Pre-pandemic antibodies screening against SARS-CoV-2 and virus detection among children diagnosed with eruptive fevers

Abstract

Objectives: This study aims to assess the seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG antibodies against the spike (S) and nucleocapsid (NP) proteins, as well as neutralizing antibodies against the receptor-binding domain (RBD). Additionally, it aims to detect viral RNA of SARS-CoV-2 in pre-pandemic archival pediatric specimens collected before the announcement of the COVID-19 pandemic spread on March 20^th, 2020, in Morocco. The objective is to investigate the existence of pre-pandemic immunity to SARS-CoV-2.

Methods: We conducted a cross-sectional study, to analyze IgG antibody levels in a cohort of 106 pre-pandemic pediatric participants. Using an indirect enzyme-linked immunosorbent assay (ELISA), we measured the IgG levels against the S and NP proteins of SARS-CoV-2. Additionally, we staged a competitive ELISA assay to evaluate the neutralizing capability of these antibodies. We used reverse transcription polymerase chain reaction (rRT-PCR) to detect viral NP and ORF1ab genes of SARS-CoV-2 in oropharyngeal swabs. Moreover, we conducted on the same specimens a multiplexed RT-PCR to detect RNA of the most common 27 pathogens involved in lower respiratory tract infections.

Results: Among the 106 serum samples, 13% (nn = =14) tested positive for SARS-CoV-2 IgG antibodies using ELISA. Temporal analysis indicated varying IgG positivity levels across 2019. Neutralizing antibodies were found in 21% of the 28 samples analyzed, including two with high inhibition rates (93%). The SARS-CoV-2 RNA was detected using rRT-PCR in 14 samples. None of the samples tested positive for the other 27 pathogens associated with lower respiratory tract infections, using multiplexed RT-PCR.

Conclusion: Our study addresses the possibility, that COVID-19 infections occurred in Morocco before the recognized outbreak. On the other hand, some of the cases might reflect cross-reactivity with other coronaviruses or be influenced by previous viral exposures or vaccinations. Understanding these factors is crucial to comprehending pediatric immune responses to newly emerging infectious diseases.

Keywords: IgG; SARS-CoV-2; cross-reactive immunity; high-affinity antibodies; human-coronaviruses; humoral immunity; neutralizing antibodies.

Figure 1.

Study Design, Population, and Results. In this investigation, 106 serum and oropharyngeal swab samples were collected from the National Institute of Hygiene virology laboratory, forming the basis of the dataset analyzed in this study. Various tests were conducted, and comprehensive details on each test and its corresponding results are elaborated in the paper.

Figure 2.

(a) Serologic positivity in different periods: Immunoglobulin G presence for SARS-CoV-2 in pre-2019 children’s serums (n = 14). The different categories of index values are determined using the test recommendations. Index= (sample OD/average serum OD Threshold) * 10. (b) Number of individuals of each IgG status; positive, negative, and ambiguous for different times of sampling. Percentages of IgG-positive samples were calculated from the total samples collected within each period.

Figure 3.

The detected levels of anti-RBD neutralizing antibodies against SARS-CoV-2 and IgG levels against the S and NP in children’s sera. (a) Neutralizing Antibodies (NAbs) levels were assessed via a competitive ELISA neutralization test. The presence of neutralizing antibodies was determined as follows: If the inhibition signal exceeded 30%, it indicated the presence of NAbs. Conversely, if the inhibition signal was less than 30%, neutralizing antibodies are undetectable. (b) Comparison of the IgG levels between two tests: the neutralization test and the Vircell ELISA.