key: cord-0770854-alicdpap authors: Wang, Ruoke; Zhang, Qi; Ge, Jiwan; Ren, Wenlin; Zhang, Rui; Lan, Jun; Ju, Bin; Su, Bin; Yu, Fengting; Chen, Peng; Liao, Huiyu; Feng, Yingmei; Li, Xuemei; Shi, Xuanling; Zhang, Zheng; Zhang, Fujie; Ding, Qiang; Zhang, Tong; Wang, Xinquan; Zhang, Linqi title: SARS-CoV-2 variants resist antibody neutralization and broaden host ACE2 usage date: 2021-03-15 journal: bioRxiv DOI: 10.1101/2021.03.09.434497 sha: 3f88b72d20157224937a7559007c5884a166e4c2 doc_id: 770854 cord_uid: alicdpap New SARS-CoV-2 variants continue to emerge from the current global pandemic, some of which can replicate faster and with greater transmissibility and pathogenicity. In particular, UK501Y.V1 identified in UK, SA501Y.V2 in South Africa, and BR501Y.V3 in Brazil are raising serious concerns as they spread quickly and contain spike protein mutations that may facilitate escape from current antibody therapies and vaccine protection. Here, we constructed a panel of 28 SARS-CoV-2 pseudoviruses bearing single or combined mutations found in the spike protein of these three variants, as well as additional nine mutations that within or close by the major antigenic sites in the spike protein identified in the GISAID database. These pseudoviruses were tested against a panel of monoclonal antibodies (mAbs), including some approved for emergency use to treat SARS-CoV-2 infection, and convalescent patient plasma collected early in the pandemic. SA501Y.V2 pseudovirus was the most resistant, in magnitude and breadth, against mAbs and convalescent plasma, followed by BR501Y.V3, and then UK501Y.V1. This resistance hierarchy corresponds with Y144del and 242-244del mutations in the N-terminal domain as well as K417N/T, E484K and N501Y mutations in the receptor binding domain (RBD). Crystal structural analysis of RBD carrying triple K417N-E484K-N501Y mutations found in SA501Y.V2 bound with mAb P2C-1F11 revealed a molecular basis for antibody neutralization and escape. SA501Y.V2 and BR501Y.V3 also acquired substantial ability to use mouse and mink ACE2 for entry. Taken together, our results clearly demonstrate major antigenic shifts and potentially broadening the host range of SA501Y.V2 and BR501Y.V3, which pose serious challenges to our current antibody therapies and vaccine protection. However, there is growing concern that the epidemic raging worldwide may be 82 generating new SARS-CoV-2 variants that are antigenically distinct from the prototype 83 Wuhan-Hu-1 strain, rendering current antibody and vaccine strategies ineffective (24). To test the impact of these mutations on neutralization sensitivity, we generated a panel with mAb P2C-1F11 revealed a molecular basis for antibody neutralization and escape. 124 We also show that mutations acquired by SA501Y.V2 and BR501Y.V3 substantially 125 improve their ability to use mouse and mink ACE2 for entry. Taken together, our results 126 clearly demonstrate major antigenic shifts and potentially broadening the host range of 127 SA501Y.V2 and BR501Y.V3, posing serious challenges to our current antibody 128 therapies and vaccine protection. (2, 3, 5, 17, 37, 38, (41) (42) (43) (44) . These epitopes assignments 137 are related to the six major sites put forward by Yuan and colleagues (45). The wildtype 138 (WT) pseudovirus used throughout the analysis was the prototype Wuhan-Hu-1 strain 139 with a D614G mutation (WT D614G). As shown in Fig. 1 D614G. The symbol "-" indicates increased resistance and "+" increased sensitivity. in the three variants and in the GISAID database ( Fig. 1 and Fig. S3 ). The IC50 of 176 REGN10933 dropped 13.0-and 8.2-fold against SA501Y.V2 and BR501Y.V3, 177 respectively, largely due to the K417N/T and E484K mutations (Fig. 1A) . Several 178 mutations within the REGN10933 epitope, such as Y453F and F486L, were also 179 associated with a substantial reduction in neutralization. N439K, located in the 180 REGN10987 epitope, showed a 26.6-fold reduction in IC50. The most dramatically 181 impacted mAb was CB6, one of the paired antibodies developed by Eli Lilly and 182 approved for EUA, for which the neutralization against SA501Y.V2 and BR501Y.V3 183 pseudoviruses was below detection limit (BDL) when the highest concentration 184 (1μg/mL) was used. The reduction and loss of neutralization are largely attributed to 185 9 K417N/T (Fig. 1A) . Interestingly, despite being a Class I or RBS-A antibody, P2C-1F11 186 was virtually unaffected by the mutations, as were the Class III and IV anti-RBD mAbs 187 REGN10987, S309, and P2B-1G5 (Fig. 1A) . (Table S1 ). Comparing with our 207 previously reported the crystal structure of the wildtype RBD with P2C-1F11 at 3.0 Å 208 (PDB ID:7CDI), we found the three mutations did not change the overall binding mode 209 of P2C-1F11 to the RBD ( Fig. 2A) , as evidenced by 0.52 Å rmsd value for all 581 Cα 210 10 atoms. However, a couple of subtle changes were identified. One is related to the 211 interactive forces with residues 417 and the other with residue at 501. Like other Class 212 I and RBS-A antibodies, P2C-1F11 binds to the wildtype K417 through hydrophobic 213 and hydrogen-bond interactions. This is largely mediated by its heavy chain germline 214 residue Y33 and Y52 (43). The K417N mutation would diminish these interactions but 215 only replaced by one hydrogen bond between Y52 and mutant N417 ( Fig. 2B and Table 216 S2). Furthermore, unlike those in Class I or RBS-A, P2C-1F11 does not form salt-217 bridge interaction between aspartic acid (D) and K417 ( Fig. 2B and 2C ). The K417N 218 mutation would therefore be less disruptive to P2C-1F11 than to those in the Class I or Structural analysis further showed that the P2C-1F11 light chain had extensive 239 interactions with RBD-3M residues Y453, Q493, T500 and Y501 around the 501 240 position but had much less with the wildtype RBD residue asparagine (N) at the 501 241 ( Fig. 2B and Table S2 ). The extra interactions between RBD-3M and P2C-1F11 around 242 the Y501 may also contribute to the retained binding and neutralization of P2C-1F11 243 against SARS-CoV-2 variants carrying the triple K417N-E484K-N501Y mutation. Susceptibility of SARS-CoV-2 variants to neutralization by convalescent plasma. 255 We next studied the degree to which major variant pseudoviruses confer resistance to increased for all plasma sample studied with an average 2.0-fold improvement. Similar 288 trend was also noticed for pseudoviruses carrying only the K417T mutant (Fig. 3C ). The single mutant N501Y pseudovirus, previously reported to enhance ACE2 binding 290 (39, 40, 52) , had limited effects on neutralizing activity of convalescent plasma. enough concern about the potential spread of these new variants to mice and beyond. Class II by the E484K mutation. Of note, we observed that the single K417N/Y mutant 403 tended to increase, rather than decrease, the neutralizing activity of non-Class I mAbs. For convalescent plasma, the most profound impact was observed on the SA501Y.V2 -CoV-2. bioRxiv, 2020 .2011 .2010 .377333 (2020 . ZF2001 vaccines. bioRxiv, 2021 .2002 .2001 .429069 (2021 . , 2021.2002.2011.430787 (2021) . crystallographic structure determination. Acta crystallographica. Section D, 599 Biological crystallography 58, 1948-1954 (2002 The authors have filed patent applications on some of the antibodies described. Hospital (LL-2020-039-K), Beijing Ditan Hospital (2020-019-01), and Shenzhen Third People's Hospital (2020-084). The research was conducted in strict accordance with the 29 rules and regulations of the Chinse government for the protection of human subjects. The study subjects agreed and signed the written informed consents for research use of 31 their blood samples. The study enrolled a total of 23 convalescent patients aged from 29 to 81 years old, 35 with an average of 56, infected with SARS-Cov-2 between January to February 2020. Endoproteinase Lys-C (Roche) with IgG to Lys-C ratio of 4000:1 (w/w) in 10 mM 62 EDTA, 100 mM Tris-HCl, pH 8.5 at 37°C overnight. Fc fragment were removed using A pneumonia outbreak associated with a new coronavirus of 439 probable bat origin Cross-neutralization of SARS-CoV-2 by a human monoclonal 441 SARS-CoV antibody Human neutralizing antibodies elicited by SARS-CoV-2 infection. 443 Potently neutralizing and protective human antibodies against 445 SARS-CoV-2 Studies in humanized mice and convalescent humans yield a 447 SARS-CoV-2 antibody cocktail Convergent antibody responses to SARS-CoV-2 in 449 convalescent individuals Potent Neutralizing Antibodies against SARS-CoV-2 Identified by 451 Single-Cell Sequencing of Convalescent Patients' B Cells Potent neutralizing antibodies against multiple epitopes on SARS CoV-2 spike A single dose of an adenovirus-vectored vaccine provides 456 protection against SARS-CoV-2 challenge Evaluation of the mRNA-1273 Vaccine against SARS-CoV-458 21 in Nonhuman Primates Development of an Inactivated Vaccine Candidate, BBIBP-CorV, 460 with Potent Protection against SARS-CoV-2 BNT162b vaccines protect rhesus macaques from SARS-462 CoV-2 COVID-19) EUA Information Effect of Bamlanivimab as Monotherapy or in Combination 468 With Etesevimab on Viral Load in Patients With Mild to Moderate COVID-19: 469 A Randomized Clinical Trial REGN-COV2, a Neutralizing Antibody Cocktail, in 471 Outpatients with Covid-19 SARS-CoV-2 Neutralizing Antibody LY 610 We acknowledge the work and contribution of all the health providers from Beijing 611 Youan Hospital, Beijing Ditan Hospital and Shenzhen Third People's Hospital. We also Protein A-Sepharose.