Metformin reduces SARS-CoV-2 in a Phase 3 Randomized Placebo Controlled Clinical Trial (preprint)

Current antiviral treatment options for SARS-CoV-2 infections are not available globally, cannot be used with many medications, and are limited to virus-specific targets.1-3 Biophysical modeling of SARS-CoV-2 replication predicted that protein translation is an especially attractive target for antiviral therapy.4 Literature review identified metformin, widely known as a treatment for diabetes, as a potential suppressor of protein translation via targeting of the host mTor pathway.5 In vitro, metformin has antiviral activity against RNA viruses including SARS-CoV-2.6,7 In the COVID-OUT phase 3, randomized, placebo-controlled trial of outpatient treatment of COVID-19, metformin had a 42% reduction in ER visits/hospitalizations/death through 14 days; a 58% reduction in hospitalizations/death through 28 days, and a 42% reduction in Long COVID through 10 months.8,9 Here we show viral load analysis of specimens collected in the COVID-OUT trial that the mean SARS-CoV-2 viral load was reduced 3.6-fold with metformin relative to placebo (-0.56 log10 copies/mL; 95%CI, -1.05 to -0.06, p=0.027) while there was no virologic effect for ivermectin or fluvoxamine vs placebo. The metformin effect was consistent across subgroups and with emerging data.10,11 Our results demonstrate, consistent with model predictions, that a safe, widely available,12 well-tolerated, and inexpensive oral medication, metformin, can be repurposed to significantly reduce SARS-CoV-2 viral load.

Fluvoxamine for Outpatient Treatment of COVID-19: A Decentralized, Placebo-controlled, Randomized, Platform Clinical Trial (preprint)

Background: The effectiveness of fluvoxamine to shorten symptom duration or prevent hospitalization among outpatients in the US with mild to moderate symptomatic coronavirus disease 2019 (COVID-19) is unclear. Design: ACTIV-6 is an ongoing, decentralized, double-blind, randomized, placebo-controlled platform trial testing repurposed medications in outpatients with mild to moderate COVID-19. A total of 1288 non-hospitalized adults aged >=30 years with confirmed COVID-19 experiencing >=2 symptoms of acute infection for <=7 days prior to randomization were randomized to receive fluvoxamine 50 mg or placebo twice daily for 10 days. The primary outcome was time to sustained recovery, defined as the third of 3 consecutive days without symptoms. Secondary outcomes included composites of hospitalization or death with or without urgent or emergency care visit by day 28. Results: Of 1331 participants randomized (mean [SD] age, 48.5 [12.8] years; 57% women; 67% reported receiving at least 2 doses of a SARS-CoV-2 vaccine), 1288 completed the trial (n=614 placebo, n=674 fluvoxamine). Median time to recovery was 13 days (IQR 12-13) in the placebo group and 12 days (IQR 11-14) in the fluvoxamine group (hazard ratio [HR] 0.96, 95% credible interval [CrI] 0.86-1.07; posterior probability for benefit [HR>1]=0.22). Twenty-six participants (3.9%) in the fluvoxamine group were hospitalized or had urgent or emergency care visits compared with 23 (3.8%) in the placebo group (HR 1.1, 95% CrI 0.6-1.8; posterior probability for benefit [HR<1]=0.340). One participant in the fluvoxamine group and 2 in the placebo group were hospitalized; no deaths occurred. Adverse events were uncommon in both groups. Conclusions: Treatment with fluvoxamine 50 mg twice daily for 10 days did not improve time to recovery, compared with placebo, among outpatients with mild to moderate COVID-19. These findings do not support the use of fluvoxamine at this dose and duration in patients with mild to moderate COVID-19.

Early Treatment with fluvoxamine among patients with COVID-19: a cost-consequence model (preprint)

ABSTRACT Background Three randomized trials have been conducted indicating a clinical benefit of early treatment with fluvoxamine versus placebo for adults with symptomatic COVID-19. We assessed the cost-consequences associated with the use of this early treatment in outpatient populations. Methods Using results from the three completed trials of fluvoxamine vs. placebo for the treatment of COVID-19, we performed a meta-analysis. We conducted a cost-consequence analysis using a decision-model to assess the health system benefits of the avoidance of progression to severe COVID-19. Outcomes of relevance to resource planning decisions in the US and elsewhere, including costs and days of hospitalization avoided, were reported. We constructed a decision-analytic model in the form of a decision tree to evaluate two treatment strategies for high-risk patients with confirmed, symptomatic COVID-19, from the perspective of a third-party payer: (1) treatment with a 10-day course of fluvoxamine (100mg twice daily); (2) current standard-of-care; (3) molnupiravir 5-day course. We used a time horizon of 28 days. Results Administration of fluvoxamine to symptomatic outpatients with COVID-19 at high-risk of developing progression to severe COVID-19 complications is substantially cost-saving in the US, in the amount of $232 per eligible patient, and saves an average of 0.15 hospital days per patient treated is likely to be similarly beneficial in other settings. Fluvoxamine is cost saving in locations where total hospital costs are >$738. Molnupiravir had an additional cost to the healthcare system of $404 per patient treated. Conclusions Fluvoxamine is cost-saving for COVID-19 outpatient therapy.

Hydroxychloroquine as pre-exposure prophylaxis for COVID-19 in healthcare workers: a randomized trial (preprint)

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a rapidly emerging virus causing the ongoing Covid-19 pandemic with no known effective prophylaxis. We investigated whether hydroxychloroquine could prevent SARS CoV-2 in healthcare workers at high-risk of exposure. Methods: We conducted a randomized, double-blind, placebo-controlled clinical trial of healthcare workers with ongoing exposure to persons with Covid-19, including those working in emergency departments, intensive care units, Covid-19 hospital wards, and first responders. Participants across the United States and in the Canadian province of Manitoba were randomized to hydroxychloroquine 400mg once weekly or twice weekly for 12 weeks. The primary endpoint was confirmed or probable Covid-19-compatible illness. We measured hydroxychloroquine whole blood concentrations. Results: We enrolled 1483 healthcare workers, of which 79% reported performing aerosol-generating procedures. The incidence of Covid-19 (laboratory-confirmed or symptomatic compatible illness) was 0.27 events per person-year with once-weekly and 0.28 events per person-year with twice-weekly hydroxychloroquine compared with 0.38 events per person-year with placebo. For once weekly hydroxychloroquine prophylaxis, the hazard ratio was 0.72 (95%CI 0.44 to 1.16; P=0.18) and for twice weekly was 0.74 (95%CI 0.46 to 1.19; P=0.22) as compared with placebo. Median hydroxychloroquine concentrations in whole blood were 98 ng/mL (IQR, 82-120) with once-weekly and 200 ng/mL (IQR, 159-258) with twice-weekly dosing. Hydroxychloroquine concentrations did not differ between participants who developed Covid-19 (154 ng/mL) versus participants without Covid-19 (133 ng/mL; P=0.08). Conclusions: Pre-exposure prophylaxis with hydroxychloroquine once or twice weekly did not significantly reduce laboratory-confirmed Covid-19 or Covid-19-compatible illness among healthcare workers.

Baseline T cell immune phenotypes predict virologic and disease control upon SARS-CoV infection (preprint)

The COVID-19 pandemic has revealed that infection with SARS-CoV-2 can result in a wide range of clinical outcomes in humans, from asymptomatic or mild disease to severe disease that can require mechanical ventilation. An incomplete understanding of immune correlates of protection represents a major barrier to the design of vaccines and therapeutic approaches to prevent infection or limit disease. This deficit is largely due to the lack of prospectively collected, pre-infection samples from indiviuals that go on to become infected with SARS-CoV-2. Here, we utilized data from a screen of genetically diverse mice from the Collaborative Cross (CC) infected with SARS-CoV to determine whether circulating baseline T cell signatures are associated with a lack of viral control and severe disease upon infection. SARS-CoV infection of CC mice results in a variety of viral load trajectories and disease outcomes. Further, early control of virus in the lung correlates with an increased abundance of activated CD4 and CD8 T cells and regulatory T cells prior to infections across strains. A basal propensity of T cells to express IFNg and IL17 over TNFa also correlated with early viral control. Overall, a dysregulated, pro-inflammatory signature of circulating T cells at baseline was associated with severe disease upon infection. While future studies of human samples prior to infection with SARS-CoV-2 are required, our studies in mice with SARS-CoV serve as proof of concept that circulating T cell signatures at baseline can predict clinical and virologic outcomes upon SARS-CoV infection. Identification of basal immune predictors in humans could allow for identification of individuals at highest risk of severe clinical and virologic outcomes upon infection, who may thus most benefit from available clinical interventions to restrict infection and disease.

Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry (preprint)

Coronavirus interaction with viral receptor is a primary genetic determinant of host range and tissue tropism. SARS-CoV-2 utilizes ACE2 as the receptor to enter the host cell in a species-specific manner. We and others have previously shown that ACE2 orthologs from New World monkeys, koala and mouse cannot interact with SARS-CoV-2 to mediate viral entry, and this defect can be restored by humanization of the restrictive residues in New World monkey ACE2. To better understand the genetic determinants of susceptibility of ACE2 orthologs to viral entry, we compared koala and mouse ACE2 sequences with human ortholog, and identified the key residues in koala or mouse ACE2 that restrict its viral receptor activity. Humanization of these critical residues could render the capabilities of koala and mouse ACE2 to bind viral spike protein and facilitate the viral entry. Our work identifies the genetic determinant of ACE2 for SARS-CoV-2 susceptibility, and a single mutation could restore the mouse ACE2 receptor activity, providing a potential avenue for the development of mouse model of SARS-CoV-2.

Cryo-EM structure of S-Trimer, a subunit vaccine candidate for COVID-19 (preprint)

Less than a year after its emergence, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 22 million people worldwide with a death toll approaching 1 million. Vaccination remains the best hope to ultimately put this pandemic to an end. Here, using Trimer-Tag technology, we produced both wild-type (WT) and furin site mutant (MT) S-Trimers for COVID-19 vaccine studies. Cryo-EM structures of the WT and MT S-Trimers, determined at 3.2 Angstrom and 2.6 Angstrom respectively, revealed that both antigens adopt a tightly closed conformation and their structures are essentially identical to that of the previously solved full-length WT S protein in detergent. These results validate Trimer-Tag as a platform technology in production of metastable WT S-Trimer as a candidate for COVID-19 subunit vaccine.

SARS-CoV-2 Nsp1 suppresses host but not viral translation through a bipartite mechanism (preprint)

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a highly contagious virus that underlies the current COVID-19 pandemic. SARS-CoV-2 is thought to disable various features of host immunity and cellular defense. The SARS-CoV-2 nonstructural protein 1 (Nsp1) is known to inhibit host protein translation and could be a target for antiviral therapy against COVID-19. However, how SARS-CoV-2 circumvents this translational blockage for the production of its own proteins is an open question. Here, we report a bipartite mechanism of SARS-CoV-2 Nsp1 which operates by: (1) hijacking the host ribosome via direct interaction of its C-terminal domain (CT) with the 40S ribosomal subunit and (2) specifically lifting this inhibition for SARS-CoV-2 via a direct interaction of its N-terminal domain (NT) with the 5 untranslated region (5 UTR) of SARS-CoV-2 mRNA. We show that while Nsp1-CT is sufficient for binding to 40S and inhibition of host protein translation, the 5 UTR of SARS-CoV-2 mRNA removes this inhibition by binding to Nsp1-NT, suggesting that the Nsp1-NT-UTR interaction is incompatible with the Nsp1-CT-40S interaction. Indeed, lengthening the linker between Nsp1-NT and Nsp1-CT of Nsp1 progressively reduced the ability of SARS-CoV-2 5 UTR to escape the translational inhibition, supporting that the incompatibility is likely steric in nature. The short SL1 region of the 5 UTR is required for viral mRNA translation in the presence of Nsp1. Thus, our data provide a comprehensive view on how Nsp1 switches infected cells from host mRNA translation to SARS-CoV-2 mRNA translation, and that Nsp1 and 5 UTR may be targeted for anti-COVID-19 therapeutics.

An immunodominance hierarchy exists in CD8+ T cell responses to HLA-A*02:01-restricted epitopes identified from the non-structural polyprotein 1a of SARS-CoV-2. (preprint)

COVID-19 vaccines are being rapidly developed and human trials are underway. Almost all of these vaccines have been designed to induce antibodies targeting spike protein of SARS-CoV-2 in expectation of neutralizing activities. However, non-neutralizing antibodies are at risk of causing antibody-dependent enhancement. Further, the longevity of SARS-CoV-2-specific antibodies is very short. Therefore, in addition to antibody-induced vaccines, novel vaccines on the basis of SARS-CoV-2-specific cytotoxic T lymphocytes (CTLs) should be considered in the vaccine development. Here, we attempted to identify HLA-A*02:01-restricted CTL epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2. Eighty-two peptides were firstly predicted as epitope candidates on bioinformatics. Fifty-four in 82 peptides showed high or medium binding affinities to HLA-A*02:01. HLA-A*02:01 transgenic mice were then immunized with each of the 54 peptides encapsulated into liposomes. The intracellular cytokine staining assay revealed that 18 out of 54 peptides were CTL epitopes because of the induction of IFN-{gamma}-producing CD8+ T cells. In the 18 peptides, 10 peptides were chosen for the following analyses because of their high responses. To identify dominant CTL epitopes, mice were immunized with liposomes containing the mixture of the 10 peptides. Some peptides were shown to be statistically predominant over the other peptides. Surprisingly, all mice immunized with the liposomal 10 peptide mixture did not show the same reaction pattern to the 10 peptides. There were three pattern types that varied sequentially, suggesting the existence of an immunodominance hierarchy, which may provide us more variations in the epitope selection for designing CTL-based COVID-19 vaccines. ImportanceFor the development of vaccines based on SARS-CoV-2-specific cytotoxic T lymphocytes (CTLs), we attempted to identify HLA-A*02:01-restricted CTL epitopes derived from the non-structural polyprotein 1a of SARS-CoV-2. Out of 82 peptides predicted on bioinformatics, 54 peptides showed good binding affinities to HLA-A*02:01. Using HLA-A*02:01 transgenic mice, 18 in 54 peptides were found to be CTL epitopes in the intracellular cytokine staining assay. Out of 18 peptides, 10 peptides were chosen for the following analyses because of their high responses. To identify dominant epitopes, mice were immunized with liposomes containing the mixture of the 10 peptides. Some peptides were shown to be statistically predominant. Surprisingly, all immunized mice did not show the same reaction pattern to the 10 peptides. There were three pattern types that varied sequentially, suggesting the existence of an immunodominance hierarchy, which may provide us more variations in the epitope selection for designing CTL-based COVID-19 vaccines.

Safety of Hydroxychloroquine among Outpatient Clinical Trial Participants for COVID-19 (preprint)

IntroductionUse of hydroxychloroquine in hospitalized patients with COVID-19, especially in combination with azithromycin, has raised safety concerns. Here, we report safety data from three outpatient randomized clinical trials. MethodsWe conducted three randomized, double-blind, placebo-controlled trials investigating hydroxychloroquine as pre-exposure prophylaxis, post-exposure prophylaxis and early treatment for COVID-19. We excluded individuals with contraindications to hydroxychloroquine. We collected side effects and serious adverse events. We report descriptive analyses of our findings. ResultsWe enrolled 2,795 participants. The median age of research participants was 40 (IQR 34-49) years, and 59% (1633/2767) reported no chronic medical conditions. Overall 2,324 (84%) participants reported side effect data, and 638 (27%) reported at least one medication side effect. Side effects were reported in 29% with daily, 36% with twice weekly, 31% with once weekly hydroxychloroquine compared to 19% with placebo. The most common side effects were upset stomach or nausea (25% with daily, 18% with twice weekly, 16% with weekly, vs. 10% for placebo), followed by diarrhea, vomiting, or abdominal pain (23% for daily, 16% twice weekly, 12% weekly, vs. 6% for placebo). Two individuals were hospitalized for atrial arrhythmias, one on placebo and one on twice weekly hydroxychloroquine. No sudden deaths occurred. ConclusionData from three outpatient COVID-19 trials demonstrated that gastrointestinal side effects were common but mild with the use of hydroxychloroquine, while serious side effects were rare. No deaths occurred related to hydroxychloroquine. Randomized clinical trials can safely investigate whether hydroxychloroquine is efficacious for COVID-19. Short SummaryData from three randomized clinical trials using hydroxychloroquine for the prevention and treatment of COVID-19 did not suggest significant safety concerns. Gastrointestinal side effects were common but arrhythmias were rare. There were no sudden deaths in any trial.

Post-exposure Prophylaxis or Preemptive Therapy for SARS-Coronavirus-2: Study Protocol for a Pragmatic Randomized Controlled Trial (preprint)

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019 causing the coronavirus disease 2019 (COVID-19) pandemic. Currently, there are a lack of evidence-based therapies to prevent COVID-19 following exposure, or to prevent worsening of symptoms following confirmed infection. We describe the design of a clinical trial of hydroxychloroquine for post-exposure prophylaxis and pre-emptive therapy for COVID-19. Methods: We will conduct two nested multicenter international double-blind randomized placebo-controlled clinical trials of hydroxychloroquine for: 1) post-exposure prophylaxis (PEP) of asymptomatic household contacts or healthcare workers exposed to COVID-19 within the past four days, and 2) pre-emptive therapy (PET) for symptomatic outpatients with COVID-19 with a total symptom duration of less than 4 days. We will recruit 1500 patients for each the PEP and PET trials. Participants will be randomized 1:1 to receive 5 days of hydroxychloroquine or placebo. The primary PEP trial outcome will be the incidence of symptomatic COVID-19 disease. The primary PET trial outcome will be an ordinal scale of disease severity (not hospitalized; hospitalized without intensive care, hospitalization with intensive care, or death). Participant screening, informed consent, and follow up will be exclusively internet-based with appropriate regulatory and research ethics board approvals in Canada and the United States. Discussion: These complementary randomized control trials are innovatively designed and adequately powered to rapidly answer urgent questions regarding the effectiveness of hydroxychloroquine to reduce transmission and disease severity of COVID-19 during a pandemic. In-person participant follow-up will not be conducted in order to facilitate social distancing strategies and reduce risks of exposure to study personnel. Innovative trial approaches are needed to urgently assess therapeutic options to mitigate the global impact of this pandemic. Trials Registration: clinicaltrials.gov (NCT04308668); 16 March 2020.