Neutralizing Potency of Prototype and Omicron RBD mRNA Vaccines Against Omicron Variant.

The newly emerged Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains more than 30 mutations on the spike protein, 15 of which are located within the receptor binding domain (RBD). Consequently, Omicron is able to extensively escape existing neutralizing antibodies and may therefore compromise the efficacy of current vaccines based on the original strain, highlighting the importance and urgency of developing effective vaccines against Omicron. Here we report the rapid generation and evaluation of an mRNA vaccine candidate specific to Omicron, and explore the feasibility of heterologous immunization with WT and Omicron RBD vaccines. This mRNA vaccine encodes the RBD of Omicron (designated as RBD-O) and is formulated with lipid nanoparticle. Two doses of the RBD-O mRNA vaccine efficiently induce neutralizing antibodies in mice; however, the antisera are effective only on the Omicron variant but not on the wildtype and Delta strains, indicating a narrow neutralization spectrum. It is noted that the neutralization profile of the RBD-O mRNA vaccine is opposite to that observed for the mRNA vaccine expressing the wildtype RBD (RBD-WT). Importantly, booster with RBD-O mRNA vaccine after two doses of RBD-WT mRNA vaccine can significantly increase neutralization titers against Omicron. Additionally, an obvious increase in IFN-γ, IL-2, and TNF-α-expressing RBD-specific CD4+ T cell responses was observed after immunization with the RBD-WT and/or RBD-O mRNA vaccine. Together, our work demonstrates the feasibility and potency of an RBD-based mRNA vaccine specific to Omicron, providing important information for further development of heterologous immunization program or bivalent/multivalent SARS-CoV-2 vaccines with broad-spectrum efficacy.

Mapping cross-variant neutralizing sites on the SARS-CoV-2 spike protein.

The emergence of multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern threatens the efficacy of currently approved vaccines and authorized therapeutic monoclonal antibodies (MAbs). It is hence important to continue searching for SARS-CoV-2 broadly neutralizing MAbs and defining their epitopes. Here, we isolate 9 neutralizing mouse MAbs raised against the spike protein of a SARS-CoV-2 prototype strain and evaluate their neutralizing potency towards a panel of variants, including B.1.1.7, B.1.351, B.1.617.1, and B.1.617.2. By using a combination of biochemical, virological, and cryo-EM structural analyses, we identify three types of cross-variant neutralizing MAbs, represented by S5D2, S5G2, and S3H3, respectively, and further define their epitopes. S5D2 binds the top lateral edge of the receptor-binding motif within the receptor-binding domain (RBD) with a binding footprint centred around the loop477-489, and efficiently neutralizes all variant pseudoviruses, but the potency against B.1.617.2 was observed to decrease significantly. S5G2 targets the highly conserved RBD core region and exhibits comparable neutralization towards the variant panel. S3H3 binds a previously unreported epitope located within the evolutionarily stable SD1 region and is able to near equally neutralize all of the variants tested. Our work thus defines three distinct cross-variant neutralizing sites on the SARS-CoV-2 spike protein, providing guidance for design and development of broadly effective vaccines and MAb-based therapies.

Yeast-produced RBD-based recombinant protein vaccines elicit broadly neutralizing antibodies and durable protective immunity against SARS-CoV-2 infection.

Massive production of efficacious SARS-CoV-2 vaccines is essential for controlling the ongoing COVID-19 pandemic. We report here the preclinical development of yeast-produced receptor-binding domain (RBD)-based recombinant protein SARS-CoV-2 vaccines. We found that monomeric RBD of SARS-CoV-2 could be efficiently produced as a secreted protein from transformed Pichia pastoris (P. pastoris) yeast. Yeast-derived RBD-monomer possessed functional conformation and was able to elicit protective level of neutralizing antibodies in mice. We further designed and expressed a genetically linked dimeric RBD protein in yeast. The engineered dimeric RBD was more potent than the monomeric RBD in inducing long-lasting neutralizing antibodies. Mice immunized with either monomeric RBD or dimeric RBD were effectively protected from live SARS-CoV-2 virus challenge even at 18 weeks after the last vaccine dose. Importantly, we found that the antisera raised against the RBD of a single SARS-CoV-2 prototype strain could effectively neutralize the two predominant circulating variants B.1.1.7 and B.1.351, implying broad-spectrum protective potential of the RBD-based vaccines. Our data demonstrate that yeast-derived RBD-based recombinant SARS-CoV-2 vaccines are feasible and efficacious, opening up a new avenue for rapid and cost-effective production of SARS-CoV-2 vaccines to achieve global immunization.

Elicitation of Broadly Neutralizing Antibodies against B.1.1.7, B.1.351, and B.1.617.1 SARS-CoV-2 Variants by Three Prototype Strain-Derived Recombinant Protein Vaccines.

The ongoing coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Most of the currently approved SARS-CoV-2 vaccines use the prototype strain-derived spike (S) protein or its receptor-binding domain (RBD) as the vaccine antigen. The emergence of several novel SARS-CoV-2 variants has raised concerns about potential immune escape. In this study, we performed an immunogenicity comparison of prototype strain-derived RBD, S1, and S ectodomain trimer (S-trimer) antigens and evaluated their induction of neutralizing antibodies against three circulating SARS-CoV-2 variants, including B.1.1.7, B.1.351, and B.1.617.1. We found that, at the same antigen dose, the RBD and S-trimer vaccines were more potent than the S1 vaccine in eliciting long-lasting, high-titer broadly neutralizing antibodies in mice. The RBD immune sera remained highly effective against the B.1.1.7, B.1.351, and B.1.617.1 variants despite the corresponding neutralizing titers decreasing by 1.2-, 2.8-, and 3.5-fold relative to that against the wild-type strain. Significantly, the S-trimer immune sera exhibited comparable neutralization potency (less than twofold variation in neutralizing GMTs) towards the prototype strain and all three variants tested. These findings provide valuable information for further development of recombinant protein-based SARS-CoV-2 vaccines and support the continued use of currently approved SARS-CoV-2 vaccines in the regions/countries where variant viruses circulate.

Development and structural basis of a two-MAb cocktail for treating SARS-CoV-2 infections.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Neutralizing antibodies against SARS-CoV-2 are an option for drug development for treating COVID-19. Here, we report the identification and characterization of two groups of mouse neutralizing monoclonal antibodies (MAbs) targeting the receptor-binding domain (RBD) on the SARS-CoV-2 spike (S) protein. MAbs 2H2 and 3C1, representing the two antibody groups, respectively, bind distinct epitopes and are compatible in formulating a noncompeting antibody cocktail. A humanized version of the 2H2/3C1 cocktail is found to potently neutralize authentic SARS-CoV-2 infection in vitro with half inhibitory concentration (IC50) of 12 ng/mL and effectively treat SARS-CoV-2-infected mice even when administered at as late as 24 h post-infection. We determine an ensemble of cryo-EM structures of 2H2 or 3C1 Fab in complex with the S trimer up to 3.8 Å resolution, revealing the conformational space of the antigen-antibody complexes and MAb-triggered stepwise allosteric rearrangements of the S trimer, delineating a previously uncharacterized dynamic process of coordinated binding of neutralizing antibodies to the trimeric S protein. Our findings provide important information for the development of MAb-based drugs for preventing and treating SARS-CoV-2 infections.

H7N9 virulent mutants detected in chickens in China pose an increased threat to humans.

Certain low pathogenic avian influenza viruses can mutate to highly pathogenic viruses when they circulate in domestic poultry, at which point they can cause devastating poultry diseases and severe economic damage. The H7N9 influenza viruses that emerged in 2013 in China had caused severe human infections and deaths. However, these viruses were nonlethal in poultry. It is unknown whether the H7N9 viruses can acquire additional mutations during their circulation in nature and become lethal to poultry and more dangerous for humans. Here, we evaluated the evolution of H7N9 viruses isolated from avian species between 2013 and 2017 in China and found 23 different genotypes, 7 of which were detected only in ducks and were genetically distinct from the other 16 genotypes that evolved from the 2013 H7N9 viruses. Importantly, some H7N9 viruses obtained an insertion of four amino acids in their hemagglutinin (HA) cleavage site and were lethal in chickens. The index strain was not lethal in mice or ferrets, but readily obtained the 627K or 701N mutation in its PB2 segment upon replication in ferrets, causing it to become highly lethal in mice and ferrets and to be transmitted efficiently in ferrets by respiratory droplet. H7N9 viruses bearing the HA insertion and PB2 627K mutation have been detected in humans in China. Our study indicates that the new H7N9 mutants are lethal to chickens and pose an increased threat to human health, and thus highlights the need to control and eradicate the H7N9 viruses to prevent a possible pandemic.