The Antiviral Mechanism of Action of Molnupiravir in Humans with COVID-19 (preprint)

Meaningful metrics of antiviral activity are essential for determining the efficacy of therapeutics in human clinical trials. Molnupiravir (MOV) is a broadly acting antiviral nucleoside analog prodrug that acts as a competitive alternative substrate for the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). We developed an assay, Culture-PCR, to better understand the impact of MOV therapy on infectious SARS-CoV-2. Culture-PCR revealed MOV eliminated infectious virus within 48 hours in the nasopharyngeal compartment, the upper airway location with the greatest levels of infectious virus. MOV therapy was associated with increases in mutations across the viral genome but select regions were completely unaffected, thus identifying regions where mutation likely abrogates infectivity. MOV therapy did not alter the magnitude or neutralization capacity of the humoral immune response, a documented correlate of protection. Thus, we provide holistic insights into the function of MOV in adults with COVID-19.

SARS-CoV-2 monoclonal antibody treatment followed by vaccination shifts human memory B cell epitope recognition suggesting antibody feedback (preprint)

Therapeutic anti-SARS-CoV-2 monoclonal antibodies (mAbs) have been extensively studied in humans, but the impact on immune memory of mAb treatment during an ongoing immune response has remained unclear. Here, we evaluated the effect of infusion of the anti-SARS-CoV-2 spike receptor binding domain (RBD) mAb bamlanivimab on memory B cells (MBCs) in SARS-CoV-2-infected individuals. Bamlanivimab treatment skewed the repertoire of memory B cells targeting Spike towards non-RBD epitopes. Furthermore, the relative affinity of RBD memory B cells was weaker in mAb-treated individuals compared to placebo-treated individuals over time. Subsequently, after mRNA COVID-19 vaccination, memory B cell differences persisted and mapped to a specific defect in recognition of the class II RBD site, the same RBD epitope recognized by bamlanivimab. These findings indicate a substantial role of antibody feedback in regulating human memory B cell responses, both to infection and vaccination. These data indicate that mAb administration can promote alterations in the epitopes recognized by the B cell repertoire, and the single administration of mAb can continue to determine the fate of B cells in response to additional antigen exposures months later. SIGNIFICANCE STATEMENTEvaluating the therapeutic use of monoclonal antibodies during SARS-CoV-2 infection requires a comprehensive understanding of their impact on B cell responses at the cellular level and how these responses are shaped after vaccination. We report for the first time the effect of bamlanivimab on SARS-CoV-2 specific human memory B cells of COVID-19 infected humans receiving, or not, mRNA immunization.

Early antiviral CD4 and CD8 T cell responses are associated with upper respiratory tract clearance of SARS-CoV-2 (preprint)

T cells are involved in protective immunity against numerous viral infections. Limited data have been available regarding roles of human T cell responses controlling SARS-CoV-2 viral clearance in primary COVID-19. Here, we examined longitudinal SARS-CoV-2 upper respiratory tract viral RNA levels and early adaptive immune responses from 95 unvaccinated individuals with acute COVID-19. Acute SARS-CoV-2-specific CD4 and CD8 T cell responses were evaluated in addition to antibody responses. Most individuals with acute COVID-19 developed rapid SARS-CoV-2-specific T cell responses during infection, and both early CD4 T cell and CD8 T cell responses correlated with reduced upper respiratory tract SARS-CoV-2 viral RNA, independent of neutralizing antibody titers. Overall, our findings indicate a distinct protective role for SARS-CoV-2-specific T cells during acute COVID-19.

Modeling the emergence of viral resistance for SARS-CoV-2 during treatment with an anti-spike monoclonal antibody (preprint)

The COVID-19 pandemic has led to over 760 million cases and 6.9 million deaths worldwide. To mitigate the loss of lives, emergency use authorization was given to several anti-SARS-CoV-2 monoclonal antibody (mAb) therapies for the treatment of mild-to-moderate COVID-19 in patients with a high risk of progressing to severe disease. Monoclonal antibodies used to treat SARS-CoV-2 target the spike protein of the virus and block its ability to enter and infect target cells. Monoclonal antibody therapy can thus accelerate the decline in viral load and lower hospitalization rates among high-risk patients with susceptible variants. However, viral resistance has been observed, in some cases leading to a transient viral rebound that can be as large as 3-4 orders of magnitude. As mAbs represent a proven treatment choice for SARS-CoV-2 and other viral infections, evaluation of treatment-emergent mAb resistance can help uncover underlying pathobiology of SARS-CoV-2 infection and may also help in the development of the next generation of mAb therapies. Although resistance can be expected, the large rebounds observed are much more difficult to explain. We hypothesize replenishment of target cells is necessary to generate the high transient viral rebound. Thus, we formulated two models with different mechanisms for target cell replenishment (homeostatic proliferation and return from an innate immune response anti-viral state) and fit them to data from persons with SARS-CoV-2 treated with a mAb. We showed that both models can explain the emergence of resistant virus associated with high transient viral rebounds. We found that variations in the target cell supply rate and adaptive immunity parameters have a strong impact on the magnitude or observability of the viral rebound associated with the emergence of resistant virus. Both variations in target cell supply rate and adaptive immunity parameters may explain why only some individuals develop observable transient resistant viral rebound. Our study highlights the conditions that can lead to resistance and subsequent viral rebound in mAb treatments during acute infection.

Statistical Challenges when Analyzing SARS-CoV-2 RNA Measurements Below the Assay Limit of Quantification in COVID-19 Clinical Trials (preprint)

Most clinical trials evaluating COVID-19 therapeutics include assessments of antiviral activity. In recently completed outpatient trials, changes in nasal SARS-CoV-2 RNA levels from baseline were commonly assessed using analysis of covariance (ANCOVA) or mixed models for repeated measures (MMRM) with single-imputation for results below assay lower limits of quantification (LLoQ). Analyzing changes in viral RNA levels with singly-imputed values can lead to biased estimates of treatment effects. In this paper, using an illustrative example from the ACTIV-2 trial, we highlight potential pitfalls of imputation when using ANCOVA or MMRM methods, and illustrate how these methods can be used when considering values

Viral and Symptom Rebound in Untreated COVID-19 Infection (preprint)

Background: There are reports of viral RNA and symptom rebound in people with COVID-19 treated with nirmatrelvir/ritonavir. Since the natural course of viral and symptom trajectories of COVID-19 has not been well described, we evaluated the incidence of viral and symptom rebound in untreated outpatients with mild-moderate COVID-19. Methods: The study population included 568 participants enrolled in the ACTIV-2/A5401 platform trial who received placebo. Anterior nasal swabs were collected for SARS-CoV-2 RNA testing on days 0-14, 21 and 28. Participants recorded the severity of 13 targeted symptoms daily from day 0 to 28. Viral rebound was defined as [≥]0.5 log10 viral RNA copies/mL increase and symptom rebound was defined as a 4-point total symptom score increase from baseline. Baseline was defined as study day 4 (primary analysis) or 8 days from symptom onset (secondary analysis). Findings: In both the primary and secondary analyses, 12% of participants had viral rebound. Viral rebounders were older than non-rebounders (median 54 vs 47 years, P=0.04). Symptom rebound occurred in 27% of participants after initial symptom improvement and in 10% of participants after initial symptom resolution. The combination of high-level viral rebound to [≥]5.0 log10 RNA copies/mL and symptom rebound after initial improvement was observed in 1-2% of participants. Interpretation: Viral RNA rebound or symptom relapse in the absence of antiviral treatment is common, but the combination of high-level viral and symptom rebound is rare. Funding: This study was supported by the National Institute of Allergy and Infectious Diseases; ACTIV-2/A5401 ClinicalTrials.gov number NCT04518410.

Bamlanivimab reduces nasopharyngeal SARS-CoV-2 RNA levels but not symptom duration in non-hospitalized adults with COVID-19 (preprint)

Importance: The antiviral activity and efficacy of anti-SARS-CoV-2 monoclonal antibody (mAb) therapies to accelerate recovery from COVID-19 is important to define. Objective: To determine safety and efficacy of the mAb bamlanivimab to reduce nasopharyngeal (NP) SARS-CoV-2 RNA levels and symptom duration. Design: ACTIV-2/A5401 is a randomized, blinded, placebo-controlled platform trial. Two dose cohorts were enrolled between August 19 and November 17, 2020 for phase 2 evaluation: in the first, participants were randomized 1:1 to bamlanivimab 7000 mg versus placebo, and in the second to bamlanivimab 700 mg versus placebo. Randomization was stratified by time from symptom onset ([≤] or >5 days) and risk of progression to severe COVID-19 ('higher' vs 'lower'). Setting: Multicenter trial conducted at U.S. sites. Participants: Non-hospitalized adults [≥]18 years of age with positive SARS-CoV-2 antigen or nucleic acid test within 7 days, [≤]10 days of COVID-19 symptoms, and with oxygen saturation [≥]92% within 48 hours prior to study entry. Intervention: Single infusion of bamlanivimab (7000 or 700 mg) or placebo. Main Outcomes and Measures: Detection of NP SARS-CoV-2 RNA at days 3, 7, 14, 21, and 28, time to improvement of all of 13 targeted COVID-19 symptoms by daily self-assessment through day 28, and grade 3 or higher treatment emergent adverse events (TEAEs) through day 28. Secondary measures included quantitative NP SARS-CoV-2 RNA, all-cause hospitalizations and deaths (composite), area under the curve of symptom scores from day 0 through day 28, plasma bamlanivimab concentrations, plasma and serum inflammatory biomarkers, and safety through week 24. Results: Ninety-four participants were enrolled to the 7000 mg cohort and 223 to the 700 mg cohort and initiated study intervention. The proportion meeting protocol criteria for 'higher' risk for COVID-19 progression was 42% and 51% for the 7000 and 700 mg cohort, respectively. Median time from symptom onset at study entry for both cohorts was 6 days. There was no difference in the proportion with undetectable NP SARS-CoV-2 RNA at any post-treatment timepoints (risk ratio compared to placebo, 0.82-1.05 for 7000 mg dose [overall p=0.88] and 0.81-1.21 for 700 mg dose [overall p=0.49]), time to symptom improvement (median of 21 vs 18.5 days, p=0.97, for 7000 mg bamlanivimab vs placebo and 24 vs 20.5 days, p=0.08, for 700 mg bamlanivimab vs placebo), or grade 3+ TEAEs with either dose compared to placebo. Median NP SARS-CoV-2 RNA levels were lower at day 3 and C-reactive protein, ferritin, and fibrinogen levels significantly reduced at days 7 and 14 for bamlanivimab 700 mg compared to placebo, with similar trends observed for bamlanivimab 7000 mg. Viral decay modeling supported more rapid decay with bamlanivimab compared to placebo. Conclusions and Relevance: Treatment with bamlanivimab 7000 mg and 700 mg was safe and compared to placebo led to more rapid reductions in NP SARS-CoV-2 RNA and inflammatory biomarkers, but did not decrease time to symptom improvement. The clinical utility of mAbs for outcomes other than hospitalizations and deaths is uncertain. Trial Registration: ClinicalTrials.gov Identifier: NCT04518410

Racial and ethnic disparities in COVID-19 disease incidence independent of comorbidities, among people with HIV in the US (preprint)

ObjectivesTo define the incidence of clinically-detected COVID-19 in people with HIV (PWH) in the US and evaluate how racial and ethnic disparities, comorbidities, and HIV-related factors contribute to risk of COVID-19. DesignObservational study within the CFAR Network of Integrated Clinical Systems cohort in 7 cities during 2020. MethodsWe calculated cumulative incidence rates of COVID-19 diagnosis among PWH in routine care by key characteristics including race/ethnicity, current and lowest CD4 count, and geographic area. We evaluated risk factors for COVID-19 among PWH using relative risk regression models adjusted with disease risk scores. ResultsAmong 16,056 PWH in care, of whom 44.5% were Black, 12.5% were Hispanic, with a median age of 52 years (IQR 40-59), 18% had a current CD4 count < 350, including 7% < 200; 95.5% were on antiretroviral therapy, and 85.6% were virologically suppressed. Overall in 2020, 649 PWH were diagnosed with COVID-19 for a rate of 4.94 cases per 100 person-years. The cumulative incidence of COVID-19 was 2.4-fold and 1.7-fold higher in Hispanic and Black PWH respectively, than non-Hispanic White PWH. In adjusted analyses, factors associated with COVID-19 included female sex, Hispanic or Black identity, lowest historical CD4 count <350 (proxy for CD4 nadir), current low CD4/CD8 ratio, diabetes, and obesity. ConclusionsOur results suggest that the presence of structural racial inequities above and beyond medical comorbidities increased the risk of COVID-19 among PWH PWH with immune exhaustion as evidenced by lowest historical CD4 or current low CD4:CD8 ratio had greater risk of COVID-19.

Factors associated with severity of COVID-19 disease in a multicenter cohort of people with HIV in the United States, March-December 2020 (preprint)

BackgroundUnderstanding the spectrum of SARS-CoV-2 infection and COVID-19 disease in people with HIV (PWH) is critical to provide clinical guidance and implement risk-reduction strategies. ObjectiveTo characterize COVID-19 in PWH in the United States and identify predictors of disease severity. DesignObservational cohort study. SettingGeographically diverse clinical sites in the CFAR Network of Integrated Clinical Systems (CNICS) ParticipantsAdults receiving HIV care through December 31, 2020. MeasurementsCOVID-19 cases and severity (hospitalization, intensive care, death). ResultsOf 16,056 PWH in care, 649 were diagnosed with COVID-19 between March-December 2020. Case fatality was 2%; 106 (16.3%) were hospitalized and 12 died. PWH with current CD4 count <350 cells/mm3 (aRR 2.68; 95%CI 1.93-3.71; P<.001) or lowest recorded CD4 count <200 (aRR 1.67; 95%CI 1.18-2.36; P<.005) had greater risk of hospitalization. HIV viral load suppression and antiretroviral therapy (ART) status were not associated with hospitalization, although the majority of PWH were suppressed (86%). Black PWH were 51% more likely to be hospitalized with COVID-19 compared to other racial/ethnic groups (aRR 1.51; 95%CI 1.04-2.19, P=.03). Chronic kidney disease (CKD), chronic obstructive pulmonary disease, diabetes, hypertension, obesity, and increased cardiovascular and hepatic fibrosis risk scores were associated with higher risk of hospitalization. PWH who were older, not on ART, with current CD4 <350, diabetes, and CKD were overrepresented amongst PWH who required intubation or died. LimitationsUnable to compare directly to persons without HIV; underestimate of total COVID-19 cases. ConclusionsPWH with CD4 <350 cells/mm3, low CD4/CD8 ratio, and history of CD4 <200, have a clear excess risk of severe COVID-19, after accounting for comorbidities also associated with severe outcomes. PWH with these risk factors should be prioritized for COVID-19 vaccination, early treatment, and monitored closely for worsening illness.

Emergence of SARS-CoV-2 Resistance with Monoclonal Antibody Therapy (preprint)

Resistance mutations to monoclonal antibody (mAb) therapy has been reported, but in the non-immunosuppressed population, it is unclear if in vivo emergence of SARS-CoV-2 resistance mutations alters either viral replication dynamics or therapeutic efficacy. In ACTIV-2/A5401, non-hospitalized participants with symptomatic SARS-CoV-2 infection were randomized to bamlanivimab (700mg or 7000mg) or placebo. Treatment-emergent resistance mutations were significantly more likely detected after bamlanivimab 700mg treatment than placebo (7% of 111 vs 0% of 112 participants, P=0.003). There were no treatment-emergent resistance mutations among the 48 participants who received bamlanivimab 7000mg. Participants with emerging mAb resistant virus had significantly higher pre-treatment nasopharyngeal and anterior nasal viral load. Intensive respiratory tract viral sampling revealed the dynamic nature of SARS-CoV-2 evolution, with evidence of rapid and sustained viral rebound after emergence of resistance mutations, and worsened symptom severity. Participants with emerging bamlanivimab resistance often accumulated additional polymorphisms found in current variants of concern/interest and associated with immune escape. These results highlight the potential for rapid emergence of resistance during mAb monotherapy treatment, resulting in prolonged high level respiratory tract viral loads and clinical worsening. Careful virologic assessment should be prioritized during the development and clinical implementation of antiviral treatments for COVID-19.

Molnupiravir, an Oral Antiviral Treatment for COVID-19 (preprint)

BackgroundEasily distributed oral antivirals are urgently needed to treat coronavirus disease-2019 (COVID-19), prevent progression to severe illness, and block transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We report the results of a Phase 2a trial evaluating the safety, tolerability, and antiviral efficacy of molnupiravir in the treatment of COVID-19 (ClinicalTrials.gov NCT04405570). MethodsEligible participants included outpatients with confirmed SARS-CoV-2 infection and symptom onset within 7 days. Participants were randomized 1:1 to 200 mg molnupiravir or placebo, or 3:1 to molnupiravir (400 or 800 mg) or placebo, twice-daily for 5 days. Antiviral activity was assessed as time to undetectable levels of viral RNA by reverse transcriptase polymerase chain reaction and time to elimination of infectious virus isolation from nasopharyngeal swabs. ResultsAmong 202 treated participants, virus isolation was significantly lower in participants receiving 800 mg molnupiravir (1.9%) versus placebo (16.7%) at Day 3 (p = 0.02). At Day 5, virus was not isolated from any participants receiving 400 or 800 mg molnupiravir, versus 11.1% of those receiving placebo (p = 0.03). Time to viral RNA clearance was decreased and a greater proportion overall achieved clearance in participants administered 800 mg molnupiravir versus placebo (p = 0.01). Molnupiravir was generally well tolerated, with similar numbers of adverse events across all groups. ConclusionsMolnupiravir is the first oral, direct-acting antiviral shown to be highly effective at reducing nasopharyngeal SARS-CoV-2 infectious virus and viral RNA and has a favorable safety and tolerability profile.

Clinical characteristics and early outcomes in patients with COVID-19 treated with tocilizumab at a United States academic center (preprint)

We describe early outcomes in 11 COVID-19 patients treated with the IL-6 receptor inhibitor tocilizumab. While C-reactive protein decreased, neither clinical improvement nor reduced temperature or oxygen requirements was observed in most patients. Our findings contrast with prior reports and raise questions about tocilizumab use in severe COVID-19.