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Infect Immun. 1999 April; 67(4): 1935–1942.
PMCID: PMC96549

Characterization of the Gene Encoding a 26-Kilodalton Protein (OMP26) from Nontypeable Haemophilus influenzae and Immune Responses to the Recombinant Protein

Editor: D. L. Burns

Abstract

A 26-kDa protein (OMP26) isolated and purified from nontypeable Haemophilus influenzae (NTHI) strain 289 has been shown to enhance clearance of infection following pulmonary challenge with NTHI in rats. DNA sequence analysis revealed that it was 99% identical to a gene encoding a cell envelope protein of the H. influenzae Rd strain (TIGR accession no. HI0916). The deduced amino acid sequence revealed a hydrophilic polypeptide rich in basic amino acids. Restriction fragment length polymorphism analysis suggested that the OMP26 gene was relatively conserved among isolates of NTHI. Analysis of the deduced amino acid sequence of the OMP26 gene from 20 different isolates showed that similarity with NTHI-289 ranged from 96.5% (1 isolate) to 99.5% (14 isolates). Two recombinant forms of OMP26, a full length 28-kDa protein (equivalent to preprotein) and a 26-kDa protein lacking a 23-amino-acid leader peptide (equivalent to processed protein), were assessed in immunization studies for the ability to induce an immune response that would be as effective as the native protein in enhancing the clearance of NTHI following pulmonary challenge in rats. Immunization with the recombinant protein that included the leader peptide was more effective in enhancing pulmonary clearance, and it induced a better cell-mediated response and higher titers of systemic and mucosal antibody. This study has characterized a 26-kDa protein from NTHI that shows significant potential as a vaccine candidate.

Nontypeable Haemophilus influenzae (NTHI) is recognized as a significant human pathogen causing mild to severe respiratory infections (24). At present, no vaccine is available for prevention of infection by this pathogen. Major outer membrane proteins (OMPs) from this group of bacteria have been examined for their potential as vaccine candidates which might provide protection against infections caused by this pathogen (5, 11, 20, 25). OMPs designated P2, P4, and P6 are among the most studied, and while some were found to elicit immune responses in animal models, not all are effective in protecting against infection with heterologous bacterial strains (5, 11, 18, 20). To date only P6 has demonstrated heterologous strain responses following mucosal immunization in a rat model of enhanced pulmonary clearance (20).

Previous investigations in this laboratory led to the isolation and characterization of a previously unidentified OMP of approximately 26 kDa (OMP26) from an NTHI biotype I strain, NTHI-289, which was found to significantly enhance bacterial clearance from the lung in an experimental animal model (19). Mucosal immunization with OMP26 antigen not only protected animals against challenge with both homologous and heterologous strains of NTHI but also resulted in significantly high levels of immunoglobulin A (IgA)-specific antibodies (19). These results identified the potential of this protein as a vaccine candidate against infections caused by NTHI. N-terminal amino acid sequence of OMP26 revealed homology to a cell envelope protein from H. influenzae Rd, identified as a Yersinia enterocolitica OmpH homologue (TIGR accession no. HI0916), and with homologies to proteins known variously as Skp, OmpH, and Hlp-1 in Pasteurella multocida, Yersinia pseudotuberculosis, Escherichia coli, and Salmonella enterica. This paper reports the cloning, nucleotide sequence, and expression of the recombinant OMP26 gene in E. coli. DNA sequences encoding OMP26 were cloned by PCR amplification using oligonucleotide primers designed from the equivalent gene of H. influenzae Rd. Recombinant forms of the OMP26 protein were isolated from E. coli and used to mucosally immunize rats. A recombinant OMP26 that included the 23-amino-acid leader sequence was a more effective antigen in enhancing pulmonary clearance of NTHI.

MATERIALS AND METHODS

Bacterial strains and plasmids.

NTHI-289 is a biotype I strain isolated from the sputum of a patient with chronic bronchitis and has been studied previously (19). An additional 20 isolates of NTHI (Table (Table1),1), collected both from healthy humans (commensal isolates) and from an incidence of NTHI infection as indicated in Table Table1,1, were examined for restriction fragment length polymorphism of OMP26 gene sequences and amplified for sequence analysis. Cells were grown on chocolate agar plates at 37°C in 5% CO2 or in brain heart infusion broth (Oxoid, Heidelberg, Victoria, Australia) supplemented with NAD and hemin.

TABLE 1
NTHI strains

E. coli XL-1 Blue (28) was used as the host strain for the recombinant plasmid DNA. Transformed E. coli was propagated in Luria-Bertani (LB) broth or on LB agar containing 50 μg of ampicillin per ml. Plasmids pQE-30 and pQE-31 were purchased from Qiagen GmbH, Hilden, Germany.

DNA preparation and PCR amplification.

Chromosomal DNA was prepared from the isolates as described by Barcak et al. (2). Plasmid DNA was prepared by the alkaline lysis method (28). DNA was digested with the endonucleases according to the conditions recommended by the manufacturer.

Sequence information for Haemophilus genes was obtained from the TIGR database. The N-terminal amino acid sequence determined for OMP26 was found to be identical to the N terminus of OmpH from H. influenzae Rd (HI0916). The oligonucleotides used for PCR amplification from NTHI-289 were 5′-GAAAAACATCGCAAAAGTAACC-3′ (5′ end) and 5′-GGAAGCTTGCATTATGAGAACC-3′ (3′ end). These primers were examined for primability and stability of match, and for amplification of the predicted PCR product in a PCR against the OMP gene from H. influenzae Rd sequence information, using the software Amplify 1 (9). The PCR mix consisted of 2 mM MgCl2, 20 mM TrisCl (pH 8.4), 25 mM KCl, 1 ng of NTHI-289 DNA, 5 pmol of each primer, 50 μM each deoxynucleoside triphosphate, and 1 U of Taq DNA polymerase (Qiagen) in a total of 25 μl. Conditions used for the amplification consisted of 1 cycle of 94°C for 3 min, 35 cycles of 94°C for 20 s, 55°C for 20 s, and 72°C for 1 min, and 1 cycle of 72°C for 5 min and 30°C for 5 min, using a Corbett FTS4000 thermal cycler (Corbett Research, Sydney, New South Wales, Australia).

For amplification of the OMP26 gene from chromosomal DNA of 20 NTHI isolates, the 5′-end primer was that described above or 5′-TGGGCCATTGGTATTCTC-3′. The 3′-end primer used was 5′-GGATTTTTGCATTATGAGAACC-3′ or 5′-TTTTTTCTCTTGTGCTTTTTCAG-3′.

Cloning of the OMP26 gene.

The PCR product was digested with HindIII and NspBII plus HindIII and ligated into SmaI/HindIII-digested pQE-30 and pQE-31, respectively. Ligated DNA was transformed into E. coli XL-1 Blue by the CaCl2 method (28).

Expression of the rOMP26 gene product.

E. coli XL-1 Blue/pP26/1 (full-length OMP26 gene) and XL-1 Blue/pP26/2 (OMP26 gene lacking its signal sequence) were grown at 37°C with vigorous shaking to an optical density at 600 nm of 0.7 to 0.9, and then isopropyl-β-d-thiogalactopyranoside (Promega, Madison, Wis.) was added to a final concentration of 2 mM. The bacteria were incubated for a further 4 h, harvested by centrifugation at 4,000 × g for 10 min, then resuspended in sonication buffer (50 mM sodium phosphate [pH 7.8], 300 mM NaCl), and subjected to brief sonication. The lysate was then centrifuged at 10,000 × g for 20 min. The soluble fraction was used for purification of recombinant OMP26 (rOMP26).

Purification of protein.

rOMP26 (from pP26/1) and rOMP26M (from pP26/2) were purified by using Ni-nitrilotriacetic acid (Qiagen) resin under conditions recommended by the manufacturer. Briefly, the protein extracts were mixed under native conditions with the resin for 60 min on ice. The resin was then loaded onto a column and washed with 80 ml of sonication buffer followed by 80 ml of wash buffer (50 mM sodium phosphate, 300 mM NaCl, 10% [wt/vol] glycerol [pH 6.0]). The histidine-tagged recombinant OMP26 proteins were eluted from the column with a 30-ml volume of wash buffer containing 250 mM imidazole.

SDS-PAGE and Western blotting.

Protein extracts were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12% (wt/vol) gels under reducing conditions, after which the proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, Mass.) (31). Nonspecific sites on the membrane were blocked with 5% (wt/vol) skim milk powder in Tris-buffered saline (TBS; 10 mM Tris HCl [pH 7.4], 50 mM NaCl) at 4°C for 16 h. Primary antibodies were diluted (1:100) in TBS containing 1% skim milk powder and 0.05% (vol/vol) Tween 20, and the membranes were incubated with the antibodies at room temperature for 2 h with gentle agitation. The membranes were then washed three times with TBS containing 0.1% skim milk powder, a 1:3,000 dilution of goat anti-rat horseradish peroxidase (HRP)-conjugated IgG (Nordic Immunological Laboratories, Tilberg, Netherlands) in TBS containing 1% skim milk powder was added, and the membranes were incubated for 1 h at room temperature with gentle agitation. The membranes were then washed three times in TBS containing 0.1% skim milk powder, and antibody binding was visualized by incubating the membranes in 0.05% diaminobenzamidine containing 0.01% (vol/vol) H2O2. The reaction was terminated by washing the membranes in water. Antiserum to native OMP26 from previous immunization studies (19) was used as a source of primary antibody. Serum from unimmunized rats was used as a control.

Determination of DNA sequence.

The double-stranded DNA from the recombinant plasmids was sequenced on an Applied Biosystems (Foster City, Calif.) model 373A version 1.2.1 system at the Biomolecular Resource Facility, John Curtin School of Medical Research, Australian National University, Canberra, Australia, using the primers listed above and primers specific for the pQE plasmid vectors (Qiagen) with Taq DyeDeoxy and Big Dye Terminator Cycle sequencing kits. Sequence analysis was with DNA Strider 1.3.

Primers used in the sequencing of the OMP26 gene of 20 NTHI strains were those described above for amplification. The PCR product was purified in a spin column (Qiagen). Cycle sequencing reaction mixtures contained 30 to 90 ng of template, 3.2 pmol of forward or reverse primer, and 12 μl of Big Dye Terminator Ready Reaction Mix (Applied Biosystems) in a 20-μl volume. Reaction conditions were 25 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min. Sequences were run at the Biomolecular Resource Facility.

Southern hybridization.

The OMP26 gene was amplified by PCR from p26/1 and labeled with digoxigenin-11-dUTP by random priming using a Dig High Prime kit (Boehringer Mannheim, Mannheim, Germany). Chromosomal DNA of NTHI strains was digested with MnlI, separated on a 1.5% (wt/vol) agarose gel, and transferred to a HybondN+ nylon membrane (Amersham Pharmacia Biotech, Uppsala, Sweden) under alkaline conditions according to the manufacturer’s protocol. Hybridization was under high-stringency conditions using the Boehringer Mannheim detection system and protocol.

Immunization and bacterial challenge.

Specific-pathogen-free DA male rats between 8 and 10 weeks of age were maintained under specific-pathogen-free conditions except for immunization, intratracheal boost, and the final live bacterial challenge. The procedure for immunization and bacterial challenge was performed as previously described (20). The proteins rOMP26 or rOMP26M were prepared by emulsification of 200 μg of protein per ml in a 1:1 ratio of incomplete Freund’s adjuvant (Sigma, St. Louis, Mo.) and phosphate-buffered saline (PBS), and approximately 10 μg of protein was administered to each animal by inoculation of intestinal Peyer’s patches (IPP). Nonimmune animals were a mix of sham-immunized (receiving the same treatment as the OMP26-immunized rats but immunized with incomplete Freund’s adjuvant and PBS only) and untreated animals. Rats received an intracheal boost on day 14 after IPP immunization. The animals were sedated with halothane, and 10 μg of OMP26 at a concentration of 200 μg per ml in PBS was introduced into the lungs via an intratracheal cannula and dispersed with two 5-ml volumes of air. The nonimmune group received 50 μl of PBS. Animals were challenged with live bacteria 21 days after the first immunization. Bacteria were prepared by overnight culture, washed, and resuspended in PBS. The concentration of inoculum was estimated by optical density at 405 nm and confirmed by counting CFU of the overnight plating of serial dilutions of the inoculum. The animals were sedated with halothane, and a bolus inoculum of 5 × 108 CFU of live NTHI-289 in 50 μl of PBS was introduced into the lungs via an intratracheal cannula and dispersed with two 5-ml volumes of air. Animals were killed by an intraperitoneal injection of pentobarbital sodium administered 4 h after lung inoculation. Blood was collected by heart puncture, and aliquots of serum were stored at −20°C for antibody analysis. Lungs were lavaged with five 2-ml volumes of PBS via the trachea, which had been exposed through an incision in the neck, and the pooled bronchoalveolar lavage fluid (BAL) was assessed for clearance by plating of serial dilutions of the washings for CFU determination. Lungs were removed following lavage and homogenized in 10 ml of PBS, and bacterial counts were determined.

Antigen-specific enzyme-linked immunosorbent assays (ELISAs).

Polysorb microtiter wells (Nunc, Roskilde, Denmark) were coated overnight at 4°C with 100 μl of coating buffer (15 mM Na2CO3, 35 mM NaHCO3 [pH 9.6]) containing either rOMP26 or rOMP26M at 1 μg/ml. The plates were washed five times in washing buffer (PBS containing 0.05% [vol/vol] Tween 20), and the wells were blocked with 100 μl of blocking buffer (5% [wt/vol] skim milk in PBS–0.05% [vol/vol] Tween 20) for 60 min at room temperature. Plates were washed five times, and serum or BAL samples were serially diluted in blocking buffer, added to the wells, and incubated at room temperature for 90 min. After removal of the samples by washing five times, 100 μl of HRP-conjugated anti-rat immunoglobulin diluted in blocking buffer was added to the wells and incubated at room temperature for 90 min. HRP-conjugated immunoglobulins used were goat anti-rat IgG (1/2,000) and IgA (1/1,000) (Fc specific; Nordic Immunological Laboratories). The plates were washed five times, and the wells were developed with 100 μl of the substrate tetramethylbenzidine (Fluka, Buchs, Switzerland) in phosphate citrate buffer (pH 5) containing 0.05% (vol/vol) H2O2. The reaction was stopped with 100 μl of 0.5 M H2SO4. Plates were read at 450 nm on a plate reader (Bio-Rad Laboratories, Hercules, Calif.). Plate background was determined by rows coated with coating buffer alone and treated the same as test wells, and between-plate variation was assessed by comparison of control samples repeated on each plate. Quantitation of anti-OMP26 IgG and IgA was achieved by inclusion of standards for rat IgG ranging from 31 to 500 ng/ml and rat IgA ranging from 7.8 to 125 ng/ml (Serotec, Oxford, England).

Antigen-specific lymphocyte assay.

The assay was performed as previously reported (18). Lymphocytes were obtained from the mesenteric lymph nodes (MLN) by passing tissue through a stainless steel sieve and washing in cold sterile buffer prepared with PBS containing calcium and magnesium supplemented with 5% (vol/vol) fetal calf serum (heat inactivated at 57°C for 30 min), penicillin (100 U/ml), streptomycin (100 μg/ml), and amphotericin B (Fungizone; 0.25 μg/ml). Viable cells were counted by trypan blue exclusion with a hemocytometer and resuspended in culture medium (Multicel RPMI 1640 [Cytosystem, Castle Hill, New South Wales, Australia] containing 0.01 M HEPES [pH 7.2], 5 × 10−5 M β-mercaptoethanol, 2 mM l-glutamine (ICN, Costa Mesa, Calif.), 5% fetal calf serum, and penicillin-streptomycin-amphotericin B [as described above]) to obtain a final concentration of 106 cells per ml. The antigen (rOMP26 or rOMP26M) was suspended in culture medium and sterile filtered to give final assay concentrations of 0.1, 1.0, and 10 μg per ml. The cell suspension and antigen were added in triplicate to flat-bottomed multiwell microculture plates (Nunc) to give a final volume of 0.2 ml/well. Lymphocyte proliferation was estimated by [3H]thymidine (Amersham) incorporation for the last 8 h of a 4-day culture. Results were calculated by subtraction of background from the mean of triplicate wells then calculation of the mean ± standard error of the entire treatment group.

Statistical analysis.

The bacterial clearance and antibody data were assessed for statistical significance by an independent t test (Macintosh Systat), and the lymphocyte proliferation data were assessed by a factorial analysis of variance (Macintosh Systat).

Nucleotide sequence accession number.

The nucleotide sequence of the NTHI-289 OMP26 gene has been assigned GenBank accession no. AF109085.

RESULTS

Strategy for cloning the gene encoding OMP26.

The N terminus of OMP26 mature protein, isolated and purified from an outer membrane extraction of NTHI-289, was previously found to be identical to that of a cell envelope protein (OmpH homologue) on the H. influenzae Rd gene database (HI0916). Oligonucleotide primers designed from 5′- and 3′-end regions of this gene were used to amplify a DNA segment from NTHI-289 genomic DNA by PCR. A HindIII restriction site was engineered into the 3′-end PCR primer to facilitate directional cloning into plasmid pQE-30. The PCR amplification generated a product of approximately 618 bp, which was in agreement with that estimated from the sequence of the gene sequence information. Following HindIII digestion, the PCR product was ligated into the SmaI/HindIII-digested pQE-30 to give pP26/1. This construct contained the full length of the OMP26 protein, including a 23-amino-acid leader peptide. Another construct, excluding the leader peptide, was generated by digesting the PCR amplification product with NspBII (5′-C[A/C]GC[G/T]G-3′), which has a site at the signal peptide cleavage position (as determined from the H. influenzae Rd sequence; data not shown), followed by HindIII digestion. The DNA insert, 552 bp in size, was ligated into the SmaI/HindIII-digested plasmid pQE-31 to give pP26/2.

Presence of the OMP26 gene in NTHI strains.

To determine if the OMP26 gene was present in different isolates of NTHI, hybridization analysis using a labeled probe generated from the recombinant plasmid pP26/1 was carried out. Genomic DNA, prepared from a collection of isolates representing clinical cases and healthy humans (commensal isolates), was digested with the restriction enzyme MnlI (5′-CCTCN7-3′), which has two sites (at positions 278 and 515 bp from the initiation codon) in both the NTHI-289 OMP26 gene and the H. influenzae Rd ompH gene. Genomic DNA of all strains hybridized with the probe, the majority giving a pattern identical to that of NTHI-289 (data not shown), indicating that similar OMP26 genes existed in all these strains.

Sequence analysis.

The nucleotide sequence of the NTHI-289 OMP26 gene (accession no. AF109085) was 99% identical to the H. influenzae Rd ompH gene (Fig. (Fig.1)1) and 66% identical to the P. multocida skp gene (data not shown) (6). The deduced amino acid sequence was 98 and 56% identical for H. influenzae Rd (Fig. (Fig.1)1) and P. multocida, respectively. Figure Figure22 shows amino acid sequence alignments of OMP26 from NTHI-289 with those of 20 other NTHI isolates (accession no. AF109086 to AF109105) and that of H. influenzae Rd. Homology to the NTHI-289 sequence ranged from 96.5% (UC9) to 99.5% (14 isolates). The amino acid sequence from the UC10 isolate had the closest homology (99.5%) to the H. influenzae Rd sequence. There were 10 different amino acid sequences among the NTHI isolates. There are 11 variant sites in the mature protein, 7 of which have substitutions occurring in more than one sequence. Only 4 of 11 substitutions; K→R at position 26, I→V at position 71, R→K at position 157, and V→I at position 170, are conservative. Six of seven nonconservative substitutions involve changes in charge, for example, E→Q at position 102. One isolate (UC18) had an insertion segment of four amino acids (12 bases) near the C terminus between positions 192 and 193.

FIG. 1
Amino acid translation (deduced from DNA sequence of NTHI-289) and nucleotide sequence of OMP26 from NTHI-289 and alignment with gene sequence (HI0916) from the H. influenzae Rd genome. Base variations from the NTHI-289 sequence in the H. influenzae Rd ...
FIG. 2
Alignment of amino acid sequences of NTHI-289 and 19 NTHI strains, a capsule-deficient type b strain (HI-CD), and the equivalent H. influenzae Rd sequence. Amino acid variations only are shown; dashes represent homology with the NTHI-289 sequence.

Analysis of the deduced amino acid sequence of OMP26.

The mature OMP26 protein contains 174 amino acids and has a calculated molecular mass of 19.5 kDa; its precursor has a 23-amino-acid signal sequence and a calculated molecular mass of 21.73 kDa. The deduced amino acid sequence predicts a pI of 8.0, indicating that OMP26 is rich in basic amino acids.

The hydrophobicity profile and suggested secondary structure generated for OMP26 are shown in Fig. Fig.3.3. The polypeptide appears to be divided into three domains; the N-terminal and C-terminal regions show a hydrophobic character, whereas the middle part shows a pronounced hydrophilicity. The first hydrophobicity peak coincides with the signal peptide region.

FIG. 3
Structure predictions for OMP26, using MacVector 3.5 software. (A) Hydrophobicity plot; (B) secondary structure predictions for helix (Hlx), sheet regions (Sht), and turns in the sequence (Trn).

Expression of the recombinant proteins in E. coli.

The two forms of recombinant protein, preprotein (rOMP26) and mature protein (rOMP26M), were expressed from plasmids pP26/1 and pP26/2, respectively, as fusions with a six-histidine tag at the N terminus of each polypeptide (Fig. (Fig.4).4). Both proteins were most abundant in the soluble fraction, from which they were then purified. Neither protein was present in the host strain transformed with pQE-30 (data not shown). This purification step resulted in OMP26 preparations which appeared to be about 80% pure (data not shown).

FIG. 4
Western blot of rOMP26 and rOMP26M used in immunizations. rOMP26 and rOMP26M were expressed in E. coli XL-1 Blue/pP26/1 and XL-1 Blue/pP26/2, respectively, and probed with polyclonal anti-OMP26 (I) or nonimmune (NI) serum as primary antibody. Molecular ...

Bacterial clearance and immune responses in rats.

The immunogenicity of the recombinant OMP26 protein was tested in a rat model, and the effectiveness of the immune response was assessed by pulmonary challenge. The two recombinant proteins expressed in E. coli were purified. The protein rOMP26M, lacking the 23-amino-acid leader sequence, was purified from XL-1 Blue induced to express plasmid pP26/2. The protein rOMP26, including the leader peptide sequence, was obtained from XL-1 Blue induced to express plasmid pP26/1. As shown in Fig. Fig.5,5, mucosal immunization with the two proteins resulted in different clearances of the bacteria from the rat lungs. rOMP26 more effectively enhanced clearance than did rOMP26M, and the rate of clearance for rOMP26 was similar to the response reported for the native protein (19). Animals were also immunized with E. coli to determine if the response could be due to traces of E. coli proteins with cross-reactive specificity. There was no difference between the E. coli-immunized group and the nonimmune animals in bacterial clearance of NTHI-289 from the lungs (data not shown).

FIG. 5
Bacterial recovery at 4 h after pulmonary challenge with NTHI-289 following immunization with rOMP26 and rOMP26M (A) or native OMP26 (previously published data [19]) (B). The y axis indicates the logarithm of CFU from the lung and BAL. ...

Immunization with both rOMP26 and rOMP26M induced significant titers of both IgG and IgA in the serum and BAL; however, the level of antibodies from animals immunized with rOMP26M was significantly lower than that following immunization with rOMP26. The antibodies recognized both recombinant proteins with similar specificities (Table (Table2).2). Thus, the higher concentration of antibody following rOMP26 immunization was measurable against both rOMP26 and rOMP26M. Antigen-specific lymphocyte responses (Fig. (Fig.6)6) also showed significant differences for the two recombinant antigens. MLN lymphocytes from both rOMP26- and rOMP26M-immunized rats were set up in culture against both antigens. rOMP26-primed cells responded significantly to rOMP26 (Fig. (Fig.6A)6A) but showed a poorer response to rOMP26M (Fig. (Fig.6B).6B). However, rOMP26M-primed cells showed a similar poor response when cultured with either antigen (Fig. (Fig.6).6). This result suggests induction of a poorer T-cell response in the lymphocytes from animals immunized with rOMP26M.

TABLE 2
Antibody responses in serum and BAL from rats mucosally immunized with rOMP26
FIG. 6
Lymphocyte proliferation of cells isolated from the MLN of nonimmune, rOMP26-immunized, and rOMP26M-immunized rats. Cells were cultured for 4 days with either rOMP26 (A) or rOMP26M (B) and pulsed with [3H]thymidine for 8 h on the last ...

DISCUSSION

NTHI is an opportunistic pathogen capable of causing respiratory infections, yet it is commonly found in a harmless association with humans. Research efforts have focused on examining the antigenicity and immunogenicity of surface antigens that might offer potential as components for an effective vaccine against infections caused by NTHI.

In this study, we have reported the cloning, sequencing, and expression of a protein which has been called OMP26. Sequence analysis has revealed that the gene for this protein is nearly identical to a gene identified in the H. influenzae Rd genome that may be related to the skp/ompH gene family (1, 6, 7, 1217, 21). The fact that a cleavable signal peptide sequence exists in the preprotein suggests that the mature protein is destined for export across the cytoplasmic membrane via the general secretory pathway (27). Previous experiments involving OMP extraction and immunolabeling of NTHI with antisera to OMP26 showed surface binding of gold-conjugated particles under electron microscopy (19), suggesting that it is a component of the outer membrane of NTHI. However, there is good evidence that in E. coli Skp/OmpH is a soluble periplasmic protein (30). Clearly, further studies are needed to determine if Skp/OmpH has undergone adaptive changes in H. influenzae so as to locate to the outer membrane or is to some extent peripherally associated with the outer membrane in all species. Skp is highly basic and in E. coli binds strongly to lipopolysaccharide (LPS) when released artificially (for example, by lysis) from the periplasm (30). Perhaps it also binds to LPS in vivo during the export of LPS and/or porin proteins (23, 30).

The role of Skp proteins and their homologues in bacterial cellular functions remains speculative. Thome and colleagues (29) presented evidence supporting a role for such proteins in the export of outer membrane proteins and/or other macromolecules, but the exact nature of the mechanism(s) involved remains to be elucidated. It has been postulated that they might behave like the chaperonins, since they appear to be active on either side of the plasma membrane in E. coli (29, 30). A recent study demonstrated that the Skp protein from E. coli appears to act as a periplasmic molecular chaperone (3) which may assist OMP transport from the cytoplasmic membrane to the outer membrane. The fact that the skp gene is located in a complex operon governing the early steps of LPS synthesis suggests a role for this family of proteins in the synthesis of LPS (7, 15, 17). Indeed, the gene organization of the skp gene and homologous sequences appears to be highly conserved in gram-negative bacteria examined to date, which suggests an important function (1, 6, 10, 12). The role of OMP26 in NTHI strains has yet to be determined.

The significance of the consistency of the relatively defined amino acid changes observed in the various strains has yet to be determined. The change to Asn and Lys from Asp and Arg at positions 146 and 157, respectively, probably does not affect the cross-protective immune response. Immunization with the native OMP26 from NTHI-289 cleared the NTHI-II strain following pulmonary challenge in a previous study (19). The significance of some other amino acid differences in under investigation. In Fig. Fig.2,2, only two of the sequences were derived from strains originating outside of Australia (however, it should be noted that the Australian strains were selected from a collection dating over a 10-year period and from two geographical locations). Two other sequences of the same regions of the Haemophilus genome appear in the GenBank database. A 129-amino-acid segment from H. influenzae Eagan (accession no. U60832) and H. influenzae isolate 33 (accession no. U60831) show 100 and 96% homologies, respectively. Interestingly, three of the substitutions in isolate 33 were shared with isolates in this study.

Immunization studies with the native OMP26 protein showed significant potential for this protein antigen as a vaccine candidate against pulmonary infections by NTHI in animal studies (19). The availability of the recombinant protein, precursor and mature, facilitates a greater understanding of the antigenicity and immunogenicity of OMP26 and provides a basis for the development of the potential of OMP26 as a candidate for a vaccine against NTHI infections. Bacterial clearance from the lungs in rats following mucosal immunization with either killed bacteria or protein has been used to identify and investigate potential vaccine antigens (4, 5, 1820, 32). Antibody responses to antigens that arise as a result of either infection or immunization play a role in facilitating bacterial clearance. This is achieved by a number of mechanisms including bactericidal activity and opsonophagocytosis by polymorphonuclear leukocytes (26); however, in mucosal defenses the role of IgA in inhibiting microbial adherence is a significant first line defense against infection (22). With respect to antibody titer and specificity, immunization with the recombinant OMP26 that included the leader sequence induced higher titers of both IgG and IgA that recognized both recombinant proteins equivalently. Immunization with the equivalent of the processed OMP26 (no leader peptide) still resulted in a significant antibody response; however, the titers were significantly lower, although the specificity for both forms of the recombinant were similar for IgG but not IgA. This finding suggests that the influence of antibody on the differences seen in the rate of the bacterial clearance may not be a result of differences in specificity but could have been associated with concentration of antibody to OMP26.

The significance of cell-mediated immunity in mucosal defenses is now well recognized (8). Enhanced pulmonary clearance of NTHI (33) and Pseudomonas aeruginosa (8) has been found in rat models following transfer of T cells from immunized rats to naive rats. A study of T- and B-cell responses following immunization with the OMP designated P2 from NTHI found that enhancement of the T-cell response in the presence of suppressed B-cell responses was capable of increasing bacterial clearance (18). The results presented in the present study also suggest that a better T-cell response following immunization, as observed with rOMP26, may have contributed to the enhanced clearance of NTHI.

In this study, we have sequenced and cloned a 26-kDa protein from NTHI, determined that the gene is present in all isolates assessed to date, and shown through sequence analysis that the protein is highly conserved, with variation occurring as specific amino acid substitutions. Immunization with rOMP26, which included the leader peptide, effectively enhanced bacterial clearance and induced a significant antibody response and a better T-cell response. Future studies will determine if immunization with this protein will clear other respiratory infections caused by NTHI, such as otitis media, and further investigate and develop the potential of OMP26 as a vaccine candidate.

ACKNOWLEDGMENTS

This work was supported by research grants from the National Health and Medical Research Council (grant 951089) and the Australian Research Council (small grant).

We thank Mark Bradley, Sandra Beaton, and José ten Have, CSIRO Division of Wildlife and Ecology, for advice and for making their facilities available. We thank Heather Domaschenz for technical assistance with the animal experiments, Jamie Baker for assisting with the ELISAs, and Pat Moor and Dianne Webb for acquiring and typing some of the H. influenzae isolates.

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