Characterization of a monoclonal antibody to the capsule of Haemophilus influenzae type b, generated by in vitro immunization.

Monoclonal antibodies to polyribosylribitolphosphate (PRP), the capsular polysaccharide of Haemophilus influenzae type b (Hib), are useful tools in the investigation of the molecular and cellular mechanisms causing Hib meningitis. A better understanding of these mechanisms may lead to improved therapeutic strategies. A number of different in vivo immunization techniques in BALB/c mice were used, which did not however reveal detectable serum levels of antibodies to PRP. Therefore a modified in vitro immunization technique, originally established for in vitro immunization of human B lymphocytes, was used for this weak immunogen in mice. After 5 days of in vitro stimulation with purified PRP the splenic lymphocytes of BALB/c mice were fused with the mouse myeloma line P3-X63-Ag8.653. One hybridoma produced an IgM antibody (12E7) which recognized the capsular polysaccharide in ELISA and specifically labelled all tested Hib strains in immune fluorescent microscopy. The blotted polysaccharide PRP was immunostained with monoclonal antibody 12E7. Preincubation of Hib with this antibody enhanced the oxygen radical metabolism of polymorphnuclear leucocytes in a chemiluminescence assay. There was no cross-reactivity with the supernatants of other Haemophilus influenzae serotypes and other bacterial species, as shown by counterimmunoelectrophoresis.

Interaction of the capsular polysaccharide of Haemophilus influenzae type B with C1q.

The Gram-negative pleomorphic bacterium Haemophilus influenza type b (Hib) is the most common cause of bacterial meningitis in children below the age of 2. Virtually all infants between 3 and 18 month of age lack anticapsular antibodies. This is typical for the response to a T-cell-independent antigen. 3-5% of this group harbour Hib in the nasopharynx, but the incidence of disease is 1000-fold less. This implicates other factors in host susceptibility in addition to the absence of such antibodies. Under physiological conditions the purified complement subcomponent C1q interacts with polyribosylribitolphosphate (PRP), the capsular polysaccharide of Hib. The complex formation of C1q, the most basic serum protein, with this polyanion was demonstrated by several methods: agarose gel electrophoresis followed by immunoprecipitation in the gel and Coomassie staining; western blot analysis of C1q-PRP complexes; complex formation in electrophoretic separation of PRP; retardation of electrophoretic mobility of PRP was checked by blotting of this polysaccharide. These results were confirmed by time- and dose-dependent alteration of antigenetic properties detected by C1q-Sandwich-ELISA after coincubation with PRP. Preincubation of serum treated Hib with C1q significantly enhanced the O2-metabolism of polymorphonuclear leucocytes in chemiluminescence assay. Infants of the susceptible age group develop antibodies to several Hib outer membrane proteins (OMP) and lipooligosaccharides (LOS) in response to infection. The complement activation by immune complexes might be inhibited by the formation of C1q-PRP complexes. Our results do not support the thesis that C1q can be activated by the interaction with PRP as shown before for other polyanions. Differing C1q to PRP ratios could be a possible explanation for different host susceptibilities.