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.

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.

Comparison of memory B cell, antibody-secreting cell, and plasma antibody responses in young children, older children, and adults with infection caused by Vibrio cholerae O1 El Tor Ogawa in Bangladesh.

Children bear a large component of the global burden of cholera. Despite this, little is known about immune responses to cholera in children, especially those under 5 years of age. Cholera vaccine studies have demonstrated lower long-term protective efficacy in young children than in older children and adults. Memory B cell (MBC) responses may correlate with duration of protection following infection and vaccination. Here we report a comparison of immune responses in young children (3 to 5 years of age; n = 17), older children (6 to 17 years of age; n = 17), and adults (18 to 60 years of age; n = 68) hospitalized with cholera in Dhaka, Bangladesh. We found that young children had lower baseline vibriocidal antibody titers and higher fold increases in titer between day 2 and day 7 than adults. Young children had higher baseline IgG plasma antibody levels to Vibrio cholerae antigens, although the magnitudes of responses at days 7 and 30 were similar across age groups. As a surrogate marker for mucosal immune responses, we assessed day 7 antibody-secreting cell (ASC) responses. These were comparable across age groups, although there was a trend for older age groups to have higher levels of lipopolysaccharide-specific IgA ASC responses. All age groups developed comparable MBC responses to V. cholerae lipopolysaccharide and cholera toxin B subunit at day 30. These findings suggest that young children are able to mount robust vibriocidal, plasma antibody, ASC, and MBC responses against V. cholerae O1, suggesting that under an optimal vaccination strategy, young children could achieve protective efficacy comparable to that induced in adults.

Characterization of anti-Salmonella enterica serotype Typhi antibody responses in bacteremic Bangladeshi patients by an immunoaffinity proteomics-based technology.

Salmonella enterica serotype Typhi is the cause of typhoid fever and a human-restricted pathogen. Currently available typhoid vaccines provide 50 to 90% protection for 2 to 5 years, and available practical diagnostic assays to identify individuals with typhoid fever lack sensitivity and/or specificity. Identifying immunogenic S. Typhi antigens expressed during human infection could lead to improved diagnostic assays and vaccines. Here we describe a platform immunoaffinity proteomics-based technology (IPT) that involves the use of columns charged with IgG, IgM, or IgA antibody fractions recovered from humans bacteremic with S. Typhi to capture S. Typhi proteins that were subsequently identified by mass spectrometry. This screening tool identifies immunogenic proteins recognized by antibodies from infected hosts. Using this technology and the plasma of patients with S. Typhi bacteremia in Bangladesh, we identified 57 proteins of S. Typhi, including proteins known to be immunogenic (PagC, HlyE, OmpA, and GroEL) and a number of proteins present in the human-restricted serotypes S. Typhi and S. Paratyphi A but rarely found in broader-host-range Salmonella spp. (HlyE, CdtB, PltA, and STY1364). We categorized identified proteins into a number of major groupings, including those involved in energy metabolism, protein synthesis, iron homeostasis, and biosynthetic and metabolic functions and those predicted to localize to the outer membrane. We assessed systemic and mucosal anti-HlyE responses in S. Typhi-infected patients and detected anti-HlyE responses at the time of clinical presentation in patients but not in controls. These findings could assist in the development of improved diagnostic assays.

Concomitant enterotoxigenic Escherichia coli infection induces increased immune responses to Vibrio cholerae O1 antigens in patients with cholera in Bangladesh.

Vibrio cholerae O1 and enterotoxigenic Escherichia coli (ETEC) are major bacterial pathogens that cause dehydrating disease requiring hospitalization of children and adults. The cholera toxin (CT) produced by V. cholerae O1 and the heat-labile toxin (LT) and/or heat-stable toxin (ST) of ETEC are responsible for secretory diarrhea. We have observed that about 13% of hospitalized diarrheal patients are concomitantly infected with V. cholerae O1 and ETEC. In order to understand the outcome of such dual infections on the clinical and immunological responses in cholera patients, we studied patients infected with V. cholerae O1 (group VC; n = 25), those infected with both V. cholerae O1 and ETEC (group VCET; n = 25), and those infected with ETEC only (group ET; n = 25). The VCET group showed more severe dehydration and had a higher intake of intravenous fluid and more vomiting than the ETEC group (P = 0.01 to 0.003). The VCET patients showed higher vibriocidal responses and increased antibody titers to cholera toxin and lipopolysaccharide (LPS) in plasma than did the V. cholerae O1 patients (P = 0.02 to <0.001). All responses in the V. cholerae O1 and in the VCET groups were more robust than those seen in the group infected with ETEC only (P = 0.01 to <0.001). We thus show that concomitant colonization with ETEC induces immune responses to V. cholerae antigens that are more robust than those seen with V. cholerae O1 infection alone. It is possible that LT or other factors expressed by ETEC may play a role as a mucosal adjuvant in enhancing the immune responses to V. cholerae O1.

Salmonella enterica serovar Typhi-specific immunoglobulin A antibody responses in plasma and antibody in lymphocyte supernatant specimens in Bangladeshi patients with suspected typhoid fever.

Many currently available diagnostic tests for typhoid fever lack sensitivity and/or specificity, especially in areas of the world where the disease is endemic. In order to identify a diagnostic test that better correlates with typhoid fever, we evaluated immune responses to Salmonella enterica serovar Typhi (serovar Typhi) in individuals with suspected typhoid fever in Dhaka, Bangladesh. We enrolled 112 individuals with suspected typhoid fever, cultured day 0 blood for serovar Typhi organisms, and performed Widal assays on days 0, 5, and 20. We harvested peripheral blood lymphocytes and analyzed antibody levels in supernatants collected on days 0, 5, and 20 (using an antibody-in-lymphocyte-supernatant [ALS] assay), as well as in plasma on these days. We measured ALS reactivity to a serovar Typhi membrane preparation (MP), a formalin-inactivated whole-cell preparation, and serovar Typhi lipopolysaccharide. We measured responses in healthy Bangladeshi, as well as in Bangladeshi febrile patients with confirmed dengue fever or leptospirosis. We categorized suspected typhoid fever individuals into different groups (groups I to V) based on blood culture results, Widal titer, and clinical features. Responses to MP antigen in the immunoglobulin A isotype were detectable at the time of presentation in the plasma of 81% of patients. The ALS assay, however, tested positive in all patients with documented or highly suspicious typhoid, suggesting that such a response could be the basis of improved diagnostic point-of-care-assay for serovar Typhi infection. It can be important for use in epidemiological studies, as well as in difficult cases involving fevers of unknown origin.

Antigen-specific memory B-cell responses to Vibrio cholerae O1 infection in Bangladesh.

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.

Mucosal and systemic immune responses in patients with diarrhea due to CS6-expressing enterotoxigenic Escherichia coli.

Colonization factor CS6 expressed by enterotoxigenic Escherichia coli (ETEC) is a nonfimbrial polymeric protein. A substantial proportion of ETEC strains isolated from patients in endemic settings and in people who travel to regions where ETEC is endemic are ETEC strains expressing CS6, either alone or in combination with fimbrial colonization factor CS5 or CS4. However, relatively little is known about the natural immune responses elicited against CS6 expressed by ETEC strains causing disease. We studied patients who were hospitalized with diarrhea (n = 46) caused by CS6-expressing ETEC (ETEC expressing CS6 or CS5 plus CS6) and had a disease spectrum ranging from severe dehydration (27%) to moderate or mild dehydration (73%). Using recombinant CS6 antigen, we found that more than 90% of the patients had mucosal immune responses to CS6 expressed as immunoglobulin (IgA) antibody-secreting cells (ASC) or antibody in lymphocyte supernatant (ALS) and that about 57% responded with CS6-specific IgA antibodies in feces. More than 80% of the patients showed IgA seroconversion to CS6. Significant increases in the levels of anti-CS6 antibodies of the IgG isotype were also observed in assays for ASC (75%), ALS (100%), and serum (70%). These studies demonstrated that patients hospitalized with the noninvasive enteric pathogen CS6-expressing ETEC responded with both mucosal and systemic antibodies against CS6. Studies are needed to determine if the anti-CS6 responses protect against reinfection and if protective levels of CS6 immunity are induced by vaccination.