Immunogenic outer-membrane proteins of Haemophilus influenzae type b in infection

Abstract

Outer-membrane proteins (OMPs) from Haemophilus i'npuenzue type b (strain Eagan), grown both in uitro (broth) and in uiuo (rat intra-peritoneal), were separated by SDS-PAGE. The major OMPs were present in both growth conditions although the amounts of OMP a and OMP d were reduced in rat-grown organisms. There were strong additional bands in in-uiuo-grown organisms at 51 and 92 kDa. Antiserum was raised in rabbits against in-uiuogrown bacteria, and absorbed with lysates of in-uitro-grown bacteria. This serum was used in Western blot analysis of OMPs from in-uitro-and in-niuo-grown cells to identify immunogenic proteins present in infection. The& infection-associated OMPs had apparent molecular masses of 43 kDa, 48 kDa, 81 kDa and > 200 kDa. Bands of reactivity, of the same molecular mass as some of these, were found on immunoblots when rat and human convalescent sera were used as the source of primary antibody. In particular, a band of 81 kDa was recognized by pooled rat and three human convalescent sera.

Evidence for in vivo expression of transferrin-binding proteins in Haemophilus influenzae type b

“…influenzae type b strain Eagan was grown in vivo and recovered without subculture from the intraperitoneal (i.p.) cavities of infant rats as described by Langford et al (16). Briefly, bacteria were grown to the log phase in sBHI, harvested, and suspended in phosphate-buffered saline (pH 7.4) at a concentration of 5 x 10 CFU/ml.…”

“…(5 x 102 CFU) or intranasally (107 CFU) and samples were collected from the nasopharynx, tail vein, CSF, and i.p. cavity at 48 h postinoculation as described previously (16,21,32). Convalescent-phase sera.…”

“…infection was used as the source of convalescent-phase sera. As a control, sera from three uninfected, age-matched infant rats were also pooled (9,16). Convalescent-phase sera were also obtained from patients with a positive diagnosis of H. influenzae type b meningitis, ranging in age from 3 to 36 months, at 3 to 5 weeks postdiagnosis.…”