Over two years into the pandemic, SARS-CoV-2—the virus causing COVID-19 disease—is still wreaking havoc worldwide, taking a heavy toll on people’s health and disrupting lives around the world. The consecutive emergence of Omicron subvariants BA.1, BA.2, BA.2.12.1, BA.4 and BA.5 poses severe challenges to the efficacy of currently established vaccines and antibody therapeutics. The receptor-binding affinity and immune evasiveness of these newly emerging mutants require immediate investigation. On June 17, 2022, a collaborative work by China’s several prominent research institutions was published online in Nature, titled “BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection”, provides comprehensive data on this front. The study is led by Xiaoliang Sunney Xie and Yunlong Cao’s team from Biomedical Pioneering Innovation Center (BIOPIC) and Beijing Advanced Innovation Center for Genomics (ICG) at Peking University and Changping Laboratory, in collaboration with Junyu Xiao’s team from School of Life Sciences at Peking University, Xiangxi Wang’s team from Institute of Biophysics at Chinese Academy of Sciences, Youchun Wang’s team from National Institutes for Food and Drug Control (NIFDC), and Zhongyang Shen’s team from Nankai University.

　　The authors found that the recently emerged Omicron sublineages BA.2.12.1 and BA.4/BA.5 display even stronger immune evasion capability than BA.2, exhibiting striking neutralization evasion against the plasma of BA.1 convalescents who had received 3-dose CoronaVac. Through high-throughput single-cell sequencing and high-throughput yeast display-based deep mutational screening, the researchers isolated and characterized over 1500 SARS-CoV-2 neutralizing antibodies (NAbs). It was found that newly evolved mutations on BA.2.12.1 and BA.4/BA.5 could specifically evade the NAbs elicited by BA.1 infection. Also, the “original antigenic sin” phenomenon was observed, meaning post-vaccination BA.1 infection mainly recalls wildtype-induced memory B cells and rarely elicits BA.1-specific NAbs. Together, these observations suggest, BA.1-derived vaccine boosters may be no longer ideal for achieving broad-spectrum protection against the newly emerged variants. The “original antigenic sin” coupled with the virus’s ability to quickly evolve immune escape mutations, means it’s extremely difficult to achieve herd immunity through Omicron infection.

　　Originally released on bioRxiv on May 2, 2022, as a preprint, this study is the world’s first comprehensive research paper to report on the spike protein structure and humoral immune escape of BA.2.12.1 and BA.4/5 while revealing the molecular mechanism underlying the “original antigenic sin” phenomenon during Omicron infection. This study, which has drawn wide attention from the international academic community, has been reported by lots of renowned media such as Science, New York Times, and ABC News.

　　By far, major Omicron variants all display high transmissibility. To examine the relations between the conformation of spike glycoprotein (S) and its receptor binding ability, the researchers determined the cryo-EM reconstructions of the S-trimers of BA.2, BA.3, BA.2.12.1, BA.2.13, and BA.4/5, and measured the binding affinity between hACE2 and receptor-binding domain (RBD) of these Omicron variants. As revealed by structural analyses, F486V mutation carried by BA.4/5 may lead to decreased ACE2-binding affinity while R493Q reversion and L452R mutation alleviate this effect. Findings from the study show hACE2-binding affinity of BA.2.12.1, BA.2.13 and BA.4/5 is comparable to that of BA.2. (Fig.1)

　　Fig.1 Structures of S protein and human ACE2 binding affinities of RBDs of Omicron variants

　　It was found that, BA.2.12.1 and BA.4/BA.5 exhibit stronger neutralization evasion, compared to BA.2, against the plasma of 3-dose vaccinees and, most strikingly, of post-vaccination BA.1 convalescents. Analyses based on flow cytometry and single-cell V(D)J sequencing (scVDJ-seq) suggest that, BA.1 breakthrough infection mainly recalls the humoral memory induced by wildtype (WT) vaccination and elicits antibodies that neutralize both WT and BA.1 but respond poorly to the newly emerged Omicron variants, consistent with the “original antigenic sin” theory. Together, these findings suggest, BA.1-derived vaccine may not achieve broad-spectrum protection against new Omicron variants and may not be suitable to serve as boosters for currently established immunity. (Fig.2)

　　Fig.2 Neutralizing titers against SARS-CoV-2 variants in plasma from vaccinees, post-vaccination BA.1 convalescents, and SARS convalescents receiving COVID-19 vaccines.

　　The strong antibody evasion capability of emerging Omicron variants was also confirmed by analyses of antibody drugs, such that the neutralizing activity of most approved antibody therapeutics is significantly abolished by Omicron variants. The S371F, D405N, and R408S mutations carried by BA.2/BA.4/BA.5 sublineages would undermine most broad sarbecovirus NAbs (such as S309), while LY-CoV1404 (Bebtelovimab) and COV2-2130 (Cilgavimab) can still effectively neutralize BA.2.12.1 and BA.4/BA.5. Notably, the researchers identified a non-competing antibody pair composed of SA58 (BD55-5840) and SA55 (BD55-5514). This antibody cocktail could potently neutralize all Omicron subvariants and sarbecoviruses like SARS-CoV-1 and Pangolin-GD, standing out as a promising drug candidate that can confer strong efficacy in both prevention and treatment. (Fig.3)

　　Fig.3 Neutralizing activity against SARS-CoV-2 variants and sarbecoviruses by therapeutic neutralizing antibodies

　　To further probe the mechanisms behind antibody evasion ability of Omicron variants, the researchers determined the escaping mutation profiles, epitope distribution, and Omicron sublineage neutralization efficacy of 1640 RBD-binding antibodies. Of the 1640 antibodies, 614 were from post-vaccination BA.1 convalescents and 411 from vaccinated SARS convalescents. The antibodies were unsupervised clustered into 12 epitope groups according to their mutational escaping profiles, namely Group A, B, C, D1, D2, E1, E2.1, E2.2, E3, F1, F2, and F3. Further analyses revealed that antibodies of the same epitope group share similar features in antigen binding and neutralization, and that the escape hotspots of each epitope group are well-matched with the binding epitopes of representative antibodies from the same epitope group as shown in complexed Cryo-EM structures. (Fig.4)

　　Fig.4 Clustering and characterization of SARS-CoV-2 wildtype RBD-binding antibodies

　　Among the 614 mAbs from post-vaccination BA.1 convalescents, 512 are cross-reactive to WT and BA.1 RBD, while 102 are BA.1-specific and do not bind to WT RBD. The cross-reactive NAbs are enriched on non-ACE2-competing epitopes, as in Groups E2.1, E2.2, E3 and F1. Group E3 and F1 antibodies demonstrated weak neutralizing activity against all variants; the neutralizing activity of E2.2 antibodies is generally moderate; although E2.1 displays significantly higher neutralizing potency than E2.2, the neutralizing activity of E2.1 is largely compromised by L452Q carried by BA.2.12.1and L452R carried by BA.4/5. By contrast, most BA.1-specific NAbs  compete with ACE2, and the binding sites are markedly different from that of WT RBD-binding antibodies. These observations suggest there is a major shift in the antigenicity of BA.1 RBD compared to that of WT RBD. Also, these BA.1-specific NAbs are largely escaped by BA.2/BA.2.12.1 due to D405N and BA.4/BA.5 due to F486V/L452R. Together, these results explained at the molecular level why the plasma of post-vaccination BA.1 convalescents is escaped by the new Omicron variants (Fig. 4,5).

　　Fig.5 Epitope groups, neutralizing activity and escaping mutations of BA.1-specific antibodies

　　Pseudovirus neutralization assays and structural analyses suggest that, the main epitopes (E1, F2, and F3) of broad neutralizing antibodies that are cross-reactive to SARS-CoV-1/2 RBD are also affected by the S371F, D405N and R408S mutations carried by BA.2, BA.2.12.1, BA.2.13 and BA.4/5. The binding affinity of NAbs of Epitope Group E1 is reduced by S371F through local conformational changes, while most of F2/F3 NAbs are escaped by D405N and R408S. These findings suggest that even the broad sarbecovirus neutralizing antibodies were largely escaped by BA.2, BA.2.12.1, BA.2.13, and BA.4/5. (Fig.6)

　　Fig.6 Neutralizing activity, structural characteristics, and escaping mutations of broad sarbecovirus neutralizing antibodies

　　In summary, this study showcased the great potential of high-throughput single-cell sequencing and high-throughput yeast display-based deep mutational screening in antibody selection and characterization. Combined with the clustering of escaping mutation profiles and structural analyses on representative antibodies of each epitope group, the researchers successfully delineated the epitope distribution of antibodies in the plasma from post-vaccination Omicron BA.1 convalescents, and deciphered the molecular mechanisms underlying the escape of emerging Omicron variants against NAbs of each epitope group. Together, this study constructed a comprehensive database on the binding epitopes, escaping mutation profile, and neutralizing activity of SARS-CoV-2 RBD-directed antibodies, providing informative data for future development of broad-spectrum sarbecovirus vaccines and therapeutic antibodies. 

　　Yunlong Cao, an associate investigator at Biomedical Pioneering Innovation Center, Peking University, is the first author of this study. Other co-first authors include Ayijiang Yisimayi, Fanchong Jian, Weiliang Song, Tianhe Xiao, Shuo Du, and Jing Wang from Peking University, Lei Wang from Institute of Biophysics at Chinese Academy of Sciences, Qianqian Li and Yuanling Yu from National Institutes for Food and Drug Control (NIFDC), and Xiaosu Chen from Nankai University. Prof. Xiaoliang Sunney Xie and Yunlong Cao from Biomedical Pioneering Innovation Center at Peking University, Prof. Junyu Xiao from School of Life Sciences at Peking University, Prof. Xiangxi Wang from Institute of Biophysics at Chinese Academy of Sciences, Prof. Youchun Wang from NIFDC, and Prof. Zhongyang Shen from Nankai University are co-corresponding authors. The study was supported by the Ministry of Science and Technology of China (CPL-1233).

　　To download this paper, please visit：https://www.nature.com/articles/s41586-022-04980-y

　　<Corresponding Author profile>

　

　　Yunlong Cao, an associate investigator at Biomedical Pioneering Innovation Center, Peking University, received his Ph.D. degree in Physical Biochemistry from Harvard University. During the COVID-19 pandemic, he made outstanding contributions to research on SARS-CoV-2-specific B cells and antibody immunity by using single-cell genomics technologies, and has published more than 10 research articles in the world’s leading science journals including Nature, Cell and Cell Research as the first author and co-corresponding author. Research findings by his team stood out as one of the “China’s top 10 scientific advances of 2020”. As an outstanding contributor to the development of SARS-CoV-2 vaccines and therapeutics, he was honored "35 Innovators Under 35" by MIT Technology Review (TR35) in 2021.

　　Junyu Xiao, a structural biologist and principal investigator at the School of Life Sciences at Peking University and the Peking University (PKU)-Tsinghua University (THU) Joint Center for Life Sciences. After receiving his B.S. degree from Peking University in 2002 and his Ph.D. degree from University of Michigan in 2008, he then obtained his two-year postdoc training with Prof. Jack Dixon at University of California, San Diego. After working as a project scientist at UC San Diego for two years, he started his own independent group at Peking University in 2014. From 2014 to 2019, he served as an assistant professor; in 2020, he was granted tenure and promoted to associate professor. He is a recipient of the National Science Fund for Distinguished Young Scholars, and an honoree of multiple top awards, including the VCANBIO Award for Biosciences and Medicine, the Gu Xiaocheng Lecture, and the WuXi AppTec Life Science and Chemistry Award.

　　

　　Xiaoliang Sunney Xie, an internationally renowned biophysical chemist, the Lee Shau-Kee Professor at Peking University, and a pioneer in the fields of single molecule biophysical chemistry, coherent Raman scattering microscopy, and single cell genomics. The whole-genome amplification technique developed by Professor Xie’s team has been used in in vitro fertilization and has benefited hundreds of couples in China in avoiding the transmission of their monogenic diseases to their newborns. He became the first tenured professor (1999) and Mallinckrodt Professor (2009) at Harvard University among Chinese Scholars who came to the US since the Reform in China. He came back to China in 2018 as a full-time professor and the Director of Biomedical Pioneering Innovation Center (BIOPIC) at Peking University. Among his numerous honors are the Albany Medical Center Prize in Medicine and Biomedical Research, the American Chemical Society’s Peter Debye Award, and the Biophysical Society Founders’ Award.