We have cloned and expressed in Escherichia coli a gene encoding a 15,000-apparent-molecular-weight peptidoglycan-associated outer membrane lipoprotein (PAL) of Haemophilus influenzae. The nucleotide sequence of this gene encodes an open reading frame of 153 codons with a predicted mature protein of 134 amino acids. The amino acid composition and sequence of the predicted mature protein agree with the chemically determined composition and partial amino acid sequence of PAL purified from H. influenzae outer membranes. We have also identified a second gene from H. influenzae that encodes a second 15,000-apparent-molecular-weight protein which is recognized by antiserum against PAL. This protein has been shown to be a lipoprotein. The nucleotide sequence of this gene encodes an open reading frame of 154 codons with a predicted mature protein of 136 amino acids and has limited sequence homology with that of the gene encoding PAL. Southern hybridization analysis indicates that both genes exist as single copies in H. influenzae chromosomal DNA. Both genes encode polypeptides which have amino-terminal sequences similar to those of reported membrane signal peptides and are associated primarily with the outer membrane when expressed in E. coli.

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,

Nontypeable Haemophilus influenzae (NTHi) is one of the leading causative agents of bacterial otitis media, and no vaccine has been shown to be effective against it. Three outer membrane lipoproteins of NTHi have been investigated extensively and are leading candidates for inclusion in a vaccine against this organism. Hi-PAL (P6), recombinant PCP (rPCP), and e (P4) proteins are antigenically conserved among NTHi strains and elicit bactericidal and protective antibodies. A genetic fusion of the rPCP and Hi-PAL proteins has also been reported. Mixtures of these proteins were used for active immunization experiments in the chinchilla model of otitis media. Chinchillas were immunized either with a mixture of all three lipoproteins or with the mixture of rPCP-PAL hybrid plus e protein. When these animals were challenged with a NTHi strain injected directly into the middle ears, no protection from infection or disease, as measured by otoscopy, was observed in either group. However, effusion and inflammation measured by tympanometry were significantly reduced in animals immunized with the three lipoproteins. Animals that had been immunized with either whole NTHi cells or total outer membranes and then challenged with the homologous strain were significantly protected from both infection and disease, as determined by tympanometry and otoscopy. Unlike other animal antisera, chinchilla antisera against the purified proteins had no bactericidal activity against NTHi but did fix complement on the cell surface. Thus, the chinchilla immune responses to mixtures of these lipoproteins differ from the immune responses observed in other animal species. Further evaluation of these proteins for their vaccine potential remains to be done.

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.