A recombinant non-fatty acylated form of the Hi-PAL (P6) protein of Haemophilus influenzae elicits biologically active antibody against both nontypeable and type b H. influenzae

Abstract

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

Nasopharyngeal Colonization with Nontypeable Haemophilus influenzae in Chinchillas

“…Two challenge methods have been used in chinchillas for NTHI-induced otitis media, one being a direct bacterial intrabulla challenge (8) and the other involving NP inoculation followed by negative-pressure aspiration of NTHI up into the middle ear through the eustachian tube (12). The chinchilla intrabulla challenge model has been widely used to test vaccine candidates against NTHI in recent years (2,6,11). However, since infection with the pathogen is not analogous to that observed in humans, interpretation of the results obtained from this model remains difficult.…”

“…Effusion is sampled by middle ear aspiration via the epitympanic bulla, the sample is cultured on chocolate agar, and bacteria are quantified 24 h later. This model has been a useful tool for investigating the importance of host immunity in the prevention of NTHI-related disease and for screening potential vaccine antigens against the bacteria (2,6,11). However, interpretation of the results obtained from this model remains difficult, since the mechanism of ear infection induced by the pathogen is not analogous to that in humans.…”

T cell cytokine responses to outer membrane proteins of Haemophilus influenzae and the house dust mite allergens Der p 1 in allergic and non‐allergic subjects

“…The measurement of immune responses to H. influenzae has been complicated by the fact that the major outer membrane protein, the porin P2, has extensive sequence and antigenic variation from isolate to isolate [10, 11]. Other membrane proteins including D15 and OMP6 have highly conserved sequences and antigenicity [12–16] and can be used to study the recall responses induced by infection with a wide range of isolates. Both antigens elicit protective immune responses [13, 16, 17] and both can be produced as recombinant polypeptides [13, 15, 17] so in vitro cytokine responses can be induced without inflammatory microbial products such as lipo‐oligosaccharide.…”

“…Other membrane proteins including D15 and OMP6 have highly conserved sequences and antigenicity [12–16] and can be used to study the recall responses induced by infection with a wide range of isolates. Both antigens elicit protective immune responses [13, 16, 17] and both can be produced as recombinant polypeptides [13, 15, 17] so in vitro cytokine responses can be induced without inflammatory microbial products such as lipo‐oligosaccharide. The whole OMP6 molecule can be produced as a soluble polypeptide and while this cannot be accomplished for D15, the peptide 6–201 is a large portion of the molecule which is soluble and can induce protective immunity [17].…”

Purification and characterization of outer membrane protein P6, a vaccine antigen of non-typeable Haemophilus influenzae

“…Mucosal immunization with P6 resulted in enhanced pulmonary clearance in the rat [14]. Active immunization of rabbits and chinchillas generates bactericidal antibodies [13,15].…”