SARS-CoV-2 Infection Biomarkers Reveal an Extended RSAD2 Dependant Metabolic Pathway (preprint)

We present compelling evidence for the existence of an evolutionary adaptive response to viral agents such as SARS-CoV-2, that results in the human in vivo biosynthesis of a family of compounds with potential antiviral activity. Using nuclear magnetic resonance (NMR) spectroscopy, we detected a characteristic spin-system motif indicative of the presence of an extended panel of urinary and serum metabolites during the acute viral phase. The structure of eight of nucleoside analogues was elucidated (six of which have not previously been reported in human urine), and subsequently confirmed by total-synthesis and matrix spiking. The molecular structures of the nucleoside analogues and their correlation with an array of serum cytokines, including IFN-2, IFN-{gamma} and IL-10, suggest an association with the viperin enzyme contributing to an endogenous innate immune defense mechanism against viral infection.

SARS-CoV-2 multi-antigen protein microarray for detailed characterization of antibody responses in COVID-19 patients (preprint)

Antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target multiple epitopes on different domains of the spike protein, and other SARS-CoV-2 proteins. We developed a SARS-CoV-2 multi-antigen protein microarray with the nucleocapsid, spike and its domains (S1, S2), and variants with single (D614G, E484K, N501Y) or double substitutions (N501Y/Deletion69/70), allowing a more detailed high-throughput analysis of the antibody repertoire following infection. The assay was demonstrated to be reliable and comparable to ELISA. We analyzed antibodies from 18 COVID-19 patients and 12 recovered convalescent donors. S IgG level was higher than N IgG in most of the COVID-19 patients, receptor-binding domain of S1 showed high reactivity, but no antibodies were detected against heptad repeat domain 2 of S2. Furthermore, antibodies were detected against S variants with single and double substitutions in COVID-19 patients who were infected with SARS-CoV-2 early in the pandemic. Here we demonstrated that SARS-CoV-2 multi-antigen protein microarray is a powerful tool for detailed characterization of antibody responses, with potential utility in understanding the disease progress and assessing current vaccines and therapies against evolving SARS-CoV-2.

Circulating ACE2-expressing Exosomes Block SARS-CoV-2 Virus Infection as an Innate Antiviral Mechanism (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease 2019 (COVID-19) with innate and adaptive immune response triggered in such patients by viral antigens. Both convalescent plasma and engineered high affinity human monoclonal antibodies have shown therapeutic potential to treat COVID-19. Whether additional antiviral soluble factors exist in peripheral blood remain understudied. Herein, we detected circulating exosomes that express the SARS-CoV-2 viral entry receptor angiotensin-converting enzyme 2 (ACE2) in plasma of both healthy donors and convalescent COVID-19 patients. We demonstrated that exosomal ACE2 competes with cellular ACE2 for neutralization of SARS-CoV-2 infection. ACE2-expressing (ACE2+) exosomes blocked the binding of the viral spike (S) protein RBD to ACE2+ cells in a dose dependent manner, which was 400- to 700-fold more potent than that of vesicle-free recombinant human ACE2 extracellular domain protein (rhACE2). As a consequence, exosomal ACE2 prevented SARS-CoV-2 pseudotype virus tethering and infection of human host cells at a 50-150 fold higher efficacy than rhACE2. A similar antiviral activity of exosomal ACE2 was further demonstrated to block wild-type live SARS-CoV-2 infection. Of note, depletion of ACE2+ exosomes from COVID-19 patient plasma impaired the ability to block SARS-CoV-2 RBD binding to host cells. Our data demonstrate that ACE2+ exosomes can serve as a decoy therapeutic and a possible innate antiviral mechanism to block SARS-CoV-2 infection.

Ca2+-dependent mechanism of membrane insertion and destabilization by the SARS-CoV-2 fusion peptide (preprint)

Cell penetration after recognition of the SARS-CoV-2 virus by the ACE2 receptor, and the fusion of its viral envelope membrane with cellular membranes, are the early steps of infectivity. A region of the Spike protein (S) of the virus, identified as the "fusion peptide" (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca2+ ions to the FPs of corona viruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca2+ binding to the SARS-CoV-2-FP, the role of Ca2+ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca2+-bound SARS-CoV-2-FP and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics (MD) simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca2+-bound peptide models with lipid membranes SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca2+ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca2+ ions. These findings provide novel mechanistic insights regarding the role of Ca2+ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry. STATEMENT OF SIGNIFICANCESARS-CoV-2, the cause of the COVID-19 pandemic, penetrates host cell membranes and uses viral-to-cellular membrane fusion to release its genetic material for replication. Experiments had identified a region termed "fusion peptide" (FP) in the Spike proteins of coronaviruses, as the spearhead in these initial processes, and suggested that Ca2+ is needed to support both functions. Absent structure and dynamics-based mechanistic information these FP functions could not be targeted for therapeutic interventions. We describe the development and determination of the missing information from analysis of extensive MD simulation trajectories, and propose specific Ca2+-dependent mechanisms of SARS-CoV-2-FP membrane insertion and destabilization. These results offer a structure-specific platform to aid the ongoing efforts to use this target for the discovery and/or of inhibitors.

Treatment of Severe COVID-19 with Convalescent Plasma in the Bronx, NYC (preprint)

Convalescent plasma with severe acute respiratory disease coronavirus 2 (SARS-CoV-2) antibodies (CCP) may hold promise as treatment for Coronavirus Disease 2019 (COVID-19). We compared the mortality and clinical outcome of patients with COVID-19 who received 200mL of CCP with a Spike protein IgG titer [≥]1:2,430 (median 1:47,385) within 72 hours of admission to propensity score-matched controls cared for at a medical center in the Bronx, between April 13 to May 4, 2020. Matching criteria for controls were age, sex, body mass index, race, ethnicity, comorbidities, week of admission, oxygen requirement, D-dimer, lymphocyte counts, corticosteroids, and anticoagulation use. There was no difference in mortality or oxygenation between CCP recipients and controls at day 28. When stratified by age, compared to matched controls, CCP recipients <65 years had 4-fold lower mortality and 4-fold lower deterioration in oxygenation or mortality at day 28. For CCP recipients, pre-transfusion Spike protein IgG, IgM and IgA titers were associated with mortality at day 28 in univariate analyses. No adverse effects of CCP were observed. Our results suggest CCP may be beneficial for hospitalized patients <65 years, but data from controlled trials is needed to validate this finding and establish the effect of ageing on CCP efficacy.

Development, clinical translation, and utility of a COVID-19 antibody test with qualitative and quantitative readouts (preprint)

The COVID-19 global pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to place an immense burden on societies and healthcare systems. A key component of COVID-19 control efforts is serologic testing to determine the community prevalence of SARS-CoV-2 exposure and quantify individual immune responses to prior infection or vaccination. Here, we describe a laboratory-developed antibody test that uses readily available research-grade reagents to detect SARS-CoV-2 exposure in patient blood samples with high sensitivity and specificity. We further show that this test affords the estimation of viral spike-specific IgG titers from a single sample measurement, thereby providing a simple and scalable method to measure the strength of an individual's immune response. The accuracy, adaptability, and cost-effectiveness of this test makes it an excellent option for clinical deployment in the ongoing COVID-19 pandemic.

Identifying zoonotic origin of SARS-CoV-2 by modeling the binding affinity between Spike receptor-binding domain and host ACE2 (preprint)

Despite considerable research progress on SARS-CoV-2, the direct zoonotic origin (intermediate host) of the virus remains ambiguous. The most definitive approach to identify the intermediate host would be the detection of SARS-CoV-2-like coronaviruses in wild animals. However, due to the high number of animal species, it is not feasible to screen all the species in the laboratory. Given that the recognition of the binding ACE2 proteins is the first step for the coronaviruses to invade host cells, we proposed a computational pipeline to identify potential intermediate hosts of SARS-CoV-2 by modeling the binding affinity between the Spike receptor-binding domain (RBD) and host ACE2. Using this pipeline, we systematically examined 285 ACE2 variants from mammals, birds, fish, reptiles, and amphibians, and found that the binding energies calculated on the modeled Spike-RBD/ACE2 complex structures correlate closely with the effectiveness of animal infections as determined by multiple experimental datasets. Built on the optimized binding affinity cutoff, we suggested a set of 96 mammals, including 48 experimentally investigated ones, which are permissive to SARS-CoV-2, with candidates from primates, rodents, and carnivores at the highest risk of infection. Overall, this work not only suggested a limited range of potential intermediate SARS-CoV-2 hosts for further experimental investigation; but more importantly, it proposed a new structure-based approach to general zoonotic origin and susceptibility analyses that are critical for human infectious disease control and wildlife protection.

Signatures and mechanisms of efficacious therapeutic ribonucleotides against SARS-CoV-2 revealed by analysis of its replicase using magnetic tweezers (preprint)

Coronavirus Disease 2019 (COVID-19) results from an infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the third coronavirus outbreak to plague humanity this century. Currently, the most efficacious therapeutic against SARS-CoV-2 infection is the Remdesivir (RDV), an adenine-like ribonucleotide analogue that is very efficiently incorporated by the SARS-CoV-2 replicase. Understanding why RDV is so well incorporated will facilitate development of even more effective therapeutics. Here, we have applied a high-throughput, single-molecule, magnetic-tweezers platform to study thousands of cycles of nucleotide addition by the SARS-CoV-2 replicase in the absence and presence of RDV, a Favipiravir-related analog (T-1106), and the endogenously produced ddhCTP. Our data are consistent with two parallel catalytic pathways of the replicase: a high-fidelity catalytic (HFC) state and a low-fidelity catalytic (LFC) state, the latter allowing the slow incorporation of both cognate and non-cognate nucleotides. ddhCTP accesses HFC, T-1106 accesses LFC as a non-cognate nucleotide, while RDV efficiently accesses both LFC pathway. In contrast to previous reports, we provide unequivocal evidence against RDV functioning as a chain terminator. We show that RDV incorporation transiently stalls the replicase, only appearing as termination events when traditional, gel-based assays are used. The efficiency of ddhCTP utilization by the SARS-CoV-2 replicase suggests suppression of its synthesis during infection, inspiring new therapeutic strategies. Use of this experimental paradigm will be essential to the development of therapeutic nucleotide analogs targeting polymerases.

Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis (preprint)

Coronavirus disease 2019 (COVID-19) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike (S) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F and ExpiCHO-S cells, two different cell lines selected for increased expression of secreted glycoproteins. We show that ExpiCHO-S cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural (cryo-EM) characterization of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high quality S protein (non-aggregated, uniform material with appropriate biochemical and biophysical properties). Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs, and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.