In vitro cell culture model of human nasal-associated lymphoid tissue (NALT) to evaluate the humoral immune response to SARS-CoV-2 spike proteins

Abstract

To date, coronavirus disease 2019 (COVID-19) continues to be considered a pandemic worldwide, with a mild to severe disease presentation that is sometimes associated with serious complications that are concerning to global health authorities. Scientists are working hard to understand the pathogenicity of this novel virus, and a great deal of attention and effort has been focused on identifying therapeutics and vaccines to control this pandemic.

Methods: This study used tonsils removed from twelve patients who underwent an elective tonsillectomy in the ear, nose, and throat (ENT) department at Saudi Germany Hospital, Madinah, Saudi Arabia. Tonsillar mononuclear cells (MNCs) were separated and co-cultured in RPMI complete medium in the presence and absence of viral spike (S) proteins (the full-length S, S1 subunit, and S2 subunit proteins). Enzyme-linked immunosorbent assay (ELISA) was used to measure secreted antibody concentrations following stimulation.

Results: The in vitro human nasal-associated lymphoid tissue (NALT) cell culture model was successfully used to evaluate the humoral immune response against SARS-CoV-2- S protein. Significant (p < 0.0001, n = 12) levels of specific, anti-S IgG, IgM, and IgA antibody responses were detected in cells culture supernatanat folloeing stimulation with the full-length S protein compared with unstimulated cells. In contrast, S1 and S2 subunit proteins alone failed to induce a mucosal humoral immune response following tonsillar MNC stimulation.

Conclusion: We demonstrated a successful human NALT in vitro cell culture model that was used to study the mucosal humoral immune response to the SARS-CoV-2 S protein. This model could be advantageous for the in-depth study of cellular immune responses to the S protein and other viral antigens, such as nucleocapsid and matrix antigen. The S protein appears to be the important viral protein that may be able to mimic the natural infection process intranasally and should be studied as a component of a candidate vaccine.

Fig. 1

Significant antibody levels were detected for isotype class IgG in stimulated cells compared with the unstimulated negative control(NC) (p < 0.0001, n = 12). Data are expressed as the mean ± standard error of the mean (SEM). Comparisons between two groups were performed using paired t-tests.

Fig. 2

Significant antibody levels were detected for isotype class IgM in stimulated cells compared with the unstimulated negative control (NC) (p < 0.0001, n = 12). Data are expressed as the mean ± standard error of the mean (SEM). Comparisons between two groups were performed using paired t-tests.

Fig. 3

Significant antibody levels were detected for isotype class IgA in stimulated cells compared with the unstimulated negative control(NC) (p < 0.0001, n = 12). Data are expressed as the mean ± standard error of the mean (SEM). Comparisons between two groups were performed using paired t-tests.