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Structural and functional analysis of an inter-Spike bivalent neutralizing antibody against SARS-CoV-2 variants

Summary The different variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have attracted most public concern because they caused “wave and wave” COVID-19 pandemic. The initial step of viral infection is mediated by the SARS-CoV-2 Spike (S) protein, which mediates the receptor recognition and membrane fusion between virus and host cells. Neutralizing antibodies (nAbs) targeting the S protein of SARS-CoV-2 have become promising candidates for clinical intervention strategy, while multiple studies have shown that different variants have enhanced infectivity and antibody resistance. Here, we explore the structure and function of STS165, a broadly inter-Spike bivalent nAb against SARS-CoV-2 variants and even SARS-CoV, contributing to further understanding of the working mechanism of nAbs.


INTRODUCTION
The ongoing coronavirus disease 2019  pandemic, caused by different variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has highlighted the need for effective therapeutic interventions . The SARS-CoV-2 is an enveloped, positive-strand RNA virus that belongs to the b-coronavirus family. Because of the selective pressure from natural and vaccine-induced immunity, SARS-CoV-2 is constantly evolving to escape the humoral immunity and creates another wave of infection, especially the Omicron variant of SARS-CoV-2. Omicron variant is a new variant of concern (VOC) identified in South Africa since November 2021 and has spread rapidly across the world.
One of the reasons for the prevalence of the Omicron variant is that over 30 mutations occur in the Spike protein on the virion surface, which is responsible for receptor recognition. During viral infection, the trimeric S protein binds to the host receptor and is cleaved into S1 and S2 subunits by host protease (Belouzard et al., 2009;Simmons et al., 2004Simmons et al., , 2013Song et al., 2018). S1 subunit contains the N-terminal domain (NTD) and receptor binding domain (RBD), which directly bind to the host receptor (Li et al., 2005;Yan et al., 2020). S2 subunit is responsible for membrane fusion between virus and host cells. The binding of S1 to the receptor and protease digestion trigger the conformational change of trimeric Spike and expose the fusion peptide of S2, enabling the virus entry into host cells (Belouzard et al., 2009;Millet and Whittaker, 2015;Simmons et al., 2005). Similar to the original SARS-CoV-2, the Omicron variant still exploits ACE2 for host infection (Cui et al., 2022).
Neutralizing antibodies (nAbs) against SARS-CoV-2 represent a promising clinical intervention. Many complex structures of nAbs targeting Spike protein have been determined, most of which used the Fab form rather than the full-length IgG form that contains two Fab fragments in a ''Y'' shape and indicates a putative bivalent binding effect. It has been reported that the bivalent binding of antibodies mediated a more potent function than Fab form in neutralizing viruses such as rhinovirus, Dengue virus, and even SARS- CoV-2 (Edeling et al., 2014;Hewat and Blaas, 1996;Yan et al., 2021). The structural and functional intra-Spike bivalent binding of nAbs has been revealed by our previous study and exhibited more potent neutralizing activity than monovalent Fab form against wild-type (WT) SARS-CoV-2 (Yan et al., 2021). However, whether there is another form of bivalent binding such as the single antibody that can bind to two Spike proteins (inter-Spike bivalent binding) remains unclear. Here, we report a neutralizing antibody STS165, which can neutralize the existing variants of SARS-CoV-2, including the Omicron variant. More

Identification of STS165 as a broadly neutralizing antibody
Briefly, STS165 is isolated from peripheral blood specific single memory B cells of a COVID-19 convalescent individual using SARS-CoV-2 RBD protein as bait. STS165 can neutralize 13 pseudoviruses of SARS-CoV-2 variants, including WT, Alpha, Beta, Gamma, Delta, Lambda, Mu, Kappa, Eta, Epsilon, Iota, C.1.2, and even Omicron, indicating its great potential. Especially, STS165 also neutralizes SARS-CoV with 50% inhibitory concentration (IC 50 ) of 2.087 mg/mL ( Figure 1A). Relatively high binding affinities to RBD proteins of SARS-CoV-2 variants and SARS-CoV in the nanomolar range or below contributed to the broadly neutralizing activities of STS165 ( Figure S1). The binding affinity of STS165 to Omicron RBD was simply decreased by 3-fold than that to WT RBD, and its neutralizing activity was largely reduced by these mutations. Similar results were also observed in the binding and neutralizing activities of the other two nAbs, AZD8895, and AZD1061 (Cao et al., 2021), suggesting that the monomeric RBD protein could not fully represent the native conformational Spike trimer on the surface of virus membrane to evaluate the interaction between nAbs and virus, especially the neutralization. These data indicated that STS165 maintained the neutralization activity against Omicron and even SARS-CoV despite the significant reduction. These data also suggest that there are many key binding sites between STS165 and Omicron that deserve further exploration.
It is widely known that RBD-specific nAbs could be divided into four classes according to the competition with ACE2 and the accessibility of epitopes recognized on the RBD in up or down conformation (Barnes et al., 2020). Using competition ELISA, we predicted the binding epitope of STS165 based on its competition with ACE2. STS165 exhibited high capacity in competing with the ACE2 receptor, which is similar to the representative Class 1 antibody P2C-1F11 and Class 2 antibody BD-368-2 ( Figure 1B, left panel). To identify a more precise epitope recognized by STS165, we further measured its competition with other 14 monoclonal antibodies whose epitopes have been well studied via structural biology technology. The 14 iScience Article antibodies included are representative ones falling in the four classical classes that have been identified: Class 1: P2C-1F11, REGN10933, CB6; Class 2: BD-368-2, C144, P2B-2F6; Class 3: S309, REGN10987, C110, C135; Class 4: EY6A, H014, S304, S2A4 (Ju et al., 2020;Li et al., 2022;Piccoli et al., 2020). As shown in Figure 1B (right panel), the STS165 antibody could compete with four Class 4 antibodies, indicating that STS165 belongs to the Class 4 antibody whose recognition site was far away from the receptor-binding motif (RBM).
Many diverse SARS-CoV-2 variants have emerged and spread around the world. These variants are resistant to specific monoclonal antibodies that have been authorized or approved for clinical use. Especially, the Omicron has escaped most of the authorized nAbs except S309 (Cao et al., 2021). Thus, it is urgent to rescue the lost neutralization of these nAbs against Omicron and ongoing variants. As STS165 can neutralize 13 variants including Beta, Delta, and Omicron, it may be an ideal partner to form a therapeutic antibody cocktail. This cocktail is not only an effective treatment but may also protect against antibody resistance caused by virus escape mutants that could arise in response to selective pressure from single antibody treatments. We evaluated the escape of the 11 pseudoviruses from neutralization by the STS165 in combination with other 3 non-competition nAbs approved for clinical use (REGN10933, REGN10987, and S309). As shown in Figures 1C and S2, the level of REGN10933 against Beta was lower than that against WT, displaying a significant decrease in IC 50 (2.318 mg/mL). Importantly, the inhibition values of REGN10933 and REGN10987 against Omicron and SARS-CoV were below 50% when tested at the highest concentration (50 mg/mL). However, the approved antibodies REGN10933 and REGN10987 in combination with STS165, respectively, restored their neutralizing activities against Omicron and SARS-CoV to some extent. Notably, for other variants, STS165 cocktail therapy with REGN10933, REGN10987, and S309 antibodies, respectively, also maintained their neutralizing activities against pseudoviruses. In summary, these results indicate that STS165 is not only a broadly neutralizing antibody but also can be used as a partner with different approved antibodies to resist constantly emerging SARS-CoV-2 variants.

Structure determination of Omicron S-6p bound with STS165
To characterize the binding epitope recognized by STS165, we aimed to determine the cryo-EM structure of the Omicron variant of SARS-CoV-2 S-6p bound with STS165. The S-6p protein of the Omicron variant was purified by Ni-NTA affinity resin. To test whether this protein is functional, we determined the structure of Omicron S-6p bound with the peptidase domain (PD) of ACE2. This structure is almost identical to the previously reported structures ( Figures S3, S4, and S5 and Table S1) (Cui et al., 2022). The IgG form of STS165 and Omicron S-6p were mixed and incubated at a molar ratio of $3.6 to 1 for 1 h and applied to size exclusion chromatography (SEC) to remove excess antibodies ( Figure S6A). The peak fractions containing the complex were concentrated for further cryo-EM sample preparation.
The cryo-EM structure of Omicron S-6p bound with STS165 was determined at an overall resolution of 3.3 Å (Figures 2A, S5, S6, and S7 and Table S1). However, the resolution at the interface between RBD and STS165 was not high enough to reveal a detailed interaction network because of the flexibility of this region even with focused refinement applied. To characterize the putative epitope, we docked an Alphafold predicted STS165 into the density for analysis. When aligned with the ACE2-binding site on the RBD, we can find the overlap between ACE2 and STS165, consistent with our competition assays ( Figure 2B).
The STS165 binding Omicron S-6p exhibits a conformation with three ''up'' RBDs, each of which is bound by an STS165 fragment ( Figure 2A). For an overall view, STS165 targets the lateral side of RBD, which is a highly conserved epitope between the SARS-CoV and SARS-CoV-2 (Lv et al., 2020;Zhou et al., 2020a). STS165 can be classified into Class 4, which also includes H014, EY6A, and S304 ( Figure 2C). We mapped the epitope of STS165 on the RBD of Omicron variant and found that the S373P and S375F might be included (Figures 2D and 2E). We propose these mutations might alter the neutralizing activities of STS165 against the Omicron variant by changing the local environment. We also mapped the epitope of STS165 on the RBD of other SARS-CoV-2 variants, including Alpha, Beta, Gamma, Delta, Lambda, Mu, Kappa, Eta, Epsilon, Iota, and C.1.2 and found that no mutated residues were involved in this region, suggesting the conservation of this epitope ( Figures S8 and S9). The Class 4 antibodies have been reported to have largely lost the neutralizing ability to the Omicron variant (Ju et al., 2022a), while STS165 still keeps its neutralizing ability. This might be attributed to the detailed epitope variation among these antibodies of Class 4.
Intriguingly, we found a special binding pattern of STS165 in our 2D classification, of which the ''head to head Spike'' is revealed for the first time ( Figure 2F). This pattern was not observed in the Fab-form of iScience Article STS165 incubated with the Omicron variant (data not shown). This binding pattern indicates that the IgG form of STS165 can bind to two distinct Spikes simultaneously, revealing the inter-Spike bivalent binding characteristics. However, we could not solve the structure of the two-Spike binding pattern because of the preferred orientation of protein particles. Based on the structure of STS165 and Omicron S-6p complex, we proposed a bivalent binding model of STS165 in which at least two STS165 in IgG form can bind to the ''up'' RBD of distinct Spikes and limit them in a straight line ( Figure 2G). This unique bivalent binding manner may enhance the neutralizing ability of STS165. Indeed, the neutralizing assay of STS165 in IgG form and Fab form also confirmed this hypothesis. Compared with the IgG form, the Fab form of STS165 nearly or totally lost the neutralizing activities against WT, Beta, Delta, and Omicron ( Figure S10). To further explore the potential mechanism, we measured the binding affinities of the Fab form of STS165 to WT and Omicron RBD proteins, respectively ( Figure S11). Compared with the IgG form ( Figure S1), the binding affinity of STS165-Fab to WT or Omicron RBD was largely decreased by more than 50-fold, which was partially responsible for the serious reduction in neutralizing activity.

DISCUSSION
The COVID-19 pandemic has lasted for more than two years around the world (Zhou et al., 2020b). As a variety of variants continue to appear, SARS-CoV-2 largely escaped the antibody immunity elicited by the natural virus infection and vaccine immunization (Dejnirattisai et al., 2021;McCallum et al., 2021;Planas et al., 2021;Wang et al., 2021). Before the Omicron emerged, any VOC or variant of interest (VOI) carried no more than three mutations in the RBD region. These mutations mainly decreased the neutralizing activities of Class 1, 2, and 3 nAbs to some extent (Cheng et al., 2021;Liu et al., 2021a;Yuan et al., 2021). None of key escape mutations was found to be located in the binding epitope of Class 4 nAbs, which were once considered as the most broad-spectrum nAbs (Barnes et al., 2020;Lv et al., 2020;Zhou et al., 2020a). However, the RBD of Omicron contained an alarming number of 15 mutations, some of which were first identified in SARS-CoV-2 variants and conferred the greater antibody resistance of Omicron (Cameroni et al., 2021;Cao et al., 2021;Dejnirattisai et al., 2022;Liu et al., 2021b). In particular, S371L appeared in the binding epitope of Class 4 nAbs and mediated the resistance of Omicron to most of these nAbs (Ju et al., 2022a;Liu et al., 2021b). Fortunately, we isolated a Class 4 nAb, named STS165, from a COVID-19 convalescent individual, retaining a degree of neutralizing activity against Omicron. Particularly, STS165 is characterized by the relatively high binding affinity to the RBD protein and the stable complex with the Spike trimer of Omicron, which facilitates the determination of the real cryo-EM structure of this class of nAbs bound with Omicron variant. The future research might well modify these Class 4 nAbs to improve their neutralizing activities based on the structural interaction. To our knowledge, this study for the first time directly observed an inter-Spike bivalent binding mode of a SARS-CoV-2 nAb through the application of the full IgG-form antibody to determine the structure. This kind of ''head to head Spike'' binding pattern in that two distinct Spike trimers are recognized and limited in a straight line by IgGs is unique. Our findings also demonstrated that this bivalent IgG form of STS165 had a higher binding affinity and neutralizing activity than its monovalent form of Fab. In addition, the bivalent binding of STS165 to distinct Spike proteins may increase the engulfing capacity and antigen-presenting function of macrophages against SARS-CoV-2 in vivo, which is needed to be further explored in the future. Overall, our functional and structural studies reveal a broadly nAb, STS165, targeting a conserved epitope on the SARS-CoV-2 RBD. The inter-Spike binding property of STS165 can enhance the neutralization activities and might mitigate the risk of viral escape. These results provide clues for antibody-based cocktail therapy interventions against COVID-19.

Limitations of study
Despite the exceptional findings for the STS165 targeting the different variants of SARS-CoV-2, there are two limitations in our study. First, the resolution of the interface between STS165 and the Spike protein is not high enough for model building, so we cannot clearly define the interaction map in this region. Second, the molecular mechanism for the inter-Spike bivalent binding mode of IgG-form STS165 remains unclear because of the preferred orientation of protein particles. We will unveil the relevant mechanisms underlying the inter-Spike bivalent binding by STS165 in the future.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

ACKNOWLEDGMENTS
We thank the Cryo-EM Facility and Supercomputer Center of Westlake University for providing cryo-EM and computation support, respectively. This work was funded by the National Science Fund for Distinguished Young Scholars (82025022) Li, X., Mooney, P., Zheng, S., Booth, C.R., Braunfeld, M.B., Gubbens, S., Agard, D.A., and Cheng, Y. (2013). Electron counting and beaminduced motion correction enable near-atomicresolution single-particle cryo-EM. Nat. To transfect one liter of cell culture, about 1.5 mg of the plasmid was premixed with 3 mg of polyethylenimines (PEIs) (Polysciences) in 50 mL of fresh medium for 15 min before adding to cell culture. Cells were removed and medium was collected by centrifugation at 40003g for 15 min after 60 h post-transfection.
The secreted S-ECD (Omicron) was purified by Ni-NTA affinity resin (Qiagen). The nickel resin loaded was rinsed with the wash buffer 1 containing 25 mM HEPES (pH 7.0), 500 mM NaCl, and washed with wash buffer 2 containing 25 mM HEPES (pH 7.0), 150 mM NaCl and 30 mM imidazole. Protein was eluted by wash buffer 2 plus 270 mM imidazole. Then the Ni-NTA eluent was subjected to size-exclusion chromatography (Superose 6 Increase 10/300 GL, GE Healthcare) in buffer containing 25 mM HEPES (pH 7.0), 150 mM NaCl. The peak fractions were collected and stored at À80 C.
The secreted PD was purified by anti-FLAG M2 affinity resin (Sigma Aldrich). After loading two times, the anti-FLAG M2 resin was washed with the wash buffer 3 containing 25 mM HEPES (pH 7.0), 150 mM NaCl.
The protein was eluted with the wash buffer 3 plus 0.2 mg/mL flag peptide. The eluent of PD was then concentrated and subjected to size-exclusion chromatography (Superdex 200 Increase 10/300 GL, GE Healthcare) in buffer containing 25 mM HEPES (pH 7.0), 150 mM NaCl.
The S-ECD (Omicron) was incubated with PD or STS165 at a molar ratio of about 1:6 or 1:3.6 for 1 h. To remove excessive PD or STS165, the mixture was subjected to size-exclusion chromatography (Superose 6 Increase 10/300 GL, GE Healthcare) in buffer containing 25 mM HEPES (pH 7.0), 150 mM NaCl. The peak fractions containing protein complexes were collected for EM analysis.

Recombinant antibody production
Paired immunoglobulin heavy and light chain genes of STS165 antibody were obtained from Smart-seq2 Genomics V(D)J sequencing, and analysis was submitted to recombinant monoclonal antibody synthesis. Fastqc was used to perform quality control on the sequenced raw data. Reads were quality filtered with the cutoff threshold for average base quality score set at 20. The filtered reads were mapped against human reference genome (version: GRCh38) using STAR software. We use Trinity software to de novo assemble the transcripts of RNA-seq data. TransDecoder was used to identify the open reading frame and annotate the BCR sequence of the assembled contig. We extracted the annotated BCR sequence from all assembled mRNA transcripts. All the BCR sequences were manually corrected. Variable genes of published nAbs were downloaded from the National Center of Biotechnology Information (NCBI) and synthesized by Sangon Biotech and GenScript. Protein data bank (  , EY6A (6ZCZ) (Zhou et al., 2020a), H014 (7CAI) (Lv et al., 2020), S304 (7JW0) and S2A4 (7JVC) (Piccoli et al., 2020), respectively. All variable genes of nAbs were cloned into IgG1 expression vectors containing heavy and light chain constant regions and then instantaneously co-transfected into 293F cells by using polyethylenimine (PEI, Polyscience). Seven days after transfection, antibodies were purified from the culture supernatants by protein A affinity chromatography.
Competitive enzyme-linked immunosorbent assay 96-well plates were coated with SARS-CoV-2 WT RBD proteins (Sino Biological) at 2 mg/mL in PBS at 4 C overnight The plates were washed with PBST (0.05% Tween-20 in PBS) buffer and blocked with 5% skim milk and 2% bovine albumin in PBS at RT for 1 h. Serially 3-fold diluted monoclonal antibodies were mixed with HRP (Abcam) conjugated human ACE2 protein or STS165 antibody in equal volume and then added to the plates and incubated at 37 C for 1 h. Finally, the TMB substrate (Sangon Biotech) was added to wells and incubated at RT for 20 min and the reaction was stopped by 2 M H 2 SO 4 . OD450 and OD630 were measured by ELISA microplate reader. The competitive effect is determined by comparing the ratio of different antibodies to control antibodies.

Binding analysis by surface plasmon resonance (SPR)
The binding assays of monoclonal antibodies to the RBD proteins of SARS-CoV-2 variants and SARS-CoV were performed using the Biacore 8K system (GE Healthcare). Specifically, one flow cell of the CM5 sensor chips were covalently coated with the RBD proteins (Sino Biological) in 10 mM sodium acetate buffer (pH 5.0) for a final RU (response units) of around 250, whereas the other flow cell was left uncoated and blocked as a control. All the assays were run at a flow rate of 30 mL/min in HBS-EP buffer (10 mM HEPES pH 7.4, ll OPEN ACCESS