An Unmutated IgM Response to the Vi Polysaccharide of Salmonella Typhi Contributes to Protective Immunity in a Murine Model of Typhoid

Abstract

T cell-dependent B cell responses typically develop in germinal centers. Abs generated during such responses are isotype switched and have a high affinity to the Ag because of somatic hypermutation of Ab genes. B cell responses to purified polysaccharides are T cell independent and do not result in the formation of bona fide germinal centers, and the dominant Ab isotype produced during such responses is IgM with very few or no somatic mutations. Activation-induced cytidine deaminase (AID) is required for both somatic hypermutation and Ig isotype switching in humans and mice. To test the extent to which unmutated polysaccharide-specific IgM confers protective immunity, we immunized wildtype and AID mice with either heat-killed serovar Typhi ( Typhi) or purified Vi polysaccharide (ViPS). We found that wildtype and AID mice immunized with heat-killed Typhi generated similar anti-ViPS IgM responses. As expected, wildtype, but not AID mice generated ViPS-specific IgG. However, the differences in the Ab-dependent killing of Typhi mediated by the classical pathway of complement activation were not statistically significant. In ViPS-immunized wildtype and AID mice, the ViPS-specific IgM levels and Typhi bactericidal Ab titers at 7 but not at 28 d postimmunization were also comparable. To test the protective immunity conferred by these immunizations, mice were challenged with a chimeric Typhimurium strain expressing ViPS. Compared with their naive counterparts, immunized wildtype and AID mice exhibited significantly reduced bacterial burden regardless of the route of infection. These data indicate that an unmutated IgM response to ViPS contributes to protective immunity to Typhi.

IL-7 Enables Antibody Responses to Bacterial Polysaccharides by Promoting B Cell Receptor Diversity

“…Bound IgM or IgG was measured using HRP-conjugated goat anti-mouse IgM or IgG (Bethyl Laboratories, Montgomery, TX). Since ViPS-specific mouse IgM and IgG reference standards are not available, and the ViPS-specific antibodies in mice are likely to be of oligoclonal nature with varying affinities, the antigen-specific antibody levels in the present study were interpreted as ng or μg/ml “equivalents” using normal mouse serum IgM or IgG standards (Bethyl Laboratories, Montgomery, TX), as described previously (13, 28–30). Measurement of dextran-specific IgM responses involved coating 96-well EIA/RIA plates (Costar 9017; Corning Incorporated, Corning, NY) with 1μg/ml or 5μg/ml of B512 dextran (α1-6-dextran) or dextran B1355 (α1-3-dextran), and incubating overnight at 4°C.…”

“…or i.v. The bacterial burden in the blood, liver, and spleen was measured 3 days later as described previously (30, 33), since mice on BALB/c or C57BL6 background succumb to Salmonella infection by 5 days post-infection at this infectious dose due to the presence of a susceptible Nramp1 allele (33–35). Tissues were processed using a Minilys tissue homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France) and bacterial burden in these tissue homogenates or in blood was measured by counting cfus on LB agar plates.…”

Terminal Deoxynucleotidyl Transferase Is Not Required for Antibody Response to Polysaccharide Vaccines against Streptococcus pneumoniae and Salmonella enterica Serovar Typhi

“…current typhoid subunit vaccine, Typhim Vi. In adult mice, administration of a single dose of ViPS induces a robust anti-ViPS IgM and IgG response (27). We found that ViPS immunization results in comparable ViPS-specific IgM and IgG responses in both TdT ϩ/Ϫ and TdT Ϫ/Ϫ mice ( Fig.…”

Chemical synthesis and immunological evaluation of entirely carbohydrate conjugate Globo H-PS A1

“…Based on the serological assay results, the IgG/IgM ratio suggests that immunoglobulin isotype switching occurred during the trials and demonstrates that T cells play a role in generating anti-Globo H antibodies. [90][91][92][93] The serological assays also revealed an interesting nding in that the anti-serum had rather high titers of anti-Gb3 antibodies (Fig. S1 †).…”