Modeling tonsil organoids for studying mucosal immune responses to SARS-CoV-2 vaccination

A key bottleneck in preclinical vaccine research is the lack of a human in vitro system that faithfully models the germinal center (GC) reactions, a critical process for generating protective antibodies. This gap hinders the accurate assessment of vaccine candidates. The tonsil is a key component of the mucosal immune system in the oropharynx, acting as its first-line immune sentinel. We established a novel human tonsil organoid platform and stimulated it with the chimpanzee adenovirus serotype 68-19 spike protein (AdC68-19S) SARS-CoV-2 vaccine candidate. Cellular immune phenotypes were analyzed by flow cytometry, while spatial organization was assessed via immunofluorescence. Functional immune responses were quantified using enzyme-linked immunosorbent assay, enzyme-linked immunospot assay, and pseudovirus neutralization tests to measure antigen-specific antibodies production and their neutralizing capacity. AdC68‑19S immunization elicited coordinated GC reactions in tonsil organoids, characterized spatially by the formation of discrete GC compartments, specifically an AICDA⁺ dark zone and a PD‑1⁺ light zone. This reorganization was accompanied by a marked expansion of key effector populations, including GC B cells and plasma cells. Functionally, the platform generated antibody‑secreting cells as well as antigen‑specific IgG and IgA antibodies, these antibodies demonstrated neutralizing capacity against the SARS‑CoV‑2 pseudovirus. The human tonsil organoids platform represents a paradigm shift in vitro immunology, establishing a system that recapitulates key features of adaptive immune responses induced by AdC68-19S vaccine. This approach provides a robust, physiologically relevant, and ethically viable model to advance our understanding of human immunity, oral immune barrier and accelerate vaccine development.