Fecal samples were immediately frozen at home by the subjects; mTOR inhibitor fecal extracts were subsequently prepared and stored at −70 °C [11]. Antibody levels in ALS specimens, fecal extracts and sera were analyzed by ELISA using plates coated with CFA/I, CS3, CS5, CS6, GM1 plus LTB or O78 LPS [9] and [11]. Fecal antibody levels were determined as the antigen-specific SIgA titer divided

by the total SIgA concentration of each sample [15]. LT toxin neutralization titers were determined using the Y1 adrenal cell assay [16]. Safety endpoints were defined as absence of any vaccine-related serious AEs and not significantly higher frequencies of vaccine-related severe AEs in each of the vaccine groups than in the placebo group. Primary immunogenicity endpoints

were defined as induction of immune responses in any of the vaccine groups in either of the primary assays proposed (fecal SIgA or ALS IgA) to at least four of the five primary vaccine components (CFA/I, CS3, CS5, CS6 and LTB). The magnitudes of immune responses (fold rises) were calculated as the post-immunization divided by pre-immunization antibody levels. Statistical differences were evaluated using t-test (magnitudes, ELISA results), Mann–Whitney test (magnitudes, toxin neutralization results) and Fisher’s exact test (frequencies) with Holm’s correction for multiple testing [17]. Differences between vaccine groups and the placebo group were evaluated using one-tailed statistical tests; all other statistical tests were two-tailed. P-values <0.05 were Proteasome inhibitor considered significant. Of 161 subjects screened, 129 were enrolled with 30–35 subjects in each of the four study groups (Table 1 and Supplementary material; Fig. 1). The age and gender distributions were comparable in Groups A, B and C, but more males

were randomized to Group D (Table 1). Overall, MEV administered alone and in Isotretinoin combination with dmLT was safe and well tolerated. No serious AEs were reported and the recorded AEs were mainly mild and not significantly different among any of the vaccine groups (B, C, D) and the placebo group (A). The addition of dmLT did not alter the safety profile. Altogether 89 solicited symptoms, deemed to be possibly or probably related to treatment, were recorded (Table 2); these AEs did not differ in either frequency or intensity between the different study groups. No significant changes of other clinical parameters, including serum chemistry and hematology, were observed in any of the volunteers. ASC responses against the primary vaccine antigens were studied by counting IgA ASCs by the ELISPOT method as well as by measuring antibody levels in lymphocyte secretions by the ALS method in the initial 43 randomized subjects. Since the frequencies of responses against all antigens were comparable using the two methods (data not shown), the ALS method was used in all subsequent study subjects as the sole measure of ASC responses.