Susceptibility of Vibrio cholerae O139 to antibody-dependent, complement-mediated bacteriolysis

Abstract

Volunteer studies with Vibrio cholerae O1 have shown that the best correlate of a vaccine's protective efficacy is its propensity to elicit serum bactericidal responses in its recipients. Attempts to detect such responses following infection with V. cholerae O139, however, have met with varying success. Using a tube-based assay which involves viable counting, we now report that strains of serogroup O139 can appear to be sensitive or resistant to a fixed concentration of complement in the presence of antibody, depending on assay conditions. Susceptibility to lysis is critically dependent on the availability of complement, but with O139 indicator strains this is not simply determined by the concentration of serum added to the reaction mix. The nature of the assay diluent and the concentration of indicator bacteria can also dramatically affect bactericidal end points, whereas such variables have minimal significance with O1 indicator bacteria. Although some laboratories use unencapsulated mutant strains to seek evidence of seroconversion following exposure to V. cholerae O139, this is not necessary, and our findings question the significance of capsule expression as a determinant of complement sensitivity when antibody is present. The medium used for growth of the indicator strain and the particular strain used appeared to be unimportant. Each of seven O139 isolates tested was found to be lysed by antibody and complement in our standard assay system, which allowed the detection of significant serum bactericidal responses in 9 of 11 cases of O139 disease.

FIG. 1

Capsule production by V. cholerae. Ferritin labeling in sections of OX-grown H1 (A) (magnification, ×52,000) LB-grown AI1838 (B) (×39,000), and OX-grown AI1838 (C to F) (×39,000, ×21,000, ×21,000, and ×16,000, respectively) are shown.

FIG. 2

Effect of assay diluent on sensitivity of complement-mediated hemolysis. Percent hemolysis versus reciprocal complement dilution using various assay diluents is shown. A, LB (50% hemolytic titer, 45); B, OX (100); C, PBS (175); D, Mg²⁺-saline (470).

FIG. 3

Effect of complement concentration on bactericidal titer. The effect of varying complement concentrations on bactericidal end points is shown. Antibodies of the IgG isotype (ICL11 versus AI1838; polyclonal anti-V. cholerae versus H1) were titrated against the appropriate indicator strain (AI1838 [○] or H1 [●]) using OX (solid lines) or LB (dotted lines) as the assay diluent.