WO2004104170A2

WO2004104170A2 - Tools for detecting moraxella catarrhalis.

Info

Publication number: WO2004104170A2
Application number: PCT/US2004/015141
Authority: WO
Inventors: Anthony A. Campagnari; Nicole Luck; Kristin Furano; Amy Howlett
Original assignee: Research Foundation of the State University of New York
Current assignee: Research Foundation of the State University of New York
Priority date: 2003-05-13
Filing date: 2004-05-13
Publication date: 2004-12-02
Anticipated expiration: 2005-11-13
Also published as: US20050142607A1; US7468256B2; WO2004104170A3

Abstract

The present invention provides compositions and methods useful for detecting Moraxella catarrhalis. The compositions are antibodies, proteins or nucleic acid sequences specific to Moraxella catarrhalis (M. catarrhalis). The method comprises the steps of obtaining a biological sample from an individual and detecting within the biological sample the presence of proteins, nucleic acid sequences and lipooligosaccharides (LOS) specific to Moraxella catarrhalis. Further, compositions and methods useful for distinguishing between M. catarrhalis serotypes are provided.

Description

TOOLS FOR DETECTING MORAXELLA CATARRHALIS

This application claims the priority of U.S. Provisional Application serial no. 60/470,022, filed on May 13, 2003, the disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to the area of Moraxella catarrhalis infections and more particularly provides tools for specifically detecting Moraxella catarrhalis.

BACKGROUND OF THE INVENTION

Moraxella catarrhalis is a Gram-negative diplococcus that primarily infects young children where it is a major cause of bacteria-induced acute otitis media (AOM). Older adults with underlying lung disease such as chronic obstructive pulmonary disease (COPD) are also frequently affected. More than 3.5 million cases of AOM are recorded every year in the United States, and it is estimated that 80% of children have experienced at least one episode of otitis before reaching the age of 3 (Klein, J O (1994) Clin.Inf.Dis 19:823). Left untreated the disease may lead to hearing loss. Most cases of AOM are caused by one of three major pathogens,

Streptococcus pneumoniae (S. pneumoniae) (30-40%), non-typeable Haemophilus influenzae (NTHi) (30%) and Moraxella catarrhalis (M. catarrhalis) (20%).

The treatment of the estimated 24 million cases of childhood AOM that occur each year in the United States is the single most prominent reason for prescribing antibiotics (Teele, et al 2001. Naccine 19:S140-S143). In the past three decades, there has been a dramatic worldwide increase in antibiotic resistance in AOM pathogens which has resulted in a reduction of the number of effective antibiotics for this disease and has begun to pose a major public health threat. Accordingly, there is a need for reliable and rapid methods for identifying Moraxella catarrhalis infections in AOM. Further, there is a need for reliable and rapid methods by which Moraxella catarrhalis can be distinguished from other strains of Moraxella and other infectious organisms known to cause AOM., as M. catarrhalis induced AOM can not be diagnosed based on symptomology alone unlike other AOM pathogens. SUMMARY OF THE INVENTION

The present invention provides compositions and methods useful for detecting Moraxella catarrhalis. The compositions are antibodies, proteins or nucleic acid sequences specific to Moraxella catarrhalis (M. catarrhalis).

The method comprises the steps of obtaining a biological sample from an individual and detecting within the biological sample the presence of proteins, nucleic acid sequences and lipooligosaccharides (LOS) specific to Moraxella catarrhalis. Further, compositions and methods useful for distinguishing between M. catarrhalis serotypes are provided.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 A and IB are representations of an SDS PAGE gel and Western blot of the gel, respectively. Fig. IB demonstrates reactivity of a monoclonal antibody (MAb) to all three M. catarrhalis serotypes. The lanes are:, a-25238 serotype A, b- CCUG 3292 serotype B, c-RS-10 serotype C, d-RS-26 serotype C, e-7169 (strain "B" from Buffalo (child), f-7431 isolate from Ohio (child), g-sk633 isolate from Buffalo (adult), h-O35E isolate from Houston (child), i-Tal 1 isolate from Philadelphia (adult), j-7482 isolate from Japan (child), k-7418 isolate from France (adult), 1-7512 isolate from Belgium (child).

Figure IB demonstrates that MAb 4G5 reacts all three the M. catarrhalis LOS A, B and C serotypes.

Figure 2 is a representation of a Western blot probed with a MAb that recognizes the LOS B serotype of M. catarrhalis. The antibody with which the Western was probed is Mab 3F7. Lane 1 demonstrates a lack of reactivity between MAb 3F7 and strain 25238 (serotype A). Lane 2 demonstrates MAb 3F7 reacting with strain CCUG3292 (serotype B). Lane 3 demonstrates MAb 3F7 reacting with strain 7169 (serotype B), and lane 4 demonstrates a lack of reactivity between MAb 3F7 and strain RS-10 (serotype C). Figure 3 A is a representation of an SDS PAGE analysis of whole cell lysates from a series of M. catarrhalis clinical isolates from various geographical locations. Figure 3B is a Western blot of the gel depicted in Fig. 3 A probed with a MAb specific to an external M. catarrhalis protein termed Mhua. The MAb with which the Western was probed is MAb 3F5-E5.

Figure 4A is a representation of an SDS PAGE analysis of outer membrane preparations from Moraxella catarrhalis strains 7169 (lanes 1, 3 and 5) and 169::mhuA (lanes 2, 4, and 6) in which the mhuA gene is deleted.

Figure 4B is a representation of a Western blot performed on the SDS PAGE gel depicted in Fig. 4A using MAb 3F5-5E5 (an IgG2a) demonstrating the loss of the 107 KDa MhuA protein band in strain 7169::mhu. Figure 5 is a representation of a Western blot of whole cell lysates from a series of M. catarrhalis clinical isolates from various geographical locations probed with a MAb specific to a M. catarrhalis internal protein termed Mcp67. The MAb with which the Western was probed is MAb 7C9 (an IgM). The strains shown are 7169, sk633, BC40 (Buffalo); Tall (Philadelphia); 7477 (Japan); 7512 (Belgium); 8063 (Germany); 10016 (Brazil); 10099 (Finland); 10113 (USA).

Figure 6 is a representation of an agarose gel showing PCR products amplified from chromosomal DNA isolated from M. catarrhalis strains. The PCR products in each lane were amplified using the primers 406/408. Lane 1 is a size ladder. Lane 2 is amplification of genomic DNA from strain 7169 and the resulting product is 3.3 Kb. Lane 3 is the negative control (no DNA in PCR reaction). Lanes 4, 5, 7, 8, 9, 11,12 and 13 contain an approximate 4.3 Kb are each serotype A or C. A 3.3 Kb band in lane 6 size was obtained by analyzing the only previously reported LOS serotype B strain, M. catarrhalis CCUG 3292, demonstrating strain 7169 as shown in Lane 2 is also serotype B. Lanes 6, 10 and 14 are other Moraxella isolates that were determined to be serotype B.

Figure 7 is a graphical representation of a PCR based scheme by which all three serotypes of M. catarrhalis can be distinguished from each other.

Figure 8 is a graphical representation of PCR primers and targets within the M. catarrhalis putative glycosyltransferase (pgt) gene cluster. Fig. 9 A is a graphical depiction of PCR amplifications of genomic DNA from various M. catarrhalis strains. The PCR amplifications in Figs. 9A and 9B were carried out with primers SEQ ID NO:3 and SEQ ID NO:4. Fig. 9B is a graphical depiction of PCR amplifications of genomic DNA from M. catarrhalis in lane 1 and various other non M. catarrhalis organisms in the remaining lanes as indicated.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods useful for detecting Moraxella catarrhalis. The compositions provided are antibodies, proteins or nucleic acid sequences unique to M. catarrhalis.

The method comprises the steps of obtaining a biological sample from an individual and detecting in the biological sample the presence of proteins, nucleic acid sequences, lipooligosaccharides (LOS) or antigens specific to Moraxella catarrhalis. The compositions and methods provided are therefore useful not only for detecting M. catarrhalis in a biological sample, but also for distinguishing M. catarrhalis from other organisms which may be present in the sample, and for further distinguishing between M. catarrhalis serotypes as will be described more fully below.

Biological Samples

A "biological sample" as used herein refers to a sample, such as tissue or fluid obtained from an individual, including without limitation urine, plasma, serum, lymph, tears, saliva, sputum, mucous, mucosal swabs, inner ear swabs and tissue sections.

Methods for obtaining biological samples are well known to those skilled in the art. For example, suitable biological samples can be obtained by swabbing mucosal surfaces such as the nasal cavity, oropharynx or throat area, inner ear swabs, sputum samples urine, blood and/or plasma samples using well known collection techniques.

Immunoassays

Once the biological sample is obtained, it can be prepared for immunological detection of M. catarrhalis by a wide variety of immunoassays using the MAbs of the present invention which are specific for Moraxella catarrhalis. By describing proteins, nucleic acid sequences lipooligosaccharides and antigens (LOS) as "specific to Moraxella catarrhalis" it is meant that the particular feature so described is unique to Moraxella catarrhalis and is not found in other tested organisms. Procedures for preparing biological samples for and performing Westerns blots, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIAs), capture assays and non-enzyme linked antibody binding assays are all well known.

In one embodiment, Western blots are performed with the MAbs of the present invention by separating whole cell lysates or outer membrane proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), after which the proteins are transferred to a suitable substrate such as nitrocellulose membrane by application of an electric field in a transfer buffer. The substrate is then incubated with a blocking buffer which allows for blocking of nonspecific adsorption sites on the immobilizing substrate and thus reduces the background caused by non-specific binding of antibodies onto the surface. The blot is then incubated with a primary antibody to induce specific immunocomplex formation and washed in a suitable washing buffer. The primary antibody may be one or a combination of the MAbs provided herein. The occurrence and amount of immunocomplex formation may be determined by subjecting the immunocomplex to a secondary antibody having specificity for the primary antibody. To provide means for detection, the second antibody may have an associated activity such as an enzymatic activity that will generate, for example, a color development upon incubating with an appropriate chromogenic substrate. Quantification may then be achieved by measuring the degree of color generation using any suitable means, such as a spectrophotometer. In another embodiment, the MAbs of the present invention are used in ELISA assays to detect M. catarrhalis from a biological sample. In general, such an ELISA assay (Coligan et al., Current Protocols in Immunology 1(2), Chapter 6, (1991)) initially comprises providing an antibody specific to an M. catarrhalis antigen, preferably a monoclonal antibody. In addition, a secondary antibody is prepared against the monoclonal antibody. To the secondary antibody is attached a detectable reagent such as radioactivity, fluorescence or colorimetric reagent such as horseradish peroxidase enzyme. The biological sample is prepared using standard techniques and incubated on a solid support, e.g. a polystyrene dish, which binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein, such as, bovine serum albumen.

Next, the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any M. catarrhalis proteins attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer. The secondary antibody linked to the detectable reagent is placed in the dish resulting in binding of the secondary antibody to any monoclonal antibody bound to M. catarrhalis proteins. Unattached secondary antibody is then washed out and any necessary reagents are then added to the dish and the amount of detectable reagent developed in a given time period is a measurement of the amount of M. catarrhalis protein present.

In another embodiment, the MAbs of the invention may be used in an immunochromatographic ("ICT") strip assay. A class of devices known as ICT devices uses immunoassay techniques in combination with a label that is conjugated with an antibody. Such devices are now commonly used for rapid, reliable field tests to determine the presence or absence of a particular analyte. The label, when attached ^: to antibody/antigen molecules that are then amassed together in a specific, restricted area, becomes readily detectable by the naked human eye, or by a scanning device, depending on the type of label used. In general, the label can be a particle of latex, gold, or carbon, a radioactive particle, a magnetic particle, or have other physical or chemical properties that allow it to be fixed or attracted to a certain defined area. ICT devices that use the sandwich technique are particularly easy to use. With this technique, labeled antibody that binds with the specific antigen to be assayed is mixed with the sample that is suspected of containing the specific antigen. If the antigen is present in the sample, the labeled antibody binds with the antigen to form a label- antibody-antigen complex. A second antibody that is immovably fixed at a test zone and that also binds with the specific antigen binds the label-antibody-antigen complex at the test zone. A positive result is made visible by the accumulation of the label at the test zone. Such devices are economical and can be used by unskilled workers. Thus, a method that uses such an ICT device to determine; in a single assay, the presence or absence of multiple enteric pathogens, in particular, multiple enteric pathogens, plus a general marker for an inflammatory condition of the intestines would provide valuable diagnostic information to a treating physician.

Several types of such ICT devices are known. Most are the "dipstick" type in which a test strip is encased in a hollow housing with a bibulous pad extending from one end. This pad is dipped into the liquid sample and draws the liquid by capillary ("wicking") action up onto a section of the test strip that contains a labeled antibody, i.e., a label conjugated to an antibody that will specifically bind with the antigen being assayed. The labeled antibody moves with the liquid that is being drawn by the capillary action further along the test strip and, if the specific antigen to which the antibody binds is present in the liquid sample, the labeled antibody will bind with the antigen, forming a labeled antibody-antigen complex. This complex continues to flow with the liquid along the test strip. Downstream from the area containing the conjugated antibody is a test zone. This test zone is typically a nitrocellulose pad into which a second binding partner, an antibody that binds to the same antigen as the ■ labeled antibody, but to a second epitope of the antigen, has been immovably fixed. The fixed antibody will attach to the labeled antibody-antigen complex that flows onto the test zone and will bind the complex to the test zone. The presence of an antigen being assayed is then visible as a stripe across the test zone or otherwise readily detectable. The excess liquid continues to flow past the test zone across a control zone. There are a number of well-known means in the field of immunoassay of creating a control zone, such as embedding into the control zone a binding partner that binds non-specifically to one or more of the labeled antibodies contained on the conjugate section, or to a labeled analyte added to the liquid sample for the purpose of binding with the non-specific binding partner at the control zone. A properly completed test will always show a visible stripe across the control zone or, if a radioisotope or magnetic particle is used as a label, an otherwise readily detectable stripe. Typically, for those devices using a colored label, the housing of the ICT device has a window through which to view the test zone and the control zone. Devices of this type have been disclosed in May et al. (U.S. Pat. No. 5,622,871; issued Apr. 22, 1997) and Charlton et al. (U.S. Pat. No. 5,714,389; issued Feb. 3, 1998).

Further, U.S. Pat. No. 5,869,345, 5,877,028, and 6,727,073 disclose ICT devices that are two-panel cards containing a test strip on one panel and a sample well on the other panel. These devices uses the sandwich technique described above with the dipstick devices, but have a particular advantage in that they allow the sample to be prepared for the test directly on the test card, rather than in a separate vessel. When the test card is closed, liquid from the sample well flows onto the test strip. As with the other devices, a window is provided through which to view the results.

Antibodies and Proteins

In various embodiments, the present invention provides antibodies that can bind surface and/or internal antigens specific to M. catarrhalis and are useful for detecting M. catarrhalis in the immunoassays as described above and as otherwise known in the art.

In one embodiment the present invention provides monoclonal antibodies

(MAbs) to M. LOS antigens, which are the predominant, surface-exposed component of the M. catarrhalis outer membrane and have been implicated as a virulence factor in the pathogenesis of M. catarrhalis infections.

Three LOS serotypes (A, B and C) have been identified on the basis of structural and immunologic analyses of the LOS terminal oligosaccharide branch extensions. The A and B LOS serotypes of M. catarrhalis account for over 90%-95% of all M. catarrhalis isolates, and approximately 30% of isolates are serotype B. In one embodiment of the invention, a MAb that can detect all three LOS serotypes is provided. In another embodiment, a MAb that is specific for a particular serotype is provided. For example, can discriminate the B serotype of M. catarrhalis is provided. While providing MAbs to LOS antigens is useful for detection of M. catarrhalis, the present invention contemplates the use of combinations of antibodies to provide more complete detection of M. catarrhalis present in a biological sample.

Accordingly, in yet another embodiment, the invention provides a novel MAb and novel M. catarrhalis surface protein to which the MAb is directed. Further, detection of surface antigens may be combined with detection of internal antigens to improve detection of M. catarrhalis in a biological sample. The present invention therefore provides in another embodiment a novel MAb and novel internal M. catarrhalis internal protein to which the MAb is directed to.

Nucleic Acids Biological samples can be prepared for detection of unique M. catarrhalis nucleic acids by a wide variety of well known techniques. For example, genomic DNA can be isolated from a biological sample using any suitable technique such as by various kits and reagents for isolation of genomic DNA which are available commercially, for example, those provided by Qiagen (Valencia, Calif.). Alternatively, genomic DNA can be isolated by standard alkaline lysis procedures followed by equilibrium ultracentrifugation in cesium chloride gradients or ethanol precipitation.

The nucleic acid molecules of the invention can be used to detect M. catarrhalis using various techniques such as PCR amplification or hybridization technologies such as Northern blotting or microarrays.

In one embodiment, DNA isolated from a biological sample is analyzed using PCR to detect the presence of M. catarrhalis DNA. PCR reactions can be carried out according to methods well known to those skilled in the art. For examples of suitable PCR protocols see Sambrook et al, "Molecular Cloning: A Laboratory Manual" Cold Spring Harbor Laboratory Press; 3rd edition (2001). Briefly, PCR is performed by subjecting isolated nucleic acids the following steps: denaturation of the nucleic acids, annealing primers to the nucleic acids, extension of the primers with polymerase, and repeating the cycle for as many repetitions as desired, with a typical number of cycles being about thirty. Accordingly, in one embodiment, the invention provides nucleic acid sequences, the detection of which definitively indicates the presence of M. catarrhalis in a biological sample.

In another embodiment, sets of PCR primers that are useful for detecting M. catarrhalis in biological samples are provided. Further, the invention provides sets of primers that can be used to determine the specific LOS serotype of M. catarrhalis in a biological sample. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitations.

EXAMPLE 1 This Example demonstrates a MAb that can recognize all three LOS serotypes of M. catarrhalis.

MAb 4G5 was developed by immunizing BALB/c mice using well known methods with iron-stressed whole bacteria of M. catarrhalis strain 25240. A fusion was performed after 28 days in which splenocytes from the immunized animals were fused to mouse myeloma cells. The resulting hybridomas were screened for the production of antibody by immunodot assay versus iron-stressed and iron-replete whole bacteria. All clones that were positive for the production of MAb to the crude antigen preparations were further tested in Western blots versus iron-stressed and iron-replete whole bacteria and subsequently against outer membrane proteins. This identified the target molecule as LOS for Mab 4G5.

These data demonstrate that MAb 4G5 recognizes LOS conserved on a diversity of clinical isolates from different geographic regions, from both adults and children, and that MAb 4G5 recognizes the M. catarrhalis LOS A, B and C serotypes. Further, additional Western blots assays with MAb 4G5 using proteinase K lysates from 8 strains of H. influenzae, 11 strains of N. meningitidis, 12 strains of N. gonorrhoeae, 2 strains of Neisserialactamica, and 2 strains of Neisseria cinerea were performed, and none reacted with MAb 4G5. (Data not shown). Thus, these data indicate that MAb 4G5 is specific for the LOS that is found on all three M. catarrhalis LOS serotypes. Further, these data demonstrate that MAb 4G5 distinguishes M. catarrhalis from pathogenic organisms that are found commensally with human biological mucus samples.

EXAMPLE 2

This Example demonstrates a MAb that can discriminate one of the three LOS serotypes.

MAb 3F7 (an IgM) is specific for M. catarrhalis LOS serotype B. Monoclonal antibodies 3F7 was developed essentially as described in Example 1 by immunizing mice with iron-stressed outer membranes proteins from M. catarrhalis strain 7169. Once reactivity was identified, further testing in Western blot versus iron-stressed and iron-replete whole bacteria was performed and subsequently outer membrane proteins which identified LOS B as the molecule targeted by Mab 3F7, as can be seen in Fig. 2. Figure 2 is a representation of a Western blot probed with a MAb that recognizes the LOS B serotype of M. catarrhalis. The antibody with which the Western was probed is Mab 3F7. Lane 1 demonstrates a lack of reactivity between MAb 3F7 and strain 25238 (serotype A). Lane 2 demonstrates MAb 3F7 reacting with strain CCUG3292 (serotype B). Lane 3 demonstrates MAb 3F7 reacting with strain 7169 (serotype B), and lane 4 demonstrates a lack of reactivity between MAb 3F7 and strain RS-10 (serotype C). Thus, MAb 3F7 recognizes only LOS B in lanes 2 and 3, and can therefore discriminate the LOS B serotype.

Further, MAb 3F7 was tested against non M. catarrhalis strains including 4 Nontypable Haemophilus influenzae, 3 Neisseria gonorrhoeae and 4 Neisseria meningitidis, all of which were negative for interaction with MAb 3F7 as confirmed by Western blotting. These results therefore demonstrate that MAb 3F7 can discriminate the B LOS serotype of M. catarrhalis and is specific for M. catarrhalis versus other organisms.

EXAMPLE 3 This Example demonstrates a MAb specific for a novel surface protein unique to M. catarrhalis.

MAb 3F5-5E5 was developed as described in Example 1. MAb 3F5-5E5 is specific for the MhuA surface protein of M. catarrhalis. MhuA is a novel putative hemoglobin (Hb)-binding protein homologue gene, and that the gene represented by SEQ ID NO: 1 encodes the Mhua protein represented by SEQ ID NO:2 is disclosed herein. The wild type MhuA gene encodes a 961 amino acid polypeptide with a predicted molecular weight of 107 KDa. The mhuA gene sequence is deposited with GenBank under accession no. AY574198.

In order to characterize MhuA as a M. catarrhalis protein, strain 7169, a middle-ear isolate from a child with otitis media was used to construct a MhuA deficient mutant termed 169::mhuA which does not express the MhuA protein. 7169::mhuA was constructed using standard molecular cloning methods. Clinical isolates of M. catarrhalis were obtained from Mark Achtman (Max Planck Institute, Germany) or from laboratory stocks. Routine culture of M. catarrhalis at 35°C in 5% CO₂ on brain heart infusion (BUT) or GC agar plates or at 37°C with rotary shaking at 225 rpm in the appropriate liquid medium was performed. The mutant strain 7169::mhuA was cultured in the presence of 20 μg kanamycin per ml.

For Western blot analysis of MhuA, M. catarrhalis strains 7169 and 7169::mhuA were cultured in 250 ml CDM broth containing 10 μM desferal plus 5 μM Hb, 8 μM Hm, 100 μM Fe(NO₃)₃, or no exogenous Hb. After 16 h, cultures were harvested and outer membrane proteins (OMPs) were isolated by standard Zwittergent extraction. Proteins were analyzed by sodium dodecyl sulfate-7% polyacrylamide gel electrophoresis (SDS-7% PAGE). Western blot analyses described below with MAb 3F5 were performed using standard methods. Figure 3 A is a representation of an SDS PAGE analysis of whole cell lysates from a series of M. catarrhalis clinical isolates from various geographical locations, while Fig. 3B is the gel from Fig. 3A probed with MAb 3F5 in a Western blot. Figs. 3 A and 3B therefore demonstrate that the 107 KDa MhuA protein is present in each M. catarrhalis strain tested.

In order to confirm that MhuA is an OMP, OMPs from wild-type 7169 and 169::mhuA were prepared. Analysis of OMP profiles are depicted in the Western blot shown in Fig. 4B. Fig. 4A represents an SDS PAGE gel of proteins isolated from 7169 (lanes 1, 3 and 5) and 7169::mhuA (lanes 2, 4 and 6). Fig. 4B is a Western blot of the gel of Fig. 4 A and demonstrates the loss of a 107 KDa band seen in lanes 2, 4, and 6 as expected for strain 7169::mhuA while MhuA is expressed in 7169 (Fig. 4B, lanes 1, 3, and 5). In order to determine the level of conservation exhibited by MhuA, whole bacterial samples were prepared from a series of M. catarrhalis clinical isolates from various geographical locations and analyzed by Western blots probed with MAb 3F5. This data is summarized in Table 1 and demonstrates that all of the isolates express MhuA. Table 1 further demonstrates that related species of Gram-negative bacteria do not express MhuA .

In addition, many of the M. catarrhalis strains in Table 1 were reactive in colony lift assays, which are indicative of surface exposed epitopes. Lift assays are performed by streaking out bacteria for single colonies on a solid media, placing a sheet of nitrocellulose over the colonies, allowing the colonies to "stick" to the sheet, lifting the sheet off and analyzing the sheet as if it was a Western blot.

Table 1

Strain Reactivity to MAb 3F5

M. catarrhalis 7169 + M. catarrhalis 7169::mhuA

M. catarrhalis isolates:

CCUG 3292 (LOS serotype B) +

O35E (Houston, TX, LOS serotype A) +

7544 (Ethiopia, LOS serotype A) +

RS10 (LOS serotype C) + genome strain +

7477 (Japan) +

7680 (United Kingdom) +

7530 (Belgium) +

7608 (Australia) + 25240 (ATCC) +

340535 (Buffalo, NY) +

CCUG 26391 (LOS serotype C) +

CCUG 26404 (LOS serotype C) +

M. lacunata

M. bovis

H. ducreyi

H. influenzae A2

N. gonorrhoeae PID2

N. gonorrhoeae F62

N. cinerea

K. pneumoniae

P. aeruginosa

P. aeruginosa PSN

Thus, the present Example demonstrates the heretofore undisclosed characterization of the MhuA gene and its expression as surface-exposed, conserved protein among M. catarrhalis strains that is recognized by MAb 3F5, which is capable of detecting the novel M. catarrhalis MhuA protein against a background of other common pathogens, some of which share a common niche and cause disease of the respiratory mucosa.

EXAMPLE 4

This Example demonstrates a MAb specific for a novel internal protein unique to M. catarrhalis.

MAb 7C9 is specific for the Mcp67 internal protein of M. catarrhalis. MAb 7C9 was developed as described essentially as described in Example 2. As represented by the Western blot depicted in Fig. 5 and the data summarized in Table 2, the Mcp67 protein is conserved in every strain of M. catarrhalis tested. By using monoclonal antibody 7C9 in Western blots performed according to standard procedures, the Mcp67 protein (approximately 67 kilodaltons) has been detected in over 50 strains of Moraxella from around the world. As can also be seen from Table 2, more than 25 various other gram negative strains have also been analyzed for reactivity with MAb 7C9 and all were negative. (The PCR results presented in Table 2 were generated using primers 537 (SEQ ID NO:3) and 538 (SEQ ID NO:4) as described more fully in Example 7 below). Table 2

Additional tests have confirmed that c protein is not present on the surface of the bacteria and the protein has no significant homology with any other protein in the current data base. Further, as can be seen from Table 3, MAb 7C9 does not react to any other bacterial strains tested.

Table 3

Thus, this Example demonstrates a MAb that can detect a novel protein unique to M. catarrhalis. That nucleotide sequence represented by SEQ ID NO:5 encodes the Mcp67 protein represented by SEQ ID NO:6 is disclosed herein.

EXAMPLE 5 This Example demonstrates DNA sequences that are unique to M. catarrhalis. The Example further describes a method for using PCR analysis of the DNA sequences to distinguish M. catarrhalis from other organisms and to distinguish between the three M. catarrhalis LOS serotypes.

M. catarrhalis chromosomal DNA was prepared using standard methods. All standard molecular biology reagents, including T4 ligase and restriction endonucleases, were purchased from either Promega (Madison, Wis.) or New England Biolabs, Inc. (Beverly, Mass.) and were utilized according to standard protocols. PCR amplification analyses were performed using genomic M. catarrhalis 7169 DNA with Platinum Taq High Fidelity Polymerase (Invitrogen Life Technologies Corp., Carlsbad, Calif.). All PCR products and plasmid constructs were purified using the MinElute kit and the QIAprep spin kit, respectively (Qiagen). DNA sequencing was performed (RPCI Biopolymer Facility, Roswell Park Cancer Institute, Buffalo, NY) and analyzed with Mac Vector software (version 7.2; Genetics Computer Group, Madison, Wis.).

Novel PCR primers were designed to amplify previously undisclosed differences in putative glycosyltransferase (pgt) genes unique to M. catarrhalis strains. Accordingly, using standard PCR protocols, combinations of the primers disclosed herein can be used to amplify regions of clustered genes from M. catarrhalis in a manner such that all three serotypes A, B and C can be distinguished from one another, and M. catarrhalis can be distinguished from other organisms. The PCR primers used in this Example are surnmarized in Table 4. A flow chart for performing the steps of this identification strategy is presented in Fig. 7. Further, the genomic organization of the Moraxella catarrhalis LOS serotype defining pgt genes is depicted in Fig. 8.

The LOS gene cluster from 25238 (type A) as shown in Fig. 8 is represented by SEQ ID NO:15. With respect to SEQ ID NO: 15, primer 406 binds from position 1-22, primer 408 binds from position 4292-4310, primer 649 binds from position 2379-2399 and primer 650 binds from position 2605-2624. Primers 750/751 do not bind to SEQ ID NO: 15.

The LOS gene cluster from strain 7169 (type B) as shown in Fig. 8 is represented by SEQ ID NO: 16. With respect to SEQ ID NO: 16, primer 406 binds from position 1-22, primer 408 binds from position 3295-3313. Primers 649/650 and 750/751 do not bind to SEQ ID NO: 16

The LOS gene cluster from RS-10 (type C) as shown in Fig. 8 is represented by SEQ ID NO: 17. With respect to SEQ ID NO: 17, primer 406 binds from position 1-22, primer 408 binds from position 4306-4324, primer 750 binds from position 822- 840, and primer 751 binds from position 2239-2258. Primers 649/650 do not bind to SEQ ID NO: 17.

Table 4

In LOS serotypes A and C primers 406 and 408 amplify an approximately 4.3 kB product, while in serotype B primers 406 and 408 amplify an approximately 3.3 kB product. This is demonstrated in Fig. 6 which is a representation of an agarose gel showing PCR products amplified from chromosomal DNA isolated from M. catarrhalis strains. The PCR products in each lane were amplified using the primers 406/408. Lane 1 is a size ladder. Lane 2 is amplification of genomic DNA from strain 7169 and the resulting product is 3.3 Kb. Lane 3 is the negative control (no DNA in PCR reaction). A 3.3 Kb band in lane 6 size was obtained by analyzing the only previously reported LOS serotype B strain, M. catarrhalis CCUG 3292, demonstrating strain 7169 is also serotype B. Lanes 4, 5, 7, 8, 9, 11,12 and 13 contain an approximate 4.3 Kb are each serotype A or C. Lanes 6, 10 and 14 are other Moraxella isolates that were determined to be serotype B.

Using primers 649 and 650 results in amplification of a 246 bp fragment from LOS serotype A and no amplification in serotypes B and C. Further, using primers 750 and 751 results in amplification of a 1.44 kB product in serotype C, but no amplification in serotypes A and B. These results are summarized in Table 5.

Table 5.

Thus, by performing PCR reactions using combinations of the primers disclosed herein, one can distinguish all three serotypes A, B and C from one another.

Primers 406 and 408 were tested on genomic DNA isolated from Neisseria meningitidis 121, Neisseria gonorrhoeae, Haemophilus influenzae (7 strains), Haemophilus ducreyi CIP542, Haemophilus ducreyi 35000, Haemophilus parainfluenzae, Klebsiella pneumoniae, Enterobacter aerogenes, Escherichia coli CP9, Psuedomonas aeruginosa, and Proteus mirabilis.

Thus, this Example demonstrates the identification of novel DNA sequences that can be used to detect M. catarrhalis in a biological sample and to discriminate between M. catarrhalis LOS serotypes. Further, the present examples illustrates primers and a PCR scheme for definitive identification of each M. catarrhalis LOS serotype.

EXAMPLE 6 This Example demonstrates DNA sequences encoding a novel surface protein unique to M. catarrhalis and methods for the identification of the DNA sequences.

M. catarrhalis chromosomal DNA was prepared and PCR reactions carried out essentially as described in Example 5 and the MhuA surface protein of M. catarrhalis was identified through BLAST searches based on homology to other TonB-dependent proteins in the National Center for Biotechnology Information (NCBI) data bank. PCR primers were designed for cloning mhuA based on its nucleotide sequence, accession number AX067431. PCR amplification of 75 bp upstream of the predicted 5' transcription start site and 197 bp upstream of the 3' predicted stop site was performed using primers 605 and 606 as depicted in Table 6.

Table 6

PCR analysis of primers 605 and 606 resulted in a 2782 bp product that was ligated into pGEM-T Easy (Promega), resulting in pTB6-AH. E. coli XL1 -Blue were transformed with pTB6-AH using electroporation. PCR analysis, restriction digestion, and sequence analysis were performed using this plasmid to confirm the nucleotide organization of the 7169 mhuA as shown in SED ID NO:l.

As indicated in Table 7, PCR reactions performed on genomic DNA of various organisms using primers 605 and 606 results in amplification of genomic

DNA in all M. catarrhalis strains tested and in no amplification in non-M. catarrhalis strains.

Table 7

Further and as will be apparent to those skilled in the art, because the DΝA sequence encoding MhuA has been shown to be unique to M. catarrhalis, amplification of any part of the sequence will specifically detect M. catarrhalis in a biological sample.

EXAMPLE 7 This Example demonstrates the detection of nucleic acid sequence encoding a novel internal protein unique to M. catarrhalis. As demonstrated in Example 4 above using MAb 7C9, the Mcp67 protein is expressed in every strain of Moraxella tested to date. This Example provides a method for identifying the DΝA sequences of the gene encoding the Mcp67 protein.

Using PCR analysis with the primers depicted in Table 8, over 50 strains of M. catarrhalis were tested and were positive for the Mcp67 gene DΝA. The gene encoding the Mcp67 protein is represented by SEQ ID ΝO:5 and the Mcp67 amino acid sequence is represented by SEQ ID NO:6. These results are presented in Table 3 and are further demonstrated in Figs. 9A and 9B.

Table 8

These primers are designed to the N-terminal internal portion of the gene that codes for the Mcp67 protein and amplify a product from nucleotide 566 to 994 of SEQ ID NO:5.

Figs. 9A and 9B depict PCR analysis using primers 537 and 538 demonstrating the presence of the Mcp67 gene in M. catarrhalis and the lack of amplification products in PCR analysis performed on a variety of other infectious bacterial species.

Further, Table 9 demonstrates results for both PCR analysis of the Mcp67 gene from clinical isolates from around the world in conjunction with antibody based

10 detection of the Mcp67 protein using MAb 7C9.

Table 9.

Moreover, Table 10 demonstrates that a wide variety of organisms other M. catarrhalis are not detected by MAb 7C9 or PCR amplification.

Table 10.

Thus, this Example demonstrates the detection of nucleic acid sequences encoding a novel internal protein unique to M. catarrhalis. Further and as will be apparent to those skilled in the art, because the DNA sequence encoding Mcp67 has been shown to be unique to M. catarrhalis, amplification of any part of the sequence will specifically detect M. catarrhalis in a biological sample.

Example 8 This Example demonstrates that using combinations of the antibodies of the present invention results in increased detection of M. catarrhalis proteins.

Table 11 summarizes data from a variety of ELISA assays using the MAbs indicated in the top row. The assays were performed as follows:

Approximately 10⁶-10⁷ M. catarrhalis 7169 whole bacteria were added to each well of a 96 well ELISA plate. The bacteria were allowed to coat the wells for 24 hours at 40C and the wells were washed and blocked for 1 hour with 2% milk in PBS. The wells were washed and 100 micro liters of each antibody was added and than removed after 1 hour.

The data summarized in Table 11 demonstrates increased detection of M. catarrhalis by using combinations of the MAbs of the present invention. The positive control was mouse sera from animals injected with M, catarrhalis strain 7169 and the negative control was normal mouse sera. Further, the data in the bottom row of Table 8 was obtained after adding a mild detergent to the bacteria in the wells and demonstrates enhanced detection, particularly when an antibody to the internal Mcp67 protein (i.e., MAb 7C9) is used.

Table 11.

Claims

What is claimed is:

1. A method for detecting the presence of Moraxella catarrhalis in a biological sample comprising the steps of: a) contacting the biological sample with at least two antibodies, wherein the epitope in Moraxella catarrhalis for each antibody is either internal or surface exposed; and b) detecting binding of the antibodies to the biological sample, wherein said binding is indicative of the presence of Moraxella catarrhalis in the sample.

2. The method of claim 1, wherein the surface exposed epitope is part of

Moraxella catarrhalis lipooligosaccharide (LOS) A, LOS B, LOS C, the protein of SEQ ID NO:2, or combinations thereof.

3. The method of claim 1, wherein the internal epitope is part of the protein of SEQ ID NO:6.

4. The method of claim 1, wherein the at least two antibodies are selected from the group consisting of 7C9, 4G5, 3F7 and 3F5.

5. The method of claim 1, wherein the binding of the antibodies is detected by a method selected from the group of Westerns blotting, enzyme-linked immunosorbent assay, radioimmunoassays, and immunochromatographic strip assay.

6. The method of claim 5, wherein the binding of the antibodies is detected by immunochromatographic strip assay.

7. A method of identifying Moraxella catarrhalis LOS serotypes in a biological sample comprising the step of: contacting a first aliquot from the sample with a monoclonal antibody which recognizes Moraxella catarrhalis serotypes A, B and C, and contacting at least a second aliquot of the sample with a monoclonal antibody that is specific for the A, B or C serotypes, wherein a comparison of the binding of the monoclonal antibody which recognizes Moraxella catarrhalis serotypes A, B and C to the monoclonal antibody that is specific for the A, B or C serotypes is indicative of the Moraxella catarrhalis LOS serotype in the sample.

8. The method of claim 7, wherein the monoclonal antibody that recognizes LOS serotypes A, B and C is 4G5.

9. The method of claim 7, wherein the monoclonal antibody that is specific for the A, B or C serotypes is monoclonal antibody 3F7.

10. The method of claim 7, wherein the binding of the monoclonal antibody which recognizes Moraxella catarrhalis serotypes A, B and C and the binding of a monoclonal antibody that is specific for the A, B or C serotypes is by measured by a method selected from the group of Westerns blotting, enzyme-linked immunosorbent assay, radioimmunoassays, and immunochromatographic strip assay.

11. The method of claim 10, wherein the binding of the monoclonal antibody which recognizes Moraxella catarrhalis serotypes A, B and C and the binding of a monoclonal antibody that is specific for the A, B or C serotypes is by measured by immunochromatographic strip assay.

12. A method for detecting the presence of Moraxella catarrhalis in a biological sample comprising the step of detecting a Moraxella catarrhalis gene in the biological sample wherein the detected gene is selected from the group consisting of: a) a gene which encodes a protein that is unique to Moraxella catarrhalis and is exposed on the surface of Moraxella catarrhalis, wherein the gene encodes the protein of SEQ ID NO:2, and b) a gene which encodes a protein that is unique to Moraxella catarrhalis and is not expressed on the surface of Moraxella catarrhalis, wherein the gene encodes a protein of SEQ ID NO:6.

13. The method of claim 12, wherein the gene of a) or b) is detected by amplifying a portion of the gene to obtain detectable amplification products and detecting the amplification products.

14. The method of claim 13, wherein the gene encoding of a) is detected by amplifying the gene by the polymerase chain reaction using the primers represented by SEQ ID NO:13 and SEQ ID NO:14.

15. The method of claim 13, wherein the gene of b) is detected by amplifying the gene by the polymerase chain reaction using the primers represented by SEQ ID NO: 3 and SEQ ID NO:4.

16. A method for determining the LOS serotype of Moraxella catarrhalis in a biological sample, the method comprising the steps of: a) amplifying Moraxella catarrhalis genomic DNA with primers represented by SEQ ID NO:7 and SEQ ID NO:8 wherein if the amplification product is 3.3 kilobases, the serotype is B and wherein if the amplification product is 4;3 kilobases, the serotype is either A or C; and b) amplifying Moraxella catarrhalis genomic DNA with primers represented by SEQ ID NO:9 and SEQ ID NO:10 wherein if the amplification product is 245 nucleotides long the serotype is A; and c) amplifying Moraxella catarrhalis genomic DNA with primers represented by SEQ ID NO:l 1 and SEQ ID NO: 12, wherein if the amplification product is 144 kilobases the serotype is C.