key: cord-0318301-dyp97u2m authors: Hurlburt, Nicholas K.; Homad, Leah J.; Sinha, Irika; Jennewein, Madeleine F.; MacCamy, Anna J.; Wan, Yu-Hsin; Boonyaratanakornkit, Jim; Sholukh, Anton M.; Zhou, Panpan; Burton, Dennis R.; Andrabi, Raiees; Stamatatos, Leonidas; Pancera, Marie; McGuire, Andrew T. title: Structural definition of a pan-sarbecovirus neutralizing epitope on the spike S2 subunit date: 2021-08-03 journal: bioRxiv DOI: 10.1101/2021.08.02.454829 sha: 80d55338de018fd8ac758cdf65159d6225f7a7d4 doc_id: 318301 cord_uid: dyp97u2m Three highly pathogenic betacoronaviruses have crossed the species barrier and established human-to-human transmission causing significant morbidity and mortality in the past 20 years. The most current and widespread of these is SARS-CoV-2. The identification of CoVs with zoonotic potential in animal reservoirs suggests that additional outbreaks are likely to occur. Evidence suggests that neutralizing antibodies are important for protection against infection with CoVs. Monoclonal antibodies targeting conserved neutralizing epitopes on diverse CoVs can form the basis for prophylaxis and therapeutic treatments and enable the design of vaccines aimed at providing pan-coronavirus protection. To this end, we previously identified a neutralizing monoclonal antibody, CV3-25 that binds to the SARS-CoV-2 fusion machinery, neutralizes the SARS-CoV-2 Beta variant comparably to the ancestral Wuhan Hu-1 strain, cross neutralizes SARS-CoV-1 and displays cross reactive binding to recombinant proteins derived from the spike-ectodomains of HCoV-OC43 and HCoV-HKU1. Here, we show that the neutralizing activity of CV3-25 is also maintained against the Alpha, Delta and Gamma variants of concern as well as a SARS-CoV-like bat coronavirus with zoonotic potential by binding to a conserved linear peptide in the stem-helix region on sarbecovirus spikes. A 1.74Å crystal structure of a CV3-25/peptide complex demonstrates that CV3-25 binds to the base of the stem helix at the HR2 boundary to an epitope that is distinct from other stem-helix directed neutralizing mAbs. Thus, CV3-25 defines a novel site of sarbecovirus vulnerability that will inform pan-CoV vaccine development. Coronaviruses (CoVs) are a large family of viruses that infect many species of 45 birds and mammals, including humans. They are subdivided into four genera; alpha, 46 beta, gamma and delta. Two alpha CoVs, NL63 and 229E, and two beta CoVs (OC43 infected ~185 million people and caused over 4 million deaths (Dong et al., 2020) . 62 SARS-CoV-2 and SARS-CoV-1 are members of the sarbecovirus subgenus and share 63 ~80% amino acid sequence identity . SARS-CoV-2 is highly similar to 64 a bat CoV, RaTG13 (Zhou et al., 2020) . Several other SARS-like bat coronaviruses 134 We previously reported that CV3-25 neutralizes the Wuhan-Hu-1 and Beta (B.1.351) 135 variants of SARS-CoV-2 with comparable potency (Jennewein et al., 2021) . Here we to an epitope on S2 that is unaffected by mutations found in these variants. 148 CV3-25 binds to a linear epitope on the SARS-CoV-2 stem helix. 149 A handful of S2-directed neutralizing mAbs that display varying degrees of CoV CoV-2 peptides or to a control peptide from HIV-1 Env. CV3-25 binding was specific, as 172 CV2-10, an S2 directed mAb that does not compete with CV3-25 binding (Jennewein et 173 al., 2021), did not bind either peptide. To confirm binding to this peptide region, we 174 synthesized a peptide spanning 1145-1167 and verified that CV3-25 bound to the 175 peptide with ~5nM affinity using biolayer interferometry (BLI). The measured affinity of 176 CV3-25 for the peptide is lower than it is for a recombinant stabilized spike protein (~0.6 177 nM) ( Fig. 2F and Table S2 ). We note that the association rate of binding to the peptide Table 1 ). The 186 structure showed that binding to this peptide is almost entirely heavy chain dependent 187 ( Fig. 3A and B). The N-terminal end of the peptide forms an α-helix that is engaged by 188 the CDRH1 and CDRH2. The CDRH3 extends under the base of the α-helix directing 189 the extended C-terminal portion of the peptide up into the CDRH1 before turning 190 downward to interact with the light chain. The Fab binds the peptide with a total buried 191 surface area (BSA) of ~594 Å 2 , of which ~516 Å 2 is from the heavy chain and ~78 Å 2 192 from the light chain (Fig. 3C ). Alanine scanning of a stem helix peptide was conducted to assess the relative 194 contributions of the interactions observed in the crystal structure (Fig. 3D ). This analysis 195 revealed that mutating Lys1157, any of the residues in 1160TSPDV1164, or Leu1166 inhibited 196 or greatly reduced binding (Fig. 3D ). This data agrees well with the structural data. Lys1157 buries ~133 Å 2 upon binding, the highest amount of BSA on the peptide, and 198 forms hydrogen bonds with two Asp residues in the CDRH2 (Fig. 3E ). 1160TSPDV1164 is 199 the segment of peptide just after the helix that interacts with CDRH3 before curving up Aligning the CV3-25-stem helix structure to this model shows good agreement (Fig. 4C ). clashing with the heavy chain (Fig. 4C) . Additionally, the alignment of the model also 245 suggests that the light chain of CV3-25 could potentially bind to the region just 246 downstream of the stem helix at the start of HR2, 1168DISGINASVVN1178 (Fig. 4D) , a 247 region that shows some sequence conservation amongst coronaviruses (Fig. 4E ). In contrast, the stem-helix directed mAbs B6 and CC40.8 showed differential 271 binding to these peptides. B6 bound most strongly to the peptide from MERS-CoV, that the lack of neutralization was not due to steric shielding of the epitope from full 289 length IgG (Fig. 5L ). CV3-25 was also unable to neutralize authentic HCoV-NL63, an 290 alpha CoV (Fig. S5 ). We conclude that although the CV3-25 epitope is present, it is not 291 equally accessible in the native conformation of the spike protein among the various 292 beta CoVs examined here. Somatic mutation leads to stronger cross-reactive binding by CV3-25 294 To assess the role of somatic mutation in CV3-25 cross-reactivity we measured 295 the binding of iGL-CV3-25 to the same linear peptides from SARS-CoV-2, MERS-CoV, 296 HCoV-OC43 and HCoV-HKU1 by ELISA (Fig. 5D ). Although the binding to the peptide 297 from SARS-CoV-1/2 and WIV1 was comparable and strong, the binding was severely 298 reduced to MERS and OC43, and to a lesser extent to HKU1. Thus, somatic mutations 299 acquired by CV3-25 lead to broad CoV-peptide reactivity. Alternatively, the CV3-25 epitope could be less exposed in the context of the 345 MERS, OC43 and HKU1 membrane-anchored spikes as compared to their 346 corresponding stabilized ectodomains or the membrane-anchored sarbecovirus spikes. 347 We note however, that the smaller size Fab domain of CV3-25 was unable to neutralize 348 the OC43 virus. It has been proposed that the B-cell lineages that gave rise to the stem helix The neutralizing potency of CV3-25 is not affected by mutations found in SARS- CoV-2 variants of concern, which harbor mutations that escape from many anti-NTD Pendleton Charitable Trust for its generous support of Formulatrix robotic instruments. 608 We thank Stephen C. 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