Variability of outer membrane protein P1 and its evaluation as a vaccine candidate against experimental otitis media due to nontypeable Haemophilus influenzae: an unambiguous, multifaceted approach

Candidate vaccine antigens for preventing otitis media caused by nontypeable Haemophilus influenzae (NTHI) should possess one or more conserved epitopes. We sought to evaluate the candidacy of P1, a surface-expressed outer membrane protein knowing that this antigen is subject to diversifying selection. Therefore, we selected NTHI strains from among >500 phylogenically variant isolates representative of the diversity found in natural populations of H. influenzae. Twenty-three variants of P1 (相似文献   

Mapping of a strain-specific bactericidal epitope to the surface-exposed loop 5 on the P2 porin protein of non-typeable Haemophilus influenzae

The P2 protein is the major outer-membrane protein of non-typeable Haemophilus influenzae (NTHI) and shows extreme heterogeneity among strains. Based on the analysis of antigenic structure, the P2 protein consists of eight potentially surface-exposed loops. Previous studies of monoclonal antibodies (mABs) to a single strain of NTHI showed that P2 contains potentially immunodominant epitopes in loop 5 of the molecule. The goal of the current work is to test the hypothesis that strain-specific and potentially immunodominant epitopes are located in loop 5 of P2 in other strains of NTHI as well. Gene fragments which encode peptides of loop 5 of strains 2019 and 5657 were cloned into an expression vector and subjected to immunoassays with mABs which recognize surface-exposed, bactericidal, strain-specific epitopes. Each mAB recognized loop 5 of the P2 protein of the homologous strain. Analysis of mutant clones with minor amino acid changes showed a loss of reactivity with the mABs. These observations indicate that loop 5 of the P2 molecule contains strain-specific, abundantly expressed surface-exposed epitopes. This further supports the hypothesis that loop 5 is an immunodominant region of the P2 molecule.     

不同佐剂及免疫途径对重组NTHi外膜蛋白P6免疫效果影响的研究

目的 研究几种佐剂及免疫途径对重组NTHi P6蛋白抗原在小鼠体内产生的免疫效果的影响.方法 以A1(OH),佐剂、CpG ODN佐剂以及两种联合的复合佐剂分别与rP6蛋白混合,经滴鼻、肌肉注射免疫途径免疫6周龄雌性BALB/c小鼠;相同剂量、免疫途径加强免疫2次.采用ELISA法检测rP6蛋白特异的血清IgG和黏膜IgA滴度;酶联免疫斑点试验(ELISPOT)检测特异性T淋巴细胞的活化.结果 3次免疫后,CpG ODN+rP6滴鼻免疫组和A1(OH)3+CpG ODN+rP6肌肉注射免疫组产生高滴度的特异性血清IgG抗体(F=41.259,P=0.000).同时ELISPOT试验显示,CpG ODN+rP6无论通过滴鼻还是肌肉注射免疫均能诱导大量抗原特异性T淋巴细胞的活化,与其他实验组比较差异有统计学意义(F=66.046,P=0.000).而且CpG ODN+rP6滴鼻免疫组能诱发高滴度的特异性黏膜IgA抗体(F=70.966,P=0.000).结论 重组NTHi外膜蛋白P6与CpG ODN佐剂联合后经黏膜途径免疫,小但能保证体液免疫的高滴度血清IgG抗体,而且能从黏膜免疫和细胞免疫两方面对该蛋白的免疫效果进行增强.

Abstract：

Objective To detect the difference of cytokines and antibodies productions by immunologic system from mice vaccinated with recombinant P6 protein with different immunization routes and adiuvants.Methods 6 weeks female BALB/c mice were vaccinated with meombinant protein P6 combined with A1(OH)3,CpG ODN or the mix of them through intramuscular and intranasal.The mice were boosted twice with the same dose by the same route.Serum and respiratory tract specimen were collected to detect rP6 specific antibodies by ELISA.Results In the 6th week after immunization.The higher titer of serum rP6 specific IgG antibody were detected from the two groups vaccinated with A1(OH)3+CpG ODN+rP6 by intramuscular and vaccinated with CpG ODN+rP6 by intranasal(F=41.259.P=0.000).The ELISPOT experiment showed that,Inoculation with the CpG ODN+rP6 either by intranasal or intramuscular immunization could induce a large number of antigen-specific T lymphocyte activation.In addition.The mice vaccinated with the CpG ODN+rP6 through intranasal can be detected high titer rP6 specific mucosal IgA antibody(F=70.966.P=0.000).Conclusion Inoculation with the CoG ODN+rP6 by jntranasal immunization not only can induce high-titer serum IgG antibody,but also can induce high-titer mucosal IsA antibody and CD4+Th1.CD8+T lymphoeyte activation.     

High-Level Expression of Truncated Surface Antigen P50 of Babesia gibsoni in Insect Cells by Baculovirus and Evaluation of Its Immunogenicity and Antigenicity

Previously, we identified an immunodominant antigen, P50 of Babesia gibsoni. In the present study, the gene encoding the truncated P50 (rP50t) without a C-terminal hydrophobic region (29 amino acids [aa]) was expressed in insect cells by a recombinant baculovirus. The highly hydrophobic C-terminal 20-aa regions seems to be a transmembrane region, which was evidenced by the fact that rP50t was effectively secreted into the supernatant of insect cells infected with the recombinant baculovirus. N-terminal amino acid sequence analysis of rP50t indicated that N-terminal 19 aa function as a signal peptide. The expression level of rP50t reached up to 2 mg per 10⁸ cells infected with the recombinant baculovirus. The immunogenic property of rP50t was evaluated by an immunization test in mice. Mice immunized with rP50t induced a high-level antibody titer against the B. gibsoni merozoite. Monoclonal antibodies (MAbs) to rP50t were produced in mice to determine the immunogenic regions of P50. The epitope(s) recognized by all five MAbs were located between aa 190 and 273, suggesting that the central part of P50 is a highly immunogenic region. The diagnostic potential of rP50t was evaluated using an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate clearly (P < 0.0001) between B. gibsoni-infected dog serum and B. canis-infected dog serum or noninfected dog serum. Our results indicated that the rP50t may provide a useful potential immunogenic reagent for use in diagnosis and as a subunit vaccine to control B. gibsoni infection in dogs.     

Antigenic characterization of the oligosaccharide portion of the lipooligosaccharide of nontypable Haemophilus influenzae

Monoclonal antibodies (MAbs) directed against epitopes in the oligosaccharide portion of the lipooligosaccharide (LOS) of nontypable Haemophilus influenzae (NTHI) were used to characterize the LOS of this pathogen. Western blot (immunoblot) analysis with four LOS-specific MAbs and proteinase K-derived LOS preparations from 69 NTHI strains allowed the classification of these strains into nine LOS antigenic groups. The use of these MAbs in a more sensitive colony blot radioimmunoassay system together with these same NTHI strains identified 14 LOS antigenic groups. Extensive cross-reactivity was detected between the LOS epitopes of these NTHI strains and the LOS of H. influenzae type b. The epitopes recognized by these MAbs were not accessible to antibody on the surface of every strain. These LOS epitopes were also not stably expressed by NTHI growing in vitro; the observed frequency of LOS antigen variation ranged from 1 to 24% when large numbers of colonies of NTHI strains were screened for reactivity with the LOS-directed MAbs in the colony blot radioimmunoassay. This LOS antigenic variation was sometimes associated with alterations in the profile of the LOS molecule as resolved by dodecyl sulfate-polyacrylamide gradient gel electrophoresis followed by staining with silver. These data indicate that considerable antigenic diversity exists among NTHI strains with regard to the oligosaccharide epitopes in their LOS molecules.     

Antibodies specific for the high-molecular-weight adhesion proteins of nontypeable Haemophilus influenzae are opsonophagocytic for both homologous and heterologous strains

The HMW1/HMW2-like adhesion proteins of nontypeable Haemophilus influenzae (NTHI) are expressed by 75% of NTHI strains. Antibodies directed against these proteins are opsonophagocytic in vitro and are protective in an animal model of infection. The objective of the present study was to determine the opsonophagocytic activity of high-titer anti-HMW1/HMW2 immune sera against both homologous and heterologous NTHI strains. Chinchillas were immunized with purified HMW1/HMW2-like proteins from five prototype NTHI strains. Serum opsonophagocytic activity was monitored in an assay that uses a human promyelocytic cell line, HL-60, as the source of phagocytic cells. Preimmune sera did not demonstrate opsonophagocytic killing of any strains. In contrast, the immune sera demonstrated killing of the five homologous NTHI strains at titers ranging from 1:320 to 1:640. The immune sera also demonstrated killing of eight heterologous NTHI strains that express HMW1/HMW2-like proteins at titers ranging from 0 to 1:640. Killing of heterologous strains sometimes demonstrated a prozone phenomenon. None of the immune sera killed NTHI strains that did not express HMW1/HMW2-like proteins. Adsorption of immune sera with HMW1/HMW2-like proteins purified from either homologous or heterologous NTHI strains eliminated opsonophagocytic killing of homologous strains in most cases. These data demonstrate that antibodies produced following immunization with the HMW1/HMW2-like proteins are opsonophagocytic for both homologous and heterologous NTHI and strongly suggest that common epitopes recognized by functionally active antibodies exist on the HMW1/HMW2-like proteins of unrelated NTHI strains. The results argue for the continued investigation of the HMW1/HMW2-like proteins as potential vaccine candidates for the prevention of NTHI disease.     

Induction of specific immunoglobulin A and Th2 immune responses to P6 outer membrane protein of nontypeable Haemophilus influenzae in middle ear mucosa by intranasal immunization

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.     

High-level expression of truncated surface antigen P50 of Babesia gibsoni in insect cells by baculovirus and evaluation of its immunogenicity and antigenicity

Previously, we identified an immunodominant antigen, P50 of Babesia gibsoni. In the present study, the gene encoding the truncated P50 (rP50t) without a C-terminal hydrophobic region (29 amino acids [aa]) was expressed in insect cells by a recombinant baculovirus. The highly hydrophobic C-terminal 20-aa regions seems to be a transmembrane region, which was evidenced by the fact that rP50t was effectively secreted into the supernatant of insect cells infected with the recombinant baculovirus. N-terminal amino acid sequence analysis of rP50t indicated that N-terminal 19 aa function as a signal peptide. The expression level of rP50t reached up to 2 mg per 10(8) cells infected with the recombinant baculovirus. The immunogenic property of rP50t was evaluated by an immunization test in mice. Mice immunized with rP50t induced a high-level antibody titer against the B. gibsoni merozoite. Monoclonal antibodies (MAbs) to rP50t were produced in mice to determine the immunogenic regions of P50. The epitope(s) recognized by all five MAbs were located between aa 190 and 273, suggesting that the central part of P50 is a highly immunogenic region. The diagnostic potential of rP50t was evaluated using an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate clearly (P < 0.0001) between B. gibsoni-infected dog serum and B. canis-infected dog serum or noninfected dog serum. Our results indicated that the rP50t may provide a useful potential immunogenic reagent for use in diagnosis and as a subunit vaccine to control B. gibsoni infection in dogs.     

Immune enhancement of pulmonary clearance of nontypable Haemophilus influenzae.

BALB/c mice systemically immunized by intraperitoneal injection with whole, viable cells of two different strains of nontypable Haemophilus influenzae (NTHI) exhibited a markedly enhanced ability to clear the homologous strain of NTHI from the lower respiratory tract. Immunization did not influence the number of phagocytic cells recovered by bronchoalveolar lavage from mice before or after intrapulmonary challenge with NTHI. Immunization also induced the synthesis of relatively large quantities of NTHI-directed antibodies which were detectable in both the bloodstream and the alveolar spaces of the lung. Radioimmunoprecipitation and Western blot (immunoblot) analyses indicated that these antibodies were directed against both the proteins and lipooligosaccharide (LOS) in the NTHI outer membrane. Bactericidal and opsonophagocytic assays determined that the NTHI-directed antibodies in the serum were functional and able to kill or opsonize the homologous NTHI strain. Mice immunized with an NTHI major outer membrane protein-LOS complex also had an increased ability to effect pulmonary clearance of NTHI. Serum and bronchoalveolar lavage fluid collected from these animals immunized with the outer membrane protein-LOS complex contained relatively high levels of antibodies to both of these antigens. The serum from these animals also possessed bactericidal and opsonic activity against the homologous NTHI strain. These results indicate that systemic immunization can enhance the ability of experimental animals to clear NTHI from the lower respiratory tract and suggest that immunoprophylaxis of NTHI pulmonary disease may be feasible.     

Identification of surface-exposed B-cell epitopes on high molecular-weight adhesion proteins of nontypeable Haemophilus influenzae.

We previously reported that two surface-exposed high-molecular-weight proteins, HMW1 and HMW2, expressed by a prototypic strain of nontypeable Haemophilus influenzae (NTHI) mediate attachment to human epithelial cells. These proteins are members of a family of highly immunogenic proteins common to most nontypeable Haemophilus strains. We also reported that immunization with an HMW1-HMW2 mixture modified the course of disease in an animal model of otitis media, suggesting the potential usefulness of these proteins as NTHI vaccine components. Identification of surface-accessible B-cell epitopes could be important to efforts to develop recombinant or synthetic peptide vaccines based upon these high-molecular-weight proteins. Thus, the purpose of the present study was to identify surface-accessible epitopes on the HMW1 and HMW2 proteins by using monoclonal antibodies (MAbs) and to determine the prevalence of these epitopes among the high-molecular-weight proteins expressed by heterologous nontypeable Haemophilus strains. MAbs were generated by immunizing mice with high-molecular-weight proteins purified from prototype strains and were screened by immunoelectron microscopy (IEM) for the ability to recognize surface epitopes. Two MAbs, designated AD6 and 10C5, that recognized surface epitopes by IEM were recovered. In order to map the epitopes recognized by these two MAbs, we constructed a set of HMW1 and HMW2 recombinant fusion proteins using the pGEMEX vectors and examined the reactivity of the MAbs with these fusion proteins. MAb AD6 recognized an epitope in both HMW1 and HMW2 which mapped to the last 75 amino acids at the carboxy termini of the two proteins. When examined for reactivity with heterologous strains, MAb AD6 recognized high-molecular-weight proteins in 75% of 125 unrelated nontypeable Haemophilus strains and, in addition, reacted with three of three such strains when examined by IEM. MAb 10C5 recognized an epitope that mapped to a 155-amino-acid segment near the carboxy terminus of HMW1. This epitope was adjacent to but distinct from the AD6 epitope and was absent from HMW2. The 10C5 epitope was expressed by 40% of the AD6 reactive strains. Identification of shared surface-exposed epitopes on the high-molecular-weight adhesion proteins suggests the possibility of developing recombinant or synthetic peptide-based vaccines protective against disease caused by the majority of NTHI strains.     

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.     

Nasal immunization with a malaria transmission-blocking vaccine candidate, Pfs25, induces complete protective immunity in mice against field isolates of Plasmodium falciparum

Malaria transmission-blocking vaccines based on antigens expressed in sexual stages of the parasites are considered one promising strategy for malaria control. To investigate the feasibility of developing noninvasive mucosal transmission-blocking vaccines against Plasmodium falciparum, intranasal immunization experiments with Pichia pastoris-expressed recombinant Pfs25 proteins were conducted. Mice intranasally immunized with the Pfs25 proteins in the presence of a potent mucosal adjuvant cholera toxin induced robust systemic as well as mucosal antibodies. All mouse immunoglobulin G (IgG) subclasses except IgG3 were found in serum at comparable levels, suggesting that the immunization induced mixed Th1 and Th2 responses. Consistent with the expression patterns of the Pfs25 proteins in the parasites, the induced immune sera specifically recognized ookinetes but not gametocytes. In addition, the immune sera recognized Pfs25 proteins with the native conformation but not the denatured forms, indicating that mucosal immunization induced biologically active antibodies capable of recognizing conformational epitopes of native Pfs25 proteins. Feeding Anopheles dirus mosquitoes with a mixture of the mouse immune sera and gametocytemic blood derived from patients infected with P. falciparum resulted in complete interference with oocyst development in mosquito midguts. The observed transmission-blocking activities were strongly correlated with specific serum antibody titers. Our results demonstrated for the first time that a P. falciparum transmission-blocking vaccine candidate is effective against field-isolated parasites and may justify the investigation of noninvasive mucosal vaccination regimens for control of malaria, a prototypical mucosa-unrelated mosquito-borne parasitic disease.     

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.     

Carrier Properties of a Protein Derived from Outer Membrane Protein A of Klebsiella pneumoniae

We have recently cloned a new protein, recombinant P40 (rP40). When tested in vivo after conjugation to a B-cell epitope, rP40 induces an important antibody response without the need for adjuvant. To characterize its potency, this carrier protein was coupled to a peptide derived from respiratory syncytial virus attachment G protein (G1'). After immunization of mice with the rP40-G1' conjugate, strong antipeptide antibodies were detected, whereas peptide alone was not immunogenic. To emphasize the carrier properties of rP40, a polysaccharide derived from Haemophilus influenzae type b (Hib) was coupled to it. Immunoglobulin G responses against the Hib polysaccharide were observed after coupling to rP40. Interestingly, an antipeptide antibody response was observed despite preexisting anti-rP40 antibodies generated by preimmunization with rP40. In addition, rP40 compares well with the reference carrier protein, tetanus toxoid (TT), since antibody responses of equal intensity were observed when a peptide or a polysaccharide was coupled to TT and rP40. Moreover, rP40 had advantages compared to TT; e.g., it induced a mixed Th1/Th2 response, whereas TT induced only a Th2 profile. Together, the results indicate that rP40 is a novel carrier protein with potential for use as an alternative carrier for human vaccination.     

Therapeutic Transcutaneous Immunization with a Band-Aid Vaccine Resolves Experimental Otitis Media

Transcutaneous immunization (TCI) is a noninvasive strategy to induce protective immune responses. We describe TCI with a band-aid vaccine placed on the postauricular skin to exploit the unique organization of the stratum corneum and to promote the development of immune responses to resolve active experimental otitis media due to nontypeable Haemophilus influenzae (NTHI). This therapeutic immunization strategy induced significantly earlier resolution of middle ear fluid and rapid eradication of both planktonic and mucosal biofilm-resident NTHI within 7 days after receipt of the first immunizing band-aid vaccine. Efficacy was ascribed to the homing of immunogen-bearing cutaneous dendritic cells to the nasal-associated lymphoid tissue, induction of polyfunctional CD4⁺ T cells, and the presence of immunogen-specific IgM and IgG within the middle ear. TCI using band-aid vaccines could expand the use of traditional parenteral preventative vaccines to include treatment of active otitis media, in addition to other diseases of the respiratory tract due to NTHI.     

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.     

Characterization and evaluation of the Moraxella catarrhalis oligopeptide permease A as a mucosal vaccine antigen

Moraxella catarrhalis is a common cause of otitis media in children and of lower respiratory tract infections in adults with chronic obstructive pulmonary disease; therefore, these two groups would benefit from a vaccine to prevent M. catarrhalis infections. A genome mining approach for vaccine antigens identified oligopeptide permease protein A (OppA), an oligopeptide binding protein of an apparent oligopeptide transport system. Analysis of the oppA gene by PCR and sequence analysis revealed that OppA is highly conserved among clinical isolates of M. catarrhalis. Recombinant OppA was expressed as a lipoprotein and purified, and an oppA knockout mutant was constructed. Antiserum raised to recombinant purified OppA recognized epitopes on the bacterial surface of the wild type but not the OppA knockout mutant. Antibodies raised to purified recombinant OppA recognized native OppA in multiple strains. Intranasal immunization of mice induced systemic and mucosal antibodies to OppA and resulted in enhanced clearance of M. catarrhalis in a mouse pulmonary clearance model. OppA is a highly conserved, immunogenic protein that expresses epitopes on the bacterial surface and that induces potentially protective immune responses in a mouse model. OppA should be evaluated further as a vaccine antigen for M. catarrhalis.     

Immunologic and Structural Relationships of the Minor Pilus Subunits among Haemophilus influenzae Isolates

Two proteins, HifD and HifE, have been identified as structural components of Haemophilus influenzae pili. Both are localized at the pilus tip, and HifE appears to mediate pilus adherence to host cells. In this study we examined the immunologic and structural diversity of these pilus subunits among type b H. influenzae (Hib) and nontypeable H. influenzae (NTHI) strains. Western immunoblot analysis revealed that antibodies directed against the C terminus of HifD and HifE from Hib strain Eagan bound to HifD and HifE proteins, respectively, of all piliated Hib and NTHI strains tested. Whole-cell enzyme-linked immunosorbent assays showed that antibodies specific for native HifD or HifE of strain Eagan also bound to all piliated Hib strains but did not bind to the piliated NTHI strains. Antibodies against HifE of strain Eagan inhibited the binding of Hib to human erythrocytes but did not inhibit the binding of NTHI strains. Restriction fragment length polymorphism (RFLP) analysis was used to determine strain-to-strain structural differences within hifD and hifE genes, either by PCR or by nucleotide sequence analysis. DNA and derived amino acid sequence analyses of HifD and HifE confirmed the uniqueness of the RFLP types. The hifD and hifE genes of all type b strains showed identical restriction patterns. Analysis of hifD and hifE genes from the NTHI strains, however, revealed seven unique RFLP patterns, suggesting that these genes encode proteins with diverse primary structures. These results indicate that HifD and HifE are immunologically and structurally similar among the Hib strains but vary among the NTHI strains.     

P27 (MBOV_RS03440) is a novel fibronectin binding adhesin of Mycoplasma bovis

Mycoplasma bovis, one of the major pathogens of bovine respiratory disease, binds to respiratory epithelial cells resulting in severe pneumonia and tissue damage. This study was designed to identify the adhesive function of a putative 27-kDa M. bovis lipoprotein, encoded by the gene MBOV_RS03440 and designated as P27. The gene was cloned and overexpressed to produce antibodies against the recombinant P27 (rP27). The western blot and flow cytometry assay confirmed P27 to be a surface-localized protein, while ELISA confirmed it to be an immunogenic protein. Confocal immunofluorescence microscopy demonstrated that rP27 bound to embryonic bovine lung (EBL) cell monolayers in a dose-dependent manner. Furthermore, anti-rP27 antiserum inhibited the attachment of M. bovis to EBL cells demonstrating the binding specificity of P27 to EBL cells. The attachment of rP27 to EBL cells was mediated by fibronectin (Fn), an extracellular matrix component. The interaction between rP27 and Fn was qualitatively and quantitatively monitored by ligand immunoblot assay, ELISA, and biolayer interferometry. Collectively, these results indicate that P27 is a novel Fn-binding, immunogenic adhesive protein of M. bovis, thereby contributing to the further understanding of the molecular pathogenesis of M. bovis.     

Nasopharyngeal Colonization with Nontypeable Haemophilus influenzae in Chinchillas

Colonization of the nasopharynx by a middle ear pathogen is the first step in the development of otitis media in humans. The establishment of an animal model of nasopharyngeal colonization would therefore be of great utility in assessing the potential protective ability of candidate vaccine antigens (especially adhesins) against otitis media. A chinchilla nasopharyngeal colonization model for nontypeable Haemophilus influenzae (NTHI) was developed with antibiotic-resistant strains. This model does not require coinfection with a virus. There was no significant difference in the efficiency of NTHI colonization between adult (1- to 2-year-old) and young (2- to 3-month-old) animals. However, the incidence of middle ear infection following nasopharyngeal colonization was significantly higher in young animals (83 to 89%) than in adult chinchillas (10 to 30%). Chinchillas that had recovered either from a previous middle ear infection caused by NTHI or from an infection by intranasal inoculation with NTHI were completely protected against nasopharyngeal colonization with a homologous strain and were found to be the best positive controls in protection studies. Systemic immunization of chinchillas with inactivated whole-cell preparations significantly protected animals not only against homologous NTHI colonization but also partially against heterologous NTHI infection. In all protected animals, significant serum anti-P6 and anti-HMW antibody responses were observed. The outer membrane P6 and high-molecular-weight (HMW) proteins appear to be promising candidate vaccine antigens to prevent nasopharyngeal colonization and middle ear infection caused by NTHI.