Dissection of Antibody Responses of Gam-COVID-Vac-Vaccinated Subjects Suggests Involvement of Epitopes Outside RBD in SARS-CoV-2 Neutralization

Abstract

Millions of people have been vaccinated with Gam-COVID-Vac but fine specificities of induced antibodies have not been fully studied. Plasma from 12 naïve and 10 coronavirus disease 2019 (COVID-19) convalescent subjects was obtained before and after two immunizations with Gam-COVID-Vac. Antibody reactivity in the plasma samples (n = 44) was studied on a panel of micro-arrayed recombinant folded and unfolded severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins and 46 peptides spanning the spike protein (S) and by immunoglobulin G (IgG) subclass enzyme-linked immunosorbent assay (ELISA). The ability of Gam-COVID-Vac-induced antibodies to inhibit binding of the receptor-binding domain (RBD) to its receptor angiotensin converting enzyme 2 (ACE2) was investigated in a molecular interaction assay (MIA). The virus-neutralizing capacity of antibodies was studied by the pseudo-typed virus neutralization test (pVNT) for Wuhan-Hu-1 and Omicron. We found that Gam-COVID-Vac vaccination induced significant increases of IgG1 but not of other IgG subclasses against folded S, spike protein subunit 1 (S1), spike protein subunit 2 (S2), and RBD in a comparable manner in naïve and convalescent subjects. Virus neutralization was highly correlated with vaccination-induced antibodies specific for folded RBD and a novel peptide (i.e., peptide 12). Peptide 12 was located close to RBD in the N-terminal part of S1 and may potentially be involved in the transition of the pre- to post-fusion conformation of the spike protein. In summary, Gam-COVID-Vac vaccination induced S-specific IgG1 antibodies in naive and convalescent subjects in a comparable manner. Besides the antibodies specific for RBD, the antibodies induced against a peptide close to the N-terminus of RBD were also associated with virus-neutralization.

Importance, Applications and Features of Assays Measuring SARS-CoV-2 Neutralizing Antibodies

“…Thus MIAs can only measure whether antibodies can prevent the interaction of RBD and ACE2. In this context, it must be mentioned that epitopes located outside RBD in the S protein may also play a role in virus-neutralization [ 45 , 46 , 47 , 48 , 49 , 53 , 126 , 127 ]. It is thus possible that certain subjects lacking antibodies blocking the RBD–ACE2 interaction may still contain NAbs.…”

“…Regarding mice, it is important to create mouse strains expressing human ACE2 to render mice susceptible for infection. The animal models show variability regarding susceptibility to different virus strains, severity of infection, symptoms and pulmonary pathology [ 127 , 128 , 129 , 130 , 131 , 132 ]. Currently the Syrian hamster model is one of the most frequently used animal models for SARS-CoV-2 infections [ 128 , 129 ].…”

Application of a Biomimetic Nanoparticle-Based Mock Virus to Determine SARS-CoV-2 Neutralizing Antibody Levels in Blood Samples Using a Lateral Flow Assay

“…A recent plaque reduction neutralization assay revealed that the neutralization activity against S1 is almost twice as high as against RBD, suggesting the presence of additional neutralizing antibodies that do not target RBD itself . Another study reported the plasma’s inability to inhibit the ACE2–RBD interaction while simultaneously showing virus neutralization activity in a pseudovirus neutralization assay . A comparative analysis of a neutralization assay with fully replicative SARS-CoV-2 and a pseudovirus with S protein on the virus’s surface demonstrated that the neutralization activity mainly depends on the S protein .…”

“… 23 Another study reported the plasma’s inability to inhibit the ACE2–RBD interaction while simultaneously showing virus neutralization activity in a pseudovirus neutralization assay. 24 A comparative analysis of a neutralization assay with fully replicative SARS-CoV-2 and a pseudovirus with S protein on the virus’s surface demonstrated that the neutralization activity mainly depends on the S protein. 25 Consequently, trimeric S proteins in surrogate neutralization assays may provide more accurate neutralization levels than in currently utilized systems.…”