A comparative study of antibody response, virus neutralization efficiency & metabolites in SARS-CoV-2-infected adults & children

Abstract

Background & objectives: COVID-19 has been a global pandemic since early 2020. It has diverse clinical manifestations, but consistent immunological and metabolic correlates of disease severity and protection are not clear. This study was undertaken to compare seropositivity rate, antibody levels against nucleocapsid and spike proteins, virus neutralization and metabolites between adult and child COVID-19 patients.

Methods: Plasma samples from naïve control (n=14) and reverse transcription (RT)-PCR positive COVID-19 participants (n=132) were tested for reactivity with nucleocapsid and spike proteins by ELISA, neutralization of SARS-CoV-2 infectivity in Vero cells and metabolites by ^[1]H nuclear magnetic resonance (NMR) spectroscopy.

Results: An ELISA platform was developed using nucleocapsid and spike proteins for COVID-19 serosurvey. The participants showed greater seropositivity for nucleocapsid (72%) than spike (55.3%), and males showed higher seropositivity than females for both the proteins. Antibody levels to both the proteins were higher in intensive care unit (ICU) than ward patients. Children showed lower seropositivity and antibody levels than adults. In contrast to ICU adults (81.3%), ICU children (33.3%) showed lower seropositivity for spike. Notably, the neutralization efficiency correlated with levels of anti-nucleocapsid antibodies. The levels of plasma metabolites were perturbed differentially in COVID-19 patients as compared with the naive controls.

Interpretation & conclusions: Our results reflect the complexity of human immune response and metabolome to SARS-CoV-2 infection. While innate and cellular immune responses are likely to be a major determinant of disease severity and protection, antibodies to multiple viral proteins likely affect COVID-19 pathogenesis. In children, not adults, lower seropositivity rate for spike was associated with disease severity.

Keywords: Antibody response; COVID-19; SARS-CoV-2; corona virus; nucleocapsid; serosurvey; spike.

Fig. 1

Production of recombinant nucleocapsid. Nucleocapsid was expressed and purified as described in the Material and Methods section. (A) The Coomassie-stained SDS-PAGE gel shows total cell lysate (TL), flow-through (FT) and elution sample (elute) containing the purified protein of about ~54 kDa. (B) The western blot shows reactivity with the nucleocapsid protein. The lanes are as in (A) and protein marker sizes (M) are in kDa.

Fig. 2

Antibody reactivity to proteins. The figure shows a comparison of reactivity of plasma samples with nucleocapsid and spike proteins in different participant groups. The Y-axis represents the reactivity of the COVID-19 plasma as times of the reactivity of naive control plasma for the same protein. P values represent the significance of the difference between two groups (P *<0.05, ** <0.01, *** <0.001, ns is non-significant). Graph A is a comparison of reactivity of all COVID-19 patients for spike and nucleocapsid. Graph B is a comparison of reactivity between male and female patients. Graph C is a comparison of reactivity between ICU and ward patients. Graph D shows a comparison of reactivity of adult and children. ICU, intensive care unit.

Fig. 3

Correlation between neutralizing antibody titres and antibody levels in paediatric patients. Antibody levels to (A) nucleocapsid and (B) spike in paediatric patients were analyzed for correlation with neutralizing antibody titres using the two-tailed Spearman rank-correlation test. The graph shows the best linear-fit relationship (red line) with R and P values.

Fig. 4

A representative ¹H NMR spectrum of naive control plasma. The ¹H NMR spectrum was obtained using pulse acquired method. The upper panel depicts expanded spectrum. Ala, alanine; Cit, citrate; Gln, glutamine; Ile, isoleucine, Lac, lactate; Leu, leucine; PC, phosphocholine; Phe, phenylalanine; Suc, succinate; Tyr, tyrosine; Val, valine.

Fig. 5

Plasma metabolite profiles in COVID-19 patients. The levels (mM) of metabolites were measured in plasma samples of naïve control (Normal), adult and paediatric (Ped) COVID-19 patients by ¹H NMR spectroscopy relative to formate added during the sample preparation. The symbol represents the level in individual participants and the bar represents mean±SD. P *<0.05, **<0.01 and ***<0.001 are for the comparison of adult COVID-19 patients with naïve controls. P ^#<0.05 and ^##<0.01 for comparison of COVID-19 children with COVID-19 adults. The metabolites on X-axis include glucose (Glc), lactate (Lac), alanine (Ala), glutamine (Gln), leucine (Leu), isoleucine (Ile), valine (Val), phenylalanine (Phe), tyrosine (Tyr), citrate (Cit), acetate (Ace), succinate (Suc), choline (Cho) and phosphocholine (PC).