4  SARS-CoV: from 2002 to 2003
SARS-CoV, first reported in 2002, belongs to the SARS-related coronavirus species that also includes many bat viruses.
Coronaviruses are spherical enveloped viruses with a diameter of 80 to 120 nm [17]. The viral capsid formed by the nucleoprotein (N) and the genome is contained in the envelope and is of helical symmetry. Three structural proteins are embedded on the surface of particles, the membrane protein (M), the envelope protein (E) and the protein spike (S). They give this aspect of crown in electron microscopy that inspired the name of this viral family.
The S protein of coronaviruses (~1255 amino acids) is a highly N-glycosylated type I transmembrane protein, from 180 to 200 kDa, that plays a major role in viral entry [18]. It insures a double function in viral entry by binding the cellular receptor before conformational changes and proceeding to the fusion of the viral envelope with the membranes of the target cells. S protein has a long N-terminal domain, a short C-terminal domain and assembles into homotrimers on the surface of the viral particle [19]. S protein has a decisive role in cellular tropism and for pathogenicity [20].
S protein of SARS-CoV is composed of two functionally distinct subunits: the globular S1 subunit (~aa 12–680) allows receptor recognition, whereas the S2 subunit (~aa 681–1255) facilitates membrane fusion and anchors S into the viral membrane. S1 is organized in four distinct domains A–D. Domains S1A and S1B may be used as a receptor-binding domain (RBD, aa 318–510) containing the highly conserved receptor-binding motif (RBM, aa 424–494) [21]. Moreover, RBD contains 3 functional glycosylation sites located at amino acids 318, 330 and 357, which are necessary for S expression but do not affect ACE2 binding [22]. S1B forms an extended loop on the viral membrane-distal side and is a hypervariable region [20]. S2 contains the fusion peptides (FP1 and FP2) [23], two heptad repeat regions (HR1, aa ~889–972 and HR2, aa ~1142–1193) and the well conserved transmembrane domain [24].
The mechanism of interactions with peptidases (aminopeptidase APN, ACE2, DPP4) as a cellular receptor for most coronaviruses is not known. Indeed, the binding of coronaviruses to their receptor is not enough and S protein on the surface of the virus must undergo proteolytic maturation. Coronaviruses do not use the catalytic activity of peptidases serving as receptors for this maturation but enter after the action of proteases located close to the receptors. The binding of SARS-CoV to its ACE2 receptor is followed by internalization and decrease in ACE2 enzyme activity on the cell surface, which may partly explain the severity of SARS-CoV infections [25].

4.1  Anti-S1 & RBD antibodies
Neutralizing Abs can fight against viral infections by blocking binding to cellular receptors or by interfering with viral fusion. Besides, in the case of enveloped viruses, the Abs can recruit effector cells or the complement, thus allowing the destruction of the infected cells or the lysis of the viral particles [6]. The S1 domain contains most of the epitopes recognized by nAbs during infection. The RBD located in this S1 domain would be the most important target for nAbs against SARS-CoV, MERS-CoV and the novel coronavirus SARS-CoV-2 [[26], [27], [28], [29]]. More specifically, certain secondary structures such as extended loops seem to be particularly immunogenic.
RBD of SARS-CoV is composed of 193 amino acids (N318-V510) within S protein. Five regions on the S glycoprotein of SARS-CoV (residues 274–306, 510–586, 587–628, 784–803 and 870–893), in which three first regions belong to S1 subunit in the CTD2 and CTD3 (C-terminal domain) and two later belong to HR1 domain of the S2 subunit, were predicted to be associated with a robust immune response to SARS-CoV [30]. Several specific-nAbs for SARS-CoV were discovered; unfortunately none of them are under clinical trial [31] (Fig. 2 ).
Fig. 2 S protein of SARS-CoV, MERS-CoV, SARS-CoV-2 with its subdomains are the target of antibodies. The antibodies cited in this review have different origins or techniques, and some of them have specific targets such as the receptor binding domain (RBD) containing the receptor binding motif (RBM), the heptad repeat regions (HR1 and HR2). Some antibodies could bind SARS-CoV and SARS-CoV-2. Background color: Black for SARS-CoV, dark grey for MERS-CoV, grey for SARS-CoV-2. SP: Signal peptide, FP: Fusion peptide, TM: Transmembrane domain, CP: Cytoplasm domain.
The human single-chain variable region fragment (scFv) antibody 80R blocked ACE-RBD interaction (epitope aa 324–503) [32] but some 80R-escape variants were found with the mutations mostly locating at lysine D480 [33]. The target epitope of 80R is not conserved in SARS-CoV-2 then it does not affect this novel virus [34]. Another nAb generated from a non-immune scFv library, named 256, could bind to an epitope of RBD but did not inhibit RBD binding. 256 is weak but specific to D480A-muted strains of 80R-escape variants. Some engineered broad nAbs, fm6 and fm39, also showed a high affinity to D480A-muted strains [33]. m396 (epitope aa 482–491) from human antibody fab library was cross-reactive [35] and used the D95 of m396 to form a salt bridge with R395 or an electrostatic interaction with D408 of SARS-CoV RBD [34]. m396 potently neutralized GD03 strain isolated from the second outbreak which resisted neutralization by 80R and S3.1. m396 also neutralized isolates from the first SARS-CoV outbreak (Urbani, Tor2) and from palm civets (SZ3, SZ16) [36]. Another human monoclonal antibody from scFv libraries CR3014 (epitope aa 318–510) showed potent effects on SARS-CoV neutralization; however, this virus can escape CR3014 upon P426L mutation in the S glycoprotein [37]. Same as 80R, m396 and CR3014 RBD-specific SARS-CoV antibodies failed to bind the S protein of SARS-CoV-2 [34]. CR3022, always from scFv libraries, could bind noncompetitively the SARS-CoV RBD (epitope aa 318–510) and had a synergistic neutralizing effect with CR3014 on SARS-CoV, even with the escaped P426L-muted variants [37].
By using Xenomouse in which mouse immunoglobulin genes were replaced by human immunoglobulin genes, 19 neutralizing mAbs bound S1 were found. 18 of them, 1B5 [38], 3A7, 3C7, 3F3, 3H12, 4A10, 4E2, 4G2, 5A5, 5A7, 5D3, 5D6, 5E4, 6B1, 6B5, 6B8, 6C1 and 6C2 bound RBD (aa 318–510) to avoid virus binding to the ACE2 receptor. The last one, 4D4, bound an epitope (aa 12–261) located on the N-terminal of RBD and inhibited post-binding event but not the RBD binding. Truncation of the first 300 amino acids of S1 blocked the trimerization and the fusion of S protein [39]. Synergistic effects in some SARS-CoV strains of 4D4 with other mAbs targeting S1 or S2 proteins such as 3C7 (S1), 1F8 (HR1) or 5E9 (HR2) were also reported [38,40]. The tri-combination of 3C7, 3H12 and 4D4 could effectively neutralize escape variants.
Other neutralizing human monoclonal Abs from transgenic mice were also reported. Ab 201 interfered with ACE2 binding by targeting S1 protein at the epitope aa 490–510. In contrast to 201, Ab 68 bound epitope aa 130–150 at the N-terminal of RBD but did not affect ACE2 binding [41].
F26 family of monoclonal Abs generated from mice (F26G9, F26G10, F26G18 and F26G19) showed neutralizing effect against SARS-CoV [42]. F26G18 binding RBD at the epitope aa 460–476 showed the most potent effect [43]. F26G19 (epitope aa 486–492 on RBD [44]) or 80R could also bind SARS-CoV by forming salt bridge R426 (RBD)-D56 or D480 (RBD)-R162, respectively [34].
SARS-CoV mouse antibody 240CD had a nanomolar affinity for the SARS-CoV-2 RBD but did not significantly block ACE-2 receptor binding [45]. As 240CD, CR3022 also has high affinity to SARS-CoV-2 and moreover, CR3022 had cross-neutralizing activity with this novel coronavirus [34].
The effects of neutralizing human monoclonal antibodies, S3.1, S215.13 [46] and S230.15, from Epstein-Bar virus transformation of human B cells were observed. As m396, S230.15 had potent inhibitory activity against isolates from the first, second SARS-CoV outbreaks and from palm civets (SZ3, SZ16) [36].

4.2  Anti-S2 antibodies
In contrast to RBD, the fusion domains are more difficult to access due to the tight folding of viral glycoproteins or the excessively transient exposure during the fusion stage. This is why few epitopes are described in these regions [6]. Interestingly, the S2 specific mAbs can neutralize pseudotyped viruses which expressing different S proteins containing RBD sequences of various clinical isolates [47]. The S2 protein is highly conserved. No mutation in HR1 was reported in an analysis of the amino acid sequences of the S protein from 94 SARS-CoV clinical isolates. Only few mutations in HR2, at amino acids K1163 or Q1183 for example, were observed in this study [47].
Some S2 epitopes inducing nAbs were reported. A peptide containing aa 1055–1192 can elicit neutralizing activity [48]. Two other proteins Trx-F3 and Trx-F9 containing linear antigenic determinants (Leu 803 to Ala 828 and Pro 1061 to Ser 1093, respectively) on the S2 domain were identified by using sera from convalescent SARS-CoV patients. Trx-F3 was capable of inducing nAbs in some animals [49].
Some human mAbs anti-HR1 (1F8, 1D12, 2A12, 2B12, 4A4, 4F9, 5C3, 6C9, 6H2) and anti-HR2 (1E10, 2D2, 2D6, 3A11, 3E10, 3H11, 5B9, 5B10, 5D7, 5E9, 5G8, 5G9, 6H1) were reported. With these Abs, the authors showed that the combination of HmAbs targeting different regions of the S protein would likely increase the broad neutralization against different isolates [47].
A human scFv antibody, named B1, showed a high affinity to an epitope (aa 1023–1189) on S2 protein. This antibody also showed potent neutralizing activities against SARS-CoV in vitro [50]. B1, 1F8 and 5E9 nAbs against epitopes on SARS-CoV S2 also showed effectiveness in neutralization [51].
The protective immunity by the time in patients after SARS-CoV natural infection was observed. After 6 years, the humoral immunity continuously decreased and eventually disappeared in most infected individuals. The IgG Ab could be an indicator of neutralizing Ab for the humoral response to SARS-CoV infection [52].