Antigenic and phenotypic variations of Haemophilus influenzae type b lipopolysaccharide and their relationship to virulence

Haemophilus influenzae type b (Hib) strains NO100 and COL10 were found to produce bacteremia in infant rats at a much lower frequency than other Hib strains previously tested. These relatively avirulent strains were the only Hib strains among 200 clinical isolates examined to date which failed to react with two Hib lipopolysaccharide (LPS)-specific monoclonal antibodies (MAbs). LPS analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that strains NO100 and COL10 possessed LPS which migrated faster than the LPS of Hib strains that reacted with one of the two or with both of these MAbs. These observations suggested that the relative lack of virulence of strains NO100 and COL10 might be related to their unusual LPS phenotype. To determine whether alteration of LPS structure would affect the virulence of these strains, we identified and isolated isogenic LPS antigenic variants of strains NO100 and COL10 using the LPS-specific MAbs 4C4 and 5G8 in a colony blot radioimmunoassay. Antigenic variation of LPS was found to occur spontaneously in these two strains at a relatively high frequency in terms of both acquisition and loss of MAb reactivity (ca. 0.2 to 16.7%). LPS antigenic variants of strains NO100 and COL10 reactive with both MAbs 4C4 and 5G8 (4C4⁺ 5G8⁺) were more virulent in the infant rat model than their respective 4C4^- 5G8^- parental strains (P < 0.01). An antigenic variant of COL10 reactive with only MAb 4C4 (4C4⁺ 5G8^-) was also significantly more virulent than its 4C4^- 5G8^- parent. These LPS antigenic variants with increased virulence synthesized altered LPS molecules which possessed apparent molecular weights higher than those of the LPS of the parental strains. Increased resistance of strain NO100 to the bactericidal activity of normal infant rat serum was associated with changes in LPS structure, while strain COL10 and its LPS variants were all uniformly resistant to serum bactericidal activity. Our results demonstrate that (i) spontaneous antigenic and phenotypic variation of LPS occurs at a relatively high frequency in some strains of Hib; (ii) the higher-molecular-weight type of LPS is associated with the full expression of Hib virulence; (iii) LPS phenotype may not correlate with Hib serum resistance; and (iv) serum resistance of Hib is not an accurate indicator of virulence.