Patent Grant
8501197
This invention relates to field of Moraxella catarrhalis infections and more particularly to an immunogenic composition against Moraxella catarrhalis.
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. Moraxella catarrhalis is a Gram-negative diplococcus frequently found as a commensal of the upper respiratory tract. However, over the past 2 to 3 decades this bacterium has emerged from being considered as a harmless commensal to being recognized as a genuine respiratory tract pathogen of serious public health concerns.
Acute otitis media is the most common bacterial infection in children with 70% experiencing at least one episode by age 3. M. catarrhalis is the third leading cause of otitis media after Streptococcus pneumoniae and nontypeable Haemophilus influenzae. M. catarrhalis is associated with 25% of acute otitis media by culture and 46.4% of chronic middle ear effusion by PCR. In addition, the nasopharyngeal carriage rate of M. catarrhalis in children is high (up to 75%) and the frequency of colonization is positively related to the development of otitis media.
In adults, M. catarrhalis is the second most common bacterial cause of exacerbations of chronic obstructive pulmonary disease (COPD) after non-typeable H. influenzae. COPD is the fourth leading cause of death in the United States, affecting 24 million Americans. M. catarrhalis causes approximately 10% of exacerbations of COPD, accounting for 2 to 4 million episodes annually. Furthermore, M. catarrhalis also colonizes the lower respiratory tract in up to 2.5 to 10% of adults with COPD at their stable states. Lower airway colonization contributes to airway inflammation in COPD as a result of sloughing of highly inflammatory bacterial cell wall antigens into the airway.
M. catarrhalis is a nonencapsulated bacterium and does not secrete exotoxin. The current vaccine studies have mostly focused on various outer membrane proteins (OMPs) as vaccine candidates. To date, a limited number of OMPs have been examined and are currently under different stages of evaluation as part of an effort to develop a multicomponent vaccine against M. catarrhalis.
The present invention provides a novel protein, Oligopeptide permease protein A (OppAs), as an antigen for use in a vaccine against M. catarrhalis. The protein is characterized in terms of sequence conservation and immunogenicity. This protein is predicted to have a periplasmic location and therefore its ability to induce an immune response and/or protection against Moraxella catarrhalis infection was surprising. However, three independent sets of experiments described below demonstrated that OppA also expresses epitopes on the bacterial surface, a novel and surprising observation. It is believed that induction of potentially protective immune responses is due to immune responses to OppAs epitopes on the bacterial surface.
In one aspect, the present invention provides a method for stimulating in an individual an immune response against M. catarrhalis. The method comprises administering to an individual a composition comprising M. catarrhalis OppA protein. The immune response stimulated in the individual may be a prophylactic or a therapeutic immune response. The stimulated immune response may comprise stimulation of an M. catarrhalis specific humoral immune response, such as by generation of antibodies in the individual against M. catarrhalis and/or M. catarrhalis specific cell-mediated immune response. In one embodiment of the invention, performing the method of the invention results in an enhanced rate of M. catarrhalis bacterial clearance from the lungs of an individual to whom the composition is administered.
In one aspect, the present invention provides immunogenic compositions which will elicit an immune response against M. catarrhalis. The compositions comprise M. catarrhalis OppA protein and optionally, an adjuvant suitable for the route of administration. For example, if the immunogenic composition is to be administered intranasally, then an adjuvant suitable for mucosal administration can be used.
The present invention provides a method for stimulating in an individual an immune response against M. catarrhalis. The method comprises administering to the individual a composition comprising M. catarrhalis OppA protein. The OppA protein is provided in an amount effective to stimulate an immune response against M. catarrhalis in the individual.
The present invention also provides immunogenic compositions comprising M. catarrhalis OppA protein, which when administered to an individual provides immunity against M. catarrhalis infection. Thus, the OppA protein in the composition comprises one or more immunogenic antigens that can stimulate an M. catarrhalis specific humoral and/or a cell mediated immune response in the individual. The amino acid sequence of OppaA protein is provided in SEQ ID NO:1. A representative cDNA sequence encoding this OppA protein is provided in SEQ ID NO:2. The immunogenic compositions may comprise one or more adjuvants or other pharmaceutical carriers.
We discovered the oppA gene in our search for surface proteins of M. catarrhalis. Homology analysis suggested that oppA encodes a soluble periplasmic protein of the oligopeptide transport system. Given its periplasmic localization, OppA did not stand out as a candidate for a potential vaccine and therefore, the observation that OppA of M. catarrhalis expresses epitopes on the bacterial surface was surprising. While not intending to be bound by any particular theory, one possibility is that OppA epitopes are intermittently exposed when they bind oligopeptides from the environment for transport across the periplasm. Alternatively, the protein may be intermittently exposed during bacterial cell wall turnover and cell division, or the protein has more than one location in the bacterial cell wall.
We found that OppA is highly conserved among strains of M. catarrhalis. Sequence conservation can be a result of selection pressure to preserve the essential physiological function of the protein. Sequence conservation of oppA gene would also be consistent with its presumed periplasmic localization. Genes that encode surface exposed proteins in H. influenzae undergo point mutations driven by immune selective pressure during colonization; these changes allow the bacterium to evade host immune responses. Therefore, again, it was surprising that OppA, which is highly conserved and predominantly present in the periplamic region, could impart immune protection to an individual against the bacterium. While not intending to be bound by any particular theory, we believe OppA is presumably not continuously displayed to the host immune system.
In this work, we evaluated the potential of M. catarrhalis OppA protein as a vaccine antigen. We showed that OppA 1) is highly conserved among strains of M. catarrhalis that cause otitis media and exacerbations of COPD; 2) is an efficient immunogen, 3) expresses epitopes that are exposed on the bacterial surface; and 4) induces potentially protective immune responses following mucosal immunization in the mouse pulmonary clearance model.
Currently the mouse pulmonary clearance model is the most widely used model to assess potential vaccine antigens of M. catarrhalis. The model is simple, reproducible, is performed in multiple centers and measures a functional response. The observation that intranasal immunization of OppA induced enhanced pulmonary clearance provides support for a mucosal route of immunization for M. catarrhalis infections which are predominantly mucosal infections.
Isolated protein used in the method of the invention may be obtained by methods known to those skilled in the art, such as by isolation of the proteins from M. catarrhalis cultures, or by producing the proteins recombinantly from expression vectors inserted into cells using conventional techniques, culturing the cells under conditions whereby the proteins are synthesized by the cells, and isolating the proteins from the cells according to established procedures.
The isolated M. catarrhalis OppA may be purified to any desired degree of purification. Methods for protein purification are well known in the art and are applicable to preparing purified M. catarrhalis OppA protein for use in the present invention. In various embodiments, the M. catarrhalis OppA protein used in the invention may be partially purified, substantially purified, or fully purified. In various embodiments, the compositions of the present invention may comprise M. catarrhalis OppA protein, may consist essentially of M. catarrhalis OppA protein, or may consist of M. catarrhalis OppA protein. In some embodiments, the OppA protein may be used with one or more of additional M. catarrhalis immunogenic proteins (such as outer membrane proteins) for an antigenic formulation. In additional embodiments, the invention provides fragments of the OppA protein for use in stimulating a specific immune response against M. catarrhalis in an individual. Given the benefit of the present disclosure, those skilled in the art will be able to recognize suitable fragments of the OppA protein so that peptides for eliciting a specific immune response against M. catarrhalis in an individual can be produced. In general, suitable peptides comprise or consist of at least 9 contiguous amino acids of SEQ ID NO:1Thus, the invention includes all peptides that comprise or consist of each 9 amino acid segment of SEQ ID NO:X across its entire length, and which can vary in length from 9 amino acids, up to one amino acid less than the full length of SEQ ID NO:X. In one embodiment, the length is from 9 to 20 amino acids, inclusive.
Homology Modeling of M. catarrhalis OppA.
Peptides of the invention can be designed based on protein homology modeling. A model of M. catarrhalis OppA was generated using the crystal structure of S. clavuligerus OppA2 (an arginine peptide binding molecule as is M. catarrhalis OppA) bound with arginine (Mackenzie A K, Valegard K, Iqbal A, Caines M E, Kershaw N J, Jensen S E, Schofield C J, Andersson I. Crystal structures of an oligopeptide-binding protein from the biosynthetic pathway of the beta-lactamase inhibitor clavulanic acid. J Mol. Biol. 2010; 396(2):332-44) (PDB ID 2WOP) as the template. Initially, the Fold and Function Assignment web server (Jaroszewski L, Rychlewski L, Li Z, Li W, Godzik A. FFAS03: a server for profile-profile sequence alignments. Nucleic Acids Res. 2005; 33 (Web Server issue):W284-8. PMCID: 1160179) was utilized to identify structural homologs based on sequence alignment. We chose S. clavuligerus OppA2 as a template given that a high-resolution crystal structure had been determined with arginine bound in the active site cleft. ProtMod, part of the FFAS server, was then used to build the all-atom homology model. The model has ˜80% coverage (538 of 679 residues) of the M. catarrhalis OppA sequence. Of the 141 residues unable to be modeled, 58 are in C-terminus of the protein, which is substantially longer than other members of the OppA family. The overall secondary structure predicted for the M. catarrhalis OppA is highly conserved and consistent with that observed for other OppA proteins whose crystal structures have been determined. Structural analyses confirmed that three domains form two lobes, with the peptide-binding site located in the cleft between the two lobes (Quiocho F A, Ledvina P S. Atomic structure and specificity of bacterial periplasmic receptors for active transport and chemotaxis: variation of common themes. Mol. Microbiol. 1996; 20(1):17-25) Our homology model is consistent with these structures. This homology model can be to design peptides for use in the invention, and in particular embodiment, the peptides are fragments of disordered regions that contain M. catarrhalis-specific sequences, which are predicted to be surface epitopes and thus are expected to contain peptide sequences that are suitable for use in making and using peptides according to the invention.
Compositions comprising the proteins, such as pharmaceutical compositions for administration to individuals, may be prepared by mixing the proteins with any suitable pharmaceutically acceptable carriers, excipients and/or stabilizers to obtain pharmaceutical compositions. Some examples of acceptable carriers, excipients and stabilizers suitable for combining with the proteins can be found in: Remington: The Science and Practice of Pharmacy (2005) 21st Edition, Philadelphia, Pa. Lippincott Williams & Wilkins.
The compositions of the invention may be administered in combination with any suitable adjuvant or adjuvant combination. In one embodiment, the mucosal adjuvant may be cholera toxin. Examples of other mucosal adjuvants include ISCOMs (immune stimulating complexes), CpG oligonucleotides, E. coli labile toxins, interleukins (including IL1α, IL5, IL6, IL12, IL15 and 18) and chemokines such as MCP1. In another embodiment, the adjuvant may be Freund's incomplete adjuvant. Examples of other adjuvants for systemic immunization include alum (aluminum salts), CpG oligonucleotides, organic compounds such as squalene, QS21, monophosphorylated lipid A and liposomes.
It is expected that the compositions used in the method of the invention may be administered to any mammal to stimulate an immune response against M. catarrhalis bacteria. In one embodiment, the mammal is a human.
The method can be performed by administering the composition to the individual via any acceptable method of delivery which enables the composition to stimulate an immune response to M. catarrhalis bacteria in the individual. Examples of acceptable administration routes include but are not limited to subcutaneous, intramuscular, intravenous, intradermal, intranasal, oral and inhalation administrations. In one embodiment, the compositions are administered subcutaneously. In another embodiment, the compositions are administered intransally.
It will be recognized by those of skill in the art that the form and character of the particular dosing regimen employed in the method of the invention will be dictated by the route of administration and other well-known variables, such as the size of the individual and the stage of the infection. It is generally considered that the amount of protein administered will range from approximately 10 micrograms to milligrams. Accordingly, in view of the present invention, one skilled in the art can determine an amount of isolated M. catarrhalis protein that is effective to stimulate an immune response against M. catarrhalis for any particular individual in need of such a stimulated immune response.
The immune response stimulated in the individual by the method of the invention may be a prophylactic or a therapeutic immune response. The stimulated immune response may comprise an M. catarrhalis specific humoral immune response, an M. catarrhalis specific cell-mediated response, or both. The humoral response may be a systemic response, a mucosal response, or both.
In one embodiment, the stimulated immune response comprises generation of antibodies in the individual that are specific to M. catarrhalis bacteria via specific binding of the antibodies to the protein to the bacteria. In one embodiment, the stimulated immune response comprises generation of antibodies that are specific to more than one strain of M. catarrhalis bacteria. The antibodies generated via stimulation of an immune response in the individual may by present in a variety of bodily fluids/tissues. Non-limiting examples of fluids in which the stimulated antibodies may be present include serum, sputum, saliva, nasopharyngeal secretions and middle ear fluid.
Stimulation of an immune response in an individual can be determined according to well known techniques. In one embodiment, stimulation of an immune response can be determined by detecting an increase in antibodies that recognize M. catarrhalis bacteria in an individual to whom a composition of the invention has been administered. An increase in antibodies in the individual that recognize M. catarrhalis bacteria can be detected using a variety of well known techniques, non-limiting examples of which include ELISAs and bactericidal assays. In one embodiment, an increase in antibodies to M. catarrhalis bacteria can be measured by determining an increase in antibodies to the isolated M. catarrhalis protein used in the composition administered to the individual.
The antibodies generated in the individual may comprise any antibody isotype. In various embodiments, the antibodies may be IgG antibodies, IgA antibodies, or combinations thereof.
In another embodiment, stimulation of an immune response to M. catarrhalis bacteria in an individual can be determined by detecting an enhanced rate of M. catarrhalis bacterial clearance from the lungs of an individual to whom the composition is administered, relative to the rate of M. catarrhalis bacterial clearance from the lungs of an individual to whom the composition has not been administered. In one embodiment, the immune response eradicates nasopharyngeal and/or or upper airway colonization by M. catarrhalis
The compositions of the invention may be administered in a single dose or in more than one dose. For example, in one embodiment, the composition may be administered as several doses over a period of time, such as by providing an initial administration, and subsequent administrations intended to boost the stimulation in immune response. The compositions of the invention may also be administered prior to, concurrently, or subsequent to conventional anti-bacterial treatments, such as antibiotic treatments.
The following Examples are meant to illustrate, but not limit the invention
Bacterial Strains and Growth.
Moraxella catarrhalis strain ATCC 43617 was obtained from the American Type Culture Collection (Manassas, Va.). Isolate 035E was provided by Eric Hansen. Strains 5P34B1, 10P58B2, 12P15B1, 14P25B1, 29P24B1, 33P25B2, 39P29B2, 55P18B3, 63P62B1, and 96P9B1 were sputum isolates obtained from adults with COPD. Strains 135, 238, 555, 2901, 3614, 3584, 5488, 8184, 9483, 6952 were middle ear fluid isolates obtained via tympanocentesis. Pulsed field gel electrophoresis of genomic DNA cut with SmaI showed that the strains are genetically diverse. M. catarrhalis strains were grown on brain heart infusion (BHI) plates at 37° C. with 5% CO2 or in BHI broth with shaking at 37° C. Chemically competent E. coli strains Top10 and BL21(DE3) were obtained from Invitrogen (Carlsbad, Calif.) and were grown at 37° C. on Luria Bertani (LB) plates or in LB broth.
Nucleotide Sequence Analysis.
M. catarrhalis genes encoding putative surface proteins including lipoproteins and outer membrane proteins with characteristic signal sequences were identified based on the genome sequence of strain ATCC 43617 using a genome mining approach. oppA gene was defined by homolog analysis using a BLAST search at the NCBI website (://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequence of oppA genes amplified from 10 clinical isolates of M. catarrhalis and the ATCC 43617 strain were determined at the Roswell Park Cancer Institute DNA sequencing facility with sequencing primers P1, P5, and P6 listed in Table 1. These sequences were assembled with the Sequencher program and aligned with the ClustalW alignment function of the MacVector program.
Genomic DNA and RNA Purification.
Genomic DNA of M. catarrhalis strains was purified with the Wizard genomic DNA purification kit (Promega, Madison, Wis.) following the manufacturer's instructions. The total RNA of M. catarrhalis 035E was isolated with the Qiagen RNeasy mini kit (Qiagen, Valencia, Calif.) followed with DNase I treatment by using the RQ1 RNase-free DNase I from Promega and finally cleaned up with the Qiagen RNeasy mini kit following the protocols provided with the kits.
Reverse Transcriptase PCR(RT-PCR).
RT-PCR was performed using the Superscript II reverse transcriptase from Invitrogen (Carlsbad, Calif.). The first-strand cDNA was synthesized with 2 pmole of the reverse primer P3 (Table 1) and 1.5 μg of total RNA extracted from M. catarrhalis 035E strain. 10% cDNA reaction was then used in the second step PCR with both forward (P2) and reverse primers (P3) (Table 1) and Hotmaster mix (5 Prime Inc, Gaithersburg, Md.) to amplify a 223 bp fragment of the oppA gene. Parallel reaction without reverse transcriptase was performed as negative control to exclude amplification from contaminating DNA. 100 ng of genomic DNA extracted from M. catarrhalis 035E strain was used in PCR as positive control.
Overlap Extension PCR.
Overlap extension PCR is a PCR based strategy to create chimeric genes by fusion of independent fragments in the absence of restriction sites. We utilized overlap extension PCR to generate the linear double stranded DNA for oppA mutant construction via homologous recombination. The transforming DNA for an oppA mutant was composed of three fragments: F1/oppAUSG (oppA upstream gene sequence), F2/aphA-3 gene (kanamycin resistance gene), and F3/oppADSG (oppA downstream gene sequence). The location of these three fragments in relation to the oppA gene is depicted in
oppA
oppA
oppA
oppA
oppA
oppA
oppA
oppA
oppA
oppA
oppAUSG/F1
aphA-3/F2
oppADSG/F3
1Restriction enzyme sites are underlined. Overlapping regions are shown in bold. Extended sequences complementary to the end of the adjacent gene are shown in italics.
2USG: upstream gene
3DSG: downstream gene.
Mutant Construction.
The isogenic oppA mutant strain was constructed by transformation of M. catarrhalis strain 035E with the overlap extension PCR product. Briefly, strain 035E was grown to OD600 nm at 0.2. A volume of 100 μl of the bacterial culture was then spread on BHI agar plate and air dried. Two circles (2 cm diameter) were marked on the plate. Either 30 μl distilled water or 30 μl of water containing 100 ng of DNA was dropped onto these circles, followed by incubation at 37° C. with 5% CO2 for 5 h. Bacteria within each circle were harvested with a cotton swab and spread on BHI plates containing 50 μg/ml kanamycin. After 24 h incubation at 37° C. with 5% CO2, numerous colonies were present on the selection plate following DNA transformation but no visible colony was present on the water control plate. The mutant colonies were examined by PCR and sequencing as described in Results.
Cloning of the oppA Gene.
The pCATCH plasmid is an expression vector engineered to express recombinant lipoprotein and has been used previously to express M. catarrhalis lipoproteins. The 1967 bp oppA gene encoding the mature OppA protein was amplified from M. catarrhalis strain ATCC 43617 with primers P7 and P8 (Table 1) and ligated into pCATCH between restriction sites NcoI and BamHI. The ligation mixture was transformed into chemically competent E. coli Top10 strain and grown on kanamycin (50 μg/ml) selection plate. The OppA expression plasmid was named pCATCH/OppA.
Southern Blot Assay.
Southern blot assays were performed with genomic DNA that was restricted with PvuII and NdeI using a Hoefer TransVac vacuum blotting unit following the manufacturer's instructions (Hoefer, San Francisco, Calif.). Probes were biotinylated with an NEBlot Phototope-Star detection kit (New England Biolabs) according to the manufacturer's instructions.
Expression and Purification of His-OppA Protein.
pCATCH/OppA was transformed into E. coli BL21(DE3) strain to express OppA as a lipoprotein with a thrombin cleavable C-terminal hexahistidine tag. A volume of 500 ml LB broth containing 50 μg/ml kanamycin was inoculated with 20 ml overnight culture of bacteria harboring the expression vector. Following growth to OD600 nm of 0.6, OppA expression was induced with 3 mM IPTG for 4 hours at 37° C. The bacteria were then harvested by centrifugation at 4,000 g for 15 min at 4° C. The pellet was suspended in 10 ml of lysis buffer (20 mM sodium phosphate, 500 mM NaCl, 1 mg/ml lysozyme, 100 μg/ml PEFABLOC (Roche, Indianapolis, Ind.), pH 7.4) and mixed with a nutator for 30 min at 4° C. The suspension was then sonicated with a Branson Sonifier 450 at setting 6, using 80% pulsed cycle of 4×30 sec bursts with 2 min pauses. The sonicated bacterial lysate was centrifuged at 10,000 g for 20 min at 4° C. The supernatant containing His-OppA protein was saved for protein purification.
His-OppA was purified with Talon metal affinity resin (BD Biosciences, Palo Alto, Calif.) following the manufacturer's instructions. Two ml of 50% suspension of the Talon resin was loaded to a 20 ml chromatography column. The resin storage buffer was drained and the remaining 1 ml beads were equilibrated with 2×10 ml binding buffer (20 mM sodium phosphate, 500 mM NaCl, PH 7.4). After equilibration, the Talon beads were incubated with 10 ml of bacterial supernatant in the column with both ends capped for 20 min at room temperature with rocking. The unbound protein was drained and the bound protein was washed with 2×20 ml wash buffer (20 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, 10 mM beta-mercaptoethanol, pH 7.4) and eluted with 5 ml elution buffer (20 mM sodium phosphate, 500 mM NaCl, 150 mM imidazole, pH 7.4) by gravity flow. The eluates were collected every 500 μl in each tube. Tubes containing concentrated protein fractions were pooled and subject to buffer exchange into phosphate buffered saline (PBS) by using a Centricon YM-10 filter device (Millipore corporation, Danvers, Mass.). The concentration of the purified protein was determined by bicinchoninic acid assay (Pierce, Rockford, Ill.). The quality of the purified protein was examined by SDS-PAGE and Coomassie blue stain.
Development of Antisera to OppA and Whole Bacteria.
Purified recombinant His-OppA protein was sent to Covance (Denva, Pa.) for antibody production in New Zealand White rabbits using a 118-day protocol. Briefly, 250 μg purified OppA was emulsified 1:1 in complete Freund's adjuvant for initial subcutaneous injection. Subsequent immunizations followed a three-week cycle of boosts with 125 μg OppA emulsified 1:1 in incomplete Freund's adjuvant. Test bleeds were taken approximately 10 days after the boosts. Final bleeds were taken 2 weeks after the 5th boosts.
Rabbit anti-035E antisera was obtained from Covance as follows. New Zealand White rabbits were injected subcutaneously and intramuscularly twice with a 4-week interval with 109 colony forming units of M. catarrhalis 035E emulsified with incomplete Freund's adjuvant (1:1). Blood samples were collected 2 weeks after the final injection. Rabbit antiserum to OMP CD was produced in our laboratory.
SDS-PAGE and Immunoblot Assay.
Whole cell extracts of M. catarrhalis clinical strains, 035E and the 035E/oppA mutant were prepared by suspending a loop of bacterial colonies grown on the BHI agar plates in 100 μl PBS. The bacterial suspension was then mixed with 100 μl 2×SDS-PAGE sample buffer (Laemmli buffer) and boiled for 10 min. Twenty μl of each whole cell extract was separated on 10% SDS-PAGE gel and transferred to nitrocellulose membrane.
For immunoblot assay with the rabbit anti-035E antiserum, the membrane was incubated with 1:5000 dilution of the primary antibody in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.1% TWEEN-20, pH 7.5) followed by 1:2000 dilution of the peroxidase labeled goat anti-rabbit IgG antibody (KPL, Gaithersburg, Md.) in TBST as secondary antibody. Bands were detected by using the SuperSignal West Pico Chemiluminescence kit (Thermo Fisher Scientific Inc, Rockford, Ill.). The image was acquired with an AlphaImager (Alpha Innotech corporation, San Leandro, Calif.).
For immunoblot with the rabbit anti-OppA or anti-CD antibody, primary antibodies used were 1:2000 dilution of test bleed anti-OppA antiserum, 1:15,000 dilution of final bleed anti-OppA antiserum, and 1:2000 dilution of anti-CD antiserum. A 1:2000 dilution of the peroxidase labeled goat anti-rabbit IgG antibody was the secondary antibody followed by colorimetric detection.
For immunoblot with the mouse antisera pooled following mucosal immunization with recombinant OppA protein, M. catarrhalis 035E, or PBS control, the membranes were incubated with 1:10,000 dilution of the antisera and probed with 1:25,000 dilution of peroxidase conjugated anti-mouse IgG and developed with chemiluminescence.
Serum from Adults with COPD.
Serum samples were obtained from adults enrolled in our COPD Study Clinic at the Buffalo Veterans Affairs Medical Center. Patients were seen monthly and at times when an exacerbation was suspected. At each visit, serum and expectorated sputum samples were collected and bacteria present in the sputum were identified. Serum samples collected ˜1 month before (pre acquisition) and ˜1 month after (post clearance) acquisition and clearance of M. catarrhalis were subjected to whole cell ELISA assays to analyze the human systemic immune response to M. catarrhalis infection. Nineteen pairs of serum samples with a positive response to homologous infecting strains were used in ELISA assays with purified recombinant OppA protein in this study to examine the OppA specific antibody response. Eight pairs of serum samples collected two months apart from patients who never had positive sputum culture for M. catarrhalis during the study were used as negative controls.
ELISA.
ELISA was carried out by coating the wells of a 96-well microtiter IMMUNOLON 4 plate (Thermo Labsystems, Franklin, Mass.) with 500 ng of purified recombinant His-OppA protein overnight in coating buffer (0.1M Na2CO3 and 0.1 M NaHCO3, pH 9.6). Equal volumes of coating buffer were added to control wells for each well that received OppA. The plate was washed once with PBST (0.5% TWEEN-20 in PBS buffer) and blocked with 3% BSA (bovine serum albumin) in PBS for 1 h at room temperature, after which the plate was washed once again with PBST. Paired COPD patient sera (pre acquisition and post clearance) described above were diluted 1:2,000 in diluent buffer (1% BSA in PBST) and added to the sham coated control wells and His-OppA coated sample wells in parallel. After incubation for 1 h at room temperature, plate was washed 4 times with PBST and 1:6000 dilution of peroxidase labeled secondary antibody, goat anti-human IgG (KPL, Gaithersburg, Md.) was added. After another 1 h incubation the plate was washed 5 times with PBST and developing reagent was added to the wells. The reaction was allowed in the dark for 10 min and stopped with 2M sulfuric acid. The absorbance at 450 nm was determined using a Bio-Rad model 3550-UV microplate reader.
The OD value of the sham coated control wells was subtracted from the OD value of each corresponding sample well to give a normalized OD of each sample. The percentage change in the normalized OD between paired samples were calculated with the following formula: (OD post clearance sample−OD pre acquisition sample)/OD pre acquisition sample)×100. ELISAs were repeated in four independent experiments. The percentage change in OD of each paired sample was determined as the average of the values derived from the four experiments. The mean and standard deviation (SD) of the percentage change of control sera were calculated. The cutoff for a significant percentage change of the sample sera was set as (Mean+2 SD) of percentage change of controls.
Whole Cell ELISA. M. catarrhalis strain 035E and the 035E/oppA mutant strain were grown in BHI broth to OD600 nm at 0.2, harvested by centrifugation and resuspended in PBS. A volume of 100 μl of the suspension was added to each well of the 96-well microtiter IMMUNOLON 4 plate (Thermo Labsystems, Franklin, Mass.). Wells with PBS alone were included as controls. The rest of the experiment was performed following the protocol described above for ELISA with OppA. Primary antibodies, either rabbit anti-OppA antibody, rabbit anti-CD antibody (positive control) or rabbit anti protein 140, a non surface exposed protein (negative control) diluted in 2 fold serial dilutions from 1:10,000 to 1:40,000, were added to the wells. Secondary antibody was 1:4000 dilution of peroxidase labeled goat anti rabbit IgG (KPL, Gaithersburg, Md.).
Flow Cytometry.
M. catarrhalis was grown to mid-logarithmic phase and 100 μl of culture was centrifuged at 4000×g for 5 minutes. The bacterial pellet was resuspended in an appropriate dilution of heat inactivated (56° C. for 30 minutes) rabbit serum in PBS. Appropriate dilutions were determined by performing initial experiments at multiple two-fold dilutions to identify the dilution of serum that yielded the greatest signal to noise ratio. Two negative controls were included: 1) bacteria incubated in PBS alone and 2) bacteria incubated with rabbit antiserum directed at a non surface protein 140. Bacteria incubated with polyclonal antiserum from rabbits immunized with whole bacterial cells were used as a positive control. Bacteria were incubated with antisera for 1 hour at 37° C. The bacteria were washed with PBS and resuspended in a 1:10 dilution of antibody conjugated to flourescein isothiocyanate (KPL). After 30 minutes of incubation at 37° C., the sample was diluted with 900 μl PBS and fluorescence was detected with a FACSCalibur flow cytometer using Cell Quest 3.1 software (BD Biosciences, San Jose, Calif.).
Mucosal Immunization of Mice.
Groups of six Balb/c mice were immunized intranasally with either 50 μg purified recombinant OppA plus 1 μg cholera toxin, or formalin-killed M. catarrhalis 035E plus 1 μg cholera toxin, or 1 μg cholera toxin alone in PBS buffer as a sham immunization control. Intranasal immunization was performed on days 0, 10 and 20 by placing a pipette tip with 5 μl immunogen at each nostril of a conscious mouse to sniff. Immunized mice were challenged on day 28 as described below.
Mouse Pulmonary Clearance Model.
The mouse pulmonary clearance model was used. Overnight culture of M. catarrhalis 035E was inoculated into 100 ml BHI broth with OD600 of 0.05 and grown to an OD600 of 0.3. Bacteria were collected by centrifugation and resuspended in 10 ml PCGM buffer (4.3 mM NaHPO4, 1.4 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 5 mM CaCl2, 0.5 mM MgCl2, 0.1% gelatin, pH 7.3). An aliquot of suspension was diluted and plated to determine the starting concentration of bacteria which was approximately 1×109 colony forming units per ml in each experiment. Ten ml of the bacterial suspension was placed in the nebulizer of a Glas-Col Inhalational Exposure System model 099C A4212 (Glas-Col, Terre Haute, Ind.). Immunized mice were challenged using this inhalation system with the following settings: 10 min preheat, 40 min nebulization, 30 min cloud decay, 10 min decontamination, vacuum flow meter at 60 cubic feet/h, compressed air flow meter at 10 cubic feet/h.
Three hours post-challenge, the mice were anesthetized by inhalation of isoflurane and bled by retro-orbital puncture. Serum was heat inactivated at 56° C. for 30 min and stored at −20° C. for later use. After serum was collected, the anesthetized mouse was euthanized by administration of additional isoflurane. Lungs were then harvested and homogenized on ice in 5 ml PCGM buffer using a tissue homogenizer. Aliquots 20 μl of undiluted and 1:10 diluted lung homogenate were plated in duplicate and incubated at 35° C. with 5% CO2 overnight. Colonies were counted the following day. Statistical significance of colony counts between groups of immunized and sham-immunized mice was determined by two-tailed t-tests. A p value of <0.05 was considered significant.
Results
Identification and Characterization of the oppA Gene.
The genomic sequence of M. catarrhalis strain ATCC 43617 was available as 41 contigs in GenBank (accession numbers AX067426 to AX067466). Computer programs predicted approximately 1,800 ORFs in the genome. These ORFs were analyzed previously in our laboratory to identify putative surface proteins as vaccine candidates leading to the identification of the oppA gene on contig 34 (AX067459, gi12545079) and was predicted to encode a lipoprotein. Homolog analysis by NCBI blastn search using the full-length sequence of the oppA gene revealed significant homology of this gene to the oppA genes in Streptococcus pyogenes strains with approximately 69% identities and 83% similarities.
The oppA gene is the last ORF in a cluster of five being translated in the same frame. The four ORFs preceding oppA gene exhibited significant homologies to the oppB, oppC, oppD, and oppF genes of S. pyogenes, respectively. Specifically, the identities of these genes between M. catarrhalis ATCC43617 (AX067459, gi12545079) and a S. pyogenes strain (X89237, gi1420857) are 68.5% for oppB, 75% for oppC, 70.8% for oppD, and 69.2% for oppF. The organization of these five genes in both strains is depicted in
Bacteria carry from 2 to 5 copies of oppA genes in their genome. However, a blast search of a database containing genome sequences of 5 M. catarrhalis strains including ATCC43617, 7169, 46P47B1, 103P14B1, and 12P80B1 identified only a single oppA gene. This database is accessible at the NCBI website: //camp.mic.med.buffalo.edu/blast/blast_cs.html. Similarly, only a single oppA gene in present in the two strains of M. catarrhalis whose genomes are published (ATCC 43617 and RH4).
To further evaluate whether strains of M. catarrhalis have one or more copies of the oppA gene, a Southern blot assay was performed with 10 clinical isolates including 5 middle ear fluid isolates that caused otitis media and 5 isolates from the sputum of adults with COPD. A single band is observed in each strain (
In bacteria carrying multiple copies of oppA genes, the oppA copies are generally not identical and the OppA proteins play both redundant and unique physiological roles. This would intuitively increase the complexity of OppA based vaccine development for that species, if applicable. A single copy of the oppA gene in M. catarrhalis was therefore considered an appealing feature in our study on vaccine investigation.
Sequence Conservation of oppA Among Strains.
The oppA gene was evaluated for sequence conservation among M. catarrhalis strains in 10 sputum isolates from adults with COPD and 10 middle ear fluid isolates from children with otitis media. The 2039 bp full length oppA genes were amplified by PCR with primers P1 and P2 (Table 1) from these 20 strains and the ATCC 43617 strain as a positive control. A single band of the expected size was present in each strain (
In order to examine the sequence conservation of the oppA gene, PCR products from the 20 clinical isolates and the ATCC 43617 strain were purified and sequenced. Nucleotide sequences and translated amino acid sequences were aligned with the MacVector program by which the oppA homology among these strains was calculated. The results showed that the DNA sequence of the PCR product from ATCC 43617 were identical to the oppA gene sequence in GenBank, which validated the sequence information derived from our PCR products. Overall, a small number of nucleotide variations were present in each strain. The nucleotide variations collected from all strains were distributed at 18 discrete and consecutive nucleotide positions scattered over the 2039 bases full length oppA gene. Nucleotide variations at 12 of these positions were silent and those at the other 6 positions gave rise to 3 amino acid changes. The gene identity scores among these strains range from 98.7%-100%. These data indicate that oppA gene is highly conserved among M. catarrhalis strains.
Of interest, sequence analysis revealed that nucleotide variations occurring at 10 of 18 positions were found exclusively in sputum isolates, whereas nucleotide variations occurring at another 5 sites were found exclusively in middle ear fluid isolates. The nucleotide variations at the remaining 3 positions were present in strains of both clinical sources. This observation suggests that while oppA varies slightly in adaptation to a different environmental niche, the gene is even more conserved within a specific ecological group of strains.
Transcription of the oppA Gene.
Once sequence conservation of the oppA gene was confirmed we examined if the oppA gene is transcribed in M. catarrhalis. M. catarrhalis 035E strain was originally isolated from the middle ear fluid of a patient with otitis media in Dallas and was subsequently widely used in M. catarrhalis studies. RT-PCR for oppA gene expression was performed with total RNA extracted from this strain. A 223 bp fragment within the oppA gene was amplified by PCR with P3 and P4 primers (Table 1), using genomic DNA extracted from 035E as template (
Recombinant His-OppA Protein.
The M. catarrhalis oppA gene encodes a predicted lipoprotein with a 23 amino acid signal peptide at the amino terminus, which contains a consensus lipoprotein signal peptidase (LSP) recognition site, LAAC. The mature OppA protein after LSP cleavage consists of 656 amino acids. The oppA gene region encoding the mature OppA protein was inserted into pCATCH vector to express recombinant OppA as a lipoprotein with a C-terminal hexahistidine tag in E. coli BL21(DE3) as described in Materials and Methods. A COOMASSIE stained SDS-PAGE gel (
Characterization of oppA Mutant.
The isogenic oppA mutant 035E strain was constructed by replacing the oppA gene with a nonpolar kanamycin resistance cassette via homologous recombination. The region of mutagenesis is illustrated in
OppA Expression in M. catarrhalis Strain 035E.
Rabbit anti-OppA antibody was raised against purified recombinant OppA as described in Materials and Methods. This antibody was used in immunoblot analysis to examine the expression of OppA in M. catarrhalis. The immunoblot assay was performed with whole cell extracts of both WT and oppA mutant 035E strain. OMP CD is a constitutively expressed M. catarrhalis protein; antiserum to OMP CD was used as a protein expression control in the assay. Antibodies raised to recombinant OppA specifically recognized the OppA protein from the whole cell extracts of the WT strain; the band is absent in the whole cell extracts of the mutant strain (
Immunogenicity of the OppA Protein.
In order to further evaluate OppA as a vaccine antigen, we characterized the immunogenicity of the OppA protein in M. catarrhalis. First, we assessed if the native OppA protein is immunogenic in a mammalian host presented with the whole organism. To answer this question, rabbit antiserum was raised against the whole organism of M. catarrhalis 035E and the presence of antibodies to OppA in the rabbit antiserum was examined by immunoblot assay.
Whole cell extracts of the 035E WT and oppA mutant strains were separated by SDS-PAGE and subjected to immunoblot assay with the rabbit anti-035E antiserum. A band of ˜84 kD was present in the WT strain but absent in the mutant strain (
To determine whether antibodies raised by immunization with recombinant OppA recognized epitopes on OppA of multiple strains of M. catarrhalis, immunoblot assays were performed with rabbit antiserum to recombinant OppA.
Human Systemic Antibody Response to OppA.
We next assessed if OppA is an antigenic protein of M. catarrhalis during infection of the human respiratory tract. Immunoassays with convalescent patient serum can not only identify antigenic proteins expressed during infection to a level sufficient to induce immune responses, but also indicate possible bacterial targets of the host immune system. To examine whether or not OppA is expressed by M. catarrhalis during infection in adults with COPD and induces an immune response, ELISAs were performed to measure the serum IgG response to OppA following M. catarrhalis infection.
ELISA was performed to measure antibodies to OppA in 19 pairs of patient serum samples (pre acquisition and post clearance) and 8 pairs of negative control samples as described in Materials and Methods. The percentage change in OD between paired samples was calculated. The mean and SD value for negative controls were −3.44% and 15.98%, respectively, representing a nonspecific percentage change distribution with an upper limit of 28.52% (mean+2SD) with 99% confidence. Therefore any percentage change in paired pre acquisition to post clearance serum samples greater than 28.52% can be regarded as significant. According to this criterion, none of the 19 pairs of patient serum samples demonstrated significant percentage change. However, all serum samples including our negative controls have high levels of anti-OppA activity when tested with the purified recombinant OppA protein in ELISAs. Two pairs of serum samples were arbitrarily selected to titrate the anti-OppA level. Both gave titers over 1:10,000, which was defined by the highest dilution of the serum in OppA coated wells giving an OD value 3 fold greater than that derived from the corresponding sham coated control wells. Based on the results of these assays, we speculate that the preexisting high level of antibody to OppA most likely masked any possible anti-OppA immune response, if present, following an episode of M. catarrhalis infection.
Surface Exposure of OppA Epitopes.
Whole cell ELISA. Surface exposure is an important property of vaccine antigens. Whole cell ELISA was performed to examine the surface exposure of OppA epitopes on M. catarrhalis. OMP CD is a surfaced exposed M. catarrhalis protein used as a control. Microtiter wells coated with both WT and oppA mutant 035E strains were incubated with the anti-OppA, anti-CD and anti protein 140 (non surface protein) antisera in ELISA. Anti-OppA antibody was captured by the WT strain but not by the oppA mutant strain (
Flow Cytometry.
As a second independent method to assess the exposure of OppA epitopes on the bacterial surface, antiserum raised to recombinant purified OppA was subjected to flow cytometry with wild type O35E and OppA mutant bacteria. Rabbit antiserum raised to OppA demonstrates an increase in median fluorescence intensity from preimmune serum to immune serum with strain O35E as indicated by a shift of the curve to the right (
Elution of Antibodies from the Bacterial Surface.
Intact log phase M. catarrhalis cells were incubated with heat inactivated antiserum to OppA to allow antibodies to bind to the bacterial cells. After washing, antibodies were eluted from the surface and tested in immunoblot assay with WT, OppA mutant and purified recombinant OppA (
Based on the results of whole cell ELISAs, flow cytometry and elution of OppA antibodies from the cell surface, we conclude that OppA expresses epitopes on the surface of the bacterial cell. This was a surprising result given the prediction of OppA as a periplasmic protein.
Protective Immune Response.
To determine if mucosal immunization with OppA induces protective immunity against M. catarrhalis infection in vivo, the mouse pulmonary clearance model was performed in which groups of mice were immunized intranasally with recombinant purified OppA. Parallel groups of mice were immunized simultaneously with either formalin-killed M. catarrhalis 035E as positive control or PBS as negative control. Mice were challenged with live M. catarrhalis 035E by an inhalation system on Day 28 post immunization. Three hours post-challenge, the lungs of mice were harvested and bacteria recovered from the lungs were quantified as colony counts. Protective immunity was evaluated as reduced number of colony counts in the OppA immunization group as compared to negative the control group. Statistical significance was analyzed by two-tailed t-tests.
Mice immunized intranasally with OppA exhibited 1) production of anti-OppA antibodies in serum that recognized both native and recombinant OppA in immunoblot assays (
While specific embodiments have been presented in this description, those skilled in the art will recognize that routine modifications can be made by those skilled in the art without departing from the scope of the invention.
This application claims the priority of U.S. Provisional application No. 61/329,825, filed on Apr. 30, 2010, the disclosure of which is incorporated herein by reference.
This invention was made with government support under Grant No. R01 AI 28304 from the National Institutes of Health. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2011/034541 | 4/29/2011 | WO | 00 | 1/25/2013 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2011/137334 | 11/3/2011 | WO | A |
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| 6673910 | Breton | Jan 2004 | B1 |
| 20070010665 | Breton | Jan 2007 | A1 |
| 20070087019 | Tucker et al. | Apr 2007 | A1 |
| 20090169577 | Murphy et al. | Jul 2009 | A1 |
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| Number | Date | Country | |
|---|---|---|---|
| 20130129765 A1 | May 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61329825 | Apr 2010 | US |
