Antibody and T Cell Immune Responses to SARS-CoV-2 Peptides in COVID-19 Convalescent Patients

Abstract

Identifying immunogenic targets of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is critical to advance diagnostic and disease control strategies. We analyzed humoral (ELISA) and T-cell (ELISpot) immune responses to spike (S) and nucleocapsid (N) SARS-CoV-2 proteins as well as to human endemic coronavirus (eCoV) peptides in serum from convalescent coronavirus disease 2019 (COVID-19) patients from Tatarstan, Russia. We identified multiple SARS-CoV-2 peptides that were reactive with serum antibodies and T cells from convalescent COVID-19. In addition, age and gender associated differences in the reactivity to S and N protein peptides were identified. Moreover, several SARS-CoV-2 peptides tested negatively correlated with disease severity and lung damage. Cross-reactivity to eCoV peptides was analyzed and found to be lower in COVID-19 compared to controls. In this study, we demonstrate the changing pattern of immunogenic peptide reactivity in COVID-19 serum based on age, gender and previous exposure to eCoVs. These data highlight how humoral immune responses and cytotoxic T cell responses to some of these peptides could contribute to SARS-CoV-2 pathogenesis.

Keywords: COVID-19; SARS-CoV-2; antibody humoral immune response; peptides; spike protein.

FIGURE 1

Schematic presentation of SARS-CoV-2 (A) S and (B) N peptide locations. Green – peptides containing both, B and T cell, epitopes. Blue – peptides containing only T cell epitopes. Orange – peptides containing only B cell epitopes.

FIGURE 2

COVID-19 serum reactivity with SARS-CoV-2 peptides. Antibody reactivity was analyzed using ELISA. Peptides were adsorbed on a 384-well plate and probed with serum samples. Data is displayed for: (A) – peptides with increased reactivity in convalescent COVID-19 samples compared to controls collected in 2020; (B) – peptides with increased reactivity in convalescent COVID-19 samples compared to controls collected in 2015; (C) – peptides with increased reactivity in acute COVID-19 samples compared to controls collected in 2015 and 2020. Asterisks indicate statistically significant differences between reactivity to SARS-CoV-2 peptides (p < 0.05, Kruskal-Wallis test).

FIGURE 3

Reactivity of COVID-19 serum samples with SARS-CoV-2 peptides depending on time after convalescence. Antibody reactivity was analyzed using ELISA. Peptides were adsorbed on a 384-well plate and probed with serum samples. (A) Reactivity to SARS-CoV-2 peptides in COVID-19 serum samples grouped as: ≤3 months, 4–6 months and ≥7 months. Asterisks indicate statistically significant differences between reactivity to SARS-CoV-2 peptides depending on time since infection (p < 0.05, Kruskal-Wallis test with Benjamini-Hochberg adjustment). (B) Detailed dynamics of COVID-19 antibody reactivity with SARS-CoV-2 peptides. Red line- mean OD₄₅₀ value in control; Dotted red line – mean ± standard error of mean of OD₄₅₀ values in control; Blue line – local regression line (LOESS) of OD₄₅₀ value in COVID-19; Gray area – standard error of mean for LOESS of OD₄₅₀ values in COVID-19.

FIGURE 4

COVID-19 serum reactivity with SARS-CoV-2 peptides depending on sex of patient. Antibody reactivity was analyzed using ELISA. Peptides were adsorbed on a 384-well plate and probed with serum samples. Asterisks indicate statistically significant differences between reactivity to SARS-CoV-2 peptides depending on sex of the patient (p < 0.05, Kruskal-Wallis test with Benjamini-Hochberg adjustment).

FIGURE 5

Reactivity of COVID-19 serum to SARS-CoV-2 peptides depending on age of patients. Patients were divided into young (<45 years old) and old (≥45 years old) groups. Antibody reactivity was analyzed using ELISA. Peptides were adsorbed on a 384-well plate and probed with serum samples. Only reactivity to peptides which significantly differed from that in corresponding age control is presented. (A) Age groups dependent difference to SARS-CoV-2 peptides reactivity in COVID-19 serum. Asterisks indicate statistically significant differences between reactivity to SARS-CoV-2 peptides depending on age of the patient (p < 0.05, Kruskal-Wallis test with Benjamini-Hochberg adjustment). (B) Age based distribution of reactivity to SARS-CoV-2 peptides in COVID-19 patient serum. Colored lines – linear regression of OD450 value.

FIGURE 6

Correlation analysis of SARS-CoV-2 peptide antibody reactivity with clinical and demographic features of COVID-19. Spearman’s rank test was used to analyze correlation between reactivity with SARS-CoV-2 peptides and clinical presentation and characteristics of patients. Asterisks indicate statistically significant correlations (p < 0.05 with Benjamini-Hochberg adjustment).

FIGURE 7

Analysis of T cell reactivity to SARS-CoV-2 peptides using ELISpot. PBMCs were collected from COVID-19 convalescent individuals and incubated with COVID-19 T cell peptides in anti–human IFN-γ mAb-coated 96-well plates. Spots were detected with biotinylated anti-human IFN-γ antibodies after 48 h. The number of spots in negative control wells was subtracted from the number of spots in stimulated wells. All experiments were done in duplicate. (A) Analysis of T cell reactivity to SARS-CoV-2 peptides in all COVID-19 patients. (B) Analysis of T cell reactivity to SARS-CoV-2 peptides in younger (<45 years old) and older (≤45 years old) COVID-19 patients. Asterisks indicate statistically significant differences between reactivity to SARS-CoV-2 peptides depending on age of the patient (p < 0.05, Kruskal-Wallis test).

FIGURE 8

Correlation analysis of COVID-19 serum reactivity with eCoV peptides and demographic and clinical COVID-19. Spearman’s rank test was used to analyze correlation between reactivity with eCoV peptides and clinical presentation and characteristics of patients. Asterisks indicate statistically significant correlations (p < 0.05 with Benjamini-Hochberg adjustment).