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Severe acute respiratory coronavirus 2 (SARS-CoV-2) is a newly identified virus that has resulted in over 2.5 million deaths globally and over 116 million cases globally in March, 2021. Small-molecule inhibitors that reverse disease severity have proven difficult to discover. One of the key approaches that has been widely applied in an effort to speed up the translation of drugs is drug repurposing. A few drugs have shown in vitro activity against Ebola viruses and demonstrated activity against SARS-CoV-2 in vivo. Most notably, the RNA polymerase targeting remdesivir demonstrated activity in vitro and efficacy in the early stage of the disease in humans. Testing other small-molecule drugs that are active against Ebola viruses (EBOVs) would appear a reasonable strategy to evaluate their potential for SARS-CoV-2. We have previously repurposed pyronaridine, tilorone, and quinacrine (from malaria, influenza, and antiprotozoal uses, respectively) as inhibitors of Ebola and Marburg viruses in vitro in HeLa cells and mouse-adapted EBOV in mice in vivo. We have now tested these three drugs in various cell lines (VeroE6, Vero76, Caco-2, Calu-3, A549-ACE2, HUH-7, and monocytes) infected with SARS-CoV-2 as well as other viruses (including MHV and HCoV 229E). The compilation of these results indicated considerable variability in antiviral activity observed across cell lines. We found that tilorone and pyronaridine inhibited the virus replication in A549-ACE2 cells with IC50 values of 180 nM and IC50 198 nM, respectively. We used microscale thermophoresis to test the binding of these molecules to the spike protein, and tilorone and pyronaridine bind to the spike receptor binding domain protein with Kd values of 339 and 647 nM, respectively. Human Cmax for pyronaridine and quinacrine is greater than the IC50 observed in A549-ACE2 cells. We also provide novel insights into the mechanism of these compounds which is likely lysosomotropic.

Zika virus (ZIKV) is a mosquito-borne positive sense RNA virus. Recently, ZIKV emerged into the Western hemisphere as a human health threat, with severe disease associated with developmental and neurological complications. The structural envelope protein of ZIKV and other neurotropic flaviviruses contains an extended CD-loop relative to non-neurotropic flaviviruses, and has been shown to augment ZIKV stability and pathogenesis. Here we show that shortening the CD-loop in ZIKV attenuates the virus in mice, by reducing the ability to invade and replicate in the central nervous system. The CD-loop mutation was genetically stable following infection in mice, though secondary site mutations arise adjacent to the CD-loop. Importantly, while shortening of the CD-loop attenuates the virus, the CD-loop mutant maintains antigenicity in immunocompetent mice, eliciting an antibody response that similarly neutralizes both the mutant and wildtype ZIKV. These findings suggest that the extended CD-loop in ZIKV is a determinant of neurotropism and may be a target in live-attenuated vaccine design, for not only ZIKV, but for other neurotropic flaviviruses.

We have developed a novel class of peptidomimetic inhibitors targeting several host cell human serine proteases including transmembrane protease serine 2 (TMPRSS2), matriptase and hepsin. TMPRSS2 is a membrane associated protease which is highly expressed in the upper and lower respiratory tract and is utilized by SARS-CoV-2 and other viruses to proteolytically process their glycoproteins, enabling host cell receptor binding, entry, replication, and dissemination of new virion particles. We have previously shown that compound MM3122 exhibited sub nanomolar potency against all three proteases and displayed potent antiviral effects against SARS-CoV-2 in a cell-viability assay. Herein, we demonstrate that MM3122 potently inhibits viral replication in human lung epithelial cells and is also effective against the EG.5.1 variant of SARS-CoV-2. Further, we have evaluated MM3122 in a mouse model of COVID-19 and have demonstrated that MM3122 administered intraperitoneally (IP) before (prophylactic) or after (therapeutic) SARS-CoV-2 infection had significant protective effects against weight loss and lung congestion, and reduced pathology. Amelioration of COVID-19 disease was associated with a reduction in pro-inflammatory cytokines and chemokines production after SARS-CoV-2 infection. Prophylactic, but not therapeutic, administration of MM3122 also reduced virus titers in the lungs of SARS-CoV-2 infected mice. Therefore, MM3122 is a promising lead candidate small molecule drug for the treatment and prevention of infections caused by SARS-CoV-2 and other coronaviruses., Importance: SARS-CoV-2 and other emerging RNA coronaviruses are a present and future threat in causing widespread endemic and pandemic infection and disease. In this paper, we have shown that the novel host-cell protease inhibitor, MM3122, blocks SARS-CoV-2 viral replication and is efficacious as both a prophylactic and therapeutic drug for the treatment of COVID-19 in mice. Targeting host proteins and pathways in antiviral therapy is an underexplored area of research but this approach promises to avoid drug resistance by the virus, which is common in current antiviral treatments.

Multivalent antigen display is a fast-growing area of interest towards broadly protective vaccines. Current nanoparticle-based vaccine candidates demonstrate the ability to confer antibody-mediated immunity against divergent strains of notably mutable viruses. In coronaviruses, this work is predominantly aimed at targeting conserved epitopes of the receptor-binding domain. However, targeting other conserved non-RBD epitopes could further limit the potential for antigenic escape. To further explore new potential targets, we engineered protein nanoparticles displaying CoV_S-2P trimers derived from MERS-CoV, SARS-CoV-1, SARS-CoV-2, hCoV-HKU1, and hCoV-OC43 and assessed their immunogenicity in mice. Monotypic SARS-1_S-2P nanoparticles elicited cross-neutralizing antibodies against MERS_S and protected against MERS-CoV challenge. MERS and SARS-I53_dn5 nanoparticles elicited S1-focused antibodies, revealing a conserved site on the NTD. Moreover, mosaic nanoparticles co-displaying distinct CoV_S-2P trimers elicited antibody responses to distant cross-group antigens while protecting against MERS challenge despite diminished valency of MERS_S-2P. Our findings will inform further efforts towards the development of pan-coronavirus vaccines., Competing Interests: Competing Interests The authors declare the existence of financial and non-financial competing interest.

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.

The portfolio of SARS-CoV-2 small molecule drugs is currently limited to a handful that are either approved (remdesivir), emergency approved (dexamethasone, baricitinib) or in advanced clinical trials. We have tested 45 FDA-approved kinase inhibitors in vitro against murine hepatitis virus (MHV) as a model of SARS-CoV-2 replication and identified 12 showing inhibition in the delayed brain tumor (DBT) cell line. Vandetanib, which targets the vascular endothelial growth factor receptor (VEGFR), the epidermal growth factor receptor (EGFR), and the RET-tyrosine kinase showed the most promising results on inhibition versus toxic effect on SARS-CoV-2-infected Caco-2 and A549-hACE2 cells (IC 50 0.79 μM) while also showing a reduction of > 3 log TCID 50 /mL for HCoV-229E. The in vivo efficacy of vandetanib was assessed in a mouse model of SARS-CoV-2 infection and statistically significantly reduced the levels of IL-6, IL-10, TNF-α, and mitigated inflammatory cell infiltrates in the lungs of infected animals but did not reduce viral load. Vandetanib rescued the decreased IFN-1β caused by SARS-CoV-2 infection in mice to levels similar to that in uninfected animals. Our results indicate that the FDA-approved vandetanib is a potential therapeutic candidate for COVID-19 positioned for follow up in clinical trials either alone or in combination with other drugs to address the cytokine storm associated with this viral infection., Competing Interests: Competing interests: SE is CEO of Collaborations Pharmaceuticals, Inc. ACP is an employee at Collaborations Pharmaceuticals, Inc. All other c-authors have no conflicts of interest.

SARS-CoV-2 is a newly identified virus that has resulted in over 1.3 M deaths globally and over 59 M cases globally to date. Small molecule inhibitors that reverse disease severity have proven difficult to discover. One of the key approaches that has been widely applied in an effort to speed up the translation of drugs is drug repurposing. A few drugs have shown in vitro activity against Ebola virus and demonstrated activity against SARS-CoV-2 in vivo . Most notably the RNA polymerase targeting remdesivir demonstrated activity in vitro and efficacy in the early stage of the disease in humans. Testing other small molecule drugs that are active against Ebola virus would seem a reasonable strategy to evaluate their potential for SARS-CoV-2. We have previously repurposed pyronaridine, tilorone and quinacrine (from malaria, influenza, and antiprotozoal uses, respectively) as inhibitors of Ebola and Marburg virus in vitro in HeLa cells and of mouse adapted Ebola virus in mouse in vivo . We have now tested these three drugs in various cell lines (VeroE6, Vero76, Caco-2, Calu-3, A549-ACE2, HUH-7 and monocytes) infected with SARS-CoV-2 as well as other viruses (including MHV and HCoV 229E). The compilation of these results indicated considerable variability in antiviral activity observed across cell lines. We found that tilorone and pyronaridine inhibited the virus replication in A549-ACE2 cells with IC 50 values of 180 nM and IC 50 198 nM, respectively. We have also tested them in a pseudovirus assay and used microscale thermophoresis to test the binding of these molecules to the spike protein. They bind to spike RBD protein with K d values of 339 nM and 647 nM, respectively. Human C max for pyronaridine and quinacrine is greater than the IC 50 hence justifying in vivo evaluation. We also provide novel insights into their mechanism which is likely lysosomotropic.

Coronaviruses, including SARS-CoV-2 the etiological agent of COVID-19 disease, have caused multiple epidemic and pandemic outbreaks in the past 20 years 1-3 . With no vaccines, and only recently developed antiviral therapeutics, we are ill equipped to handle coronavirus outbreaks 4 . A better understanding of the molecular mechanisms that regulate coronavirus replication and pathogenesis is needed to guide the development of new antiviral therapeutics and vaccines. RNA secondary structures play critical roles in multiple aspects of coronavirus replication, but the extent and conservation of RNA secondary structure across coronavirus genomes is unknown 5 . Here, we define highly structured RNA regions throughout the MERS-CoV, SARS-CoV, and SARS-CoV-2 genomes. We find that highly stable RNA structures are pervasive throughout coronavirus genomes, and are conserved between the SARS-like CoV. Our data suggests that selective pressure helps preserve RNA secondary structure in coronavirus genomes, suggesting that these structures may play important roles in virus replication and pathogenesis. Thus, disruption of conserved RNA secondary structures could be a novel strategy for the generation of attenuated SARS-CoV-2 vaccines for use against the current COVID-19 pandemic., Competing Interests: Competing Interest Declaration The authors declare they have no competing interests.

A mechanistic understanding of the SARS-CoV-2 viral replication cycle is essential to develop new therapies for the COVID-19 global health crisis. In this study, we show that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with the viral genome, and propose a model of viral packaging through LLPS. N-protein condenses with specific RNA sequences in the first 1000 nts (5'-End) under physiological conditions and is enhanced at human upper airway temperatures. N-protein condensates exclude non-packaged RNA sequences. We comprehensively map sites bound by N-protein in the 5'-End and find preferences for single-stranded RNA flanked by stable structured elements. Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules thus presenting screenable processes for identifying antiviral compounds effective against SARS-CoV-2., Competing Interests: Competing interests: K.M.W. is an advisor to and holds equity in Ribometrix, to which mutational profiling (MaP) technologies have been licensed. All other authors declare that they have no competing interests.

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., Competing Interests: Competing Interest Declaration K.S.C., N.W., J.S.M., and B.S.G. are inventors on International Patent Application No. WO/2018/081318 entitled “Prefusion Coronavirus Spike Proteins and Their Use.” K.S.C., O.M.A., G.B.H., N.W., D.W., J.S.M, and B.S.G. are inventors on US Patent Application No. 62/972,886 entitled “2019-nCoV Vaccine”. R.S.B. filed an invention report for the SARS-CoV-2 MA virus (UNC ref. #18752).