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Structural and antigenic variations in the spike protein of emerging SARS-CoV-2 variants

['Anshumali Mittal', 'Department Of Structural Biology', 'University Of Pittsburgh School Of Medicine', 'Pittsburgh', 'Pennsylvania', 'United States Of America', 'Arun Khattri', 'Department Of Pharmaceutical Engineering', 'Technology', 'Indian Institute Of Technology']

Date: 2022-05

Abstract The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus is continuously evolving, and this poses a major threat to antibody therapies and currently authorized Coronavirus Disease 2019 (COVID-19) vaccines. It is therefore of utmost importance to investigate and predict the putative mutations on the spike protein that confer immune evasion. Antibodies are key components of the human immune system’s response to SARS-CoV-2, and the spike protein is a prime target of neutralizing antibodies (nAbs) as it plays critical roles in host cell recognition, fusion, and virus entry. The potency of therapeutic antibodies and vaccines partly depends on how readily the virus can escape neutralization. Recent structural and functional studies have mapped the epitope landscape of nAbs on the spike protein, which illustrates the footprints of several nAbs and the site of escape mutations. In this review, we discuss (1) the emerging SARS-CoV-2 variants; (2) the structural basis for antibody-mediated neutralization of SARS-CoV-2 and nAb classification; and (3) identification of the RBD escape mutations for several antibodies that resist antibody binding and neutralization. These escape maps are a valuable tool to predict SARS-CoV-2 fitness, and in conjunction with the structures of the spike-nAb complex, they can be utilized to facilitate the rational design of escape-resistant antibody therapeutics and vaccines.

Citation: Mittal A, Khattri A, Verma V (2022) Structural and antigenic variations in the spike protein of emerging SARS-CoV-2 variants. PLoS Pathog 18(2): e1010260. https://doi.org/10.1371/journal.ppat.1010260 Editor: Kenneth Stapleford, NYU Langone Health, UNITED STATES Published: February 17, 2022 Copyright: © 2022 Mittal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist.

1. Introduction Enveloped viruses are found across diverse viral families and are characterized by having a lipid bilayer (envelope) derived from the host cell membrane, which contains virus-encoded membrane protein (M), envelope protein (E), and glycoprotein peplomer or commonly known as spike protein (S). These peplomers are often seen as projections from the outer surface of the envelope and are essential for host cell recognition, fusion, and virus entry [1]. The spike glycoprotein is the dominant exposed antigen on enveloped viruses, which can trigger a series of adaptive immune responses mediated by 3 major cell types: B cells (humoral immunity) and CD4+ and CD8+ T cells (cell-mediated immunity) [1,2]. B cells activation in response to viral infection results in the production of antigen-specific antibodies that can neutralize and clear the virus particle. Viruses also develop immune evasion strategies for escaping these responses through several ways, such as antigenic shielding by the addition of complex glycans [3,4], by secreting truncated viral glycoproteins that share viral spike epitopes for subverting the host immune response [5], by antigenic variations [6], and by blocking complement activation and neutralization of virus particles [6]. On the other hand, activated B cells within germinal centers of secondary lymphoid organs produce class-switched memory B cells, which undergo rounds of population expansion, somatic hypermutation, and selection for improved antigen binding. These evolutionary strategies are regularly used by our immune system to protect us against virus variants and other pathogens [7–9]. In the ongoing Coronavirus Disease 2019 (COVID-19) pandemic, we are witnessing all these features of the human immune system along with continuously emerging Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants. The COVID-19 pandemic caused by the SARS-CoV-2 virus has claimed over 5 million lives worldwide since the first reported case in Wuhan, China [10,11]. Each SARS-CoV-2 contains 24 to 40 randomly arranged spike-like projections on its surface [12,13], which resemble a solar corona under the electron microscope, from which they get their name and are referred to as coronaviruses. The SARS-CoV-2 spike glycoprotein is a homotrimer composed of S1 and S2 subunits separated by a furin cleavage site (RRAR) that modulates its fusogenic activity [14]. The receptor-binding domain (RBD) and N-terminal domain (NTD) of the S1 subunit guide the SARS-COV-2 attachment to host cells, and the S2 subunit drives fusion between virus and host cell membrane [15–18]. Cryo-EM studies have indicated that the SARS-CoV-2 spike protein is wildly flexible and it exhibits several prefusion conformations where 3 RBDs adopt distinct orientations: “up” (receptor-accessible state) and “down” (receptor-inaccessible). Protomers with the up-conformation can facilitate the binding between the spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor (Fig 1). The RBD and ACE2 binding interface shows highly complementary electrostatic surface potentials with the RBD and ACE2 being positively and negatively charged, respectively [13,17,19–21]. The antigenic nature together with multiple conformations of the trimeric spike protein make it an attractive target for drug and vaccine development projects. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Structure of the SARS-CoV-2 spike protein trimer. (A) Left: side view of the trimeric spike ectodomain with 3 RBDs in the down-conformation; right: top view of the trimeric spike protein showing RBDs in gray, forest green, and orchid (PDB: 6VXX). (B) Left: side view of the trimeric spike ectodomain with 1 RBD in the up-conformation; right: top view of the trimeric spike protein showing 1 up RBD in gray (PDB: 7BNN). (C) A schematic layout of the spike protein is shown at the top. Right: structure of a monomer displaying the RBD in the open conformation. Spike protein structure shows the receptor-binding subunit S1 and the membrane-fusion subunit S2 separated by the furin-like protease site (S1/S2). Different subdomains of the spike protein are the NTD in green, RBD in gray containing RBM in cyan at their top, the fusion peptide in pink, second cleavage site S2’ in red, and HR1 and HR2 in olive (PBB: 7BNN). The scissors represent the S1/S2 boundary at amino acid position 685. Left: The open conformation RBD highlights the 3 different regions: receptor-binding ridge, flat surface, and 443–450 loop of the RBM that form the ACE2-binding region. ACE2, angiotensin-converting enzyme 2; FP, fusion peptide; NTD, N-terminal domain; RBD, receptor-binding domain; RBM, receptor-binding motif; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2. https://doi.org/10.1371/journal.ppat.1010260.g001 RNA viruses, usually approximately 10,000 nucleotides long, exploit various tools of genetic variation for ensuring their survival, which are characterized by high mutation rates, high yields, and short replication times [22]. The low replicative fidelity armors RNA viruses to adapt to different replicative environments and selective pressures, which, in turn, enable them to escape host immunity and develop drug resistance. SARS-CoV-2 encodes an RNA proofreading system (nsp14-ExoN), but despite this, more than 25,000 mutations have been reported in the SARS-CoV-2 genome (https://ngdc.cncb.ac.cn/ncov/variation/annotation). The first notable mutation discovered was D614G in early 2020 [23], and since then, several SARS-CoV-2 variants have emerged periodically, including the Delta (B.1.612.2) [24] and Omicron (B.1.1.529) [25] variants. It is critical to identify these mutations on the RBD that escape antibody-mediated SARS-CoV-2 neutralization. Mutational antigenic profiling has successfully been used for identifying such hotspots in envelope or spike proteins that escape antibody binding in several viruses, including SARS-CoV-2 [26–36]. These antibody escape maps define functional binding epitopes and predict which mutations are selected when the virus is exposed to convalescent plasma or monoclonal antibodies (mAbs). These escape maps in combination with atomic structures could be of great importance from a therapeutic standpoint. Structural studies of the SARS-COV-2 variants spike protein have revealed that they exhibit higher conformational heterogeneity than observed for the Wuhan-Hu-1 isolate or D614G, which might play key roles in binding and locating the ACE2 receptors on cell surfaces [21]. These variants appear to have increased transmissibility, potentially higher pathogenicity and show reduced sensitivity to neutralization by therapeutic mAbs and serum-derived polyclonal antibodies [37–41]. In this review, we discuss the molecular characteristics of naturally occurring SARS-CoV-2 variants including the Delta and Omicron variants that armor them for escaping the therapeutic antibodies, natural infections, or even current vaccines. We define the structural analysis and classification of the RBD targeting antibodies to understand the molecular mechanisms of neutralization and potency differences among them. We further describe the identification of the RBD escape mutations for several antibodies that resist vaccine-elicited and therapeutically relevant antibodies binding with a focus on the SARS-CoV-2 variants.

5. Conclusions Francis Crick famously said, “if you want to understand the function, study structure”. In the last more than a year, an unprecedented number of structures of the SARS-CoV-2 spike protein, including its variants, in complex with numerous mAbs have been determined to understand the molecular mechanism of antibody-mediated neutralization of SARS-CoV-2. The structural analyses of these complexes provide a comprehensive map of nAb epitopes on the spike protein. However, these analyses do not directly measure how/which mutations will escape from antibody binding. Going forward, mutations in the RBD that escape antibody binding were identified by different methods [27,28,30,32–34,114]. An analysis between the structural contact sites and escape maps suggested that these maps complement each other; therefore, comprehensive knowledge of immunodominant epitopes on the spike protein and escape mutations would aid efforts to understand viral evolution and rational design of antibody therapeutics, vaccines, and other countermeasures (Tables 1 and S1). Antibodies that were isolated from the early COVID-19 phase were found to have a relatively low number of somatic mutations. However, it has been recently demonstrated that these antibodies show affinity maturation by somatic hypermutation in the lymphoid germinal centers, which empower them for increased binding affinity and neutralization potency for SARS-CoV-2 [84]. For example, C051 and C052 exhibit about 4-fold higher somatic hypermutation than the clonally related class 2 C144 antibody, and mutations in the RBD at L455, F456, F490, Q493, and S494 do not confer resistance anymore. These antibodies display an increased binding affinity for the RBD with Q493 mutation as compared to C144 but show resistance in binding to the RBD with E484K mutation [84]. It is important to note here that most of the affinity matured antibodies of class 2 were unable to neutralize SARS-CoV-2 pseudovirus with an E484K mutation alone or in combination with K417N and N501Y substitutions (B1.351 VOC) [84]. Overall, these results indicate that serological immunity is evolving to fight against the continuously evolving SARS-CoV-2 virus [84,115]. SARS-CoV-2 will continue to evolve, and it is nearly impossible to predict future variants; therefore, identification of bnAbs is a pressing need of the hour due to their strength of neutralizing multiple variants of a virus by recognizing evolutionarily conserved epitope. In this direction, the RBD-targeting antibodies, A23-58.1 and B1-182.1, were identified by Wang and colleagues, which neutralize multiple SARS-CoV-2 variants, including the B.1.1.7, B.1.351, P.1, and B.1.617 variants at subnanomolar concentration [116]. They bind to an invariant region of the RBD tip that is offset from major mutational hotspots (K417, E484, and N501), which increases their breadth and potency (Fig 6). These mutations are the major determinants conferring resistance in the B.1.1.7, B.1.351, P.1, and B.1.617 variants (Fig 2). The mAb 222 antibody is another notable example that binds both P.1 (K417T, E484K, and N501Y) and the ancestral Wuhan-Hu-1 RBD with similar affinities despite differences in the binding site. Notably, mAb 222, a Class 1 antibody, can neutralizes all 3 B.1.1.7, B.1.351, and P.1 variants despite its interactions with 2 of the ACE2-binding site mutations [60] (Fig 4E). The RBD-Fab 222 structure (PDB: 7NX6) indicates that the CDRH1 and CDRH2 weakly interact with K417N/T of the RBD, and CDRL1 contains a proline (residue P30) due to a rare somatic mutation that forms a cis-amide bond [89] with N501Y of the RBD [60] (Fig 4E). The 7D6 is a recently isolated bnAb that binds away from the RBM and is not sensitive to currently circulating SARS-CoV-2 variants. The crystal structure shows that 7D6 binds a novel cryptic site located behind the receptor-binding ridge of the RBD that faces toward the NTD. The 7D6 antibody binding causes interference with the adjacent NTD and appears to destabilize the spike protein. Structural analysis has shown that the 7D6 binding site residues are nevertheless conserved within the Sarbecovirus subgenus and the 7D6-epitope location is distinct from those bound by most antibodies that are insensitive to the mutations [117] (Fig 6C). These antibodies are classified as bnAbs based on the data available and can be further modified and evaluated for their use in cocktail therapy. PPT PowerPoint slide

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TIFF original image Download: Fig 6. Structural analysis of bnAbs binding to the RBD. The epitope surface of the A23-58.1 antibody is shown in red. A23-58.1 targets the supersite with minimal contacts to major mutational hotspots (K417, L452R, E484, S494P, and N501) of current VOCs, which are shown in blue. The binding mode of A23-58.1 is very similar to those of class 1 antibodies. The CDR H3 of A23-58.1 contains 14 residues and can only bind an RBD in the up-conformation (PDB: 7LRT). The RBD is shown in gray containing the RBM in cyan at their top. (B, C) S309 is a class 3 antibody while 7D6 binds proximal to the S309 epitope. S309 recognizes an epitope containing a glycan in white (N343 in SARS-CoV-2). 7D6 binds a novel cryptic site located behind the receptor-binding ridge of the RBD that faces toward the NTD. The S309 and 7D6 binding site residues are conserved within the Sarbecovirus subgenus. Both antibodies are resistant to mutations that emerged in the SARS-CoV-2 variants. The footprint residues on the RBD have been defined as those residues, which were within 4 Å of a Fab atom. bnAb, broadly neutralizing antibody; NTD, N-terminal domain; RBD, receptor-binding domain; RBM, receptor-binding motif; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; VOC, variant of concern. https://doi.org/10.1371/journal.ppat.1010260.g006 In addition to bnAbs, broadly neutralizing nanobodies are also identified that are capable of neutralizing B.1.1.7 (Alpha), B.1.351 (Beta), and P.1 (Gamma) variants [118]. Xu and colleagues identified 2 groups of neutralizing nanobodies from llama and an engineered mouse (nanomouse). Nanomouse nanobodies, Nb12 and Nb30, target a conserved region on the RBD that is largely inaccessible to human antibodies and is located outside the ACE2-binding motif (PDB: 7MY3 and 7MY2), which explains why their binding is not affected by E484K (Beta and Gamma) or N501Y (Alpha) substitutions [118]. Going forward with the identification of more effective therapies against SARS-CoV-2, multiple laboratories have reported the development of bispecific antibodies by combining 2 antibodies that target nonoverlapping epitopes on the spike protein and multivalent nanobodies. These engineered molecules have been described to be highly potent in neutralizing the SARS-CoV-2 variants and suppressing mutational escape [119–122]. Alongside this, antibodies from the NTD and community RBD-1 to RBD-4 are an attractive choice for the therapeutic cocktails being more potent than others, but the data suggest that emerging SARS-CoV-2 variants tend to escape binding by many members of the groups associated with most potent neutralizers [80,81]. Notably, the epitopes targeted by RBD-5 to RBD-7 antibodies are highly conserved but less potent in the Sarbecovirus subgenus of Betacoronavirus; therefore, they can be engineered in multivalent format to achieve enhanced potency and further use in the variant-resistant cocktails formulation [80]. For example, RBD-5 to RBD-7 or Class 4 antibodies, such as S2X259 (PDB: 7RA8 and 7RAL) [103] and DH1047 [104], are broadly protective antibodies, which bind to an epitope that is highly conserved among the Sarbecovirus subgenus. Although nAbs are an important defense against viral infections, they are not the only mechanism of protection. The T cells responses and non-nAbs are additional measures to protect from viral infections [123,124]. Pfizer-BioNTech and Moderna mRNA vaccines for SARS-CoV-2 have been shown to elicit cross-protective B and T cell responses, which can recognize different SARS-CoV-2 variants and are minimally affected by mutations in the spike protein [125–128]. This suggests that vaccine-induced immunity will continue to protect against currently circulating SARS-CoV-2 variants. The SARS-CoV-2 virus will continue to evolve, resulting in the emergence of escape variants; therefore, worldwide genomic surveillance, better vaccination drive, development of bnAbs, and new drugs are vital to combat COVID-19.

Supporting information S1 Table. Therapeutic countermeasures against COVID-19 under development. The data have been taken from the US Department of Health and Human Services (https://www.medicalcountermeasures.gov/app/barda/coronavirus/COVID19.aspx?filter=therapeutic). https://doi.org/10.1371/journal.ppat.1010260.s001 (TIF)

Acknowledgments We thank Prof. Dr. Jonathan Coleman for critical reading the manuscript.

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