Immunogenicity in Weanling Rabbits of a Polyribophosphate Complex from Haemophilus influenzae Type b

Polyribophosphate (PRP), the capsular polysaccharide of Haemophilus influenzae type b, is more effectively immunogenic when it is associated with the bacterium than when it is in the purified form that is being tested as a vaccine for humans. In an effort to analyze this difference, we isolated from H. influenzae type b a highmolecular-weight, soluble complex, in which PRP appears to be combined with protein (about 7% protein). The pyrogenicity and limulus lysate gelation activity of the complex suggest that a small amount of lipopolysaccharide also is present. The protein was resolved into five polypeptides by electrophoresis in polyacrylamide gel containing sodium dodecyl sulfate. In weanling rabbits, which do not respond to purified PRP, the complex induces high titers of antibody to PRP, in an anamnestic pattern. Bactericidal antibody to other bacterial components was also elicited. Equilibrium density gradient centrifugation of the complex indicated that most of the immunogenicity of PRP resides in the least dense fractions, which are high in protein, low in polysaccharide, and active in the limulus lysate test; denser fractions that react strongly with limulus lysate but are poor in protein were much less immunogenic. Although purified polyribophosphate (PRP), the capsular polysaccharide of Haemnophilus influenzae type b (H.i.b.), is a poor immunogen in human infants, systemic infection with H.i.b. does elicit antibodies to PRP in infants [1]. This dichotomy between the immunogenicity of purified bacterial capsular polysaccharides and that of such polysaccharides as bacterial components has been observed previously. Whole cells of Streptococcus pneumoniae elicit antibodies to capsular polysaccharides in rabbits, but the purified polysaccharides are not primary immunogens [2]. In the case of PRP, even preparations of high molecular weight and substantial immunogenicity for adult humans are inactive in rabbits, whereas live or formalin-killed H.i.b. induce