Student’s t-test was employed to determine the significance of differences between the studied groups. p values <0.05 (*) were

considered to be significant. DNA fragments encoding bfpA (600 bp) and intimin (eae388–667) (840 bp), were amplified by PCR from EPEC (E2348/69) and ligated into the KpnI and BamHI sites of the pMIP12 vector under the control of the pblaF* promoter IGF-1R inhibitor ( Supplementary Figure); the constructs were named pMH12-bfpA and pMH12-intimin, respectively. The plasmids were electroporated into BCG and Smeg, and the resulting strains were examined for BfpA and intimin expression. Expression of both bfpA and intimin (eae) was confirmed by immunoblotting bacterial whole-cell extracts using anti-BfpA or anti-intimin antisera. As observed in Fig. 1A and B, the antisera specifically recognized bands of approximately 19.5 and 34 kDa, corresponding to BfpA and intimin, respectively, from both rBCG and rSmeg strains. No proteins were recognized by the antisera in whole-cell lysates from BCG or Smeg controls without the plasmid vectors ( Fig. 1A and B). C57BL/6 mice were immunized by oral gavage or intraperitoneal injection with 4 doses of 1 × 108 CFU in 200 μL of rBCG-bfpA, rSmeg-bfpA, rBCG-intimin or rSmeg-intimin at two-week intervals. As a mucosal adjuvant, SBA-15 Hydroxychloroquine solubility dmso silica was used. Control mice were immunized with

non-recombinant BCG or Smeg or with PBS following the same immunization schedule. A significantly higher level of anti-BfpA and anti-intimin IgA or IgG antibodies was observed in

both the feces and serum of mice immunized with rBCG or rSmeg as compared with that of serum collected in the groups that received non-recombinant BCG or Smeg or PBS (p < 0.001) ( Fig. 2A and B). Pre-immune sera and feces that were collected and pooled were evaluated, and presented no reactivity to BfpA or intimin (data not shown), suggesting the absence of anti-BfpA or anti-intimin antibodies prior to immunization. Our analysis of serum IgG subclass Rolziracetam responses also revealed that mice subjected to intraperitoneal immunization predominantly developed an IgG2a response, indicating a Th1-type cell response ( Fig. 2C). To evaluate the involvement of Th1-type cells on the immune responses induced by recombinant BCG-bfpA, BCG-intimin, Smeg-bfpA and Smeg-intimin, spleen cells were recovered 15 days after the final immunization and treated in vitro with the corresponding recombinant protein expressed in the vaccine used. We assayed the supernatants for the presence of the cytokines TNF-α, IFN-γ, IL-4 and IL-5. As is shown in Fig. 2A–C, anti-BfpA and anti-intimin, respectively, IgA and IgG antibodies were detected in feces and serum. Immunization with recombinant vaccine expressing BfpA induced higher production of IFN-γ, in vitro, by spleen cells (Fig. 3).