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Abstract Despite antiretroviral therapy (ART), HIV persists in latently-infected cells (the HIV reservoir) which decay slowly over time. Here, leveraging >500 longitudinal samples from 67 people living with HIV (PLWH) treated during acute infection, we developed a mathematical model to predict reservoir decay from peripheral CD4 + T cells. Nonlinear generalized additive models demonstrated rapid biphasic decay of intact DNA (week 0-5: t1/2 ~ 2.83 weeks; week 5-24: t1/2 ~ 15.4 weeks) that extended out to 1 year. These estimates were ~5-fold faster than prior decay estimates among chronic treated PLWH. Defective DNA had a similar biphasic pattern, but data were more variable. Predicted intact and defective decay rates were faster for PLWH with earlier timing of ART initiation, higher initial CD4 + T cell count, and lower pre-ART viral load. In this study, we advanced our limited understanding of HIV reservoir decay at the time of ART initiation, informing future curative strategies targeting this critical time.

Abstract Introduction Vesatolimod is a Toll-like receptor-7 (TLR7) agonist in clinical development as part of a combination regimen for human immunodeficiency virus (HIV) cure. Influenza-like symptoms associated with TLR7-mediated immune activation have been reported in clinical trials of vesatolimod. Therefore, a broader understanding of the safety profile of vesatolimod and association with dose and mechanism of action will help inform future clinical studies. Methods In this analysis, data on flu-like adverse events of interest (AEIs) were pooled from eight clinical studies in which 606 participants either received single or multiple doses of vesatolimod (0.3–12 mg; n = 505) or placebo (n = 101). Vesatolimod pharmacokinetics, inflammatory responses, and pharmacodynamics were assessed. Results The incidence of flu-like AEIs was higher with vesatolimod versus placebo (19% [96/505] vs. 8% [8/101]) and increased with vesatolimod dose and exposure. Most flu-like AEIs with vesatolimod were grade 1 or 2 severity (55% [53 of 96] grade 1; 35% [34 of 96] grade 2) with onset primarily after the first and second dose. Occurrence of flu-like AEIs after doses 1–3 was predictive of reoccurrence after later doses. Dose-dependent elevations of pharmacodynamic biomarkers (interferon-stimulated gene 15, 2′-5′-oligoadenylate synthetase 1, myxovirus resistance-1, interferon-α, interleukin-1 receptor antagonist, interferon-γ-induced protein 10, interferon-inducible T-cell-α chemoattractant) observed in participants with flu-like AEIs suggest a link with vesatolimod mechanism of action. Conclusions Flu-like AEIs associated with vesatolimod administration were typically mild but increased with exposure, which may be predicted by the response to initial doses. The data suggest that adaptive clinical monitoring could help maximize pharmacodynamic responses and balance adverse events in future clinical trials of vesatolimod.

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.

The US National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institute of Health (NIH), convened a virtual workshop on August 8-9th, 2023 to explore potential synergies between HIV vaccine approaches that are designed to induce cellular or humoral immune responses. The goal of this workshop was to review data on leading vaccine candidates and to discuss the best strategies for combining these approaches to optimize immunity against HIV. Here, we summarize the findings reviewed at the workshop and discuss the knowledge gaps and priorities for future studies that will help accelerate the development of a preventive HIV vaccine.

BackgroundInterleukin-17–producing CD4 T cells contribute to the control of Mycobacterium tuberculosis (Mtb) infection in humans; whether infection with human immunodeficiency virus (HIV) disproportionately affects distinct Th17-cell subsets that respond to Mtb is incompletely defined.MethodsWe performed high-definition characterization of circulating Mtb-specific Th17 cells by spectral flow cytometry in people with latent TB and treated HIV (HIV-ART). We also measured kynurenine pathway activity by liquid chromatography-mass spectrometry (LC/MS) on plasma and tested the hypothesis that tryptophan catabolism influences Th17-cell frequencies in this context.ResultsWe identified two subsets of Th17 cells: subset 1 defined as CD4+Vα7.2−CD161+CD26+and subset 2 defined as CD4+Vα7.2−CCR6+CXCR3−cells of which subset 1 was significantly reduced in latent tuberculosis infection (LTBI) with HIV-ART, yet Mtb-responsive IL-17–producing CD4 T cells were preserved; we found that IL-17–producing CD4 T cells dominate the response to Mtb antigen but not cytomegalovirus (CMV) antigen or staphylococcal enterotoxin B (SEB), and tryptophan catabolism negatively correlates with both subset 1 and subset 2 Th17-cell frequencies.ConclusionsWe found differential effects of ART-suppressed HIV on distinct subsets of Th17 cells, that IL-17–producing CD4 T cells dominate responses to Mtb but not CMV antigen or SEB, and that kynurenine pathway activity is associated with decreases of circulating Th17 cells that may contribute to tuberculosis immunity.

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.

Abstract Most proviruses persisting in people living with HIV (PWH) on antiretroviral therapy (ART) are defective. However, rarer intact proviruses almost always reinitiate viral rebound if ART stops. Therefore, assessing therapies to prevent viral rebound hinges on specifically quantifying intact proviruses. We evaluated the same samples from 10 male PWH on ART using the two-probe intact proviral DNA assay (IPDA) and near full length (nfl) Q4PCR. Both assays admitted similar ratios of intact to total HIV DNA, but IPDA found ~40-fold more intact proviruses. Neither assay suggested defective proviruses decay over 10 years. However, the mean intact half-lives were different: 108 months for IPDA and 65 months for Q4PCR. To reconcile this difference, we modeled additional longitudinal IPDA data and showed that decelerating intact decay could arise from very long-lived intact proviruses and/or misclassified defective proviruses: slowly decaying defective proviruses that are intact in IPDA probe locations (estimated up to 5%, in agreement with sequence library based predictions). The model also demonstrates how misclassification can lead to underestimated efficacy of therapies that exclusively reduce intact proviruses. We conclude that sensitive multi-probe assays combined with specific nfl-verified assays would be optimal to document absolute and changing levels of intact HIV proviruses.

Summary: Deciphering the mechanisms underlying viral persistence is critical to achieving a cure for human immunodeficiency virus (HIV) infection. Here, we implement a systems approach to discover molecular signatures of HIV latently infected CD4+ T cells, identifying the immunosuppressive, adenosine-producing ectonucleotidase CD73 as a key surface marker of latent cells. Hypoxic conditioning, reflecting the lymphoid tissue microenvironment, increases the frequency of CD73+ CD4+ T cells and promotes HIV latency. Transcriptomic profiles of CD73+ CD4+ T cells favor viral quiescence, immune evasion, and cell survival. CD73+ CD4+ T cells are capable of harboring a functional HIV reservoir and reinitiating productive infection ex vivo. CD73 or adenosine receptor blockade facilitates latent HIV reactivation in vitro, mechanistically linking adenosine signaling to viral quiescence. Finally, tissue imaging of lymph nodes from HIV-infected individuals on antiretroviral therapy reveals spatial association between CD73 expression and HIV persistence in vivo. Our findings warrant development of HIV-cure strategies targeting the hypoxia-CD73-adenosine axis.

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.

Summary: Host repressors mediate HIV latency, but how they interactively silence the virus remains unclear. Here, we develop “reiterative enrichment and authentication of CRISPRi targets for synergies (REACTS)” to probe the genome for synergies between HIV transcription repressors. Using eight known host repressors as queries, we identify 32 synergies involving eleven repressors, including BCL7C, KANSL2, and SIRT2. Overexpression of these three proteins reduces HIV reactivation in Jurkat T cells and in CD4 T cells from people living with HIV on antiretroviral therapy (ART). We show that the BCL7C-containing BAF complex and the KANSL2-containing NSL complex form a “supercomplex” that increases inhibitory histone acetylation of the HIV long-terminal repeat (LTR) and its occupancy by the short variant of the acetyl-lysine reader Brd4. Collectively, we provide a validated platform for defining gene synergies genome wide, and the BAF-NSL “supercomplex” represents a potential target for overcoming HIV rebound after ART cessation.

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.

IntroductionIn people with HIV (PWH) both off and on antiretroviral therapy (ART), the expression of immune checkpoint (IC) proteins is elevated on the surface of total and HIV-specific T-cells, indicating T-cell exhaustion. Soluble IC proteins and their ligands can also be detected in plasma, but have not been systematically examined in PWH. Since T-cell exhaustion is associated with HIV persistence on ART, we aimed to determine if soluble IC proteins and their ligands also correlated with the size of the HIV reservoir and HIV-specific T-cell function.MethodsWe used multiplex bead-based immunoassay to quantify soluble programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), T cell immunoglobulin domain and mucin domain 3 (TIM-3), PD-1 Ligand 1 (PD-L1) and PD-1 Ligand 2 (PD-L2) in plasma from PWH off ART (n=20), on suppressive ART (n=75) and uninfected controls (n=20). We also quantified expression of membrane-bound IC and frequencies of functional T-cells to Gag and Nef peptide stimulation on CD4+ and CD8+ T-cells using flow cytometry. The HIV reservoir was quantified in circulating CD4+ T-cells using qPCR for total and integrated HIV DNA, cell-associated unspliced HIV RNA and 2LTR circles.ResultsSoluble (s) PD-L2 level was higher in PWH off and on ART compared to uninfected controls. Higher levels of sPD-L2 correlated with lower levels of HIV total DNA and higher frequencies of gag-specific CD8+ T-cells expressing CD107a, IFNγ or TNFα. In contrast, the concentration of sLAG-3 was similar in uninfected individuals and PWH on ART, but was significantly elevated in PWH off ART. Higher levels of sLAG-3 correlated with higher levels of HIV total and integrated DNA, and lower frequency of gag-specific CD4+ T cells expressing CD107a. Similar to sLAG-3, levels of sPD-1 were elevated in PWH off ART and normalized in PWH on ART. sPD-1 was positively correlated with the frequency of gag-specific CD4+ T cells expressing TNF-a and the expression of membrane-bound PD-1 on total CD8+ T-cells in PWH on ART.DiscussionPlasma soluble IC proteins and their ligands correlate with markers of the HIV reservoir and HIV-specific T-cell function and should be investigated further in in large population-based studies of the HIV reservoir or cure interventions in PWH on ART.

Abstract Background HIV cure research involving cell and gene therapy has intensified in recent years. There is a growing need to identify ethical standards and safeguards to ensure cell and gene therapy (CGT) HIV cure research remains valued and acceptable to as many stakeholders as possible as it advances on a global scale. Methods To elicit preliminary ethical and practical considerations to guide CGT HIV cure research, we implemented a qualitative, in-depth interview study with three key stakeholder groups in the United States: (1) biomedical HIV cure researchers, (2) bioethicists, and (3) community stakeholders. Interviews permitted evaluation of informants’ perspectives on how CGT HIV cure research should ethically occur, and were transcribed verbatim. We applied conventional content analysis focused on inductive reasoning to analyze the rich qualitative data and derive key ethical and practical considerations related to CGT towards an HIV cure. Results We interviewed 13 biomedical researchers, 5 community members, and 1 bioethicist. Informants generated considerations related to: perceived benefits of CGT towards an HIV cure, perceived risks, considerations necessary to ensure an acceptable benefit/risk balance, CGT strategies considered unacceptable, additional ethical considerations, and considerations for first-in-human CGT HIV cure trials. Informants also proposed important safeguards to developing CGT approaches towards an HIV cure, such as the importance of mitigating off-target effects, mitigating risks associated with long-term duration of CGT interventions, and mitigating risks of immune overreactions. Conclusion Our study identified preliminary considerations for CGT-based HIV cure across three key stakeholder groups. Respondents identified an ideal cure strategy as one which would durably control HIV infection, protect the individual from re-acquisition, and eliminate transmission to others. Known and unknown risks should be anticipated and perceived as learning opportunities to preserve and honor the altruism of participants. Preclinical studies should support these considerations and be transparently reviewed by regulatory experts and peers prior to first-in-human studies. To protect the public trust in CGT HIV cure research, ethical and practical considerations should be periodically revisited and updated as the science continues to evolve. Additional ethics studies are required to expand stakeholder participation to include traditionally marginalized groups and clinical care providers.

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.

Abstract Cognitive post‐acute sequelae of SARS‐CoV‐2 (PASC) can occur after mild COVID‐19. Detailed clinical characterizations may inform pathogenesis. We evaluated 22 adults reporting cognitive PASC and 10 not reporting cognitive symptoms after mild SARS‐CoV‐2 infection through structured interviews, neuropsychological testing, and optional cerebrospinal fluid (CSF) evaluations (53%). Delayed onset of cognitive PASC occurred in 43% and associated with younger age. Cognitive PASC participants had a higher number of pre‐existing cognitive risk factors (2.5 vs. 0; p = 0.03) and higher proportion with abnormal CSF findings (77% vs. 0%; p = 0.01) versus controls. Cognitive risk factors and immunologic mechanisms may contribute to cognitive PASC pathogenesis.

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.

Abstract Background An increasing number of HIV cure trials involve combining multiple potentially curative interventions. Until now, considerations for designing and implementing complex combination HIV cure trials have not been thoroughly considered. Methods We used a purposive method to select key informants for our study. Informants included biomedical HIV cure researchers, regulators, policy makers, bioethicists, and community members. We used in-depth interviews to generate ethical and practical considerations to guide the design and implementation of combination HIV cure research. We analyzed the qualitative data using conventional content analysis focused on inductive reasoning. Results We interviewed 11 biomedical researchers, 4 community members, 2 regulators, 1 policy researcher, and 1 bioethicist. Informants generated considerations for designing and implementing combination interventions towards an HIV cure, focused on ethical aspects, as well as considerations to guide trial design, benefit/risk determinations, regulatory requirements, prioritization and sequencing and timing of interventions, among others. Informants also provided considerations related to combining specific HIV cure research modalities, such as broadly neutralizing antibodies (bNAbs), cell and gene modification products, latency-reversing agents and immune-based interventions. Finally, informants provided suggestions to ensure meaningful therapeutic improvements over standard antiretroviral therapy, overcome challenges of designing combination approaches, and engage communities around combination HIV cure research. Conclusion The increasing number of combination HIV cure trials brings with them a host of ethical and practical challenges. We hope our paper will inform meaningful stakeholder dialogue around the use of combinatorial HIV cure research approaches. To protect the public trust in HIV cure research, considerations should be periodically revisited and updated with key stakeholder input as the science continues to advance.

HIV-specific chimeric antigen receptor–T cell (CAR T cell) therapies are candidates to functionally cure HIV infection in people with HIV (PWH) by eliminating reactivated HIV-infected cells derived from latently infected cells within the HIV reservoir. Paramount to translating such therapeutic candidates successfully into the clinic will require anti-HIV CAR T cells to localize to lymphoid tissues in the body and eliminate reactivated HIV-infected cells such as CD4+ T cells and monocytes/macrophages. Here we show that i.v. injected anti-HIV duoCAR T cells, generated using a clinical-grade anti-HIV duoCAR lentiviral vector, localized to the site of active HIV infection in the spleen of humanized mice and eliminated HIV-infected PBMCs. CyTOF analysis of preinfusion duoCAR T cells revealed an early memory phenotype composed predominantly of CCR7+ stem cell–like/central memory T cells (TSCM/TCM) with expression of some effector-like molecules. In addition, we show that anti-HIV duoCAR T cells effectively sense and kill HIV-infected CD4+ T cells and monocytes/macrophages. Furthermore, we demonstrate efficient genetic modification of T cells from PWH on suppressive ART into anti-HIV duoCAR T cells that subsequently kill autologous PBMCs superinfected with HIV. These studies support the safety and efficacy of anti-HIV duoCAR T cell therapy in our presently open phase I/IIa clinical trial (NCT04648046).

ABSTRACT HIV infects long-lived CD4 memory T cells, establishing a latent viral reservoir that necessitates lifelong antiretroviral therapy (ART). How this reservoir is formed so quickly after infection remains unclear. We now show the innate inflammatory response to HIV infection results in CCL2 chemokine release, leading to recruitment of cells expressing the CCR2 receptor, including a subset of central memory CD4 T cells. Supporting a role for the CCL2/CCR2 axis in rapid reservoir formation, we find (i) treatment of humanized mice with anti-CCL2 antibodies during early HIV infection decreases reservoir seeding and preserves CCR2/5+ cells and (ii) CCR2/5+ cells from the blood of HIV-infected individuals on long-term ART contain significantly more integrated provirus than CCR2/5-negative memory or naive cells. Together, these studies support a model where the host’s innate inflammatory response to HIV infection, including CCL2 production, leads to the recruitment of CCR2/5+ central memory CD4 T cells to zones of virus-associated inflammation, likely contributing to rapid formation of the latent HIV reservoir. IMPORTANCE There are currently over 35 million people living with HIV worldwide, and we still have no vaccine or scalable cure. One of the difficulties with HIV is its ability to rapidly establish a viral reservoir in lymphoid tissues that allows it to elude antivirals and the immune system. Thus, it is important to understand how HIV accomplishes this so we can develop preventive strategies. Our current results show that an early inflammatory response to HIV infection includes production of the chemokine CCL2, which recruits a unique subset of CCR2/5+ CD4+ T cells that become infected and form a significant reservoir for latent infection. Furthermore, we show that blockade of CCL2 in humanized mice significantly reduces persistent HIV infection. This information is relevant to the development of therapeutics to prevent and/or treat chronic HIV infections.

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.

Immunogenicity of HIV-1 mRNA vaccine regimens was analyzed in a non-human primate animal model. Rhesus macaques immunized with mRNA in lipid nanoparticle (mRNA/LNP) formulation expressing HIV-1 Gag and Gag conserved regions (CE) as immunogens developed robust, durable antibody responses but low adaptive T-cell responses. Augmentation of the dose resulted in modest increases in vaccine-induced cellular immunity, with no difference in humoral responses. The gag mRNA/lipid nanoparticle (LNP) vaccine provided suboptimal priming of T cell responses for a heterologous DNA booster vaccination regimen. In contrast, a single immunization with gag mRNA/LNP efficiently boosted both humoral and cellular responses in macaques previously primed by a gag DNA-based vaccine. These anamnestic cellular responses were mediated by activated CD8+ T cells with a phenotype of differentiated T-bet+ cytotoxic memory T lymphocytes. The heterologous prime/boost regimens combining DNA and mRNA/LNP vaccine modalities maximized vaccine-induced cellular and humoral immune responses. Analysis of cytokine responses revealed a transient systemic signature characterized by the release of type I interferon, IL-15 and IFN-related chemokines. The pro-inflammatory status induced by the mRNA/LNP vaccine was also characterized by IL-23 and IL-6, concomitant with the release of IL-17 family of cytokines. Overall, the strong boost of cellular and humoral immunity induced by the mRNA/LNP vaccine suggests that it could be useful as a prophylactic vaccine in heterologous prime/boost modality and in immune therapeutic interventions against HIV infection or other chronic human diseases.

Inflammation associated with increased risk of comorbidities persists in people living with HIV (PWH) on combination antiretroviral therapy (ART). A recent placebo-controlled trial of low-dose methotrexate (MTX) in PWH found that numbers of total CD4 and CD8 T cells decreased in the low-dose MTX arm. In this report we analyzed T cell phenotypes and additional plasma inflammatory indices in samples from the trial. We found that cycling (Ki67+) T cells lacking Bcl-2 were reduced by MTX but plasma inflammatory cytokines were largely unaffected. In a series of in vitro experiments to further investigate the mechanisms of MTX activity, we found that MTX did not inhibit effector cytokine production but inhibited T cell proliferation downstream of mTOR activation, mitochondrial function, and cell cycle entry. This inhibitory effect was reversible with folinic acid, suggesting low-dose MTX exerts anti-inflammatory effects in vivo in PWH largely by blocking T cell proliferation via dihydrofolate reductase inhibition, yet daily administration of folic acid did not rescue this effect in trial participants. Our findings identify the main mechanism of action of this widely used anti-inflammatory medicine in PWH and may provide insight into how MTX works in the setting of other inflammatory conditions.

Background: Efforts to understand the impact of SARS-CoV-2 variants, vaccine status, and treatment on the development and persistence of Long COVID have intensified. Methods: We report 4 sequential cases from a post-COVID cohort study demonstrating variability in outcomes following differentially timed nirmatrelvir therapy, received as part of clinical care. Results: In the first case, the participant experienced symptomatic rebound and developed Long COVID despite early initiation of antiviral therapy. In the next 2 cases, participants reported improvement in persistent COVID symptoms when nirmatrelvir was taken 25 and 60 days following initial symptom onset. In the final case, an individual with presumed Long COVID for 2 years reported substantial improvement in chronic symptoms when taking nirmatrelvir following SARS-CoV-2 re-infection. Conclusions: These anecdotes suggest that systematic study of antiviral therapy for Long COVID is warranted.

Summary: Background: In HIV infection, even under long-term antiretroviral therapy (ART), up to 20% of HIV-infected individuals fail to restore CD4+ T cell counts to the levels similar to those of healthy controls. The mechanisms of poor CD4+ T cell reconstitution on suppressive ART are not fully understood. Methods: Here, we tested the hypothesis that lipopolysaccharide (LPS) from bacteria enriched in the plasma from immune non-responders (INRs) contributes to blunted CD4+ T cell recovery on suppressive ART in HIV. We characterized plasma microbiome in HIV INRs (aviremic, CD4+ T cell counts < 350 cells/μl), immune responders (IRs, CD4+ T cell counts > 500 cells/μl), and healthy controls. Next, we analyzed the structure of the lipid A domain of three bacterial species identified by mass spectrometry (MS) and evaluated the LPS function through LPS induced proinflammatory responses and CD4+ T cell apoptosis in PBMCs. In comparison, we also evaluated plasma levels of proinflammatory cytokine and chemokine patterns in these three groups. At last, to study the causality of microbiome-blunted CD4+ T cell recovery in HIV, B6 mice were intraperitoneally (i.p.) injected with heat-killed Burkholderia fungorum, Serratia marcescens, or Phyllobacterium myrsinacearum, twice per week for total of eight weeks. Findings: INRs exhibited elevated plasma levels of total microbial translocation compared to the IRs and healthy controls. The most enriched bacteria were Burkholderia and Serratia in INRs and were Phyllobacterium in IRs. Further, unlike P. myrsinacearum LPS, B. fungorum and S. marcescens LPS induced proinflammatory responses and CD4+ T cell apoptosis in PBMCs, and gene profiles of bacteria-mediated cell activation pathways in THP-1 cells in vitro. Notably, LPS structural analysis by mass spectrometry revealed that lipid A from P. myrsinacearum exhibited a divergent structure consistent with weak toll-like receptor (TLR) 4 agonism, similar to the biological profile of probiotic bacteria. In contrast, lipid A from B. fungorum and S. marcescens showed structures more consistent with canonical TLR4 agonists stemming from proinflammatory bacterial strains. Finally, intraperitoneal (i.p.) injection of inactivated B. fungorum and S. marcescens but not P. myrsinacearum resulted in cell apoptosis in mesenteric lymph nodes of C57BL/6 mice in vivo. Interpretation: These results suggest that the microbial products are causally associated with INR phenotype. In summary, variation in blood microbial LPS immunogenicity may contribute to immune reconstitution in response to suppressive ART. Collectively, this work is consistent with immunologically silencing microbiome being causal and targetable with therapy in HIV. Funding: This work was supported by the National Institute of Allergy and Infectious Diseases (NIAID; R01 AI128864, Jiang) (NIAID; P30 AI027767, Saag/Health), the Medical Research Service at the Ralph H. Johnson VA Medical Center (merit grant VA CSRD MERIT I01 CX-002422, Jiang), and the National Institute of Aging (R21 AG074331, Scott). The SCOPE cohort was supported by the UCSF/Gladstone Institute of Virology & Immunology CFAR (P30 AI027763, Gandhi) and the CFAR Network of Integrated Clinical Systems (R24 AI067039, Saag). The National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR001450 (the pilot grant, Jiang). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Summary: Background: While increased CD8 counts and low CD4/CD8 ratio during treated HIV correlate with immunosenescence, their additional predictive values to identify individuals with HIV at higher risk of clinical events remain controversial. Methods: We selected treatment-naive individuals initiating ART from ACTG studies 384, 388, A5095, A5142, A5202, and A5257 who had achieved viral suppression at year 2. We examined the effect of CD8+ T cell counts and CD4/CD8 at year 2 on the probability of AIDS and serious non-AIDS events in years 3−7. We used inverse probability weighting methods to address informative censoring, combined with multivariable logistic regression models. Findings: We analyzed 5133 participants with a median age of 38 years; 959 (19%) were female, pre-ART median CD4 counts were 249 (Q1-Q3 91–372) cell/µL. Compared to participants with CD8 counts between 500/µL and 1499/µL, those with >1500/µL had a higher risk of clinical events during years 3−7 (aOR 1.75; 95%CI 1.33−2.32). CD4/CD8 ratio was not predictive of greater risk of events through year 7. Additional analyses revealed consistent CD8 count effect sizes for the risk of AIDS events and noninfectious non-AIDS events, but opposite effects for the risk of severe infections, which were more frequent among individuals with CD8 counts

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.

People living with HIV (PLWH) who are immune nonresponders (INRs) are at greater risk of comorbidity and mortality than are immune responders (IRs) who restore their CD4+ T cell count after antiretroviral therapy (ART). INRs have low CD4+ T cell counts (

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.

Type I IFNs (TI-IFNs) drive immune effector functions during acute viral infections and regulate cell cycling and systemic metabolism. That said, chronic TI-IFN signaling in the context of HIV infection treated with antiretroviral therapy (ART) also facilitates viral persistence, in part by promoting immunosuppressive responses and CD8+ T cell exhaustion. To determine whether inhibition of IFN-α might provide benefit in the setting of chronic, ART-treated SIV infection of rhesus macaques, we administered an anti–IFN-α antibody followed by an analytical treatment interruption (ATI). IFN-α blockade was well-tolerated and associated with lower expression of TI-IFN–inducible genes (including those that are antiviral) and reduced tissue viral DNA (vDNA). The reduction in vDNA was further accompanied by higher innate proinflammatory plasma cytokines, expression of monocyte activation genes, IL-12–induced effector CD8+ T cell genes, increased heme/metabolic activity, and lower plasma TGF-β levels. Upon ATI, SIV-infected, ART-suppressed nonhuman primates treated with anti–IFN-α displayed lower levels of weight loss and improved erythroid function relative to untreated controls. Overall, these data demonstrated that IFN-α blockade during ART-treated SIV infection was safe and associated with the induction of immune/erythroid pathways that reduced viral persistence during ART while mitigating the weight loss and anemia that typically ensue after ART interruption.

People with HIV (PWH) experience accentuated biological aging, as defined by markers of inflammation, immune dysfunction, and the epigenetic clock. They also have an elevated risk of multiple age-associated comorbidities. To discuss current knowledge, research gaps, and priorities in aging and age-related comorbidities in treated HIV infection, the NIH program staff organized a workshop held in Bethesda, Maryland in September 2019. This review article describes highlights of discussions led by the Pathogenesis/Basic Science Research working group that focused on three high priority topics: immunopathogenesis; the microbiome/virome; and aging and senescence. We summarize knowledge in these fields and describe key questions for research on the pathogenesis of aging and age-related comorbidities in PWH. Understanding the drivers and mechanisms underlying accentuated biological aging is a high priority that will help identify potential therapeutic targets to improve healthspan in older PWH.

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.

Objectives: The aim of this study was to assess soluble CD30 (sCD30), a protein that colocalises with HIV-1 RNA and DNA in lymphoid cells and tissues, in cerebrospinal fluid (CSF) as a marker of HIV-1 infection in the central nervous system (CNS). Methods: This was a cross-sectional study using archived samples from two clinical cohorts. Soluble CD30 concentrations were measured in paired CSF and plasma from untreated viraemic individuals (n=52), individuals on suppressive antiretroviral therapy (ART) (n=33), HIV-1 controllers (n=10), participants with CSF HIV-1 ‘escape’ (n=11) and controls without HIV-1 infection (n=16). Nonparametric tests were used to compare levels across groups and evaluate correlations with HIV-1 RNA, CSF neurofilament light chain protein (NFL) and neopterin. Results: Compared with controls (median 30 ng/mL, interquartile range [IRQ] 23–50), plasma sCD30 levels were elevated in viraemic participants (75 ng/mL, 52–116; P

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.

HIV infection induces a robust T cell response that is sustained by high viremia, but falls following the onset of antiretroviral therapy (ART). Relatively little has been reported on the subsequent stability of the HIV-specific T cell response in individuals on durable therapy. Such data are critical for powering clinical trials testing T cell-based immunotherapies. In a cross-sectional study, HIV-specific T cell responses were detectable by ex vivo interferon (IFN)-γ ELISpot (average ∼1,100 spot-forming units [SFUs]/106 peripheral blood mononuclear cells) in persons living with HIV (PLWH; n = 34), despite median durable ART suppression of 5.0 years. No substantial association was detected between the summed HIV-specific T cell response and the size of the replication-competent HIV reservoir. T cell responses were next measured in participants sampled weekly, monthly, or yearly. HIV-specific T cell responses were highly stable over the time periods examined; within-individual variation ranged from 16% coefficient of variation (CV) for weekly to 27% CV for yearly sampling. These data were used to generate power calculations for future immunotherapy studies. The stability of the HIV-specific T cell response in suppressed PLWH will enable powered studies of small sizes (e.g., n = 6–12), facilitating rapid and iterative testing for T cell-based immunotherapies against HIV. Keywords: HIV, T cell, immunotherapy, CD8

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.

IntroductionLow HIV viral load is associated with delayed disease progression and reduced HIV transmission. HIV controllers suppress viral load to low levels in the absence of antiretroviral treatment (ART). We used an antibody profiling system, VirScan, to compare antibody reactivity and specificity in HIV controllers, non-controllers with treatment-induced viral suppression, and viremic non-controllers.MethodsThe VirScan library contains 3,384 phage-displayed peptides spanning the HIV proteome. Antibody reactivity to these peptides was measured in plasma from a Discovery Cohort that included 13 elite controllers, 27 viremic controllers, 12 viremic non-controllers, and 21 non-controllers who were virally suppressed on ART. Antibody reactivity to selected peptides was also assessed in an independent cohort of 29 elite controllers and 37 non-controllers who were virally suppressed on ART (Validation Cohort) and in a longitudinal cohort of non-controllers.ResultsIn the Discovery Cohort, 62 peptides were preferentially targeted in HIV controllers compared to non-controllers who were virally suppressed on ART. These specificities were not significantly different when comparing controllers versus viremic non-controllers. Aggregate reactivity to these peptides was also high in elite controllers from the independent Validation Cohort. The 62 peptides formed seven clusters of homologous epitopes in env, gag, integrase, and vpu. Reactivity to one of these clusters located in gag p17 was inversely correlated with viral load set point in an independent cohort of non-controllers.ConclusionsAntibody reactivity was low in non-controllers suppressed on ART, but remained high in viremic controllers despite viral suppression. Antibodies in controllers and viremic non-controllers were directed against epitopes in diverse HIV proteins; higher reactivity against p17 peptides was associated with lower viral load set point. Further studies are needed to determine if these antibodies play a role in regulation of HIV viral load.

Observational and interventional studies for HIV cure research often use single-copy assays to quantify rare entities in blood or tissue samples. Statistical analysis of such measurements presents challenges due to tissue sampling variability and frequent findings of 0 copies in the sample analysed. We examined four approaches to analysing such studies, reflecting different ways of handling observations of 0 copies: (A) replace observations of 0 copies with 1 copy; (B) add 1 to all observed numbers of copies; (C) treat observations of 0 copies as left-censored at 1 copy; and (D) leave the data unaltered and apply a method for count data, negative binomial regression. Because research seeks to estimate general patterns rather than individuals’ values, we argue that unaltered use of 0 copies is suitable for research purposes and that altering those observations can introduce bias. When applied to a simulated study comparing preintervention to postintervention measurements within 12 participants, methods A–C showed more attenuation than method D in the estimated intervention effect, less chance of finding P

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.

Summary: Quantifying the replication-competent HIV reservoir is essential for evaluating curative strategies. Viral outgrowth assays (VOAs) underestimate the reservoir because they fail to induce all replication-competent proviruses. Single- or double-region HIV DNA assays overestimate it because they fail to exclude many defective proviruses. We designed two triplex droplet digital PCR assays, each with 2 unique targets and 1 in common, and normalize the results to PCR-based T cell counts. Both HIV assays are specific, sensitive, and reproducible. Together, they estimate the number of proviruses containing all five primer-probe regions. Our 5-target results are on average 12.1-fold higher than and correlate with paired quantitative VOA (Spearman's ρ = 0.48) but estimate a markedly smaller reservoir than previous DNA assays. In patients on antiretroviral therapy, decay rates in blood CD4+ T cells are faster for intact than for defective proviruses, and intact provirus frequencies are similar in mucosal and circulating T cells.

Background: One strategy being pursued to clear latently infected cells that persist in people living with HIV (PLWH) on antiretroviral therapy (ART) is to activate latent HIV infection with a latency reversing agent (LRA). Surrogate markers that accurately measure virus production following an LRA are needed. Methods: We quantified cell-associated unspliced (US), multiply spliced (MS) and supernatant (SN) HIV RNA by qPCR from total and resting CD4+ T cells isolated from seven PLWH on ART before and after treatment ex vivo with different LRAs, including histone deacetylase inhibitors (HDACi). MS and plasma HIV RNA were also quantified from PLWH on ART (n-11) who received the HDACi panobinostat. Findings: In total and resting CD4+ T cells from PLWH on ART, detection of US RNA was common while detection of MS RNA was infrequent. Primers used to detect MS RNA, in contrast to US RNA, bound sites of the viral genome that are commonly mutated or deleted in PLWH on ART. Following ex vivo stimulation with LRAs, we identified a strong correlation between the fold change increase in SN and MS RNA, but not the fold change increase in SN and US RNA. In PLWH on ART who received panobinostat, MS RNA was significantly higher in samples with detectable compared to non0detectable plasma HIV RNA. Interpretation: Following administration of an LRA, quantification of MS RNA is more likely to reflect an increase in virion production and is therefore a better indicator of meaningful latency reversal. Funding: NHMRC, NIH DARE collaboratory.

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.

Thinking about public health impact should inform HIV curative investigations. Should an effective HIV cure or sustained viral remission intervention emerge from ongoing investigations, implementation strategies aimed at ensuring global access will be needed if these approaches are to be impactful, and planning accordingly makes sense now. Specifically, we discuss three key access barriers to future cure-related interventions: high cost of the strategy; non-financial challenges to procurement, distribution and point-of-care delivery; and non-adherence and the need for long-term monitoring. As we argue, plans and decision-making for overcoming each of these barriers will need to be developed in advance. An evaluation of remaining barriers and likely global impact of the leading strategies under investigation should inform decisions on which strategy might receive funding priority. Among the strategies being investigated, implementation barriers for latency-reversing agents, immunotherapy and combination antiretroviral therapy (ART) may be overcome on a global scale with some effort. Overcoming implementation barriers for medically complex and high-risk interventions, such as stem cell and, to some degree, gene therapy, may be less feasible.

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.