Antibodies to Loop 6 of the P2 Porin Protein of Nontypeable Haemophilus influenzae Are Bactericidal against Multiple Strains

Abstract

The P2 porin protein is the most abundant protein in the outer membrane of nontypeable Haemophilus influenzae (NTHI). Analysis of sequences of P2 from different strains reveals the presence of both heterogeneous and conserved surface-exposed loops of the P2 molecule among strains. The present study was undertaken to test the hypothesis that antibodies to a conserved surface-exposed loop are bactericidal for multiple strains of NTHI and could thus form the basis of vaccines to prevent infection due to NTHI. Polyclonal antiserum to a peptide corresponding to loop 6 was raised and was immunopurified over a loop 6 peptide column. Analysis of the antibodies to whole organisms and peptides corresponding to each of the eight loops of P2 by immunoassays revealed that the antibodies were highly specific for loop 6 of P2. The immunopurified antibodies bound to P2 of 14 of 15 strains in immunoblot assays. These antibodies to loop 6 demonstrated complement-mediated bactericidal killing of 8 of 15 strains. These results support the concept of using conserved regions of the P2 protein as a vaccine antigen.Nontypeable Haemophilus influenzae (NTHI) is a small, gram-negative bacillus which causes otitis media in children and lower respiratory infections in adults with chronic obstructive pulmonary disease (COPD). In both otitis media and COPD, patients routinely suffer recurrent episodes of disease (15,21). Factors such as health care costs, pain and suffering, and lost work time underscore the need for a vaccine against NTHI (10,14,22).The ability of NTHI to cause recurrent infections is in part attributable to antigenic variability in several surface-exposed loops of major outer membrane protein P2 (2, 5, 26). The P2 protein is a homotrimeric porin which constitutes approximately one-half of the total outer membrane protein of the organism. The loop 5 region is highly heterogeneous among strains and contains almost all of the epitopes to which an antibody response is mounted when animals are immunized with the whole organism (30). Adults with COPD make new antibodies to strain-specific epitopes on P2 following infection by NTHI (31). Thus, immunity against NTHI is most often strain specific, leaving the patient vulnerable to reinfection by other strains.One approach to vaccine development for NTHI has been to study antigenically conserved outer membrane proteins as potential vaccine antigens. In view of the abundant expression of P2 on the bacterial surface, identification of a conserved region on the P2 molecule to which immune responses could be directed would be a significant step towards developing a vaccine against NTHI.In this study, antibodies to a conserved loop of the P2 molecule of NTHI (loop 6) were raised and studied for their ability to recognize the P2 molecules of heterologous strains. Since bactericidal antibody is associated with protection from otitis media due to NTHI (8, 25), antibodies to loop 6 were also assessed for their ability to direct killing of heterologous strains. MATERIALS AND METHODSBacterial s...

Characterization and comparison of Pasteurella multocida strains associated with porcine pneumonia and atrophic rhinitis

“…Molecular mass heterogeneity also occurs in the corresponding P2 (OmpH) and P5 (OmpA) proteins of Haemophilus influenzae and is also due to differences in the size of hypervariable surface-exposed loop regions (Sikkema & Murphy, 1992;Duim et al, 1997). These surface-exposed loops are thought to interact with the host immune system and, by undergoing antigenic variation, provide the bacterium with an important defence mechanism (Yi & Murphy, 1997;Neary et al, 2001). The observed heterogeneity of the OmpA and OmpH proteins of porcine P. multocida strains might also be due to variation in the size of hypervariable surface-exposed loop regions and might play important roles in the pathogenesis of pneumonic pasteurellosis and PAR.…”

Epitope-specific immune recognition of the nontypeableHaemophilus influenzaeouter membrane protein 26

“…Duangkamol Kunthalert, 1 Laura a. Novotny, 2 Helen M. Massa, 3 Glen c. Ulett, 3 Lauren O. Bakaletz, 2 Jennelle M. Kyd 4 and allan W. cripps 3, samples masked any specific responses and thus only rat data are presented. To localize the immunologically important regions within OMP26 in this response, a series of overlapping OMP26 peptides spanning the entire sequence of full-length OMP26 was used as the in vitro proliferation stimulus.…”

“…Several outer membrane proteins (OMPs) of NTHi and its oligosaccharides have been investigated as possible vaccine antigens against NTHi. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] One OMP that has shown promise as a potential vaccine candidate is OMP26. The amino acid sequence of this 26kDa OMP 16 is conserved among NTHi isolates from various disease states.…”

Strain-Specific Pulmonary Defense Achieved after Repeated Airway Immunizations with Non-Typeable Haemophilus Influenzae in a Mouse Model

“…The authors also demonstrated that a strain-specific immune response to NTHi was directed to epitopes on the loop 5 region of the P2 molecule and was associated with bactericidal activity in animal experiments . Another study also reported the loop 6 of the P2 molecule was capable of inducing bactericidal antibodies (Neary et al 2001). Accumulated evidence from clinical and laboratory investigations by Murphy and coworkers suggest that the P2 molecule is one of surface antigens that participate in strain-specific immune responses in the acute exacerbation of COPD by NTHi Neary et al 2001;Sethi et al 2004).…”

“…Another study also reported the loop 6 of the P2 molecule was capable of inducing bactericidal antibodies (Neary et al 2001). Accumulated evidence from clinical and laboratory investigations by Murphy and coworkers suggest that the P2 molecule is one of surface antigens that participate in strain-specific immune responses in the acute exacerbation of COPD by NTHi Neary et al 2001;Sethi et al 2004). This study was, therefore, conducted to determine whether repeated airway immunizations with NTHi strains bearing different P2 molecules were capable of inducing protective immunity in lungs against homologous or heterologous strains in a mouse model.…”