SARS-CoV-2 Viral Shedding and Rapid Antigen Test Performance - Respiratory Virus Transmission Network, November 2022-May 2023.

As population immunity to SARS-CoV-2 evolves and new variants emerge, the role and accuracy of antigen tests remain active questions. To describe recent test performance, the detection of SARS-CoV-2 by antigen testing was compared with that by reverse transcription-polymerase chain reaction (RT-PCR) and viral culture testing during November 2022-May 2023. Participants who were enrolled in a household transmission study completed daily symptom diaries and collected two nasal swabs (tested for SARS-CoV-2 via RT-PCR, culture, and antigen tests) each day for 10 days after enrollment. Among participants with SARS-CoV-2 infection, the percentages of positive antigen, RT-PCR, and culture results were calculated each day from the onset of symptoms or, in asymptomatic persons, from the date of the first positive test result. Antigen test sensitivity was calculated using RT-PCR and viral culture as references. The peak percentage of positive antigen (59.0%) and RT-PCR (83.0%) results occurred 3 days after onset, and the peak percentage of positive culture results (52%) occurred 2 days after onset. The sensitivity of antigen tests was 47% (95% CI = 44%-50%) and 80% (95% CI = 76%-85%) using RT-PCR and culture, respectively, as references. Clinicians should be aware of the lower sensitivity of antigen testing compared with RT-PCR, which might lead to false-negative results. This finding has implications for timely initiation of SARS-CoV-2 antiviral treatment, when early diagnosis is essential; clinicians should consider RT-PCR for persons for whom antiviral treatment is recommended. Persons in the community who are at high risk for severe COVID-19 illness and eligible for antiviral treatment should seek testing from health care providers with the goal of obtaining a more sensitive diagnostic test than antigen tests (i.e., an RT-PCR test).

AS03 adjuvant enhances the magnitude, persistence, and clonal breadth of memory B cell responses to a plant-based COVID-19 vaccine in humans.

Vaccine adjuvants increase the breadth of serum antibody responses, but whether this is due to the generation of antigen-specific B cell clones with distinct specificities or the maturation of memory B cell clones that produce broadly cross-reactive antibodies is unknown. Here, we longitudinally analyzed immune responses in healthy adults after two-dose vaccination with either a virus-like particle COVID-19 vaccine (CoVLP), CoVLP adjuvanted with AS03 (CoVLP+AS03), or a messenger RNA vaccination (mRNA-1273). CoVLP+AS03 enhanced the magnitude and durability of circulating antibodies and antigen-specific CD4+ T cell and memory B cell responses. Antigen-specific CD4+ T cells in the CoVLP+AS03 group at day 42 correlated with antigen-specific memory B cells at 6 months. CoVLP+AS03 induced memory B cell responses, which accumulated somatic hypermutations over 6 months, resulting in enhanced neutralization breadth of monoclonal antibodies. Furthermore, the fraction of broadly neutralizing antibodies encoded by memory B cells increased between day 42 and 6 months. These results indicate that AS03 enhances the antigenic breadth of B cell memory at the clonal level and induces progressive maturation of the B cell response.

Single-cell analysis reveals an antiviral network that controls Zika virus infection in human dendritic cells.

Zika virus (ZIKV) is a mosquito-borne flavivirus that caused an epidemic in the Americas in 2016 and is linked to severe neonatal birth defects, including microcephaly and spontaneous abortion. To better understand the host response to ZIKV infection, we adapted the 10× Genomics Chromium single-cell RNA sequencing (scRNA-seq) assay to simultaneously capture viral RNA and host mRNA. Using this assay, we profiled the antiviral landscape in a population of human monocyte-derived dendritic cells infected with ZIKV at the single-cell level. The bystander cells, which lacked detectable viral RNA, expressed an antiviral state that was enriched for genes coinciding predominantly with a type I interferon (IFN) response. Within the infected cells, viral RNA negatively correlated with type I IFN-dependent and -independent genes (the antiviral module). We modeled the ZIKV-specific antiviral state at the protein level, leveraging experimentally derived protein interaction data. We identified a highly interconnected network between the antiviral module and other host proteins. In this work, we propose a new paradigm for evaluating the antiviral response to a specific virus, combining an unbiased list of genes that highly correlate with viral RNA on a per-cell basis with experimental protein interaction data. IMPORTANCE: Zika virus (ZIKV) remains a public health threat given its potential for re-emergence and the detrimental fetal outcomes associated with infection during pregnancy. Understanding the dynamics between ZIKV and its host is critical to understanding ZIKV pathogenesis. Through ZIKV-inclusive single-cell RNA sequencing (scRNA-seq), we demonstrate on the single-cell level the dynamic interplay between ZIKV and the host: the transcriptional program that restricts viral infection and ZIKV-mediated inhibition of that response. Our ZIKV-inclusive scRNA-seq assay will serve as a useful tool for gaining greater insight into the host response to ZIKV and can be applied more broadly to the flavivirus field.

Maternal COVID-19 Vaccination and Prevention of Symptomatic Infection in Infants.

BACKGROUND AND OBJECTIVES: Maternal vaccination may prevent infant coronavirus disease 2019 (COVID-19). We aimed to quantify protection against infection from maternally derived vaccine-induced antibodies in the first 6 months of an infant's life. METHODS: Infants born to mothers vaccinated during pregnancy with 2 or 3 doses of a messenger RNA COVID-19 vaccine (nonboosted or boosted, respectively) had full-length spike (Spike) immunoglobulin G (IgG), pseudovirus 614D, and live virus D614G, and omicron BA.1 and BA.5 neutralizing antibody (nAb) titers measured at delivery. Infant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection was determined by verified maternal-report and laboratory confirmation through prospective follow-up to 6 months of age between December 2021 and July 2022. The risk reduction for infection by dose group and antibody titer level was estimated in separate models. RESULTS: Infants of boosted mothers (n = 204) had significantly higher Spike IgG, pseudovirus, and live nAb titers at delivery than infants of nonboosted mothers (n = 271), and were 56% less likely to acquire infection in the first 6 months (P = .03). Irrespective of boost, for each 10-fold increase in Spike IgG titer at delivery, the infant's risk of acquiring infection was reduced by 47% (95% confidence interval 8%-70%; P = .02). Similarly, a 10-fold increase in pseudovirus titers against Wuhan Spike, and live virus nAb titers against D614G, and omicron BA.1 and BA.5 at delivery were associated with a 30%, 46%, 56%, and 60% risk reduction, respectively. CONCLUSIONS: Higher transplacental binding and nAb titers substantially reduced the risk of SARS-CoV-2 infection in infants, and a booster dose amplified protection during a period of omicron predominance. Until infants are age-eligible for vaccination, maternal vaccination provides passive protection against symptomatic infection during early infancy.

CD4+ and CD8+ T cells are required to prevent SARS-CoV-2 persistence in the nasal compartment.

SARS-CoV-2 is the causative agent of COVID-19 and continues to pose a significant public health threat throughout the world. Following SARS-CoV-2 infection, virus-specific CD4+ and CD8+ T cells are rapidly generated to form effector and memory cells and persist in the blood for several months. However, the contribution of T cells in controlling SARS-CoV-2 infection within the respiratory tract are not well understood. Using C57BL/6 mice infected with a naturally occurring SARS-CoV-2 variant (B.1.351), we evaluated the role of T cells in the upper and lower respiratory tract. Following infection, SARS-CoV-2-specific CD4+ and CD8+ T cells are recruited to the respiratory tract and a vast proportion secrete the cytotoxic molecule Granzyme B. Using antibodies to deplete T cells prior to infection, we found that CD4+ and CD8+ T cells play distinct roles in the upper and lower respiratory tract. In the lungs, T cells play a minimal role in viral control with viral clearance occurring in the absence of both CD4+ and CD8+ T cells through 28 days post-infection. In the nasal compartment, depletion of both CD4+ and CD8+ T cells, but not individually, results in persistent and culturable virus replicating in the nasal compartment through 28 days post-infection. Using in situ hybridization, we found that SARS-CoV-2 infection persisted in the nasal epithelial layer of tandem CD4+ and CD8+ T cell-depleted mice. Sequence analysis of virus isolates from persistently infected mice revealed mutations spanning across the genome, including a deletion in ORF6. Overall, our findings highlight the importance of T cells in controlling virus replication within the respiratory tract during SARS-CoV-2 infection.

XBB.1.5 monovalent booster improves antibody binding and neutralization against emerging SARS-CoV-2 Omicron variants.

The rapid emergence of divergent SARS-CoV-2 variants has led to an update of the COVID-19 booster vaccine to a monovalent version containing the XBB.1.5 spike. To determine the neutralization breadth following booster immunization, we collected blood samples from 24 individuals pre- and post-XBB.1.5 mRNA booster vaccination (∼1 month). The XBB.1.5 booster improved both neutralizing activity against the ancestral SARS-CoV-2 strain (WA1) and the circulating Omicron variants, including EG.5.1, HK.3, HV.1, XBB.1.5 and JN.1. Relative to the pre-boost titers, the XBB.1.5 monovalent booster induced greater total IgG and IgG subclass binding, particular IgG4, to the XBB.1.5 spike as compared to the WA1 spike. We evaluated antigen-specific memory B cells (MBCs) using either spike or receptor binding domain (RBD) probes and found that the monovalent booster largely increases non-RBD cross-reactive MBCs. These data suggest that the XBB.1.5 monovalent booster induces cross-reactive antibodies that neutralize XBB.1.5 and related Omicron variants.

Single cell analysis reveals an antiviral network that controls Zika virus infection in human dendritic cells.

Zika virus (ZIKV) is a mosquito-borne flavivirus that caused an epidemic in the Americas in 2016 and is linked to severe neonatal birth defects, including microcephaly and spontaneous abortion. To better understand the host response to ZIKV infection, we adapted the 10x Genomics Chromium single cell RNA sequencing (scRNA-seq) assay to simultaneously capture viral RNA and host mRNA. Using this assay, we profiled the antiviral landscape in a population of human moDCs infected with ZIKV at the single cell level. The bystander cells, which lacked detectable viral RNA, expressed an antiviral state that was enriched for genes coinciding predominantly with a type I interferon (IFN) response. Within the infected cells, viral RNA negatively correlated with type I IFN dependent and independent genes (antiviral module). We modeled the ZIKV specific antiviral state at the protein level leveraging experimentally derived protein-interaction data. We identified a highly interconnected network between the antiviral module and other host proteins. In this work, we propose a new paradigm for evaluating the antiviral response to a specific virus, combining an unbiased list of genes that highly correlate with viral RNA on a per cell basis with experimental protein interaction data. Our ZIKV-inclusive scRNA-seq assay will serve as a useful tool to gaining greater insight into the host response to ZIKV and can be applied more broadly to the flavivirus field.

BCG vaccination stimulates integrated organ immunity by feedback of the adaptive immune response to imprint prolonged innate antiviral resistance.

Bacille Calmette-Guérin (BCG) vaccination can confer nonspecific protection against heterologous pathogens. However, the underlying mechanisms remain mysterious. We show that mice vaccinated intravenously with BCG exhibited reduced weight loss and/or improved viral clearance when challenged with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 B.1.351) or PR8 influenza. Protection was first evident between 14 and 21 d post-vaccination and lasted ∼3 months. Notably, BCG induced a biphasic innate response and robust antigen-specific type 1 helper T cell (TH1 cell) responses in the lungs. MyD88 signaling was essential for innate and TH1 cell responses, and protection against SARS-CoV-2. Depletion of CD4+ T cells or interferon (IFN)-γ activity before infection obliterated innate activation and protection. Single-cell and spatial transcriptomics revealed CD4-dependent expression of IFN-stimulated genes in lung myeloid and epithelial cells. Notably, BCG also induced protection against weight loss after mouse-adapted SARS-CoV-2 BA.5, SARS-CoV and SHC014 coronavirus infections. Thus, BCG elicits integrated organ immunity, where CD4+ T cells feed back on tissue myeloid and epithelial cells to imprint prolonged and broad innate antiviral resistance.

Mucosal boosting enhances vaccine protection against SARS-CoV-2 in macaques.

A limitation of current SARS-CoV-2 vaccines is that they provide minimal protection against infection with current Omicron subvariants1,2, although they still provide protection against severe disease. Enhanced mucosal immunity may be required to block infection and onward transmission. Intranasal administration of current vaccines has proven inconsistent3-7, suggesting that alternative immunization strategies may be required. Here we show that intratracheal boosting with a bivalent Ad26-based SARS-CoV-2 vaccine results in substantial induction of mucosal humoral and cellular immunity and near-complete protection against SARS-CoV-2 BQ.1.1 challenge. A total of 40 previously immunized rhesus macaques were boosted with a bivalent Ad26 vaccine by the intramuscular, intranasal and intratracheal routes, or with a bivalent mRNA vaccine by the intranasal route. Ad26 boosting by the intratracheal route led to a substantial expansion of mucosal neutralizing antibodies, IgG and IgA binding antibodies, and CD8+ and CD4+ T cell responses, which exceeded those induced by Ad26 boosting by the intramuscular and intranasal routes. Intratracheal Ad26 boosting also led to robust upregulation of cytokine, natural killer, and T and B cell pathways in the lungs. After challenge with a high dose of SARS-CoV-2 BQ.1.1, intratracheal Ad26 boosting provided near-complete protection, whereas the other boosting strategies proved less effective. Protective efficacy correlated best with mucosal humoral and cellular immune responses. These data demonstrate that these immunization strategies induce robust mucosal immunity, suggesting the feasibility of developing vaccines that block respiratory viral infections.

Durable immunity to SARS-CoV-2 in both lower and upper airways achieved with a gorilla adenovirus (GRAd) S-2P vaccine in non-human primates.

SARS-CoV-2 continues to pose a global threat, and current vaccines, while effective against severe illness, fall short in preventing transmission. To address this challenge, there's a need for vaccines that induce mucosal immunity and can rapidly control the virus. In this study, we demonstrate that a single immunization with a novel gorilla adenovirus-based vaccine (GRAd) carrying the pre-fusion stabilized Spike protein (S-2P) in non-human primates provided protective immunity for over one year against the BA.5 variant of SARS-CoV-2. A prime-boost regimen using GRAd followed by adjuvanted S-2P (GRAd+S-2P) accelerated viral clearance in both the lower and upper airways. GRAd delivered via aerosol (GRAd(AE)+S-2P) modestly improved protection compared to its matched intramuscular regimen, but showed dramatically superior boosting by mRNA and, importantly, total virus clearance in the upper airway by day 4 post infection. GrAd vaccination regimens elicited robust and durable systemic and mucosal antibody responses to multiple SARS-CoV-2 variants, but only GRAd(AE)+S-2P generated long-lasting T cell responses in the lung. This research underscores the flexibility of the GRAd vaccine platform to provide durable immunity against SARS-CoV-2 in both the lower and upper airways.

Mucosal Adenoviral-vectored Vaccine Boosting Durably Prevents XBB.1.16 Infection in Nonhuman Primates.

Waning immunity and continued virus evolution have limited the durability of protection from symptomatic infection mediated by intramuscularly (IM)-delivered mRNA vaccines against COVID-19 although protection from severe disease remains high. Mucosal vaccination has been proposed as a strategy to increase protection at the site of SARS-CoV-2 infection by enhancing airway immunity, potentially reducing rates of infection and transmission. Here, we compared protection against XBB.1.16 virus challenge 5 months following IM or mucosal boosting in non-human primates (NHP) that had previously received a two-dose mRNA-1273 primary vaccine regimen. The mucosal boost was composed of a bivalent chimpanzee adenoviral-vectored vaccine encoding for both SARS-CoV-2 WA1 and BA.5 spike proteins (ChAd-SARS-CoV-2-S) and delivered either by an intranasal mist or an inhaled aerosol. An additional group of animals was boosted by the IM route with bivalent WA1/BA.5 spike-matched mRNA (mRNA-1273.222) as a benchmark control. NHP were challenged in the upper and lower airways 18 weeks after boosting with XBB.1.16, a heterologous Omicron lineage strain. Cohorts boosted with ChAd-SARS-CoV-2-S by an aerosolized or intranasal route had low to undetectable virus replication as assessed by levels of subgenomic SARS-CoV-2 RNA in the lungs and nose, respectively. In contrast, animals that received the mRNA-1273.222 boost by the IM route showed minimal protection against virus replication in the upper airway but substantial reduction of virus RNA levels in the lower airway. Immune analysis showed that the mucosal vaccines elicited more durable antibody and T cell responses than the IM vaccine. Protection elicited by the aerosolized vaccine was associated with mucosal IgG and IgA responses, whereas protection elicited by intranasal delivery was mediated primarily by mucosal IgA. Thus, durable immunity and effective protection against a highly transmissible heterologous variant in both the upper and lower airways can be achieved by mucosal delivery of a virus-vectored vaccine. Our study provides a template for the development of mucosal vaccines that limit infection and transmission against respiratory pathogens.

BA.5 bivalent booster vaccination enhances neutralization of XBB.1.5, XBB.1.16 and XBB.1.9 variants in patients with lung cancer.

This study reports that most patients with NSCLC had a significant increase in the nAb response to the currently circulating Omicron variants after bivalent booster vaccination and had Ab titers comparable to healthy participants. Interestingly, though the durability of the nAb response persisted in most of the healthy participants, patients with NSCLC had significantly reduced nAb titers after 4-6 months of vaccination. Our data highlight the importance of COVID-19 bivalent booster vaccination as the standard of care for patients with NSCLC given the evolution of new variants of concern.

Divergence of variant binding/neutralizing antibodies following SARS-CoV-2 booster vaccines in myeloma: Impact of hybrid immunity.

We characterized virus-neutralization and spike-binding antibody profiles in myeloma patients following monovalent or bivalent-SARS-CoV-2 booster vaccination. Vaccination improves the breadth of binding antibodies but not neutralization activity against current variants. Hybrid immunity and immune imprinting impact vaccine-elicited immunity.

Mechanisms by which the cystic fibrosis transmembrane conductance regulator may influence SARS-CoV-2 infection and COVID-19 disease severity.

Patients with cystic fibrosis (CF) exhibit pronounced respiratory damage and were initially considered among those at highest risk for serious harm from SARS-CoV-2 infection. Numerous clinical studies have subsequently reported that individuals with CF in North America and Europe-while susceptible to severe COVID-19-are often spared from the highest levels of virus-associated mortality. To understand features that might influence COVID-19 among patients with cystic fibrosis, we studied relationships between SARS-CoV-2 and the gene responsible for CF (i.e., the cystic fibrosis transmembrane conductance regulator, CFTR). In contrast to previous reports, we found no association between CFTR carrier status (mutation heterozygosity) and more severe COVID-19 clinical outcomes. We did observe an unexpected trend toward higher mortality among control individuals compared with silent carriers of the common F508del CFTR variant-a finding that will require further study. We next performed experiments to test the influence of homozygous CFTR deficiency on viral propagation and showed that SARS-CoV-2 production in primary airway cells was not altered by the absence of functional CFTR using two independent protocols. On the contrary, experiments performed in vitro strongly indicated that virus proliferation depended on features of the mucosal fluid layer known to be disrupted by absent CFTR in patients with CF, including both low pH and increased viscosity. These results point to the acidic, viscous, and mucus-obstructed airways in patients with cystic fibrosis as unfavorable for the establishment of coronaviral infection. Our findings provide new and important information concerning relationships between the CF clinical phenotype and severity of COVID-19.

Infants and young children generate more durable antibody responses to SARS-CoV-2 infection than adults.

As SARS-CoV-2 becomes endemic, it is critical to understand immunity following early-life infection. We evaluated humoral responses to SARS-CoV-2 in 23 infants/young children. Antibody responses to SARS-CoV-2 spike antigens peaked approximately 30 days after infection and were maintained up to 500 days with little apparent decay. While the magnitude of humoral responses was similar to an adult cohort recovered from mild/moderate COVID-19, both binding and neutralization titers to WT SARS-CoV-2 were more durable in infants/young children, with spike and RBD IgG antibody half-life nearly 4X as long as in adults. IgG subtype analysis revealed that while IgG1 formed the majority of the response in both groups, IgG3 was more common in adults and IgG2 in infants/young children. These findings raise important questions regarding differential regulation of humoral immunity in infants/young children and adults and could have broad implications for the timing of vaccination and booster strategies in this age group.

A wild boar cathelicidin peptide derivative inhibits severe acute respiratory syndrome coronavirus-2 and its drifted variants.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a clear threat to humanity. It has infected over 200 million and killed 4 million people worldwide, and infections continue with no end in sight. To control the pandemic, multiple effective vaccines have been developed, and global vaccinations are in progress. However, the virus continues to mutate. Even when full vaccine coverage is achieved, vaccine-resistant mutants will likely emerge, thus requiring new annual vaccines against drifted variants analogous to influenza. A complimentary solution to this problem could be developing antiviral drugs that inhibit SARS CoV-2 and its drifted variants. Host defense peptides represent a potential source for such an antiviral as they possess broad antimicrobial activity and significant diversity across species. We screened the cathelicidin family of peptides from 16 different species for antiviral activity and identified a wild boar peptide derivative that inhibits SARS CoV-2. This peptide, which we named Yongshi and means warrior in Mandarin, acts as a viral entry inhibitor. Following the binding of SARS-CoV-2 to its receptor, the spike protein is cleaved, and heptad repeats 1 and 2 multimerize to form the fusion complex that enables the virion to enter the cell. A deep learning-based protein sequence comparison algorithm and molecular modeling suggest that Yongshi acts as a mimetic to the heptad repeats of the virus, thereby disrupting the fusion process. Experimental data confirm the binding of Yongshi to the heptad repeat 1 with a fourfold higher affinity than heptad repeat 2 of SARS-CoV-2. Yongshi also binds to the heptad repeat 1 of SARS-CoV-1 and MERS-CoV. Interestingly, it inhibits all drifted variants of SARS CoV-2 that we tested, including the alpha, beta, gamma, delta, kappa and omicron variants.

Divergence of variant binding/neutralizing antibodies following SARS-CoV-2 booster vaccines in myeloma: Impact of hybrid immunity.

We characterized virus-neutralization and spike-binding antibody profiles in myeloma patients following monovalent or bivalent-SARS-CoV-2 booster vaccination. Vaccination improves the breadth of binding antibodies but not neutralization activity against current variants. Hybrid immunity and immune imprinting impact vaccine-elicited immunity.

IFN-λ derived from nonsusceptible enterocytes acts on tuft cells to limit persistent norovirus.

Norovirus is a leading cause of epidemic viral gastroenteritis, with no currently approved vaccines or antivirals. Murine norovirus (MNoV) is a well-characterized model of norovirus pathogenesis in vivo, and persistent strains exhibit lifelong intestinal infection. Interferon-λ (IFN-λ) is a potent antiviral that rapidly cures MNoV. We previously demonstrated that IFN-λ signaling in intestinal epithelial cells (IECs) controls persistent MNoV, and here demonstrate that IFN-λ acts on tuft cells, the exclusive site of MNoV persistence, to limit infection. While interrogating the source of IFN-λ to regulate MNoV, we confirmed that MDA5-MAVS signaling, required for IFN-λ induction to MNoV in vitro, controls persistent MNoV in vivo. We demonstrate that MAVS in IECs and not immune cells controls MNoV. MAVS in nonsusceptible enterocytes, but not in tuft cells, restricts MNoV, implicating noninfected cells as the IFN-λ source. Our findings indicate that host sensing of MNoV is distinct from cellular tropism, suggesting intercellular communication between IECs for antiviral signaling induction in uninfected bystander cells.

Light chain of a public SARS-CoV-2 class-3 antibody modulates neutralization against Omicron.

The pairing of antibody genes IGHV2-5/IGLV2-14 is established as a public immune response that potently cross-neutralizes SARS-CoV-2 variants, including Omicron, by targeting class-3/RBD-5 epitopes in the receptor binding domain (RBD). LY-CoV1404 (bebtelovimab) exemplifies this, displaying exceptional potency against Omicron sub-variants up to BA.5. Here, we report a human antibody, 002-S21B10, encoded by the public clonotype IGHV2-5/IGLV2-14. While 002-S21B10 neutralized key SARS-CoV-2 variants, it did not neutralize Omicron, despite sharing >92% sequence similarity with LY-CoV1404. The structure of 002-S21B10 in complex with spike trimer plus structural and sequence comparisons with LY-CoV1404 and other IGHV2-5/IGLV2-14 antibodies revealed significant variations in light-chain orientation, paratope residues, and epitope-paratope interactions that enable some antibodies to neutralize Omicron but not others. Confirming this, replacing the light chain of 002-S21B10 with the light chain of LY-CoV1404 restored 002-S21B10's binding to Omicron. Understanding such Omicron evasion from public response is vital for guiding therapeutics and vaccine design.

Atovaquone and Pibrentasvir Inhibit the SARS-CoV-2 Endoribonuclease and Restrict Infection In Vitro but Not In Vivo.

The emergence of SARS-CoV-1 in 2003 followed by MERS-CoV and now SARS-CoV-2 has proven the latent threat these viruses pose to humanity. While the SARS-CoV-2 pandemic has shifted to a stage of endemicity, the threat of new coronaviruses emerging from animal reservoirs remains. To address this issue, the global community must develop small molecule drugs targeting highly conserved structures in the coronavirus proteome. Here, we characterized existing drugs for their ability to inhibit the endoribonuclease activity of the SARS-CoV-2 non-structural protein 15 (nsp15) via in silico, in vitro, and in vivo techniques. We have identified nsp15 inhibition by the drugs pibrentasvir and atovaquone which effectively inhibit SARS-CoV-2 and HCoV-OC43 at low micromolar concentrations in cell cultures. Furthermore, atovaquone, but not pibrentasvir, is observed to modulate HCoV-OC43 dsRNA and infection in a manner consistent with nsp15 inhibition. Although neither pibrentasvir nor atovaquone translate to clinical efficacy in a murine prophylaxis model of SARS-CoV-2 infection, atovaquone may serve as a basis for the design of future nsp15 inhibitors.