Dynamics of T cellular immunity to nucleocapsid protein (N-protein) of SARS-CoV- 2 assessed through 9 months after infection

ABSTRACT

Background: The nucleocapsid protein (N-protein) of SARS-CoV- 2 regulates transcription, replication and packaging of the viral genome. Potentially, each of the structural proteins can act as an antigen for the production of specific antibodies and T-cell stimulation. This study evaluates T-cellular immunity to N-protein after infection caused by the SARS-CoV- 2 for up to 9 months. Method(s) Patients who had COVID-19 in 2020 were divided by severity into 3 groups mild (n = 41), moderate (n = 46), and severe (n = 29). Blood samples were taken 3 times at 3, 6 and 9 months after infection. Human peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation and stained with Tag-it Violet vital dye. PBMCs were stimulated with PepTivator SARS-CoV- 2 ProtN (pool of peptides, covering the complete sequence of N-protein);unstimulated cells served as negative control. PBMCs were cultured in AIM-V media for 5-7 days. Cell colonies were detected by microscopy. Relative content (%) of proliferating live T cells (CD3+7AAD-) were evaluated by flow cytometry. Samples were counted positive for antigen-specific T cells (ASCs) to N-protein when both proliferation was >1% compared with negative control and microcolony were observed. Result(s) There were 58.5% positive samples with ASCs to the N-protein in mild, 60.9% in moderate and 48.3% in severe group in 3 months after recovery from COVID-19. There was no difference between groups at this point (p > 0.05, chi2). After 6 months results were 26.7% positive samples in mild, 21.9% in moderate and 35.0 % in severe group. No differences between groups were observed after 6 months, but percentages of positive samples were less in mild and moderate groups when compared 3d and 6th months (p3-6 < 0.05, chi2). Drastic decreases of ASCs-positives in the mild group was observed after 9 month -only 4.2% (p6-9 < 0.05, chi2). Further decrease in moderate (25.0%) and severe (41.7%) groups was not observed after 9 months. Conclusion(s) T cell immunity to the N-protein of SARS-CoV- 2 was formed after mild, moderate and severe forms of COVID-19 with different effectiveness. The duration of immunity depends on the form of the disease and was stronger in severe group. By the 9th month, the number of positive samples in the group with mild COVID-19 was significantly lower when compared with the moderate and severe groups.