Optimization and deoptimization of codons in SARS-CoV-2 and the implications for vaccine development

The spread of Coronavirus Disease 2019 (COVID-19), caused by the SARS-CoV-2 coronavirus, has progressed into a global pandemic. To date, thousands of genetic variants have been identified across SARS-CoV-2 isolates from patients. Sequence analysis reveals that the codon usage of viral sequences decreased over time but fluctuated from time to time. In this study, through evolution modeling, we found that this phenomenon might result from the virus preference for mutations during transmission. Using dual luciferase assays, we further discovered that the deoptimization of codons on viruses might weaken protein expression during the virus evolution, indicating that the choice of codon usage might play important role in virus fitness. Finally, given the importance of codon usage in protein expression and particularly for mRNA vaccine, we designed several omicron BA.2.12.1 and BA.4/5 spike mRNA vaccine candidates based on codon optimization, and experimentally validated their high levels of expression. Our study highlights the importance of codon usage in virus evolution and mRNA vaccine development.

A Bispecific Antibody Targeting RBD and S2 Potently Neutralizes SARS-CoV-2 Omicron and Other Variants of Concern

Emerging severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) variants, especially the Omicron variant, have impaired the efficacy of existing vaccines and most therapeutic antibodies, highlighting the need for additional antibody-based tools that can efficiently neutralize emerging SARS-CoV-2 variants. The use of a "single" agent to simultaneously target multiple distinct epitopes on the spike is desirable to overcome the neutralizing escape of SARS-CoV-2 variants. Herein, we generated a human-derived IgG-like bispecific antibody (bsAb), Bi-Nab35B5-47D10, which successfully retained the specificity and simultaneously bound to the two distinct epitopes on RBD and S2. Bi-Nab35B5-47D10 showed improved spike binding breadth among wild-type (WT) SARS-CoV-2, variants of concern (VOCs) and variants being monitored (VBMs) compared with its parental mAbs. Furthermore, pseudotyped virus neutralization demonstrated that Bi-Nab35B5-47D10 can efficiently neutralize VBMs including Alpha (B.1.1.7), Beta (B.1.351) and Kappa (B.1.617.1) and VOCs including Delta (B.1.617.2), Omicron BA.1 and Omicron BA.2. Crucially, Bi-Nab35B5-47D10 substantially improved neutralizing activity against Omicron BA.1 (IC50= 27.3 ng/mL) and Omicron BA.2 (IC50= 121.1 ng/mL) compared with their parental mAbs. Therefore, Bi-Nab35B5-47D10 represents a potential effective countermeasure against SARS-CoV-2 Omicron and other variants of concern. ImportanceThe new highly contagious SARS-CoV-2 Omicron variant caused substantial breakthrough infections and has become the dominant strain in countries across the world. Omicron variants usually bear high mutations in the spike protein and exhibit considerable escape of most potent neutralization monoclonal antibodies and reduced efficacy of current COVID-19 vaccines. The development of neutralizing antibodies with potent efficacy against the Omicron variant is still an urgent priority. Here, we generated a bsAb, Bi-Nab35B5-47D10, that simultaneously targets SARS-CoV-2 RBD and S2 and improved neutralizing potency and breadth against SARS-CoV-2 WT and the tested variants compared with their parental antibodies. Notably, Bi-Nab35B5-47D10 has more potent neutralizing activity against the VOC Omicron pseudotyped virus. Therefore, Bi-Nab35B5-47D10 is a feasible and potentially effective strategy to treat and prevent COVID-19.

The Rhinolophus affinis bat ACE2 and multiple animal orthologs are functional receptors for bat coronavirus RaTG13 and SARS-CoV-2

Bat coronavirus (CoV) RaTG13 shares the highest genome sequence identity with SARS-CoV-2 among all known coronaviruses, and also uses human angiotensin converting enzyme 2 (hACE2) for virus entry. Thus, SARS-CoV-2 is thought to have originated from bat. However, whether SARS-CoV-2 emerged from bats directly or through an intermediate host remains elusive. Here, we found that Rhinolophus affinis bat ACE2 (RaACE2) is an entry receptor for both SARS-CoV-2 and RaTG13, although RaACE2 binding to the receptor binding domain (RBD) of SARS-CoV-2 is markedly weaker than that of hACE2. We further evaluated the receptor activities of ACE2s from additional 16 diverse animal species for RaTG13, SARS-CoV, and SARS-CoV-2 in terms of S protein binding, membrane fusion, and pseudovirus entry. We found that the RaTG13 spike (S) protein is significantly less fusogenic than SARS-CoV and SARS-CoV-2, and seven out of sixteen different ACE2s function as entry receptors for all three viruses, indicating that all three viruses might have broad host rages. Of note, RaTG13 S pseudovirions can use mouse, but not pangolin ACE2, for virus entry, whereas SARS-CoV-2 S pseudovirions can use pangolin, but limited for mouse, ACE2s enter cells. Mutagenesis analysis revealed that residues 484 and 498 in RaTG13 and SARS-CoV-2 S proteins play critical roles in recognition of mouse and human ACE2. Finally, two polymorphous Rhinolophous sinicus bat ACE2s showed different susceptibilities to virus entry by RaTG13 and SARS-CoV-2 S pseudovirions, suggesting possible coevolution. Our results offer better understanding of the mechanism of coronavirus entry, host range, and virus-host coevolution.

Human monoclonal antibodies block the binding of SARS-CoV-2 spike protein to angiotensin converting enzyme 2 receptor

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of novel corona virus disease (COVID-19). To date, no prophylactic vaccines or approved therapeutic agents are available for preventing and treating this highly transmittable disease. Here we report two monoclonal antibodies (mAbs) cloned from memory B cells of patients recently recovered from COVID-19, and both mAbs specifically bind to the spike (S) protein of SARS-CoV-2, block the binding of receptor binding domain (RBD) of SARS-CoV-2 to human angiotensin converting enzyme 2 (hACE2), and effectively neutralize S protein-pseudotyped virus infection. These human mAbs hold the promise for the prevention and treatment of the ongoing pandemic of COVID-19.