Identification and differential usage of a host metalloproteinase entry pathway by SARS-CoV-2 Delta and Omicron.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike glycoprotein (S) binds to angiotensin-converting enzyme 2 (ACE2) to mediate membrane fusion via two distinct pathways: 1) a surface, serine protease-dependent or 2) an endosomal, cysteine protease-dependent pathway. In this study, we found that SARS-CoV-2 S has a wider protease usage and can also be activated by TMPRSS13 and matrix metalloproteinases (MMPs). We found that MMP-2 and MMP-9 played roles in SARS-CoV-2 S cell-cell fusion and TMPRSS2- and cathepsin-independent viral entry in cells expressing high MMP levels. MMP-dependent viral entry required cleavage at the S1/S2 junction in viral producer cells, and differential processing of variants of concern S dictated its usage; the efficiently processed Delta S preferred metalloproteinase-dependent entry when available, and less processed Omicron S was unable to us metalloproteinases for entry. As MMP-2/9 are released during inflammation, they may play roles in S-mediated cytopathic effects, tropism, and disease outcome.

Arsenal of nanobodies shows broad-spectrum neutralization against SARS-CoV-2 variants of concern in vitro and in vivo in hamster models.

Nanobodies offer several potential advantages over mAbs for the control of SARS-CoV-2. Their ability to access cryptic epitopes conserved across SARS-CoV-2 variants of concern (VoCs) and feasibility to engineer modular, multimeric designs, make these antibody fragments ideal candidates for developing broad-spectrum therapeutics against current and continually emerging SARS-CoV-2 VoCs. Here we describe a diverse collection of 37 anti-SARS-CoV-2 spike glycoprotein nanobodies extensively characterized as both monovalent and IgG Fc-fused bivalent modalities. The nanobodies were collectively shown to have high intrinsic affinity; high thermal, thermodynamic and aerosolization stability; broad subunit/domain specificity and cross-reactivity across existing VoCs; wide-ranging epitopic and mechanistic diversity and high and broad in vitro neutralization potencies. A select set of Fc-fused nanobodies showed high neutralization efficacies in hamster models of SARS-CoV-2 infection, reducing viral burden by up to six orders of magnitude to below detectable levels. In vivo protection was demonstrated with anti-RBD and previously unreported anti-NTD and anti-S2 nanobodies. This collection of nanobodies provides a potential therapeutic toolbox from which various cocktails or multi-paratopic formats could be built to combat multiple SARS-CoV-2 variants.

Relative Ratios of Human Seasonal Coronavirus Antibodies Predict the Efficiency of Cross-Neutralization of SARS-CoV-2 Spike Binding to ACE2.

BACKGROUND: Antibodies raised against human seasonal coronaviruses (sCoVs), which are responsible for the common cold, are known to cross-react with SARS-CoV-2 antigens. This prompts questions about their protective role against SARS-CoV-2 infections and COVID-19 severity. However, the relationship between sCoVs exposure and SARS-CoV-2 correlates of protection are not clearly identified. METHODS: We performed a cross-sectional analysis of cross-reactivity and cross-neutralization to SARS-CoV-2 antigens (S-RBD, S-trimer, N) using pre-pandemic sera from four different groups: pediatrics and adolescents, individuals 21 to 70 years of age, older than 70 years of age, and individuals living with HCV or HIV. Data was then further analysed using machine learning to identify predictive patterns of neutralization based on sCoVs serology. FINDINGS: Antibody cross-reactivity to SARS-CoV-2 antigens varied between 1.6% and 15.3% depending on the cohort and the isotype-antigen pair analyzed. We also show a range of neutralizing activity (0-45%) with median inhibition ranging from 17.6 % to 23.3 % in serum that interferes with SARS-CoV-2 spike attachment to ACE2 independently of age group. While the abundance of sCoV antibodies did not directly correlate with neutralization, we show that neutralizing activity is rather dependent on relative ratios of IgGs in sera directed to all four sCoV spike proteins. More specifically, we identified antibodies to NL63 and OC43 as being the most important predictors of neutralization. INTERPRETATION: Our data support the concept that exposure to sCoVs triggers antibody responses that influence the efficiency of SARS-CoV-2 spike binding to ACE2, which may potentially impact COVID-19 disease severity through other latent variables. FUNDING: This study was supported by a grant by the CIHR (VR2 -172722) and by a grant supplement by the CITF, and by a NRC Collaborative R&D Initiative Grant (PR031-1).

Identification of a High-Frequency Intrahost SARS-CoV-2 Spike Variant with Enhanced Cytopathic and Fusogenic Effects.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a virus that is continuously evolving. Although its RNA-dependent RNA polymerase exhibits some exonuclease proofreading activity, viral sequence diversity can be produced by replication errors and host factors. A diversity of genetic variants can be observed in the intrahost viral population structure of infected individuals. Most mutations will follow a neutral molecular evolution and will not make significant contributions to variations within and between infected hosts. Herein, we profiled the intrasample genetic diversity of SARS-CoV-2 variants, also known as quasispecies, using high-throughput sequencing data sets from 15,289 infected individuals and infected cell lines. Despite high mutational background, we identified recurrent intragenetic variable positions in the samples analyzed, including several positions at the end of the gene encoding the viral spike (S) protein. Strikingly, we observed a high frequency of C→A missense mutations resulting in the S protein lacking the last 20 amino acids (SΔ20). We found that this truncated S protein undergoes increased processing and increased syncytium formation, presumably due to escaping M protein retention in intracellular compartments. Our findings suggest the emergence of a high-frequency viral sublineage that is not horizontally transmitted but potentially involved in intrahost disease cytopathic effects. IMPORTANCE The mutation rate and evolution of RNA viruses correlate with viral adaptation. While most mutations do not make significant contributions to viral molecular evolution, some are naturally selected and produce variants through positive selection. Many SARS-CoV-2 variants have been recently described and show phenotypic selection toward more infectious viruses. Our study describes another type of variant that does not contribute to interhost heterogeneity but rather phenotypic selection toward variants that might have increased cytopathic effects. We identified that a C-terminal truncation of the spike protein removes an important endoplasmic reticulum (ER) retention signal, which consequently results in a spike variant that easily travels through the Golgi complex toward the plasma membrane in a preactivated conformation, leading to increased syncytium formation.