an HIV cure. Based on our recent finding that the host factor p21 regulates HIV transcription during antiretroviral therapy (ART), and published data demonstrating that the human carbohydrate-binding immunomodulatory protein galectin-9 regulates p21, we hypothesized that galectin-9 modulates HIV transcription. We report that the administration of a recombinant, stable form of galectin-9 (rGal-9) potently reverses HIV latency in vitro in the J-Lat HIV latency model. Furthermore, rGal-9 reverses HIV latency ex vivo in primary CD4+ T cells from HIV-infected, ART-suppressed individuals (p = 0.002), more potently than vorinostat (p = 0.02). rGal-9 co-administration with the latency reversal agent "JQ1", a bromodomain inhibitor, exhibits synergistic activity (p

The ability to reconstitute a normal immune system with antiretroviral therapy in the setting of HIV infection remains uncertain. This study aimed to characterize quantitative and qualitative aspects of various T cell subpopulations that do not improve despite effective ART. CD4∶CD8 ratio was evaluated in HIV-infected subjects with viral loads >10,000 copies/µl ("non-controllers", n = 42), those with undetectable viral loads on ART ("ART-suppressed", n = 53), and HIV-uninfected subjects (n = 22). In addition, T cell phenotype and function were examined in 25 non-controllers, 18 ART-suppressed, and 7 HIV-uninfected subjects. CD4∶CD8 ratio in non-controllers, ART-suppressed, and HIV-uninfected subjects was 0.25, 0.48, and 1.95 respectively (P

HIV-1 reservoirs preclude virus eradication in patients receiving highly active antiretroviral therapy (HAART). The best characterized reservoir is a small, difficult-to-quantify pool of resting memory CD4(+) T cells carrying latent but replication-competent viral genomes. Because strategies targeting this latent reservoir are now being tested in clinical trials, well-validated high-throughput assays that quantify this reservoir are urgently needed. Here we compare eleven different approaches for quantitating persistent HIV-1 in 30 patients on HAART, using the original viral outgrowth assay for resting CD4(+) T cells carrying inducible, replication-competent viral genomes as a standard for comparison. PCR-based assays for cells containing HIV-1 DNA gave infected cell frequencies at least 2 logs higher than the viral outgrowth assay, even in subjects who started HAART during acute/early infection. This difference may reflect defective viral genomes. The ratio of infected cell frequencies determined by viral outgrowth and PCR-based assays varied dramatically between patients. Although strong correlations with the viral outgrowth assay could not be formally excluded for most assays, correlations achieved statistical significance only for integrated HIV-1 DNA in peripheral blood mononuclear cells and HIV-1 RNA/DNA ratio in rectal CD4(+) T cells. Residual viremia was below the limit of detection in many subjects and did not correlate with the viral outgrowth assays. The dramatic differences in infected cell frequencies and the lack of a precise correlation between culture and PCR-based assays raise the possibility that the successful clearance of latently infected cells may be masked by a larger and variable pool of cells with defective proviruses. These defective proviruses are detected by PCR but may not be affected by reactivation strategies and may not require eradication to accomplish an effective cure. A molecular understanding of the discrepancy between infected cell frequencies measured by viral outgrowth versus PCR assays is an urgent priority in HIV-1 cure research.

The study of HIV-infected "controllers" who are able to maintain low levels of plasma HIV RNA in the absence of antiretroviral therapy (ART) may provide insights for HIV cure and vaccine strategies. Despite maintaining very low levels of plasma viremia, controllers have elevated immune activation and accelerated atherosclerosis. However, the degree to which low-level replication contributes to these phenomena is not known. Sixteen asymptomatic controllers were prospectively treated with ART for 24 weeks. Controllers had a statistically significant decrease in ultrasensitive plasma and rectal HIV RNA levels with ART. Markers of T cell activation/dysfunction in blood and gut mucosa also decreased substantially with ART. Similar reductions were observed in the subset of "elite" controllers with pre-ART plasma HIV RNA levels below conventional assays (

The rates of immunologic and clinical progression are lower in patients with drug-resistant HIV compared to wild-type HIV. This difference is not fully explained by viral load. It has been argued that reductions in T cell activation and/or viral fitness might result in preserved target cells and an altered relationship between the level of viremia and the rate of CD4+ T cell loss. We tested this hypothesis over time in a cohort of patients with highly resistant HIV. Fifty-four antiretroviral-treated patients with multi-drug resistant HIV and detectable plasma HIV RNA were followed longitudinally. CD4+ T cell counts and HIV RNA levels were measured every 4 weeks and T cell activation (CD38/HLA-DR) was measured every 16 weeks. We found that the levels of CD4+ T cell activation over time were a strong independent predictor of CD4+ T cell counts while CD8+ T cell activation was more strongly associated with viremia. Using spectral analysis, we found strong evidence for oscillatory (or cyclic) behavior in CD4+ T cell counts, HIV RNA levels, and T cell activation. Each of the cell populations exhibited an oscillatory behavior with similar frequencies. Collectively, these data suggest that there may be a mechanistic link between T cell activation, CD4+ T cell counts, and viremia and lends support for the hypothesis of altered predator-prey dynamics as a possible explanation of the stability of CD4+ T cell counts in the presence of sustained multi-drug resistant viremia.