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.

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.

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.

Common and Divergent Features of T Cells from Blood, Gut, and Genital Tract of Antiretroviral Therapy-Treated HIV+ Women.

T cells residing in mucosal tissues play important roles in homeostasis and defense against microbial pathogens. The gut and female reproductive tract (FRT) are both tolerogenic environments, but they differ in the kinds of foreign Ags they need to tolerate. How these different environments influence the properties of their T cells is poorly understood, but important for understanding women's health. We recruited antiretroviral therapy-suppressed women living with HIV who donated, within one visit, blood and tissue samples from the ileum, colon, rectosigmoid, endometrium, endocervix, and ectocervix. With these samples, we conducted 36-parameter cytometry by time of flight phenotyping of T cells. Although gut and FRT T cells shared features discriminating them from their blood counterparts, they also harbored features distinguishing them from one another. These included increased proportions of CD69+ T resident memory cells of the T effector memory phenotype, as well as preferential coexpression of CD69 and CD103, on the gut-derived cells. In contrast, CD69+CD103+ T resident memory CD8+ T cells from FRT, but not those from gut, preferentially expressed PD1. We further determined that a recently described population of CXCR4+ T inflammatory mucosal cells differentially expressed multiple other chemokine receptors relative to their blood counterparts. Our findings suggest that T cells resident in different tolerogenic mucosal sites take on distinct properties.

Protracted yet Coordinated Differentiation of Long-Lived SARS-CoV-2-Specific CD8+ T Cells during Convalescence.

CD8+ T cells can potentiate long-lived immunity against COVID-19. We screened longitudinally-sampled convalescent human donors against SARS-CoV-2 tetramers and identified a participant with an immunodominant response against residues 322 to 311 of nucleocapsid (Nuc322-331), a peptide conserved in all variants of concern reported to date. We conducted 38-parameter cytometry by time of flight on tetramer-identified Nuc322-331-specific CD8+ T cells and on CD4+ and CD8+ T cells recognizing the entire nucleocapsid and spike proteins, and took 32 serological measurements. We discovered a coordination of the Nuc322-331-specific CD8+ T response with both the CD4+ T cell and Ab pillars of adaptive immunity. Over the approximately six month period of convalescence monitored, we observed a slow and progressive decrease in the activation state and polyfunctionality of Nuc322-331-specific CD8+ T cells, accompanied by an increase in their lymph node-homing and homeostatic proliferation potential. These results suggest that following a typical case of mild COVID-19, SARS-CoV-2-specific CD8+ T cells not only persist but continuously differentiate in a coordinated fashion well into convalescence into a state characteristic of long-lived, self-renewing memory.

Novel RT-ddPCR assays for measuring the levels of subgenomic and genomic SARS-CoV-2 transcripts.

The replication of SARS-CoV-2 and other coronaviruses depends on transcription of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and multiple different subgenomic mRNAs (sgRNAs) encompassing fragments arising from discontinuous transcription. Recent studies have aimed to characterize the expression of subgenomic SARS-CoV-2 transcripts in order to investigate their clinical significance. Here, we describe a novel panel of reverse transcription droplet digital PCR (RT-ddPCR) assays designed to specifically quantify multiple different subgenomic SARS-CoV-2 transcripts and distinguish them from transcripts that do not arise from discontinuous transcription at each locus. These assays can be applied to samples from SARS-CoV-2 infected patients to better understand the regulation of SARS-CoV-2 transcription and how different sgRNAs may contribute to viral pathogenesis and clinical disease severity.

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.

No detectable alloreactive transcriptional responses under standard sample preparation conditions during donor-multiplexed single-cell RNA sequencing of peripheral blood mononuclear cells.

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) provides high-dimensional measurements of transcript counts in individual cells. However, high assay costs and artifacts associated with analyzing samples across multiple sequencing runs limit the study of large numbers of samples. Sample multiplexing technologies such as MULTI-seq and antibody hashing using single-cell multiplexing kit (SCMK) reagents (BD Biosciences) use sample-specific sequence tags to enable individual samples to be sequenced in a pooled format, markedly lowering per-sample processing and sequencing costs while minimizing technical artifacts. Critically, however, pooling samples could introduce new artifacts, partially negating the benefits of sample multiplexing. In particular, no study to date has evaluated whether pooling peripheral blood mononuclear cells (PBMCs) from unrelated donors under standard scRNA-seq sample preparation conditions (e.g., 30 min co-incubation at 4 °C) results in significant changes in gene expression resulting from alloreactivity (i.e., response to non-self). The ability to demonstrate minimal to no alloreactivity is crucial to avoid confounded data analyses, particularly for cross-sectional studies evaluating changes in immunologic gene signatures. RESULTS: Here, we applied the 10x Genomics scRNA-seq platform to MULTI-seq and/or SCMK-labeled PBMCs from a single donor with and without pooling with PBMCs from unrelated donors for 30 min at 4 °C. We did not detect any alloreactivity signal between mixed and unmixed PBMCs across a variety of metrics, including alloreactivity marker gene expression in CD4+ T cells, cell type proportion shifts, and global gene expression profile comparisons using Gene Set Enrichment Analysis and Jensen-Shannon Divergence. These results were additionally mirrored in publicly-available scRNA-seq data generated using a similar experimental design. Moreover, we identified confounding gene expression signatures linked to PBMC preparation method (e.g., Trima apheresis), as well as SCMK sample classification biases against activated CD4+ T cells which were recapitulated in two other SCMK-incorporating scRNA-seq datasets. CONCLUSIONS: We demonstrate that (i) mixing PBMCs from unrelated donors under standard scRNA-seq sample preparation conditions (e.g., 30 min co-incubation at 4 °C) does not cause an allogeneic response, and (ii) that Trima apheresis and PBMC sample multiplexing using SCMK reagents can introduce undesirable technical artifacts into scRNA-seq data. Collectively, these observations establish important benchmarks for future cross-sectional immunological scRNA-seq experiments.

Gut and blood differ in constitutive blocks to HIV transcription, suggesting tissue-specific differences in the mechanisms that govern HIV latency.

Latently-infected CD4+ T cells are widely considered to be the major barrier to a cure for HIV. Much of our understanding of HIV latency comes from latency models and blood cells, but most HIV-infected cells reside in lymphoid tissues such as the gut. We hypothesized that tissue-specific environments may impact the mechanisms that govern HIV expression. To assess the degree to which different mechanisms inhibit HIV transcription in the gut and blood, we quantified HIV transcripts suggestive of transcriptional interference (U3-U5; "Read-through"), initiation (TAR), 5' elongation (R-U5-pre-Gag; "Long LTR"), distal transcription (Nef), completion (U3-polyA; "PolyA"), and multiple splicing (Tat-Rev) in matched peripheral blood mononuclear cells (PBMCs) and rectal biopsies, and matched FACS-sorted CD4+ T cells from blood and rectum, from two cohorts of ART-suppressed individuals. Like the PBMCs, rectal biopsies showed low levels of read-through transcripts (median = 23 copies/106 cells) and a gradient of total (679)>elongated(75)>Nef(16)>polyadenylated (11)>multiply-spliced HIV RNAs(<1) [p<0.05 for all], demonstrating blocks to HIV transcriptional elongation, completion, and splicing. Rectal CD4+ T cells showed a similar gradient of total>polyadenylated>multiply-spliced transcripts, but the ratio of total to elongated transcripts was 6-fold lower than in blood CD4+ T cells (P = 0.016), suggesting less of a block to HIV transcriptional elongation in rectal CD4+ T cells. Levels of total transcripts per provirus were significantly lower in rectal biopsies compared to PBMCs (median 3.5 vs. 15.4; P = 0.008) and in sorted CD4+ T cells from rectum compared to blood (median 2.7 vs. 31.8; P = 0.016). The lower levels of HIV transcriptional initiation and of most HIV transcripts per provirus in the rectum suggest that this site may be enriched for latently-infected cells, cells in which latency is maintained by different mechanisms, or cells in a "deeper" state of latency. These are important considerations for designing therapies that aim to disrupt HIV latency in all tissue compartments.

Recent stimulant use and leukocyte gene expression in methamphetamine users with treated HIV infection.

Stimulant use may accelerate HIV disease progression through biological and behavioral pathways. However, scant research with treated HIV-positive persons has examined stimulant-associated alterations in pathophysiologic processes relevant to HIV pathogenesis. In a sample of 55 HIV-positive, methamphetamine-using sexual minority men with a viral load less than 200 copies/mL, we conducted RNA sequencing to examine patterns of leukocyte gene expression in participants who had a urine sample that was reactive for stimulants (n = 27) as compared to those who tested non-reactive (n = 28). Results indicated differential expression of 32 genes and perturbation of 168 pathways in recent stimulant users. We observed statistically significant differential expression of single genes previously associated with HIV latency, cell cycle regulation, and immune activation in recent stimulant users (false discovery rate p < 0.10). Pathway analyses indicated enrichment for genes associated with inflammation, innate immune activation, neuroendocrine hormone regulation, and neurotransmitter synthesis. Recent stimulant users displayed concurrent elevations in plasma levels of tumor necrosis factor - alpha (TNF-α) but not interleukin 6 (IL-6). Further research is needed to examine the bio-behavioral mechanisms whereby stimulant use may contribute to HIV persistence and disease progression.

Immunologic Pathways That Predict Mortality in HIV-Infected Ugandans Initiating Antiretroviral Therapy.

The plasma kynurenine/tryptophan (KT) ratio, a marker of adaptive immune defects, strongly predicts mortality during treated human immunodeficiency virus (HIV) disease in Ugandans as compared to US-based populations. Here, the KT ratio and T-cell and plasma biomarkers of immune activation were measured among 535 HIV-infected Ugandans prior to ART initiation and at month 6 of viral suppression. The month 6 KT ratio (adjusted hazard ratio [aHR], 2.74), soluble CD14 level (aHR, 2.32), interleukin 6 level (aHR, 2.34), and D-dimer level (aHR, 1.95) were associated with mortality occurring ≥6 months after ART initiation. The KT ratio remained significantly predictive of mortality even after adjustment for the additional biomarkers, suggesting an independent contribution to clinical outcomes in resource-limited settings.

Immunologic Biomarkers, Morbidity, and Mortality in Treated HIV Infection.

Despite marked improvements in the modern treatment era, human immunodeficiency virus (HIV)-infected individuals, particularly those who initiated antiretroviral therapy (ART) at advanced disease stages, continue to have increased age-related morbidity and mortality, compared with the general population. Immune activation and inflammation persist despite suppressive ART and predict many of these morbidities. The goal of this review is to examine the evidence suggesting a link between the persistent inflammatory state and morbidity and mortality in this setting, to describe the impact of early ART initiation on these factors, and to highlight important unanswered questions for the field. We also advance a hypothesis to explain why some morbidities-and their root inflammatory drivers-may be prevented more than others by early ART initiation.

CD8 T-Cell Expansion and Inflammation Linked to CMV Coinfection in ART-treated HIV Infection.

BACKGROUND: Persistent CD8 T-cell expansion, low CD4/CD8 T-cell ratios, and heightened inflammation persist in antiretroviral therapy (ART)-treated human immunodeficiency virus (HIV) infection and are associated with increased risk of morbid outcomes. We explored the role of cytomegalovirus (CMV) infection in CD8 lymphocytosis and inflammation in ART-treated HIV infection. METHODS: Absolute CD4 and CD8 T-cell counts were abstracted from clinical records and compared among 32 HIV-infected CMV-seronegative subjects, 126 age, CD4 and gender-matched HIV-infected CMV-seropositive subjects, and among 21 HIV-uninfected controls (9 CMV-negative, 12 CMV-positive). Plasma inflammatory indices were measured in a subset by ELISA. RESULTS: Median CD8 counts/µL were higher in HIV-positive/CMV-positive patients (795) than in HIV-positive/CMV-negative subjects (522, P = .006) or in healthy controls (451, P = .0007), whereas CD8 T-cell counts were similar to controls' levels in HIV-positive/CMV-negative subjects. Higher plasma levels of IP-10 (P = .0011), TNF-RII (P = .0002), and D-dimer (P = .0444) were also found in coinfected patients than in HIV-positive/CMV-negative subjects. CONCLUSIONS: CMV infection is associated with higher CD8 T-cell counts, resultant lower CD4/CD8 ratios, and increased systemic inflammation in ART-treated HIV infection. CMV infection may contribute to risk for morbid outcomes in treated HIV infection.

HIV-infected individuals with low CD4/CD8 ratio despite effective antiretroviral therapy exhibit altered T cell subsets, heightened CD8+ T cell activation, and increased risk of non-AIDS morbidity and mortality.

A low CD4/CD8 ratio in elderly HIV-uninfected adults is associated with increased morbidity and mortality. A subset of HIV-infected adults receiving effective antiretroviral therapy (ART) fails to normalize this ratio, even after they achieve normal CD4+ T cell counts. The immunologic and clinical characteristics of this clinical phenotype remain undefined. Using data from four distinct clinical cohorts and three clinical trials, we show that a low CD4/CD8 ratio in HIV-infected adults during otherwise effective ART (after CD4 count recovery above 500 cells/mm3) is associated with a number of immunological abnormalities, including a skewed T cell phenotype from naïve toward terminally differentiated CD8+ T cells, higher levels of CD8+ T cell activation (HLADR+CD38+) and senescence (CD28- and CD57+CD28-), and higher kynurenine/tryptophan ratio. Changes in the peripheral CD4/CD8 ratio are also reflective of changes in gut mucosa, but not in lymph nodes. In a longitudinal study, individuals who initiated ART within six months of infection had greater CD4/CD8 ratio increase compared to later initiators (>2 years). After controlling for age, gender, ART duration, nadir and CD4 count, the CD4/CD8 ratio predicted increased risk of morbidity and mortality. Hence, a persistently low CD4/CD8 ratio during otherwise effective ART is associated with increased innate and adaptive immune activation, an immunosenescent phenotype, and higher risk of morbidity/mortality. This ratio may prove useful in monitoring response to ART and could identify a unique subset of individuals needed of novel therapeutic interventions.

Bartonella quintana Aortitis in a Man with AIDS, Diagnosed by Needle Biopsy and 16S rRNA Gene Amplification.

A man with newly diagnosed AIDS presented with months of back pain and fever. Computed tomography (CT) results demonstrated aortitis with periaortic tissue thickening. DNA amplification of biopsy tissue revealed Bartonella quintana, and Bartonella serologies were subsequently noted to be positive. The patient improved with prolonged doxycycline and rifabutin treatment. This case illustrates how molecular techniques are increasingly important in diagnosing Bartonella infections.

Low proportions of CD28- CD8+ T cells expressing CD57 can be reversed by early ART initiation and predict mortality in treated HIV infection.

BACKGROUND: Unlike cytomegalovirus (CMV) infection and aging, human immunodeficiency virus (HIV) decreases the proportion of CD28(-)CD8(+) T cells expressing CD57. Whether this abnormality predicts mortality in treated HIV infection and can be reversed by early antiretroviral therapy (ART) remains unknown. METHODS: We sampled recently HIV-infected individuals (<6 months) and HIV-uninfected controls and compared longitudinal changes in the proportion of CD28(-)CD8(+) T cells expressing CD57 between those who initiated ART early (<6 months) vs later (≥2 years). We also assessed the relationship between this phenotype and mortality in a nested case-control study of ART-suppressed chronically infected individuals. RESULTS: Compared to HIV-uninfected controls (n = 15), individuals who were recently infected with HIV had lower proportions of CD28(-)CD8(+) T cells expressing CD57 (P < .001), and these proportions increased during ART. The early ART group (n = 33) achieved normal levels, whereas the later ART group (n = 30) continued to have lower levels than HIV-uninfected controls (P = .02). Among 141 ART-suppressed participants in the SOCA study, those in the lowest quartile of CD28(-)CD8(+) T cells expressing CD57 had 5-fold higher odds of mortality than those in the highest quartile (95% CI, 1.6-15.9, P = .007). CONCLUSIONS: Abnormally low proportions of CD28(-)CD8(+) T cells expressing CD57 predict increased mortality during treated HIV infection and may be reversed with early ART initiation.

Autologous neutralizing antibody responses after antiretroviral therapy in acute and early HIV-1.

BACKGROUNDEarly antiretroviral therapy initiation (ARTi) in HIV-1 restricts reservoir size and diversity while preserving immune function, potentially improving opportunities for immunotherapeutic cure strategies. For antibody-based cure approaches, the development of autologous neutralizing antibodies (anAbs) after acute/early ARTi is relevant but is poorly understood.METHODSWe characterized antibody responses in a cohort of 23 participants following ARTi in acute HIV (<60 days after acquisition) and early HIV (60-128 days after acquisition).RESULTSPlasma virus sequences at the time of ARTi revealed evidence of escape from anAbs after early, but not acute, ARTi. HIV-1 envelopes representing the transmitted/founder virus(es) (acute ARTi) or escape variants (early ARTi) were tested for sensitivity to longitudinal plasma IgG. After acute ARTi, no anAb responses developed over months to years of suppressive ART. In 2 of the 3 acute ARTi participants who experienced viremia after ARTi, however, anAbs arose shortly thereafter. After early ARTi, anAbs targeting those early variants developed between 12 and 42 weeks of ART and continued to increase in breadth and potency thereafter.CONCLUSIONResults indicate a threshold of virus replication (~60 days) required to induce anAbs, after which they continue to expand on suppressive ART to better target the range of reservoir variants.TRIAL REGISTRATIONClinicalTrials.gov NCT02656511.FUNDINGNIH grants U01AI169767, R01AI162646, UM1AI164570, UM1AI164560, U19AI096109, K23GM112526, T32AI118684, P30AI045008, P30AI027763, R24AI067039; Gilead Sciences grant INUS2361354; Viiv Healthcare grant A126326.

Rapid Biphasic Decay of Intact and Defective HIV DNA Reservoir During Acute Treated HIV Disease.

Despite antiretroviral therapy (ART), HIV persists in latently-infected cells ("the reservoir") which decay slowly over time. Here, leveraging >500 longitudinal samples from 67 people with HIV (PWH) treated during acute infection, we developed a novel 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 PWH. Defective DNA had a similar biphasic pattern, but data were more variable. Predicted intact and defective decay rates were faster for PWH with earlier timing of ART initiation, higher initial CD4+ T cell count, and lower pre-ART viral load. These data add to our limited understanding of HIV reservoir decay at the time of ART initiation, informing future curative strategies targeting this critical time.

Mild SARS-CoV-2 infection results in long-lasting microbiota instability.

Viruses targeting mammalian cells can indirectly alter the gut microbiota, potentially compounding their phenotypic effects. Multiple studies have observed a disrupted gut microbiota in severe cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that require hospitalization. Yet, despite demographic shifts in disease severity resulting in a large and continuing burden of non-hospitalized infections, we still know very little about the impact of mild SARS-CoV-2 infection on the gut microbiota in the outpatient setting. To address this knowledge gap, we longitudinally sampled 14 SARS-CoV-2-positive subjects who remained outpatient and 4 household controls. SARS-CoV-2 cases exhibited a significantly less stable gut microbiota relative to controls. These results were confirmed and extended in the K18-humanized angiotensin-converting enzyme 2 mouse model, which is susceptible to SARS-CoV-2 infection. All of the tested SARS-CoV-2 variants significantly disrupted the mouse gut microbiota, including USA-WA1/2020 (the original variant detected in the USA), Delta, and Omicron. Surprisingly, despite the fact that the Omicron variant caused the least severe symptoms in mice, it destabilized the gut microbiota and led to a significant depletion in Akkermansia muciniphila. Furthermore, exposure of wild-type C57BL/6J mice to SARS-CoV-2 disrupted the gut microbiota in the absence of severe lung pathology. IMPORTANCE Taken together, our results demonstrate that even mild cases of SARS-CoV-2 can disrupt gut microbial ecology. Our findings in non-hospitalized individuals are consistent with studies of hospitalized patients, in that reproducible shifts in gut microbial taxonomic abundance in response to SARS-CoV-2 have been difficult to identify. Instead, we report a long-lasting instability in the gut microbiota. Surprisingly, our mouse experiments revealed an impact of the Omicron variant, despite producing the least severe symptoms in genetically susceptible mice, suggesting that despite the continued evolution of SARS-CoV-2, it has retained its ability to perturb the intestinal mucosa. These results will hopefully renew efforts to study the mechanisms through which Omicron and future SARS-CoV-2 variants alter gastrointestinal physiology, while also considering the potentially broad consequences of SARS-CoV-2-induced microbiota instability for host health and disease.

Assessing the associations between known genetic variants and substance use in people with HIV in the United States.

BACKGROUND: The prevalence of substance use in people with HIV (PWH) in the United States is higher than in the general population and is an important driver of HIV-related outcomes. We sought to assess if previously identified genetic associations that contribute to substance use are also observed in a population of PWH. METHODS: We performed genome-wide association studies (GWAS) of alcohol, smoking, and cannabis use phenotypes in a multi-ancestry population of 7,542 PWH from the Center for AIDS Research Network of Integrated Clinical Systems (CNICS). We conducted multi-ancestry GWAS for individuals of African (n = 3,748), Admixed American (n = 1,334), and European (n = 2,460) ancestry. Phenotype data were self-reported and collected using patient reported outcomes (PROs) and three questions from AUDIT-C, an alcohol screening tool. We analyzed nine phenotypes: 1) frequency of alcohol consumption, 2) typical number of drinks on a day when drinking alcohol, 3) frequency of five or more alcoholic drinks in a 30-day period, 4) smoking initiation, 5) smoking cessation, 6) cigarettes per day, 7) cannabis use initiation, 8) cannabis use cessation, 9) frequency of cannabis use during the previous 30 days. For each phenotype we considered a) variants previously identified as associated with a substance use trait and b) novel associations. RESULTS: We observed evidence for effects of previously reported single nucleotide polymorphisms (SNPs) related to alcohol (rs1229984, p = 0.001), tobacco (rs11783093, p = 2.22E-4), and cannabis use (rs2875907, p = 0.005). We also report two novel loci (19p13.2, p = 1.3E-8; and 20p11.21, p = 2.1E-8) associated with cannabis use cessation. CONCLUSIONS: Our analyses contribute to understanding the genetic bases of substance use in a population with relatively higher rates of use compared to the general population.