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Understanding the complexity of the long-lived HIV reservoir during antiretroviral therapy (ART) remains a considerable impediment in research towards a cure for HIV. To address this, we developed a single-cell strategy to precisely define the unperturbed peripheral blood HIV-infected memory CD4+ T cell reservoir from ART-treated people living with HIV (ART-PLWH) via the presence of integrated accessible proviral DNA in concert with epigenetic and cell surface protein profiling. We identified profound reservoir heterogeneity within and between ART-PLWH, characterized by new and known surface markers within total and individual memory CD4+ T cell subsets. We further uncovered new epigenetic profiles and transcription factor motifs enriched in HIV-infected cells that suggest infected cells with accessible provirus, irrespective of reservoir distribution, are poised for reactivation during ART treatment. Together, our findings reveal the extensive inter- and intrapersonal cellular heterogeneity of the HIV reservoir, and establish an initial multiomic atlas to develop targeted reservoir elimination strategies.

BackgroundThe COVID-19 standard of care (SOC) evolved rapidly during 2020 and 2021, but its cumulative effect over time is unclear.ObjectiveTo evaluate whether recovery and mortality improved as SOC evolved, using data from ACTT (Adaptive COVID-19 Treatment Trial).DesignACTT is a series of phase 3, randomized, double-blind, placebo-controlled trials that evaluated COVID-19 therapeutics from February 2020 through May 2021. ACTT-1 compared remdesivir plus SOC to placebo plus SOC, and in ACTT-2 and ACTT-3, remdesivir plus SOC was the control group. This post hoc analysis compared recovery and mortality between these comparable sequential cohorts of patients who received remdesivir plus SOC, adjusting for baseline characteristics with propensity score weighting. The analysis was repeated for participants in ACTT-3 and ACTT-4 who received remdesivir plus dexamethasone plus SOC. Trends in SOC that could explain outcome improvements were analyzed. (ClinicalTrials.gov: NCT04280705 [ACTT-1], NCT04401579 [ACTT-2], NCT04492475 [ACTT-3], and NCT04640168 [ACTT-4]).Setting94 hospitals in 10 countries (86% U.S. participants).ParticipantsAdults hospitalized with COVID-19.InterventionSOC.Measurements28-day mortality and recovery.ResultsAlthough outcomes were better in ACTT-2 than in ACTT-1, adjusted hazard ratios (HRs) were close to 1 (HR for recovery, 1.04 [95% CI, 0.92 to 1.17]; HR for mortality, 0.90 [CI, 0.56 to 1.40]). Comparable patients were less likely to be intubated in ACTT-2 than in ACTT-1 (odds ratio, 0.75 [CI, 0.53 to 0.97]), and hydroxychloroquine use decreased. Outcomes improved from ACTT-2 to ACTT-3 (HR for recovery, 1.43 [CI, 1.24 to 1.64]; HR for mortality, 0.45 [CI, 0.21 to 0.97]). Potential explanatory factors (SOC trends, case surges, and variant trends) were similar between ACTT-2 and ACTT-3, except for increased dexamethasone use (11% to 77%). Outcomes were similar in ACTT-3 and ACTT-4. Antibiotic use decreased gradually across all stages.LimitationUnmeasured confounding.ConclusionChanges in patient composition explained improved outcomes from ACTT-1 to ACTT-2 but not from ACTT-2 to ACTT-3, suggesting improved SOC. These results support excluding nonconcurrent controls from analysis of platform trials in rapidly changing therapeutic areas.Primary funding sourceNational Institute of Allergy and Infectious Diseases.

BackgroundBaricitinib and dexamethasone have randomised trials supporting their use for the treatment of patients with COVID-19. We assessed the combination of baricitinib plus remdesivir versus dexamethasone plus remdesivir in preventing progression to mechanical ventilation or death in hospitalised patients with COVID-19.MethodsIn this randomised, double-blind, double placebo-controlled trial, patients were enrolled at 67 trial sites in the USA (60 sites), South Korea (two sites), Mexico (two sites), Singapore (two sites), and Japan (one site). Hospitalised adults (≥18 years) with COVID-19 who required supplemental oxygen administered by low-flow (≤15 L/min), high-flow (>15 L/min), or non-invasive mechanical ventilation modalities who met the study eligibility criteria (male or non-pregnant female adults ≥18 years old with laboratory-confirmed SARS-CoV-2 infection) were enrolled in the study. Patients were randomly assigned (1:1) to receive either baricitinib, remdesivir, and placebo, or dexamethasone, remdesivir, and placebo using a permuted block design. Randomisation was stratified by study site and baseline ordinal score at enrolment. All patients received remdesivir (≤10 days) and either baricitinib (or matching oral placebo) for a maximum of 14 days or dexamethasone (or matching intravenous placebo) for a maximum of 10 days. The primary outcome was the difference in mechanical ventilation-free survival by day 29 between the two treatment groups in the modified intention-to-treat population. Safety analyses were done in the as-treated population, comprising all participants who received one dose of the study drug. The trial is registered with ClinicalTrials.gov, NCT04640168.FindingsBetween Dec 1, 2020, and April 13, 2021, 1047 patients were assessed for eligibility. 1010 patients were enrolled and randomly assigned, 516 (51%) to baricitinib plus remdesivir plus placebo and 494 (49%) to dexamethasone plus remdesivir plus placebo. The mean age of the patients was 58·3 years (SD 14·0) and 590 (58%) of 1010 patients were male. 588 (58%) of 1010 patients were White, 188 (19%) were Black, 70 (7%) were Asian, and 18 (2%) were American Indian or Alaska Native. 347 (34%) of 1010 patients were Hispanic or Latino. Mechanical ventilation-free survival by day 29 was similar between the study groups (Kaplan-Meier estimates of 87·0% [95% CI 83·7 to 89·6] in the baricitinib plus remdesivir plus placebo group and 87·6% [84·2 to 90·3] in the dexamethasone plus remdesivir plus placebo group; risk difference 0·6 [95% CI -3·6 to 4·8]; p=0·91). The odds ratio for improved status in the dexamethasone plus remdesivir plus placebo group compared with the baricitinib plus remdesivir plus placebo group was 1·01 (95% CI 0·80 to 1·27). At least one adverse event occurred in 149 (30%) of 503 patients in the baricitinib plus remdesivir plus placebo group and 179 (37%) of 482 patients in the dexamethasone plus remdesivir plus placebo group (risk difference 7·5% [1·6 to 13·3]; p=0·014). 21 (4%) of 503 patients in the baricitinib plus remdesivir plus placebo group had at least one treatment-related adverse event versus 49 (10%) of 482 patients in the dexamethasone plus remdesivir plus placebo group (risk difference 6·0% [2·8 to 9·3]; p=0·00041). Severe or life-threatening grade 3 or 4 adverse events occurred in 143 (28%) of 503 patients in the baricitinib plus remdesivir plus placebo group and 174 (36%) of 482 patients in the dexamethasone plus remdesivir plus placebo group (risk difference 7·7% [1·8 to 13·4]; p=0·012).InterpretationIn hospitalised patients with COVID-19 requiring supplemental oxygen by low-flow, high-flow, or non-invasive ventilation, baricitinib plus remdesivir and dexamethasone plus remdesivir resulted in similar mechanical ventilation-free survival by day 29, but dexamethasone was associated with significantly more adverse events, treatment-related adverse events, and severe or life-threatening adverse events. A more individually tailored choice of immunomodulation now appears possible, where side-effect profile, ease of administration, cost, and patient comorbidities can all be considered.FundingNational Institute of Allergy and Infectious Diseases.

Despite the success of antiretroviral therapy (ART) for people living with HIV, lifelong treatment is required and there is no cure. HIV can integrate in the host genome and persist for the life span of the infected cell. These latently infected cells are not recognized as foreign because they are largely transcriptionally silent, but contain replication-competent virus that drives resurgence of the infection once ART is stopped. With a combination of immune activators, neutralizing antibodies, and therapeutic vaccines, some nonhuman primate models have been cured, providing optimism for these approaches now being evaluated in human clinical trials. In vivo delivery of gene-editing tools to either target the virus, boost immunity or protect cells from infection, also holds promise for future HIV cure strategies. In this Review, we discuss advances related to HIV cure in the last 5 years, highlight remaining knowledge gaps and identify priority areas for research for the next 5 years. An effective and scalable cure strategy is a top priority for the HIV research field; this Review discusses recent advances, knowledge gaps, and priority research areas for the next 5 years.

Background The Adaptive Coronavirus Disease 2019 (COVID-19) Treatment Trial-1 (ACTT-1) found that remdesivir therapy hastened recovery in patients hospitalized with COVID-19, but the pathway for this improvement was not explored. We investigated how the dynamics of clinical progression changed along 4 pathways: recovery, improvement in respiratory therapy requirement, deterioration in respiratory therapy requirement, and death. Methods We analyzed trajectories of daily ordinal severity scores reflecting oxygen requirements of 1051 patients hospitalized with COVID-19 who participated in ACTT-1. We developed competing risks models that estimate the effect of remdesivir therapy on cumulative incidence of clinical improvement and deterioration, and multistate models that utilize the entirety of each patient’s clinical course to characterize the effect of remdesivir on progression along the 4 pathways above. Results Based on a competing risks analysis, remdesivir reduced clinical deterioration (hazard ratio [HR], 0.73; 95% confidence interval [CI]: .59–.91) and increased clinical improvement (HR, 1.22; 95% CI: 1.08, 1.39) relative to baseline. Our multistate models indicate that remdesivir inhibits worsening to ordinal scores of greater clinical severity among patients on room air or low-flow oxygen (HR, 0.74; 95% CI: .57–.94) and among patients receiving mechanical ventilation or high-flow oxygen/noninvasive positive-pressure ventilation (HR, 0.73; 95% CI: .53–1.00) at baseline. We also find that remdesivir reduces expected intensive care respiratory therapy utilization among patients not mechanically ventilated at baseline. Conclusions Remdesivir speeds time to recovery by preventing worsening to clinical states that would extend the course of hospitalization and increase intensive respiratory support, thereby reducing the overall demand for hospital care., Remdesivir altered dynamics of clinical progression and sped recovery in hospitalized coronavirus disease 2019 (COVID-2019) patients in Adaptive COVID-19 Treatment Trial-1 (ACTT-1) by preventing clinical deterioration and reducing demand for supplemental oxygen and other respiratory therapies. Remdesivir may therefore improve outcomes and alleviate healthcare system capacity strain.

The potential of immunotherapy strategies utilizing BNAbs such as 3BNC117 and 10-1074 to limit viral replication while also facilitating clearance of HIV infected cells has heightened interest in identifying the predominant NK effector subset(s) capable of mediating Antibody Dependent Cellular Cytotoxicity (ADCC). Utilizing Advanced Poly-chromatic Flow Cytometry, we identified that CD57 positive NK cells from ART-suppressed PLWH expressed significantly higher levels of the CD16 FcγR Receptor, 2B4 ADCC co-receptor and HLA-DR activation marker while NKG2C positive NK cells expressed significantly higher levels of the CD2 ADCC co-receptor (p

In the pilot NCT01935089 trial, we tested whether pegylated interferon alpha2b (Peg-IFN-α2b) with antiretroviral therapy (ART) was safe and could impact HIV and immune measures in blood and in gut-associated lymphoid tissue (GALT). Twenty HIV-1(+) ART-suppressed individuals received 1 μg/kg/week Peg-IFN-α2b with ART for 20 weeks, with intermediate 4-week analytical ART interruption (ATI). Safety, immune activation, HIV viral load and integrated HIV DNA in blood, and HIV RNA and DNA in gut biopsies were measured. A total of 7/20 participants experienced grade 3–4 adverse events, while 17/20 participants completed the study. Of the 17 participants who completed the study, 8 remained suppressed during ATI, while all 17 were suppressed at end of treatment (EoT). As expected, treatment increased activation of T and natural killer (NK) cells and IFN-stimulated molecule expression on monocytes in periphery. While circulating CD4(+) T cells showed a trend for a decrease in integrated HIV DNA, GALT showed a significant decrease in HIV-1 RNA(+) cells as measured by in situ hybridization along with a reduction in total HIV DNA and cell-associated RNA by EoT. The observed decrease in HIV-1 RNA(+) cells in GALT was positively associated with the decrease in activated NK cells and macrophages. This study documents for the first time that 20 weeks of immunotherapy with Peg-IFN-α2b+ART (inclusive of a 4-week ATI) is safe and results in an increase in blood and GALT immune activation and in a significant decrease in HIV-1 RNA(+) cells in GALT in association with changes in innate cell activation.

Background Cytomegalovirus (CMV) infection is thought to result in increased immune activation in people with human immunodeficiency virus (HIV, PWH). Although some data have linked asymptomatic CMV infection to cardiovascular disease among PWH, it remains unknown whether CMV is associated with increased or high-risk coronary plaque. Methods The Randomized Trial to Prevent Vascular Events in HIV (REPRIEVE) enrolled PWH aged 40–75 years on stable antiretroviral therapy (ART) with low-to-moderate atherosclerotic cardiovascular disease (ASCVD) risk. Among a subset of US REPRIEVE participants, coronary plaque was assessed by coronary computed tomography angiography. Here, we assessed the relationship between CMV immunoglobulin G (IgG) titer and (1) levels of immune activation, (2) inflammatory biomarkers, and (3) coronary plaque phenotypes at study entry. Results Of 672 participants, mean age was 51 years, 83% were men, median ASCVD risk score was 4.5%, and 66% had current CD4+ T-cell count ≥500 cells/mm3. Higher CMV IgG quartile group was associated with older age and lower current and nadir CD4+ T-cell counts. CMV IgG titer was associated with specific inflammatory biomarkers (sCD163, MCP-1, interleukin [IL]-6, hsCRP) in univariate analysis, but not after controlling for HIV-specific factors. In contrast, CMV IgG titer was not associated with coronary artery disease indexes, including presence of plaque, coronary artery calcium (CAC) score >0, vulnerable plaque presence, or Leaman score >5. Conclusions No meaningful association was seen between CMV IgG titer and coronary artery disease indexes among ART-treated PWH at study enrollment. Longitudinal assessments in REPRIEVE will determine the relationship of CMV IgG titer to plaque progression and cardiovascular events. Clinical Trials Registration NCT02344290.

Despite effective antiretroviral therapy, HIV-1 persistence in latent reservoirs remains a major obstacle to a cure. We postulate that HIV-1 silencing factors suppress HIV-1 reactivation and that inhibition of these factors will increase HIV-1 reactivation. To identify HIV-1 silencing factors, we conducted a genome-wide CRISPR inhibition (CRISPRi) screen using four CRISPRi-ready, HIV-1-d6-GFP-infected Jurkat T cell clones with distinct integration sites. We sorted cells with increased green fluorescent protein (GFP) expression and captured single guide RNAs (sgRNAs) via targeted deep sequencing. We identified 18 HIV-1 silencing factors that were significantly enriched in HIV-1-d6-GFP(high) cells. Among them, SLTM (scaffold attachment factor B-like transcription modulator) is an epigenetic and transcriptional modulator having both DNA and RNA binding capacities not previously known to affect HIV-1 transcription. Knocking down SLTM by CRISPRi significantly increased HIV-1-d6-GFP expression (by 1.9- to 4.2-fold) in three HIV-1-d6-GFP-Jurkat T cell clones. Furthermore, SLTM knockdown increased the chromatin accessibility of HIV-1 and the gene in which HIV-1 is integrated but not the housekeeping gene POLR2A. To test whether SLTM inhibition can reactivate HIV-1 and further induce cell death of HIV-1-infected cells ex vivo, we established a small interfering RNA (siRNA) knockdown method that reduced SLTM expression in CD4(+) T cells from 10 antiretroviral therapy (ART)-treated, virally suppressed, HIV-1-infected individuals ex vivo. Using limiting dilution culture, we found that SLTM knockdown significantly reduced the frequency of HIV-1-infected cells harboring inducible HIV-1 by 62.2% (0.56/10(6) versus 1.48/10(6) CD4(+) T cells [P = 0.029]). Overall, our study indicates that SLTM inhibition reactivates HIV-1 in vitro and induces cell death of HIV-1-infected cells ex vivo. Our study identified SLTM as a novel therapeutic target. IMPORTANCE HIV-1-infected cells, which can survive drug treatment and immune cell killing, prevent an HIV-1 cure. Immune recognition of infected cells requires HIV-1 protein expression; however, HIV-1 protein expression is limited in infected cells after long-term therapy. The ways in which the HIV-1 provirus is blocked from producing protein are unknown. We identified a new host protein that regulates HIV-1 gene expression. We also provided a new method of studying HIV-1–host factor interactions in cells from infected individuals. These improvements may enable future strategies to reactivate HIV-1 in infected individuals so that infected cells can be killed by immune cells, drug treatment, or the virus itself.

In untreated HIV-1 infection, rapid viral evolution allows escape from immune responses. Viral replication can be blocked by antiretroviral therapy. However, HIV-1 persists in a latent reservoir in resting CD4(+) T cells, and rebound viremia occurs following treatment interruption. The reservoir, which is maintained in part by clonal expansion, can be measured using quantitative viral outgrowth assays (QVOAs) in which latency is reversed with T cell activation to allow viral outgrowth. Recent studies have shown that viruses detected in QVOAs prior to treatment interruption often differ from rebound viruses. We hypothesized that autologous neutralizing antibodies directed at the HIV-1 envelope (Env) protein might block outgrowth of some reservoir viruses. We modified the QVOA to reflect pressure from low concentrations of autologous antibodies and showed that outgrowth of a substantial but variable fraction of reservoir viruses is blocked by autologous contemporaneous immunoglobulin G (IgG). A reduction in outgrowth of >80% was seen in 6 of 15 individuals. This effect was due to direct neutralization. We established a phylogenetic relationship between rebound viruses and viruses growing out in vitro in the presence of autologous antibodies. Some large infected cell clones detected by QVOA carried neutralization-sensitive viruses, providing a cogent explanation for differences between rebound virus and viruses detected in standard QVOAs. Measurement of the frequency of reservoir viruses capable of outgrowth in the presence of autologous IgG might allow more accurate prediction of time to viral rebound. Ultimately, therapeutic immunization targeting the subset of variants resistant to autologous IgG might contribute to a functional cure.

Accurate characterization of the human immunodeficiency virus (HIV) reservoir is imperative to develop an effective cure. HIV was measured in antiretroviral therapy-suppressed individuals using the intact proviral DNA assay (IPDA), along with assays for total or integrated HIV DNA, and inducible HIV RNA or p24. Intact provirus correlated with total and integrated HIV.

Understanding the drivers and markers of clonally expanding HIV-1-infected CD4(+) T cells is essential for HIV-1 eradication. We used single-cell ECCITE-seq, which captures surface protein expression, cellular transcriptome, HIV-1 RNA, and TCR sequences within the same single cell to track clonal expansion dynamics in longitudinally archived samples from six HIV-1-infected individuals (during viremia and after suppressive antiretroviral therapy) and two uninfected individuals, in unstimulated conditions and after CMV and HIV-1 antigen stimulation. Despite antiretroviral therapy, persistent antigen and TNF responses shaped T cell clonal expansion. HIV-1 resided in Th1 polarized, antigen-responding T cells expressing BCL2 and SERPINB9 that may resist cell death. HIV-1 RNA(+) T cell clones were larger in clone size, established during viremia, persistent after viral suppression, and enriched in GZMB(+) cytotoxic effector memory Th1 cells. Targeting HIV-1-infected cytotoxic CD4(+) T cells and drivers of clonal expansion provides another direction for HIV-1 eradication.

Background The Adaptive COVID Treatment Trial-2 (ACTT-2) found that baricitinib in combination with remdesivir therapy (BCT) sped recovery in hospitalized coronavirus disease 2019 (COVID-19) patients vs remdesivir monotherapy (RMT). We examined how BCT affected progression throughout hospitalization and utilization of intensive respiratory therapies. Methods We characterized the clinical trajectories of 891 ACTT-2 participants requiring supplemental oxygen or higher levels of respiratory support at enrollment. We estimated the effect of BCT on cumulative incidence of clinical improvement and deterioration using competing risks models. We developed multistate models to estimate the effect of BCT on clinical improvement and deterioration and on utilization of respiratory therapies. Results BCT resulted in more linear improvement and lower incidence of clinical deterioration compared with RMT (hazard ratio [HR], 0.74; 95% CI, 0.58 to 0.95). The benefit was pronounced among participants enrolled on high-flow oxygen or noninvasive positive-pressure ventilation. In this group, BCT sped clinical improvement (HR, 1.21; 95% CI, 0.99 to 1.51) while slowing clinical deterioration (HR, 0.71; 95% CI, 0.48 to 1.02), which reduced the expected days in ordinal score (OS) 6 per 100 patients by 74 days (95% CI, −8 to 154 days) and the expected days in OS 7 per 100 patients by 161 days (95% CI, 46 to 291 days) compared with RMT. BCT did not benefit participants who were mechanically ventilated at enrollment. Conclusions Compared with RMT, BCT reduces the clinical burden and utilization of intensive respiratory therapies for patients requiring low-flow oxygen or noninvasive positive-pressure ventilation compared with RMT and may thereby improve care for this patient population.

Coronavirus disease 2019 (COVID-19) convalescent plasma (CCP), a passive polyclonal antibody therapeutic agent, has had mixed clinical results. Although antibody neutralization is the predominant approach to benchmarking CCP efficacy, CCP may also influence the evolution of the endogenous antibody response. Using systems serology to comprehensively profile severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) functional antibodies of hospitalized people with COVID-19 enrolled in a randomized controlled trial of CCP (ClinicalTrials.gov: NCT04397757), we find that the clinical benefits of CCP are associated with a shift toward reduced inflammatory Spike (S) responses and enhanced nucleocapsid (N) humoral responses. We find that CCP has the greatest clinical benefit in participants with low pre-existing anti-SARS-CoV-2 antibody function and that CCP-induced immunomodulatory Fc glycan profiles and N immunodominant profiles persist for at least 2 months. We highlight a potential mechanism of action of CCP associated with durable immunomodulation, outline optimal patient characteristics for CCP treatment, and provide guidance for development of a different class of COVID-19 hyperinflammation-targeting antibody therapeutic agents.

Frequent viral load testing is necessary during analytical treatment interruptions (ATIs) in HIV cure-directed clinical trials, though such may be burdensome and inconvenient to trial participants. We implemented a national, cross-sectional survey in the United States to examine the acceptability of a novel home-based peripheral blood collection device for HIV viral load testing. Between June and August 2021, we distributed an online survey to people with HIV (PWH) and community members, biomedical HIV cure researchers and HIV care providers. We performed descriptive analyses to summarize the results. We received 73 survey responses, with 51 from community members, 12 from biomedical HIV cure researchers and 10 from HIV care providers. Of those, 51 (70%) were cisgender men and 50 (68%) reported living with HIV. Most (>80% overall) indicated that the device would be helpful during ATI trials and they would feel comfortable using it themselves or recommending it to their patients/participants. Of the 50 PWH, 42 (84%) indicated they would use the device if they were participating in an ATI trial and 27 (54%) also expressed a willingness to use the device outside of HIV cure studies. Increasing sensitivity of viral load tests and pluri-potency of the device (CD4 count, chemistries) would augment acceptability. Survey findings provide evidence that viral load home testing would be an important adjunct to ongoing HIV cure-directed trials involving ATIs. Survey findings may help inform successful implementation and uptake of the device in the context of personalized HIV care.

Key Points Question What is the pooled evidence from high-quality randomized clinical trials regarding the safety and potential benefit of convalescent plasma to treat hospitalized patients with COVID-19? Findings In this meta-analysis of 8 randomized clinical trials enrolling 2341 participants, individual patient data were monitored in real time and analyzed using a robust bayesian framework and advanced statistical modeling. No association of convalescent plasma with clinical outcomes was found. Meaning These findings suggest that real-time individual patient data pooling and meta-analysis during a pandemic are feasible, offering a model for future research and providing a rich data resource., This meta-analysis examines individual patient data from 8 randomized clinical trials of COVID-19 convalescent plasma to determine its safety and efficacy in treating hospitalized patients with COVID-19., Importance COVID-19 convalescent plasma (CCP) is a potentially beneficial treatment for COVID-19 that requires rigorous testing. Objective To compile individual patient data from randomized clinical trials of CCP and to monitor the data until completion or until accumulated evidence enables reliable conclusions regarding the clinical outcomes associated with CCP. Data Sources From May to August 2020, a systematic search was performed for trials of CCP in the literature, clinical trial registry sites, and medRxiv. Domain experts at local, national, and international organizations were consulted regularly. Study Selection Eligible trials enrolled hospitalized patients with confirmed COVID-19, not receiving mechanical ventilation, and randomized them to CCP or control. The administered CCP was required to have measurable antibodies assessed locally. Data Extraction and Synthesis A minimal data set was submitted regularly via a secure portal, analyzed using a prespecified bayesian statistical plan, and reviewed frequently by a collective data and safety monitoring board. Main Outcomes and Measures Prespecified coprimary end points—the World Health Organization (WHO) 11-point ordinal scale analyzed using a proportional odds model and a binary indicator of WHO score of 7 or higher capturing the most severe outcomes including mechanical ventilation through death and analyzed using a logistic model—were assessed clinically at 14 days after randomization. Results Eight international trials collectively enrolled 2369 participants (1138 randomized to control and 1231 randomized to CCP). A total of 2341 participants (median [IQR] age, 60 [50-72] years; 845 women [35.7%]) had primary outcome data as of April 2021. The median (IQR) of the ordinal WHO scale was 3 (3-6); the cumulative OR was 0.94 (95% credible interval [CrI], 0.74-1.19; posterior probability of OR

SummaryCOVID-19 convalescent plasma (CCP), a passive polyclonal antibody therapeutic, has exhibited mixed results in the treatment of COVID-19. Given that the therapeutic effect of CCP may extend beyond the ability of SARS-CoV-2-specific antibody binding and neutralization to influence the evolution of the endogenous antibody response, we took a systematic and comprehensive approach to analyze SARS-CoV-2 functional antibody profiles of participants in a randomized controlled trial of CCP treatment of individuals hospitalized with COVID-19 pneumonia where CCP was associated with both decreased mortality and improved clinical severity. Using systems serology, we found that the clinical benefit of CCP is related to a shift towards reduced inflammatory Spike (S) responses and enhanced Nucleocapsid (N) humoral responses. We found CCP had the greatest clinical benefit in participants with low pre-existing anti-SARS-CoV-2 antibody function, rather than S or N antibody levels or participant demographic features. Further, CCP induced immunomodulatory changes to recipient humoral profiles persisted for at least two months, marked by the selective evolution of anti-inflammatory Fc-glycan profiles and persistently expanded nucleocapsid-specific humoral immunity following CCP therapy. Together, our findings identify a novel mechanism of action of CCP, suggest optimal patient characteristics for CCP treatment, identify long-last immunomodulatory effects of CCP, and provide guidance for development of novel N-focused antibody therapeutics for severe COVID-19 hyperinflammation.

BACKGROUND: Antibody-based strategies for COVID-19 have shown promise in prevention and treatment of early disease. COVID-19 convalescent plasma (CCP) has been widely used but results from randomized trials supporting its benefit in hospitalized patients with pneumonia are limited. Here, we assess the efficacy of CCP in severely ill, hospitalized adults with COVID-19 pneumonia. METHODS: We performed a randomized control trial (PennCCP2), with 80 adults hospitalized with COVID-19 pneumonia, comparing up to 2 units of locally sourced CCP plus standard care versus standard care alone. The primary efficacy endpoint was comparison of a clinical severity score. Key secondary outcomes include 14- and 28-day mortality, 14- and 28-day maximum 8-point WHO ordinal score (WHO8) score, duration of supplemental oxygenation or mechanical ventilation, respiratory SARS-CoV-2 RNA, and anti–SARS-CoV-2 antibodies. RESULTS: Eighty hospitalized adults with confirmed COVID-19 pneumonia were enrolled at median day 6 of symptoms and day 1 of hospitalization; 60% were anti–SARS-CoV-2 antibody seronegative. Participants had a median of 3 comorbidities, including risk factors for severe COVID-19 and immunosuppression. CCP treatment was safe and conferred significant benefit by clinical severity score (median [MED] and interquartile range [IQR] 10 [5.5–30] vs. 7 [2.75–12.25], P = 0.037) and 28-day mortality (n = 10, 26% vs. n = 2, 5%; P = 0.013). All other prespecified outcome measures showed weak evidence toward benefit of CCP. CONCLUSION: Two units of locally sourced CCP administered early in hospitalization to majority seronegative participants conferred a significant benefit in clinical severity score and 28-day mortality. Results suggest CCP may benefit select populations, especially those with comorbidities who are treated early. TRIAL REGISTRATION: ClinicalTrials.gov NCT04397757. FUNDING: University of Pennsylvania.

Antivirals are urgently needed to combat the global SARS-CoV-2/COVID-19 pandemic, supplement existing vaccine efforts, and target emerging SARS-CoV-2 variants of concern. Small molecules that interfere with binding of the viral spike receptor binding domain (RBD) to the host angiotensin-converting enzyme II (ACE2) receptor may be effective inhibitors of SARS-CoV-2 cell entry. Here, we screened 512 pure compounds derived from natural products using a high-throughput RBD/ACE2 binding assay and identified (–)-hopeaphenol, a resveratrol tetramer, in addition to vatalbinoside A and vaticanol B, as potent and selective inhibitors of RBD/ACE2 binding and viral entry. For example, (–)-hopeaphenol disrupted RBD/ACE2 binding with a 50% inhibitory concentration (IC50) of 0.11 μM, in contrast to an IC50 of 28.3 μM against the unrelated host ligand/receptor binding pair PD-1/PD-L1 (selectivity index, 257.3). When assessed against the USA-WA1/2020 variant, (–)-hopeaphenol also inhibited entry of a VSVΔG-GFP reporter pseudovirus expressing SARS-CoV-2 spike into ACE2-expressing Vero-E6 cells and in vitro replication of infectious virus in cytopathic effect and yield reduction assays (50% effective concentrations [EC50s], 10.2 to 23.4 μM) without cytotoxicity and approaching the activities of the control antiviral remdesivir (EC50s, 1.0 to 7.3 μM). Notably, (–)-hopeaphenol also inhibited two emerging variants of concern, B.1.1.7/Alpha and B.1.351/Beta in both viral and spike-containing pseudovirus assays with similar or improved activities over the USA-WA1/2020 variant. These results identify (–)-hopeaphenol and related stilbenoid analogues as potent and selective inhibitors of viral entry across multiple SARS-CoV-2 variants of concern.

Summary Background Functional impairment of interferon, a natural antiviral component of the immune system, is associated with the pathogenesis and severity of COVID-19. We aimed to compare the efficacy of interferon beta-1a in combination with remdesivir compared with remdesivir alone in hospitalised patients with COVID-19. Methods We did a double-blind, randomised, placebo-controlled trial at 63 hospitals across five countries (Japan, Mexico, Singapore, South Korea, and the USA). Eligible patients were hospitalised adults (aged ≥18 years) with SARS-CoV-2 infection, as confirmed by a positive RT-PCR test, and who met one of the following criteria suggestive of lower respiratory tract infection: the presence of radiographic infiltrates on imaging, a peripheral oxygen saturation on room air of 94% or less, or requiring supplemental oxygen. Patients were excluded if they had either an alanine aminotransferase or an aspartate aminotransferase concentration more than five times the upper limit of normal; had impaired renal function; were allergic to the study product; were pregnant or breast feeding; were already on mechanical ventilation; or were anticipating discharge from the hospital or transfer to another hospital within 72 h of enrolment. Patients were randomly assigned (1:1) to receive intravenous remdesivir as a 200 mg loading dose on day 1 followed by a 100 mg maintenance dose administered daily for up to 9 days and up to four doses of either 44 μg interferon beta-1a (interferon beta-1a group plus remdesivir group) or placebo (placebo plus remdesivir group) administered subcutaneously every other day. Randomisation was stratified by study site and disease severity at enrolment. Patients, investigators, and site staff were masked to interferon beta-1a and placebo treatment; remdesivir treatment was given to all patients without masking. The primary outcome was time to recovery, defined as the first day that a patient attained a category 1, 2, or 3 score on the eight-category ordinal scale within 28 days, assessed in the modified intention-to-treat population, defined as all randomised patients who were classified according to actual clinical severity. Safety was assessed in the as-treated population, defined as all patients who received at least one dose of the assigned treatment. This trial is registered with ClinicalTrials.gov , NCT04492475 . Findings Between Aug 5, 2020, and Nov 11, 2020, 969 patients were enrolled and randomly assigned to the interferon beta-1a plus remdesivir group (n=487) or to the placebo plus remdesivir group (n=482). The mean duration of symptoms before enrolment was 8·7 days (SD 4·4) in the interferon beta-1a plus remdesivir group and 8·5 days (SD 4·3) days in the placebo plus remdesivir group. Patients in both groups had a time to recovery of 5 days (95% CI not estimable) (rate ratio of interferon beta-1a plus remdesivir group vs placebo plus remdesivir 0·99 [95% CI 0·87–1·13]; p=0·88). The Kaplan-Meier estimate of mortality at 28 days was 5% (95% CI 3–7%) in the interferon beta-1a plus remdesivir group and 3% (2–6%) in the placebo plus remdesivir group (hazard ratio 1·33 [95% CI 0·69–2·55]; p=0·39). Patients who did not require high-flow oxygen at baseline were more likely to have at least one related adverse event in the interferon beta-1a plus remdesivir group (33 [7%] of 442 patients) than in the placebo plus remdesivir group (15 [3%] of 435). In patients who required high-flow oxygen at baseline, 24 (69%) of 35 had an adverse event and 21 (60%) had a serious adverse event in the interferon beta-1a plus remdesivir group compared with 13 (39%) of 33 who had an adverse event and eight (24%) who had a serious adverse event in the placebo plus remdesivir group. Interpretation Interferon beta-1a plus remdesivir was not superior to remdesivir alone in hospitalised patients with COVID-19 pneumonia. Patients who required high-flow oxygen at baseline had worse outcomes after treatment with interferon beta-1a compared with those given placebo. Funding The National Institute of Allergy and Infectious Diseases (USA).

Summary Background Infection with influenza virus causes substantial morbidity and mortality globally, although antiviral treatments are available. Previous studies have suggested that anti-influenza immune plasma could be beneficial as treatment, but they were not designed as randomised, blinded, placebo-controlled trials. Therefore, we aimed to prospectively evaluate the clinical efficacy of high-titre immune plasma compared with standard low-titre plasma to improve outcomes in patients with severe influenza A infection. Methods We did this randomised, double-blind, phase 3 trial at 41 US medical centres to assess the efficacy of high-titre anti-influenza plasma (haemagglutination inhibition antibody titre ≥1:80) compared with low-titre plasma (≤1:10). Children and adults with PCR-confirmed influenza A infection, a National Early Warning score of 3 or greater, and onset of illness within 6 days before randomisation were eligible. Patients were randomly assigned (2:1) using an interactive web response system to receive either two units (or paediatric equivalent) of high-titre plasma (high-titre group) or low-titre plasma (low-titre group), and were followed up for 28 days from randomisation. High-titre and low-titre plasma had the same appearance. Randomisation was stratified by severity (in intensive care unit, not in intensive care but requiring supplemental oxygen, or not in intensive care and not requiring supplemental oxygen) and age ( 1 indicates improvement in clinical status across all categories for the high-titre vs the low-titre group). The primary analysis was done in the intention-to-treat population, excluding two participants who did not receive plasma. This trial is registered with ClinicalTrials.gov , NCT02572817 . Findings Participants were recruited between Jan 26, 2016, and April 19, 2018. Of 200 participants enrolled (177 adults and 23 children), 140 met the criteria for randomisation and were assigned to the high-titre group (n=92) or to the control low-titre group (n=48). One participant from each group did not receive plasma. At baseline, 60 (43%) of 138 participants were in intensive care and 55 (71%) of 78 participants who were not in intensive care required oxygen. 93% of planned plasma infusions were completed. The study was terminated in July, 2018, when independent efficacy analysis showed low conditional power to detect an effect of high-titre plasma even if full accrual (150 participants) was achieved. The proportional OR for improved clinical status on day 7 was 1·22 (95% CI 0·65–2·29, p=0·54). 47 (34%) of 138 participants experienced 88 serious adverse events: 32 (35%) with 60 events in the high-titre group and 15 (32%) with 28 events in the low-titre group. The most common serious adverse events were acute respiratory distress syndrome (ARDS; four [4%] vs two [4%]), allergic transfusion reactions (two [2%] vs two [4%]), and respiratory distress (three [3%] vs none). 65 (47%) participants experienced 183 adverse events: 42 (46%) with 126 events in the high-titre group and 23 (49%) with 57 events in the low-titre group. The most common adverse events were anaemia (four [3%] vs two [4%]) and ARDS (four [3%] vs three [5%]). Ten patients died during the study (six [7%] in the high-titre group vs four [9%] in the low-titre group, p=0·73). The most common cause of death was worsening of acute respiratory distress syndrome (two [2%] vs two [4%] patients). Interpretation High-titre anti-influenza plasma conferred no significant benefit over non-immune plasma. Although our study did not have the precision to rule out a small, clinically relevant effect, the benefit is insufficient to justify the use of immune plasma for treating patients with severe influenza A. Funding National Institute of Allergy and Infectious Diseases of the National Institutes of Health (Bethesda, MD, USA).

Background Statins exert pleiotropic anti-inflammatory and immune-modulatory effects, which might translate into antiviral activity. We evaluated whether reported current statin exposure is associated with lower levels of markers of HIV persistence and immune activation/inflammation. Methods We compared levels of markers of HIV viral persistence [cell-associated HIV RNA (CA-RNA), CA-DNA, and single copy assay plasma HIV RNA] and immune activation/inflammation (IL-6, IP-10, neopterin, sCD14, sCD163, and TNF-alpha) between statin users and nonusers among participants of ACTG A5321 who initiated antiretroviral therapy (ART) during chronic infection and maintained virologic suppression (HIV-1 RNA levels ≤50 copies/mL) for ≥3 years. Results A total of 303 participants were analyzed. Median time on the current statin was 2.9 years (1.2-5.1). There were no differences between statin users and nonusers in levels of CA-DNA (median 650 vs. 540 copies/10 CD4 T cells; P = 0.58), CA-RNA (53 vs. 37 copies/10 CD4 T cells; P = 0.12), or single copy assay (0.4 vs. 0.4 copies/mL; P = 0.45). Similarly, there were no significant differences between statin users and nonusers in markers of inflammation/activation, except for IP-10 (137 vs. 118 pg/mL; P = 0.028). Findings were unchanged after adjustment for factors including pre-ART CD4 and HIV RNA, and years on ART. Conclusions In this cohort of persons on long-term suppressive ART, current statin use was not associated with lower levels of HIV persistence or immune activation/inflammation. These results do not support a major role for statins in reducing HIV persistence, although an early transient effect cannot be excluded. Prospective, randomized studies are needed to confirm these findings.

We previously reported that pegylated IFN-α2a (Peg–IFN-α2a) added to antiretroviral therapy (ART)–suppressed, HIV-infected subjects resulted in plasma HIV control and integrated HIV DNA decrease. We now evaluated whether innate NK cell activity or PBMC transcriptional profiles were associated with decreases in HIV measures. Human peripheral blood was analyzed prior to Peg–IFN-α2a administration (ART, baseline), after 5 wk of ART+Peg–IFN-α2a, and after 12 wk of Peg–IFN-α2a monotherapy (primary endpoint). After 5 wk of ART+Peg–IFN-α2a, immune subset frequencies were preserved, and induction of IFN-stimulated genes was noted in all subjects except for a subset in which the lack of IFN-stimulated gene induction was associated with increased expression of microRNAs. Viral control during Peg–IFN-α2a monotherapy was associated with 1) higher levels of NK cell activity and IFN-γ–induced protein 10 (IP-10) on ART (preimmunotherapy) and 2) downmodulation of NK cell KIR2DL1 and KIR2DL2/DL3 expression, transcriptional enrichment of expression of genes associated with NK cells in HIV controller subjects, and higher ex vivo IFN-α–induced NK cytotoxicity after 5 wk of ART+Peg–IFN-α2a. Integrated HIV DNA decline after immunotherapy was also associated with gene expression patterns indicative of cell-mediated activation and NK cytotoxicity. Overall, an increase in innate activity and NK cell cytotoxicity were identified as correlates of Peg–IFN-α2a–mediated HIV control.