Vaccination against ETEC diarrhea in Bangladeshi participants and the influence of age on vaccine induced immune responses

Abstract

Enterotoxigenic Escherichia coli (ETEC) is one of the leading bacterial causes of diarrhea in children in low and middle income countries including Bangladesh as well as in travelers. No effective ETEC vaccine is yet available, but efforts are ongoing to develop such a vaccine since the burden of ETEC diarrhea is very high. Most oral vaccines induce lower immune responses in young children in low-resource countries than in developed countries. Several strategies are currently used to enhance ETEC vaccine immunogenicity, including addition of the mucosal adjuvant double mutant heat labile toxin (dmLT). The main objectives of this dissertation were to analyze mucosal immune responses induced by the oral inactivated ETEC candidate vaccine ETVAX in Bangladeshi adults and the influence of age on vaccine induced immune responses. In preparations to test ETVAX in Bangladesh, optimal sampling time points for evaluation of mucosal immune responses after oral vaccination were determined. Adults, toddlers, young children and infants were given two doses of the licensed oral cholera vaccine Dukoral. Cholera toxin B subunit (CTB) and Vibrio cholerae membrane protein specific IgA in antibodies in lymphocyte supernatant (ALS) specimens, IgA and IgG in plasma and secretory IgA (SIgA) in fecal samples were evaluated 4/5 and 7 days after each vaccination using ELISA. After the first dose, most adults and toddlers developed high and comparable anti- CTB ALS IgA responses on day 5 and 7, while very few young children responded. After the second dose, highest ALS responses were observed on day 5 in all age groups and the responses declined on day 7. In contrast, plasma antibody responses were high on both day 5 and day 7 after the second dose. Fecal SIgA responses were increased on day 7 in young children and infants after the second dose. To evaluate the safety and immunogenicity of ETVAX in Bangladeshi adults, participants were given two doses of ETVAX±dmLT or placebo (n=15/group) and samples were collected according to the optimal sampling schedule previously identified. To facilitate the analysis of ALS responses against multiple vaccine antigens in small sample volumes for subsequent analyses in younger age groups, a highly sensitive electrochemiluminescence (ECL) assay was established. Magnitudes of IgA ALS responses determined by ECL and ELISA correlated very well and ECL was used as a primary readout method for ALS responses against colonization factors (CFs) and LTB in the trial. ETVAX±dmLT was safe and well tolerated in adults. After the first dose, IgA ALS responses against each of the primary antigens were detected in 87-100% and after the second dose in 100% of vaccinees. Plasma IgA responses against different CFs and LTB, as determined by ELISA, were observed in 62-93% and 100% of vaccinees, respectively. No statistically significant adjuvant effect of dmLT on responses was detected but the overall antigenic breadth of the plasma IgA responses tended to favor the adjuvanted vaccine. The adjuvant effect of dmLT was also studied in vitro in peripheral blood mononuclear cells from infants and adults. dmLT enhanced IL-17A responses in both infant and adult cells. Blocking experiments showed that the IL-17A enhancing effect may at least partly be mediated by IL1β

in both age groups. dmLT significantly enhanced IL-1β responses to low amounts of ETVAX vaccine in cells from infants but not from adults.

Collectively, the results and methodological improvements obtained from this dissertation have facilitated the design and execution of an ongoing trial of the ETVAX vaccine in young children and infants. The results demonstrate that the ETEC vaccine is safe in adults and induce strong mucosal and plasma antibody responses. The in vitro results show that dmLT has potential to enhance IL-17A responses in both infants and adults and may particularly promote IL-1β responses to low ETVAX doses in infants. These results are important for the clinical evaluation of ETVAX as well as other mucosal vaccines in adults and infants.