Characterization of SARS-CoV-2 Convalescent Patients’ Serological Repertoire Reveals High Prevalence of Iso–RBD Antibodies

Abstract

While our understanding of SARS-CoV-2 pathogenesis and antibody responses following infection and vaccination has improved tremendously since the outbreak in 2019, the sequence identities and relative abundances of the individual constituent antibody molecules in circulation remain understudied. Using Ig-Seq, we proteomically profiled the serological repertoire specific to the whole ectodomain of SARS-CoV-2 prefusion-stabilized spike (S) as well as to the receptor binding domain (RBD) over a 6-month period in four subjects following SARS-CoV-2 infection before SARS-CoV-2 vaccines were available. In each individual, we identified between 59 and 167 unique IgG clonotypes in serum. To our surprise, we discovered that ∼50% of serum IgG specific for RBD did not recognize prefusion-stabilized S (referred to as iso–RBD antibodies), suggesting that a significant fraction of serum IgG targets epitopes on RBD inaccessible on the prefusion-stabilized conformation of S. On the other hand, the abundance of iso–RBD antibodies in nine individuals who received mRNA-based COVID-19 vaccines encoding prefusion-stabilized S was significantly lower (∼8%). We expressed a panel of 12 monoclonal antibodies (mAbs) that were abundantly present in serum from two SARS-CoV-2 infected individuals, and their binding specificities to prefusion-stabilized S and RBD were all in agreement with the binding specificities assigned based on the proteomics data, including 1 iso–RBD mAb which bound to RBD but not to prefusion-stabilized S. 2 of 12 mAbs demonstrated neutralizing activity, while other mAbs were non-neutralizing. 11 of 12 mAbs also bound to S (B.1.351), but only 1 maintained binding to S (B.1.1.529). This particular mAb binding to S (B.1.1.529) 1) represented an antibody lineage that comprised 43% of the individual’s total S-reactive serum IgG binding titer 6 months post-infection, 2) bound to the S from a related human coronavirus, HKU1, and 3) had a high somatic hypermutation level (10.9%), suggesting that this antibody lineage likely had been elicited previously by pre-pandemic coronavirus and was re-activated following the SARS-CoV-2 infection. All 12 mAbs demonstrated their ability to engage in Fc-mediated effector function activities. Collectively, our study provides a quantitative overview of the serological repertoire following SARS-CoV-2 infection and the significant contribution of iso–RBD antibodies, demonstrating how vaccination strategies involving prefusion-stabilized S may have reduced the elicitation of iso–RBD serum antibodies which are unlikely to contribute to protection.

Characteristics and functions of infection-enhancing antibodies to the N-terminal domain of SARS-CoV-2

“…These results are consistent with a prior study of FcR-mediated effector function by an NTD-binding mAb (21) and further suggest that NTD-binding antibodies that display infection-enhancing properties in vitro also have the capacity to activate FcγRIIIa and may therefore have the potential to mediate ADCC. These results also align with data demonstrating additional FcγRmediated effector functions associated with these same serum-derived mAbs, including antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent complement deposition (ADCD) (30).…”

“…We sought to examine the functionality of serum antibodies from two of the eight COVID-19 convalescent patients in our cohort. These two patients (3546, 3548) had moderate and severe illness, respectively, and were evaluated within the context of a study of their serum antibody repertoires to SARS-CoV-2 (30). A total of twelve unique mAbs, representing the most abundant serum clonotypes in each subject, were recombinantly expressed in Expi293 cells (30).…”

“…These two patients (3546, 3548) had moderate and severe illness, respectively, and were evaluated within the context of a study of their serum antibody repertoires to SARS-CoV-2 (30). A total of twelve unique mAbs, representing the most abundant serum clonotypes in each subject, were recombinantly expressed in Expi293 cells (30). These antibodies are fully glycosylated and capable of engagement with FcγR.…”

“…These antibodies are fully glycosylated and capable of engagement with FcγR. Five were found to bind the NTD, of which four enhanced infection with SARS-CoV-2 PVs in vitro (30) ( Fig. 5A ).…”

“… A. IgG mAbs representing major serum clonotypes from two donors (3546, 3548) were characterized for SARS CoV-2 binding specificity, variable heavy chain (VH) germline use, percent VH somatic hypermutation (IGHM SHM) and categorized as lasting (detected at Visit 3), intermediate (detected at 2, but not Visit 3), or transient (detected only at Visit 1) as described (30). Functional activity was determined using a VSV-SARS CoV-2-S (Wuhan) pseudovirus (PV) assay to measure neutralization or infection-enhancement as reported (30). B. Serum mAbs binding NTD (46 S-1, 48 S-4, 48 S-5, 48-S-M3-5), RBD (46 S-2, 48 S-2), or control mAbs (4A8, C105, CR3022) were tested for activation of FcγRIIIa in a luciferase reporter-cell assay with recombinant S from Wuhan, Beta, Delta or Omicron (BA.1, BA.2).…”