Fig. 1: Unadjuvanted nasal booster elicits generation of antigen-specific IgA⁺ plasma cells at the respiratory mucosa.

a, Schematic of the experimental setup showing C57BL/6J mice immunized i.m. with mRNA–LNPs encoding the full-length SARS-CoV-2 spike protein at day 0, i.n. boosted with an unadjuvanted SARS-CoV-2 spike protein at day 14, followed by lung, BALF and serum collection at days 16, 18, 21 and 28 post-priming i.m. b, Number of extravascular CD44⁺Tet⁺CD4⁺ T cells, CD44⁺Tet⁺CD8⁺ T cells, RBD⁺CD38⁻GL7⁺ GC-like B cells, RBD⁺CD38⁺GL7⁻IgM⁻IgD⁻ class-switched (CS) B cells and RBD⁺IgA⁺ ASCs in the lungs of naive C57BL/6J mice (day 0 (D0), n = 9); intramuscular prime-only mice (D16^P, n = 10) and intranasal booster-only mice (D16^S, n = 10) at day 16 post-priming i.m. and P + S mice at day 16 (D16^P+S, n = 10), day 18 (D18^P+6, n = 10), day 21 (D21^P+S, n = 8) and day 28 (D128^P, n = 10) post-priming i.m., analyzed by flow cytometry as in a. c, ELISA measurements of SARS-CoV-2 spike S1 subunit-specific IgA and IgG in the BALF and serum from mice as in b. AUC, area under the curve. d, Schematic of experiment showing Aicda-tdTomato-Prdm1-EYFP mice immunized i.m. with mRNA–LNPs at day 0, treated with 10 mg of tamoxifen via oral gavage at days 4, 8 and 12, boosted i.n. with a spike protein at day 35, followed by lung collection at day 42 post-priming. e, Immunofluorescence of IgA staining (left) and quantification (right) of colocalization of tdTomato⁺EYFP⁺ and tdTomato⁺EYFP⁺IgA⁺ cells in the lungs of intramuscular prime-only (D42^P, n = 5) or P + S (D42^P+S, n = 5) Aicda-tdTomato-Prdm1-EYFP mice at day 42 post-priming i.m. (day 7 post-boosting i.n.) as in d. Scale bar, 100 μm. f, Representative flow cytometry plots and frequency of EYFP⁺, tdTomato⁺ and tdTomato⁺EYFP⁺ cells in the lungs of intramuscular prime-only (D42^P, n = 7) and P + S (D42^P+S, n = 8) Aicda-tdTomato-Prdm1-EYFP mice analyzed by flow cytometry at day 42 post-priming i.m. as in d. g, Representative flow cytometry plots and frequency of tdTomato⁺ cells among EYFP⁺IgA⁺ cells in the lungs of mice as in f (mean ± s.e.m.). Statistical significance was calculated using one-way ANOVA (b,c,e) and unpaired Student’s t-test (f,g). Tukey’s multiple comparisons (b,c,e) were performed: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NS, not significant. Data were pooled from three (b,c) independent experiments or represented (e–g) two independent experiments. Values indicated as zero represent the absence of detectable cells (b,e–g).

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