Bispecific antibodies promote natural killer cell-mediated elimination of HIV-1 reservoir cells.

The persistence of CD4+ T cells carrying latent human immunodeficiency virus-1 (HIV-1) proviruses is the main barrier to a cure. New therapeutics to enhance HIV-1-specific immune responses and clear infected cells will probably be necessary to achieve reduction of the latent reservoir. In the present study, we report two single-chain diabodies (scDbs) that target the HIV-1 envelope protein (Env) and the human type III Fcγ receptor (CD16). We show that the scDbs promoted robust and HIV-1-specific natural killer (NK) cell activation and NK cell-mediated lysis of infected cells. Cocultures of CD4+ T cells from people with HIV-1 on antiretroviral therapy (ART) with autologous NK cells and the scDbs resulted in marked elimination of reservoir cells that was dependent on latency reversal. Treatment of human interleukin-15 transgenic NSG mice with one of the scDbs after ART initiation enhanced NK cell activity and reduced reservoir size. Thus, HIV-1-specific scDbs merit further evaluation as potential therapeutics for clearance of the latent reservoir.

Long COVID manifests with T cell dysregulation, inflammation and an uncoordinated adaptive immune response to SARS-CoV-2.

Long COVID (LC) occurs after at least 10% of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, yet its etiology remains poorly understood. We used 'omic" assays and serology to deeply characterize the global and SARS-CoV-2-specific immunity in the blood of individuals with clear LC and non-LC clinical trajectories, 8 months postinfection. We found that LC individuals exhibited systemic inflammation and immune dysregulation. This was evidenced by global differences in T cell subset distribution implying ongoing immune responses, as well as by sex-specific perturbations in cytolytic subsets. LC individuals displayed increased frequencies of CD4+ T cells poised to migrate to inflamed tissues and exhausted SARS-CoV-2-specific CD8+ T cells, higher levels of SARS-CoV-2 antibodies and a mis-coordination between their SARS-CoV-2-specific T and B cell responses. Our analysis suggested an improper crosstalk between the cellular and humoral adaptive immunity in LC, which can lead to immune dysregulation, inflammation and clinical symptoms associated with this debilitating condition.

Multiple Origins of Virus Persistence during Natural Control of HIV Infection.

Targeted HIV cure strategies require definition of the mechanisms that maintain the virus. Here, we tracked HIV replication and the persistence of infected CD4 T cells in individuals with natural virologic control by sequencing viruses, T cell receptor genes, HIV integration sites, and cellular transcriptomes. Our results revealed three mechanisms of HIV persistence operating within distinct anatomic and functional compartments. In lymph node, we detected viruses with genetic and transcriptional attributes of active replication in both T follicular helper (TFH) cells and non-TFH memory cells. In blood, we detected inducible proviruses of archival origin among highly differentiated, clonally expanded cells. Linking the lymph node and blood was a small population of circulating cells harboring inducible proviruses of recent origin. Thus, HIV replication in lymphoid tissue, clonal expansion of infected cells, and recirculation of recently infected cells act together to maintain the virus in HIV controllers despite effective antiviral immunity.

HIV silencing and cell survival signatures in infected T cell reservoirs.

Rare CD4 T cells that contain HIV under antiretroviral therapy represent an important barrier to HIV cure1-3, but the infeasibility of isolating and characterizing these cells in their natural state has led to uncertainty about whether they possess distinctive attributes that HIV cure-directed therapies might exploit. Here we address this challenge using a microfluidic technology that isolates the transcriptomes of HIV-infected cells based solely on the detection of HIV DNA. HIV-DNA+ memory CD4 T cells in the blood from people receiving antiretroviral therapy showed inhibition of six transcriptomic pathways, including death receptor signalling, necroptosis signalling and antiproliferative Gα12/13 signalling. Moreover, two groups of genes identified by network co-expression analysis were significantly associated with HIV-DNA+ cells. These genes (n = 145) accounted for just 0.81% of the measured transcriptome and included negative regulators of HIV transcription that were higher in HIV-DNA+ cells, positive regulators of HIV transcription that were lower in HIV-DNA+ cells, and other genes involved in RNA processing, negative regulation of mRNA translation, and regulation of cell state and fate. These findings reveal that HIV-infected memory CD4 T cells under antiretroviral therapy are a distinctive population with host gene expression patterns that favour HIV silencing, cell survival and cell proliferation, with important implications for the development of HIV cure strategies.

A common allele of HLA is associated with asymptomatic SARS-CoV-2 infection.

Studies have demonstrated that at least 20% of individuals infected with SARS-CoV-2 remain asymptomatic1-4. Although most global efforts have focused on severe illness in COVID-19, examining asymptomatic infection provides a unique opportunity to consider early immunological features that promote rapid viral clearance. Here, postulating that variation in the human leukocyte antigen (HLA) loci may underly processes mediating asymptomatic infection, we enrolled 29,947 individuals, for whom high-resolution HLA genotyping data were available, in a smartphone-based study designed to track COVID-19 symptoms and outcomes. Our discovery cohort (n = 1,428) comprised unvaccinated individuals who reported a positive test result for SARS-CoV-2. We tested for association of five HLA loci with disease course and identified a strong association between HLA-B*15:01 and asymptomatic infection, observed in two independent cohorts. Suggesting that this genetic association is due to pre-existing T cell immunity, we show that T cells from pre-pandemic samples from individuals carrying HLA-B*15:01 were reactive to the immunodominant SARS-CoV-2 S-derived peptide NQKLIANQF. The majority of the reactive T cells displayed a memory phenotype, were highly polyfunctional and were cross-reactive to a peptide derived from seasonal coronaviruses. The crystal structure of HLA-B*15:01-peptide complexes demonstrates that the peptides NQKLIANQF and NQKLIANAF (from OC43-CoV and HKU1-CoV) share a similar ability to be stabilized and presented by HLA-B*15:01. Finally, we show that the structural similarity of the peptides underpins T cell cross-reactivity of high-affinity public T cell receptors, providing the molecular basis for HLA-B*15:01-mediated pre-existing immunity.

Distinct viral reservoirs in individuals with spontaneous control of HIV-1.

Sustained, drug-free control of HIV-1 replication is naturally achieved in less than 0.5% of infected individuals (here termed 'elite controllers'), despite the presence of a replication-competent viral reservoir1. Inducing such an ability to spontaneously maintain undetectable plasma viraemia is a major objective of HIV-1 cure research, but the characteristics of proviral reservoirs in elite controllers remain to be determined. Here, using next-generation sequencing of near-full-length single HIV-1 genomes and corresponding chromosomal integration sites, we show that the proviral reservoirs of elite controllers frequently consist of oligoclonal to near-monoclonal clusters of intact proviral sequences. In contrast to individuals treated with long-term antiretroviral therapy, intact proviral sequences from elite controllers were integrated at highly distinct sites in the human genome and were preferentially located in centromeric satellite DNA or in Krüppel-associated box domain-containing zinc finger genes on chromosome 19, both of which are associated with heterochromatin features. Moreover, the integration sites of intact proviral sequences from elite controllers showed an increased distance to transcriptional start sites and accessible chromatin of the host genome and were enriched in repressive chromatin marks. These data suggest that a distinct configuration of the proviral reservoir represents a structural correlate of natural viral control, and that the quality, rather than the quantity, of viral reservoirs can be an important distinguishing feature for a functional cure of HIV-1 infection. Moreover, in one elite controller, we were unable to detect intact proviral sequences despite analysing more than 1.5 billion peripheral blood mononuclear cells, which raises the possibility that a sterilizing cure of HIV-1 infection, which has previously been observed only following allogeneic haematopoietic stem cell transplantation2,3, may be feasible in rare instances.

A cohort-based study of host gene expression: tumor suppressor and innate immune/inflammatory pathways associated with the HIV reservoir size.

The major barrier to an HIV cure is the HIV reservoir: latently-infected cells that persist despite effective antiretroviral therapy (ART). There have been few cohort-based studies evaluating host genomic or transcriptomic predictors of the HIV reservoir. We performed host RNA sequencing and HIV reservoir quantification (total DNA [tDNA], unspliced RNA [usRNA], intact DNA) from peripheral CD4+ T cells from 191 ART-suppressed people with HIV (PWH). After adjusting for nadir CD4+ count, timing of ART initiation, and genetic ancestry, we identified two host genes for which higher expression was significantly associated with smaller total DNA viral reservoir size, P3H3 and NBL1, both known tumor suppressor genes. We then identified 17 host genes for which lower expression was associated with higher residual transcription (HIV usRNA). These included novel associations with membrane channel (KCNJ2, GJB2), inflammasome (IL1A, CSF3, TNFAIP5, TNFAIP6, TNFAIP9, CXCL3, CXCL10), and innate immunity (TLR7) genes (FDR-adjusted q<0.05). Gene set enrichment analyses further identified significant associations of HIV usRNA with TLR4/microbial translocation (q = 0.006), IL-1/NRLP3 inflammasome (q = 0.008), and IL-10 (q = 0.037) signaling. Protein validation assays using ELISA and multiplex cytokine assays supported these observed inverse host gene correlations, with P3H3, IL-10, and TNF-α protein associations achieving statistical significance (p<0.05). Plasma IL-10 was also significantly inversely associated with HIV DNA (p = 0.016). HIV intact DNA was not associated with differential host gene expression, although this may have been due to a large number of undetectable values in our study. To our knowledge, this is the largest host transcriptomic study of the HIV reservoir. Our findings suggest that host gene expression may vary in response to the transcriptionally active reservoir and that changes in cellular proliferation genes may influence the size of the HIV reservoir. These findings add important data to the limited host genetic HIV reservoir studies to date.

Low Prevalence of Interferon α Autoantibodies in People Experiencing Symptoms of Post-Coronavirus Disease 2019 (COVID-19) Conditions, or Long COVID.

Interferon (IFN)-specific autoantibodies have been implicated in severe coronavirus disease 2019 (COVID-19) and have been proposed as a potential driver of the persistent symptoms characterizing "long COVID," a type of postacute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We report that only 2 of 215 participants with convalescent SARS-CoV-2 infection tested over 394 time points, including 121 people experiencing long COVID symptoms, had detectable IFN-α2 antibodies. Both had been hospitalized during the acute phase of the infection. These data suggest that persistent anti-IFN antibodies, although a potential driver of severe COVID-19, are unlikely to contribute to long COVID symptoms in the postacute phase of the infection.

Reduced Exercise Capacity, Chronotropic Incompetence, and Early Systemic Inflammation in Cardiopulmonary Phenotype Long Coronavirus Disease 2019.

BACKGROUND: Mechanisms underlying persistent cardiopulmonary symptoms after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (postacute sequelae of coronavirus disease 2019 [COVID-19; PASC] or "long COVID") remain unclear. This study sought to elucidate mechanisms of cardiopulmonary symptoms and reduced exercise capacity. METHODS: We conducted cardiopulmonary exercise testing (CPET), cardiac magnetic resonance imaging (CMR) and ambulatory rhythm monitoring among adults >1 year after SARS-CoV-2 infection, compared those with and those without symptoms, and correlated findings with previously measured biomarkers. RESULTS: Sixty participants (median age, 53 years; 42% female; 87% nonhospitalized; median 17.6 months after infection) were studied. At CPET, 18/37 (49%) with symptoms had reduced exercise capacity (<85% predicted), compared with 3/19 (16%) without symptoms (P = .02). The adjusted peak oxygen consumption (VO2) was 5.2 mL/kg/min lower (95% confidence interval, 2.1-8.3; P = .001) or 16.9% lower percent predicted (4.3%-29.6%; P = .02) among those with symptoms. Chronotropic incompetence was common. Inflammatory markers and antibody levels early in PASC were negatively correlated with peak VO2. Late-gadolinium enhancement on CMR and arrhythmias were absent. CONCLUSIONS: Cardiopulmonary symptoms >1 year after COVID-19 were associated with reduced exercise capacity, which was associated with earlier inflammatory markers. Chronotropic incompetence may explain exercise intolerance among some with "long COVID."

SARS-CoV-2 Serology Across Scales: A Framework for Unbiased Estimation of Cumulative Incidence Incorporating Antibody Kinetics and Epidemic Recency.

Serosurveys are a key resource for measuring severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) population exposure. A growing body of evidence suggests that asymptomatic and mild infections (together making up over 95% of all infections) are associated with lower antibody titers than severe infections. Antibody levels also peak a few weeks after infection and decay gradually. We developed a statistical approach to produce estimates of cumulative incidence from raw seroprevalence survey results that account for these sources of spectrum bias. We incorporate data on antibody responses on multiple assays from a postinfection longitudinal cohort, along with epidemic time series to account for the timing of a serosurvey relative to how recently individuals may have been infected. We applied this method to produce estimates of cumulative incidence from 5 large-scale SARS-CoV-2 serosurveys across different settings and study designs. We identified substantial differences between raw seroprevalence and cumulative incidence of over 2-fold in the results of some surveys, and we provide a tool for practitioners to generate cumulative incidence estimates with preset or custom parameter values. While unprecedented efforts have been launched to generate SARS-CoV-2 seroprevalence estimates over this past year, interpretation of results from these studies requires properly accounting for both population-level epidemiologic context and individual-level immune dynamics.

New Assay Reveals Vast Excess of Defective over Intact HIV-1 Transcripts in Antiretroviral Therapy-Suppressed Individuals.

Most of the HIV DNA in infected individuals is noninfectious because of deleterious mutations. However, it is unclear how much of the transcribed HIV RNA is potentially infectious or defective. To address this question, we developed and validated a novel intact viral RNA assay (IVRA) that uses droplet digital reverse transcriptase PCR (dd-RT-PCR) for the commonly mutated packaging signal (Psi) and Rev response element (RRE) regions (from the intact proviral DNA assay [IPDA]) to quantify likely intact (Psi+ RRE+), 3' defective (Psi+ RRE-), and 5' defective (Psi- RRE+) HIV RNA. We then applied the IPDA and IVRA to quantify intact and defective HIV DNA and RNA from peripheral CD4+ T cells from 9 antiretroviral therapy (ART)-suppressed individuals. Levels of 3' defective HIV DNA were not significantly different from those of 5' defective HIV DNA, and both were higher than intact HIV DNA. In contrast, 3' defective HIV RNA (median 86 copies/106 cells; 94% of HIV RNA) was much more abundant than 5' defective (2.1 copies/106 cells; 5.6%) or intact (0.6 copies/106 cells; <1%) HIV RNA. Likewise, the frequency of CD4+ T cells with 3' defective HIV RNA was greater than the frequency with 5' defective or intact HIV RNA. Intact HIV RNA was transcribed by a median of 0.018% of all proviruses and 2.2% of intact proviruses. The vast excess of 3' defective RNA over 5' defective or intact HIV RNA, which was not observed for HIV DNA, suggests that HIV transcription is completely blocked prior to the RRE in most cells with intact proviruses and/or that cells transcribing intact HIV RNA are cleared at very high rates. IMPORTANCE We developed a new assay that can distinguish and quantify intact (potentially infectious) as well as defective HIV RNA. In ART-treated individuals, we found that the vast majority of all HIV RNA is defective at the 3' end, possibly due to incomplete transcriptional processivity. Only a very small percentage of all HIV RNA is intact, and very few total or intact proviruses transcribe intact HIV RNA. Though rare, this intact HIV RNA is tremendously important because it is necessary to serve as the genome of infectious virions that allow transmission and spread, including rebound after stopping ART. Moreover, intact viral RNA may contribute disproportionately to the immune activation, inflammation, and organ damage observed with untreated and treated HIV infection. The intact viral RNA assay can be applied to many future studies aimed at better understanding HIV pathogenesis and barriers to HIV cure.

Relationship between CD4 T cell turnover, cellular differentiation and HIV persistence during ART.

The precise role of CD4 T cell turnover in maintaining HIV persistence during antiretroviral therapy (ART) has not yet been well characterized. In resting CD4 T cell subpopulations from 24 HIV-infected ART-suppressed and 6 HIV-uninfected individuals, we directly measured cellular turnover by heavy water labeling, HIV reservoir size by integrated HIV-DNA (intDNA) and cell-associated HIV-RNA (caRNA), and HIV reservoir clonality by proviral integration site sequencing. Compared to HIV-negatives, ART-suppressed individuals had similar fractional replacement rates in all subpopulations, but lower absolute proliferation rates of all subpopulations other than effector memory (TEM) cells, and lower plasma IL-7 levels (p = 0.0004). Median CD4 T cell half-lives decreased with cell differentiation from naïve to TEM cells (3 years to 3 months, p<0.001). TEM had the fastest replacement rates, were most highly enriched for intDNA and caRNA, and contained the most clonal proviral expansion. Clonal proviruses detected in less mature subpopulations were more expanded in TEM, suggesting that they were maintained through cell differentiation. Earlier ART initiation was associated with lower levels of intDNA, caRNA and fractional replacement rates. In conclusion, circulating integrated HIV proviruses appear to be maintained both by slow turnover of immature CD4 subpopulations, and by clonal expansion as well as cell differentiation into effector cells with faster replacement rates.

The breadth of the neutralizing antibody response to original SARS-CoV-2 infection is linked to the presence of Long COVID symptoms.

The associations between longitudinal dynamics and the breadth of SARS-CoV-2 neutralizing antibody (nAb) response with various Long COVID phenotypes before vaccination are not known. The capacity of antibodies to cross-neutralize a variety of viral variants may be associated with ongoing pathology and persistent symptoms. We measured longitudinal neutralizing and cross-neutralizing antibody responses to pre- and post-SARS-CoV-2 Omicron variants in participants infected early in the COVID-19 pandemic, before widespread rollout of SARS-CoV-2 vaccines. Cross-sectional regression models adjusted for clinical covariates and longitudinal mixed-effects models were used to determine the impact of the breadth and rate of decay of neutralizing responses on the development of Long COVID symptoms, as well as Long COVID phenotypes. We identified several novel relationships between SARS-CoV-2 antibody neutralization and the presence of Long COVID symptoms. Specifically, we show that, although nAb responses to the original, infecting strain of SARS-CoV-2 were not associated with Long COVID in cross-sectional analyses, cross-neutralization ID50 levels to the Omicron BA.5 variant approximately 4 months following acute infection was independently and significantly associated with greater odds of Long COVID and with persistent gastrointestinal and neurological symptoms. Longitudinal modeling demonstrated significant associations in the overall levels and rates of decay of neutralization capacity with Long COVID phenotypes. A higher proportion of participants had antibodies capable of neutralizing Omicron BA.5 compared with BA.1 or XBB.1.5 variants. Our findings suggest that relationships between various immune responses and Long COVID are likely complex but may involve the breadth of antibody neutralization responses.

SARS-CoV-2 and Mitochondrial Proteins in Neural-Derived Exosomes of COVID-19.

OBJECTIVE: As SARS-CoV-2 is known to invade neural cell mitochondria, a plasma system for quantifying central nervous system proteins in living humans was used to investigate neuropathogenic mechanisms of long-COVID-19. METHODS: SARS-CoV-2 proteins and mitochondrial proteins (MPs) in enriched plasma neuron-derived extracellular vesicles (NDEVs) and astrocyte-derived EVs (ADEVs) were quantified in resolved acute COVID-19 without post-acute sequelae of SARS-CoV-2 (PASC), PASC without neuropsychiatric manifestations (NP), PASC with NP and healthy controls. RESULTS: NDEV and ADEV mean levels of SARS-CoV-2 S1 and nucleocapsid (N) proteins were higher in all PASC sub-groups than controls, but only N levels were higher in PASC with than without NP. Exosome marker CD81-normalized NDEV mean levels of subunit 6 of MP respiratory chain complex I and subunit 10 of complex III, and neuroprotective MPs Humanin and mitochondrial open-reading frame of the 12S rRNA-c (MOTS-c) all were decreased significantly in PASC with NP but not in PASC without NP relative to controls. NDEV levels of MPs voltage-dependent anion-selective channel protein 1 (VDAC1) and N-methyl-D-aspartate receptor 1 (NMDAR1) were decreased in PASC without and with NP, whereas those of calcium channel MPs mitochondrial calcium uniporter (MCU), sodium/calcium exchanger (NCLX) and leucine zipper EF-hand containing transmembrane 1 protein (LETM1) were decreased only in PASC with NP. ADEV levels of MCU and NCLX only were increased in PASC without and with NP. INTERPRETATION: Abnormal NDEV and ADEV levels of SARS-CoV-2 N and S1 protein and MPs correlate with NP and may be biomarkers for long-COVID prognostics and therapeutic trials. ANN NEUROL 2022;91:772-781.

Cellular Activation, Differentiation, and Proliferation Influence the Dynamics of Genetically Intact Proviruses Over Time.

Human immunodeficiency virus (HIV) persists in cells despite antiretroviral therapy; however, the influence of cellular mechanisms such as activation, differentiation, and proliferation upon the distribution of proviruses over time is unclear. To address this, we used full-length sequencing to examine proviruses within memory CD4+ T-cell subsets longitudinally in 8 participants. Over time, the odds of identifying a provirus increased in effector and decreased in transitional memory cells. In all subsets, more activated (HLA-DR-expressing) cells contained a higher frequency of intact provirus, as did more differentiated cells such as transitional and effector memory subsets. The proportion of genetically identical proviruses increased over time, indicating that cellular proliferation was maintaining the persistent reservoir; however, the number of genetically identical proviral clusters in each subset was stable. As such, key biological processes of activation, differentiation, and proliferation influence the dynamics of the HIV reservoir and must be considered during the development of any immune intervention.

Tissue memory CD4+ T cells expressing IL-7 receptor-alpha (CD127) preferentially support latent HIV-1 infection.

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

Characterizing the COVID-19 Illness Experience to Inform the Study of Post-acute Sequelae and Recovery.

BACKGROUND: There is an urgent need to fully understand the impact of variable COVID-19 experiences and the optimal management of post-acute sequelae of SARS-CoV-2 infection. We characterized the variability in the acute illness experience and ongoing recovery process from participants in a COVID-19 recovery cohort study in Northern California in 2020. METHOD: We completed 24 semi-structured in-depth interviews with adults with confirmed positive SARV-CoV-2 nucleic acid amplification test result, had recovered or were recovering from acute infection, and underwent serial evaluations. We purposefully sampled English- and Spanish-speaking adults with asymptomatic, mild, and severe symptomatic infection, including those who were hospitalized and those with HIV co-infection. We used a thematic analysis to analyze interviews and identify salient themes. RESULTS: After integrating the thematic analysis with clinical data, we identified key themes: (1) across symptom profiles and severity, experiencing COVID-19 was associated with psychological distress; (2) symptomatic infection carried uncertainty in symptom presentation and ongoing recovery (e.g., long COVID); and (3) health information-seeking behavior was facilitated by access to medical care and uncertainty with the recovery process. CONCLUSION: Our data informs the emerging field of "long COVID" research and shows a need to provide information and continuous support to persons with post-acute sequelae to ensure they feel secure along the path to recovery.

Combined HIV-1 sequence and integration site analysis informs viral dynamics and allows reconstruction of replicating viral ancestors.

Understanding HIV-1 persistence despite antiretroviral therapy (ART) is of paramount importance. Both single-genome sequencing (SGS) and integration site analysis (ISA) provide useful information regarding the structure of persistent HIV DNA populations; however, until recently, there was no way to link integration sites to their cognate proviral sequences. Here, we used multiple-displacement amplification (MDA) of cellular DNA diluted to a proviral endpoint to obtain full-length proviral sequences and their corresponding sites of integration. We applied this method to lymph node and peripheral blood mononuclear cells from 5 ART-treated donors to determine whether groups of identical subgenomic sequences in the 2 compartments are the result of clonal expansion of infected cells or a viral genetic bottleneck. We found that identical proviral sequences can result from both cellular expansion and viral genetic bottlenecks occurring prior to ART initiation and following ART failure. We identified an expanded T cell clone carrying an intact provirus that matched a variant previously detected by viral outgrowth assays and expanded clones with wild-type and drug-resistant defective proviruses. We also found 2 clones from 1 donor that carried identical proviruses except for nonoverlapping deletions, from which we could infer the sequence of the intact parental virus. Thus, MDA-SGS can be used for "viral reconstruction" to better understand intrapatient HIV-1 evolution and to determine the clonality and structure of proviruses within expanded clones, including those with drug-resistant mutations. Importantly, we demonstrate that identical sequences observed by standard SGS are not always sufficient to establish proviral clonality.

Markers of Immune Activation and Inflammation in Individuals With Postacute Sequelae of Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

BACKGROUND: The biological processes associated with postacute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (PASC) are unknown. METHODS: We measured soluble markers of inflammation in a SARS-CoV-2 recovery cohort at early (<90 days) and late (>90 days) timepoints. We defined PASC as the presence of 1 or more coronavirus disease 2019 (COVID-19)-attributed symptoms beyond 90 days. We compared fold-changes in marker values between those with and without PASC using mixed-effects models with terms for PASC and early and late recovery time periods. RESULTS: During early recovery, those who went on to develop PASC generally had higher levels of cytokine biomarkers including tumor necrosis factor-α (1.14-fold higher mean ratio [95% confidence interval {CI}, 1.01-1.28]; P = .028) and interferon-γ-induced protein 10 (1.28-fold higher mean ratio [95% CI, 1.01-1.62]; P = .038). Among those with PASC, there was a trend toward higher interleukin 6 levels during early recovery (1.29-fold higher mean ratio [95% CI, .98-1.70]; P = .07), which became more pronounced in late recovery (1.44-fold higher mean ratio [95% CI, 1.11-1.86]; P < .001). These differences were more pronounced among those with a greater number of PASC symptoms. CONCLUSIONS: Persistent immune activation may be associated with ongoing symptoms following COVID-19. Further characterization of these processes might identify therapeutic targets for those experiencing PASC.

Impact of Antiretroviral Therapy Duration on HIV-1 Infection of T Cells within Anatomic Sites.

Understanding the impact of antiretroviral therapy (ART) duration on HIV-infected cells is critical for developing successful curative strategies. To address this issue, we conducted a cross-sectional/inter-participant genetic characterization of HIV-1 RNA from pre- and on-therapy plasmas and HIV-1 DNA from CD4+ T cell subsets derived from peripheral blood (PB), lymph node (LN), and gut tissues of 26 participants after 3 to 17.8 years of ART. Our studies revealed in four acute/early participants who had paired PB and LN samples a substantial reduction in the proportion of HIV-infected cells per year on therapy within the LN. Extrapolation to all 12 acute/early participants estimated a much smaller reduction in the proportion of HIV-1-infected cells within LNs per year on therapy that was similar to that in the participants treated during chronic infection. LN-derived effector memory T (TEM) cells contained HIV-1 DNA that was genetically identical to viral sequences derived from pre- and on-therapy plasma samples. The proportion of identical HIV-1 DNA sequences increased within PB-derived TEM cells. However, the infection frequency of TEM cells in PB was stable, indicating that cellular proliferation that compensates for T cell loss over time contributes to HIV-1 persistence. This study suggests that ART reduces HIV-infected T cells and that clonal expansion of HIV-infected cells maintains viral persistence. Importantly, LN-derived TEM cells are a probable source of HIV-1 genomes capable of producing infectious HIV-1 and should be targeted by future curative strategies.IMPORTANCE HIV-1 persists as an integrated genome in CD4+ memory T cells during effective therapy, and cessation of current treatments results in resumption of viral replication. To date, the impact of antiretroviral therapy duration on HIV-infected CD4+ T cells and the mechanisms of viral persistence in different anatomic sites is not clearly elucidated. In the current study, we found that treatment duration was associated with a reduction in HIV-infected T cells. Our genetic analyses revealed that CD4+ effector memory T (TEM) cells derived from the lymph node appeared to contain provirus that was genetically identical to plasma-derived virions. Moreover, we found that cellular proliferation counterbalanced the decay of HIV-infected cells throughout therapy. The contribution of cellular proliferation to viral persistence is particularly significant in TEM cells. Our study emphasizes the importance of HIV-1 intervention and provides new insights into the location of memory T cells infected with HIV-1 DNA, which is capable of contributing to viremia.