Experimental studies on cholera immunization. 4. The antibody response to formalinized Vibrio cholerae and purified endotoxin with special reference to protective capacity.

The primary and secondary antibody responses in rabbits, intravenously immunized with formalin-killed Vibrio cholerae, were studied with regard to amount, immunoglobulin class distribution and binding qualities of the antibodies to the bacterial endotoxin. Further the protective capacity of antisera and purified antibodies against experimental cholera infection was analyzed and related to the in vitro characteristics of the immune preparations. For the employed 1,000-fold varied range of bacteria used for immunization, the dose dependance of the amount and binding qualities of the antibodies, formed after a single antigen injection, was negligible. In the secondary responses, however, slightly increasing antibody titres and avidities as well as moderately rising protective titres were registered with increasing immunization doses. In the primary response to an optimally immunogenic dose of 1 X 10(10) bacteria the highest IgM titres were noted after 1 week and the maximal IgG titres after 6 weeks, whereas the avidity of vibriocidal antibodies and the protective titres of sera increased for at least 3 months. A booster injection 4 weeks after the primary immunization gave the highest IgG, IgM and protective titres within 1 week, whereafter all titres gradually decreased. Moreover, higher maximal antibody and protective titres were noted in the secondary than in the primary response. An immunological memory could also be induced by purified V. cholerae lipopolysaccharide (LPS), since a booster injection of this antigen gave rise to higher and earlier appearing antibody titres than the first immunization. The influence of the interval between a first and a second immunization on the amount and the avidity of the secondary response antibodies was comparatively small, whereas the protective titres increased markedly with shortening interval. The relation between protection against experimental cholera, as tested in the rabbit small bowel loop system using live vibrios for challenge, and the in vitro estimates of antibody amount and binding properties was poor. However, the IgM antibody titres, determined with an immunosorbent assay, and the antibody binding qualities, as measured by quantitative inhibition in a vibriocidal assay, showed statistically significant correlations with the protective titres. Purification of anti-endotoxin antibodies was achieved by affinity chromatography using columns with V. cholerae LPS covalently coupled to Sepharose beads. The antibodies bound specifically to the gel and could later be eluted by acid buffers. Decreasing pH of the buffer released antibodies with increasing avidity. Antiserum taken early after a primary immunization had a higher proportion of antibodies, eluted at a high pH, than antisera taken late in the primary and secondary responses.