OUTER-MEMBRANE PROTEIN D15 IS CONSERVED AMONG HAEMOPHILUS-INFLUENZAE SPECIES AND MAY REPRESENT A UNIVERSAL PROTECTIVE ANTIGEN AGAINST INVASIVE DISEASE

We have cloned and sequenced the d15 gene from two strains of Haemophi lus influenzae type b (Hib) and two strains of nontypeable H. influenz ae (NTHI), The nucleotide and deduced protein sequences of d15 are hig hly conserved, with only a small variable region identified near the c arboxyl terminus of the protein, Analysis of upstream sequences reveal ed that the H. influenzae d15 gene may be part of a large potential op eron of closely spaced open reading frames, including one with signifi cant homology to the Escherichia coli cds gene encoding CDP-diglycerid e synthetase. Southern blot analysis demonstrated that the d15 gene is also present in H. influenzae types a, c, d, e, and f and in Haemophi lus parainfluenzae. A recombinant D15 (rD15) protein was expressed in good quantity in E. coli from the inducible T7 promoter, and monospeci fic anti-rD15 antibodies were raised, Immunoblot analysis of H. influe nzae serotypes a, b, c, d, e, and f, NTHI, and H. parainfluenzae lysat es revealed that they all expressed a cross-reactive D15-like protein. Purified rD15 was found to be highly immunogenic in mice, guinea pigs , and rabbits, and passive transfer of anti-rD15 antibodies protected infant rats from challenge with H. influenzae type b or type a in infa nt rat models of bacteremia. Thus, D15 is a highly conserved antigen t hat is protective in animal models and it may be a useful component of a universal subunit vaccine against Haemophilus infection and disease .