Antigen-specific memory T cell responses after vaccination with an oral killed cholera vaccine in Bangladeshi children and comparison to responses in patients with naturally acquired cholera

Abstract

Young children, older children, and adults develop comparable levels and durations of immunity following cholera. In comparison, young children receiving oral killed cholera vaccines (OCV) develop a lower level and shorter duration of protection than those of older children and adults. The reasons for this are unclear. We investigated OCV-induced memory T cell responses in younger and older children and compared responses to those in children with cholera. We found that patients with cholera developed significant levels of toxin-specific effector memory T cells (T(EM)) with follicular helper and gut-homing characteristics. Older children (6 to 14 years of age) receiving two doses of OCV containing recombinant cholera toxin B subunit (rCTB) had more modest T(EM) responses with follicular helper and gut-homing characteristics, but younger vaccinees (24 to 71 months of age) did not develop T(EM) responses. The T(EM) response correlated positively with subsequent IgG memory B cell responses specific to rCTB in older vaccinees. Cytokine analyses indicated that cholera patients developed significant Th1, Th17, and Th2 responses, while older children receiving vaccine developed more modest increases in Th1 and Th17 cells. Younger vaccinees had no increase in Th1 cells, a decrease in Th17 cells, and an increase in regulatory T (Treg) cells. Our findings suggest that T cell memory responses are markedly diminished in children receiving OCV, especially young children, compared to responses following naturally acquired cholera, and that these differences affect subsequent development of memory B cell responses. These findings may explain the lower efficacy and shorter duration of protection afforded by OCV in young children.

Fig 1

Time line for vaccination, blood draws, and immunological assays. For vaccinees, d21 and d42 indicate 7 days and 4 weeks from the second dose of vaccine, respectively. For patients, d2 indicates the acute stage of infection; d7 and d30 indicate the early and late convalescent phases, respectively. (d, day; MBC, memory B cell).

Fig 2

Comparison of different toxin derivatives as T cell stimulatory antigens. Stimulation indices of patients (n = 9) at different time points are shown, with horizontal bars representing median stimulation indices. The toxin antigens include the following: recombinant cholera toxin B subunit (rCTB) (a gift from AM Svennerholm), the G33D variant of CTB (mCTB) (48), native CT, G33D variant of CT holotoxin (mCT) (48), double mutant LT (dmLT) (30), and membrane preparation of V. cholerae O1 (MP).

Fig 3

Comparison of different toxin derivatives as T cell stimulatory antigens in younger children receiving vaccine (n = 20), older children receiving vaccine (n = 20), and patients with naturally acquired cholera (n = 8). Mean stimulation indexes of T effector memory cells and their subtypes, with error bars representing standard errors of the means, are given. An asterisk (*) denotes a significant difference (P ≤ 0.05) from the baseline (day 2 for patients and day 0 for vaccinees). “∧” indicates a significant increase (P ≤ 0.05) in responses on day 7 for patients compared to day 21 responses in older vaccinees. The G33D variant of CT holotoxin (mCT), membrane preparation of V. cholerae O1 (MP), T effector memory cells (T_EM), and T follicular helper cells (T_FH) are analyzed.

Fig 4

Correlation between cholera toxin-specific early T cell responses and later B cell memory responses. Percentages of rCTB-specific IgG memory B cells in older vaccinees (n = 9) on day 42 correlated with stimulation indices of G33D variant of CT holotoxin (mCT) specific T effector memory (T_EM) cells on day 21.

Fig 5

Cytokines in cell culture supernatants of older (n = 20) and younger (n = 20) children receiving vaccine and patients (n = 6) stimulated with mCT. Cytokine concentrations at different time points are shown for each vaccinee and patient, with horizontal bars representing medians. A single asterisk (*) indicates P ≤ 0.05, and double asterisks (**) indicate P ≤ 0.01 for later time points compared to earlier time points. Cytokines were measured 7 days after the second dose of vaccine on day 14 (i.e., on day 21) or 7 days after presenting for care for naturally acquired cholera (day 7).