Human bactericidal antibody response to outer membrane protein P2 of nontypeable Haemophilus influenzae.

The human bactericidal antibody response to the major outer membrane protein, P2, of nontypeable Haemophilus influenzae was studied. P2 was isolated from two strains of nontypeable H. influenzae and coupled to affinity columns. Pooled normal human serum was subjected to affinity chromatography with the P2 columns and a control column. Reducing the titer of antibody to P2 resulted in reduced bactericidal activity of that serum for the organism. Immunopurified antibody to P2 from human serum was bactericidal for the homologous strain. The extent to which these bactericidal determinants on P2 are conserved among strains was investigated. Immunopurified antibodies to P2 of two epidemiologically unrelated isolates were bactericidal for four of six strains tested. We conclude that P2 is a target for human bactericidal antibody and that some of these determinants that are recognized by human bactericidal antibody are conserved among strains of nontypeable H. influenzae.     

不可分型流感嗜血杆菌外膜蛋白P6的重组表达及免疫保护作用研究

目的 研究不可分型流感嗜血杆菌(NTHi)重组外膜蛋白P6的理化性质和对小鼠的免疫保护作用.方法 利用PCR技术,以NTHi基因组为模板,扩增出编码外膜蛋白P6的基因片段;构建pET-32a-P16重组质粒;转化到大肠杆菌BL21(DE3)中进行表达,并对表达条件进行了优化.通过阴离子交换和凝胶过滤层析对蛋白进行纯化.经SDS-PAGE、Western blot等对蛋白的理化性质进行初步分析,继而免疫BALB/c小鼠,用同源菌经腹腔攻击,比较免疫组和对照组小鼠死亡率.结果 实现了该蛋白在大肠杆菌中的高效表达,产物表达形式为可溶性表达,纯化后蛋白纯度可达95%,收率可达5 mg/g(蛋白/湿菌),相对分子质量(M_r)为14 145.848,Western blot证实重组P6蛋白能与菌源提纯P6蛋白免疫小鼠后的抗血清发生特异性反应.动物实验结果表明重组P6蛋白在小鼠体内能诱生高滴度的抗体,显示免疫组小鼠存活率明显高于对照组(P<0.01).结论 重组NTHi外膜蛋白P6的原核表达产物为可溶性蛋白,在小鼠体内能诱生高效价抗体,在小鼠感染模型中具有保护作用,为NTHi疫苗的研发提供了基础资料.     

不可分型流感嗜血杆菌P6 DNA疫苗与蛋白疫苗序贯免疫效应研究

观察不可分型流感嗜血杆菌P6DNA疫苗初免蛋白疫苗加强免疫方式的免疫效果。大量扩增pcDNA3-P6。将65只BALB/c小鼠随机分PBS对照组、pcDNA3.1对照组、pcDNA3.1-P6组、P6蛋白组、pcDNA3.1-P6/P6蛋白组,分别于0、14、28d免疫。质粒每次每只接种100μg,蛋白每次每只接种10μg。末次免疫后14d,每组取10只小鼠取血,ELISA检测血清中特异性IgG抗体滴度。每组取3只小鼠处死,制备脾淋巴细胞,ELISA检测IL-4和IFN-γ水平,CCK-8法检测脾淋巴细胞增殖指数。用15LD50NTHi攻击每组剩余10只小鼠,观察免疫保护作用。结果:pcDNA3.1-P6/P6蛋白组小鼠的脾淋巴增殖指数和IFN-γ水平明显高于质粒组和蛋白组(P0.05)。pcDNA3.1-P6/P6蛋白组小鼠IgG抗体滴度、IL-4水平和动物生存率明显高于质粒组(P0.05)但和蛋白组相比无明显差异(P0.05)。因此pcDNA3.1-P6初免P6蛋白加强免疫的方式可以提高疫苗的免疫效果。     

Immunochemical characterization of variable epitopes of outer membrane protein P2 of nontypeable Haemophilus influenzae.

Monoclonal antibodies (MAbs) were elicited to the nontypeable Haemophilus influenzae variants d1 to d4, which differ in the outer membrane protein P2 to analyze the immunological properties of the variable parts of this protein. Five MAbs reacted in a whole-cell enzyme-linked immunosorbent assay (ELISA) only with the homologous strain and in some cases with its variants, but not with 69 unrelated nonencapsulated H. influenzae isolates; nine MAbs also reacted with some other H. influenzae isolates, and four MAbs showed broad cross-reactivity. All of the MAbs reacted with purified protein P2 in ELISAs and immunoblotting. The five MAbs which reacted with the homologous strain d3 and not with the variants d1, d2, and d4 promoted complement-dependent bactericidal activity against strain d3. These and four other MAbs reacted with the intact bacteria of strain d3 in immunogold electron microscopy, indicating that they were directed against surface-exposed epitopes of outer membrane protein P2. A mutant of strain d3 was isolated as a survivor from bacterial killing by complement and MAb 30DA5. This mutant had an altered P2 protein on sodium dodecyl sulfate-polyacrylamide gels and had lost its reactivity with all of the five H. influenzae d3-specific MAbs but not with the other MAbs. From these results, we conclude that the variable parts of outer membrane protein P2 of nonencapsulated H. influenzae from the sputum of patients with chronic obstructive pulmonary disease are immunogenic and mostly surface exposed. Only strain-specific MAbs promoted complement-dependent killing of the bacteria, which was abolished in a spontaneous mutant with an altered P2 protein.     

Importance of an immunodominant surface-exposed loop on outer membrane protein P2 of nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHI) frequently causes recurrent infections of the respiratory tract in humans. Previous indirect evidence suggested that a strain-specific immune response occurs following infection and that this immune response is directed at an immunodominant epitope on the bacterial surface. To test this hypothesis, mice and rabbits were immunized with whole cells of a strain of NTHI and the antiserum was characterized to identify the antigens to which antibodies were directed. All animals made a prominent antibody response to the loop 5 region of the P2 molecule, which is the major outer membrane protein. Rabbit serum showed complement-dependent bactericidal activity. Adsorption of the immune serum with the loop 5 fusion peptide removed bactericidal activity and also abolished reactivity to P2 detected by an immunoblot assay, an enzyme-linked immunosorbent assay, and a radioimmunoprecipitation assay. These data indicate that immunization with whole cells of NTHI results in a prominent antibody response which is directed at epitopes on the loop 5 region of the P2 molecule. Thus, a strain-specific immune response to NTHI occurs as a result of the expression of an immunodominant epitope on the P2 molecule.     

Elicitation of epithelial cell-derived immune effectors by outer membrane vesicles of nontypeable Haemophilus influenzae

Outer membrane vesicles (OMVs) are produced by all Gram-negative microorganisms studied to date. The contributions of OMVs to biological processes are diverse and include mediation of bacterial stress responses, selective packaging and secretion of virulence determinants, modulation of the host immune response, and contributions to biofilm formation and stability. First characterized as transformasomes in Haemophilus, these membranous blebs facilitate transfer of DNA among bacteria. Nontypeable Haemophilus influenzae (NTHI), an opportunistic pathogen of the upper and lower respiratory tracts, produces OMVs in vivo, but there is a paucity of information regarding both the composition and role of OMVs during NTHI colonization and pathogenesis. We demonstrated that purified NTHI vesicles are 20 to 200 nm in diameter and contain DNA, adhesin P5, IgA endopeptidase, serine protease, and heme utilization protein, suggesting a multifaceted role in virulence. NTHI OMVs can bind to human pharyngeal epithelial cells, resulting in a time- and temperature-dependent aggregation on the host cell surface, with subsequent internalization. OMVs colocalize with the endocytosis protein caveolin, indicating that internalization is mediated by caveolae, which are cholesterol-rich lipid raft domains. Upon interaction with epithelial cells, NTHI OMVs stimulate significant release of the immunomodulatory cytokine interleukin-8 (IL-8) as well as the antimicrobial peptide LL-37. Thus, we demonstrated that NTHI OMVs contain virulence-associated proteins that dynamically interact with and invade host epithelial cells. Beyond their ability to mediate DNA transfer in Haemophilus, OMV stimulation of host immunomodulatory cytokine and antimicrobial peptide release supports a dynamic role for vesiculation in NTHI pathogenesis and clinically relevant disease progression.     

Epitope mapping of the outer membrane protein P5-homologous fimbrin adhesin of nontypeable Haemophilus influenzae

To identify potential immunodominant and/or adhesin binding domains of the outer membrane protein P5-homologous fimbrin adhesin of nontypeable Haemophilus influenzae (NTHI), three sets of synthetic peptides were synthesized and assayed in an adherence inhibition assay, by Western blotting, and in a biomolecular interaction analysis (BIA) system. The first series of 34 8- to 10-mer peptides represented the entire mature protein sequentially. The second set of four peptides (each 19 to 28 residues) represented the four predicted major surface-exposed regions (or loops) of this adhesin. The third series of seven peptides (each 27 to 34 residues) were specifically designed to map the third surface-exposed region. Data obtained by BIA indicated limited reactivity of a panel of high-titered immune chinchilla sera to the 8- to 10-mer peptides representing the mature protein, likely because these linear peptides did not represent continuous epitopes. However, several of these short peptides did inhibit adherence of multiple NTHI strains to a human respiratory epithelial cell. Overall, greatest relative reactivity in both BIA and adherence inhibition assays was demonstrated against, or shown by, peptides mapping to the third and fourth predicted surface-exposed regions of this adhesin, thereby indicating the presence of immunodominant and adhesin binding domains at these sites. Middle ear fluids sequentially recovered from a chinchilla with an ongoing NTHI-induced otitis media (OM) as well as sera from children with OM due to NTHI also reacted exclusively with peptides representing the third and fourth surface-exposed regions of the P5-fimbrin adhesin, indicating a similarity in immune recognition of this bacterial protein by these two hosts. Collectively, these data together with the previously demonstrated protective efficacy of immunogens derived from this adhesin in chinchilla models support the continued development of P5-fimbrin based vaccine components.     

Conservation of immune responses to proteins isolated by preparative polyacrylamide gel electrophoresis from the outer membrane of nontypeable Haemophilus influenzae.

Outer membrane proteins P2, P4, and P6 and two with molecular masses of 26 and 28 kDa have been purified from a strain of nontypeable Haemophilus influenzae by a preparative form of polyacrylamide gel electrophoresis (PAGE). Outer membrane protein P6, with a molecular mass of 16 kDa (determined by sodium dodecyl sulfate [SDS]-PAGE) was purified by both native PAGE and SDS-PAGE from three strains of nontypeable H. influenzae and one strain of type b H. influenzae. The same conditions were required for purification from each strain. The suitability of proteins isolated by these methods was assessed by studying the immune response of rats immunized with P6 in incomplete Freund's adjuvant into the Peyer's patches. P6 purified by either native PAGE or SDS-PAGE did not differ significantly from P6 purified by gel filtration and anion-exchange chromatography in the ability to enhance pulmonary clearance of live bacteria. This study also investigated the effects of SDS on P2 immunological responses in vivo and the effects of the reagents Zwittergent and sodium lauryl sarcosinate on outer membrane protein lymphocyte-proliferative responses in vitro. It was found that the presence of SDS in the immunization emulsion enhanced the antigen-specific cell-mediated response but suppressed the antigen-specific antibody responses. The presence of residual traces of Zwittergent in an outer membrane protein preparation inhibited antigen-specific cell-mediated proliferation, whereas extraction of outer membrane proteins with sodium lauryl sarcosinate did not inhibit antigen-specific proliferation. These results demonstrate that preparative PAGE is a suitable method for the purification of proteins from the outer membrane of H. influenzae required for investigation of their immunological significance as vaccine candidates and that traces of reagents used during protein purification may play an important role in determining the success of in vivo and in vitro studies.     

Neonatal, urogenital isolates of biotype 4 nontypeable Haemophilus influenzae express a variant P6 outer membrane protein molecule.

The P6 outer membrane protein is a highly conserved molecule which is present on the surface of all strains of Haemophilus influenzae. Sixty strains of nontypeable H. influenzae which caused invasive disease or colonized the female urogenital tract were studied with monoclonal antibodies 7F3 and 4G4, which recognize different surface-exposed epitopes on the P6 molecule. All 60 strains expressed the epitope recognized by 4G4, whereas 47 of 60 strains expressed the epitope recognized by antibody 7F3. The 7F3-nonreactive strains were all biotype 4 and were recovered from the blood of neonates or postpartum women or from the female urogenital tract. The P6 genes from two 7F3-nonreactive strains were cloned, and the nucleotide sequences were determined. Analysis of amino acid sequences, immunoassays with synthetic peptides, and site-directed mutation of the P6 gene indicate that the epitope recognized by antibody 7F3 is conformational and that the sequence Asp-Ile-Thr is critical in maintaining the conformation of the epitope. We conclude that the unusually virulent clone family of biotype 4 strains of nontypeable H. influenzae express a variant P6 molecule which has an alteration in a highly conserved surface-exposed epitope.     

Identification of an outer membrane protein involved in utilization of hemoglobin-haptoglobin complexes by nontypeable Haemophilus influenzae.

A recombinant plasmid containing a 6.5-kb fragment of nontypeable Haemophilus influenzae (NTHI) chromosomal DNA was shown to confer a hemoglobin-haptoglobin-binding phenotype on Escherichia coli. Use of a mini-Tn10kan transposon for random insertion mutagenesis of this recombinant plasmid allowed localization of the NTHI DNA responsible for this hemoglobin-haptoglobin-binding phenotype to a 3.5-kb PstI-XhoI fragment within the 6.5-kb NTHI DNA insert. When this mutagenized NTHI DNA fragment was used to transform the wild-type NTHI strain, the resultant kanamycin-resistant mutant exhibited significantly decreased abilities to bind hemoglobin-haptoglobin and utilize it as a source of heme for aerobic growth in vitro. This mutant also lacked expression of a 115-kDa outer membrane protein that was present in the wild-type parent strain. Transformation of this mutant with wild-type NTHI chromosomal DNA restored the abilities to bind and utilize hemoglobin-haptoglobin and to express the 115-kDa outer membrane protein. Nucleotide sequence analysis of the relevant NTHI DNA revealed the presence of a gene, designated hhuA, that encoded a predicted 117,145-Da protein. The HhuA protein exhibited features typical of a TonB-dependent outer membrane receptor and had significant identity with the hemoglobin receptors of both Haemophilus ducreyi and Neisseria meningitidis.     

Diversity of the P2 protein among nontypeable Haemophilus influenzae isolates.

The genes for outer membrane protein P2 of four nontypeable Haemophilus influenzae strains were cloned and sequenced. The derived amino acid sequences were compared with the outer membrane protein P2 sequence from H. influenzae type b MinnA and the sequences of P2 from three additional nontypeable H. influenzae strains. The sequences were 76 to 94% identical. The sequences had regions with considerable variability separated by regions which were highly conserved. The variable regions mapped to putative surface-exposed loops of the protein.     

Pasteurella multocida produces a protein with homology to the P6 outer membrane protein of Haemophilus influenzae.

An antibody specific for a 16-kDa outer membrane protein of a rabbit strain of Pasteurella multocida was used to probe representatives of all 16 somatic serotypes of P. multocida, as well as the vaccine strains CU and M9, and all were shown to express the protein. The gene encoding this protein was cloned and sequenced and found to have extensive sequence homology with the gene encoding the P6 protein of Haemophilus influenzae. The protein in P. multocida has been designated P6-like. The gene encoding the P6-like protein was used to probe members of the family Pasteurellaceae and other gram-negative bacteria. Representatives of all 16 somatic serotypes (as well as the vaccine strains CU and M9) of P. multocida hybridized with the P6-like gene under conditions of high stringency. The DNA from H. influenzae hybridized weakly with the P6-like gene under these conditions, but Pasteurella haemolytica (representatives of A and T biotypes), Bordetella bronchiseptica, B. avium, Actinobacillus suis, A. suis-like, A. lignieresii, A. ureae, A. rossii, A. pleuropneumoniae, A. equuli, and various members of the family Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae, and Salmonella typhimurium) did not hybridize detectably. Under conditions of lower stringency, the P6-like gene also hybridized strongly with DNA from P. multocida, H. influenzae, and A. rossii but weakly with DNA from P. haemolytica and members of the genus Actinobacillus. These results suggest that the P6-like protein of P. multocida might be useful as an immunizing product to protect poultry from avian cholera. This suggestion stems from (i) our finding that the P6-like protein in P. multocida is widely distributed among all the somatic serotypes and (ii) the previous work of others demonstrating that the P6 protein of H. influenzae elicits a protective immune response in animal models of human disease.     

Efficacy of the 26-kilodalton outer membrane protein and two P5 fimbrin-derived immunogens to induce clearance of nontypeable Haemophilus influenzae from the rat middle ear and lungs as well as from the chinchilla middle ear and nasopharynx

The rat middle ear and lung clearance model has been used to show that the nontypeable Haemophilus influenzae 26-kDa outer membrane protein OMP26 is highly efficacious as a mucosal immunogen, inducing significantly enhanced clearance in immunized rats upon direct challenge of these two anatomic sites. Similarly, the chinchilla model of middle ear and nasopharyngeal clearance has been used to show that two P5 fimbrin adhesin-derived immunogens, LB1 and lipoprotein D (LPD)-LB1(f)(2,1,3), are highly efficacious as parenteral immunogens. Both induced significantly augmented clearance of nontypeable H. influenzae upon challenge of these sites. Here, these three nontypeable H. influenzae immunogens in addition to six bovine serum albumin and keyhole limpet hemocyanin conjugates of the synthetic peptide LB1(f) were assayed for relative efficacy in the reciprocal rodent model system. OMP26 was assayed in the chinchilla host by a parenteral immunization route, with clearance of the middle ear and nasopharynx used as outcome measures. Both LB1 and LPD-LB1(f)(2,1,3) were assayed in the rat host with a mucosal immunization route and clearance of nontypeable H. influenzae from the lungs and middle ears as outcome measures. Both of the immunogens were found to induce a high-titered and specific immune responses in the heterologous host system. Moreover, each was found to be highly efficacious in the reciprocal host system, providing strong support for the continued development and inclusion of both OMP26 and P5 fimbrin-derived peptides as candidate vaccine antigens directed at otitis media caused by nontypeable H. influenzae.     

Nonculture detection of Haemophilus influenzae in sputum with monoclonal antibodies specific for outer membrane lipoprotein P6

Isolation of Haemophilus influenzae from sputum is hampered by overgrowth by other microorganisms or by antibiotic treatment of the patient. To overcome this problem in the detection of H. influenzae, an in situ immunoperoxidase staining technique was developed with monoclonal antibody (MAb) 8BD9, immunoglobulin subclass G2a. MAb 8BD9 appeared to be directed to an epitope on the outer membrane lipoprotein P6 of H. influenzae. The species specificity of MAb 8BD9 was analyzed by staining isolates from different bacterial species. MAb 8BD9 reacted with all 300 H. influenzae strains tested and with H. aegyptius and H. haemolyticus. Twenty-six of 30 H. parainfluenzae strains, other Haemophilus species, and other bacterial species often isolated from sputum were not stained. The staining technique was compared with culture of 845 routinely obtained sputum samples from patients with respiratory tract infections. In 829 sputa (98.1%), the results of both techniques were in agreement; 173 were positive for H. influenzae, and 656 were negative. With 14 sputum samples, the staining method gave a positive result for H. influenzae, but the bacterium was not cultured. This could be ascribed to antibiotic treatment of the patient (n = 7), the presence of other MAb 8BD9-positive Haemophilus species in the sputum (n = 5), and overgrowth by swarming Proteus mirabilis or by Branhamella catarrhalis. In the immunoperoxidase- and Gram-stained smears of two sputum samples, no bacteria were seen, although some H. influenzae was cultured. On the basis of these results, we conclude that immunoperoxidase staining with MAb 8BD9 is a fast and reliable extension of the available detection techniques for H. influenzae.     

Molecular analysis of the P2 porin protein of nontypeable Haemophilus influenzae.

The P2 porin protein is the most abundant outer membrane protein (OMP) of nontypeable Haemophilus influenzae (NTHI) and shows extensive antigenic heterogeneity among strains. To study the molecular basis of this heterogeneity, the DNA sequences of the genes encoding the P2 proteins of three unrelated strains of NTHI were determined, and restriction fragment length polymorphisms around the P2 genes of 35 strains were analyzed. The deduced amino acid sequences of the P2 genes from the three strains of NTHI revealed four major (12 to 35 amino acids long) and several smaller (2 to 7 amino acids) hypervariable regions in each protein. The major variations occurred in identical portions of the genes, and these regions showed a high antigenic index and surface exposure probability in computer modeling analysis. Differences in the molecular mass of the P2 protein correlate with differences in the size of the variable region in each strain. Oligonucleotide primers suitable for amplification of the P2 genes by polymerase chain reaction were developed. Restriction fragment length polymorphism analysis showed marked heterogeneity in and around the ompP2 locus of 35 NTHI strains. These results contrast with the high degree of conservation of the P2 genes in H. influenzae type b strains. We conclude that the molecular mass and antigenic heterogeneity of the P2 molecule of NTHI is due to variations in gene sequence that are clustered primarily in four large hypervariable regions of the gene.     

Immunization with outer membrane protein P6 from nontypeable Haemophilus influenzae induces bactericidal antibody and affords protection in the chinchilla model of otitis media.

The role of nontypeable Haemophilus influenzae (NTHi) outer membrane protein (OMP) P6 in the pathogenesis of otitis media (OM) has not been defined. OMPs, fimbriae, pili, and lipooligosaccharide are several types of surface antigens of NTHi that are currently being evaluated as potential vaccine candidates. P6 is antigenically conserved among both nontypeable and type b H. influenzae strains and elicits bactericidal as well as protective antibodies; however, initial evaluation of a vaccine mixture of P6 combined with other NTHi OMPs failed to induce bactericidal antibody or protection in the chinchilla model of OM. We undertook an assessment of the ability of immunization with isolated P6 lipoprotein alone to confer protection. Chinchillas were immunized with P6 and challenged 10 days after the final immunization with either 3 x 10(3) CFU of NTHi delivered directly into the middle ear to induce OM or 5 x 10(8) CFU of NTHi delivered intranasally to establish nasopharyngeal colonization. All immunized animals responded with elevated serum titers of anti-P6 antibody, which also demonstrated bactericidal activity against homologous as well as a heterologous NTHi isolate. By 14 days post-transbullar challenge, the number of chinchillas with middle ear fluid and the incidence of NTHi culture-positive middle ear fluids were reduced 48 and 51%, respectively, in the P6-immunized chinchillas relative to the sham-immunized cohort. Nasopharyngeal colonization levels were comparable in the two cohorts. These data demonstrate that active immunization with P6 results in the production of NTHi-specific bactericidal antibody in the chinchilla and also affords a reduction in the incidence of NTHi-induced OM; however, parenteral immunization does not appear to affect the extent or duration of nasopharyngeal colonization by NTHi.     

Immunological responsiveness of chinchillas to outer membrane and isolated fimbrial proteins of nontypeable Haemophilus influenzae.

Thin, nonhemagglutinating fimbriae have been demonstrated on 100% of the clinical isolates of nontypeable Haemophilus influenzae recovered from children with chronic otitis media tested in this laboratory (L. O. Bakaletz, B.M. Tallan, T.M. Hoepf, T.F. DeMaria, H.G. Birck, and D.J. Lim, Infect. Immun. 56:331-335, 1988). Chinchillas with induced otitis media responded to this surface-located antigen of both the infecting and a heterologous strain. Antibodies were found in both serum and middle ear fluids.     

Construction of a mutant and characterization of the role of the vaccine antigen P6 in outer membrane integrity of nontypeable Haemophilus influenzae

Outer membrane protein P6 is the subject of investigation as a vaccine antigen to prevent infections caused by nontypeable Haemophilus influenzae, which causes otitis media in children and respiratory tract infections in adults with chronic lung disease. P6 induces protective immune responses in animal models and is the target of potentially protective immune responses in humans. P6 is a 16-kDa lipoprotein that shares homology with the peptidoglycan-associated lipoproteins of gram-negative bacteria and is highly conserved among strains of H. influenzae. To characterize the function of P6, an isogenic mutant was constructed by replacing the P6 gene with a chloramphenicol resistance cassette. The P6 mutant showed altered colony morphology and slower growth in vitro than that of the parent strain. By electron microscopy, the P6 mutant cells demonstrated increased size, variability in size, vesicle formation, and fragility compared to the parent cells. The P6 mutant showed hypersensitivity to selected antibiotics with different mechanisms of action, indicating increased accessibility of the agents to their targets. The P6 mutant was more sensitive to complement-mediated killing by normal human serum. Complementation of the mutation in trans completely or partially restored the phenotypes. We concluded that P6 plays a structural role in maintaining the integrity of the outer membrane by anchoring the outer membrane to the cell wall. The observation that the absence of expression of P6 is detrimental to the cell is a highly desirable feature for a vaccine antigen, supporting further investigation of P6 as a vaccine candidate for H. influenzae.     

Antibodies to loop 6 of the P2 porin protein of nontypeable Haemophilus influenzae are bactericidal against multiple strains

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.