Experimental Lung Inflammation Induced by Inactivated SARS-CoV-2 is Controlled by Intranasal Instillation of Vitamin D (preprint)

COVID-19 is a pandemic triggered by the coronavirus SARS-CoV-2 whose peak occurred in the years 2020 and 2021. The main target of the virus is the lung and infection is associated to an accentuated inflammatory process involving mainly the innate arm of the immune system. Here, we described the induction of a pulmonary inflammatory process triggered by the intranasal (IN) instillation of UV-inactivated SARS-CoV-2 in C57BL/6 mice and then the evaluation of vitamin D (VitD) ability to control this process. The assays used to estimate the severity of lung involvement included total and differential number of cells in the BALF, histopathological analysis, quantification of T cell subsets and inflammatory mediators by RT-PCR, cytokine quantification in lung homogenates and flow cytometric analysis of cells recovered from lung parenchyma. IN instillation of inactivated SARS-CoV-2 triggered a pulmonary inflammatory process, consisting of various cell types and mediators, resembling the typical inflammation found in COVID-19 patients. This inflammatory process was significantly decreased by IN delivery of vitD, but not by its IP administration, suggesting that this hormone has therapeutic potential in COVID-19 if locally applied.

Discovery and structural characterization of chicoric acid as a SARS-CoV-2 nucleocapsid protein ligand and RNA binding disruptor (preprint)

The nucleocapsid (N) protein plays critical roles in coronavirus genome transcription and packaging, representing a key target for the development of novel antivirals, and for which structural information on ligand binding is scarce. We used a novel fluorescence polarization assay to identify small molecules that disrupt the binding of the N protein to a target RNA derived from the SARS-CoV-2 genome packaging signal. Several phenolic compounds, including L-chicoric acid (CA), were identified as high-affinity N-protein ligands. The binding of CA to the N protein was confirmed by isothermal titration calorimetry, 1H-STD NMR, and by the crystal structure of CA bound to the N protein C-terminal domain (CTD), further revealing a new modulatory site in the SARS-CoV-2 N protein. Moreover, CA reduced SARS-CoV-2 replication in cell cultures. These data thus open venues for the development of new antivirals targeting the N protein, an essential and yet underexplored coronavirus target.

Characterization of the interaction between SARS-CoV-2 Membrane Protein and Proliferating Cell Nuclear Antigen (PCNA) as a Potential Therapeutic Target (preprint)

SARS-CoV-2 is an emerging virus from the Coronaviridae family and is responsible for the ongoing COVID-19 pandemic. In this work, we explored the previously reported SARS-CoV-2 structural membrane protein (M) interaction with human Proliferating Cell Nuclear Antigen (PCNA). The M protein is responsible for maintaining virion shape, and PCNA is a marker of DNA damage which is essential for DNA replication and repair. We validated the M PCNA interaction through immunoprecipitation, immunofluorescence co-localization, and a PLA assay. In cells infected with SARS-CoV-2 or transfected with M protein, using immunofluorescence and cell fractioning, we documented a reallocation of PCNA from the nucleus to the cytoplasm and the increase of PCNA and {gamma}H2AX (another DNA damage marker) expression. We also observed an increase of PCNA and {gamma}H2AX expression in the lung of a COVID-19 patient by immunohistochemistry. In addition, the inhibition of PCNA translocation by PCNA I1 and Verdinexor led to a reduction of plaque formation in an in vitro assay. We, therefore, propose that the transport of PCNA to the cytoplasm and its association with M could be a virus strategy to manipulate cell functions and may be considered a target for COVID-19 therapy.

Increased mTOR signaling, impaired autophagic flux and cell-to-cell viral transmission are hallmarks of SARS-CoV-2 infection. (preprint)

The COVID-19 disease caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has two characteristics that distinguish it from other viral infections. It affects more severely people with pre-existing comorbidities and viral load peaks prior to the onset of the symptoms. Investigating factors that could contribute to these characteristics, we found increased mTOR signaling and suppressed genes related to autophagy, lysosome, and vesicle fusion in Vero E6 cells infected with SARS-CoV-2. Transcriptomic data mining of bronchoalveolar epithelial cells from severe COVID-19 patients revealed that COVID-19 severity is associated with increased expression of genes related to mTOR signaling and decreased expression of genes related to au-tophagy, lysosome function, and vesicle fusion. SARS-CoV-2 infection in Vero E6 cells also re-sulted in virus retention inside the cells and trafficking of virus-bearing vesicles between neighboring cells. Our findings support a scenario where SARS-CoV-2 benefits from compromised autophagic flux and inhibited exocytosis in individuals with chronic hyperactivation of mTOR signaling, which might relate to undetectable proliferation and evasion of the immune system.

Levels of SARS-CoV-2 Lineage P.1 Neutralization by Antibodies Elicited after Natural Infection and Vaccination (preprint)

Background: A new SARS-CoV-2 lineage, named P.1 (20J/501Y.V3), has recently been detected in Brazil. Mutations accrued by the P.1 lineage include amino acid changes in the receptor-binding domain of the spike protein that also are reported in variants of concern in the United Kingdom (B.1.1.7) and South Africa (B.1.325).Methods: We isolated two P.1-containing specimens from nasopharyngeal and bronchoalveolar lavage samples of patients of Manaus, Brazil. We measured neutralization of the P.1 virus after incubation with the plasma of 19 COVID-19 convalescent blood donors and recipients of the chemically-inactivated CoronaVac vaccine and compared these results to neutralization of a SARS-CoV-2 B-lineage previously circulating in Brazil.Findings: The immune plasma of COVID-19 convalescent blood donors had 6-fold less neutralizing capacity against the P.1 than against the B-lineage. Moreover, five months after booster immunization with CoronaVac, plasma from vaccinated individuals failed to efficiently neutralize P.1 lineage isolates.Interpretation: These data indicate that the P.1 lineage may escape from neutralizing antibodies generated in response to polyclonal stimulation against previously circulating variants of SARS-CoV-2.Funding: São Paulo Research Foundation, MCTI/FINEP, Medical Research Council, National Council for Scientific and Technological Development, National Institutes of Health.Conflict of Interest: M.S.D. is a consultant for Inbios, Vir Biotechnology, NGMBiopharmaceuticals, and Carnival Corporation, and on the Scientific Advisory Boards of Moderna and Immunome. The Diamond laboratory has received funding support in sponsored research agreements from Moderna, Vir Biotechnology, and Emergent BioSolutions.Ethical Approval: All procedures followed the ethical standards of the responsible committee on humanexperimentation and approved by the ethics committees from the University of Campinas, Brazil (Approval number CONEP 4.021.484 for plasma collection of blood donors, CAEE32078620.4.0000.5404 and 30227920.9.0000.5404 for the sampling of vaccinated and viral genome sequencing, respectively). All patient data were anonymized before study inclusion.Informed consent was obtained from all subjects for being included in the study.

SARS-CoV-2 infects brain astrocytes of COVID-19 patients and impairs neuronal viability (preprint)

COVID-19 patients may exhibit neuropsychiatric and neurological symptoms. We found that anxiety and cognitive impairment are manifested by 28-56% of SARS-CoV-2-infected individuals with mild respiratory symptoms and are associated with altered cerebral cortical thickness. Using an independent cohort, we found histopathological signs of brain damage in 25% of individuals who died of COVID-19. All of the affected brain tissues exhibited foci of SARS-CoV-2 infection and replication, particularly in astrocytes. Infection of neural stem cell-derived astrocytes changed energy metabolism, altered key proteins and metabolites used to fuel neurons and for biogenesis of neurotransmitters, and elicited a secretory phenotype that reduces neuronal viability. Our data support the model where SARS-CoV-2 reaches the brain, infects astrocytes and triggers neuropathological changes that contribute to the structural and functional alterations in the brain of COVID-19 patients.

Early use of nitazoxanide in mild Covid-19 disease: randomized, placebo-controlled trial (preprint)

The antiparasitic drug nitazoxanide is widely available and exerts broad-spectrum antiviral activity in vitro. However, there is no evidence of its impact on SARS-CoV-2 infection. In a multicenter, randomized, double-blind, placebo-controlled trial, adult patients who presented up to 3 days after onset of Covid-19 symptoms (dry cough, fever, and/or fatigue) were enrolled. After confirmation of SARS-CoV2 infection by RT-PCR on nasopharyngeal swab, patients were randomized 1:1 to receive either nitazoxanide (500 mg) or placebo, TID, for 5 days. The primary outcome was complete resolution of symptoms. Secondary outcomes were viral load, general laboratory tests, serum biomarkers of inflammation, and hospitalization rate. Adverse events were also assessed. From June 8 to August 20, 2020, 1,575 patients were screened. Of these, 392 (198 placebo, 194 nitazoxanide) were analyzed. Median time from symptom onset to first dose of study drug was 5 (4-5) days. At the 5-day study visit, symptom resolution did not differ between the nitazoxanide and placebo arms. However, at the 1-week follow-up, 78% in the nitazoxanide arm and 57% in the placebo arm reported complete resolution of symptoms (p=0.048). Swabs collected were negative for SARS-CoV-2 in 29.9% of patients in the nitazoxanide arm versus 18.2% in the placebo arm (p=0.009). Viral load was also reduced after nitazoxanide compared to placebo (p=0.006). No serious adverse events were observed. In patients with mild Covid-19, symptom resolution did not differ between the nitazoxanide and placebo groups after 5 days of therapy. However, early nitazoxanide therapy was safe and reduced viral load significantly.

SARS-CoV-2 infects brain astrocytes of COVID-19 patients and impairs neuronal viability (preprint)

COVID-19 patients may exhibit neuropsychiatric and/or neurological symptoms. We found that anxiety and cognitive impairment are manifested by 28-56% of SARS-CoV-2-infected individuals with mild or no respiratory symptoms and are associated with altered cerebral cortical thickness. Using an independent cohort, we found histopathological signs of brain damage in 19% of individuals who died of COVID-19. All of the affected brain tissues exhibited foci of SARS-CoV-2 infection, particularly in astrocytes. Infection of neural stem cell-derived astrocytes changed energy metabolism, altered key proteins and metabolites used to fuel neurons and for biogenesis of neurotransmitters, and elicited a secretory phenotype that reduces neuronal viability. Our data support the model where SARS-CoV-2 reaches the brain, infects astrocytes and triggers neuropathological changes that contribute to the structural and functional alterations in the brain of COVID-19 patients.

Sequential infection with influenza A virus followed by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to more severe disease and encephalitis in a mouse model of COVID-19. (preprint)

COVID-19 is a spectrum of clinical symptoms in humans caused by infection with SARS-CoV-2, a recently emerged coronavirus that has rapidly caused a pandemic. Coalescence of a second wave of this virus with seasonal respiratory viruses, particularly influenza virus is a possible global health concern. To investigate this, transgenic mice expressing the human ACE2 receptor driven by the epithelial cell cytokeratin-18 gene promoter (K18-hACE2) were first infected with IAV followed by SARS-CoV-2. The host response and effect on virus biology was compared to K18-hACE2 mice infected with IAV or SARS-CoV-2 only. Infection of mice with each individual virus resulted in a disease phenotype compared to control mice. Although, SARS-CoV-2 RNA synthesis appeared significantly reduced in the sequentially infected mice, these mice had a more rapid weight loss, more severe lung damage and a prolongation of the innate response compared to singly infected or control mice. The sequential infection also exacerbated the extrapulmonary manifestations associated with SARS-CoV-2. This included a more severe encephalitis. Taken together, the data suggest that the concept of "twinfection" is deleterious and mitigation steps should be instituted as part of a comprehensive public health response to the COVID-19 pandemic.

A 3D Structural Interactome to Explore the Impact of Evolutionary Divergence, Population Variation, and Small-molecule Drugs on SARS-CoV-2-Human Protein-Protein Interactions (preprint)

The recent COVID-19 pandemic has sparked a global public health crisis. Vital to the development of informed treatments for this disease is a comprehensive understanding of the molecular interactions involved in disease pathology. One lens through which we can better understand this pathology is through the network of protein-protein interactions between its viral agent, SARS-CoV-2, and its human host. For instance, increased infectivity of SARS-CoV-2 compared to SARS-CoV can be explained by rapid evolution along the interface between the Spike protein and its human receptor (ACE2) leading to increased binding affinity. Sequence divergences that modulate other protein-protein interactions may further explain differences in transmission and virulence in this novel coronavirus. To facilitate these comparisons, we combined homology-based structural modeling with the ECLAIR pipeline for interface prediction at residue resolution, and molecular docking with PyRosetta. This enabled us to compile a novel 3D structural interactome meta-analysis for the published interactome network between SARS-CoV-2 and human. This resource includes docked structures for all interactions with protein structures, enrichment analysis of variation along interfaces, predicted {Delta}{Delta}G between SARS-CoV and SARS-CoV-2 variants for each interaction, predicted impact of natural human population variation on binding affinity, and a further prioritized set of drug repurposing candidates predicted to overlap with protein interfaces. All predictions are available online for easy access and are continually updated when new interactions are published. NOTE: Some sections of this pre-print have been redacted to comply with current bioRxiv policy restricting the dissemination of purely in silico results predicting potential therapies for SARS-CoV-2 that have not undergone thorough peer-review. The results section titled 'Prioritization of Candidate Inhibitors of SARS-CoV-2-Human Interactions Through Binding Site Comparison,' Figure 4, Supplemental Table 9, and all links to our web resource have been removed. Blank headers left in place to preserve structure and item numbering. Our full manuscript will be published in an appropriate journal following peer-review.

Molecular characterization of SARS-CoV-2 from Bangladesh: Implications in genetic diversity, possible origin of the virus, and functional significance of the mutations (preprint)

In a try to understand the pathogenesis, evolution, and epidemiology of the SARS-CoV-2 virus, scientists from all over the world are tracking its genomic changes in real-time. Genomic studies can be helpful in understanding the disease dynamics. We have downloaded 324 complete and near-complete SARS-CoV-2 genomes submitted in the GISAID database from Bangladesh which were isolated between 30 March to 7 September 2020. We then compared these genomes with the Wuhan reference sequence and found 4160 mutation events including 2253 missense single nucleotide variations, 38 deletions, and 10 insertions. The C>T nucleotide change was most prevalent possibly due to selective mutation pressure to reduce CpG sites to evade CpG targeted host immune response. The most frequent mutation that occurred in 98% of the isolates was 3037C>T which is a synonymous change that almost always accompanied 3 other mutations that include 241C>T, 14408C>T (P323L in RdRp), and 23403A>G (D614G in spike protein). The P323L was reported to increase mutation rate and D614G is associated with increased viral replication and currently the most prevalent variant circulating all over the world. We identified multiple missense mutations in B-cell and T-cell predicted epitope regions and/or PCR target regions (including R203K and G204R that occurred in 86% of the isolates) that may impact immunogenicity and/or RT-PCR based diagnosis. Our analysis revealed 5 large deletion events in ORF7a and ORF8 gene products that may be associated with less severity of the disease and increased viral clearance. Our phylogeny analysis identified most of the isolates belonged to the Nextstrain clade 20B (86%) and GISAID clade GR (88%). Most of our isolates shared common ancestors either directly with European countries or jointly with middle eastern countries as well as Australia and India. Interestingly, the 19B clade (GISAID S clade) was unique to Chittagong which was originally prevalent in China. This reveals possible multiple introductions of the virus in Bangladesh via different routes. Hence more genome sequencing and analysis with related clinical data are needed to interpret the functional significance and better predict the disease dynamics that may be helpful for policymakers to control the COVID-19 pandemic in Bangladesh.

Structural basis for bivalent binding and inhibition of SARS-CoV-2 infection by human potent neutralizing antibodies (preprint)

Neutralizing monoclonal antibodies (nAbs) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represent promising candidates for clinical intervention against coronavirus virus diseases 2019 (COVID-19). We isolated a large number of nAbs from SARS-CoV-2 infected individuals capable of disrupting proper interaction between the receptor binding domain (RBD) of the viral spike (S) protein and the receptor angiotensin converting enzyme 2 (ACE2). In order to understand the mechanism of these nAbs on neutralizing SARS-CoV-2 virus infections, we have performed cryo-EM analysis and here report cryo-EM structures of the ten most potent nAbs in their native full-length IgG or Fab forms bound to the trimeric S protein of SARS-CoV-2. The bivalent binding of the full-length IgG is found to associate with more RBD in the "up" conformation than the monovalent binding of Fab, perhaps contributing to the enhanced neutralizing activity of IgG and triggering more shedding of the S1 subunit from the S protein. Comparison of large number of nAbs identified common and unique structural features associated with their potent neutralizing activities. This work provides structural basis for further understanding the mechanism of nAbs, especially through revealing the bivalent binding and their correlation with more potent neutralization and the shedding of S1 subunit.

Degradation of SARS-CoV-2 receptor ACE2 by tobacco carcinogen-induced Skp2 in lung epithelial cells (preprint)

An unexpected observation among the COVID-19 pandemic is that smokers constituted only 1.4-18.5% of hospitalized adults, calling for an urgent investigation to determine the role of smoking in SARS-CoV-2 infection. Here, we show that cigarette smoke extract (CSE) and carcinogen benzo(a)pyrene (BaP) increase ACE2 mRNA but trigger ACE2 protein catabolism. BaP induces an aryl hydrocarbon receptor (AhR)-dependent upregulation of the ubiquitin E3 ligase Skp2 for ACE2 ubiquitination. ACE2 in lung tissues of non-smokers is higher than in smokers, consistent with the findings that tobacco carcinogens downregulate ACE2 in mice. Tobacco carcinogens inhibit SARS-CoV-2 Spike protein pseudovirions infection of the cells. Given that tobacco smoke accounts for 8 million deaths including 2.1 million cancer deaths annually and Skp2 is an oncoprotein, tobacco use should not be recommended and cessation plan should be prepared for smokers in COVID-19 pandemic.

Structural impact on SARS-CoV-2 spike protein by D614G substitution (preprint)

Substitution for aspartic acid by glycine at position 614 in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the ongoing pandemic, appears to facilitate rapid viral spread. The G614 variant has now replaced the D614-carrying virus as the dominant circulating strain. We report here cryo-EM structures of a full-length S trimer carrying G614, which adopts three distinct prefusion conformations differing primarily by the position of one receptor-binding domain (RBD). A loop disordered in the D614 S trimer wedges between domains within a protomer in the G614 spike. This added interaction appears to prevent premature dissociation of the G614 trimer, effectively increasing the number of functional spikes and enhancing infectivity. The loop transition may also modulate structural rearrangements of S protein required for membrane fusion. These findings extend our understanding of viral entry and suggest an improved immunogen for vaccine development.

SARS-CoV-2 Uses CD4 to Infect T Helper Lymphocytes (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent of a major global outbreak of respiratory tract disease known as coronavirus disease-2019 (COVID-19). SARS-CoV-2 infects the lungs and may cause several immune-related complications such as lymphocytopenia and cytokine storm which are associated with the severity of the disease and predict mortality . The mechanism by which SARS-CoV-2 infection may result in immune system dysfunction is not fully understood. Here we show that SARS-CoV-2 infects human CD4+ T helper cells, but not CD8+ T cells, and is present in blood and bronchoalveolar lavage T helper cells of severe COVID-19 patients. We demonstrated that SARS-CoV-2 spike glycoprotein (S) directly binds to the CD4 molecule, which in turn mediates the entry of SARS- CoV-2 in T helper cells in a mechanism that also requires ACE2 and TMPRSS2. Once inside T helper cells, SARS-CoV-2 assembles viral factories, impairs cell function and may cause cell death. SARS-CoV-2 infected T helper cells express higher amounts of IL-10, which is associated with viral persistence and disease severity. Thus, CD4-mediated SARS-CoV-2 infection of T helper cells may explain the poor adaptive immune response of many COVID- 19 patients.

A Tethered Ligand Assay to Probe the SARS-CoV-2 ACE2 Interaction under Constant Force (preprint)

The current COVID-19 pandemic has a devastating global impact and is caused by the SARS-CoV-2 virus. SARS-CoV-2 attaches to human host cells through interaction of its receptor binding domain (RBD) located on the viral Spike (S) glycoprotein with angiotensin converting enzyme-2 (ACE2) on the surface of host cells. RBD binding to ACE2 is a critical first step in SARS-CoV-2 infection. Viral attachment occurs in dynamic environments where forces act on the binding partners and multivalent interactions play central roles, creating an urgent need for assays that can quantitate SARS-CoV-2 interactions with ACE2 under mechanical load and in defined geometries. Here, we introduce a tethered ligand assay that comprises the RBD and the ACE2 ectodomain joined by a flexible peptide linker. Using specific molecular handles, we tether the fusion proteins between a functionalized flow cell surface and magnetic beads in magnetic tweezers. We observe repeated interactions of RBD and ACE2 under constant loads and can fully quantify the force dependence and kinetics of the binding interaction. Our results suggest that the SARS-CoV-2 ACE2 interaction has higher mechanical stability, a larger free energy of binding, and a lower off-rate than that of SARS-CoV-1, the causative agents of the 2002-2004 SARS outbreak. In the absence of force, the SARS-CoV-2 RBD rapidly (within [≤]1 ms) engages the ACE2 receptor if held in close proximity and remains bound to ACE2 for 400-800 s, much longer than what has been reported for other viruses engaging their cellular receptors. We anticipate that our assay will be a powerful tool investigate the roles of mutations in the RBD that might alter the infectivity of the virus and to test the modes of action of neutralizing antibodies and other agents designed to block RBD binding to ACE2 that are currently developed as potential COVID-19 therapeutics.

Discovery and Development of Human SARS-CoV-2 Neutralizing Antibodies using an Unbiased Phage Display Library Approach (preprint)

SARS-CoV-2 neutralizing antibodies represent an important component of the ongoing search for effective treatment of and protection against COVID-19. We report here on the use of a naive phage display antibody library to identify a panel of fully human SARS-CoV-2 neutralizing antibodies. Following functional profiling in vitro against an early pandemic isolate as well as a recently emerged isolate bearing the D614G Spike mutation, the clinical candidate antibody, STI-1499, and the affinity-engineered variant, STI-2020, were evaluated for in vivo efficacy in the Syrian golden hamster model of COVID-19. Both antibodies demonstrated potent protection against the pathogenic effects of the disease and a dose-dependent reduction of virus load in the lungs, reaching undetectable levels following a single dose of 500 micrograms of STI-2020. These data support continued development of these antibodies as therapeutics against COVID-19 and future use of this approach to address novel emerging pandemic disease threats.

No evidence that plasmablasts transdifferentiate into developing neutrophils in severe COVID-19 disease (preprint)

A recent study by Wilk et al. of the transcriptome of peripheral blood mononuclear cells (PBMCs) in seven patients hospitalized with COVID-19 described a population of 'developing neutrophils' that were 'phenotypically related by dimensionality reduction' to plasmablasts, and that these two cell populations represent a 'linear continuum of cellular phenotype'. The authors suggest that, in the setting of acute respiratory distress syndrome (ARDS) secondary to severe COVID-19, a 'differentiation bridge from plasmablasts to developing neutrophils' connected these distantly related cell types. This conclusion is controversial as it appears to violate several basic principles in cell biology relating to cell lineage identity and fidelity. Correctly classifying cells and their developmental history is an important issue in cell biology and we suggest that this conclusion is not supported by the data as we show here that: (1) regressing out covariates such as unique molecular identifiers (UMIs) can lead to overfitting; and (2) that UMAP embeddings may reflect the expression of similar genes but not necessarily direct cell lineage relationships.

Identification of TMEM106B as proviral host factor for SARS-CoV-2 (preprint)

The ongoing COVID-19 pandemic is responsible for worldwide economic damage and nearly one million deaths. Potent drugs for the treatment of severe SARS-CoV-2 infections are not yet available. To identify host factors that support coronavirus infection, we performed genome-wide functional genetic screens with SARS-CoV-2 and the common cold virus HCoV-229E in non-transgenic human cells. These screens identified PI3K type 3 as a potential drug target against multiple coronaviruses. We discovered that the lysosomal protein TMEM106B is an important host factor for SARS-CoV-2 infection. Furthermore, we show that TMEM106B is required for replication in multiple human cell lines derived from liver and lung and is expressed in relevant cell types in the human airways. Our results identify new coronavirus host factors that may potentially serve as drug targets against SARS-CoV-2 or to quickly combat future zoonotic coronavirus outbreaks.