A monoclonal antibody that neutralizes SARS-CoV-2 variants, SARS-CoV, and other sarbecoviruses

ABSTRACT

The repeated emergence of highly pathogenic human coronaviruses as well as their evolving variants highlight the need to develop potent and broad-spectrum antiviral therapeutics and vaccines. By screening monoclonal antibodies (mAbs) isolated from COVID-19-convalescent patients, we found one mAb, 2-36, with cross-neutralizing activity against SARS-CoV. We solved the cryo-EM structure of 2–36 in complex with SARS-CoV-2 or SARS-CoV spike, revealing a highly conserved epitope in the receptor-binding domain (RBD). Antibody 2–36 neutralized not only all current circulating SARS-CoV-2 variants and SARS-COV, but also a panel of bat and pangolin sarbecoviruses that can use human angiotensin-converting enzyme 2 (ACE2) as a receptor. We selected 2-36-escape viruses in vitro and confirmed that K378 T in SARS-CoV-2 RBD led to viral resistance. Taken together, 2–36 represents a strategic reserve drug candidate for the prevention and treatment of possible diseases caused by pre-emergent SARS-related coronaviruses. Its epitope defines a promising target for the development of a pan-sarbecovirus vaccine.

KEYWORDS:

• SARS-CoV-2
• antibody
• variants
• sarbecovirus
• SARS-CoV

Data availability

The cryo-EM structure of antibody 2–36 in complex with prefusion SARS-CoV-2 spike glycoprotein has been deposited in the PDB ID: 7N5H and EMDB ID: 24190.

Results

Identification of a SARS-CoV-2 and SARS-CoV cross-reactive mAb from a COVID-19 patient

By single B-cell sorting and 10X Genomics sequencing, we have previously recovered 252 mAb sequences from five COVID-19 patients and isolated 19 potent neutralizing mAbs [10] targeting different epitopes on SARS-CoV-2 spike [10, 20–22]. To identify mAbs with broad reactivity against other coronaviruses, we screened these 252 mAb transfection supernatants for neutralization against SARS-CoV pseudovirus in the same way we did for SARS-CoV-2 pseudovirus. Although about one-fifth of mAbs showed neutralization activities against SARS-CoV-2 [10], only one, 2-36, had an appreciable neutralization potency against SARS-CoV (Figure 1A).

Figure 1. 2–36 neutralizes both SARS-CoV-2 and SARS-CoV. (A) Screening of mAb transfection supernatant for neutralizing activities against SARS-CoV-2 and SARS-CoV pseudoviruses. (B) 2–36 neutralization IC₅₀ (µg/mL) against SARS-CoV-2 and SARS-CoV pseudoviruses (PV) as well as the live viruses (LV).

We then focused on 2-36, by carefully characterizing it on SARS-CoV-2 and SARS-CoV with purified antibody. It neutralized SARS-CoV-2 pseudovirus at IC₅₀ ∼0.04 µg/mL and the authentic virus (WA1 strain) at IC₅₀ ∼0.1 µg/mL. Its potency versus SARS-CoV was lower, with IC₅₀ ∼0.2 µg/mL against the pseudovirus and IC₅₀ ∼7.5 µg/mL against the authentic virus (GZ50 strain) (Figure 1B). While 2–36 could bind to both SARS-CoV-2 and SARS-CoV spike trimer (Supplementary Figure 1), its binding affinity to SARS-CoV-2 spike measured by SPR was much higher than that to SARS-CoV (Supplementary Figure 2A). As our previous study already showed 2–36 is an RBD-directed mAb [10], we also compared its binding affinity to SARS-CoV-2 and SARS-CoV RBDs, and observed similar trend as seen for the spike proteins (Supplementary Figure 2B), with higher affinity found for SARS-CoV-2 RBD.

Cryo-EM structure of 2–36 in complex with SARS-CoV-2 and SARS-CoV spikes

To gain insight into the epitope of 2-36, we first evaluated its competition with other mAbs in binding to SARS-CoV-2 spike trimer by ELISA. Two mAbs isolated from SARS patients and showed cross-reactivity against SARS-CoV-2 by targeting distinct epitopes, CR3022 [23] and S309 [24], together with our SARS-CoV-2 mAb targeting the RBM, 2–4 [10], were used in the competition experiments. 2–36 binding to SARS-CoV-2 spike was inhibited by CR3022, but not by S309 or 2–4 (Supplementary Figure 3A), suggesting 2–36 targeted a region similar to the “highly conserved cryptic epitope” of CR3022 [23]. To further investigate the molecular nature of the 2–36 epitope, we determined cryo-EM structures for 2–36 Fab in complex with both SARS-CoV-2 and SARS-CoV spike (S2P-prefusion-stabilized trimers). In the SARS-CoV-2 structure, a single predominant population was observed wherein three 2–36 Fabs were bound per spike in a 3-RBD-up conformation (Figure 2A and Supplementary Figure 4). In the SARS-CoV structure, two 3D classes were observed: one Fab bound per spike in a 1-RBD-up conformation and two Fabs bound per spike in a 2-RBD-up conformation (Figure 2B and Supplementary Figure 5). For the SARS-CoV-2 complex structure, local refinement provided side chain resolution for much of the interface, allowing construction of a molecular model.

Figure 2. Cryo-EM structure of 2–36 in complex with SARS-CoV-2 and SARS-CoV Spike. (A) Cryo-EM reconstruction of 2–36 Fab in complex with the SARS-CoV-2 S trimer at 3.4 Å. RBD is coloured in green, the 2–36 Fab heavy chain in darker blue, the light chain in lighter blue, and the rest of the spike is coloured in light purple with glycans in darker purple. (B) Cryo-EM reconstructions of 2–36 Fab in complex with the SARS-CoV S trimer reveal two major classes: one 2–36 Fab bound to spike with 1-RBD up, and two 2–36 Fabs bound to spike with 2-RBD up. Reconstructions are shown in two different orientations. (C) The 2–36 interface model superposed onto an ACE2-RBD complex model (pdb: 6M0J) shows an ACE2 clash with the light chain. (D) Conservation analysis on the RBD among SARS-CoV-2, SARS-CoV, and bat and pangolin sarbecoviruses show 2–36 binding site is highly conserved, with regions of high conservation in grey and low conservation in red. The 2–36 epitope is outlined in dark blue, the epitope for COVA1-16 in cyan, and the interface with ACE2 in orange. (E) The interface model depicted in ribbon representation, with the CDR1 loops in gold, CDR2 loops in orange, and CDR3 loops in red. The interface residue contacts are shown for CDR H1, H3, and L2 complemented with the electron density map for a 4.1 Å local reconstruction. Hydrogen bonds between CDR H3 and RBD are depicted as dashed yellow lines.

Antibody 2–36 recognizes a region on the “inner-side” of RBD that is buried in the RBD-down conformation of the spike [6]. Thus, 2–36 recognizes RBD only in the up position. This is similar to the antibody epitopes previously defined for antibodies CR3022 [23] and COVA1-16 [25]. The positioning of the 2–36 light chain causes a clash with ACE2 in the spike-ACE2 complex (Figure 2C), which is consistent with competition ELISA data (Supplementary Figure 3B), suggesting that blockage of receptor binding likely accounts for neutralization by 2-36. The 2–36 epitope on RBD is highly conserved among sarbecoviruses that utilize ACE2 for binding, including SARS-CoV-2, SARS-CoV, and a panel of SARS-related coronaviruses (Figure 2D). For example, 24 out of 27 amino acids that contact 2–36 are identical based on the sequence alignment of SARS-CoV-2 Wuhan-Hu-1 and SARS-CoV BJ01 (Supplementary Figure 6), consistent with a similar binding mode between SARS-CoV-2 and SARS-CoV (Figure 2A and B).

The interaction of 2–36 with RBD is dominated by CDR H3 (472 Ų buried in the interface), with minor contributions from CDR H1 (176 Ų buried) and CDR L2 (145 Ų buried) (Figure 2E). CDR H3 forms most of the interactions by interacting with a loop on RBD comprising residues 369-385. Residue 99 in the CDR H3 forms backbone hydrogen bonds with RBD residue 379 to extend an RBD β-sheet, much like antibodies C118 and C022 [26] (Figure 2E, bottom right). For 2-36, interactions were primarily hydrophobic, with CDR H3 residues Tyr98, Tyr99, the aliphatic chain of Arg100a, and Tyr100e all making significant contacts with hydrophobic residues on RBD.

2–36 Broadly neutralizes SARS-CoV-2 variants and SARS-related sarbecoviruses

Given the high conservation of the 2–36 epitope, we went on to test its breadth on the recent emerging SARS-CoV-2 variants first. Our previous studies already showed 2–36 retained its activities against SARS-CoV-2 variants B.1.1.7, B.1.351 [6], P.1 [7] and B.1.526 [8], on both authentic viruses and pseudoviruses (replotted in Figure 3A). Here we assessed 2–36 activity on more variants, including pseudoviruses representing the combination of key spike mutations of B.1.427/B.1.429, R.1, B.1.1.1, B.1.525, B.1.617.1, B.1.617.2 and B.1.1.7 with E484 K, as well as many pseudoviruses with single spike mutations which are naturally circulating in COVID-19 patients with high frequency and located in the N-terminal domain, RBD, or S2. As shown in Figure 3(A), 2–36 could neutralize all the variants tested and maintained its potency against most of the variants with IC₅₀ below 0.1 µg/mL.

Figure 3. 2–36 neutralizes SARS-CoV-2 variants and SARS-CoV-like coronaviruses. (A) 2–36 neutralization IC₅₀ (µg/mL) against SARS-CoV-2 variants. (B) Phylogenetic tree of SARS-CoV-2- and SARS-CoV-related lineages and other coronaviruses constructed via MEGA7 and maximum likelihood analysis of spike amino acid sequences extracted from the NCBI and GISAID database. Representative viruses selected for further testing are denoted in colour same as in (C). (C) Heatmap showing the neutralization IC₅₀ values of the indicated antibodies against SARS-CoV-2, SARS-CoV and their related sarbecoviruses.

We further explored 2-36’s potential as a broadly neutralizing antibody against bat or pangolin coronaviruses (Figure 3B) in the SARS-CoV-2-related lineage (bat coronavirus RaTG13, pangolin coronavirus Guangdong and pangolin coronavirus Guangxi) and SARS-CoV-related lineage (bat WIV1, SHC014, LYRa11, Rs7327, Rs4231 and Rs4084), each of which can use human ACE2 as receptor [2,5]. For comparison, we also tested COVA1-16, CR3022 and S309 in parallel with 2-36. As indicated by the heatmap in Figure 3C and neutralization curves in Supplementary Figure 7, 2–36 could neutralize all these sarbecoviruses. COVA1-16, with a similar epitope as 2–36 (Figure 2D), neutralized most of the viruses but with lower potency. S309 and CR3022 could only neutralize part of this panel of viruses. None of the four antibodies could neutralize MERS (merbecovirus) or 229E (alpha-coronavirus), reflecting their longer genetic distance from SARS-CoV-2 and SARS-CoV (Figure 3B and C).

In vitro selection of 2–36 escape virus

To test whether SARS-CoV-2 can escape from 2–36 neutralization, we co-incubated the authentic SARS-CoV-2 (WA1 strain) with serially diluted 2-36, and repeatedly passaged the virus from wells showing 50% cytopathic effect (CPE) again in serial dilutions of the antibody. 2–36 retained its neutralization activities on the serially passaged viruses until passage 10 (Supplementary Figure 8), and then at passage 12, the virus became resistant to the antibody (Figure 4A). Sequence analyses of the passage 12 virus revealed four single point spike mutations (T284I, K378 T, H655Y, V1128A), all of which were found at 100% frequency. And when we went back to sequence the viruses from earlier passages, only H655Y, which has also been found in other circulating variants such as P.1 [27,28], and V1128A were found. The T284I and K378 T mutations appeared only in the 2-36-resistant virus (Figure 4B). We localized the selected mutations in the model of 2–36 in complex with SARS-CoV-2 S trimer (Figure 4C), only K378 resided in RBD and showed a strong van der Waals contact with Y98 of the 2–36 heavy chain, while all the other three residues were distal from the antibody interface. Indeed, when these mutations were introduced into pseudoviruses and tested for their sensitivity to 2-36, only K378 T alone or in combination with the other mutations was found to be resistant to 2-36, whereas viruses with T284I, H655Y, or V1128A alone remained sensitive (Figure 4D). Interestingly, although the K378 position in SARS-CoV-2 spike can be mutated to other residues at very low frequency, we could not find any K378 T mutation circulating in patient viruses to date (Figure 4E), further demonstrating the conserved nature of the region recognized by 2-36.

Figure 4. In vitro selection of 2–36 escape viruses. (A) Neutralizing activity of 2–36 against viruses at different passages. (B) Spike mutations found in viruses at different passages (C) Model of 2–36 in complex with the SARS-CoV-2 S trimer highlighting mutations in red. CDR H3 Tyrosine 98 makes van der Waals contacts with RBD residue 378 as shown as electron density map mesh in the subpanel. (D) The selected mutations were introduced into pseudoviruses and then tested for neutralization sensitivity to 2-36. (E) The frequency of the selected mutations in circulating in infected patients (data updated to Oct 13th, 2021).

Discussion

We have seen the repeated emergence of novel highly pathogenic human coronaviruses that seriously threaten public health and the global economy in the last two decades. While we are still in the midst of the COVID-19 pandemic, other animal coronaviruses with the potential to cross the species-barrier to infect humans need to be considered as potential threats in the future. Therefore, the development of potent and broad-spectrum antiviral interventions against current and emerging coronaviruses is critical. Here, in this study, we described a mAb, 2-36, that exhibited broadly neutralizing activity against not only SARS-CoV-2 variants and SARS-CoV but also a panel of related sarbecoviruses.

Several other studies have also reported the discovery of broadly neutralizing mAbs against sarbecoviruses in addition to the controls used in this study, COVA1-16 and S309. For example, H014 [29] and Ey6a [30] were reported to neutralize SARS-CoV and SARS-CoV-2. Wec et al isolated several antibodies from a SARS survivor that neutralized SARS-CoV, SARS-CoV-2, and the bat SARS-CoV-like virus WIV1 with modest potency [31]. In addition, antibodies C118 and C022 were also shown to neutralize another bat SARS-CoV-like virus SHC014 [26]. Compared to these studies, we have included a much larger panel of viruses for testing the neutralization breadth of 2-36, with 4 viruses in the SARS-CoV-2-related lineage and 7 viruses in the SARS-CoV-related lineage (Figure 3C). Very recently, additional antibodies with broad activities, such as S2X259 [32] and DH1047 [33], targeting a similar surface on the inner side of RBD as 2–36 (Supplementary figure 9), have been reported, again highlighting that the conserved region of the inner face of RBD could represent a target for a pan-sarbecovirus vaccine.

Apart from these RBD-directed mAbs, antibodies targeting the conserved S2 stem-helix region of the coronavirus spike fusion machinery have been shown to possess even broader reactivity against more beta-coronaviruses including MERS [34,35]. However, their relatively low neutralization potency may limit their clinical utility. Using an in vitro affinity maturation strategy, CR3022 has been re-engineered to neutralize SARS-CoV-2 more potently [36]. Similarly, Rappazzo et al engineered a SARS-CoV-2 and SARS-CoV mAb into a better version, ADG-2, with enhanced neutralization breadth and potency [37]. Perhaps 2–36 and S2-specific mAbs could be improved in the same way without sacrificing neutralization breadth.

The cryo-EM structures of 2–36 in complex with both SARS-CoV-2 and SARS-CoV spike trimers revealed a highly conserved epitope. This region on the inner side of RBD is also targeted by several other mAbs [25,26,30]. The structural information from these studies of broadly neutralizing antibodies isolated from natural infection could be valuable in guiding immunogen design for the development of pan-sarbecovirus vaccines. Such vaccines and 2-36-like broadly neutralizing antibodies could be developed and stockpiled to prevent or mitigate future outbreaks of sarbecoviruses.

Acknowledgements

We thank Bob Grassucci and Chi Wang for help with cryo-EM data collection at the Columbia University cryo-EM Center at the Zuckerman Institute. This study was supported by funding from Andrew & Peggy Cherng, Samuel Yin, Barbara Picower and the JPB Foundation, Brii Biosciences, Roger & David Wu, the Bill and Melinda Gates Foundation, and the SAVE programme from the NIH. Support was also provided by the Intramural Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, and Health@InnoHK, Innovation and Technology Commission, the Government of the Hong Kong Special Administrative Region. The study was conceptualized by D.D.H. The biological experiments and analyses were carried out by P.W., M.S.N., J.Y., M.W., J.F.-W.C., S.I., L.L., Z.C., K-Y.Y., and Y.H. The structural experiment and analyses were carried out by R.G.C., G.C., Y.G., Z.S., P.D.K., and L.S. The manuscript was written by P.W., R.G.C., L.S., and D.D.H. and reviewed, commented, and approved by all the authors.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Competing interests

P.W., J.Y., M.N., Y.H., L.L., and D.D.H. are inventors on a provisional patent application on mAbs to SARS-CoV-2.