Fig. 5: r.i. vaccination imbues durable colonization and infection protection at 60 and 200 days after boost.

a, Experimental schematic. The diagram was created using BioRender. b,c, Survival of WT mice after infection with C. difficile R20291 at 60 (b) and 200 (c) days after boost. d,e, Weight loss at 2 d.p.i. (d) and 3 d.p.i. (e) after infection at 60 and 200 days after boost. f–h, Percentages of T cells (CD8⁺ (f), CD8⁺ T_RM (g) and CD4⁺ T_RM (h) LPLs) in the LPL compartment of the colon. i–t, Enumeration of total C. difficile R20291bacteria (vegetative cells and spores; i–k and o–q) and spores (l–n and r–t) in the faeces of mice 60 (i–n) and 200 (o–t) days after boosting, for the dmLT only (i,l,o,r), toxins + dmLT (j,m,p,s) and toxins + NTAs + dmLT (k,n,q,t) groups. For i–t, P values were calculated relative to the control at the same timepoint. Individual lines correspond to individual mice. The limit of detection is shown by the dotted line: 500 CFU per g faeces. For d–h, data are mean ± s.e.m. Statistical significance was calculated using log-rank Mantel–Cox tests (b and c) and one-way ANOVA with Tukey’s correction (d–t; for i–t, each day was analysed individually). For b–e, n = 15 per group; f–h, n = 5 per group; i–t, n = 10 per group; decreases in n were due to animal mortality during infection. Individual datapoints are represented and were pooled from two independent experiments. FACS, fluorescence-activated cell sorting.

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