Fig. 6: THX mice vaccinated with flagellin mount a mature neutralizing antibody response to S. Typhimurium.

a–i, THX mice were injected i.p. with S. Typhimurium flagellin (50 μg in 100 μl alum) or nil (100 μl alum) on day 0, boosted (50 μg in 100 μl PBS or 100 μl PBS) on day 14 and euthanized on day 28. a, Total serum immunoglobulin concentration (μg eq ml⁻¹) and flagellin-specific human antibodies (RUs) in flagellin-vaccinated (n = 5) and non-vaccinated (nil, n = 5) THX mice measured by specific ELISAs (NS, not significant; ***P < 0.001, two-sided Student’s unpaired t-test). b,c, Dose-dependent antibody S. Typhimurium neutralizing activity of sera from flagellin-vaccinated THX mice (n = 5), non-vaccinated THX mice (n = 5) and healthy humans (n = 5); representative Luria-Bertoni (LB)-agar plates showing (residual) S. Typhimurium colony-forming units (CFUs) at each serum dilution. d, Survival of flagellin-vaccinated (n = 5) and non-vaccinated (n = 5) THX mice infected orally with S. Typhimurium (1 × 10⁵ CFUs, day 21; Kaplan–Meier curves, P = 0.0026, log-rank Mantel–Cox test). e, Flagellin-specific huCD19⁺ B cells, huIgG⁺ B cells, huIgA⁺ B cells and class-switched memory huCD19⁺CD27⁺ B cells in flagellin-vaccinated THX mouse spleen and healthy human blood, as identified by binding of Andy Fluor 647 (AF647)-labeled flagellin (AF647 alone as negative control); identification of huCD19⁺CD138⁺ PBs and huCD19⁻CD138⁺ PCs among pre-gated huCD27⁺CD38⁺ cells. f, Blood and spleen MZ huCD19⁺IgM⁺IgD⁺CD27⁺ B cells (41.9% ± 1.9% and 20.7% ± 3.0% huB cells, respectively) in flagellin-vaccinated THX mice (n = 4). g, Point mutation frequencies (changes per base) in sorted spleen huB cell huV_HDJ_H-Cγ (V1: 4.0 ± 1.4 × 10⁻³, V2: 8.6 ± 0.9 × 10⁻³, V3: 1.3 ± 0.2 × 10⁻²) and huV_HDJ_H-Cα1 (V1: 4.5 ± 0.9 × 10⁻³, V3: 9.3 ± 2.3 × 10⁻³, V4: 1.1 ± 0.4 × 10⁻²) transcripts of additional flagellin-vaccinated THX mice (n = 3, THX 450, 451, 452) depicted as scatterplots and pie charts. Each dot represents a single sequence and bars depict the mean with s.e.m. h, huV_HDJ_H-Cγ and huV_HDJ_H-Cα1 huB cell clones and intraclonal diversification in THX mice as in g, depicted by TreeMaps and phylogenetic trees. Individual rectangle or square (unique color) area reflects huB cell clone size. In THX 450, 451 and 452 mice, the three largest huV_HDJ_H-Cγ and huV_HDJ_H-Cα1 clones accounted for 18.6%, 9.2% and 18.4% of huV_HDJ_H-Cγ huB cells and 19.6%, 33.4% and 57.9% of huV_HDJ_H-Cα1 huB cells. i, Evolutive lineage of a huB cell clone that underwent SHM and CSR in a flagellin-vaccinated THX mouse (THX 450). huCD45⁺ cells were pre-gated in all FACS analyses. In the histograms (a and f), each dot represents an individual mouse and bars depict the mean with s.e.m.

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