Comprehensive mapping of SARS-CoV-2 peptide epitopes for development of a highly sensitive serological test for total and neutralizing antibodies

Abstract

Quantification of the anti-SARS-CoV-2 antibody response has proven to be a prominent diagnostic tool during the COVID-19 pandemic. Antibody measurements have aided in the determination of humoral protection following infection or vaccination and will likely be essential for predicting the prevalence of population level immunity over the next several years. Despite widespread use, current tests remain limited in part, because antibody capture is accomplished through the use of complete spike and nucleocapsid proteins that contain significant regions of overlap with common circulating coronaviruses. To address this limitation, a unique epitope display platform utilizing monovalent display and protease-driven capture of peptide epitopes was used to select high affinity peptides. A single round of selection using this strategy with COVID-19 positive patient plasma samples revealed surprising differences and specific patterns in the antigenicity of SARS-CoV-2 proteins, especially the spike protein. Putative epitopes were assayed for specificity with convalescent and control samples, and the individual binding kinetics of peptides were also determined. A subset of prioritized peptides was used to develop an antibody diagnostic assay that showed low cross reactivity while detecting 37% more positive antibody cases than a gold standard FDA EUA test. Finally, a subset of peptides were compared with serum neutralization activity to establish a 2 peptide assay that strongly correlates with neutralization. Together, these data demonstrate a novel phage display method that is capable of comprehensively and rapidly mapping patient viral antibody responses and selecting high affinity public epitopes for the diagnosis of humoral immunity.

Keywords: antibodies; epitope mapping; neutralizing; phage display; serological assay; serum ELISA.

Fig. 1

(A) Schematic for library generation. Codon optimized genes of spike FX1, envelope FX2, membrane FX3 and nucleocapsid FX4, were digested in 50–200 base pair fragments to generate SARS-CoV-2 epitope-displaying phage library. (B) Graph showing the size distribution of the ligated fragments where x-axis shows the number of nucleotides in each fragment sequenced and y-axis represents the number of reads. (C) Graph representing the number of times each base in the spike, envelope, membrane and nucleocapsid genes was sequenced, where x-axis is the nucleotide number and y-axis is the number of times it was read. (D) Selection of the phage library against commercial anti SARS-CoV-2 antibodies. Bar graph denotes mean recovered phage ± standard error measurement. The sample graph displays the frequency of nucleotides of positional nucleotides following against anti-spike antibodies. (E–H) Pie charts demonstrating distribution of sequenced epitopes following selection against respective labeled antibodies.

Fig. 2

(A) Schematic for selection against COVID-19 positive patient serum samples. (B) Heat map representing the relative abundance of the sequences recovered in each selection. Columns represent individual patient serum samples and rows represent sequences recovered in increments of 50 nucleotides, starting from the first nucleotide position. (C–F) Representative plots of total enrichment value for selected epitopes that were chosen for peptide synthesis.

Fig. 3

Differential enrichment of sequences in serum selections. Total enrichment against all selections in (A) spike and nucleocapsid proteins, (B) S1 (AA 1–320), RBD (321–543) and S2 (544–1276) domains and (C) RBM (438–506), FCS (680–687) and S2′ CS (811–818) sequences.

Fig. 4

(A) Heat map displaying anti-SARS-CoV-2 antibody binding for 23 COVID-19 positive and 1 negative human serum samples against the 14 enriched epitope peptides sequences. (B) Heat map displaying the anti-SARS-CoV-2 antibody binding for 45 COVID-19 positive human sera samples and 1 negative against 5 prioritized peptides PepS2-1, PepS2-2, PepS2-3, PepS2-4 and PepN-12 individually. (C) Plot representing the anti-SARS-CoV-2 antibodies for 43 COVID-19 positive and 4 negative human sera samples that were detected by the GALL-5P ELISA. Each dot represents the mean ELISA signal, and bars denote the mean of all patient samples. (D) Normalized count of anti-SARS-CoV-2 antibodies for 151 patient plasma samples and their comparison with the standard Abbott antibody detection ELISA. Gray lines indicate agreement; green Abbott, positive GALL-5P negative; and blue GALL-5P positive, Abbott negative.

Fig. 5

Peptide S2-1 and S2-4 correlation with serum neutralization. (A) Non-parametric linear correlation curve between ACE2 binding and the sum of the background corrected absorbance for peptides S2-1 and S2-4. (B) Box and whiskers plot comparing summed absorbance values for neutralizing (>20% inhibition of ACE2/RBD binding) and non-neutralizing COVID-19 positive serum samples. (C) Receiver-operating characteristic analysis for detecting neutralization using S2-1 and S2-4. (D) Table of diagnostic values for the peptide neutralization assay. ^****P < 0.0001.

Fig. 6

SPR to identify the binding affinity of peptide PepS2-1, PepS2-2, PepS2-3, PepS2-4 and PepN-12 with the antibodies present in COVID-19 positive patient serum samples. The plots show the binding response of (A) PepS2-1 with a 1:75 dilution of patient number CR0074, (B) PepS2-2 with a 1:300 dilution of patient number CR0025, (C) PepS2-3 with a 1:300 dilution of patient number CR0011, (D) PepS2-4 with a 1:300 dilution of patient number CR0011 and (E) PepN-12 with a 1:75 dilution of patient number CR0045. (F) The approximate serum k_on, k_off and K_D values for all the peptide-serum binding interactions. Edited by: Chica, Roberto.