IMMUNOLOGICAL PROPERTIES OF RECOMBINANT PORIN OF HAEMOPHILUS-INFLUENZAE TYPE-B EXPRESSED IN BACILLUS-SUBTILIS

The major surface-located, channel-forming protein in the outer membra ne of Haemophilus influenzae type b (Hib) is porin (341 amino acids; M (r), 37,782). In order to generate Hib porin that is devoid of lipooli gosaccharides and capsular polysaccharide, the Hib porin gene ompP2 wa s subcloned into a plasmid vector and recombinant Hib porin was expres sed in Bacillus subtilis. Recombinant porin was produced in large quan tities in B. subtilis and formed intracellular inclusion bodies. Recom binant porin was extracted from inclusion bodies and shown to be activ e in forming pores in synthetic black lipid membranes. However, these pores demonstrated different pore characteristics than wild-type Hib p orin. Mouse hyperimmune sera against recombinant porin were generated and subjected to epitope scanning with a library of 336 overlapping sy nthetic hexapeptides that corresponded to the entire sequence of Hib p orin. The epitope specificities of the anti-recombinant porin antibodi es were similar to those of antibodies against Hib porin: selected reg ions near the amino terminus which include a buried loop in the native structure of Hib porin were more immunogenic than regions at the carb oxy terminus. Although some mouse anti-recombinant porin antibodies me diated complement-dependent binding to Hib by polymorphonuclear leucoc ytes in opsonophagocytosis assays, the antibodies were not bactericida l, nor did they abrogate bacteremia in the infant rat model of infecti on. It was concluded that the native state of Hib porin is required fo r the generation of a protective immune response against the bacterium .