Immune profiling of SARS-CoV-2 epitopes in asymptomatic and symptomatic pediatric and adult patients

Abstract

Background: The infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has unpredictable manifestations of coronavirus disease (COVID-19) and variable clinical course with some patients being asymptomatic whereas others experiencing severe respiratory distress, or even death. We aimed to evaluate the immunoglobulin G (IgG) response towards linear peptides on a peptide array containing sequences from SARS-CoV-2, Middle East respiratory syndrome-related coronavirus (MERS) and common-cold coronaviruses 229E, OC43, NL63 and HKU1 antigens, in order to identify immunological indicators of disease outcome in SARS-CoV-2 infected patients.

Methods: We included in the study 79 subjects, comprising 19 pediatric and 30 adult SARS-CoV-2 infected patients with increasing disease severity, from mild to critical illness, and 30 uninfected subjects who were vaccinated with one dose of SARS-CoV-2 spike mRNA BNT162b2 vaccine. Serum samples were analyzed by a peptide microarray containing 5828 overlapping 15-mer synthetic peptides corresponding to the full SARS-CoV-2 proteome and selected linear epitopes of spike (S), envelope (E) and membrane (M) glycoproteins as well as nucleoprotein (N) of MERS, SARS and coronaviruses 229E, OC43, NL63 and HKU1 (isolates 1, 2 and 5).

Results: All patients exhibited high IgG reactivity against the central region and C-terminus peptides of both SARS-CoV-2 N and S proteins. Setting the threshold value for serum reactivity above 25,000 units, 100% and 81% of patients with severe disease, 36% and 29% of subjects with mild symptoms, and 8% and 17% of children younger than 8-years reacted against N and S proteins, respectively. Overall, the total number of peptides in the SARS-CoV-2 proteome targeted by serum samples was much higher in children compared to adults. Notably, we revealed a differential antibody response to SARS-CoV-2 peptides of M protein between adults, mainly reacting against the C-terminus epitopes, and children, who were highly responsive to the N-terminus of M protein. In addition, IgG signals against NS7B, NS8 and ORF10 peptides were found elevated mainly among adults with mild (63%) symptoms. Antibodies towards S and N proteins of other coronaviruses (MERS, 229E, OC43, NL63 and HKU1) were detected in all groups without a significant correlation with SARS-CoV-2 antibody levels.

Conclusions: Overall, our results showed that antibodies elicited by specific linear epitopes of SARS-CoV-2 proteome are age dependent and related to COVID-19 clinical severity. Cross-reaction of antibodies to epitopes of other human coronaviruses was evident in all patients with distinct profiles between children and adult patients. Several SARS-CoV-2 peptides identified in this study are of particular interest for the development of vaccines and diagnostic tests to predict the clinical outcome of SARS-CoV-2 infection.

Keywords: COVID-19; Children SARS-CoV-2 infection; Neutralizing antibodies; Peptide biomarkers; Peptide microarray; Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2).

Fig. 1

Barplot A showing the sum of signals for each protein. Samples labeled as control are samples incubated with the secondary antibody only (n = 3), the PDC are from SARS-CoV-2 positive children, INT indicates adults with mild/moderate symptoms and HOS identifies hospitalized patients with severe symptoms. Samples labeled as VAX-B1, VAX-B2 and VAX-B3 are each a pool of sera from 10 vaccinated subjects. Barplot B showing the fraction of signals above the threshold of 25'000. Samples are labelled as in A

Fig. 2

The heatmap depicts the humoral response of COVID-19 patients to the epitopes of N and S proteins of human coronaviruses with a signal threshold above 25'000. Samples labeled as PDC are children below 8 years of age, PDC.8-9p and PDC.10-12p are pools of sera from children in the range 8–9 and 10–12 years of age, respectively. Samples labeled as INT are from patients with mild or moderate symptoms, HOS are from patients with severe symptoms. Samples labeled as INT-B1, INT-B2 and INT-B3 are each a pool of sera from 10 vaccinated subjects. The color intensity designates the number of peptides of the indicated coronaviruses with a signal intensity threshold above 25'000

Fig. 3

A SARS-CoV-2 N protein antibodies cross-react with SARS-CoV N protein (panels A1, A2, A3) and S protein (panel A4) in adults; B SARS-CoV-2 N protein antibodies cross-react with SARS-CoV N (panel B1 and S B2 proteins in children