Cholera Toxin–Specific Memory B Cell Responses Are Induced in Patients with Dehydrating Diarrhea Caused byVibrio choleraeO1

Abstract

Background Infection with Vibrio cholerae induces durable immunity against subsequent disease, a process hypothesized to reflect anamnestic immune responses at the intestinal mucosa. The presence of antigen-specific memory B cells may therefore be a more direct measure of protection than serum antibody responses. Methods We measured immunoglobulin (Ig) G memory B cells specific to cholera toxin B subunit (CTB) in 14 patients up to 90 days after V. cholerae O1 infection, by polyclonal stimulation of peripheral blood mononuclear cells followed by standard enzyme-linked immunospot assay. Results All patients generated CTB-specific IgG memory B cell responses by day 30 (mean, 0.10% of total circulating IgG memory B cells; range, 0.037%−0.28%), which persisted to day 90 (mean, 0.07%; range, 0.003%−0.27%). In contrast, circulating CTB-specific IgG antibody-secreting cells and serum vibriocidal and anti-CTB antibody responses peaked on day 7 and declined to undetectable or significantly lower levels by day 90. Conclusions CTB-specific IgG memory B cell responses are detectable in the circulation at least 3 months after V. cholerae O1 infection and remain measurable even after serum antibody titers have declined to undetectable or considerably lower levels. This suggests that antigen-specific memory B cells may be an important long-term marker of the immune response to cholera.

Antigen-Specific Memory B-Cell Responses in Bangladeshi Adults after One- or Two-Dose Oral Killed Cholera Vaccination and Comparison with Responses in Patients with Naturally Acquired Cholera

“…We then added 100 l of plasma (diluted 1:100 for CtxB and 1:25 for LPS in 0.1% bovine serum albumin in phosphate-buffered saline-Tween)/well and used horseradish peroxidase-conjugated secondary antibodies to human IgG or IgA (Jackson Immunoresearch, West Grove, PA), developing the samples with ortho-phenylene diamine (Sigma, St. Louis, MO) in 0.1 M sodium citrate buffer (pH 4.5) and 0.1% hydrogen peroxide. We read the plates kinetically at 450 nm for 5 min (12) and normalized the maximal rate of change in optical density in milli-absorbance units per minute across plates by calculating the ratio of the test sample to a standard of pooled convalescent-phase serum from previously infected cholera patients included on each plate. We considered individuals with a Ն2-fold increase in anti-CtxB and LPS responses as responders.…”

“…We performed memory B-cell assays using PBMC recovered on days 0, 30, 90, 180, 270, and 360, using a previously described method (5,6,8,12). For this assay, we placed 500,000 PBMC/well in 24-well cell culture plates (BD Biosciences, San Jose, CA) containing culture medium optimized to stimulate antigen-independent proliferation and differentiation of memory B cells into ASC.…”

“…The cholera patients were adults presenting with diarrhea at the ICDDR,B hospital and with a positive microbiologic culture of stool for V. cholerae O1 (8,12,25). Of the vaccinees, 92% (n ϭ 55) completed follow-up to day 360; of the cholera patients, 53% (n ϭ 37) completed follow-up to day 360.…”

“…We have recently shown that anti-V. cholerae memory B-cell responses occur following natural cholera (8,12) and that these responses may be important mediators of the duration of protection against subsequent cholera. Therefore, we were interested in whether oral cholera vaccination also induced anti-V. cholerae memory B-cell responses, whether these responses differed following one or two doses of vaccine, and whether the responses differed from those seen following natural disease.…”

Study of Avidity of Antigen-Specific Antibody as a Means of Understanding Development of Long-Term Immunological Memory after Vibrio cholerae O1 Infection

“…However, the currently available licensed vaccines give protection for between 2 to 3 years, shorter than that following natural infection (4,5). Several markers of protection against cholera on exposure, such as plasma IgA against V. cholerae O1 lipopolysaccharide (LPS) (6), vibriocidal antibodies (7,8), and memory B cell responses to cholera antigens (9,10), have been suggested as potential markers of protective immunity. However, plasma antibody responses decrease in the 6 to 9 months after cholera, before protective immunity wanes, and while memory B cell responses persist longer, assay of these responses is more complicated.…”