A Genome-Wide Arrayed CRISPR Screen Reveals PLSCR1 as an Intrinsic Barrier to SARS-CoV-2 Entry (preprint)

Interferons (IFNs) play a crucial role in the regulation and evolution of host-virus interactions. Here, we conducted a genome-wide arrayed CRISPR knockout screen in the presence and absence of IFN to identify human genes that influence SARS-CoV-2 infection. We then performed an integrated analysis of genes interacting with SARS-CoV-2, drawing from a selection of 67 large-scale studies, including our own. We identified 28 genes of high relevance in both human genetic studies of COVID-19 patients and functional genetic screens in cell culture, with many related to the IFN pathway. Among these was the IFN-stimulated gene PLSCR1. PLSCR1 did not require IFN induction to restrict SARS-CoV-2 and did not contribute to IFN signaling. Instead, PLSCR1 specifically restricted spike-mediated SARS-CoV-2 entry. The PLSCR1-mediated restriction was alleviated by TMPRSS2 over-expression, suggesting that PLSCR1 primarily restricts the endocytic entry route. In addition, recent SARS-CoV-2 variants have adapted to circumvent the PLSCR1 barrier via currently undetermined mechanisms. Our study contributes to understanding the association between PLSCR1 variants and severe COVID-19 cases reported in a recent GWAS.

Mouse Adapted SARS-CoV-2 Model Induces "Long-COVID" Neuropathology in BALB/c Mice (preprint)

The novel coronavirus SARS-CoV-2 has caused significant global morbidity and mortality and continues to burden patients with persisting neurological dysfunction. COVID-19 survivors develop debilitating symptoms to include neuro-psychological dysfunction, termed "Long COVID", which can cause significant reduction of quality of life. Despite vigorous model development, the possible cause of these symptoms and the underlying pathophysiology of this devastating disease remains elusive. Mouse adapted (MA10) SARS-CoV-2 is a novel mouse-based model of COVID-19 which simulates the clinical symptoms of respiratory distress associated with SARS-CoV-2 infection in mice. In this study, we evaluated the long-term effects of MA10 infection on brain pathology and neuroinflammation. 10-week and 1-year old female BALB/cAnNHsd mice were infected intranasally with 104 plaque-forming units (PFU) and 103 PFU of SARS-CoV-2 MA10, respectively, and the brain was examined 60 days post-infection (dpi). Immunohistochemical analysis showed a decrease in the neuronal nuclear protein NeuN and an increase in Iba-1 positive amoeboid microglia in the hippocampus after MA10 infection, indicating long-term neurological changes in a brain area which is critical for long-term memory consolidation and processing. Importantly, these changes were seen in 40-50% of infected mice, which correlates to prevalence of LC seen clinically. Our data shows for the first time that MA10 infection induces neuropathological outcomes several weeks after infection at similar rates of observed clinical prevalence of "Long COVID". These observations strengthen the MA10 model as a viable model for study of the long-term effects of SARS-CoV-2 in humans. Establishing the viability of this model is a key step towards the rapid development of novel therapeutic strategies to ameliorate neuroinflammation and restore brain function in those suffering from the persistent cognitive dysfunction of "Long-COVID".

Fc mediated pan-sarbecovirus protection after alphavirus vector vaccination (preprint)

Two group 2B beta-coronaviruses (sarbecoviruses) have caused regional and global epidemics in modern history. The mechanisms of cross protection driven by the sarbecovirus spike, a dominant immunogen, are less clear yet critically important for pan-sarbecovirus vaccine development. We evaluated the mechanisms of cross-sarbecovirus protective immunity using a panel of alphavirus-vectored vaccines covering bat to human strains. While vaccination did not prevent virus replication, it protected against lethal heterologous disease outcomes in both SARS-CoV-2 and clade 2 bat sarbecovirus HKU3-SRBD challenge models. The spike vaccines tested primarily elicited a highly S1-specific homologous neutralizing antibody response with no detectable cross-virus neutralization. We found non-neutralizing antibody functions that mediated cross protection in wild-type mice were mechanistically linked to FcgR4 and spike S2-binding antibodies. Protection was lost in FcR knockout mice, further supporting a model for non-neutralizing, protective antibodies. These data highlight the importance of FcR-mediated cross-protective immune responses in universal pan-sarbecovirus vaccine designs.

A C57BL/6 Mouse model of SARS-CoV-2 infection recapitulates age- and sex-based differences in human COVID-19 disease and recovery (preprint)

We present a comprehensive analysis of SARS-CoV-2 infection and recovery in wild type C57BL/6 mice, demonstrating that this is an ideal model of infection and recovery that accurately phenocopies acute human disease arising from the ancestral SARS-CoV-2. Disease severity and infection kinetics are age- and sex-dependent, as has been reported for humans, with older mice and males in particular exhibiting decreased viral clearance and increased mortality. We identified key parallels with human pathology, including intense virus positivity in bronchial epithelial cells, wide-spread alveolar involvement, recruitment of immune cells to the infected lungs, and acute bronchial epithelial cell death. Moreover, older animals experienced increased virus persistence, delayed dispersal of immune cells into lung parenchyma, and morphologic evidence of tissue damage and inflammation. Parallel analysis of SCID mice revealed that the adaptive immune response was not required for recovery from COVID disease symptoms nor early phase clearance of virus but was required for efficient clearance of virus at later stages of infection. Finally, transcriptional analyses indicated that induction and duration of key innate immune gene programs may explain differences in age-dependent disease severity. Importantly, these data demonstrate that SARS-CoV-2-mediated disease in C57BL/6 mice accurately phenocopies human disease across ages and establishes a platform for future therapeutic and genetic screens for not just SARS-CoV-2 but also novel coronaviruses that have yet to emerge.

A model of persistent post SARS-CoV-2 induced lung disease for target identification and testing of therapeutic strategies (preprint)

COVID-19 survivors develop post-acute sequelae of SARS-CoV-2 (PASC), but the mechanistic basis of PASC-associated lung abnormalities suffers from a lack of longitudinal samples. Mouse-adapted SARS-CoV-2 MA10 produces an acute respiratory distress syndrome (ARDS) in mice similar to humans. To investigate PASC pathogenesis, studies of MA10-infected mice were extended from acute disease through clinical recovery. At 15-120 days post-virus clearance, histologic evaluation identified subpleural lesions containing collagen, proliferative fibroblasts, and chronic inflammation with tertiary lymphoid structures. Longitudinal spatial transcriptional profiling identified global reparative and fibrotic pathways dysregulated in diseased regions, similar to human COVID-19. Populations of alveolar intermediate cells, coupled with focal upregulation of pro-fibrotic markers, were identified in persistently diseased regions. Early intervention with antiviral EIDD-2801 reduced chronic disease, and early anti-fibrotic agent (nintedanib) intervention modified early disease severity. This murine model provides opportunities to identify pathways associated with persistent SARS-CoV-2 pulmonary disease and test countermeasures to ameliorate PASC.

SARS-CoV-2 D614G Variant Exhibits Enhanced Replication ex vivo and Earlier Transmission in vivo (preprint)

The D614G substitution in the S protein is most prevalent SARS-CoV-2 strain circulating globally, but its effects in viral pathogenesis and transmission remain unclear. We engineered SARS-CoV-2 variants harboring the D614G substitution with or without nanoluciferase. The D614G variant replicates more efficiency in primary human proximal airway epithelial cells and is more fit than wildtype (WT) virus in competition studies. With similar morphology to the WT virion, the D614G virus is also more sensitive to SARS-CoV-2 neutralizing antibodies. Infection of human ACE2 transgenic mice and Syrian hamsters with the WT or D614G viruses produced similar titers in respiratory tissue and pulmonary disease. However, the D614G variant exhibited significantly faster droplet transmission between hamsters than the WT virus, early after infection. Our study demonstrated the SARS-CoV2 D614G substitution enhances infectivity, replication fitness, and early transmission.

A Newcastle disease virus (NDV) expressing membrane-anchored spike as a cost-effective inactivated SARS-CoV-2 vaccine (preprint)

A successful SARS-CoV-2 vaccine must be not only safe and protective but must also meet the demand on a global scale at low cost. Using the current influenza virus vaccine production capacity to manufacture an egg-based inactivated Newcastle disease virus (NDV)/SARS-CoV-2 vaccine would meet that challenge. Here, we report pre-clinical evaluations of an inactivated NDV chimera stably expressing the membrane-anchored form of the spike (NDV-S) as a potent COVID-19 vaccine in mice and hamsters. The inactivated NDV-S vaccine was immunogenic, inducing strong binding and/or neutralizing antibodies in both animal models. More importantly, the inactivated NDV-S vaccine protected animals from SARS-CoV-2 infections or significantly attenuated SARS-CoV-2 induced disease. In the presence of an adjuvant, antigen-sparing could be achieved, which would further reduce the cost while maintaining the protective efficacy of the vaccine.

SARS-CoV-2 mRNA Vaccine Development Enabled by Prototype Pathogen Preparedness (preprint)

A SARS-CoV-2 vaccine is needed to control the global COVID-19 public health crisis. Atomic-level structures directed the application of prefusion-stabilizing mutations that improved expression and immunogenicity of betacoronavirus spike proteins. Using this established immunogen design, the release of SARS-CoV-2 sequences triggered immediate rapid manufacturing of an mRNA vaccine expressing the prefusion-stabilized SARS-CoV-2 spike trimer (mRNA-1273). Here, we show that mRNA-1273 induces both potent neutralizing antibody and CD8 T cell responses and protects against SARS-CoV-2 infection in lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a Phase 2 clinical trial with a trajectory towards Phase 3 efficacy evaluation.

A mouse-adapted SARS-CoV-2 model for the evaluation of COVID-19 medical countermeasures (preprint)

Coronaviruses are prone to emergence into new host species most recently evidenced by SARS-CoV-2, the causative agent of the COVID-19 pandemic. Small animal models that recapitulate SARS-CoV-2 disease are desperately needed to rapidly evaluate medical countermeasures (MCMs). SARS-CoV-2 cannot infect wildtype laboratory mice due to inefficient interactions between the viral spike (S) protein and the murine ortholog of the human receptor, ACE2. We used reverse genetics to remodel the S and mACE2 binding interface resulting in a recombinant virus (SARS-CoV-2 MA) that could utilize mACE2 for entry. SARS-CoV-2 MA replicated in both the upper and lower airways of both young adult and aged BALB/c mice. Importantly, disease was more severe in aged mice, and showed more clinically relevant phenotypes than those seen in hACE2 transgenic mice. We then demonstrated the utility of this model through vaccine challenge studies in immune competent mice with native expression of mACE2. Lastly, we show that clinical candidate interferon (IFN) lambda-1a can potently inhibit SARS-CoV-2 replication in primary human airway epithelial cells in vitro, and both prophylactic and therapeutic administration diminished replication in mice. Our mouse-adapted SARS-CoV-2 model demonstrates age-related disease pathogenesis and supports the clinical use of IFN lambda-1a treatment in human COVID-19 infections.

Remdesivir potently inhibits SARS-CoV-2 in human lung cells and chimeric SARS-CoV expressing the SARS-CoV-2 RNA polymerase in mice. (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 as the causative agent of the novel pandemic viral disease COVID-19. With no approved therapies, this pandemic illustrates the urgent need for safe, broad-spectrum antiviral countermeasures against SARS-CoV-2 and future emerging CoVs. We report that remdesivir (RDV), a monophosphoramidate prodrug of an adenosine analog, potently inhibits SARS-CoV-2 replication in human lung cells and primary human airway epithelial cultures (EC50 = 0.01 M). Weaker activity was observed in Vero E6 cells (EC50 = 1.65 M) due to their low capacity to metabolize RDV. To rapidly evaluate in vivo efficacy, we engineered a chimeric SARS-CoV encoding the viral target of RDV, the RNA-dependent RNA polymerase, of SARS-CoV-2. In mice infected with chimeric virus, therapeutic RDV administration diminished lung viral load and improved pulmonary function as compared to vehicle treated animals. These data provide evidence that RDV is potently active against SARS-CoV-2 in vitro and in vivo, supporting its further clinical testing for treatment of COVID-19.