Protection of infant rats from Haemophilus influenzae type b infection by antiserum to purified outer membrane...

Protein a (46,000 molecular weight [46K]) was purified from outer membranes of Haemophilus influenzae type b by a relatively simple procedure. Spontaneously shed outer membranes from a 24-h, 12-liter culture of an unencapsulated variant of strain Eag were combined with outer membranes released from the cells by Tris buffer and extracted with the nonionic detergent octylpolyoxyethylene. The extract was then subjected to open column chromatography on Sephacryl S-200 and Trisacryl-carboxymethyl to yield 7.5 mg of protein a from 180 mg of outer membrane protein. Approximately 99% of the protein in this preparation was protein a; in addition, the preparation contained 1.25% (wt/wt) lipopolysaccharide and had a residual detergent/protein ratio of 1.6:1 (wt/wt). Antibodies to the preparation were induced in rabbits by using alum as an adjuvant. As determined by immunoblotting, the great preponderance of antibodies induced were specific for protein a. However, very low levels of antibodies to several other outer membrane components, which were not apparent on gels of the pure preparation of protein a, were also induced. Preimmune and postimmune sera, after depletion of antibodies to capsular polysaccharide and lipopolysaccharide, were tested for biological activity against H. influenzae type b. Compared with preimmune serum, postimmune serum was bactericidal in vitro against strain Eag (the only strain tested) and offered significant protection (P less than 0.01) to infant rats against infection by all four strains tested, two of which had a protein a that was larger (47K) than the 46K protein a in the preparation. These results indicate that protein a should be considered as a vaccine to prevent H. influenzae type b disease.