The global COVID-19 pandemic has caused massive disruptions in every society around the world. To help fight COVID-19, new molecular tools specifically targeting critical components of the causative agent of COVID-19, SARS-Coronavirus-2 (SARS-CoV-2), are desperately needed. The SARS-CoV-2 nucleocapsid protein is a major component of the viral replication processes, integral to viral particle assembly, and is a major diagnostic marker for infection and immune protection. Currently available antibody reagents targeting the nucleocapsid protein were primarily developed against the related SARS-CoV virus and are not specific to SARS-CoV-2 nucleocapsid protein. Therefore, in this work we developed and characterized a series of new mouse monoclonal antibodies against the SARS-CoV-2 nucleocapsid protein. The anti-nucleocapsid monoclonal antibodies were tested in ELISA, western blot, and immunofluorescence analyses. The variable regions from the heavy and light chains from five select clones were cloned and sequenced, and preliminary epitope mapping of the sequenced clones was performed. Overall, the new antibody reagents described here will be of significant value in the fight against COVID-19.

Many efforts to design and screen therapeutics for severe acute respiratory syndrome coronavirus (SARS-CoV-2) have focused on inhibiting viral cell entry by disrupting ACE2 binding with the SARS-CoV-2 spike protein. This work focuses on inhibiting SARS-CoV-2 entry through a hypothesized α5β1 integrin-based mechanism, and indicates that inhibiting the spike protein interaction with α5β1 integrin (+/- ACE2), and the interaction between α5β1 integrin and ACE2 using a molecule ATN-161 represents a promising approach to treat COVID-19.

SARS-CoV2, the etiologic agent of COVID-19, uses ACE2 as a cell entry receptor. Soluble ACE2 has been shown to have neutralizing antiviral activity but has a short half-life and no active transport mechanism from the circulation into the alveolar spaces of the lung. To overcome this, we constructed an ACE2-human IgG1 fusion protein with mutations in the catalytic domain of ACE2. This fusion protein contained a LALA mutation that abrogates Fcrγ binding, but retains FcRN binding to prolong the half-life, as well as achieve therapeutic concentrations in the lung lavage. Interestingly, a mutation in the catalytic domain of ACE2, MDR504, completely abrogated catalytic activity, but significantly increased binding to SARS-CoV2 spike protein in vitro. This feature correlated with more potent viral neutralization in a plaque assay. Parental administration of the protein showed stable serum concentrations with a serum half-life of ~ 145 hours with excellent bioavailability in the epithelial lining fluid of the lung. Prophylactic administration of MDR504 significantly attenuated SARS-CoV2 infection in a murine model. These data support that the MDR504 hACE2-Fc is an excellent candidate for pre or post-exposure prophylaxis or treatment of COVID-19.

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.

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.

17 years after the SARS-CoV epidemic, the world is facing the COVID-19 pandemic. COVID-19 is caused by a coronavirus named SARS-CoV-2. Given the most optimistic projections estimating that it will take over a year to develop a vaccine, the best short-term strategy may lie in identifying virus-specific targets for small molecule interventions. All coronaviruses utilize a molecular mechanism called -1 PRF to control the relative expression of their proteins. Prior analyses of SARS-CoV revealed that it employs a structurally unique three-stemmed mRNA pseudoknot to stimulate high rates of -1 PRF, and that it also harbors a -1 PRF attenuation element. Altering -1 PRF activity negatively impacts virus replication, suggesting that this molecular mechanism may be therapeutically targeted. Here we present a comparative analysis of the original SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar rates of -1 PRF and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablated -1 PRF activity. The upstream attenuator hairpin activity has also been functionally retained. Small-angle x-ray scattering indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 had the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit -1 PRF was similarly effective against -1 PRF in SARS-CoV-2, suggesting that such frameshift inhibitors may provide promising lead compounds to counter the current pandemic.

The novel Corona Virus Disease 2019 (COVID-19) pandemic has set the fatality rates ablaze across the world. So, to combat this disease, we have designed a multi-epitope vaccine from various proteins of Severe Acute Respiratory Syndrome Corona virus 2 (SARS-CoV-2) with an immuno-informatics approach, validated in silico to be stable, non-allergic and antigenic. Cytotoxic T-cell, helper T-cell, and B-cell epitopes were computationally predicted from six conserved protein sequences among four viral strains isolated across the world. The T-cell epitopes, overlapping with the B-cell epitopes, were included in the vaccine construct to assure the humoral and cell-mediated immune response. The beta-subunit of cholera toxin was added as an adjuvant at the N-terminal of the construct to increase immunogenicity. Interferon-gamma inducing epitopes were even predicted in the vaccine. Molecular docking and binding energetics studies revealed strong interactions of the vaccine with immune-stimulatory toll-like receptors (TLR) -2, 3, 4. Molecular dynamics simulation of the vaccine ensured in vivo stability in the biological system. The immune simulation of vaccine evinced elevated immune response. The efficient translation of the vaccine in an expression vector was assured utilizing in silico cloning approach. Certainly, such a vaccine construct could reliably be effective against COVID-19.

The severity of COVID-19 lung disease is higher in the elderly and people with pre-existing co-morbidities. People who were born preterm may be at greater risk for COVID-19 because their early exposure to oxygen at birth increases their risk of being hospitalized when infected with RSV and other respiratory viruses. Our prior studies in mice showed how high levels of oxygen (hyperoxia) between postnatal days 0-4 increases the severity of influenza A virus infections by reducing the number of alveolar epithelial type 2 (AT2) cells. Because AT2 cells express the SARS-CoV-2 receptors angiotensin converting enzyme (ACE2) and transmembrane protease/serine subfamily member 2 (TMPRSS2), we expected their expression would decline as AT2 cells were depleted by hyperoxia. Instead, we made the surprising discovery that expression of Ace2 and Tmprss2 mRNA increases as mice age and is accelerated by exposing mice to neonatal hyperoxia. ACE2 is primarily expressed at birth by airway Club cells and becomes detectable in AT2 cells by one year of life. Neonatal hyperoxia increases ACE2 expression in Club cells and makes it detectable in 2-month-old AT2 cells. This early and increased expression of SARS-CoV-2 receptors was not seen in adult mice who had been administered the mitochondrial superoxide scavenger mitoTEMPO during hyperoxia. Our finding that early life insults such as hyperoxia enhances the age-dependent expression of SARS-CoV-2 receptors in the respiratory epithelium helps explain why COVID-19 lung disease is greater in the elderly and people with pre-existing co-morbidities.

SARS-CoV-2, the causative agent of COVID-19, emerged at the end of 2019 and by mid-June 2020, the virus has spread to at least 215 countries, caused more than 8,000,000 confirmed infections and over 450,000 deaths, and overwhelmed healthcare systems worldwide. Like SARS-CoV, which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low passage (P) strains of SARS-CoV-2 (Wash1: P4 and Munich: P1) were cultured twice in Vero-E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1: P6 and minor variants in the Munich: P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero-E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies we investigated the development of neutralizing antibody titers in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.

Decontamination helps limit environmental transmission of infectious agents. It is required for the safe re-use of contaminated medical, laboratory and personal protective equipment, and for the safe handling of biological samples. Heat treatment is a common decontamination method, notably used for viruses. We show that for liquid specimens (here, solution of SARS-CoV-2 in cell culture medium), virus inactivation rate under heat treatment at 70°C can vary by almost two orders of magnitude depending on the treatment procedure, from a half-life of 0.86 min (95% credible interval: [0.09, 1.77]) in closed vials in a heat block to 37.00 min ([12.65, 869.82]) in uncovered plates in a dry oven. These findings suggest a critical role of evaporation in virus inactivation via dry heat. Placing samples in open or uncovered containers may dramatically reduce the speed and efficacy of heat treatment for virus inactivation. Given these findings, we reviewed the literature temperature-dependent coronavirus stability and found that specimen containers, and whether they are closed, covered, or uncovered, are rarely reported in the scientific literature. Heat-treatment procedures must be fully specified when reporting experimental studies to facilitate result interpretation and reproducibility, and must be carefully considered when developing decontamination guidelines.

The spike (S) glycoprotein in the envelope of SARS-CoV-2 is densely glycosylated but the functions of its glycosylation are unknown. Here we demonstrate that S is recognized in a glycan-dependent manner by multiple innate immune receptors including the mannose receptor MR/CD206, DC-SIGN/CD209, L-SIGN/CD209L, and MGL/CLEC10A/CD301. Single-cell RNA sequencing analyses indicate that such receptors are highly expressed in innate immune cells in tissues susceptible to SARS-CoV-2 infection. Binding of the above receptors to S is characterized by affinities in the picomolar range and consistent with S glycosylation analysis demonstrating a variety of N- and O-glycans as receptor ligands. These results indicate multiple routes for SARS-CoV-2 to interact with human cells and suggest alternative strategies for therapeutic intervention.

Characterization of the T cell response in individuals who recover from SARS-CoV-2 infection is critical to understanding its contribution to protective immunity. A multiplexed peptide-MHC tetramer approach was used to screen 408 SARS-CoV-2 candidate epitopes for CD8+ T cell recognition in a cross-sectional sample of 30 COVID-19 convalescent individuals. T cells were evaluated using a 28-marker phenotypic panel, and findings were modelled against time from diagnosis, humoral and inflammatory responses. 132 distinct SARS-CoV-2-specific CD8+ T cell epitope responses across six different HLAs were detected, corresponding to 52 unique reactivities. T cell responses were directed against several structural and non-structural virus proteins. Modelling demonstrated a coordinated and dynamic immune response characterized by a decrease in inflammation, increase in neutralizing antibody titer, and differentiation of a specific CD8+ T cell response. Overall, T cells exhibited distinct differentiation into stem-cell and transitional memory states, subsets, which may be key to developing durable protection.

Coronavirus disease 2019 (COVID-19) was initially described as a viral infection of the respiratory tract. It is now known, however, that several other organs are affected, including the brain. Neurological manifestations such as stroke, encephalitis, and psychiatric conditions have been reported in COVID-19 patients, but the neurotropic potential of the virus is still debated. Herein, we sought to investigate SARS-CoV-2 infection in human neural cells. We demonstrated that SARS-CoV-2 infection of neural tissue is non-permissive, however, it can elicit inflammatory response and cell damage. These findings add to the hypothesis that most of the neural damage caused by SARS-CoV-2 infection is due to a systemic inflammation leading to indirect harmful effects on the central nervous system despite the absence of local viral replication.

The sex discordance in COVID-19 outcomes has been widely recognized, with males generally faring worse than females and a potential link to sex steroids. A plausible mechanism is androgen-induced expression of TMPRSS2 and/or ACE2 in pulmonary tissues that may increase susceptibility or severity in males. This hypothesis is the subject of several clinical trials of anti-androgen therapies around the world. Here, we investigated the sex-associated TMPRSS2 and ACE2 expression in human and mouse lungs and interrogated the possibility of pharmacologic modification of their expression with anti-androgens. We found no evidence for increased TMPRSS2 expression in the lungs of males compared to females in humans or mice. Furthermore, in male mice, treatment with the androgen receptor antagonist enzalutamide did not decrease pulmonary TMPRSS2. On the other hand, ACE2 and AR expression was sexually dimorphic and higher in males than females. ACE2 was moderately suppressible with enzalutamide therapy. Our work suggests that sex differences in COVID-19 outcomes attributable to viral entry are independent of TMPRSS2. Modest changes in ACE2 could account for some of the sex discordance.

Understanding the trends in SARS-CoV-2 evolution is paramount to control the COVID-19 pandemic. We analyzed more than 300,000 high quality genome sequences of SARS-CoV-2 variants available as of January 2021. The results show that the ongoing evolution of SARS-CoV-2 during the pandemic is characterized primarily by purifying selection, but a small set of sites appear to evolve under positive selection. The receptor-binding domain of the spike protein and the nuclear localization signal (NLS) associated region of the nucleocapsid protein are enriched with positively selected amino acid replacements. These replacements form a strongly connected network of apparent epistatic interactions and are signatures of major partitions in the SARS-CoV-2 phylogeny. Virus diversity within each geographic region has been steadily growing for the entirety of the pandemic, but analysis of the phylogenetic distances between pairs of regions reveals four distinct periods based on global partitioning of the tree and the emergence of key mutations. The initial period of rapid diversification into region-specific phylogenies that ended in February 2020 was followed by a major extinction event and global homogenization concomitant with the spread of D614G in the spike protein, ending in March 2020. The NLS associated variants across multiple partitions rose to global prominence in March-July, during a period of stasis in terms of inter-regional diversity. Finally, beginning July 2020, multiple mutations, some of which have since been demonstrated to enable antibody evasion, began to emerge associated with ongoing regional diversification, which might be indicative of speciation.

Hydroxychloroquine (HCQ) has been proposed in the treatment of SARS-coronavirus 2 (SARS-CoV-2) infection, albeit with much controversy. In vitro, HCQ effectively inhibits viral entry, but its use in the clinic has been hampered by conflicting results. A better understanding of HCQ's mechanism of actions in vitro is needed to resolve these conflicts. Recently, anesthetics were shown to disrupt ordered monosialotetrahexosylganglioside1 (GM1) lipid rafts. These same lipid rafts recruit the SARS-CoV-2 surface receptor angiotensin converting enzyme 2 (ACE2) to an endocytic entry point, away from phosphatidylinositol 4,5 bisphosphate (PIP2) domains. Here we employed super resolution imaging of cultured mammalian cells to show HCQ directly perturbs GM1 lipid rafts and inhibits the ability of ACE2 receptor to associate with the endocytic pathway. HCQ also disrupts PIP2 domains and their ability to cluster and sequester ACE2. Similarly, the antibiotic erythromycin inhibits viral entry and both HCQ and erythromycin decrease the antimicrobial host defense peptide amyloid beta in cultured cells. We conclude HCQ is an anesthetic-like compound that disrupts GM1 lipid rafts similar to anesthetics. The disruption likely decreases viral clustering at both endocytic and putative PIP2 entry points.

We studied the activity of a range of weakly basic and moderately lipophilic drugs against SARS CoV2 in Vero E6 cells, using Vero E6 survival, qPCR of viral genome and plaque forming assays. No clear relationship between their weakly basic and hydrophobic nature upon their activity was observed. However, the approved drugs ambroxol and ciprofloxacin showed potent activity at concentrations that are clinically relevant and within their known safety profiles, and so may provide potentially useful agents for preclinical and clinical studies in COVID-19.

A safe, effective, and scalable vaccine is urgently needed to halt the ongoing SARS-CoV-2 pandemic. Here, we describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 copies of the SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD) in a highly immunogenic array and induce neutralizing antibody titers roughly ten-fold higher than the prefusion-stabilized S ectodomain trimer despite a more than five-fold lower dose. Antibodies elicited by the nanoparticle immunogens target multiple distinct epitopes on the RBD, suggesting that they may not be easily susceptible to escape mutations, and exhibit a significantly lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the protein components and assembled nanoparticles, especially compared to the SARS-CoV-2 prefusion-stabilized S trimer, suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms for inducing potent neutralizing antibody responses and have launched cGMP manufacturing efforts to advance the lead RBD nanoparticle vaccine into the clinic.

Neutralizing agents against SARS-CoV-2 are urgently needed for treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domain binders with high affinity toward neutralizing epitopes without the need for high-resolution structural information. We constructed a VH-phage library and targeted a known neutralizing site, the angiotensin-converting enzyme 2 (ACE2) binding interface of the trimeric SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified 85 unique VH binders to two non-overlapping epitopes within the ACE2 binding site on Spike-RBD. This enabled us to systematically link these VH domains into multivalent and bi-paratopic formats. These multivalent and bi-paratopic VH constructs showed a marked increase in affinity to Spike (up to 600-fold) and neutralization potency (up to 1400-fold) on pseudotyped SARS-CoV-2 virus when compared to the standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with half-minimal inhibitory concentration (IC 50 ) of 4.0 nM (180 ng/mL). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain bound an RBD at the ACE2 binding site, explaining its increased neutralization potency and confirming our original design strategy. Our results demonstrate that targeted selection and engineering campaigns using a VH-phage library can enable rapid assembly of highly avid and potent molecules towards therapeutically important protein interfaces.

Global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues unabated. Binding of SARS-CoV-2's Spike protein to host angiotensin converting enzyme 2 triggers viral entry, but other proteins may participate, including neuropilin-1 receptor (NRP-1). As both Spike protein and vascular endothelial growth factor-A (VEGF-A) - a pro-nociceptive and angiogenic factor, bind NRP-1, we tested if Spike could block VEGF-A/NRP-1 signaling. VEGF-A-triggered sensory neuronal firing was blocked by Spike protein and NRP-1 inhibitor EG00229. Pro-nociceptive behaviors of VEGF-A were similarly blocked via suppression of spontaneous spinal synaptic activity and reduction of electrogenic currents in sensory neurons. Remarkably, preventing VEGF-A/NRP-1 signaling was antiallodynic in a neuropathic pain model. A 'silencing' of pain via subversion of VEGF-A/NRP-1 signaling may underlie increased disease transmission in asymptomatic individuals.