Immunogenicity and protective potential of Protein D of Haemophilus influenzae against H. influenzae infection

Abstract: Both Haemophilus influenzae type b (Hib) and nontypeable H. influenzae (NTHi) are important human pathogens causing invasive and mucosal infections. Currently there are number of vaccines available against Hib infections. Hib vaccines consist of the capsular polysaccharide antigen, polyribosyl ribitol phosphate (PRP) of Hib, coupled to different protein carriers. These vaccines are not effective against NTHi infections. A vaccine formulation including the PRP of Hib strains coupled to common membrane antigens of NTHi strains may be effective against both encapsulated and unencapsulated strains, as immunization with membrane proteins of NTHi have been shown to have protective effect against NTHi infections. The aim of this study have been to evaluate the vaccine potential of a 42-kDa membrane lipoprotein (protein D) of H. influenzae as a conjugate to PRP. Screening of 127 H. influenzae strain in a direct binding assay and in Western blot assay with a human IgD myeloma which was previously shown to bind to protein D and with three different monoclonal antibodies to protein D demonstrated that protein D is a well conserved protein. Immunization of rats through gut or nasal flora with bacteria carrying protein D manifested saliva IgA antibodies to protein D, but no antibodies to protein D were detected in the middle ear lavage material. Experimentally induced acute otitis media (AOM) with NTHi or Hib yielded serum antibodies to protein D in rats, but unlike per oral or intranasal immunization with NTHi, there were no saliva antibodies to protein D. Screening the serum of individuals from different age groups showed that only IgG antibodies to protein D are present in infants less than 6 months old, whereas IgM antibodies seroconverge after 6 months old. A significant increase in the levels of IgA antibodies were detected only after 10 years of age. Sera of rats immunized with the native form of protein D (LPD) yielded higher serum IgG and IgA antibody levels than PDm nonacylated form of protein D (PDm) immunized rats and they had higher bactericidal activity levels against NTHi strains. Immunization with PDm or LPD failed to protect rats in the experimental AOM model induced with a NTHi strain, although there was a difference in the resolution rate of AOM infection between the LPD immunized rats and the sham immunized rats. Immunization of rats with conjugated PRP vaccines yielded higher anti-PRP antibodies than PDm conjugated PRP vaccines. Rats with higher serum antibodies to PRP had the highest level of bactericidal activity and the highest protection rate in experimental AOM induced with a Hib strain. In conclusion, protein D is a well conserved membrane protein which can induce antibodies with biological activity against NTHi strains. LPD is more immunogenic than PDm in inducing antibodies to protein D and when conjugated to PRP it induces higher levels of anti-PRP antibodies than PDm conjugated PRP vaccines. Thus, LPD may be exerting an adjuvant effect besides providing T cell help to PRP. These results merit further consideration of protein D as a carrier for a PRP vaccine.