Monoclonal antibodies (MAbs) that retain neutralizing activity against distinct coronavirus (CoV) lineages and variants of concern (VoC) must be developed to protect against future pandemics. These broadly neutralizing MAbs (BNMAbs) may be used as therapeutics and/or to assist in the rational design of vaccines that induce BNMAbs. 1249A8 is a BNMAb that targets the stem helix (SH) region of CoV spike (S) protein and neutralizes Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) original strain, delta, and omicron VoC, Severe Acute Respiratory Syndrome CoV (SARS-CoV) and Middle East Respiratory Syndrome CoV (MERS-CoV). To understand its mechanism of action, the crystal structure of 1249A8 bound to a MERS-CoV SH peptide was determined at 2.1Å resolution. BNMAb 1249A8 mimics the SARS-CoV-2 S loop residues 743-749, which interact with the C-terminal end of the SH helix in the S postfusion conformation. The crystal structure shows that BNMAb 1249A8 disrupts SH secondary structure and packing rearrangements required for CoV S to adopt its prefusion conformation that mediates membrane fusion and ultimately infection. The mechanisms regulating BNMAb 1249A8 CoV S specificity are also defined. This study provides novel insights into the neutralization mechanisms of SH-targeting CoV BNMAbs that may inform vaccine development and the design of optimal BNMAb therapeutics.

Monoclonal antibodies that retain neutralizing activity against multiple coronavirus (CoV) lineages and variants of concern (VoC) must be developed to protect against future pandemics. These broadly neutralizing MAbs (BNMAbs) may be used as therapeutics and/or to assist in the rational design of vaccines that induce BNMAbs. 1249A8 is a BNMAb that targets the stem helix (SH) region of CoV spike (S) protein and neutralizes Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2) original strain, delta, and omicron VoC, Severe Acute Respiratory Syndrome CoV (SARS‐CoV), and Middle East Respiratory Syndrome CoV (MERS‐CoV). To understand its mechanism of action, the crystal structure of 1249A8 bound to a MERS‐CoV SH peptide was determined at 2.1 Å resolution. BNMAb 1249A8 mimics the SARS‐CoV‐2 S loop residues 743–749, which interacts with the N‐terminal end of the SH helix in the S post‐fusion conformation. The conformation of 1249A8‐bound SH is distinct from the SH conformation observed in the post‐fusion SARS‐CoV‐2 S structure, suggesting 1249A8 disrupts the secondary structure and refolding events required for CoV post‐fusion S to initiate membrane fusion and ultimately infection. This study provides novel insights into the neutralization mechanisms of SH‐targeting CoV BNMAbs that may inform vaccine development and the design of optimal BNMAb therapeutics.