Early trajectories of virological and immunological biomarkers and clinical outcomes in patients admitted to hospital for COVID-19: an international, prospective cohort study.

BACKGROUND: Serial measurement of virological and immunological biomarkers in patients admitted to hospital with COVID-19 can give valuable insight into the pathogenic roles of viral replication and immune dysregulation. We aimed to characterise biomarker trajectories and their associations with clinical outcomes. METHODS: In this international, prospective cohort study, patients admitted to hospital with COVID-19 and enrolled in the Therapeutics for Inpatients with COVID-19 platform trial within the Accelerating COVID-19 Therapeutic Interventions and Vaccines programme between Aug 5, 2020 and Sept 30, 2021 were included. Participants were included from 108 sites in Denmark, Greece, Poland, Singapore, Spain, Switzerland, Uganda, the UK, and the USA, and randomised to placebo or one of four neutralising monoclonal antibodies: bamlanivimab (Aug 5 to Oct 13, 2020), sotrovimab (Dec 16, 2020, to March 1, 2021), amubarvimab-romlusevimab (Dec 16, 2020, to March 1, 2021), and tixagevimab-cilgavimab (Feb 10 to Sept 30, 2021). This trial included an analysis of 2149 participants with plasma nucleocapsid antigen, anti-nucleocapsid antibody, C-reactive protein (CRP), IL-6, and D-dimer measured at baseline and day 1, day 3, and day 5 of enrolment. Day-90 follow-up status was available for 1790 participants. Biomarker trajectories were evaluated for associations with baseline characteristics, a 7-day pulmonary ordinal outcome, 90-day mortality, and 90-day rate of sustained recovery. FINDINGS: The study included 2149 participants. Participant median age was 57 years (IQR 46-68), 1246 (58·0%) of 2149 participants were male and 903 (42·0%) were female; 1792 (83·4%) had at least one comorbidity, and 1764 (82·1%) were unvaccinated. Mortality to day 90 was 172 (8·0%) of 2149 and 189 (8·8%) participants had sustained recovery. A pattern of less favourable trajectories of low anti-nucleocapsid antibody, high plasma nucleocapsid antigen, and high inflammatory markers over the first 5 days was observed for high-risk baseline clinical characteristics or factors related to SARS-CoV-2 infection. For example, participants with chronic kidney disease demonstrated plasma nucleocapsid antigen 424% higher (95% CI 319-559), CRP 174% higher (150-202), IL-6 173% higher (144-208), D-dimer 149% higher (134-165), and anti-nucleocapsid antibody 39% lower (60-18) to day 5 than those without chronic kidney disease. Participants in the highest quartile for plasma nucleocapsid antigen, CRP, and IL-6 at baseline and day 5 had worse clinical outcomes, including 90-day all-cause mortality (plasma nucleocapsid antigen hazard ratio (HR) 4·50 (95% CI 3·29-6·15), CRP HR 3·37 (2·30-4·94), and IL-6 HR 5·67 (4·12-7·80). This risk persisted for plasma nucleocapsid antigen and CRP after adjustment for baseline biomarker values and other baseline factors. INTERPRETATION: Patients admitted to hospital with less favourable 5-day biomarker trajectories had worse prognosis, suggesting that persistent viral burden might drive inflammation in the pathogenesis of COVID-19, identifying patients that might benefit from escalation of antiviral or anti-inflammatory treatment. FUNDING: US National Institutes of Health.

SARS-CoV-2 RNA and nucleocapsid antigen are blood biomarkers associated with severe disease outcomes that improve in response to remdesivir.

BACKGROUND: Although antivirals remain important for the treatment COVID-19, methods to assess treatment efficacy are lacking. Here, we investigated the impact of remdesivir on viral dynamics and their contribution to understanding antiviral efficacy in the multicenter ACTT-1 clinical trial that randomized patients to remdesivir or placebo. METHODS: Longitudinal specimens collected during hospitalization from a substudy of 642 COVID-19 patients were measured for viral RNA (upper respiratory tract and plasma), viral nucleocapsid antigen (serum), and host immunologic markers. Associations with clinical outcomes and response to therapy were assessed. RESULTS: Higher baseline plasma viral loads were associated with poorer clinical outcomes, and decreases in viral RNA and antigen in blood but not the upper respiratory tract correlated with enhanced benefit from remdesivir. The treatment effect of remdesivir was most pronounced in patients with elevated baseline nucleocapsid antigen levels: the recovery rate ratio was 1.95 (95%CI 1.40-2.71) for levels >245 pg/ml vs 1.04 (95%CI 0.76-1.42) for levels < 245 pg/ml. Remdesivir also accelerated the rate of viral RNA and antigen clearance in blood, and patients whose blood levels decreased were more likely to recover and survive. CONCLUSIONS: Reductions in SARS-CoV-2 RNA and antigen levels in blood correlated with clinical benefit from antiviral therapy.

Novel subtypes of severe COVID-19 respiratory failure based on biological heterogeneity: a secondary analysis of a randomized controlled trial.

BACKGROUND: Despite evidence associating inflammatory biomarkers with worse outcomes in hospitalized adults with COVID-19, trials of immunomodulatory therapies have met with mixed results, likely due in part to biological heterogeneity of participants. Latent class analysis (LCA) of clinical and protein biomarker data has identified two subtypes of non-COVID acute respiratory distress syndrome (ARDS) with different clinical outcomes and treatment responses. We studied biological heterogeneity and clinical outcomes in a multi-institutional platform randomized controlled trial of adults with severe COVID-19 hypoxemic respiratory failure (I-SPY COVID). METHODS: Clinical and plasma protein biomarker data were analyzed from 400 trial participants enrolled from September 2020 until October 2021 with severe COVID-19 requiring ≥ 6 L/min supplemental oxygen. Seventeen hypothesis-directed protein biomarkers were measured at enrollment using multiplex Luminex panels or single analyte enzyme linked immunoassay methods (ELISA). Biomarkers and clinical variables were used to test for latent subtypes and longitudinal biomarker changes by subtype were explored. A validated parsimonious model using interleukin-8, bicarbonate, and protein C was used for comparison with non-COVID hyper- and hypo-inflammatory ARDS subtypes. RESULTS: Average participant age was 60 ± 14 years; 67% were male, and 28-day mortality was 25%. At trial enrollment, 85% of participants required high flow oxygen or non-invasive ventilation, and 97% were receiving dexamethasone. Several biomarkers of inflammation (IL-6, IL-8, IL-10, sTNFR-1, TREM-1), epithelial injury (sRAGE), and endothelial injury (Ang-1, thrombomodulin) were associated with 28- and 60-day mortality. Two latent subtypes were identified. Subtype 2 (27% of participants) was characterized by persistent derangements in biomarkers of inflammation, endothelial and epithelial injury, and disordered coagulation and had twice the mortality rate compared with Subtype 1. Only one person was classified as hyper-inflammatory using the previously validated non-COVID ARDS model. CONCLUSIONS: We discovered evidence of two novel biological subtypes of severe COVID-19 with significantly different clinical outcomes. These subtypes differed from previously established hyper- and hypo-inflammatory non-COVID subtypes of ARDS. Biological heterogeneity may explain inconsistent findings from trials of hospitalized patients with COVID-19 and guide treatment approaches.

Effect of Neutralizing Monoclonal Antibody Treatment on Early Trajectories of Virologic and Immunologic Biomarkers in Patients Hospitalized With COVID-19.

BACKGROUND: Neutralizing monoclonal antibodies (nmAbs) failed to show clear benefit for hospitalized patients with coronavirus disease 2019 (COVID-19). Dynamics of virologic and immunologic biomarkers remain poorly understood. METHODS: Participants enrolled in the Therapeutics for Inpatients with COVID-19 trials were randomized to nmAb versus placebo. Longitudinal differences between treatment and placebo groups in levels of plasma nucleocapsid antigen (N-Ag), anti-nucleocapsid antibody, C-reactive protein, interleukin-6, and D-dimer at enrollment, day 1, 3, and 5 were estimated using linear mixed models. A 7-point pulmonary ordinal scale assessed at day 5 was compared using proportional odds models. RESULTS: Analysis included 2149 participants enrolled between August 2020 and September 2021. Treatment resulted in 20% lower levels of plasma N-Ag compared with placebo (95% confidence interval, 12%-27%; P < .001), and a steeper rate of decline through the first 5 days (P < .001). The treatment difference did not vary between subgroups, and no difference was observed in trajectories of other biomarkers or the day 5 pulmonary ordinal scale. CONCLUSIONS: Our study suggests that nmAb has an antiviral effect assessed by plasma N-Ag among hospitalized patients with COVID-19, with no blunting of the endogenous anti-nucleocapsid antibody response. No effect on systemic inflammation or day 5 clinical status was observed. CLINICAL TRIALS REGISTRATION: NCT04501978.

Long-term persistence of transcriptionally active 'defective' HIV-1 proviruses: implications for persistent immune activation during antiretroviral therapy.

OBJECTIVES: People with HIV-1 (PWH) on effective antiretroviral therapy (ART) continue to exhibit chronic systemic inflammation, immune activation, and persistent elevations in markers of HIV-1 infection [including HIV-DNA, cell-associated HIV-RNA (CA HIV-RNA), and antibodies to HIV-1 proteins] despite prolonged suppression of plasma HIV-RNA levels less than 50 copies/ml. Here, we investigated the hypothesis that nonreplicating but transcriptionally and translationally competent 'defective' HIV-1 proviruses may be one of drivers of these phenomena. DESIGN: A combined cohort of 23 viremic and virologically suppressed individuals on ART were studied. METHODS: HIV-DNA, CA HIV-RNA, western blot score (measure of anti-HIV-1 antibodies as a surrogate for viral protein expression in vivo ), and key biomarkers of inflammation and coagulation (IL-6, hsCRP, TNF-alpha, tissue factor, and D-dimer) were measured in peripheral blood and analyzed using a combined cross-sectional and longitudinal approaches. Sequences of HIV-DNA and CA HIV-RNA obtained via 5'-LTR-to-3'-LTR PCR and single-genome sequencing were also analyzed. RESULTS: We observed similar long-term persistence of multiple, unique, transcriptionally active 'defective' HIV-1 provirus clones (average: 11 years., range: 4-20 years) and antibody responses against HIV-1 viral proteins among all ART-treated participants evaluated. A direct correlation was observed between the magnitude of HIV-1 western blot score and the levels of transcription of 'defective' HIV-1 proviruses ( r  = 0.73, P  < 0.01). Additional correlations were noted between total CD8 + T-cell counts and HIV-DNA ( r  = 0.52, P  = 0.01) or CA HIV-RNA ( r  = 0.65, P  < 0.01). CONCLUSION: These findings suggest a novel interplay between transcription and translation of 'defective' HIV-1 proviruses and the persistent immune activation seen in the setting of treated chronic HIV-1 infection.

Immune reconstitution inflammatory syndrome drives emergence of HIV drug resistance from multiple anatomic compartments in a person living with HIV.

Reservoirs of HIV maintained in anatomic compartments during antiretroviral therapy prevent HIV eradication. However, mechanisms driving their persistence and interventions to control them remain elusive. Here we report the presence of an inducible HIV reservoir within antigen-specific CD4+T cells in the central nervous system of a 59-year-old male with progressive multifocal leukoencephalopathy immune reconstitution inflammatory syndrome (PML-IRIS). HIV production during PML-IRIS was suppressed by modulating inflammation with corticosteroids; selection of HIV drug resistance caused subsequent breakthrough viremia. Therefore, inflammation can influence the composition, distribution and induction of HIV reservoirs, warranting it as a key consideration for developing effective HIV remission strategies.

Clinical evaluation of commercial SARS-CoV-2 serological assays in a malaria endemic setting.

The levels of immune response to SARS-CoV-2 infection or vaccination are poorly understood in African populations and is complicated by cross-reactivity to endemic pathogens as well as differences in host responsiveness. To begin to determine the best approach to minimize false positive antibody levels to SARS-CoV-2 in an African population, we evaluated three commercial assays, namely Bio-Rad Platelia SARS-CoV-2 Total Antibody (Platelia), Quanterix Simoa Semi-Quantitative SARS-CoV-2 IgG Antibody Test (anti-Spike), and the GenScript cPass™ SARS-CoV-2 Neutralization Antibody Detection Kit (cPass) using samples collected in Mali in West Africa prior to the emergence of SARS-CoV-2. A total of one hundred samples were assayed. The samples were categorized in two groups based on the presence or absence of clinical malaria. Overall, thirteen out of one hundred (13/100) samples were false positives with the Bio-Rad Platelia assay and one of the same one hundred (1/100) was a false positive with the anti-Spike IgG Quanterix assay. None of the samples tested with the GenScript cPass assay were positive. False positives were more common in the clinical malaria group, 10/50 (20%) vs. the non-malaria group 3/50 (6%); p = 0.0374 using the Bio-Rad Platelia assay. Association between false positive results and parasitemia by Bio-Rad remained evident, after adjusting for age and sex in multivariate analyses. In summary, the impact of clinical malaria on assay performance appears to depend on the assay and/or antigen being used. A careful evaluation of any given assay in the local context is a prerequisite for reliable serological assessment of anti-SARS-CoV-2 humoral immunity.

Vaccination Ameliorates Cellular Inflammatory Responses in SARS-CoV-2 Breakthrough Infections.

BACKGROUND: Data on cellular immune responses in persons with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection following vaccination are limited. The evaluation of these patients with SARS-CoV-2 breakthrough infections may provide insight into how vaccinations limit the escalation of deleterious host inflammatory responses. METHODS: We conducted a prospective study of peripheral blood cellular immune responses to SARS-CoV-2 infection in 21 vaccinated patients, all with mild disease, and 97 unvaccinated patients stratified based on disease severity. RESULTS: We enrolled 118 persons (aged 50 years [SD 14.5 years], 52 women) with SARS-CoV-2 infection. Compared to unvaccinated patients, vaccinated patients with breakthrough infections had a higher percentage of antigen-presenting monocytes (HLA-DR+), mature monocytes (CD83+), functionally competent T cells (CD127+), and mature neutrophils (CD10+); and lower percentages of activated T cells (CD38+), activated neutrophils (CD64+), and immature B cells (CD127+CD19+). These differences widened with increased disease severity in unvaccinated patients. Longitudinal analysis showed that cellular activation decreased over time but persisted in unvaccinated patients with mild disease at 8-month follow-up. CONCLUSIONS: Patients with SARS-CoV-2 breakthrough infections exhibit cellular immune responses that limit the progression of inflammatory responses and suggest mechanisms by which vaccination limits disease severity. These data may have implications for developing more effective vaccines and therapies. Clinical Trials Registration. NCT04401449.

Clinical, Virologic, and Immunologic Evaluation of Symptomatic Coronavirus Disease 2019 Rebound Following Nirmatrelvir/Ritonavir Treatment.

BACKGROUND: Nirmatrelvir/ritonavir, the first severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protease inhibitor, reduces the risk of hospitalization and death by coronavirus disease 2019 (COVID-19) but has been associated with symptomatic rebound after therapy completion. METHODS: Six individuals with relapse of COVID-19 symptoms after treatment with nirmatrelvir/ritonavir, 2 individuals with rebound symptoms without prior antiviral therapy and 7 patients with acute Omicron infection (controls) were studied. Soluble biomarkers and serum SARS-CoV-2 nucleocapsid protein were measured. Nasal swabs positive for SARS-CoV-2 underwent viral isolation and targeted viral sequencing. SARS-CoV-2 anti-spike, anti-receptor-binding domain, and anti-nucleocapsid antibodies were measured. Surrogate viral neutralization tests against wild-type and Omicron spike protein, as well as T-cell stimulation assays, were performed. RESULTS: High levels of SARS-CoV-2 anti-spike immunoglobulin G (IgG) antibodies were found in all participants. Anti-nucleocapsid IgG and Omicron-specific neutralizing antibodies increased in patients with rebound. Robust SARS-CoV-2-specific T-cell responses were observed, higher in rebound compared with early acute COVID-19 patients. Inflammatory markers mostly decreased during rebound. Two patients sampled longitudinally demonstrated an increase in activated cytokine-producing CD4+ T cells against viral proteins. No characteristic resistance mutations were identified. SARS-CoV-2 was isolated by culture from 1 of 8 rebound patients; Polybrene addition increased this to 5 of 8. CONCLUSIONS: Nirmatrelvir/ritonavir treatment does not impede adaptive immune responses to SARS-CoV-2. Clinical rebound corresponds to development of a robust antibody and T-cell immune response, arguing against a high risk of disease progression. The presence of infectious virus supports the need for isolation and assessment of longer treatment courses. CLINICAL TRIALS REGISTRATION: NCT04401436.

Design of an observational multi-country cohort study to assess immunogenicity of multiple vaccine platforms (InVITE).

In response to the COVID-19 pandemic, COVID-19 vaccines have been developed, and the World Health Oraganization (WHO) has granted emergency use listing to multiple vaccines. Studies of vaccine immunogenicity data from implementing COVID-19 vaccines by national immunization programs in single studies spanning multiple countries and continents are limited but critically needed to answer public health questions on vaccines, such as comparing immune responses to different vaccines and among different populations.

Plasma SARS-CoV-2 nucleocapsid antigen levels are associated with progression to severe disease in hospitalized COVID-19.

BACKGROUND: Studies quantifying SARS-CoV-2 have focused on upper respiratory tract or plasma viral RNA with inconsistent association with clinical outcomes. The association between plasma viral antigen levels and clinical outcomes has not been previously studied. Our aim was to investigate the relationship between plasma SARS-CoV-2 nucleocapsid antigen (N-antigen) concentration and both markers of host response and clinical outcomes. METHODS: SARS-CoV-2 N-antigen concentrations were measured in the first study plasma sample (D0), collected within 72 h of hospital admission, from 256 subjects admitted between March 2020 and August 2021 in a prospective observational cohort of hospitalized patients with COVID-19. The rank correlations between plasma N-antigen and plasma biomarkers of tissue damage, coagulation, and inflammation were assessed. Multiple ordinal regression was used to test the association between enrollment N-antigen plasma concentration and the primary outcome of clinical deterioration at one week as measured by a modified World Health Organization (WHO) ordinal scale. Multiple logistic regression was used to test the association between enrollment plasma N-antigen concentration and the secondary outcomes of ICU admission, mechanical ventilation at 28 days, and death at 28 days. The prognostic discrimination of an externally derived "high antigen" cutoff of N-antigen ≥ 1000 pg/mL was also tested. RESULTS: N-antigen on D0 was detectable in 84% of study participants. Plasma N-antigen levels significantly correlated with RAGE (r = 0.61), IL-10 (r = 0.59), and IP-10 (r = 0.59, adjusted p = 0.01 for all correlations). For the primary outcome of clinical status at one week, each 500 pg/mL increase in plasma N-antigen level was associated with an adjusted OR of 1.05 (95% CI 1.03-1.08) for worse WHO ordinal status. D0 plasma N-antigen ≥ 1000 pg/mL was 77% sensitive and 59% specific (AUROC 0.68) with a positive predictive value of 23% and a negative predictive value of 93% for a worse WHO ordinal scale at day 7 compared to baseline. D0 N-antigen concentration was independently associated with ICU admission and 28-day mechanical ventilation, but not with death at 28 days. CONCLUSIONS: Plasma N-antigen levels are readily measured and provide important insight into the pathogenesis and prognosis of COVID-19. The measurement of N-antigen levels early in-hospital course may improve risk stratification, especially for identifying patients who are unlikely to progress to severe disease.

Safety and Efficacy of Dupilumab for the Treatment of Hospitalized Patients With Moderate to Severe Coronavirus Disease 2019: A Phase 2a Trial.

Background: Based on studies implicating the type 2 cytokine interleukin 13 (IL-13) as a potential contributor to critical coronavirus disease 2019 (COVID-19), this trial was designed as an early phase 2 study to assess dupilumab, a monoclonal antibody that blocks IL-13 and interleukin 4 signaling, for treatment of inpatients with COVID-19. Methods: We conducted a phase 2a randomized, double-blind, placebo-controlled trial (NCT04920916) to assess the safety and efficacy of dupilumab plus standard of care vs placebo plus standard of care in mitigating respiratory failure and death in those hospitalized with COVID-19. Results: Forty eligible subjects were enrolled from June to November of 2021. There was no statistically significant difference in adverse events nor in the primary endpoint of ventilator-free survival at day 28 between study arms. However, for the secondary endpoint of mortality at day 60, there were 2 deaths in the dupilumab group compared with 5 deaths in the placebo group (60-day survival: 89.5% vs 76.2%; adjusted hazard ratio [HR], 0.05 [95% confidence interval {CI}, .004-.72]; P = .03). Among subjects who were not in the intensive care unit (ICU) at randomization, 3 subjects in the dupilumab arm were admitted to the ICU compared to 6 in the placebo arm (17.7% vs 37.5%; adjusted HR, 0.44 [95% CI, .09-2.09]; P = .30). Last, we found evidence of type 2 signaling blockade in the dupilumab group through analysis of immune biomarkers over time. Conclusions: Although the primary outcome of day 28 ventilator-free survival was not reached, adverse events were not observed and survival was higher in the dupilumab group by day 60. Clinical Trials Registration: NCT04920916.

The Association of Baseline Plasma SARS-CoV-2 Nucleocapsid Antigen Level and Outcomes in Patients Hospitalized With COVID-19.

BACKGROUND: Levels of plasma SARS-CoV-2 nucleocapsid (N) antigen may be an important biomarker in patients with COVID-19 and enhance our understanding of the pathogenesis of COVID-19. OBJECTIVE: To evaluate whether levels of plasma antigen can predict short-term clinical outcomes and identify clinical and viral factors associated with plasma antigen levels in hospitalized patients with SARS-CoV-2. DESIGN: Cross-sectional study of baseline plasma antigen level from 2540 participants enrolled in the TICO (Therapeutics for Inpatients With COVID-19) platform trial from August 2020 to November 2021, with additional data on day 5 outcome and time to discharge. SETTING: 114 centers in 10 countries. PARTICIPANTS: Adults hospitalized for acute SARS-CoV-2 infection with 12 days or less of symptoms. MEASUREMENTS: Baseline plasma viral N antigen level was measured at a central laboratory. Delta variant status was determined from baseline nasal swabs using reverse transcriptase polymerase chain reaction. Associations between baseline patient characteristics and viral factors and baseline plasma antigen levels were assessed using both unadjusted and multivariable modeling. Association between elevated baseline antigen level of 1000 ng/L or greater and outcomes, including worsening of ordinal pulmonary scale at day 5 and time to hospital discharge, were evaluated using logistic regression and Fine-Gray regression models, respectively. RESULTS: Plasma antigen was below the level of quantification in 5% of participants at enrollment, and 1000 ng/L or greater in 57%. Baseline pulmonary severity of illness was strongly associated with plasma antigen level, with mean plasma antigen level 3.10-fold higher among those requiring noninvasive ventilation or high-flow nasal cannula compared with room air (95% CI, 2.22 to 4.34). Plasma antigen level was higher in those who lacked antispike antibodies (6.42 fold; CI, 5.37 to 7.66) and in those with the Delta variant (1.73 fold; CI, 1.41 to 2.13). Additional factors associated with higher baseline antigen level included male sex, shorter time since hospital admission, decreased days of remdesivir, and renal impairment. In contrast, race, ethnicity, body mass index, and immunocompromising conditions were not associated with plasma antigen levels. Plasma antigen level of 1000 ng/L or greater was associated with a markedly higher odds of worsened pulmonary status at day 5 (odds ratio, 5.06 [CI, 3.41 to 7.50]) and longer time to hospital discharge (median, 7 vs. 4 days; subhazard ratio, 0.51 [CI, 0.45 to 0.57]), with subhazard ratios similar across all levels of baseline pulmonary severity. LIMITATIONS: Plasma samples were drawn at enrollment, not hospital presentation. No point-of-care test to measure plasma antigen is currently available. CONCLUSION: Elevated plasma antigen is highly associated with both severity of pulmonary illness and clinically important patient outcomes. Multiple clinical and viral factors are associated with plasma antigen level at presentation. These data support a potential role of ongoing viral replication in the pathogenesis of SARS-CoV-2 in hospitalized patients. PRIMARY FUNDING SOURCE: U.S. government Operation Warp Speed and National Institute of Allergy and Infectious Diseases.

High-level dolutegravir resistance can emerge rapidly from few variants and spread by recombination: implications for integrase strand transfer inhibitor salvage therapy.

The integrase strand transfer inhibitor (INSTI) dolutegravir is commonly used in combination antiretroviral therapy regimens and retains strong potency even with primary resistance mutations to some other INSTIs. Acquisition of accessory mutations to primary mutations results in significant increases in dolutegravir resistance. Previously, we reported that addition of the secondary mutation T97A can result in rapid treatment failure in individuals with INSTI mutations at positions 140 and 148. Here, we conducted a detailed case study of one of these individuals and find that T97A-containing HIV emerged from a large replicating population from only a few (≤4) viral lineages. When combined with primary INSTI resistance mutations, T97A provides a strong selective advantage; the finding that T97A-containing variants spread by replication and recombination, and persisted for months after discontinuing dolutegravir, has important implications as dolutegravir is rolled out worldwide.

COVID-19 redux: clinical, virologic, and immunologic evaluation of clinical rebound after nirmatrelvir/ritonavir.

Clinical rebound of COVID-19 after nirmatrelvir/ritonavir treatment has been reported. We performed clinical, virologic, and immune measurements in seven patients with symptomatic rebound, six after nirmatrelvir/ritonavir treatment and one without previous treatment. There was no evidence of severe disease or impaired antibody and T-cell responses in people with rebound symptoms.

QuasiSeq: profiling viral quasispecies via self-tuning spectral clustering with PacBio long sequencing reads.

MOTIVATION: The existence of quasispecies in the viral population causes difficulties for disease prevention and treatment. High-throughput sequencing provides opportunity to determine rare quasispecies and long sequencing reads covering full genomes reduce quasispecies determination to a clustering problem. The challenge is high similarity of quasispecies and high error rate of long sequencing reads. RESULTS: We developed QuasiSeq using a novel signature-based self-tuning clustering method, SigClust, to profile viral mixtures with high accuracy and sensitivity. QuasiSeq can correctly identify quasispecies even using low-quality sequencing reads (accuracy <80%) and produce quasispecies sequences with high accuracy (≥99.55%). Using high-quality circular consensus sequencing reads, QuasiSeq can produce quasispecies sequences with 100% accuracy. QuasiSeq has higher sensitivity and specificity than similar published software. Moreover, the requirement of the computational resource can be controlled by the size of the signature, which makes it possible to handle big sequencing data for rare quasispecies discovery. Furthermore, parallel computation is implemented to process the clusters and further reduce the runtime. Finally, we developed a web interface for the QuasiSeq workflow with simple parameter settings based on the quality of sequencing data, making it easy to use for users without advanced data science skills. AVAILABILITY AND IMPLEMENTATION: QuasiSeq is open source and freely available at https://github.com/LHRI-Bioinformatics/QuasiSeq. The current release (v1.0.0) is archived and available at https://zenodo.org/badge/latestdoi/340494542. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A Longitudinal Study of COVID-19 Sequelae and Immunity: Baseline Findings.

BACKGROUND: A substantial proportion of persons who develop COVID-19 report persistent symptoms after acute illness. Various pathophysiologic mechanisms have been implicated in the pathogenesis of postacute sequelae of SARS-CoV-2 infection (PASC). OBJECTIVE: To characterize medical sequelae and persistent symptoms after recovery from COVID-19 in a cohort of disease survivors and controls. DESIGN: Cohort study. (ClinicalTrials.gov: NCT04411147). SETTING: National Institutes of Health Clinical Center, Bethesda, Maryland. PARTICIPANTS: Self-referred adults with laboratory-documented SARS-CoV-2 infection who were at least 6 weeks from symptom onset were enrolled regardless of presence of PASC. A control group comprised persons with no history of COVID-19 or serologic evidence of SARS-CoV-2 infection, recruited regardless of their current health status. Both groups were enrolled over the same period and from the same geographic area. MEASUREMENTS: All participants had the same evaluations regardless of presence of symptoms, including physical examination, laboratory tests and questionnaires, cognitive function testing, and cardiopulmonary evaluation. A subset also underwent exploratory immunologic and virologic evaluations. RESULTS: 189 persons with laboratory-documented COVID-19 (12% of whom were hospitalized during acute illness) and 120 antibody-negative control participants were enrolled. At enrollment, symptoms consistent with PASC were reported by 55% of the COVID-19 cohort and 13% of control participants. Increased risk for PASC was noted in women and those with a history of anxiety disorder. Participants with findings meeting the definition of PASC reported lower quality of life on standardized testing. Abnormal findings on physical examination and diagnostic testing were uncommon. Neutralizing antibody levels to spike protein were negative in 27% of the unvaccinated COVID-19 cohort and none of the vaccinated COVID-19 cohort. Exploratory studies found no evidence of persistent viral infection, autoimmunity, or abnormal immune activation in participants with PASC. LIMITATIONS: Most participants with COVID-19 had mild to moderate acute illness that did not require hospitalization. The prevalence of reported PASC was likely overestimated in this cohort because persons with PASC may have been more motivated to enroll. The study did not capture PASC that resolved before enrollment. CONCLUSION: A high burden of persistent symptoms was observed in persons after COVID-19. Extensive diagnostic evaluation revealed no specific cause of reported symptoms in most cases. Antibody levels were highly variable after COVID-19. PRIMARY FUNDING SOURCE: Division of Intramural Research, National Institute of Allergy and Infectious Diseases.

Safety and Efficacy of Dupilumab for the Treatment of Hospitalized Patients with Moderate to Severe COVID 19: A Phase IIa Trial.

Background: A profound need remains to develop further therapeutics for treatment of those hospitalized with COVID-19. Based on data implicating the type 2 cytokine interleukin (IL)-13 as a significant factor leading to critical COVID-19, this trial was designed to assess dupilumab, a monoclonal antibody that blocks IL-13 and IL-4 signaling, for treatment of inpatients with COVID-19. Methods: We conducted a phase IIa randomized double-blind placebo-controlled trial to assess the safety and efficacy of dupilumab plus standard of care versus placebo plus standard of care in mitigating respiratory failure and death in those hospitalized with COVID-19. Subjects were followed prospectively for 60 days. The primary endpoint was the proportion of patients alive and free of invasive mechanical ventilation at 28 days. Findings: Forty eligible subjects were enrolled from June to November of 2021. There was no difference in adverse events nor in ventilator free survival at day 28 between study arms. However, for the secondary endpoint of mortality at day 60, subjects randomized to dupilumab had a higher survival rate compared to the placebo group (89.5% vs 76.2%, adjusted HR 0.05, 95% CI: 0.0-0.72, p=0.03). There were fewer subjects admitted to the ICU in the dupilumab group compared to placebo (33.3% vs 66.7%; adjusted HR 0.44, 95% CI: 0.09-2.09, p=0.30). Lastly, we saw downstream evidence of IL-4 and IL-13 signaling blockade in the dupilumab group through analysis of immune biomarkers over time. Interpretation: Dupilumab was well tolerated and improved 60-day survival in patients hospitalized with moderate to severe COVID-19. Trial Registration: This trial is registered with ClinicalTrials.gov, NCT04920916 . Funding: Virginia Biosciences Health Research Corporation, PBM C19, Henske Family Foundation, National Institutes of Health, National Cancer Institute.

RAGE has potential pathogenetic and prognostic value in nonintubated hospitalized patients with COVID-19.

BackgroundThe value of the soluble receptor for advanced glycation end-products (sRAGE) as a biomarker in COVID-19 is not well understood. We tested the association between plasma sRAGE and illness severity, viral burden, and clinical outcomes in hospitalized patients with COVID-19 who were not mechanically ventilated.MethodsBaseline sRAGE was measured among participants enrolled in the ACTIV-3/TICO trial of bamlanivimab for hospitalized patients with COVID-19. Spearman's rank correlation was used to assess the relationship between sRAGE and other plasma biomarkers, including viral nucleocapsid antigen. Fine-Gray models adjusted for baseline supplemental oxygen requirement, antigen level, positive endogenous anti-nucleocapsid antibody response, sex, age, BMI, diabetes mellitus, renal impairment, corticosteroid treatment, and log2-transformed IL-6 level were used to assess the association between baseline sRAGE and time to sustained recovery. Cox regression adjusted for the same factors was used to assess the association between sRAGE and mortality.ResultsAmong 277 participants, baseline sRAGE was strongly correlated with viral plasma antigen concentration (ρ = 0.57). There was a weaker correlation between sRAGE and biomarkers of systemic inflammation, such as IL-6 (ρ = 0.36) and CRP (ρ = 0.20). Participants with plasma sRAGE in the highest quartile had a significantly lower rate of sustained recovery (adjusted recovery rate ratio, 0.64 [95% CI, 0.43-0.90]) and a higher unadjusted risk of death (HR, 4.70 [95% CI, 2.01-10.99]) compared with participants in the lower quartiles.ConclusionElevated plasma sRAGE in hospitalized, nonventilated patients with COVID-19 was an indicator of both clinical illness severity and plasma viral load. Plasma sRAGE in the highest quartile was associated with a lower likelihood of sustained recovery and higher unadjusted risk of death. These findings, which we believe to be novel, indicate that plasma sRAGE may be a promising biomarker for COVID-19 prognostication and clinical trial enrichment.Trial RegistrationClinicalTrials.gov NCT04501978.FundingNIH (5T32GM008440-24, 18X107CF6, HHSN261201500003I, R35HL140026, and OT2HL156812).

A Combination of Amino Acid Mutations Leads to Resistance to Multiple Nucleoside Analogs in Reverse Transcriptases from HIV-1 Subtypes B and C.

Resistance to anti-HIV drugs has been a problem from the beginning of antiviral drug treatments. The recent expansion of combination antiretroviral therapy worldwide has led to an increase in resistance to antiretrovirals; understanding the mechanisms of resistance is increasingly important. In this study, we analyzed reverse transcriptase (RT) variants based on sequences derived from an individual who had low-level rebound viremia while undergoing therapy with abacavir, azidothymidine (AZT) (zidovudine), and (-)-l-2',3'-dideoxy-3'-thiacytidine (3TC) (lamivudine). The RT had mutations at positions 64, 67, 70, 184, and 219 and a threonine insertion after amino acid 69 in RT. The virus remained partially susceptible to the nucleoside RT inhibitor (NRTI) regimen. We show how these mutations affect the ability of NRTIs to inhibit DNA synthesis by RT. The presence of the inserted threonine reduced the susceptibility of the RT mutant to inhibition by tenofovir.