A peptidoglycan-associated lipoprotein of about 15 kilodaltons was purified from the outer membranes of Haemophilus influenzae by using nondenaturing detergents. To assess its vaccine potential, rabbit antiserum to the purified protein was obtained. The antiserum was specific for the peptidoglycan-associated lipoprotein in whole cell lysates of H. influenzae and was bactericidal for H. influenzae types a, b, d, e, and f and for 181 of 182 H. influenzae type b clinical strains isolated in widely dispersed geographic areas. The antibody protected infant rats from challenge with each of five clinical H. influenzae type b isolates and was additive to and did not

Outer membrane proteins of nontypeable (NT) Haemophilus influenzae are among the major candidates for inclusion in vaccines against these organisms. This article reports the purification of the e (P4) lipoprotein of H. influenzae and the subsequent production of antiserum directed against this protein. The anti-e polyclonal serum cross-reacted with e protein in multiple clinical NT H. influenzae isolates. Monoclonal antibody analysis of e protein showed at least one surface-exposed epitope to be conserved among NT H. influenzae strains. Anti-e serum also had bactericidal activity against multiple clinical isolates of NT H. influenzae. These results are in contrast to previous reports in the literature that purified P4 protein did not elicit biologically active antibodies. Anti-e antibodies exhibited synergistic bactericidal activity directed against NT H. influenzae when mixed with antibodies directed against another Haemophilus lipoprotein, PCP. This bactericidal synergy was observed against a variety of NT clinical isolates. We also report the cloning of the Haemophilus e lipoprotein, or hel, gene encoding the e protein and its expression and processing in Escherichia coli. The nucleotide sequence of the gene and deduced amino acid sequence of the protein are given. These results demonstrate that e protein is a viable candidate to be a component of a vaccine against NT H. influenzae. * Corresponding author. tective against Hib meningitis. To further investigate this protein and to determine the degree of antigenic conservation among NT H. influenzae and the biologic activity of anti-e serum against NT H. influenzae, we purified the e protein and produced an anti-e polyclonal antiserum. This article reports that the purified protein elicited biologically active antibodies against NT H. influenzae and that anti-e OMP serum showed synergistic BC activity when mixed with antiserum against the recombinant PCP protein. We also report the cloning and sequencing of the gene encoding this protein and its expression in Escherichia coli as a lipoprotein. These results show that the e protein is a vaccine candidate against NT H. influenzae. MATERIALS AND METHODS Bacteria. Clinical isolates of NT H. influenzae P860295,

A gene from Haemophilus influenzae encoding an outer membrane lipoprotein of about 15,000 daltons and which comigrates with the peptidoglycan-associated lipoprotein (PAL) of H. influenzae on sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been previously reported and designated pcp gene, and its product has been designated PCP. In order to obtain specific immunologic probes for the analysis of PCP expression, cellular location, and antigenic conservation in H. influenzae, pcp was fused to the lac polylinker region of plasmid pUCl9 and the hybrid gene was expressed in Escherichia coli. PCP purified from these cells was used to generate rabbit and mouse polyclonal antisera and mouse monoclonal antibody against PCP. Western immunoblot analysis with anti-PCP monoclonal antibody demonstrated that PCP is present and antigenically conserved in 30 tested strains of H. influenzae, including 27 clinical nontypeable strains. Polyclonal antiserum against PCP killed 9 of 11 clinical H. influenzae strains in a complement-mediated bactericidal assay, and bactericidal activity was additive with bactericidal activity of antisera against PAL. These results indicate that PCP is a potentially valuable component for a subunit vaccine against nontypeable H. influenzae disease, especially in combination with PAL or other components.

An approximately 15,000-dalton outer membrane lipoprotein of Haemophilus influenzae, the Hi-PAL (P6) protein, has been shown to elicit bactericidal and protective antibodies against both type b and nontypeable H. influenzae strains and is a vaccine candidate for these organisms. To determine whether the lipid modification of this protein is required for immunogenicity or the elicitation of biologically active antibodies, a genetic fusion was constructed that contains the sequence of mature Hi-PAL fused to the polylinker region of pUCl9. The protein expressed by this clone does not contain detectable lipid and was purified to homogeneity. This recombinant fusion protein, rPAL, elicited a strong immune response when injected into rabbits, and the antiserum reacted well with native Hi-PAL. The antiserum was bactericidal against a number of clinical nontypeable strains, duplicating the activity of anti-Hi-PAL. The anti-rPAL antiserum was also protective against type b bacteremia in the infant rat model. These results demonstrate that purified rPAL elicits antibodies with biological activities that are similar to those of anti-Hi-PAL antibodies. Thus, the lipid component of Hi-PAL is not required for either immunogenicity or elicitation of biologically active antibodies.Haemophilus influenzae strains represent a major cause of morbidity and mortality in infants and elderly adults (2,19,24). H. influenzae type b (Hib) strains are the leading causative agents of neonatal meningitis (21), and nontypeable (NT) H. influenzae strains are among the major causative agents of acute otitis media (2). The current vaccines for Hib consist of the polysaccharide capsule from Hib, polyribosyl-ribitol phosphate or oligosaccharides derived from polyribosyl-ribitol phosphate coupled to protein carriers. The protective efficacy of anti-polyribosyl-ribitol phosphate antibody is demonstrated in the infant rat meningitis model system, in which antiserum is passively transferred and the animals are challenged with virulent Hib (23). Presently, the protein-polysaccharide conjugate vaccines are licensed for use in 18-month-old children. Clinical trials of saccharide-protein vaccines are currently underway to demonstrate efficacy in infants. Although anti-polyribosyl-ribitol phosphate antibody appears to be sufficient to protect humans from Hib disease, it has no efficacy against diseases caused by NT H. influenzae. Recent attempts to identify potential vaccine components