| Abstract: | ![]()
Cholera, caused by Vibrio cholerae, is a noninvasive dehydrating enteric disease with a high mortality rate if untreated. Infection with V. cholerae elicits long-term protection against subsequent disease in countries where the disease is endemic. Although the mechanism of this protective immunity is unknown, it has been hypothesized that a protective mucosal response to V. cholerae infection may be mediated by anamnestic responses of memory B cells in the gut-associated lymphoid tissue. To characterize memory B-cell responses to cholera, we enrolled a cohort of 39 hospitalized patients with culture-confirmed cholera and evaluated their immunologic responses at frequent intervals over the subsequent 1 year. Memory B cells to cholera antigens, including lipopolysaccharide (LPS), and the protein antigens cholera toxin B subunit (CTB) and toxin-coregulated pilus major subunit A (TcpA) were enumerated using a method of polyclonal stimulation of peripheral blood mononuclear cells followed by a standard enzyme-linked immunospot procedure. All patients demonstrated CTB, TcpA, and LPS-specific immunoglobulin G (IgG)and IgA memory responses by day 90. In addition, these memory B-cell responses persisted up to 1 year, substantially longer than other traditional immunologic markers of infection with V. cholerae. While the magnitude of the LPS-specific IgG memory B-cell response waned at 1 year, CTB- and TcpA-specific IgG memory B cells remained significantly elevated at 1 year after infection, suggesting that T-cell help may result in a more durable memory B-cell response to V. cholerae protein antigens. Such memory B cells could mediate anamnestic responses on reexposure to V. cholerae.Vibrio cholerae, the etiologic agent of cholera, causes an estimated 3 to 5 million cases of secretory diarrhea, resulting in over 100,000 deaths annually (24). Strains of V. cholerae can be differentiated serologically by the O side chain of the lipopolysaccharide (LPS) component of the outer membrane. Although more than 200 different serogroups have been isolated from the environment, the vast majority of strains that produce cholera belong to serogroup O1 or O139, both of which consist of noninvasive pathogens that colonize the mucosal surface of the small intestine (19). V. cholerae O1 biotype El Tor is currently the predominant cause of cholera globally and in Bangladesh.The mechanisms of protective immunity to cholera are not known. Volunteer and epidemiologic studies demonstrate that clinically apparent infection with V. cholerae confers long-term protection of at least 3 years against subsequent disease (7, 12, 13). The best-studied marker of protective immunity is the vibriocidal antibody, a complement-dependent bactericidal antibody; however, there is no vibriocidal antibody titer at which complete protection is achieved (20). Furthermore, the vibriocidal response wanes rapidly, and it is hypothesized that the vibriocidal antibody may reflect other longer-lasting, protective immune responses occurring at the mucosal surface (3).Patients with cholera develop additional humoral immune responses to several antigens including cholera toxin subunit B (CTB), toxin-coregulated pilus major subunit A (TcpA), and LPS (1). We have recently shown that serum anti-CTB immunoglobulin A (IgA) antibody levels are also associated with protective immunity independent of the vibriocidal antibody on exposure to cholera, but serum IgA levels also wane rapidly after infection (10). Although levels of serum anti-LPS and anti-CTB IgG antibodies increase considerably after infection, these have not been shown to correlate with protection from V. cholerae infection in humans (8, 10).Cholera patients develop substantial mucosal immune responses after infection. These can be measured by the transient increase of antigen-specific IgA antibody-secreting cells (ASC) in the circulation. The ASC assay quantifies lymphocytes that are activated in the gut-associated lymphoid tissue (GALT) when they transiently circulate in blood before rehoming to mucosal effector sites (6, 16, 17). These predominantly gut-homing ASC peak in the circulation between 5 and 10 days after onset of illness but are no longer detected during late convalescence as they return to populate the GALT (1, 11). Because V. cholerae is a noninvasive pathogen, it is hypothesized that protective immunity is derived from the activity of the secretory IgA system of the GALT (14, 22, 23). Volunteer studies of subjects receiving CTB orally demonstrate local and systemic generation of anti-CTB IgA antibodies that peak at 7 days following ingestion but decline to baseline by 15 months; however, these volunteers mount anamnestic responses with a rapid return to peak mucosal antibody titers in as few as 3 days after subsequent challenge with oral CTB (22, 23). It is thus hypothesized that protection from cholera may be mediated by rapid anamnestic responses of memory B cells in the GALT to V. cholerae antigens.In this study, we examined the memory B-cell immune responses to V. cholerae infection, using a polyclonal stimulation method to enhance the detection of memory B cells in the circulation by inducing their proliferation and differentiation into antibody-secreting plasmablasts (4, 5). A standardized two-color enzyme-linked immunospot (ELISPOT) assay allows for the quantification of small numbers of circulating V. cholerae antigen-specific memory B cells as a proportion of total memory B cells (2, 4, 5, 21). Using this system, we have previously shown that cholera patients develop CTB-specific IgG memory B-cell responses that persist for at least 3 months after infection (11). The present study further characterizes memory B-cell responses to CTB, TcpA, and LPS for both IgA and IgG isotypes for a period of 1 year following acute infection and examines differences between the memory B-cell responses to the T-cell-dependent protein antigens CTB and TcpA and the T-cell-independent antigen LPS. |
