key: cord-0987460-lry247t4
authors: Ghotloo, Somayeh; Maghsood, Faezeh; Golsaz‐Shirazi, Forough; Amiri, Mohammad Mehdi; Moog, Christiane; Shokri, Fazel
title: Epitope mapping of neutralising anti‐SARS‐CoV‐2 monoclonal antibodies: Implications for immunotherapy and vaccine design
date: 2022-04-08
journal: Rev Med Virol
DOI: 10.1002/rmv.2347
sha: 6ee24c5d6015523ff5de64f07e7db85232d42d3e
doc_id: 987460
cord_uid: lry247t4

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the causative agent of the coronavirus disease 2019 (COVID‐19) pandemic. This disease has currently affected more than 346 million people and resulted in more than 5.5 million deaths in many countries. Neutralising monoclonal antibodies (MAbs) against the SARS‐CoV‐2 virus could serve as prophylactic/therapeutic agents in COVID‐19 infection by providing passive protection against the virus in individuals. Until now, no Food and Drug Administration/European Medicines Agency‐approved neutralising MAb against SARS‐CoV‐2 virus exists in the market, though a number of MAbs have been authorised for emergency use. Therefore, there is an urgent need for development of efficient anti‐SARS‐CoV‐2 neutralising MAbs for use in the clinic. Moreover, neutralising anti‐SARS‐CoV‐2 MAbs could be used as beneficial tools for designing epitope‐based vaccines against the virus. Given that the target epitope of a MAb is a crucial feature influencing its neutralising potency, target epitopes of neutralising anti‐SARS‐CoV‐2 MAbs already reported in the literature and reactivity of these MAbs with SARS‐CoV‐2 variants are reviewed herein.

COVID-19 requiring hospitalisation. The FDA and the World Health Organization (WHO) recommend several therapeutics for COVID -19 such as IL-6 receptor blockers (tocilizumab or sarilumab) as well as systemic corticosteroids in patients with severe or critical disease. [3] [4] [5] [6] [7] Meanwhile, many efforts have been made to produce SARS-CoV-2 neutralising monoclonal antibodies (MAbs). Sotrovimab, REGN-CoV-2, and the cocktail of bamlanivimab and etesevimab have been authorised for emergency use as post-exposure prophylaxis for COVID-19 in adults and children at high risk for progression to severe COVID-19. [8] [9] [10] Considering the emergence of new variants and lack of efficacy of a number of the neutralising MAbs against the newly emerged Omicron variant, there is imperative need for MAbs able to efficiently cross-neutralise various variants to be used as passive immunotherapy for the control SARS-CoV-2 infection and/or disease severity. 

The S protein of SARS-CoV-2 virus (1273 amino acids (aa)) is a cloveshaped, type I Transmembrane (TM) protein and contains a large N-terminal extracellular domain (aa: 1-1212), a TM domain (TM; aa: 1213-1237), and a short C-terminal intracellular domain (aa: 1238-1273). It consists of a signal peptide (aa: 1-13), S1 subunit (aa: , and an S2 subunit (aa: 686-1273). 12 Moreover, the S1 subunit, responsible for binding the virus to host cell receptors, is composed of the N-terminal domain (NTD; aa: 18-305), the C-terminal receptor-binding domain (RBD; aa: 329-528), subdomain-1 (SD1; aa: 529-589), and SD2 (aa: 590-686; Figure 1b) . 12 The Receptor binding domain (RBD) is composed of two sub-domains including core subdomain composed of a β-sheet with 5 anti-parallel strands (β1, β2, β3, β4, and β7) in the inner side of the S protein and receptor-binding motif (RBM) from the outer side that extends from the core subdomain and consists of β5 and β6 strands. 13, 14 Angiotensin-converting enzyme 2 (ACE2), a membrane-bound zinc-containing enzyme expressed on many tissues including lungs, arteries, heart, kidneys, and intestines, has been demonstrated to be the main receptor for virus attachment to target cells. 15 Moreover, transmembrane protease serine 2 (TMPRSS2) has been proposed for S protein priming. 16 The RBM sub-domain of RBD, which forms a concave surface accommodating the N-terminal α-helix of the ACE2, is responsible for the virus binding to the ACE2. 12 The SARS-CoV virus also employs a similar mechanism to bind to host cells. On the other hand, the S2 subunit mediates fusion of the viral membrane with the host cell membrane allowing virus entry into target cells. 12 The S2 subunit consists of upstream helix (UH; aa: The S protein possesses two distinct conformational states including prefusion and postfusion conformations. The prefusion state of the S protein, composed of three S1 subunits and three S2 subunits, exists in two conformations: (1) a closed conformation in which all three protomers of RBDs are hidden and thus preventing RBD-ACE2 interaction (down conformation of RBD or receptor inaccessible state), (2) an open conformation in which one protomer of RBD is exposed allowing for RBD-ACE2 interaction (up conformation of RBD or receptor accessible state; Figure 1c) . Indeed, the up conformation of RBD provides the surface required for RBD interaction with the ACE2. 18, 19 Upon RBD binding to the ACE2, a conformational change in S protein structure occurs allowing for proteolytic cleavage of the protein at the S1-S2 boundary by host proteases. This converts S protein from the inactive prefusion state into the active postfusion state resulting in fusion of the viral membrane with the host cell membrane and entrance into the cell. 20 Though TMPRSS2 has been proven as the protease responsible for cleavage of the S protein, other host proteases such as trypsin were also recognized for their function in cleavage of the S protein. 21 Serum levels of antibodies specific for the spike RBD increase during the two weeks after onset of symptom. Higher levels of RBD-specific IgM have been shown in deceased COVID-19 patients rather than recovered patients. Also, a significant correlation was reported between RBD-specific IgG and IgM in both groups of patients. 22, 23 Furthermore, several studies found a positive correlation between serum neutralising capacity and disease severity in recovered patients with a wide range of disease severity (severe, moderate, mild, and asymptomatic). 24 29, 37, 38 Interestingly, neutralising MAbs targeting microbial antigens including COVID-19 lack these limitations and could therefore be considered as prophylactic/therapeutic alternative for the passive immunotherapy. 29, 37 Until now, no FDA/European Medicines Agency (EMA)-approved neutralising MAb for COVID-19 infection has entered in clinic, although a few number of the MAbs have been authorised for emergency use. 39, 40 Therefore, there is an urgent need for development of efficient neutralising anti-SARS-CoV-2 MAbs.

Given that the target epitope of a MAb is a crucial feature influencing its neutralising potency, herein, epitope specificity of the neutralising anti-SARS-CoV-2 MAbs already reported in the literature is delineated and discussed.

Identification of the target epitope of an antibody molecule is instrumental in development of effective prophylactic therapeutics and epitope-based vaccines as well as molecular elucidation of MAb neutralising activities. 41, 42 In a recent study, we performed epitope mapping of RBD in COVID-19 patients' sera using a panel of linear Table 1 ).

Given the crucial role of the RBD fragment of the S protein in binding of the virus to its receptor on target cells, it is not surprizing that a major proportion of neutralising anti-SARS-CoV-2 antibodies are directed against RBD ( Figure 3 , Table 1 ). Analysis of MAbs isolated from 25 COVID-19-infected patients showed that a majority of the nAbs recognized the S1 subunit of the virus. Removal of anti-RBD antibodies from sera of patients abolished their neutralising activity, highlighting the dependency of the neutralising activity to anti-RBD antibodies. 44 Accordingly, analysis of anti-SARS-CoV-2 MAbs for their neutralising activities against the virus showed that a large number of neutralising MAbs (67/70) recognise the RBD fragment. 45 In line with these findings, none of the non-RBD-binding MAbs showed neutralising activities in a different study. 46 Therefore, RBD seems to be the most crucial domain of the S protein for eliciting nAbs against the virus.

Considering that the up conformation of RBD provides the surface required for RBD interaction with the ACE2, it is assumed that neutralising anti-RBD MAbs should recognise S protein in the up conformation. 47 Barnes 49 We have recently generated a panel of mouse MAbs against RBD and observed that less than half of these MAbs display neutralising activity in pseudovirus-based neutralising assays, suggesting that recognising RBD is not necessarily sufficient for virus neutralisation (unpublished data). These findings indicate that RBD contains potent neutralising epitopes even if not all RBD epitopes contribute to virus neutralisation. Robbiani et al. identified three distinct neutralising epitopes on RBD including C144 and C101 in group 1; C121 and C119 in group 2 and C135 in group 3. They showed that groups 1 and 2 antibodies could bind to the RBD immunocomplexed with group 3 antibodies. Of note, groups 1 and 2 displayed different properties in binding specificity, so that group 1 could bind to the RBD immunocomplexed with group 2, but not vice versa. 32 These MAbs also revealed in vivo efficiency when administered either prophylactically or therapeutically (the antibody administration after the virus challenge). BD23, another nAb in their panel that bound the "down" conformation of RBD also competed with ACE2. 46 Moreover, the MAbs that interrupted RBD-ACE2 interaction imposed neutralising activity. Among the panel of human neutralising MAbs targeting the SARS-CoV-2 RBD isolated from patients at the acute phase, a subset of them inhibited binding to the human ACE2. 50 Also, a large number of neutralising anti-RBD MAbs obtained by Zost et al. interfered with RBD-ACE2 interaction. 51 The neutralising anti-RBD MAb, rRBD-15, inhibited binding of RBD to ACE2. 52 Neutralising anti-RBD MAb LY-CoV555, that prevented RBD-ACE2 interaction, was successfully used in a phase two clinical trial conducted on outpatients with mild or moderate COVID-19 disease. A single dose (2800 mg) administration of this MAb, also known as bamlanivimab, significantly improved clinical outcomes in patients by reducing severity of symptoms and viral load. 53, 54 However, bamlanivimab was revoked by FDA because of increased risk for treatment failure due to continued development of SARS-CoV-2 escape variants. 55 Altogether, these findings indicate that the most potent neutralising epitopes are the epitopes involved in RBD binding to the ACE2. Based on this notion, Liu et al. used an innovative approach to isolate neutralising anti-SARS-CoV-2 MAbs. In this approach, they initially isolated MAbs based on positive selection for RBD followed by negative selection of the isolated MAbs for a mutant RBD in which RBD residues that contribute to ACE2 binding were deleted. Indeed, these selections ensured isolation of neutralising MAbs including 4A2, 4A12, 4D5, and 4A10 accurately recognising RBD epitopes involved in ACE2 binding. 35 Cryo-electron microscopy studies showed that RBD interact with ACE2 through hydrogen and ionic bonds. Residues A475, N487, E484, and Y453 in RBM interact with residues S19, Q24, K31, and H34 of ACE2, respectively. Moreover, the residues Q498, T500, and N501 form hydrogen bonds of RBD interact with Y41, Q42, K353, and R357 of ACE2. 13, 14 In another study, RBD residues of Y505, Y449, G496, F497, and G502 bound to ACE2 residues including E37, D38, D38, K353, and G354, respectively. An ionic bond between P491 of RBD with K31 of ACE2 also contributed to RBD-ACE2 interaction. 56 The other RBD residues, including T470, F486, Y489, and Q493, were also identified as crucial residues of RBD for interaction with the ACE2. 14,57 Furthermore, the SARS-CoV-2 RBM provides a larger and more favourable contact interface with ACE2 in comparison to the SARS-CoV RBM. 58 In sum, RBD residues, including of S protein have also been reported. 33, 34, 74, 75 Sera from 40% of COVID-19-infected patients contained both neutralising anti-S1 and neutralising anti-S2 antibodies, and only 4% of patients with neutralising activity developed only anti-S2 antibodies. 44 Rogers et al. 

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We would like to thank Dr Mina Tabrizi for technical and linguistic revision of the manuscript. This study was partially supported by a grant from the National Institute for Medical Research Development (NIMAD) of Iran (Grant No. 993421) and ANRS COVID Sud project (ECTZ144757).

The authors declare no conflict of interest. 

Data sharing is not applicable to this article as no datasets were generated or analysed during the current study.

https://orcid.org/0000-0002-0576-3360Fazel Shokri https://orcid.org/0000-0001-5157-5603