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  • Subjects, vaccination and sample collection: This open, interventional study was approved by the Ethics Commission of the Canton St. Gallen (EKSG 15/085) and has been published as ISRCTN18360696 (DOI 10.1186/ISRCTN18360696). Before enrolment, subjects were informed about the purpose of the study and written informed consent was obtained in compliance with local and global regulations (S1 File and S2 File). A physical examination and a medical questionnaire to ensure the health status of the participants were performed. Healthy volunteers enrolled in the study were 18–50 years old, without any previous vaccination against typhoid fever or infection with S. Typhi, with a negative test for HIV infection and a negative pregnancy test. In order to minimize bias block randomization was used. Randomization sequence was created with a 1:3 allocation using random block sizes of 4. Twenty volunteers were enrolled for the study from these, five volunteers were assigned as controls (all females, mean age 29.4 ± 6.4 years old), and 15 volunteers were assigned to the vaccinated group (10 females and 5 males, mean age 35.9 ± 8.4 years old) (Table 1). One volunteer of the vaccinated group abandoned the study before completion (Fig 1). Three enteric-coated capsules each containing 2–10×109 live and 5–50×109 dead lyophilized Ty21a bacteria (Crucell) [24] were administered orally every other day with lukewarm water; the subjects of the control group received no treatment. Blood and stool samples were collected before vaccination (day 0) and on days 11, 25 and 60 after application of the first vaccine dose (Table 1) and tested in a blinded fashion in regards to study group and tested in a blinded fashion in regards to study group assignment. PBMCs were isolated using Vacutainer CPT Ficoll tubes (Becton Dickinson) and frozen at -150°C in FCS with 10% DMSO (Sigma-Aldrich). Feces were mechanically disrupted, diluted (50% w/v) in a PBS solution containing 10% FCS and a protease inhibitor cocktail (1:500, Sigma-Aldrich) and centrifuged at 14,000 rpm at 4°C for 10 min. Clarified supernatants were collected and stored at -70°C until IgA measurement by ELISA. This study took place in the Kantonsspital St. Gallen, St. Gallen, Switzerland. The recruitment of the volunteers for the study started on June 15th, 2015, and ended on September 15th, 2015. The consort checklist of information to include when reporting a randomized trial is shown in S3 File. Figure data removed from full text. Figure identifier and caption: 10.1371/journal.pone.0178669.g001 Study design.CONSORT flow diagram indicating the number of participants that have been included and have completed the study. Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0178669.t001 Study demographic details. # Mean ± standard deviation. Production of S. Typhi Omps: OmpC and F proteins were purified from S. Typhi ATCC 9993 as previously described [21, 22]. LPS content was determined using the limulus amebocyte lysate (LAL) assay (Charles River Endosafe Laboratories), and all batches were found to be negative with a detection limit 0.2 ng LPS/mg protein. Western blot analysis using anti-LPS polyclonal sera confirmed that LPS was not detectable by these means. Antibody titers against S. Typhi OmpC/F in sera were determined as previously described [22]. Briefly, high-binding 96-well polystyrene plates (Corning) were coated with 10 μg/ml of the protein preparation in 0.1 M carbonate-bicarbonate buffer, pH 9.5. Plates were incubated for 1 h at 37°C and then overnight at 4°C. Before use, plates were washed three times with PBS containing 0.05% Tween-20 (PBS-T) (Sigma–Aldrich). Non-specific binding was blocked with 5% non-fat dry milk diluted in PBS (PBS-M) for 1 h at 37°C. After washing, sera were diluted 1:40 and stool extracts 1:2, both in PBS-M and twofold serial dilutions were added to the wells. Plates were incubated for 1 h at 37°C, followed by four washes with PBS-T. After 1 h of incubation at 37°C with peroxidase-conjugated rabbit anti-human IgG (1:10,000) or IgM (1:5000) antibody (in PBS-M, Jackson Immuno Research), four washes with PBS-T, ortho-phenylenediamine (0.5 mg/ml; Sigma) in 0.1 M citrate buffer, pH 5.6, containing 0.08% H2O2 was used to develop the reaction. Optical density was read at 492 nm using an automated ELISA plate reader (Tecan). Antibody titers are given as -log2 dilution × 40. Antibody titers were defined as the highest dilution of the sample at which the OD was higher than the mean ± 3 SD of the negative sample values. Identification of antibody secreting cells and activated B cells by flow cytometry: Activated B cells (ABC) and antibody secreting cells (ASC) were measured in blood as previously described [25]. Briefly, 106 PBMCs were stained using the following: PerCP anti-human CD14, PerCP anti-human CD16 and APC-Cy7 anti-human CD20 from Biolegend; PerCP anti-human CD3, PE anti-human IgD, APC anti-human CD38, FITC anti-human CD19, and PECy7 anti-human CD71 from eBiosciences; the fixable viability stain 510 (e-Biosciences) was used to discriminate dead cells. Samples were stained for 30 min on ice with the viability dye, washed and stain for 20 min at 4°C with the required antibodies. Flow cytometric analysis was performed using a LSR-FORTESSA (Becton Dickinson). Data were analyzed using FlowJo software 10 (Tree Star, USA). In vitro stimulation and assessment CD4+ T cell responses: CD4+ T cell activation profile was assessed as previously described [26]. Briefly, 106 PBMCs in RPMI 1640 medium containing 5% FCS, 1% penicillin-streptomycin were stimulated with 10 μg/ml of S. Typhi OmpC/F or 50 μg/ml of tetanus toxoid for 24h at 37°C. After in vitro stimulation, surface staining was performed and the frequency of CD4+ T cells and intracellular expression of CD40L, IFN- γ and TNF was assessed by flow cytometry using the following antibodies: PerCP/Cy5.5 anti-human CD3, PE/Cy7 anti-human CD4 and FITC anti-human CD154, PE anti-human IFN- γ and APC anti-human TNF (all from Biolegend); the fixable viability stain 780 (e-Biosciences) was used to discriminate dead cells. Samples were analyzed using a FACS Canto flow cytometer (Becton Dickinson), and data were analyzed using FlowJo software version 10 (Tree Star, USA). Statistical analyses were performed with Graphpad Prism 5.0 (GraphPad Software Inc. USA) using two tailed Student’s t test with Welch’s correction. Statistical analysis was performed using one way ANOVA with Dunnett’s multiple comparison test for comparisons between individuals of the same group at different time points (pre- versus post-vaccination) Statistical significance was defined as p < 0.05. Raw data is available as S4 File.
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