High-Resolution Linear Epitope Mapping of the Receptor Binding Domain of SARS-CoV-2 Spike Protein in COVID-19 mRNA Vaccine Recipients

Abstract

The prompt rollout of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccine is facilitating population immunity, which is becoming more dominant than natural infection-mediated immunity. In the midst of coronavirus disease 2019 (COVID-19) vaccine deployment, understanding the epitope profiles of vaccine-elicited antibodies will be the first step in assessing the functionality of vaccine-induced immunity. In this study, the high-resolution linear epitope profiles of Pfizer-BioNTech COVID-19 mRNA vaccine recipients and COVID-19 patients were delineated by using microarrays mapped with overlapping peptides of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The vaccine-induced antibodies targeting the RBD had a broader distribution across the RBD than that induced by the natural infection. Half-maximal neutralization titers were measured in vitro by live virus neutralization assays. As a result, relatively lower neutralizability was observed in vaccine recipient sera, when normalized to a total anti-RBD IgG titer. However, mutation panel assays targeting the SARS-CoV-2 variants of concern have shown that the vaccine-induced epitope variety, rich in breadth, may grant resistance against future viral evolutionary escapes, serving as an advantage of vaccine-induced immunity. IMPORTANCE Establishing vaccine-based population immunity has been the key factor in attaining herd protection. Thanks to expedited worldwide research efforts, the potency of mRNA vaccines against the coronavirus disease 2019 (COVID-19) is now incontestable. The next debate is regarding the coverage of SARS-CoV-2 variants. In the midst of vaccine deployment, it is of importance to describe the similarities and differences between the immune responses of COVID-19 vaccine recipients and naturally infected individuals. In this study, we demonstrated that the antibody profiles of vaccine recipients are richer in variety, targeting a key protein of the invading virus, than those of naturally infected individuals. Vaccine-elicited antibodies included more nonneutralizing antibodies than infection-elicited antibodies, and their breadth in antibody variations suggested possible resilience against future SARS-CoV-2 variants. The antibody profile achieved by vaccinations in naive individuals provides important insight into the first step toward vaccine-based population immunity.

Keywords: COVID-19; RBD; SARS-CoV-2; immunoserology; neutralizing antibodies; serology; spike; spike protein.

FIG 1

Total antibody titers targeting the RBD and neutralization of live SARS-CoV-2. (a) Anti-RBD IgG titers of vaccine (BNT162b2) recipients (n = 21) and COVID-19 patients (n = 19) were depicted. No significant difference in the level of total anti-RBD IgG titers was observed. (b) The half-maximal neutralization titers (NT50s) were remarkably lower in the vaccinated group than those in patients. (c) The anti-RBD IgG/NT50 ratio was plotted for both groups. Black horizontal bars indicate geometric mean with geometric standard deviation. Red dots denote the subset of samples chosen for the microarray analysis. For detailed information on subjects, see Table S1.

FIG 2

High-resolution linear epitope mapping of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. (a) Overlapping 15-mer peptides (shift by 3 amino acids) of the RBD was sequentially synthesized on a cellulose membrane. Sera of vaccine recipients and COVID-19 patients were incubated with the microarray, followed by the procedure mentioned in the Materials and Methods to detect the reactive peptides. (b) Heat map identifying peptides recognized by IgG, IgA, and IgM in sera of vaccine recipients (samples A to E) and COVID-19 patients (samples F to O). The signal of each peptide was normalized to the maximum signal in each subject. Raw signal the data can be found in Table S3.

FIG 3

Comparison of epitope profiles between the following two groups: BNT162b2 vaccine recipients (n = 5) and COVID-19 patients (n = 10). (a) Thin red lines denote peptide signals of individuals. Bold red lines depict the mean values of the peptide signals of the COVID-19 patient sera (n = 10). (b) Thin gray lines denote peptide signals of individuals. Bold black lines denote the mean values of the peptide signals of the vaccine recipient sera (n = 5). (c) Red arrows denote epitopes recognized in the sera of both groups. Black arrows denote epitopes identified only in the vaccine recipient sera. Designated peptide numbers are shown above the arrows.

FIG 4

Linear epitopes in the receptor binding domain (RBD) identified in both groups of vaccine recipients and COVID-19 patients. Angiotensin converting enzyme2 (ACE2), green; the RBD, tint blue; linear epitope T415 to F429 (peptide no. 33), cyan (a); linear epitope R457 to S477 (peptide no. 47 to 49), yellow (b); linear epitope V433 to N450 (peptide no. 39 to 40), blue (c).

FIG 5

Linear epitopes in the receptor binding domain (RBD) identified only in vaccine recipient sera. Angiotensin converting enzyme2 (ACE2) is shown in green. (a) The RBD is shown in tint blue. Linear epitope N334 to A348 (peptide no. 6), magenta. (b) SARS-CoV-2 spike trimer in one open, two closed (one RBD up, two RBD up) composition. Spike subunit 2 and N-terminal domain are in the same color, light blue, yellow, and tint blue. Up RBD is in dark blue. Down RBDs are in yellow. Linear epitope N334 to A348 has good accessibility in both the up and down composition of the RBD. (c) The RBD, green. Linear epitope S373 to L390 (peptide no. 19 and 20), orange.

FIG 6

Mutation peptide panels showed more reactivity to vaccine recipient sera than to patient sera. Heat maps identifying peptides carrying the single mutations, recognized by IgG, IgA, and IgM in sera of vaccine recipients (samples A to E) and COVID-19 patients (samples F to O). The signal of each peptide was calculated as a relative value to the maximum signal of each subject of 100.