Immunodominant Linear B-Cell Epitopes of SARS-CoV-2 Spike, Identified by Sera from K18-hACE2 Mice Infected with the WT or Variant Viruses

Abstract

SARS-CoV-2 surface spike protein mediates the viral entry into the host cell and represents the primary immunological target of COVID-19 vaccines as well as post-exposure immunotherapy. Establishment of the highly immunogenic B-cell epitope profile of SARS-CoV-2 proteins in general, and that of the spike protein in particular, may contribute to the development of sensitive diagnostic tools and identification of vaccine` candidate targets. In the current study, the anti-viral antibody response in transgenic K18-hACE-2 mice was examined by implementing an immunodominant epitope mapping approach of the SARS-CoV-2 spike. Serum samples for probing an epitope array covering the entire spike protein were collected from mice following infection with the original SARS-CoV-2 strain as well as the B.1.1.7 Alpha and B.1.351 Beta genetic variants of concern. The analysis resulted in distinction of six linear epitopes common to the humoral response against all virus variants inspected at a frequency of more than 20% of the serum samples. Finally, the universality of the response was probed by cross-protective in vitro experiments using plaque-reducing neutralization tests. The data presented here has important implications for prediction of the efficacy of immune countermeasures against emerging SARS-CoV-2 variants.

Keywords: COVID-19; K18-hACE2; SARS-CoV-2; epitope mapping; linear epitopes.

Figure 1

Humoral response against the spike glycoprotein among surviving mice and human patients. Endpoint values at 405 nm (−650 nm) representing the binding of naïve mouse sera (n = 12; open circles) and the sera obtained from mice surviving the infection with WT (n = 15; black circles), B.1.1.7 (n = 17; blue circles) and B.1.351 (n = 22; red circles) SARS-CoV-2 isolates towards the spike glycoprotein [28]. Human patients’ sera recovering from COVID-19 was also included (n = 6; green circles). Statistical differences were determined by a non-parametric Kruskal–Wallis with Dunn’s post hoc test. * p < 0.05, *** p < 0.005, ns = nonsignificant. The average background value based on 10 independent measurements was 0.0087 (range 0.004–0.014).

Figure 2

Heatmap plot of the binding results for the 240 peptides with various sera samples. Absorbance values at 405 nm marking the binding of convalescent serum antibodies to each one of the 240 peptides is presented by a heatmap. A graded 3-color scale of black (minimum—10th percentile), dark blue (midpoint—50th percentile) and yellow (maximum—98th percentile) was used. The peptide number is indicated on the top and the related spike domains depicted on the bottom. A hierarchal clustering using the average linkage method [29] was applied to the samples for each group in separate (top to bottom: naïve, COVID-19 patients, WT-infected mice, B.1.1.7-infected mice and B.1.351-infected mice).

Figure 3

Spatial localization of the two most abundant cross-variant peptides on the spike trimer. Top (A) and side (B) Semi-transparent surface view of SARS-CoV-2 spike trimer (PBD 7C2l stripped off the 4A8 antibody). The N-terminal domain (NTD) is brown-colored and the receptor binding domain (RBD) is shown in purple. Other parts of the spike are colored in gray. The spatial localization of epitope 134QFCNDF139 is shown in green and epitope 501NGVGYQP507 is highlighted in dark pink. Black arrows mark the surface exposed epitope (when the RBD assumes the “up” position) and black-framed white arrows mark this epitope within the “closed” inaccessible position of the RBD. All analyses were performed by using the PyMol Molecular Graphics System (Version 1.7 Schrödinger, LLC (Portland, OR, USA)).

Figure 4

Cross-neutralization of SARS-CoV-2 WT, B.1.1.7 and B.1.351 variants. The in vitro neutralization capacity of serum samples collected from WT, B.1.1.7 or B.1.351 SARS-CoV-2 infected mice, was assessed by plaque reduction neutralization test (PRNT). The neutralization ability of each sample (at indicated dilutions) was assessed in duplicates against SARS-CoV-2 WT (black), B.1.1.7 (blue) and B.1.351 (red) variant. (A) Results are expressed as percent inhibition compared to control without serum. (B) Summary of the calculated NT50 values (dilution-1). NT50 <100 indicates low neutralization capacity, emphasized by gray shading.