Discovery and Characterization of a Pan-betacoronavirus S2-binding antibody (preprint)

SUMMARY/ABSTRACTThree coronaviruses have spilled over from animal reservoirs into the human population and caused deadly epidemics or pandemics. The continued emergence of coronaviruses highlights the need for pan-coronavirus interventions for effective pandemic preparedness. Here, using LIBRA-seq, we report a panel of 50 coronavirus antibodies isolated from human B cells. Of these antibodies, 54043-5 was shown to bind the S2 subunit of spike proteins from alpha-, beta-, and deltacoronaviruses. A cryo-EM structure of 54043-5 bound to the pre-fusion S2 subunit of the SARS-CoV-2 spike defined an epitope at the apex of S2 that is highly conserved among betacoronaviruses. Although non-neutralizing, 54043-5 induced Fc-dependent antiviral responses, including ADCC and ADCP. In murine SARS-CoV-2 challenge studies, protection against disease was observed after introduction of Leu234Ala, Leu235Ala, and Pro329Gly (LALA-PG) substitutions in the Fc region of 54043-5. Together, these data provide new insights into the protective mechanisms of non-neutralizing antibodies and define a broadly conserved epitope within the S2 subunit.

LGBTQ2S+ Youth Perspectives on Mental Healthcare Provider Bias, Standards of Care, and Accountability

This study explores the experiences of LGBTQ2S+ youth while accessing mental health and substance use care services during the COVID-19 pandemic. Through a series of facilitated virtual meetings, 33 LGBTQ2S+ youth from across Ontario participated in collaborative activities to identify barriers they have experienced when accessing mental health services, as well as potential solutions to these barriers. Discussions were recorded, transcribed, and analyzed using thematic analysis. The study revealed that LGBTQ2S+ youth disproportionately experience bias, discrimination, and heteronormative assumptions when accessing mental health services, resulting in negative care experiences. Youth also reported insufficient availability of quality care, little continuity in care, and a lack of educated providers capable of effectively addressing the needs of the community. Potential solutions proposed by youth include training resources for providers, LGBTQ2S+ specific care centers, better continuity of care, and assessments to ensure care providers are culturally competent. These results show the COVID-19 pandemic has exacerbated the disparities LGBTQ2S+ youth experience when accessing mental health services and highlight the urgent need to implement policies and programs that will advance the standards of care for LGBTQ2S+ youth.

Genetic and structural basis for recognition of SARS-CoV-2 spike protein by a two-antibody cocktail (preprint)

The SARS-CoV-2 pandemic has led to an urgent need to understand the molecular basis for immune recognition of SARS-CoV-2 spike (S) glycoprotein antigenic sites. To define the genetic and structural basis for SARS-CoV-2 neutralization, we determined the structures of two human monoclonal antibodies COV2-2196 and COV2-21301, which form the basis of the investigational antibody cocktail AZD7442, in complex with the receptor binding domain (RBD) of SARS-CoV-2. COV2-2196 forms an 'aromatic cage' at the heavy/light chain interface using germline-encoded residues in complementarity determining regions (CDRs) 2 and 3 of the heavy chain and CDRs 1 and 3 of the light chain. These structural features explain why highly similar antibodies (public clonotypes) have been isolated from multiple individuals1-4. The structure of COV2-2130 reveals that an unusually long LCDR1 and HCDR3 make interactions with the opposite face of the RBD from that of COV2-2196. Using deep mutational scanning and neutralization escape selection experiments, we comprehensively mapped the critical residues of both antibodies and identified positions of concern for possible viral escape. Nonetheless, both COV2-2196 and COV2130 showed strong neutralizing activity against SARS-CoV-2 strain with recent variations of concern including E484K, N501Y, and D614G substitutions. These studies reveal germline-encoded antibody features enabling recognition of the RBD and demonstrate the activity of a cocktail like AZD7442 in preventing escape from emerging variant viruses.

Evaluation of the effects of SARS-CoV-2 genetic mutations on diagnostic RT-PCR assays (preprint)

Several mutant strains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are emerging. Mismatch(es) in primer/probe binding regions would decrease the detection sensitivity of the PCR test, thereby affecting the results of clinical testing. In this study, we conducted an in silico survey on SARS-CoV-2 sequence variability within the binding regions of primer/probe published by the Japan National Institute of Infectious Diseases (NIID) and Centers for Disease Control and Prevention (CDC). In silico analysis revealed the presence of mutations in the primer/probe binding regions. We performed RT-PCR assays using synthetic RNAs containing the mutations and showed that some mutations significantly decreased the detection sensitivity of the RT-PCR assays. Our results highlight the importance of genomic monitoring of SARS-CoV-2 and evaluating the effects of mismatches on PCR testing sensitivity.

A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering (preprint)

A universal vaccine design platform that can rapidly generate multiplex vaccine candidates is critically needed to control future pandemics. Here, using SARS-CoV-2 pandemic virus as a model, we have developed such a platform by CRISPR engineering of bacteriophage T4. A pipeline of vaccine candidates were engineered by incorporating various viral components into appropriate compartments of phage nanoparticle structure. These include: expressible spike genes in genome, spike and envelope epitopes as surface decorations, and nucleocapsid proteins in packaged core. Phage decorated with spike trimers is found to be the most potent vaccine candidate in mouse and rabbit models. Without any adjuvant, this vaccine stimulated robust immune responses, both TH1 and TH2 IgG subclasses, blocked virus-receptor interactions, neutralized viral infection, and conferred complete protection against viral challenge. This new type of nanovaccine design framework might allow rapid deployment of effective phage-based vaccines against any emerging pathogen in the future.

Neutralizing and protective human monoclonal antibodies recognizing the N-terminaldomain of the SARS-CoV-2 spike protein (preprint)

Most human monoclonal antibodies (mAbs) neutralizing SARS-CoV-2 recognize the spike (S) protein receptor-binding domain and block virus interactions with the cellular receptor angiotensin-converting enzyme 2. We describe a panel of human mAbs binding to diverse epitopes on the N-terminal domain (NTD) of S protein from SARS-CoV-2 convalescent donors and found a minority of these possessed neutralizing activity. Two mAbs (COV2-2676 and COV2-2489) inhibited infection of authentic SARS-CoV-2 and recombinant VSV/SARS-CoV-2 viruses. We mapped their binding epitopes by alanine-scanning mutagenesis and selection of functional SARS-CoV-2 S neutralization escape variants. Mechanistic studies showed that these antibodies neutralize in part by inhibiting a post-attachment step in the infection cycle. COV2-2676 and COV2-2489 offered protection either as prophylaxis or therapy, and Fc effector functions were required for optimal protection. Thus, natural infection induces a subset of potent NTD-specific mAbs that leverage neutralizing and Fc-mediated activities to protect against SARS-CoV-2 infection using multiple functional attributes.

Cross-reactive coronavirus antibodies with diverse epitope specificities and extra-neutralization functions (preprint)

ABSTRACT The continual emergence of novel coronavirus (CoV) strains, like SARS-CoV-2, highlights the critical need for broadly reactive therapeutics and vaccines against this family of viruses. Coronavirus spike (S) proteins share common structural motifs that could be vulnerable to cross-reactive antibody responses. To study this phenomenon in human coronavirus infection, we applied a high-throughput sequencing method called LIBRA-seq (Linking B cell receptor to antigen specificity through sequencing) to a SARS-CoV-1 convalescent donor sample. We identified and characterized a panel of six monoclonal antibodies that cross-reacted with S proteins from the highly pathogenic SARS-CoV-1 and SARS-CoV-2 and demonstrated a spectrum of reactivity against other coronaviruses. Epitope mapping revealed that these antibodies recognized multiple epitopes on SARS-CoV-2 S, including the receptor binding domain (RBD), N-terminal domain (NTD), and S2 subunit. Functional characterization demonstrated that the antibodies mediated a variety of Fc effector functions in vitro and mitigated pathological burden in vivo . The identification of cross-reactive epitopes recognized by functional antibodies expands the repertoire of targets for pan-coronavirus vaccine design strategies that may be useful for preventing potential future coronavirus outbreaks.