Marginal zone B cells mediate humoral immunity in artificial and natural active immunization against COVID-19

Background The role of marginal zone B (MZB) cells in humoral immunity against SARS-CoV-2, particularly in the context of vaccination and natural infection, remains incompletely understood. MZB cells are critical for rapid antibody responses to pathogens, yet their contribution to vaccine-induced immunity versus natural infection, especially in vulnerable populations like the elderly has not been fully elucidated. Methods MZB-deficient (FcμR ^-/- ) mice were immunized with SARS-CoV-2 spike protein-loaded virus-like particles (VLPs) to simulate infection. Mouse serum antibodies and BCR distribution were analyzed by ELISA, flow cytometry, and single-cell sequencing. In human studies, peripheral blood samples were collected from two groups: vaccinated volunteers (artificial active immunity) and unvaccinated elderly with Omicron infection (natural active immunity). Volunteers' blood was taken at pre-vaccination (Pre), 7 days after the second dose (Post 1), 6 months post-vaccination (Post 2), and 7 days after the third booster (Post 3). Elderly patients' blood was collected during acute and recovery phases. Samples were analyzed using hypersensitive chemiluminescence immunoassay, protein microarray, and flow cytometry. Results MZB deficiency restricted the splenic BCR repertoire diversity in mice. In vaccinated volunteers, total SARS-CoV-2 antibodies, IgG, IgM, and receptor-binding domain (RBD)-specific neutralizing antibodies peaked at Post 1, declined at Post 2, and significantly increased at Post 3. Vaccine-induced IgM/IgG against RBD/S protein was confirmed via protein microarray. Compared to Pre, Post 3 saw expanded MZB (CD21 ⁺ CD23 ^- ) and Tfh (ICOS ⁺ CXCR5 ⁺ ) cells in PBMCs. In unvaccinated elderly with Omicron infection, MZB cells in PBMCs rose significantly during recovery versus acute phase. Conclusions MZB cells mediate humoral protection against COVID-19 vaccination and natural infection. However, the elderly natural infection group had a delayed response, highlighting the need to prioritize vaccination protection for this population.