Antibody escape and cryptic cross-domain stabilization in the SARS CoV-2 Omicron spike protein (preprint)

The worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the repeated emergence of variants of concern. The Omicron variant has two dominant sub-lineages, BA.1 and BA.2, each with unprecedented numbers of nonsynonymous and indel spike protein mutations: 33 and 29, respectively. Some of these mutations individually increase transmissibility and enhance immune evasion, but their interactions within the Omicron mutational background is unknown. We characterize the molecular effects of all Omicron spike mutations on expression, human ACE2 receptor affinity, and neutralizing antibody recognition. We show that key mutations enable escape from neutralizing antibodies at a variety of epitopes. Stabilizing mutations in the N-terminal and S2 domains of the spike protein compensate for destabilizing mutations in the receptor binding domain, thereby enabling the record number of mutations in Omicron sub-lineages. Taken together, our results provide a comprehensive account of the mutational effects in the Omicron spike protein and illuminate previously unknown mechanisms of how the N-terminal domain can compensate for destabilizing mutations within the more evolutionarily constrained RBD.

Synthetic repertoires derived from convalescent COVID-19 patients enable discovery of SARS-CoV-2 neutralizing antibodies and a novel quaternary binding modality (preprint)

The ongoing evolution of SARS-CoV-2 into more easily transmissible and infectious variants has sparked concern over the continued effectiveness of existing therapeutic antibodies and vaccines. Hence, together with increased genomic surveillance, methods to rapidly develop and assess effective interventions are critically needed. Here we report the discovery of SARS-CoV-2 neutralizing antibodies isolated from COVID-19 patients using a high-throughput platform. Antibodies were identified from unpaired donor B-cell and serum repertoires using yeast surface display, proteomics, and public light chain screening. Cryo-EM and functional characterization of the antibodies identified N3-1, an antibody that binds avidly (Kd,app = 68 pM) to the receptor binding domain (RBD) of the spike protein and robustly neutralizes the virus in vitro. This antibody likely binds all three RBDs of the trimeric spike protein with a single IgG. Importantly, N3-1 equivalently binds spike proteins from emerging SARS-CoV-2 variants of concern, neutralizes UK variant B.1.1.7, and binds SARS-CoV spike with nanomolar affinity. Taken together, the strategies described herein will prove broadly applicable in interrogating adaptive immunity and developing rapid response biological countermeasures to emerging pathogens.

Rapid characterization of spike variants via mammalian cell surface display (preprint)

The SARS-CoV-2 spike (S) protein is a critical component of subunit vaccines and a target for neutralizing antibodies. Spike is also undergoing immunogenic selection with clinical variants that increase infectivity and partially escape convalescent plasma. Here, we describe spike display, a high-throughput platform to rapidly characterize glycosylated spike ectodomains across multiple coronavirus-family proteins. We assayed ~200 variant SARS-CoV-2 spikes for their expression, ACE2 binding, and recognition by thirteen neutralizing antibodies (nAbs). An alanine scan of the N-terminal domain (NTD) highlights a public class of epitopes in the N3 and N5 loops that are recognized by most of the NTD-binding nAbs assayed in this study. Some clinical NTD substitutions abrogate binding to these epitopes but are circulating at low frequencies around the globe. NTD mutations in variants of concern B.1.1.7 (United Kingdom), B.1.351 (South Africa), B.1.1.248 (Brazil), and B.1.427/B.1.429 (California) impact spike expression and escape most NTD-targeting nAbs. However, two classes of NTD nAbs still bind B.1.1.7 spikes and neutralize in pseudoviral assays. B.1.1351 and B.1.1.248 include compensatory mutations that either increase spike expression or increase ACE2 binding affinity. Finally, B.1.351 and B.1.1.248 completely escape a potent ACE2 peptide mimic. We anticipate that spike display will be useful for rapid antigen design, deep scanning mutagenesis, and epitope mapping of antibody interactions for SARS-CoV-2 and other emerging viral threats.

Changes in SARS-CoV-2 before the peak in each country, producing unique variants. (preprint)

The second and third waves of coronavirus disease 2019 (COVID-19) have caused problems worldwide. Those are often thought to have resulted from people's carelessness or people not following restrictions, but in reality, the cause remains unclear. Here, using an objective analytical method, we present the changes in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing COVID-19 over time. The virus has mutated in three major directions, with three groups remaining to date. The basic structure of the groups was completed by April and shared across all continents. However, the virus continued to mutate independently in each country after the borders were closed. In particular, the virus mutated before the occurrence of a second or third peak. It seems that the mutations conferred higher infectivity to the virus, because of which the virus overcame previously effective protections. Currently, each country may possess such a unique stronger variant, which may cause another peak in other countries. These viruses could also serve as sources of mutations by exchanging parts of the genome. Such mutations could create a variant with superior infectivity.

The SARS-CoV-2 spike protein disrupts the cooperative function of human cardiac pericytes - endothelial cells through CD147 receptor-mediated signalling: a potential non-infective mechanism of COVID-19 microvascular disease (preprint)

Background: Severe coronavirus disease 2019 (COVID-19) manifests as a life-threatening microvascular syndrome. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses primarily the capsid spike (S) protein to engage with its receptors and infect host cells. To date, it is still not known if the S protein alone, without the other viral elements, is able to trigger vascular cell signalling and provoke cell dysfunction. Methods: We investigated the effects of the recombinant, stabilised S protein on primary human cardiac pericytes (PCs) signalling and function. Endpoints included cell viability, proliferation, migration, cooperation with endothelial cells (ECs) in angiogenesis assays, and release of pro-inflammatory cytokines. Adopting a blocking strategy against the S protein receptors ACE2 and CD147, we explored which receptor mediates the S protein signalling in PCs. Findings: We show, for the first time, that the recombinant S protein alone elicits functional alterations in cardiac PCs. This was documented as: (1) increased migration, (2) reduced ability to support EC network formation on Matrigel, (3) secretion of pro-inflammatory molecules typically involved in the cytokine storm; and (4) production of pro-apoptotic factors responsible for EC death. Furthermore, the S protein stimulates the phosphorylation/activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) through the CD147 receptor, but not ACE2, in cardiac PCs. Accordingly, the neutralization of CD147, using a blocking antibody, prevented the activation of ERK1/2 and partially rescued the PC function in the presence of the S protein. Interpretation: Our findings suggest the new, intriguing hypothesis that the S protein may elicit vascular cell dysfunction, potentially amplifying, or perpetuating, the damage caused by the whole coronavirus. This mechanism may have clinical and therapeutic implication.

Cetylpyridinium chloride-containing mouthwashes reduce in vitro SARS-CoV-2 infectivity (preprint)

Oral mouthwashes decrease the infectivity of several respiratory viruses including SARS-CoV-2. However, the precise agents with antiviral activity present in these oral rinses and their exact mechanism of action remain unknown. Here we show that Cetylpyridinium chloride (CPC), a quaternary ammonium compound present in many oral mouthwashes, reduces SARS-CoV-2 infectivity by inhibiting viral fusion with target cells. We also found that CPC and CPC-containing mouth rinses decreased a thousand times the infectivity of SARS-CoV-2 in vitro, while the corresponding vehicles had no effect. CPC-containing mouth rinses could represent a cost-effective measure to reduce SARS-CoV-2 infectivity in saliva, aiding to reduce viral transmission from infected individuals.

Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes in COVID-19 convalescent plasma (preprint)

Although humoral immunity is essential for control of SARS-CoV-2, the molecular composition, binding epitopes and effector functions of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following infection are unknown. Proteomic deconvolution of the circulating IgG repertoire (Ig-Seq1) to the spike ectodomain (S-ECD2) in four convalescent study subjects revealed that the plasma response is oligoclonal and directed predominantly (>80%) to S-ECD epitopes that lie outside the receptor binding domain (RBD). When comparing antibodies directed to either the RBD, the N-terminal domain (NTD) or the S2 subunit (S2) in one subject, just four IgG lineages (1 anti-S2, 2 anti-NTD and 1 anti-RBD) accounted for 93.5% of the repertoire. Although the anti-RBD and one of the anti-NTD antibodies were equally potently neutralizing in vitro, we nonetheless found that the anti-NTD antibody was sufficient for protection to lethal viral challenge, either alone or in combination as a cocktail where it dominated the effect of the other plasma antibodies. We identified in vivo protective plasma anti-NTD antibodies in 3/4 subjects analyzed and discovered a shared class of antibodies targeting the NTD that utilize unmutated or near-germline IGHV1-24, the most electronegative IGHV gene in the human genome. Structural analysis revealed that binding to NTD is dominated by interactions with the heavy chain, accounting for 89% of the entire interfacial area, with germline residues uniquely encoded by IGHV1-24 contributing 20% (149 [A]2). Together with recent reports of germline IGHV1-24 antibodies isolated by B-cell cloning3,4 our data reveal a class of shared IgG antibodies that are readily observed in convalescent plasma and underscore the role of NTD-directed antibodies in protection against SARS-CoV-2 infection.

Binding strength and hydrogen bond numbers between Covid-19 RBD and HVR of antibody (preprint)

The global battle against the Covid-19 pandemic relies strongly on the human defence of antibody, which is assumed to bind the Receptor Binding Domain of the antigen with its Hypervariable Region. Due to the similarity to other viruses such as SARS, however, our understanding of the antibody-virus interaction has been limited to the genomic sequencing, which poses serious challenges to the containment, vaccine exploration and rapid serum testing. Based on the physical/chemical nature of the interaction, infrared spectroscopy was employed to reveal the binding disparity, when unusual temperature dependence was discovered from the 1550cm 1 absorption band, attributed to the hydrogen bonds by carboxyl/amino groups, binding the SARS-CoV-2 spike protein and closely resembled SARS-CoV-2 or SARS-CoV-1 antibodies. The infrared absorption intensity, associated with the number of hydrogen bonds, was found to increase sharply between 27C and 31C, with the relative absorbance matches at 37C the hydrogen bonding numbers of the two antibody types (19 vs 12). As a result, the specificity of the SARS-CoV-2 antibody will be more conclusive beyond 31C, instead of at the usual room temperature of 20C - 25C, when the vaccine research and antibody diagnosis would likely be undermined.

Predicting COVID-19 cases with unknown homogeneous or heterogeneous resistance to infectivity (preprint)

This article constructs a restricted infection rate inverse binomial-based approach to predict COVID-19 cases after a family gathering. The traditional inverse binomial (IB) model is unqualified to match the reality of COVID-19, because the data contradicts the models requirement that variance should be greater than expected value. A refined version of the IB model is a necessity to predict COVID-19 cases after family gatherings. Our refined version of an IB model is more appropriate and versatile, as it accommodates all potential data scenarios: equal, lesser, or greater variance than expected value. Application of the approach is based on a restricted infectivity rate and methodology on Fan et al.s COVID-19 data, which exhibits two clusters of infectivity. Cluster 1 has a smaller number of primary cases and exhibits larger variance than the expected cases with a negative correlation of 28%, implying that the number of secondary cases is lesser when the number of primary cases increases and vice versa. The traditional inverse binomial (IB) model is appropriate for Cluster 1. The probability of contracting COVID-19 is estimated to be 0.13 among the primary, but is 0.75 among the secondary in Cluster 1, with a wider gap. Conversely, Cluster 2, exhibits smaller variance than the expected cases with a correlation of 79%, implying the number of primary and secondary cases increase or decrease together. Cluster 2 disqualifies the traditional IB model and demands its refined version. Probability of contracting COVID-19 is estimated to be 0.74 among the primary, but is 0.72 among the secondary in Cluster 2, with a narrower gap. The models ability to estimate the communitys health system memory for future policies to be developed is an asset of this approach. The current hazard level to be infected with COVID-19 among the primary and secondary groups are estimable and interpretable. Author SummaryCurrent statistical models are not able to accurately predict disease infection spread in the COVID-19 pandemic. We have applied a widely-used inverse binomial method to predict rates of infection after small gatherings, going from primary (original) cases to secondary (later) cases after family gatherings or social events, using the data from the Wuhan and Gansu provinces in China, where the virus first spread. The advantages of the proposed approach include that the models ability to estimate the communitys health system memory for future policies to be developed, as such policies might reduce COVIDs spread if not its control. In our approach, as demonstrated, the current hazard level of becoming infected with COVID-19 and the odds of contracting COVID-19 among the primary in comparison to the secondary groups are estimable and interpretable. We hope the proposed approach will be used in future epidemics.

In vitro characterization of engineered red blood cells as potent viral traps against HIV-1 and SARS-CoV-2 (preprint)

Engineered red blood cells (RBCs) expressing viral receptors could be used therapeutically as viral traps as RBCs lack nuclei and other organelles required for viral replication. Here we show that the combination of a powerful erythroid-specific expression system and transgene codon optimization yields high expression levels of the HIV-1 receptors CD4 and CCR5, as well as a CD4-glycophorin A (CD4-GpA) fusion protein on enucleated RBCs. Engineered RBCs expressing CD4 and CCR5 were efficiently infected by HIV-1, but CD4 or CD4-GpA expression in the absence of CCR5 was sufficient to potently neutralize HIV-1 in vitro. To facilitate continuous large-scale production of engineered RBCs, we generated erythroblast cell lines stably expressing CD4-GpA or ACE2-GpA fusion proteins, which produced potent RBC viral traps against HIV-1 and SARS-CoV-2. Our results suggest that this approach warrants further investigation as a potential treatment against viral infections.

pH and Receptor Induced Confirmational Changes- Implications Towards S1 Dissociation of SARS-CoV2 Spike Glycoprotein (preprint)

Viruses, being obligate intracellular parasites, must first attach themselves and gain entry into host cells. Viral fusion machinery is the central player in the viral attachment process in almost every viral disease. Viruses have incorporated an array of efficient fusion proteins on their surfaces to bind efficiently to host cell receptors. They make use of the host proteolytic enzymes to rearrange their surface protein(s) into the form which facilitates their binding to host-cell membrane proteins and subsequently, fusion. This stage of viral entry is very critical and has many therapeutic implications. The current global pandemic of COVID-19 has sparked severe health crisis and economic shutdowns. SARS-CoV2, the etiological agent of the disease has led to millions of deaths and brought the scientific community together in an attempt to understand the mechanisms of SARS-CoV2 pathogenesis and mortality. Like other viral fusion machinery, CoV2 spike (S) glycoprotein- 'The Demogorgon' poses the same questions about viral-host cell fusion. The intermediate stages of S protein-mediated viral fusion are unclear owing to the lack of structural insights and concrete biochemical evidence. The mechanism of conformational transition is still unclear. S protein binding and fusion with host cell receptors, Eg., angiotensin-converting enzyme-2 (ACE2) is accompanied by cleavage of S1/S2 subunits. To track the key events of viral-host cell fusion, we have identified (in silico) that low pH-induced conformational change and ACE-2 binding events promote S1 dissociation. Deciphering key mechanistic insights of SARS-CoV2 fusion will further our understanding of other class- I fusion proteins.

Mutation Landscape of SARS COV2 in Africa (preprint)

COVID-19 disease has had a relatively less severe impact in Africa. To understand the role of SARS CoV2 mutations on COVID-19 disease in Africa, we analysed 282 complete nucleotide sequences from African isolates deposited in the NCBI Virus Database. Sequences were aligned against the prototype Wuhan sequence (GenBank accession: NC_045512.2) in BWA v. 0.7.17. SAM and BAM files were created, sorted and indexed in SAMtools v. 1.10 and marked for duplicates using Picard v. 2.23.4. Variants were called with mpileup in BCFtools v. 1.11. Phylograms were created using Mr. Bayes v 3.2.6. A total of 2,349 single nucleotide polymorphism (SNP) profiles across 294 sites were identified. Clades associated with severe disease in the United States, France, Italy, and Brazil had low frequencies in Africa (L84S=2.5%, L3606F=1.4%, L3606F/V378I/=0.35, G251V=2%). Sub Saharan Africa (SSA) accounted for only 3% of P323L and 4% of Q57H mutations in Africa. Comparatively low infections in SSA were attributed to the low frequency of the D614G clade in earlier samples (25% vs 67% global). Higher disease burden occurred in countries with higher D614G frequencies (Egypt=98%, Morocco=90%, Tunisia=52%, South Africa) with D614G as the first confirmed case. V367F, D364Y, V483A and G476S mutations associated with efficient ACE2 receptor binding and severe disease were not observed in Africa. 95% of all RdRp mutations were deaminations leading to CpG depletion and possible attenuation of virulence. More genomic and experimental studies are needed to increase our understanding of the temporal evolution of the virus in Africa, clarify our findings, and reveal hot spots that may undermine successful therapeutic and vaccine interventions.