Protective neutralizing epitopes in SARS‐CoV‐2

Abstract The COVID‐19 pandemic has caused an unprecedented health crisis and economic burden worldwide. Its etiological agent SARS‐CoV‐2, a new virus in the coronavirus family, has infected hundreds of millions of people worldwide. SARS‐CoV‐2 has evolved over the past 2 years to increase its transmissibility as well as to evade the immunity established by previous infection and vaccination. Nevertheless, strong immune responses can be elicited by viral infection and vaccination, which have proved to be protective against the emergence of variants, particularly with respect to hospitalization or severe disease. Here, we review our current understanding of how the virus enters the host cell and how our immune system is able to defend against cell entry and infection. Neutralizing antibodies are a major component of our immune defense and have been extensively studied for SARS‐CoV‐2 and its variants. Structures of these neutralizing antibodies have provided valuable insights into epitopes that are protective against the original ancestral virus and the variants that have emerged. The molecular characterization of neutralizing epitopes as well as epitope conservation and resistance are important for design of next‐generation vaccines and antibody therapeutics.


| INTRODUC TI ON
The ongoing pandemic coronavirus disease 2019 (COVID-19) has caused over 400 million infections and 6 million deaths since the first identification of the etiological agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in late 2019. 1,2 COVID-19 is a communicable disease, where the most common symptoms are fever, cough, and shortness of breath. 3,4 A number of complications, such as pneumonia, acute respiratory distress syndrome, sepsis, and cardiac injury, can lead to severe illness and death.
The disease spreads mainly via droplet and aerosol transmission and also through direct or indirect contact with respiratory secretions. 3,5,6,7,8 Hence, social distancing, masking, and frequent hand washing reduce the opportunity for viral transmission. SARS-CoV-2 is a member of the betacoronaviruses in the coronavirus family.
Its relatives, such as SARS-CoV-1 and MERS-CoV, were responsible for two human epidemics: severe acute respiratory syndrome (SARS) in 2003 and Middle East respiratory syndrome (MERS) in 2012. Overall, these viruses are highly transmissible with fatality rates ranging from 1%-35%. Here we assess the immune response to SARS-CoV-2, focusing mainly on the antibody response, and how the already impressive and constantly growing database of information on antibody isolation, characterization, and epitope identification can be used to guide design of next-generation vaccines and antibody therapeutics not only to SARS-CoV-2 but to coronaviruses in general.

| B RIEF MOLECUL AR VIROLOGY
SARS-CoV-2 is an enveloped RNA virus with a mainly spherical, crown-shaped morphology of about 104 nm in diameter on average (around 92 nm if produced in Vero cells 9,10 ). 11 Like many other coronaviruses, the SARS-CoV-2 virion contains single-stranded positive RNA as its genome wrapped around viral nucleocapsid (N) protein.
Its membrane is derived from the host cell in which the viral membrane (M), small envelop (E) and spike (S) proteins are embedded.
Its genomic RNA encodes another 16 non-structural proteins and several other regulatory proteins. Once the virus enters a receptive host cell, its viral RNA undergoes transcription and translation to produce the viral proteins required for both host immune evasion and self-replication. 3

| S PIKE PROTEIN AND VIR AL ENTRY MECHANIS M
The virion surface is dominated by the viral spike protein that is responsible for attachment to the host cell surface and for mediating membrane fusion between virus and host cell. [9][10][11] Unlike most coronaviruses, the spikes of SARS-CoV-2, as well as MERS-CoV, are cleaved by a proprotein convertase, presumably furin, during biogenesis into two non-covalently linked subunits, S1 and S2 ( Figure 1). 52,53,71,72 The cleaved spike proteins in prefusion and postfusion states, as well as the non-cleaved form (S0), appear to be present on mature SARS-CoV-2 virons. 9,11,73 Structures of the spike in prefusion and postfusion forms were rapidly determined after SARS-CoV-2 was identified. 53,71,74,75,76,77 S1 consists of an Nterminal domain (NTD) and receptor binding domain (RBD) followed by two subdomains SD-1 and SD-2 ( Figure 1B). S2 consists of several regions including the N-terminal fusion peptide and its proximity region, heptad repeat 1 (HR1), central helix, stem helix, HR2, transmembrane region, and cytoplasmic tail ( Figure 1B). The virus binds human receptor ACE2 on the target cell through its RBD on the spike S1. Structural studies have shown that the RBDs in spike can have down, up, and intermediate conformations where the predominant conformations are all down and one up when the RBD is in a native unliganded conformation. 9,10,11,53,65,71,78,79,80,81 However, the ACE2 receptor binding site (RBS) on the spike is not exposed when the RBD is in a down conformation ( Figure 2). 53,71 As the spike has to expose its RBS to bind ACE2, such exposure can also lead to RBS recognition by antibodies in the immune system.
After engagement with the human receptor, transmembrane serine protease 2 (TMPRSS2) in target cells cleave the spike protein at residue R815, 16,72,82 leaving a processed S2' that renders the fusion peptide accessible for membrane fusion with the host target cell (Figure 1). 77 This process is similar to that observed for SARS-CoV-1 [83][84][85] and validated by the TMPRSS2 inhibitor camostat, which inhibits virus infection of TMPRSS2-positive cells. 16,72 Precleavage of the SARS-CoV-2 spike by proprotein convertase is beneficial to SARS-CoV-2 infection of TMPRSS2-positive cells. 52,72 Nevertheless, SARS-CoV-2 can also infect TMPRSS2-negative cells.
Reagents such as ammonium chloride and hydroxychloroquine that inhibit endosomal acidification can suppress SARS-CoV-2 infection in cell-based assays but not in the clinic. 76,86 In this case, after engagement by human receptor ACE2 on the target cell surface, the virus through its spike protein is internalized via clathrin-mediated endocytosis. 73,87,88 In the endolysosomes, the spike is presumed to be cleaved by proteases cathepsin B/L in a similar way to SARS-CoV-1. 16,52,76,86,89 However, it is not clear whether endocytosis has a major role in SARS-CoV-2 pathogenesis, although TMPRSS2 appears to be essential in mouse models of MERS-CoV and SARS-CoV-1 infection. 90 Recent studies have shown that the mutations in SARS-CoV-2 may change the disease severity. The recent Omicron variant (BA.1) replicates faster in upper-airway bronchi but less efficiently in lung parenchyma or lower-airway tissues compared to other variants of concern or ancestral strain, which may lead to more dependence on entry through the endocytosis pathway in the upper airway. 73,91 Another study under review reports that Omicron BA.2 has similar infectivity and pathology in mice and hamsters. 92 The fusogenic process in respiratory viruses is highly similar and has been widely reviewed for influenza virus, [93][94] HIV, 95,96 paramyxoviruses, 97 and coronaviruses including SARS-CoV-1, SARS-CoV-2, and MERS-CoV. [98][99][100] The viral spike is thought to contain a spring-loaded fusion machinery. In case of SARS-CoV-2, binding of ACE2 leads to cleavage at the R815 site, either by TMPRSS2 or cathepsin B/L, and is akin to releasing the safety bolt and liberating the fusion peptide for membrane fusion. The S2' region then undergoes dramatic structural reorganization to form a super-long helix that contains HR1 and the central helix (CH) (Figure 1). The fusion peptide is now relocated atop the long helix (approximately 180Å) in the six-helix bundle in the spike trimer and poised to target the host cell membrane. 77,100,101 Overall, these concerted conformational changes bring the cell and viral membranes into close proximity that ultimately leads to membrane fusion, which is critical for releasing the viral genome into the target cell. Inhibitors that in- The postfusion spike is shown as a ribbon. Domains within the protomer are colored separately. The spike is first cleaved by a proprotein convertase, such as furin, during biogenesis into two subunits, S1 and S2, that are non-covalently bound to each other. A secondary cleavage at the S2' site by TMPRSS2 or cathepsin B/L liberates the fusion peptide (FP) sequence for membrane insertion. Glycans on the spike surface are not shown for simplicity. NTD, N-terminal domain; RBD, receptor binding domain; SD-1 and SD-2, two subdomains in S1 followed RBD. CH, central helix, forms a long helix with HR1, the heptad repeat region 1, in the postfusion state. HR2, heptad repeat region 2 in the prefusion structure (A) and the fusion peptide in the postfusion structure (B) have not been resolved yet and are shown as dashed spheroids. PDBs 6XR8 and 6XRA were used to represent the prefusion and postfusion structures to D) based on their epitope preference as we previously proposed (and updated in Figure S1). 145 Binding sites on the RBD other than the RBS have also been identified. A cryptic epitope site on one of the lateral faces of the RBD was first identified as a binding site for antibody CR3022, a cross-reactive antibody isolated from a SARS patient. 146 The N343 proteoglycan site on the opposite lateral face was identified by antibody S309 (Figure 3), a cross-neutralizing antibody also isolated from a SARS patient. 147 More recently, another lateral RBD site was identified by antibodies COVOX-45 148 and S2H97 148 ( Figure 3). In general, RBS antibodies are usually more potent, while antibodies to the CR3022, N343 proteoglycan, and lateral RBD sites tend to have greater breadth. 149 However, there are some exceptions of RBS antibodies that have breadth as well as potency, and antibodies to other sites that are potent as well as broad in neutralizing SARS-CoV-2. Two linear epitope sites on the NTD ( Figure 4A) are often targeted by neutralizing antibodies but less frequently compared to the RBD. When comparing antibodies to these various sites, RBS-A and NTD antibodies usually have larger antibody-antigen interfaces, that is, buried surface areas (BSAs), versus other RBD antibodies ( Figure 4B). Many antibodies also target the S2 subunit but most are not neutralizing, although some show moderate protection in animal models. [150][151][152][153][154] We will now discuss the characteristics of these epitopes and the propensity for antibodies to target these sites.

| RBD epitope sites
RBD is the domain on the spike protein that binds human receptor ACE2. However, the region on the RBD where ACE2 binds (RBS) is not fully accessible when the RBDs are in the down conformation ( Figure 2). RBD in the up conformation exposes the receptor binding site and binds ACE2 at nanomolar affinities in the wild-type and different SARS-CoV-2 variants identified so far. [155][156][157] The virus has retained if not increased its affinity to ACE2 in the emerging variants through mutations within the RBD interface with ACE2; some of these mutations also aid in escape from host immunity. In general, the RBD is highly immunogenic since both SARS-

CoV-2 infection and vaccination elicit robust antibody responses
to the RBD. 123,127,134,158,159,160,161,162,163,164,165,166,167,168 In many cases, vaccination such as with first-generation mRNA vaccines elicit higher levels of neutralizing antibodies compared to natural infection. 123,136,158,164,166 Antibodies targeting the RBS generally compete with ACE2 binding if they bind with high enough affinity. Electrostatic surface of SARS-CoV-2 RBD. Charge potential was calculated using APBS plugin in PyMol software. The perspective view is the same as G for easy comparison Indeed, many antibody studies have shown that the most potent antibodies target the RBS. 131,139,140,143,145,162,169,170 Currently, more than 150 neutralizing antibodies with atomic structures have been reported that bind the RBS and block ACE2 binding. The epitopes of these antibodies can be clustered into at least four subgroups. 145,155 One aim of finely differentiating the epitope sites is to inform on the characteristics and properties of antibodies that bind to each subsite, including germline usage, susceptibility to mutations, where next-generation vaccines should be targeted, and what is the best combination of antibodies as therapeutics. Antibodies that target different epitope sites have also been combined to reduce the chance of escape mutations during antibody treatment. 171 170 The epitope residues of these antibodies cluster to a specific region roughly corresponding to RBD residues 400-425, 444-460, and 473-506 (except 479-483), which define an antibodytargeting subsite in the RBS ( Figure S1). This epitope site, designated as RBS-A, 145 is largely buried when RBD is in the down state and becomes fully accessible when RBD is in the up conformation. 170 However, antibodies such as S2H14 182 191,192 and NT-193 180 are also broad but less potent in neutralizing SARS-CoV-2 VOCs or SARS-CoV-1. Overall, these findings suggest RBS-A is capable of eliciting antibodies with both potent and broad protection, although strain-specific antibodies are more predominant at this epitope site.

| RBS-B epitope site
Structural studies showed that some IGHV3-53/3-66 antibodies with long CDRH3 (15 amino acids or longer) or specific somatic hypermutations bind RBS in a distinct conformation compared to those with short CDRH3 or lower somatic hypermutation. These antibodies defined a new epitope site 145,169,175,193 that we termed RBS-B. 145,175 Later, many other germline antibodies were found to target this site and refined the definition of RBS-B epitope site that mainly covers the RBD ridge (470-491) and its nearby regions (approximately residues 446-457 and 492-505) ( Figure S1). The RBS-B epitope residues have some overlap with both RBS-A and RBS-C epitopes as their fairly large footprints encroach to some extent on these adjacent sites. However, the essential epitope residues that interact with neutralizing antibodies are quite distinct ( Figure 3B).
Residues F486, Y489, E484/K484, Q493, and Y449 generally contribute most to neutralizing antibody binding with F486, Y489, and E484/K484 that are located on the prominent RBD ridge. In almost all of these antibodies, F486 is buried in a pocket at the heavy-light chain interface. 145,193 Thus, RBS-B antibodies favor interaction with the RBD ridge. The shape of the RBS-B surface renders a relatively smaller interface area for antibodies compared to RBS-A and RBS-C ( Figure 4B). The RBD ridge is also exposed on the surface of the spike regardless of whether RBD is in the up or down conformation.
A substantial number of neutralizing antibodies, including C144 169 and S2M11, 194 can bind RBD in both up and down states and also interact with the conserved N343 glycan and residues from a neighboring RBD in the spike trimer. Interaction with the neighboring RBD sometimes can lock the spike trimer in a closed RBD down state, which prevents human receptor engagement with the neighboring RBD. 169,194 Other antibodies, such as COVA2-39 and S2E12, however, may require additional space for binding RBS-B and thus can only bind RBD in the up conformation. 169,175,182 Many neutralizing antibodies targeting RBS-B are extremely potent against specific SARS-CoV-2 strains and show efficacy in protection from SARS-CoV-2 infection or severe disease in animal models or humans. For instance, CV07-209, S2E12, S2M11, CT-P59, and J08 provide protection in the Syrian hamster model 140,182,195,196 and AZD7442 in non-human primates. 197 155,168,193 However, accumulating evidence suggests that the RBS-B epitope can elicit both potent and broad antibodies against SARS-CoV-2 variants. The structure of RBD ridge is retained between SARS-CoV-1 and SARS-CoV-2, where a disulfide bond between C480 and C488 helps maintain the structural integrity and its conservation. A small patch in the RBD ridge is moderately conserved across SARS-CoV-2 strains and even other sarbecoviruses that may account for elicitation of broadly neutralizing antibodies to this region. 202 Antibodies J08, AZD8895, S2E12, and BRII-198 broadly neutralize a broad spectrum of SARS-CoV-2 variants, 148,192,203,204 whereas antibodies β47, COVOX-253, A23-58.1, and B1-182.1, also broadly neutralize variants including Omicron VOC (BA.1). 168,205,206 Some RBS-B antibodies, such as CS44, and CV07-287, can neutralize many variants but Omicron only weakly. 167 Interestingly, all of these antibodies (except BRII-198) are encoded by a IGHV1-58 and IGKV3-20 public clonotype for their heavy and light chain variable regions, respectively. The germline-encoded paratope residues of these IGHV1-58 antibodies, such as W50 and Y52 in heavy chain CDR2, and the disulfide bond between C97 and C100b in the heavy chain CDR3 region, favor interaction with the protruding RBD ridge, especially engagement with F486. 167,207,208 Collectively, the RBS-B epitope also seems a promising site for therapeutic antibodies and next-generation vaccine design.

| RBS-C epitope
The RBS-C epitope is located on the other side on the RBS from RBS-A and overlaps partially with RBS-B, N343 proteoglycan, and lateral RBD epitope sites ( Figure 3C) RBS-C contains a region roughly corresponding to residues 340-360 (except 343 and 350) ( Figure S1). Specifically, residues Y449, F490, R346, E484/K484, N450, and R346 are the key residues that interact with neutralizing antibodies to RBS-C. Since RBS-C is exposed on the spike surface regardless of RBD conformation, antibodies targeting RBS-C, such as AZD1061, C104, P36-5D2, BG1-24, BG7-20, and N-612-017, can usually bind RBD in both up and down states. 169,173,209,210 RBS-C antibodies, such as BG1-24 and BG7-20, can bind the RBD in down state while interacting with glycans on NTD and a neighboring "up"-RBD. 173 However, some RBS-C antibodies, such as COVOX-58, only bind RBD in the down state due to its close proximity to NTD. 204 Antibodies targeting RBS-C, such as AZD1061, may synergize with RBS-B antibodies, such as AZD8895, in neutralization against SARS-CoV-2 including Omicron. 204,211 RBS-C antibodies can also be very potent, for example 1-57 (8 ng/mL), β38 (11 ng/mL), S2D106 (7 ng/ mL) and BG1-24 (2 ng/mL). 148,168,173,212 Notwithstanding that RBS-C antibodies are also sensitive to mutations in VOCs at E484 and L452, broad neutralization against several variants of concern has been observed in a few antibodies. 204 Combination of these mutations in the context of the different variants retains if not increases the binding affinity between RBD and ACE2. [155][156][157] We and others have shown that K417N leads to decrease in ACE2 binding, whereas N501Y increases binding affinity between RBD and ACE2. 155,214,215,216 Thus, K417N is most often accompanied by a concomitant mutation N501Y. 155,215,216 These mutations are also at sites that represent essential epitope residues for neutralizing antibodies targeting the RBS and their mutation can lead to escape from immunity established by prior infection or vaccination. Nevertheless, several potent RBS antibodies such as S2K146 and S2E12 are highly resistant to mutations in VOCs. 148,186 Although broad RBS antibodies are much less frequently isolated, they seem to be more abundant in a small fraction of individuals as reported in a recent study. 183 Moreover, recent studies on antibodies isolated from patients infected by Beta VOC showed that the RBS sites can still elicit both broad and potent antibodies, such as β40 and β55, which neutralize SARS-CoV-2 variants including Omicron VOC (BA.1). 167,168 These findings suggest that the RBS, or at least components of it, can be considered to be important for antibody targeting by vaccines and therapeutics regardless of antigenic drift.

| CR3022 cryptic epitope site
At the very start of the pandemic, we reported the structure of a SARS-CoV-1 antibody, CR3022, in complex with SARS-CoV-2 RBD. 146 This structure revealed a cryptic antigenic site that is not exposed when the RBD is in the down state on the spike. However, CR3022 does not neutralize SARS-CoV-2, but this is likely due to its modest binding affinity (~100 nM) compared to SARS-CoV-1 (~1 nM). 146 CR3022 site, such as S2A4 and S2X259, can induce S1 shedding and premature conversion to the postfusion conformation of the spike protein, which could offer another mechanism of protection. 185,220 The CR3022 cryptic site is located in the intramolecular interface within a spike trimer. RBD residues K378, R408, F377, Y369, and T385 are the most favored epitope residues targeted by neutralizing antibodies to this site ( Figure 3E). Amino acid sequence analysis shows that the CR3022 epitope site is highly conserved across sarbecoviruses, a subgroup of betacoronaviruses including SARS-CoV-2 and SARS-CoV-1 viruses. 202 This high sequence similarity indicates functional conservation of this region among these viruses.
Many residues at this site are involved in intramolecular interactions among the RBDs within a spike trimer as well as between the S1 and S2 subunits. 219 For instance, RBD residues R408, K378, K386, and variants and other related sarbecoviruses. 148,192,202,221,222,223,224,225 Although highly conserved residues render CR3022 epitope site an ideal target for broad neutralizing antibodies, relatively few potent antibodies to this site have been isolated. One possibility would be the cryptic nature of this site, which may be less visible to the immune system. The other may be the specific approach angle required to effectively compete with ACE2 binding. All of the most potent neutralizing antibodies observed so far to this site interact with highly similar epitope residues ( Figure S1). Thus, relatively high conservation of these residues across SARS-CoV-2 variants and other coronaviruses, seems ideal for pan-sarbecovirus vaccine design, although how to specifically target this site needs to be resolved.  219 We recently reported that the immunoglobulin D gene, IGHD3-22, encodes the YYDRxG motif, and is responsible for the highly similar binding mode used by these antibodies. 202,222 YYDRxG antibodies have been elicited in both COVID-19 patients and vaccinees, 202 albeit at low frequency. Hence, tuning the immune system to elicit such YYDRxG antibodies would be highly beneficial to broad protection against SARS-CoV-2 variants and other related viruses.

| N343 proteoglycan epitope site
The N343 proteoglycan site is on the opposite face from the CR3022 cryptic site ( Figure 3F). It is characterized by N-glycosylation at residue N343 of SARS-CoV-2 RBD. Most residues at this site are more highly conserved compared to the RBS site but less so than the CR3022 site. Unlike the CR3022 site, this site is exposed regardless of whether the RBD is in up, down, or other intermediate states.
However, fewer neutralizing antibodies have been isolated to this epitope, possibly due to shielding by the N343 glycan in the center of the epitope. This N-glycosylation also seems to be important for stability of the RBD. Starr et al. for example reported that mutations at N343 or T345, which remove the glycosylation sequon, lead to decreased expression of the RBD. 214 We also observed a decrease in protein yield in mutation of the sequon at this site. Protein dynamics simulations have shown that the N343 glycan is important in modulating the dynamics of the RBD conformation. 79 As this glycan site is highly conserved across different sarbecoviruses, it suggests a vital role for this region of the RBD in viral evolution and function.
Despite the extra barrier to the immune system generated by the N343 glycan, neutralizing antibodies isolated from SARS or COVID-19 patients have been isolated that target this epitope site.
The first neutralizing antibody structurally characterized to target this site was S309, an antibody isolated from a SARS patient. 147 226 Next-generation vaccine design should therefore take both N343 proteoglycan and CR3022 sites into consideration since these sites can elicit antibodies that neutralize SARS-CoV-2 variants, as well as other sarbecoviruses.

| Lateral RBD epitope site
Recently, a lateral RBD site has been shown to be a promising epitope site in eliciting neutralizing antibodies ( Figure 3G).  Figure 3I). Although this site barely overlaps with the RBS, antibody S2H97 can induce premature transition of the spike to the postfusion state, S1 shedding, and low levels of syncytia formation, and thus contribute to the neutralization activity. 148 Further antibody discovery may identity more potent neutralizing antibodies to this site.

| NTD epitope site
NTD is mainly constituted of β-sheets and connecting loops and positioned proximal to the neighboring RBD in the spike trimer like the petals of a flower (Figure 2). While the exact biological role of the domain remains elusive, several reports suggest that NTD plays a role in binding attachment factors on host cell surface or recruiting heme metabolites to evade antibody immunity. 229,230 Although NTD is more exposed on the virion surface compared to other components of the S1 subunit, it is highly glycosylated, which probably decreases its overall immunogenicity. 53

| Neutralizing epitopes in S2 subunit
Many studies have shown that the S2 subunit elicits a substantial portion of SARS-CoV-2 specific antibodies. 121,143,183,236,237,238,239,240 However, most antibodies targeting the S2 subunit are not neutralizing. 150 Two recent studies reported that stem helix antibodies could also be isolated from vaccinated COVID-19 patients and exhibit protection against SARS-CoV-2 and MERS-CoV in animal models, further suggesting universal vaccine design to this stem helix epitope site is promising for eliciting pan-betacoronavirus protection if potency can be increased. 154,244 Since the S2 stem helix is highly conserved across betacoronaviruses whether there is a germline convergent response with conserved motifs to betacoronaviruses, warrants further investigation.

| Fusion peptide
Antibodies targeting the fusion peptide in S2 that can neutralize viral infection are not uncommon for HIV, [245][246][247] although this region was not one of the early epitopes to be identified. The fusion peptide in SAR-CoV-2 has to be cleaved by either TMPRSS2 or cathepsin B/L to allow membrane fusion between virus and host cell.
Antibodies targeting the fusion peptide could block either protease cleavage or insertion of cleaved fusion peptide into host membrane.
Interestingly, recent studies have revealed that some antibodies do indeed target the fusion peptide and contribute to SARS-CoV-2 neutralization. 242,243,248 Thus, this fusion peptide region is also a very promising target for pan-coronavirus vaccine and therapeutic design.

| IMPLI C ATI ON S FOR VACCINE DE S I G N
In general, SARS-CoV-2 infection and vaccination can elicit a robust immune response and provide protective immunity. We reviewed here the characteristics of over 200 neutralizing human antibodies whose structures have also been determined. A number of neutralizing epitopes have now been discovered on the RBD, NTD, and S2 (stem helix and fusion peptide) of the spike protein.
The most desirable antibodies that have both breadth and potency are indeed being discovered, but they have been much more difficult to find, particularly as the SARS-CoV-2 virus continues to evolve with greater and greater antigenic variation.
Notwithstanding, a few rare antibodies have been isolated that have both breadth and reasonable potency to SARS-CoV-2 and variants of concern, including Omicron. The most highly conserved sites in the RBS include a small region of the RBD ridge, the CR3022 site, and N343 proteoglycan site seem to be promising epitope sites for next-generation vaccine design and therapeutic antibody development. Vaccines that specifically target a combination of these broadly neutralizing epitopes while not eliciting an overabundance of antibodies against the other more variable or non-neutralizing epitopes will likely be the best strategy against SARS-CoV-2 and variants. Given the extraordinary progress over the past two years, it is now possible to consider pan-coronavirus vaccines and therapeutics with even greater breadth. A number of neutralizing antibodies to the highly conserved S2 domain of the spike have recently demonstrated that regions such as the fusion peptide and stem helix are promising neutralizing epitopes as they are highly conserved in coronaviruses. Thus, it is now possible to capitalize on these advances to pursue pan-coronavirus vaccines and therapeutics to protect not only from current SARS-CoV-2 strains but also from SARS-CoV-1 and MERS-CoV like viruses and other zoonotic coronaviruses with pandemic potential.

Writing of his review was supported in part by the Bill and Melinda
Gates Foundation INV-004923 (I.A.W.).

CO N FLI C T O F I NTE R E S T
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analyzed in this study.