HIV rebound in HIV controllers is associated with a specific fecal microbiome profile.

HIV infection is associated with gut dysbiosis, and microbiome variability may affect HIV control when antiretroviral therapy (ART) is stopped. The TLR7 agonist, vesatolimod, was previously associated with a modest delay in viral rebound following analytical treatment interruption in HIV controllers (HCs). Using a retrospective analysis of fecal samples from HCs treated with vesatolimod or placebo (NCT03060447), people with chronic HIV (CH; NCT02858401) or without HIV (PWOH), we examined fecal microbiome profile in HCs before/after treatment, and in CH and PWOH. Microbiome diversity and abundance were compared between groups to investigate the association between specific phyla/species, immune biomarkers, and viral outcomes during treatment interruption. Although there were no significant differences in gut microbiome diversity between people with and without HIV, HCs, and CH shared common features that distinguished them from PWOH. there was a trend toward greater microbiome diversity among HCs. Treatment with vesatolimod reduced dysbiosis in HCs. Firmicutes positively correlated with T-cell activation, while Bacteroidetes and Euryarchaeota inversely correlated with TLR7-mediated immune activation. Specific types of fecal microbiome abundance (e.g. Alistipes putredinis) positively correlated with HIV rebound. In conclusion, variability in the composition of the fecal microbiome is associated with markers of immune activation following vesatolimod treatment and ART interruption.

High-parameter phenotypic characterization reveals a subset of human Th17 cells that preferentially produce IL-17 against M. tuberculosis antigen.

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

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

Antiretroviral therapy (ART) is not a cure. Upon ART cessation, virus rapidly rebounds from latently-infected cells ("the HIV reservoir"). The reservoir is largely stabilized at the time of ART initiation and then decays slowly. 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 using the intact proviral DNA assay (IPDA) from peripheral CD4+ T cells. Nonlinear generalized additive models adjusted for initial CD4+ T count, pre-ART viral load, and timing of ART initiation demonstrated rapid biphasic decay of intact DNA (week 0-5: t1/2 ~0.71 months; week 5-24: t1/2 ~3.9 months) that extended out to 1 year of ART, with similar trends for defective DNA. Predicted reservoir decay were faster for participants individuals with earlier timing of ART initiation, higher initial CD4+ T cell count, and lower pre-ART viral load. These estimates are ~5-fold faster than prior reservoir decay estimates among chronic-treated PWH. Thus, these data add to our limited understanding of host viral control at the earliest stages of HIV reservoir stabilization, potentially informing future HIV cure efforts aimed at diverse, global population of PWH initiating ART at varying stages of disease.

AMPLON: Amplifying DNA with Multiarm Priming and Looping Optimization of Nucleic Acid.

Nucleic acid amplification, the bedrock of biotechnology and molecular diagnostics, surges in applications-especially isothermal approaches-heightening the demand for advanced and precisely engineered methods. Here, a novel approach for amplifying DNA with multiarm priming and looping optimization of nucleic acid (AMPLON) is presented. AMPLON relies on a novel polymeric material with unique set of multiarm polyethylene glycol-DNA primers for efficient DNA amplification under isothermal conditions. Each arm carries single-stranded DNA complementing the sense or antisense sequence of the target DNA. The amplification reaction begins with antisense arms binding to the target DNA, forming a template for sense-carrying arms to direct multiarm large DNA amplicon synthesis through successive DNA looping and unlooping steps. Using human immunodeficiency virus type 1 (HIV-1) as a model clinical target, AMPLON exhibits high sensitivity, detecting target concentrations as low as 100 copies mL-1. Compared to a quantitative real-time polymerase chain reaction assay using sensitive primers, AMPLON reliably identifies HIV-1 RNA in plasma samples (n = 20) with a significant agreement rate of 95%. With its ability to achieve highly specific and sensitive target amplification within 30 min, AMPLON holds immense potential to transform the field of nucleic acid research and unleashing new possibilities in medicine and biotechnology.

Long COVID and post-acute sequelae of SARS-CoV-2 pathogenesis and treatment: A Keystone Symposia report.

In 2023, the Keystone Symposia held the first international scientific conference convening research leaders investigating the pathology of post-acute sequelae of COVID-19 (PASC) or Long COVID, a growing and urgent public health priority. In this report, we present insights from the talks and workshops presented during this meeting and highlight key themes regarding what researchers have discovered regarding the underlying biology of PASC and directions toward future treatment. Several themes have emerged in the biology, with inflammation and other immune alterations being the most common focus, potentially related to viral persistence, latent virus reactivation, and/or tissue damage and dysfunction, especially of the endothelium, nervous system, and mitochondria. In order to develop safe and effective treatments for people with PASC, critical next steps should focus on the replication of major findings regarding potential mechanisms, disentangling pathogenic mechanisms from downstream effects, development of cellular and animal models, mechanism-focused randomized, placebo-controlled trials, and closer collaboration between people with lived experience, scientists, and other stakeholders. Ultimately, by learning from other post-infectious syndromes, the knowledge gained may help not only those with PASC/Long COVID, but also those with other post-infectious syndromes.

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

BACKGROUND: Early 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 (anAb) after acute/early ARTi is relevant, but poorly understood. METHODS: We characterize antibody responses in a cohort of 23 participants following ARTi in acute HIV (<60 days after infection) and early HIV (60-128 days after infection). RESULTS: Plasma virus sequences at the time of ARTi revealed evidence of escape from anAbs after early, but not acute, ARTi. HIV-1 Envs 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 two of the three 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. CONCLUSIONS: Results 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 REGISTRATION: NCT02656511FUNDING. National Institutes of Health grants U01AI169767; R01AI162646; UM1AI164570; UM1AI164560; U19AI096109; K23GM112526; T32AI118684, P30-AI-045008, P30 AI027763, R24 AI067039. Gilead Sciences grant INUS2361354; Viiv healthcare grant A126326.

Mild HIV-specific selective forces overlaying natural CD4+ T cell dynamics explain the clonality and decay dynamics of HIV reservoir cells.

The latent reservoir of HIV persists for decades in people living with HIV (PWH) on antiretroviral therapy (ART). To determine if persistence arises from the natural dynamics of memory CD4+ T cells harboring HIV, we compared the clonal dynamics of HIV proviruses to that of memory CD4+ T cell receptors (TCRß) from the same PWH and from HIV-seronegative people. We show that clonal dominance of HIV proviruses and antigen-specific CD4+ T cells are similar but that the field's understanding of the persistence of the less clonally dominant reservoir is significantly limited by undersampling. We demonstrate that increasing reservoir clonality over time and differential decay of intact and defective proviruses cannot be explained by mCD4+ T cell kinetics alone. Finally, we develop a stochastic model of TCRß and proviruses that recapitulates experimental observations and suggests that HIV-specific negative selection mediates approximately 6% of intact and 2% of defective proviral clearance. Thus, HIV persistence is mostly, but not entirely, driven by natural mCD4+ T cell kinetics.

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.

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.

Association of nirmatrelvir for acute SARS-CoV-2 infection with subsequent Long COVID symptoms in an observational cohort study.

Oral nirmatrelvir/ritonavir is approved as treatment for acute COVID-19, but the effect of treatment during acute infection on risk of Long COVID is unknown. We hypothesized that nirmatrelvir treatment during acute SARS-CoV-2 infection reduces risk of developing Long COVID and rebound after treatment is associated with Long COVID. We conducted an observational cohort study within the Covid Citizen Science (CCS) study, an online cohort study with over 100 000 participants. We included vaccinated, nonhospitalized, nonpregnant individuals who reported their first SARS-CoV-2 positive test March-August 2022. Oral nirmatrelvir/ritonavir treatment was ascertained during acute SARS-CoV-2 infection. Patient-reported Long COVID symptoms, symptom rebound and test-positivity rebound were asked on subsequent surveys at least 3 months after SARS-CoV-2 infection. A total of 4684 individuals met the eligibility criteria, of whom 988 (21.1%) were treated and 3696 (78.9%) were untreated; 353/988 (35.7%) treated and 1258/3696 (34.0%) untreated responded to the Long COVID survey (n = 1611). Among 1611 participants, median age was 55 years and 66% were female. At 5.4 ± 1.3 months after infection, nirmatrelvir treatment was not associated with subsequent Long COVID symptoms (odds ratio [OR]: 1.15; 95% confidence interval [CI]: 0.80-1.64; p = 0.45). Among 666 treated who answered rebound questions, rebound symptoms or test positivity were not associated with Long COVID symptoms (OR: 1.34; 95% CI: 0.74-2.41; p = 0.33). Within this cohort of vaccinated, nonhospitalized individuals, oral nirmatrelvir treatment during acute SARS-CoV-2 infection and rebound after nirmatrelvir treatment were not associated with Long COVID symptoms more than 90 days after infection.

Impact of HLA class I functional divergence on HIV control.

Heterozygosity of Human leukocyte antigen (HLA) class I genes is linked to beneficial outcomes after HIV infection, presumably through greater breadth of HIV epitope presentation and cytotoxic T cell response. Distinct allotype pairs, however, differ in the extent to which they bind shared sets of peptides. We developed a functional divergence metric that measures pairwise complementarity of allotype-associated peptide binding profiles. Greater functional divergence for pairs of HLA-A and/or HLA-B allotypes was associated with slower AIDS progression and independently with enhanced viral load control. The metric predicts immune breadth at the peptide level rather than gene level and redefines HLA heterozygosity as a continuum differentially affecting disease outcome. Functional divergence may affect response to additional infections, vaccination, immunotherapy, and other diseases where HLA heterozygote advantage occurs.

High-parameter phenotypic characterization reveals a subset of human Th17 cells that preferentially produce IL17 against M. tuberculosis antigen.

Background: Interleukin 17 producing CD4 T cells contribute to the control of Mycobacterium tuberculosis (Mtb) infection in humans; whether infection with Human Immunodeficiency Virus (HIV) disproportionately affects distinct Th17 cell subsets that respond to Mtb is incompletely defined. Methods: We performed high-definition characterization of circulating Mtb-specific Th17 cells by spectral flow cytometry in people with latent TB and treated HIV (HIV-ART). We also measured kynurenine pathway activity by LC/MS on plasma and tested the hypothesis that tryptophan catabolism influences Th17 cell frequencies in this context. Results: We identified two subsets of Th17 cells: subset 1 defined as CD4+Vα7.2-CD161+CD26+ and subset 2 defined as CD4+Vα7.2-CCR6+CXCR3- cells of which subset 1 was significantly reduced in LTBI with HIV-ART, yet Mtb-responsive IL17-producing CD4 T cells were preserved; we found that IL17-producing CD4 T cells dominate the response to Mtb antigen but not CMV antigen or staphylococcal enterotoxin B (SEB); and tryptophan catabolism negatively correlates with both subset 1 and subset 2 Th17 cell frequencies. Conclusions: We found differential effects of ART-suppressed HIV on distinct subsets of Th17 cells, that IL17-producing CD4 T cells dominate responses to Mtb but not CMV antigen or SEB, and that kynurenine pathway activity is associated with decreases of circulating Th17 cells that may contribute to tuberculosis immunity.

Viral and host mediators of non-suppressible HIV-1 viremia.

Non-suppressible HIV-1 viremia (NSV) is defined as persistent low-level viremia on antiretroviral therapy (ART) without evidence of ART non-adherence or significant drug resistance. Unraveling the mechanisms behind NSV would broaden our understanding of HIV-1 persistence. Here we analyzed plasma virus sequences in eight ART-treated individuals with NSV (88% male) and show that they are composed of large clones without evidence of viral evolution over time in those with longitudinal samples. We defined proviruses that match plasma HIV-1 RNA sequences as 'producer proviruses', and those that did not as 'non-producer proviruses'. Non-suppressible viremia arose from expanded clones of producer proviruses that were significantly larger than the genome-intact proviral reservoir of ART-suppressed individuals. Integration sites of producer proviruses were enriched in proximity to the activating H3K36me3 epigenetic mark. CD4+ T cells from participants with NSV demonstrated upregulation of anti-apoptotic genes and downregulation of pro-apoptotic and type I/II interferon-related pathways. Furthermore, participants with NSV showed significantly lower HIV-specific CD8+ T cell responses compared with untreated viremic controllers with similar viral loads. We identified potential critical host and viral mediators of NSV that may represent targets to disrupt HIV-1 persistence.

The hypoxia-regulated ectonucleotidase CD73 is a host determinant of HIV latency.

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

Plasma-based antigen persistence in the post-acute phase of SARS-CoV-2 infection.

BACKGROUND: Persistent symptoms among some persons who develop COVID-19 has led to the hypothesis that SARS-CoV-2 may, in some form or location, persist for long periods following acute infection. Several studies have shown data in this regard but are limited by non-representative and small study populations, short duration since acute infection, and lack of a true-negative comparator group to assess assay specificity. METHODS: We evaluated adults with RNA-confirmed COVID-19 at multiple time points following acute infection (pandemic-era participants) and adults with specimens collected prior to 2020 (pre-pandemic era). Using once-thawed plasma, we employed the Simoa® (Quanterix) single molecule array detection platform to measure SARS-CoV-2 spike, S1, and nucleocapsid antigens. RESULTS: Compared to 250 pre-pandemic participants who had 2% assay positivity, detection of any SARS-CoV-2 antigen was significantly more frequent among 171 pandemic-era participants at three different time periods in the post-acute phase of infection. The absolute difference in SARS-CoV-2 plasma antigen prevalence was +11% (95% CI: +5.0% to +16%) at 3.0-6.0 months post-onset of COVID-19; +8.7% (95% CI: +3.1% to +14%) at 6.1 to 10.0 months; and +5.4% (95% CI: +0.42% to +10%) at 10.1-14.1 months. Hospitalization for acute COVID-19 and, among the non-hospitalized, worse self-reported health during acute COVID-19 were associated with greater post-acute phase antigen detection. CONCLUSIONS: Compared to uninfected persons, there is an excess prevalence of SARS-CoV-2 antigenemia in SARS-CoV-2-infected individuals up to 14 months after acute COVID-19. These findings motivate an urgent research agenda regarding the short-term and long-term clinical manifestations of this viral persistence.

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.

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.