CD8+ T cells have an essential role in pulmonary clearance of nontypeable Haemophilus influenzae following mucosal immunization

A rodent respiratory experimental model has proved useful for investigating the immune mechanisms responsible for clearance of bacteria from the lungs. Immunohistochemical studies in immune and nonimmune rats have identified the cellular kinetics of response to bacterial pulmonary infection for CD8+, CD4+, and gammadelta+ T cells; B cells; and the expression of major histocompatibility complex class II (MHC-II). During the course of bacterial clearance, there was no apparent proliferation or extravasation of lymphocytes, nor was there increased expression of MHC-II in nonimmune animals despite an influx of polymorphonuclear leukocytes, whereas in immunized animals there was an early influx of CD8+ and gammadelta+ T cells, followed by enhanced expression of the MHC-II marker, cellular infiltration by polymorphonuclear leukocytes, and finally an increased number of CD4+ T cells. Depletion of CD8+ T cells confirmed their vital contribution in the preprimed immune response to pulmonary infection by significantly decreasing the animals' ability to clear bacteria following challenge.     

Occult bacteremia with nontypeable Haemophilus influenzae.

A 2-year-old boy had occult bacteremia with nontypeable Haemophilus influenzae 6 weeks after receiving H. influenzae type b polysaccharide vaccine. Evaluation of his host defense was normal. As determined by outer membrane protein electrophoresis and Southern hybridization analysis, this strain was not related to type b strains. Its virulence in rats was similar to that of another nontypeable strain and less than that of a type b strain.     

Lipooligosaccharides containing phosphorylcholine delay pulmonary clearance of nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi) causes pulmonary infections in patients with chronic obstructive pulmonary disease and other mucociliary clearance defects. Like many bacteria inhabiting mucosal surfaces, NTHi produces lipooligosaccharide (LOS) endotoxins that lack the O side chain. Persistent NTHi populations express a discrete subset of LOS glycoforms, including those containing phosphorylcholine (PCho). In this study, we compared two NTHi strains with isogenic mutants lacking PCho for clearance from mice following pulmonary infection. Consistent with data from other model systems, populations of the strains NTHi 2019 and NTHi 86-028NP recovered from mouse lung contained an increased proportion of PCho+ variants compared to that in the inocula. PCho- mutants were more rapidly cleared. Serial passage of NTHi increased both PCho content and bacterial resistance to clearance, and no such increases were observed for PCho- mutants. Increased PCho content was also observed in NTHi populations within non-endotoxin-responsive C3H/HeJ and Toll-like receptor 4 null (TLR4-/-) mice, albeit at later times postinfection. Changes in bacterial subpopulations and clearance were unaffected in TLR2-/- mice compared to the subpopulations in and clearance from mice of the parental strain. The clearance of PCho- mutants occurred at earlier time points in both strain backgrounds and in all types of mice. Comparison of bacterial populations in lung tissue cryosections by immunofluorescent staining showed sparse bacteria within the air spaces of C57BL/6 mice and large bacterial aggregates within the lungs of MyD88-/- mice. These results indicate that PCho promotes bacterial resistance to pulmonary clearance early in infection in a manner that is at least partially independent of the TLR4 pathway.     

Long PCR-ribotyping of nontypeable Haemophilus influenzae.

PCR-ribotyping, a new typing method based on long PCR, has been developed for nontypeable Haemophilus influenzae (NTHi). Ribosomal operons of NTHi were amplified by long PCR and were found to be highly polymorphic for internal HaeIII sites. The technique was applied to 49 isolates previously subjected to conventional ribotyping, and the two methods showed a high level of concordance for serial isolates from individual subjects. PCR-ribotyping provides a powerful new typing tool for strain characterization in epidemiological investigations of NTHi.     

Immunization with recombinant transferrin binding protein B enhances clearance of nontypeable Haemophilus influenzae from the rat lung

Nontypeable Haemophilus influenzae (NTHI) is an opportunistic pathogen, and heterogeneity in the surface-exposed immunodominant domains of NTHI proteins is thought to be associated with the failure of an infection to stimulate an immune response that is cross-protective against heterologous NTHI strains. The aim of this study was to assess the vaccine potential of a surface-exposed component of the NTHI human transferrin receptor, TbpB, and to determine if the antibody response elicited was cross-reactive with heterologous strains of NTHI. The efficacy of immunization with a recombinant form of TbpB (rTbpB) was determined by assessing the pulmonary clearance of viable bacteria 4 h after a live challenge with NTHI. There was a significant reduction in the number of viable bacteria in both the bronchoalveolar lavage fluid (34% for the 20-microgram dose and 58% for the 40-microgram dose) and lung homogenates (26% for the 20-microgram dose and 60% for the 40-microgram dose) of rats immunized with rTbpB compared to the control animals. While rTbpB-specific antibodies from immunized rats were nonspecific in the recognition of TbpB from six heterologous NTHI strains on Western blots, these antibodies differed in their ability to block transferrin binding to heterologous strains and to cross-react in bactericidal assays. If bactericidal antibodies are key indicators of the efficacy of the immune response in eliminating NTHI, this data suggests that while immunization with rTbpB stimulates protective responses against the homologous isolate, variability in the recognition of TbpB from heterologous isolates may limit the potential of rTbpB as an NTHI vaccine component.     

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.     

Intranasal immunization enhances clearance of nontypeable Haemophilus influenzae and reduces stimulation of tumor necrosis factor alpha production in the murine model of otitis media

Nontypeable Haemophilus influenzae (NTHi) is a major pathogen causing otitis media (OM). One of the outer membrane proteins of NTHi, P6, is a common antigen to all strains and is considered a candidate for mucosal vaccine. We have previously reported that intranasal immunization with P6 and cholera toxin (CT) could induce P6-specific immunoglobulin A (IgA) antibodies in the middle ear. In the present study, we assessed the effect of intranasal immunization for the protection against NTHi-induced OM. Mice were immunized intranasally with P6 and CT as an adjuvant on days 0, 7, and 14. Control mice were given phosphate-buffered saline (PBS) without antigen. One week after the final immunization, a suspension of live NTHi (10(7) CFU) was injected into the tympanic cavity to induce experimental OM. On days 3 and 7 after bacterial challenge, mice were killed and middle ear effusions (MEEs) were collected. All immunized mice showed elevated titers of P6-specific antibodies in MEEs. The rank order of specific antibody included, from highest to lowest levels, IgG, IgA, and IgM. In addition, immunized mice showed enhanced clearance of NTHi from the middle ear and the number of NTHi in MEEs of immunized mice was reduced by 97% on day 3 and by 92% on day 7 after bacterial challenge relative the number in the MEEs of control mice. The protective effect of intranasal immunization on the incidence of NTHi-induced experimental OM was evident on day 7 after challenge. By day 7, the number of MEEs in immunized mice was 64% less than that in control mice and the incidence of NTHi culture-positive MEEs in immunized mice was 56% less than that in control mice. Less stimulation of tumor necrosis factor alpha (TNF-alpha) production in the middle ear was evident on day 3 after challenge. Immunized mice showed lower concentrations of TNF-alpha in MEEs. These results indicate that intranasal immunization affords protection against experimental OM as evidenced by enhanced clearance of NTHi and less stimulation of TNF-alpha production in the middle ear. These findings suggest that a nasal vaccine might be useful for preventing OM.     

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.     

Clonality of multidrug-resistant nontypeable strains of Haemophilus influenzae.

The genetic structure of a population of multidrug-resistant nontypeable (unencapsulated) Haemophilus influenzae strains isolated at a hospital in Barcelona, Spain, was investigated by using multilocus enzyme electrophoresis to determine the allelic variation in 15 structural loci. In our study we have also included some antimicrobial agent-susceptible strains isolated at the same hospital. All enzymes were polymorphic for two to eight electromorphs, and the analysis revealed 43 distinct electrophoretic types among the 44 isolates. The mean genetic diversity of the entire population was 0.55. Multilocus linkage disequilibrium analysis of the isolates revealed a strong association between alleles, suggesting little possibility of recombination. Furthermore, the dendrogram and the allele mismatch distribution are typical of a population with no extensive genetic mixing.     

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.     

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.     

Effect of systemic immunization on pulmonary clearance of Haemophilus influenzae type b.

The effect of systemic immunization on pulmonary clearance of Haemophilus influenzae type b (Hib) was studied in a mouse model system. Immunization of mice by intraperitoneal injection of viable Hib cells resulted in the appearance of Hib-directed antibodies in both serum and bronchoalveolar lavage fluid. The development of this Hib-directed antibody activity was associated with significant enhancement of early pulmonary clearance of Hib. Systemic immunization did not affect the recruitment of polymorphonuclear leukocytes to the alveoli, suggesting that the enhanced clearance of Hib observed in immunized animals was due to specific antibodies which promote either phagocytosis or extracellular killing of Hib. The spectrum of Hib-directed antibody specificities detected in sera from immunized animals was essentially identical to that detected in bronchoalveolar lavage fluids from these same animals. Similarly, intravenous administration of an immunoglobulin G monoclonal antibody specific for Hib lipopolysaccharide resulted in the subsequent appearance of this antibody in the alveolar spaces where it enhanced pulmonary clearance of Hib. This study shows that this mouse model system can be used to measure the effect of both active and passive immunization on the clearance of Hib from the lower respiratory tract.     

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。     

Effect of primary immunization on pulmonary clearance of nontypable Haemophilus influenzae

Nontypable Haemophilus influenzae (NTHI) is being increasingly recognized as a cause of both adult pneumonia and acute infectious exacerbations in chronic bronchitis. We used a mouse model to study the immune enhancement of pulmonary clearance of NTHI after a primary immunization. BALB/c mice were immunized with whole NTHI either by intraperitoneal (i.p.) or intratracheal (i.t.) routes. There was 10-fold more NTHI-directed antibody detected in the serum of the i.p.-immunized mice than in the serum from the i.t.-immunized animals. Western blot analysis revealed that these antibodies were directed against both NTHI lipooligosaccharide and the various outer membrane proteins of NTHI. The development of NTHI-directed antibodies in serum was associated with significant enhancement of early pulmonary clearance of NTHI. Six hours after delivery of an endobronchial challenge with NTHI, the i.p.-immunized mice had cleared most of the organisms from their lungs, while the i.t.-immunized mice did not clear NTHI any more rapidly than did unimmunized mice. Serum from the i.p.-immunized mice caused more than 99% uptake of NTHI in an in vitro opsonophagocytic assay, while serum from i.t.-immunized mice stimulated little or no phagocytosis of this organism. Opsonophagocytosis of NTHI was obtained with bronchoalveolar lavage (BAL) fluid collected from i.p.-immunized mice 6 h after, but not before, an endobronchial challenge with NTHI. Intravenous injection of an opsonic IgG monoclonal antibody directed against NTHI lipooligosaccharide resulted in both the appearance of this antibody in the alveolar spaces of the unperturbed lung and enhanced pulmonary clearance of NTHI. These data indicate that the i.p. (systemic) route of immunization is more effective than the i.t. route in establishing pulmonary immunity to NTHI in this model system. Furthermore, immune enhancement of clearance of NTHI from the lungs after a primary immunization apparently results from the exudation of opsonic and bactericidal antibodies from the serum into the alveolae in response to the inflammatory challenge.     

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.     

Reconstitution of a porin-deficient mutant of Haemophilus influenzae type b with a porin gene from nontypeable H. influenzae.

The major outer membrane protein (OmpP2) of nontypeable Haemophilus influenzae (NTHI) has been shown to vary markedly with respect to both size and the presence of specific surface-exposed epitopes among strains of this unencapsulated pathogen. In contrast, the OmpP2 proteins of H. influenzae type b (Hib) strains are well conserved at the level of primary protein structure and have in common several surface-exposed antigenic determinants that have not been detected in NTHI strains. The availability of an isogenic, avirulent Hib ompP2 mutant made it possible to investigate whether an NTHI OmpP2 protein could function properly in the Hib outer membrane. A plasmid shuttle vector (pGJB103) was used to clone the ompP2 gene from NTHI TN106 into a recombination-deficient H. influenzae strain in which expression of the NTHI OmpP2 protein was detected by means of an NTHI TN106 OmpP2-specific monoclonal antibody. The amino acid sequence of this NTHI OmpP2 protein, as deduced from the nucleotide sequence of the NTHI TN106 ompP2 gene, was determined to be 83% identical to that of the Hib OmpP2 protein. Transformation of this cloned NTHI ompP2 gene into the Hib ompP2 mutant yielded a Hib transformant strain that expressed the NTHI OmpP2 protein. Expression of this NTHI OmpP2 protein allowed the Hib ompP2 mutant, which normally grows poorly in vitro, to grow in a manner indistinguishable from that of the wild-type Hib strain. More importantly, the introduction of this functional NTHI ompP2 gene into the avirulent Hib ompP2 mutant restored the virulence of this strain to wild-type levels. These results indicate that an NTHI OmpP2 protein can be expressed and function properly in the Hib outer membrane.     

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.     

Mapping of bactericidal epitopes on the P2 porin protein of nontypeable Haemophilus influenzae.

The P2 porin protein is the major outer membrane protein of nontypeable Haemophilus influenzae and is a potential target of a protective immune response. Nine monoclonal antibodies (MAbs) to P2 were developed by immunizing mice with nontypeable H. influenzae whole organisms. Each MAb reacted exclusively with the homologous strain in a whole-cell immunodot assay demonstrating exquisite strain specificity. All nine MAbs recognized abundantly expressed surface-exposed epitopes on the intact bacterium by immunofluorescence and immunoelectron microscopy. Each MAb was bactericidal to the homologous strain in an in vitro complement-mediated killing assay. Immunoblot assay of cyanogen bromide cleavage products of purified P2 indicated that MAb 5F2 recognized the 10-kDa fragment, and the other eight MAbs recognized the 32-kDa fragment. Competitive ELISAs confirmed that 5F2 recognized an epitope that is different from the other eight MAbs. To further localize epitopes, MAbs 5F2 and 6G3 were studied in protein footprinting by using reversed-phase high-performance liquid chromatography. Three potential epitope-containing peptides which were reactive in an enzyme-linked immunosorbent assay with both 5F2 and 6G3 were isolated. These peptides were identified by N-terminal amino acid sequence and localized to loops 5 and 8 of the proposed model for P2. Fusion proteins consisting of glutathione S-transferase fused with variable-length peptides from loops 5 and 8 were expressed in the pGEX-2T vector. Immunoblot assay of fusion peptides of loops 5 and 8 confirmed that 5F2 recognized an epitope within residues 338 to 354 of loop 8; 6G3 and the remaining MAbs recognized an epitope within residues 213 to 229 of loop 5. These studies indicate that nontypeable H. influenzae contains bactericidal epitopes which have been mapped to two different surface-exposed loops of the P2 molecule. These potentially protective epitopes are strain specific and abundantly expressed on the surface of the intact bacterium.     

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.     

Strain-specific and immunodominant surface epitopes of the P2 porin protein of nontypeable Haemophilus influenzae.

The P2 porin protein is the major outer membrane protein of nontypeable Haemophilus influenzae. Five monoclonal antibodies to P2 of four strains of nontypeable H. influenzae were developed by immunizing mice with whole bacterial cells. All five antibodies recognized epitopes on P2 in immunoblot assays of whole organism lysates, purified outer membrane, and purified P2. Competitive enzyme-linked immunosorbent assays and immunoblot assays of cyanogen bromide-digested P2 showed that two antibodies to the P2 protein of strain 1479 recognized different epitopes on the molecule. Immunofluorescence and immunoelectron microscopy demonstrated that each of the five antibodies recognized epitopes that were abundantly expressed on the bacterial surface. Analysis of 120 H. influenzae strains indicated that three of the five antibodies were reactive exclusively with the homologous strain. The remaining two antibodies were reactive with less than 3% of the strains. These studies indicate that the P2 protein expresses a highly strain-specific and immunodominant epitope on the bacterial surface. The expression of strain-specific and immunodominant epitopes on the bacterial surface may represent a mechanism by which the bacterium induces antibodies that will protect against recurrent infection by the homologous strain but will not protect against infection by heterologous strains.