Identification of a 16,600-dalton outer membrane protein on nontypeable Haemophilus influenzae as a target for human serum bactericidal antibody.

Murphy, Timothy F.; Bartos, L C; Rice, Peter A.; Nelson, Michael; Dudas, Kathleen C.; Apicella, Michael A.

doi:10.1172/jci112656

Cited by 124 publications

(96 citation statements)

References 32 publications

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“…At the 30-min time point, there was consistently more killing in the normal human serum control than in the anti-loop 6 reaction mixture. Interestingly, these kinetics are similar to results with immunopurified antibodies to P6 used in bactericidal assays (19). No bactericidal activity was apparent in a control reaction mixture in which both complement and the anti-loop 6 antibody were omitted.…”

Section: Specificity Of Immunopurified Antibodies To Loopsupporting

confidence: 75%

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

Neary¹,

Yi²,

Karalus³

et al. 2001

Infect Immun

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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...

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Section: Specificity Of Immunopurified Antibodies To Loopsupporting

confidence: 75%

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

Neary¹,

Yi²,

Karalus³

et al. 2001

Infect Immun

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“…Since NTHI lacks capsular antigens, the chief antigenicity is present in the outer membrane proteins (OMPs). One of the OMPs of NTHI, P6, is a common antigen to all strains and is considered as a candidate for mucosal vaccine (7,9,10,11,30,31).In the mucosal surface, secretory immunoglobulin A (IgA) plays a major role in protective immunity. We previously demonstrated that intranasal immunization was an effective regimen for inducing mucosal IgA immune responses in the upper respiratory tract (26) and that the nasal mucosal IgA immune responses induced by intranasal immunization were effective for the clearance of bacteria in the nasopharynx.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Induction of Specific Immunoglobulin A and Th2 Immune Responses to P6 Outer Membrane Protein of NontypeableHaemophilus influenzaein Middle Ear Mucosa by Intranasal Immunization

et al. 2000

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Nontypeable Haemophilus influenzae (NTHI) is a major pathogen of otitis media. One of the outer membrane proteins of NTHI, P6, is an antigen common to all strains and is considered as a candidate for mucosal vaccine. To elucidate the possibility of developing a nasal vaccine against nontypeable Haemophilus influenzae (NTHI) and to investigate mucosal immune responses in the middle ear, mice were immunized intranasally with the P6 outer membrane protein of NTHI, and P6-specific immune responses in the middle ear mucosa were examined. Mice were given with P6 and cholera toxin intranasally as an adjuvant on days 0, 7, and 14 and were killed on day 21. The P6-specific immunoglobulin A (IgA) antibody titer in ear wash was significantly elevated. Mononuclear cells were isolated from middle ear mucosa, and an increase in P6-specific IgA-producing cells was shown with an enzyme-linked immunospot assay. In addition, an increase in memory T cells in middle ear mucosa was detected with flow cytometric analysis after intranasal immunization. Moreover, in vitro stimulation with P6 resulted in proliferation of purified CD4 ؉ T cells from immunized mice, and these T cells expressed Th2 cytokine mRNA. These results indicate that P6-specific IgA-B-cell immune responses and selected Th2 cytokine expressing Th cells were induced in middle ear mucosa by intranasal immunization. These findings suggest that a nasal vaccine is useful for preventing otitis media with effusion.Nontypeable Haemophilus influenzae (NTHI) is a major pathogen of otitis media with effusion (OME) and other upper respiratory tract diseases (10, 30). In patients with OME, this bacterium is frequently isolated from the nasopharynx, as well as from middle ear effusions, and the inhibition of NTHI colonization in the upper respiratory tract is considered effective in preventing OME. Due to the increase of antibiotic-resistant strains of NTHI in recent years, the development of a vaccine against this bacterium is considered an important goal for public health. Since NTHI lacks capsular antigens, the chief antigenicity is present in the outer membrane proteins (OMPs). One of the OMPs of NTHI, P6, is a common antigen to all strains and is considered as a candidate for mucosal vaccine (7,9,10,11,30,31).In the mucosal surface, secretory immunoglobulin A (IgA) plays a major role in protective immunity. We previously demonstrated that intranasal immunization was an effective regimen for inducing mucosal IgA immune responses in the upper respiratory tract (26) and that the nasal mucosal IgA immune responses induced by intranasal immunization were effective for the clearance of bacteria in the nasopharynx.The mucosal immune system is considered as a separate functional entity quite independent of the systemic immune system because the mucosal immune system possesses unique anatomic features and is composed of specialized subsets of lymphoid cells (21,27,34). Despite the recent emphasis on a better understanding of molecular and cellular aspects of the mucosal immune system,...

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“…The interstrain variation of P5 seen in NTHi (136) (10,115,143,145). It elicits bactericidal antibody in both its native (135,141) and recombinant (71) forms. Since this protein is highly conserved among encapsulated and nonencapsulated H. influenzae strains (140,148), surface exposed, and immunogenic (31,112,135), there has been much interest in it as a potential vaccine candidate.…”

Section: Outer Membrane Proteinsmentioning

confidence: 99%

Lower Respiratory Tract Infections

Oakes¹

2003

The Infectious Diseases Manual

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Identification of a 16,600-dalton outer membrane protein on nontypeable Haemophilus influenzae as a target for human serum bactericidal antibody.

Cited by 124 publications

References 32 publications

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

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

Induction of Specific Immunoglobulin A and Th2 Immune Responses to P6 Outer Membrane Protein of NontypeableHaemophilus influenzaein Middle Ear Mucosa by Intranasal Immunization

Lower Respiratory Tract Infections

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