Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes coronavirus disease (COVID-19), is a novel betacoronavirus similar to SARS-CoV and the Middle East respiratory syndrome (MERS)-CoV.
SARS-CoV-2 infects host cells by binding to the angiotensin convertase 2 receptor (ACE2) on the cell surface through its trimeric spike (S) protein. SARS-CoV enters host human cells through endosomes, while SARS-CoV-2 enters through the cell surface and endosomes. The present study published in the journal “Cell Reports” aimed to understand the mechanisms of SARS-CoV-2 antibody neutralization to facilitate the development of therapeutic interventions for COVID-19.
Public antibody response against SARS-CoV-2 involving shared and structural elements in IGHV3-53 and IGHV3-66 heavy-chain V-gene has been reported. The target of this antibody class is a conserved receptor-binding domain (RBD) epitope region that also overlaps with the ACE2 binding site.
Scientists in this study have discovered monoclonal antibody (mAb) 910-30 possessing moderate neutralization capacity as a new member of the IGHV3-53/3-66 antibody class and have further investigated the molecular and genetic aspects of 910-30 and related antibodies.
mAb 910-30
A new member of the public IGHV3-53/3-66 antibody class was identified by screening the immune repertoire of a convalescent COVID-19 patient identified as donor 910. The serum from donor 910 potently exhibited S trimer recognition in enzyme-linked immunosorbent assays (ELISA) and SARS-CoV-2 serum neutralization in pseudovirus neutralization assays.
In order to select the donor 910 cryo-preserved peripheral blood mononuclear cells (PBMCs) for performing analysis, functional screening of yeast antibody display libraries was performed for their binding against SARS-CoV-2 S protein probes. mAb 910-30 was identified to produce a strong effect, and it was expressed as immunoglobulin (IgG) in HEK293 cells and subjected to neutralization assays. It exhibited an IC50 of 0.071 mg/mL against the SARS-CoV-2 pseudovirus and 0.142 mg/mL against the authentic SARA-CoV-2 virus.
RBD exists in the “up” position or “down” position and it has been found that the SARS-CoV-2 spike causes a pH-dependent conformational change in RBD where the “down” position is favored. ACE2 and IGHV3-53/3-66 class antibody binding requires RBD “up” conformation.
Cryo-electron microscopy revealed that 910-30 binds to SARS-CoV-2 S2P at its RBD in the “up” position. Structural modeling studies show that 910-30 recognizes the ACE2 binding site. 910-30 IgG was found to bind to RBD with a binding affinity (KD) of 230 pM, and as observed with other IGHV3-53/3-66 class antibodies, it competes with human ACE2 for RBD binding.
IGHV3-53/3-66 class antibodies, which are potent neutralizers, exhibit strong competition with ACE2 for binding to spike protein
IGHV3-53/ 3-66 antibody class members showed potent antibody neutralization against SARA-CoV-2 in neutralization assays. The molecular features of the neutralization process were further assessed using a panel of antibodies, 1-20 (a potent neutralizer), 910-30 (a moderate neutralizer), and B38 (a weak neutralizer). Analysis using ELISA revealed that the potent neutralizer 1-20 and the moderate neutralizer 910-30 exhibited high binding to full-length spike proteins. However, all members exhibited similar binding to RBD.
Neutralization assays using pseudo and authentic SARS-CoV-2 showed differences in IC50 values across the antibodies tested, suggesting the involvement of other factors apart from recognition of ACE2 binding site as determinants of neutralization potency. It was also found that the ability of the antibodies to compete with dimerized human ACE2 (dhACE2) to bind to SARS-CoV-2 S2P correlated with their neutralizing potencies. The most potent mAB (1-20) was sixfold more competitive than the moderate neutralizer 910-30 and 150 fold more competitive than B38, which is a poor neutralizer. Similar to previous findings with the IGHV3-53/3-66 antibody class, the panel of antibodies assessed exhibited high sequence similarity and low levels of somatic hypermutation (SHM), which is the process of accumulation of point mutations in the variable regions of immunoglobulin genes.
Sequence-structure analyses was performed and it identified that residues 27a/28[GDS]xSx{1,2}[FY] (kappa) and 29GY[KN] (lambda) in CDR-L1 influence RBD recognition. Aromatic or hydrophobic residues 28Val, 29Ile/Val, or 30/32Tyr30/32 present in light chains interact with 505Tyr in the RBD, which is a component of the overlapping binding region of ACE2 and IGHV3-53/3-66 antibody class.
The scientists also attempted to assess the prevalence of antibody class precursors in immune repertoires of healthy individuals. Antibody lineages containing anti-SARS-CoV-2 IGHV3-53/3-66 signature prevalent in 1 in 44,000 reported human antibody sequences. This high prevalence corroborates the finding that antibodies from this class have been found in multiple convalescent COVID-19 patients.
Native light and heavy chain pairings are essential for efficient antibody neutralization performance
The scientists further attempted to explore the characteristics of the antibodies that differentiated them as potent neutralizers and poor neutralizers. In the IGHV3-53/IGHV3-66 anti- SARS-CoV-2 antibodies, two primary heavy chain genes (IGHV3-53 and IGHV3-66) pair with at least 14 light-chain genes to create a diverse repertoire of antibodies. 12 Antibody variants were constructed by swapping the native heavy, and light chain combinations of 4 IGHV3-53/3-66 encoded mAbs 1-20, 910-30, B38, and 4-3 which is treated as control.
Except in the case of combinations of strongly potent neutralizing antibody (1-20) heavy chains with light chains, reduced neutralization was observed in almost all non-native heavy and light chain combinations. Similar to the native combination antibodies, the ability of non-native combination antibodies to compete with dhACE2 also showed a correlation with their neutralization potential, and all four heavy chains genes exhibited sequence similarity and low SHM. This suggests that native light and heavy chain pairings are essential for efficient antibody neutralization performance. However, even though high sequence similarity was observed in the heavy chains and light chains, non-native combinations reduced antibody neutralization.
Data from examining the in-silico models of non-native combination antibodies suggested that native heavy and light chain combinations were essential for the right complementary determining region orientation and RBD recognition.
RBD conformation determines IGHV3-53/3-66 antibody class spike protein recognition
The RBD should be in the “up” position to facilitate both IGHV3-53/3-66 class antibody and ACE2 binding. A change in pH enables the change between the two RBD conformations, and it has been found that endosomal pH between 4.5 -5.5 facilitates the “down” conformation. D614G mutations in SARS-CoV-2 variants also influence the RBD conformational changes.
The effect of pH-mediated RBD conformational change on IGHV3-53/3-66 class recognition was assessed using dhACE2 competition ELISA. The IGHV3-53/3-66 class members competed in a concentration-dependent manner with dhACE2 to bind to SARS-CoV-2 S2P spike or D614G S2P spike at endosomal pH. Single-cycle surface plasmon resonance studies were performed. The findings revealed that unlike the 910-30 and B38 antibodies with low neutralizing capacity, the highly potent mAB 1-20 did not show reduced recognition or binding to spike or RBD at endosomal pH.
Further, it was also seen at all the tested pH values, high mAb neutralization correlated with mAb affinities. Octet pH series analysis shows that, the potent neutralizer 1-20 bound to D614 S2P spike potently at reduced pH while the other less potent neutralizers showed reduced binding. This is in contrast to that seen with D614G S2P where all members exhibited strong binding and also effectively recognized the RBD “up” conformation at broad pH ranges.
The data from the studies suggest that the IGHV3-53/3-66 public antibody class performs neutralization by competing with ACE2 and that the conformation of RBD significantly determines their spike protein recognition.
Implications of the study
The present study aimed to understand recognition and interaction between IGHV3-53/3-66 antibody class and SARS-CoV-2 spike protein. The findings from this study will contribute towards improving health care for COVID-19 patients by enabling antibody discovery, identification of novel vaccine candidates and enhance knowledge for monitoring their immune system.
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