Values are expressed as the percentage of the parent CD4+ or CD8+ population. . Thus, polyfunctional CD8+ T cells do appear to play a dominant role in protection against typhoid fever in humans. We have previously demonstrated that vaccination with Ty21a generates robust, polyfunctional CD4+ and CD8+ T-cell responses at the duodenal mucosa and in peripheral blood at day 18 ; however, whether early duodenal responses persist in the long-term has yet to be determined. An increased understanding of the longevity of immune responses both at the intestinal mucosa and in peripheral blood may allow us to identify both early and late functional correlates of vaccine-mediated protection, which are currently unknown. Here, we have assessed humoral immunity in peripheral blood and cellular immunity at the duodenal mucosa and in peripheral blood approximately 1.5?years following oral vaccination with Ty21a, and compared responses with those observed in a control group. These data provide a unique insight into the longevity of human mucosal and peripheral immune defence. 2.?Materials and methods 2.1. Ethical approval, recruitment, and study protocol All volunteers provided written informed consent. This study was approved by the BMS-707035 United Kingdom National Research Ethics Service (13/NW/0282). Eighteen healthy adult volunteers were enrolled into the study. Ten volunteers (5 males and 5 females; median age 24?years) were recruited to BMS-707035 an unvaccinated control group. Eight volunteers (3 males and 5 females; median Efnb2 age 23.5?years) who had previously been vaccinated with live-attenuated Typhi Ty21a (Vivotif; suspended in Dulbeccos PBS, quantified using the Miles and Misra technique, and killed by incubation at 95?C for 30?min) or 10?ng FliC protein flagella. One positive control well was stimulated with 100?ng staphylococcal enterotoxin B (SEB; Sigma-Aldrich). One negative control well was left untreated to adjust for non-antigen-specific background cytokine production. Cells were then incubated at 37?C in 5% CO2. After 2?h, 1 L brefeldin A (BD GolgiPlug; BD Biosciences) and 1 L monensin (BD GolgiStop; BD Biosciences) was added to each well, and the plate incubated for a further 16?h at BMS-707035 37?C in 5% CO2. 2.5. Flow cytometric analyses Following incubation, PBMCs and MMCs were washed, stained for viability and surface phenotype and, following fixation and permeabilisation, stained for intracellular cytokine production. Details of the antibodies that were used are presented in the Supplementary Materials and Methods. Cells were washed, resuspended and stored in the absence of light at 4?C until data were acquired using a LSR II flow cytometer (BD Biosciences). Compensation beads (BD Biosciences) were used to create compensation matrices and sequential cell isolation used to identify populations of interest (Fig. 2). Full details are presented BMS-707035 in the Supplementary Materials and Methods. Open in a separate window Fig. 2 Representative flow cytometric gating strategy for intracellular cytokine analysis. Dot plots are shown for cells isolated from (A) peripheral blood and (B) the duodenal mucosa. Dead cells were removed by staining for viability (LIVE/DEAD) and gating on the negative population. T cells were identified according to the expression of CD3. T cells were classified according to the expression of CD4 and CD8 and the expression of IFN-, TNF-, IL-2, IL-17A, and MIP-1 assessed in non-stimulated (NS) and in Ty21a, FliC and SEB stimulated samples. Values were expressed as the percentage of the parent CD4+ or CD8+ population. 2.6. Enzyme-linked immunosorbent assay (ELISA) Each well in flat-bottomed 96-well microtitre plates (Nunc) was coated with 100?L carbonate-bicarbonate buffer containing either 50?ng tests based on 1000 bootstrapped samples, as indicated. Statistical analyses were performed using SPSS v22 (IBM). Differences were considered significantly different if bootstrapped confidence intervals did not cross zero. 3.?Results 3.1. Recruitment and sampling Eight volunteers who had previously been vaccinated as part of past studies were recalled for sampling. The period between vaccination and sampling varied, with the median period between vaccination and sampling at 1.5?years (Table 1; range from 1.1 to 3.7?years). 3.2. Serum immunoglobulin specificity Ty21a-mediated protection is dependent upon the expression of LPS . Although humoral responses to LPS are not believed to confer protection at an individual level, in field trials, they have been shown to correlate with overall vaccine efficacy and are useful measures of immunogenicity , , . We measured levels of serum anti-LPS IgG and IgA in vaccinated volunteers and controls. At day 0 (baseline), levels of BMS-707035 anti-LPS IgG and IgA did not differ between vaccinated and unvaccinated volunteers (Fig. 1). Among vaccinated volunteers, levels of anti-LPS serum IgG were 6-fold higher at day 11 (T1), 5-fold higher at day 18.