Infection with wild-type SARS-CoV-2 elicits broadly neutralizing and protective antibodies against omicron subvariants

The omicron variants of SARS-CoV-2 have substantial ability to escape infection- and vaccine-elicited antibody immunity. Here, we investigated the extent of such escape in nine convalescent patients infected with the wild-type SARS-CoV-2 during the first wave of the pandemic. Among the total of 476 monoclonal antibodies (mAbs) isolated from peripheral memory B cells, we identified seven mAbs with broad neutralizing activity to all variants tested, including various omicron subvariants. Biochemical and structural analysis indicated the majority of these mAbs bound to the receptor-binding domain, mimicked the receptor ACE2 and were able to accommodate or inadvertently improve recognition of omicron substitutions. Passive delivery of representative antibodies protected K18-hACE2 mice from infection with omicron and beta SARS-CoV-2. A deeper understanding of how the memory B cells that produce these antibodies could be selectively boosted or recalled can augment antibody immunity against SARS-CoV-2 variants.

The omicron variants of SARS-CoV-2 have substantial ability to escape infection-and vaccine-elicited antibody immunity. Here, we investigated the extent of such escape in nine convalescent patients infected with the wild-type SARS-CoV-2 during the first wave of the pandemic. Among the total of 476 monoclonal antibodies (mAbs) isolated from peripheral memory B cells, we identified seven mAbs with broad neutralizing activity to all variants tested, including various omicron subvariants. Biochemical and structural analysis indicated the majority of these mAbs bound to the receptor-binding domain, mimicked the receptor ACE2 and were able to accommodate or inadvertently improve recognition of omicron substitutions. Passive delivery of representative antibodies protected K18-hACE2 mice from infection with omicron and beta SARS-CoV-2. A deeper understanding of how the memory B cells that produce these antibodies could be selectively boosted or recalled can augment antibody immunity against SARS-CoV-2 variants.
The rapid emergence and turnover of SARS-CoV-2 variants of concern (VOCs) and progressive waning of antibody immunity have raised serious concerns that the current vaccine strategies based on the wild-type spike (S) protein would not be able to provide sufficient protection against the antigenically distinct VOCs, particularly in the case of omicron [1][2][3][4] . The omicron subvariants are the most efficient in transmission and have not only been associated with steeply increased new infections among unvaccinated individuals, Article https://doi.org/10.1038/s41590-023-01449-6 the wild-type SARS-CoV-2 was circulating 13 , we analyzed the memory B cell receptor repertoires specific to the wild-type S (S WT ) trimer (Extended Data Fig. 1). We were able to obtain a total of 771 paired antibody heavy and light variable genes from flow cytometry-sorted single B cells. Once expressed in HEK 293T cells, 476 of the 771 anti bodies bound to the recombinant S WT trimer (Fig. 1a). The number of such high-binding mAbs varied from patient to patient (Fig. 1b), perhaps due to variability in cell conditions and time points when samples were collected. Among the 476 mAbs, 214 (45%) were S2-specific, 132 (28%) were RBD-specific and 130 (27%) were either NTD-specific or targeting the quaternary epitopes that only exist in the form of S WT trimer 14 ( Fig. 1b and Extended Data Fig. 2). S2-specific mAbs tended to crossreact with the recombinant S trimer from SARS-CoV-1 and with only a few from MERS-CoV (Extended Data Fig. 2). In contrast, RBD-specific and the remaining mAbs were largely SARS-CoV-2-specific, with only a few cross-reacting with SARS-CoV-1 and even fewer with MERS-CoV (Extended Data Fig. 2). Thus, S2-binding mAbs dominated among the isolated 476 mAbs specific to the SARS-CoV-2 S WT trimer.

Broad and potent nAbs neutralize various SARS-CoV-2 variants
We next found that approximately 30% (40 of 132) of RBD-specific mAbs demonstrated neutralizing activity against wild-type SARS-CoV-2 pseudovirus, whereas this was detected for only 13% (17 of 130) of the other mAbs and less than 1% (1 of 214) of the S2-specific antibodies (Fig. 1c,d). Furthermore, through screening against a panel of 17 pseudoviruses bearing the S protein from the VOCs, including omicron subvariants and other variants under global surveillance, we were able to categorize the top 40 RBD-specific mAbs into five major groups based on their distinctive neutralizing breadth and potency ( Fig. 2 and Extended Data Fig. 3). Group 1 included seven mAbs (P2-1B1, P5-1C8, P5S-2B10, P5S-2B6, P5-1H1, P5S-2A9 and P2S-2E9) capable of neutralizing the entire panel of pseudoviruses, despite some having a marked reduction in neutralization against omicron (Fig. 2). Among the seven group 1 mAbs, P5-1C8 demonstrated an extremely broad and potent but also with break-through infections among those vaccinated [5][6][7] . The exceptionally high number of mutations in the S protein of omicron has resulted in considerable antigenic shift from the wild-type and previously identified VOCs [8][9][10][11] and renders strong capability to escape from neutralization of convalescent, vaccinated and hybrid immunized (convalescent/vaccinated or vaccinated/convalescent) individuals 3,4,8,12 . However, one of the major questions that remains to be answered is the extent of such escape and whether antibodies in the infected or vaccinated individuals can neutralize the VOCs identified so far. If such neutralizing antibodies (nAbs) do exist, it is important to identify their specific features and whether they could be selectively boosted by vaccines to confer broader and more sufficient protection against VOCs, particularly omicron subvariants.
The current work aimed to dissect the antibody repertoire of nine convalescent patients infected with the wild-type SARS-CoV-2, enrolled during the first wave of the COVID-19 pandemic. The naïve background of these individuals allowed us to evaluate the antibody imprint of the wild-type SARS-CoV-2 infection, as well as the immunological potential of these antibodies against subsequently emerged VOCs. We isolated and characterized a total of 476 mAbs from the peripheral memory B cells 16-111 d post symptom onset, and identified seven receptor-binding domain (RBD)-specific mAbs able to neutralize all variants tested, including omicron subvariants. Passive delivery of four representative antibodies of the broad nAbs (bnAbs) protected K18-hACE2 mice from infection with omicron or beta. Biochemical, structural and functional characterization of these bnAbs revealed unique as well as common features associated with their neutralizing breadth and potency.
Group 2 to group 5 antibodies appeared to be specifically affected by the different VOCs and variants tested (Fig. 2). Before the emergence of omicron variants, group 2 antibodies had impressive neutralizing breadth and potency across all variants tested (Fig. 2). However, widespread mutations found on the RBD of omicron have facilitated escape from the neutralizing activity of this group of antibodies (Fig. 2). One antibody, P5S-3B11, failed to compete with any of the five representative mAbs used (Fig. 2), indicating its epitope was beyond the regions these antibodies recognized. Group 3 and group 4 antibodies, while maintaining some activity against omicron, were essentially uniform in competing with known antibodies (Fig. 2). Group 3 antibodies competed with P2C-1F11 and P2B-2F6, whereas group 4 antibodies competed with S309 and P2B-2F6 (Fig. 2). Such uniformity indicated the epitopes of group 3 and group 4 antibodies were rather focused, as opposed to those widely distributed found in group 2 antibodies (Fig. 2). As a result, a few mutations, such as K417N/T, E484K and N501Y found in beta and gamma and L452R and/or E484Q in delta plus, delta, epsilon and kappa, respectively, had led to severe reduction or complete loss of neutralization in group 3 and 4 antibodies. Two antibodies in group 3 (P186-2B1 and P186-1H2) and one in group 4 (P2-1D5) improved their neutralizing activity to some omicron subvariants relative to wild-type (D614G). Finally, group 5 antibodies were most vulnerable to the mutations found in VOCs and the other variants. While the majority of group 5 antibodies maintained neutralizing activity to the wild-type (D614G) and alpha, almost all of them failed to neutralize omicron subvariants, beta and gamma (Fig. 2). Close to 29% (4 of 14) and 50% (7 of 14) of group 5 antibodies  VOCs and VOIs, indicated by antibody concentration required to achieve 50% reduction in viral infectivity (IC 50 , ng ml −1 ). The neutralizing activity is colored from red, orange, yellow, green to gray, with red being the strongest, while gray failed to reach IC 50 at the highest concentration tested (10,000 ng ml −1 ). A few representative mutations that potentially facilitate viral escape from antibodies are indicated below each of the VOCs and VOIs. The complete set of mutations for each of the VOCs and VOIs can be found in the Methods. Epitope specificity was determined by competition with typical class 1 (P2C-1F11 and REGN10933), class 2 (P2B-2F6) and class 3 (REGN10987 and S309) antibodies measured by surface plasmon resonance (SPR). All results were calculated from at least two independent experiments. The germline gene usage (IGHV, IGKV, IGLV), the length of complementarity determining region (CDR) 3 and the proportion of somatic hypermutation (SHM) were estimated using the IMGT program. Antibodies highlighted in red text are those competed with typical class 1 antibody P2C-1F11 and which preferentially used germline IGHV3-53/66. For clarity, the five antibodies that had their crystal or cryo-EM structures resolved in complex with RBD or S trimer are labeled with either red (class 1) or black (other classes) background. G1 to G5, group 1 to group 5; VOIs, variants of interest.
Article https://doi.org/10.1038/s41590-023-01449-6 lost their neutralizing activity to L452R-and E484K-containing variants, respectively (Fig. 2). Epitope mapping through competition also indicated the epitope distributions of group 5 antibodies were rather broad and diverse on the RBD (Fig. 2). Taken together, these results indicated that there was temporal and progressive loss of nAbs in convalescent patients as the new VOCs and variants continued to emerge and circulate in the population; however, antibodies such as those in group 1, which constituted about 18% (7 of 40) of the top RBD nAbs and 1.5% (7 of 476) of total S protein-binding antibodies, maintained broad and potent neutralizing activity.

bnAbs disproportionally favor IGHV3-53 germline
To characterize the genetic features of the five groups of RBD-specific nAbs, we analyzed their heavy and light chain repertories using the IMGT/V-QUEST program (http://www.imgt.org/IMGT_vquest/vquest).

bnAbs protect K18-hACE2 mice against omicron and beta
We next studied the protective potential of four antibodies in group 1 (P2-1B1, P5S-2B10, P5-1H1 and P2S-2E9) against infection with omicron BA.1 or beta variant in a model of SARS-CoV-2 infection in K18-hACE2 mice. In mice infected with omicron BA.1, 12 mice per group were intraperitoneally administered with P2-1B1, P5S-2B10 or P5-1H1 at a dose of 10 mg per kg body weight. In untreated controls, 12 mice were included for P2-1B1 whereas another 12 were shared by P5S-2B10 and P5-1H1. In mice infected with the beta variant, eight mice each received P2S-2E9 intraperitoneally at a dose of 10 mg per kg body weight or remained untreated. At 24 h later, all mice were intranasally challenged with 1.7 × 10 3 plaque-forming units (PFU) of omicron BA.1 or beta variant. Half of the mice in each group were monitored daily for body weight and survival for 14 d. The remaining half were analyzed for viral titers in the lungs and brain and underwent histopathological analysis on day 3 (omicron BA.1) or day 4 (beta) post infection.
In mice infected with omicron BA.1, those treated with P2-1B1, P5S-2B10 or P5-1H1 remained healthy, maintained body weight and survived infection, while 1 of 12 untreated control mice succumbed to disease on day 11 after challenge ( Fig. 4a-c). P2-1B1 or P5S-2B10 treated mice had no detectable levels of live viruses in the lungs on day 3 post infection while untreated mice reached an average of 10 3 PFU per lung (Fig. 4d,e). P5-1H1 protected against disease and body weight loss (Fig. 4c), but had moderate effect on reducing viral load in the lungs, consistent with its reduced neutralizing activity against BA.1 in vitro (Fig. 4f). No detectable levels of live viruses were found in the brain in P2-1B1, P5S-2B10 or P5-1H1 treated or untreated mice (Fig. 4d-f). These results indicated that P2-1B1 and P5S-2B10 conferred stronger protection than P5-1H1 against omicron BA.1 infection in vivo via prophylactic interventions.
In mice infected with beta, P2S-2E9 treated mice remained healthy and survived infection for 14 d, while the untreated group exhibited progressive body weight loss from 3 to 5 d post infection and all succumbed to infection by 5 d post infection (Fig. 4g). No detectable levels of live viruses were found in the lungs or brain of P2S-2E9 treated mice on day 4 post infection (Fig. 4h). By contrast, substantial levels of live viruses were detected in the lungs and brain in three of four untreated mice analyzed (Fig. 4h).
Immunohistochemistry analysis on day 3 (omicron BA.1) post infection showed that the lung tissue of P2-1B1, P5S-2B10 or P5-1H1 treated mice remained intact, while the lung sections of untreated mice presented moderate damage and inflammation, with marked infiltration of inflammatory cells ( Fig. 4i-k). In the P2S-2E9 group, no damage or pulmonary inflammation was found in lung tissues, while untreated mice showed severe inflammatory infiltrates and edema (Fig. 4l). Infected cells in the lung sections of mice were detected using an N protein-specific antibody ( Fig. 4i-l). These results indicated the broad and potent neutralizing activity of bnAbs in vitro translated into strong protection in vivo.
P5S-3B11 buried a surface area of ~810 Å 2 and consisted of 14 residues on the cryptic inner face of beta RBD (Fig. 5j). Most of the omicron variants had two substitutions (S371L/F and S375F) in the P5S-3B11 epitope and were expected to affect 11 of the total 13 hydrogen bonds and salt bridges formed with RBD (Extended Data Fig. 8). As these mutations were located within the epitope, their impact on P5S-3B11 was more likely mediated through a direct rather than indirect fashion, similar to that found for P2S-2E9 (Extended Data Fig. 9 and Fig. 2). Many antibodies with similar epitopes to P5S-3B11, such as S2X259 and DH1047, were also compromised by substitutions found in omicron subvariants (Fig. 5h,j) 3,5 , largely due to the main-chain conformational change of the residing loop (Y369-C379) by triple S371L/S373P/S375F substitutions 9,30 . Overall, the structural basis for five representative bnAbs (P5S-2B10, P5-1H1, P2-1B1, P2S-2E9 and P5S-3B11) revealed the binding modes and potential mechanisms of action against SARS-CoV-2 variants.

Discussion
In this report, we dissected the antibody responses of nine convalescent patients enrolled during the first wave of the COVID-19 pandemic in early 2020. We provided strong and solid evidence of the existence of a small number (7 of 476) of S WT -specific antibodies with broad neutralizing activities against all variants tested, including omicron subvariants BA.1, BA.2, BA.2.12.1, BA.2.75, BA.3 and BA.4/5, despite marked reduction or complete loss of neutralizing activity in the overwhelming majority of the remaining mAbs. Of the seven antibodies in group 1, five bound to the receptor-binding motif surface mimicking that of ACE2 and belonged to the class 1/RBS-A/RBD-2 community antibodies 15,16,20,26 . Four of these five antibodies favored the usage of the germline region IGHV3-53, while one used IGHV1-69. The preferential use of IGHV3-53 has been well documented among convalescent, messenger RNA-vaccinated and convalescent vaccinated individuals 16,18,19 . Such common features suggest that the IGHV3-53-encoded antibodies possess unique biochemical and structural features rendering them naturally strong in binding and highly complementary in shape to the receptor-binding motif surface. However, additional features are required to achieve broad and potent neutralizing activity, as IGHV3-53 usage alone does not guarantee their full activity. The other two antibodies in group 1 bound to the outer surface of RBD and belonged to the class 2/RBS-C/RBD-4 community (P5S-2A9) or class 3/RBS-D/ RBD-5 community (P2S-2E9) 15,20,26 .
Crystal or cryo-EM structural analysis indicated these antibodies maintained their neutralizing breadth and potency either through tolerating substitutions within or close to their epitopes (P2-1B1, P5-1H1 and P2S-2E9) or through inadvertently increasing their binding potency to the mutated epitopes (P5S-2B10). It is possible that some of the mutations in omicron subvariants changed the local structure of the epitope, allowing better exposure and accessibility for the nAbs. Similar enhanced activities to omicron subvariants have also been reported for antibodies isolated or tested by other groups 5,10 . P2S-2E9 extensively overlapped in the footprints of and shared the same germlines, IGHV2-5 and IGLV2-14, with LY-CoV1404, the parental antibody of Bebtelovimab by Eli Lilly, which was also isolated from an early convalescent patient and demonstrates exceptional neutralizing breadth and potency against all VOCs identified, including omicron subvariants 5,[9][10][11]24 . A single intraperitoneal injection of representative antibodies P2-1B1, P5S-2B10 and P5-1H1 in class 1 and P2S-2E9 in class 3 as a prophylactic treatment protected mice from infection with the most antigenically divergent variants, omicron BA.1 and beta. These results indicated that natural infection with wild-type SARS-CoV-2 could generate broadly neutralizing and protective mAbs targeting to multiple regions of the RBD, including omicron subvariants.
It is expected that more antibodies similar to the ones identified here and those targeting other regions of the RBD will be identified as more studies continue to dig deeper into the antibody repertories in convalescent and vaccinated individuals. Among these bnAbs are the IGHV1-58 supersite antibodies and those targeting the most conserved cryptic inner face of the RBD of SARS-CoV-2 variants, as well as other sarbecoviruses (class 4/RBD-6/RBD-7) 9,18,21,31 . However, the latter antibodies would have to overcome substitutions such as S371L/F, S373P, S375F and T376A found in the omicron subvariants in this region to achieve broad and potent neutralizing activity 3,5,8,9 . Nevertheless, identification of broadly neutralizing and protective antibodies in naturally infected individuals highlights the broad and diverse nAb repertoire established and imprinted by the wild-type SARS-CoV-2 infection [3][4][5]8,32 . Particularly, it also points to the RBD as a viable and critical target to induce a broad and protective response against VOCs, as recently demonstrated by several RBD-based vaccine strategies either in the form of a trimer or nanoparticles [33][34][35] . While studies on booster immunity are ongoing, it is reasonable to speculate that the broad and potent antibodies identified here and elsewhere in convalescent patients likely contribute to the hybrid immunity augmented by the vaccine booster among previously infected and recovered patients [36][37][38][39][40] . While these antibodies could serve as promising candidates for the development of next-generation antibody therapies, a deeper understanding of how the memory B cells producing these antibodies are initially induced, maintained and recalled will hold the key to rational design of vaccines capable of selectively boosting such desirable antibodies against SARS-CoV-2 variants and beyond.
While selectively boosting and recalling antibody response is practically challenging, several strategies are being proposed and evaluated in the field. Among these, a germline targeting strategy is being tested for HIV-1 vaccines (426c and eOD-GT8) to selectively target the antibody germline IGHV1-2 to induce VRC01-like antibodies, which showed broadly neutralizing activities against a diverse panel of HIV-1 variants around the world [41][42][43][44] . Analogously, designing SARS-CoV-2 antigens to target the germline IGHV3-53 or IGHV2-5 would be expected to stimulate the type of rare but broadly neutralizing antibodies found in the current study. Glycan engineering is another strategy being tested to reduce the immunogenicity of regions outside the conserved epitopes. This could be achieved through the addition of N-linked glycan and/or polyethylene glycol (PEG). Particularly, as the N-linked glycosylation motif (Asn-X-Ser/Thr) can be introduced into the spike or RBD of SARS-CoV-2, glycan silencing could be precisely designed to shield unwanted epitopes, while improving the exposure of desirable epitopes for broadly neutralizing antibodies [45][46][47][48][49] . Finally, as our understanding of the structure of antigens and immunity to antigens improves, it will provide additional concepts and technologies to selectively target and amplify the type of broadly neutralizing antibodies identified here.

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Study approval, convalescent patients and blood samples
The study was approved by the Research Ethics Committee of Shenzhen Third People's Hospital, China (approval no.: 2020-084). The research was conducted in strict accordance with the rules and regulations of the Chinse government for the protection of human subjects. All participants had provided written, informed consents for sample collection and subsequent analysis. Detailed information of participants in this study is provided in Supplementary Table 1. The study enrolled a total of nine patients aged 32-73 yr and recovered from infection with wild-type SARS-CoV-2 in January 2020. Of these, three (P2, P5 and P10) once developed severe pneumonia whereas the remaining six (P43, P75, P104, P140, P186 and P195) only had mild symptoms during hospitalization at Shenzhen Third People's Hospital. P2 and P5 donated their blood samples twice and the remaining patient once during a 16-111-d recovery period post symptom onset. According to the policies at that time, local patients with COVID-19 were given free treatments and follow-up visits. The collected blood samples were separated into plasma and PBMCs through Ficoll-Hypaque density gradient centrifugation. The plasma samples were heat-inactivated at 56 °C for 1 h and stored at −80 °C, whereas the PBMCs were maintained in freezing media and stored in liquid nitrogen until use.

Plasma and antibody binding analyzed by ELISA
The recombinant S, S1, RBD and S2 proteins derived from the wild-type SARS-CoV-2, and S1 proteins or S proteins of SARS-CoV-1 and MERS-CoV (Sino Biological), were diluted to final concentrations of 0.5 or 2 µg ml −1 , coated onto 96-well plates and incubated overnight at 4 °C. The plates were washed with PBS-T (PBS containing 0.05% Tween 20) and blocked with blocking buffer (PBS containing 5% skim milk and 2% BSA) at room temperature for 1 h. Serially diluted plasma samples or mAbs were added to the plates and incubated at 37 °C for 1 h. After extensive washing, the plates were then incubated with secondary anti-human IgG labeled with HRP (1:5,000 dilution) (ZSGB-BIO) at 37 °C for 30 min or 1 h before incubation with TMB substrate (Kinghawk) at room temperature for 5 min or 20 min. Optical density was measured by a spectrophotometer at 450 nm.

Isolation of S WT -specific single B cells by FACS
SARS-CoV-2 S WT -specific B cells were sorted as previously described 53,54 . In brief, PBMCs from convalescent individuals were collected and incubated with an antibody and recombinant S WT trimer cocktail for identification of S WT -specific B cells. The cocktail consisted of CD19-PE-Cy7 96-well PCR plates containing 20 µl of lysis buffer (5 µl of 5 × first strand buffer, 0.5 µl of RNaseOUT, 1.25 µl of 0.1 M dithiothreitol (Invitrogen) and 0.0625 µl of Igepal (Sigma) per well). Plates were then snap-frozen on dry ice and stored at −80 °C until the reverse transcription reaction.

Single B cell PCR, cloning and expression of mAbs
The IgG heavy and light chain variable genes were amplified by nested PCR and cloned into linear expression cassettes to produce full IgG1 antibodies as previously described 54,55 . Specifically, all second round PCR primers containing tag sequences were used to produce the linear immunoglobulin expression cassettes by overlapping PCR. Meanwhile, the variable genes of heavy and light chain were sequenced, synthesized and then cloned into the backbone of antibody expression vectors containing the constant regions of human IgG1 by GenScript 56 .
Overlapping PCR products of paired heavy and light chain expression cassettes were cotransfected into the HEK 293T cells (ATCC) to produce antibodies for binding analysis. Large quantities of mAbs were produced by transient transfection of 293F cells (Life Technologies) with equal amounts of paired heavy and light chain plasmids. Antibodies in the culture supernatant were purified by affinity chromatography using Protein A bead columns (GE Healthcare) and their concentrations were determined by a NanoDrop2000 (Thermo Scientific).

Gene family usage and recombination analysis of mAbs
The IMGT/V-QUEST program (http://www.imgt.org/IMGT_vquest/ vquest) was used to analyze the germline gene, degree of somatic hypermutation, framework region and the loop lengths of complementarity determining region 3 (CDR3) of each antibody. Chord diagrams showing the germline gene usages of paired heavy and light chain were analyzed and presented by the R package circlize v.0.4.14. The width of the linking arc is proportional to the number of antibodies identified.

Epitope mapping by competition surface plasmon resonance
For epitope mapping, two different mAbs were sequentially injected and monitored for binding activity to determine whether the two mAbs recognized separate or closely situated epitopes. To determine competition with the human ACE2, antibodies (1 µM) were injected onto the RBD-immobilized CM5 chip until the binding steady-state was reached. ACE2 (2 µM) was then injected for 60 s. Blocking efficacy was determined by comparison of response units with and without previous antibody incubation. Biacore 8K Control Software v.3.0.12.15655 was used for binding competition studies.

Crystallization and data collection
The SARS-CoV-2 RBD and the Fab fragment were mixed at a molar ratio of 1:1.2, incubated at 4 °C for 2 h and further purified by gel-filtration chromatography. The purified complex concentrated to approximately 10-15 mg ml −1 in HBS buffer (10 mM HEPES, pH 7.2, 150 mM NaCl) was used for crystallization. The screening trials were performed at 18 °C using the sitting-drop vapor-diffusion method by mixing 0.2 µl of protein with 0.2 µl of reservoir solution. Crystals of P5S-2B10 Fab and RBD complex were successfully obtained in 0.2 M magnesium sulfate heptahydrate, 17% w/v PEG 3350, whereas P5-1H1 Fab and RBD complex was obtained in 2% v/v Tacsimate pH 5.0, 0.1 M sodium citrate tribasic dihydrate pH 5.4 and 13% w/v PEG 3350. P2S-2E9 Fab and RBD complex was obtained in 15% v/v 2-Propanol, 0.1 M sodium citrate tribasic dihydrate pH 4.8, 11% w/v PEG 10000, and P5S-3B11 Fab and RBD complex in 0.05 M citric acid pH 4.4, 0.05 M BIS-TRIS propane, 16% w/v PEG 3350. All crystals were collected, soaked briefly in mother liquid with 20% glycerol and flash-frozen in liquid nitrogen. Diffraction data were collected at a wavelength of 0.987 Å on the BL18U1 beam line of the Shanghai Synchrotron Research Facility and processed by HKL2000. The data processing statistics are listed in Extended Data Fig. 5.

Structure determination and refinement
The structure was determined by the molecular replacement method with PHASER in CCP4 suite 7.1.007 (ref. 57 ). The search models were the SARS-CoV-2 RBD structure (PDB: 6M0J) and the structures of the variable domains of the heavy and light chains available in the PDB with the highest sequence identities. Subsequent model building and refinement were performed using COOT v.0.9.2 and PHENIX v.1.18.2, respectively 58,59 . The structural refinement statistics are listed in Extended Data Fig. 5. All structural figures were generated using PyMOL 2.0 and Chimera v.1.15.

Binding of mAbs to cell-surface-expressed S proteins
The entire procedure was conducted as previously published 51,52,65 . Specifically, HEK 293T cells were transfected with expression plasmids encoding either wild-type or omicron variant S proteins, and incubated at 37 °C for 36 h. Cells were digested from the plate with trypsin and distributed onto 96-well plates. Cells were washed twice with 200 µl of staining buffer (PBS with 2% heated-inactivated FBS) between each of the following steps. First, cells were stained with each antibody (1 µg ml −1 ), or ACE2 (1 µg ml −1 ), or S2-specific mAb (1 µg ml −1 ) (1:200 dilution) (MP Biomedicals) at 4 °C for 30 min. PE-labeled anti-human IgG Fc (1:40 dilution; BioLegend), anti-his PE secondary antibody (1:200 dilution; Miltenyi) or anti-mouse IgG FITC (1:200 dilution; Thermo Fisher Scientific) was added and incubated at 4 °C for 30 min. After extensive washes, the cells were resuspended and analyzed with BD LSRFortessa (BD Biosciences) and FlowJo 10 software (FlowJo). HEK 293T cells with mock transfection were stained as background control. Fold changes in antibody binding were calculated by the ratio of the total fluorescence intensity (TFI) of omicron over wild-type, normalized by that of S2-specific antibody (nTFI). TFI was calculated by multiplying the mean fluorescence intensity (MFI) and the number of positive cells in the selected gates. For example, the fold change in BA.1 of P5S-2B10 was calculated by the following formula: fold change = (BA.1 of TFI/BA.1 of nTFI)/(wild-type of TFI/wild-type of nTFI). TFI = MFI × subset frequency.

Antibody protection in hACE2 transgenic mice
Mouse experiments were performed in a Biosafety Level 3 (BSL-3) facility in accordance with the National University of Singapore (NUS) Nature Immunology Article https://doi.org/10.1038/s41590-023-01449-6 Institutional Animal Care and Use Committee (protocol no. R20-0504), and the NUS Institutional Biosafety Committee and NUS Medicine BSL-3 Biosafety Committee approved SOPs. As previously described 66 , 8-week-old female K18-hACE2 transgenic mice (InVivos) were utilized for this study. The mice were housed and acclimatized in a BSL-3 facility for 72 h before the start of the experiment. The housing conditions were 23 ± 2 °C (high/low temperature), 50 ± 10% (high/low humidity) and 12-h light/12-h dark (light cycle). K18-hACE2 transgenic mice were subjected to P2-1B1, P5S-2B10, P5-1H1 or P2S-2E9 (10 mg kg −1 ) delivered through intraperitoneal injection a day before infection or left untreated. P5S-2B10 and P5-1H1 experiments shared a group of untreated mice. The viral challenge was conducted through intranasal delivery in 25 µl of 1.7 × 10 3 PFU of the infectious SARS-CoV-2 omicron BA.1 or beta variant. Body weights were measured before infection as baseline and monitored daily throughout the following 14 d. Mice were euthanized when their body weight fell below 80% of their baseline body weight. Some of the mice from each experimental group were killed at 3 d for omicron BA.1 or 4 d for the beta variant post infection, and lung and brain tissues were collected. Each organ was halved for the plaque assay and histology analysis, respectively.
For virus titer determination, supernatants from homogenized tissues were tenfold serially diluted in DMEM supplemented with antibiotic and antimycotic, and added to A549-hACE2 cells (omicron virus) or Vero E6 cells (beta virus) in 12-well plates. The inoculum was removed after 1 h of incubation for virus adsorption. Cells were washed once with PBS before 1.2% MCC-DMEM overlay medium was added to each well. Then cells were incubated at 37 °C, 5% CO 2 for 72 h for plaque formation. Cells were fixed in 10% formalin overnight and counterstained with crystal violet. The number of plaques was determined and the virus titers of individual samples were expressed as logarithm of PFU per organ.
For histopathological analyses, lung lobes were fixed in 3.7% formaldehyde solution before removal from BSL-3 containment. Tissues were routinely processed, embedded in paraffin blocks (Leica Surgipath Paraplast), sectioned at 4-µm thickness and stained with hematoxylin and eosin (Thermo Scientific) following standard histological procedures. For immunohistochemistry, the sections were deparaffinized and rehydrated, followed by heat-mediated antigen retrieval, quenching of endogenous peroxidases and protein blocking. Sections were then covered with rabbit anti-SARS-CoV-2 N protein mAb (Abcam, 1:1,000 dilution) for 1 h at room temperature. Subsequently, sections were incubated with rabbit-specific HRP polymer secondary antibody (Abcam, no dilution), visualized using chromogenic substrate DAB solution (Abcam) and counterstained with hematoxylin.

Data reporting
No statistical methods were used to predetermine sample sizes but our sample sizes are similar to those reported in previous publications. The sample size of nine COVID-19 convalescent patients is sufficient for isolating nAbs in the field (PMID: 32454513 and PMID: 32698192). For animal experiments, the number of mice for the in vivo protection assay in each group was 4-6, which is acceptable in the field (PMID: 33657424 and PMID: 33431856). Data distribution was assumed to be normal but this was not formally tested. The experiments for antibody isolation from COVID-19 convalescents were not randomized. Mice in antibody protection experiments were randomly divided into treated and untreated groups. Data collection and analysis were not performed blind to the conditions of the experiments. No animals or data points were excluded.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
Structure coordinates have been deposited in the Protein Data Bank under accession codes 7XSC (P5S-2B10:WT-RBD), 7XS8 (P5-1H1:WT-RBD), 7XSA (P2S-2E9:Beta-RBD) and 7XSB (P5S-3B11:Beta-RBD). Sequences of 40 RBD-specific mAbs have been provided in Supplementary Table 2. All data generated or analyzed during this study are available within the paper and the Supplementary Information files. Source data are provided with this paper. Binding of the five testing mAbs and ACE2 to the S protein expressed on the surface of HEK 293T, measured by flow cytometry. Fold changes in binding activity were measured by mean fluorescence intensity (MFI) relative to that of wild-type and summarized in (a). The symbol '+' and '−' indicate increase and decrease in binding, respectively. The result was calculated from two independent experiments. (b) The flow cytometry shown was representative of two independent experiments. Anti-S2 is a S2-specific antibody used for positive and normalized control. This mouse anti-S2 monoclonal antibody specifically targeted the epitope (1129-VIGIVNNTVYDPLQPELDSF-1148), which was identical among all tested SARS-CoV-2 variants. The numbers highlighted in the gates represent the percent of positive cells detected by indicated antibodies or ACE2.