Effect of O acetylation of Neisseria meningitidis serogroup A capsular polysaccharide on development of functional immune responses

Abstract

The importance of O-acetyl groups to the immunogenicity of Neisseria meningitidis serogroup A polysaccharide (PS) was examined in studies using human sera and mouse immunization. In 17 of 18 postimmunization human sera, inhibition enzyme-linked immunosorbent assay indicated that the majority of antibodies binding to serogroup A PS were specific for epitopes involving O-acetyl groups. Studies with mice also showed an essential role for O-acetyl groups, where serum bactericidal titers following immunization with de-O-acetylated (de-O-Ac) conjugate vaccine were at least 32-fold lower than those following immunization with O-Ac PS-conjugate vaccine and 4-fold lower than those following immunization with native capsular PS. Inhibition studies using native and de-O-Ac PS confirmed the specificity of murine antibodies to native PS. The dramatic reduction in immunogenicity associated with removal of O-acetyl groups indicates that O acetylation is essential to the immunogenic epitopes of serogroup A PS. Since levels of bactericidal antibodies are correlated with protection against disease, O-acetyl groups appear to be important in protection.

FIG. 1.

Inhibition of human antibodies binding to N. meningitidis serogroup A capsular PS by native PS (A) and de-O-Ac PS (B). Four representative sera from individuals after vaccination with serogroup A PS vaccine are shown. Fivefold dilutions of inhibiting PS were incubated with a single serum dilution previously shown by ELISA to be in the linear range of the titration curve. Percent inhibition was calculated by comparison of the mean of two OD values for each inhibitor concentration to the mean of two OD values for sera and buffer only.

FIG. 2.

Inhibition of serum IJ binding to serogroup A capsular PS by native, de-O-Ac, and size-reduced PS. Note that the polymer length is inversely related to the K_d value.

FIG. 3.

Antibody responses in mice immunized with 2 μg of native PS, O-Ac-TT, de-O-Ac PS, de-O-Ac-TT, or adjuvant alone (control), as measured by ELISA (A) and by SBA (B). (A) PS ELISA, using native PS mixed with mHSA as the coating antigen, was performed with individual sera. The geometric mean concentration and 95% confidence intervals determined by one-way analysis of variance are shown for each group. Only the geometric mean concentrations in native PS- and O-Ac-TT-immunized animals are significantly different from those in the control mice by Dunnett's test with a significance level of 0.05. (B) The SBA titers shown are for pooled sera for each immunization group. Data from a representative assay are shown; titers obtained from duplicate sera and repeat assays and from an independent immunization experiment were within one twofold dilution for each group.

FIG. 4.

Inhibition of mouse postimmunization sera binding to native PS in the PS ELISA with native and de-O-Ac PS as inhibitors. Anti-serogroup A PS antibodies in sera from mice immunized with PS (diamonds) and O-Ac-TT (squares) are inhibited by native (solid symbols) but not de-O-Ac (open symbols) PS.