Spike substitution T813S increases Sarbecovirus fusogenicity by enhancing the usage of TMPRSS2.

SARS-CoV Spike (S) protein shares considerable homology with SARS-CoV-2 S, especially in the conserved S2 subunit (S2). S protein mediates coronavirus receptor binding and membrane fusion, and the latter activity can greatly influence coronavirus infection. We observed that SARS-CoV S is less effective in inducing membrane fusion compared with SARS-CoV-2 S. We identify that S813T mutation is sufficient in S2 interfering with the cleavage of SARS-CoV-2 S by TMPRSS2, reducing spike fusogenicity and pseudoparticle entry. Conversely, the mutation of T813S in SARS-CoV S increased fusion ability and viral replication. Our data suggested that residue 813 in the S was critical for the proteolytic activation, and the change from threonine to serine at 813 position might be an evolutionary feature adopted by SARS-2-related viruses. This finding deepened the understanding of Spike fusogenicity and could provide a new perspective for exploring Sarbecovirus' evolution.

A triple-RBD-based mucosal vaccine provides broad protection against SARS-CoV-2 variants of concern.

The rapid mutation and spread of SARS-CoV-2 variants urge the development of effective mucosal vaccines to provide broad-spectrum protection against the initial infection and thereby curb the transmission potential. Here, we designed a chimeric triple-RBD immunogen, 3Ro-NC, harboring one Delta RBD and two Omicron RBDs within a novel protein scaffold. 3Ro-NC elicits potent and broad RBD-specific neutralizing immunity against SARS-CoV-2 variants of concern. Notably, intranasal immunization with 3Ro-NC plus the mucosal adjuvant KFD (3Ro-NC + KFDi.n) elicits coordinated mucosal IgA and higher neutralizing antibody specificity (closer antigenic distance) against the Omicron variant. In Omicron-challenged human ACE2 transgenic mice, 3Ro-NC + KFDi.n immunization significantly reduces the tissue pathology in the lung and lowers the viral RNA copy numbers in both the lung (85.7-fold) and the nasal turbinate (13.6-fold). Nasal virologic control is highly correlated with RBD-specific secretory IgA antibodies. Our data show that 3Ro-NC plus KFD is a promising mucosal vaccine candidate for protection against SARS-CoV-2 Omicron infection, pathology and transmission potential.

Comparative global B cell receptor repertoire difference induced by SARS-CoV-2 infection or vaccination via single-cell V(D)J sequencing.

Dynamic changes of the paired heavy and light chain B cell receptor (BCR) repertoire provide an essential insight into understanding the humoral immune response post-SARS-CoV-2 infection and vaccination. However, differences between the endogenous paired BCR repertoire kinetics in SARS-CoV-2 infection and previously recovered/naïve subjects treated with the inactivated vaccine remain largely unknown. We performed single-cell V(D)J sequencing of B cells from six healthy donors with three shots of inactivated SARS-CoV-2 vaccine (BBIBP-CorV), five people who received the BBIBP-CorV vaccine after having recovered from COVID-19, five unvaccinated COVID-19 recovered patients and then integrated with public data of B cells from four SARS-CoV-2-infected subjects. We discovered that BCR variable (V) genes were more prominently used in the SARS-CoV-2 exposed groups (both in the group with active infection and in the group that had recovered) than in the vaccinated groups. The VH gene that expanded the most after SARS-CoV-2 infection was IGHV3-33, while IGHV3-23 in the vaccinated groups. SARS-CoV-2-infected group enhanced more BCR clonal expansion and somatic hypermutation than the vaccinated healthy group. A small proportion of public clonotypes were shared between the SARS-CoV-2 infected, vaccinated healthy, and recovered groups. Moreover, several public antibodies had been identified against SARS-CoV-2 spike protein. We comprehensively characterize the paired heavy and light chain BCR repertoire from SARS-CoV-2 infection to vaccination, providing further guidance for the development of the next-generation precision vaccine.

Rapid isolation and immune profiling of SARS-CoV-2 specific memory B cell in convalescent COVID-19 patients via LIBRA-seq.

B cell response plays a critical role against SARS-CoV-2 infection. However, little is known about the diversity and frequency of the paired SARS-CoV-2 antigen-specific BCR repertoire after SARS-CoV-2 infection. Here, we performed single-cell RNA sequencing and VDJ sequencing using the memory and plasma B cells isolated from five convalescent COVID-19 patients, and analyzed the spectrum and transcriptional heterogeneity of antibody immune responses. Via linking BCR to antigen specificity through sequencing (LIBRA-seq), we identified a distinct activated memory B cell subgroup (CD11chigh CD95high) had a higher proportion of SARS-CoV-2 antigen-labeled cells compared with memory B cells. Our results revealed the diversity of paired BCR repertoire and the non-stochastic pairing of SARS-CoV-2 antigen-specific immunoglobulin heavy and light chains after SARS-CoV-2 infection. The public antibody clonotypes were shared by distinct convalescent individuals. Moreover, several antibodies isolated by LIBRA-seq showed high binding affinity against SARS-CoV-2 receptor-binding domain (RBD) or nucleoprotein (NP) via ELISA assay. Two RBD-reactive antibodies C14646P3S and C2767P3S isolated by LIBRA-seq exhibited high neutralizing activities against both pseudotyped and authentic SARS-CoV-2 viruses in vitro. Our study provides fundamental insights into B cell response following SARS-CoV-2 infection at the single-cell level.