When SARS-CoV-2 infects cells, the viral S protein binds to human angiotensin converting enzyme 2 (hACE2) and is proteolytically activated by human proteases, furin and TMPRSS2, which respectively cleave the S1/S2 and the S2’ sites of the S protein to cause dissociation the S1 and S2 subunits. In contrast to the frequently mutated S1, the S2 subunit is more conserved among SARS-CoV-2 variants. The BA.1 sublineage of Omicron harbors 28 altered amino acid residues in the S1 subunit and 6 altered amino acid residues in the S2 subunit compared to the original Wuhan strain. The SARS-CoV-2 S protein is composed of S1 and S2 subunits, which adopt a trimeric conformation with three receptor-targeting S1 heads and an S2-trimer stalk on the surface of the virus particle. Notably, all of these Omicron variants carry multiple mutations in the viral spike (S) protein that significantly reduce vaccine efficacies and markedly raise the risk of breakthrough infections. Since its emergence, the initial Omicron variant BA.1 has split into several other sublineages, including BA.2, BA.3, BA.4 and BA.5. The SARS-CoV-2 Omicron variants were first detected in November 2021 and quickly outcompeted other variants, such as Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617), due to their especially high transmissibility and immune evasion. As SARS-CoV-2 continues to evolve through mutations in its viral genomic RNA, viral variants of concern periodically emerge and pose new threats to public health.

Since the end of 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread throughout the world in an immunologically naïve population, causing significant morbidity and mortality. Importantly, our study results demonstrate that the viral S2 subunit can be targeted for the production of cross-reactive antibodies, which may be used for coronavirus detection and neutralization. Since there are not many mAbs that can bind the S2 subunit of SARS-CoV-2 variants, our set of B-S2-mAbs may provide important materials for basic research and potential clinical applications. The neutralizing epitope of B-S2-mAb-2 was identified by phage display as amino acid residues 1146–1152 (DSFKEEL) in the S2 subunit HR2 domain of SARS-CoV-2.

In particular, B-S2-mAb-2 exhibited potent neutralization of Gamma variant (IC 50 = 0.048 µg/ml) in a pseudovirus neutralization assay. Furthermore, we demonstrated the broad reactivities of these B-S2-mAbs in several different applications, including immunosorbent, immunofluorescence and immunoblotting assays. ResultsĪmong these mAbs, five exhibited strong neutralizing ability toward the Gamma variant and also recognized viral S proteins from the Wuhan, Alpha, Beta, Gamma, Delta and Omicron (BA.1, BA.2 and BA.5) variants. These mAbs were assessed for their neutralizing activity with pseudotyped viruses and binding ability for SARS-CoV-2 variants. Using an mRNA-LNP immunization strategy, we generated several mAbs that specifically target the conserved S2 subunit of SARS-CoV-2 (B-S2-mAbs). Therefore, it would be of great value to develop neutralizing monoclonal antibodies (mAbs) that can broadly recognize multiple variants.

The variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) harbor diverse spike (S) protein sequences, which can greatly influence the efficacies of therapeutics.

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