NOVEL SURFACE EXPOSED IMMUNOGLOBULIN D-BINDING PROTEIN FROM MORAXELLA CATARRHALIS

Abstract

The present invention relates to a surface exposed protein, which can be detected in Moraxella catarrhalis, having an amino acid sequence as described in SEQ ID NO: 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, to an immunogenic or IgD-binding fragment of said surface exposed protein, and to an immunogenic and adhesive fragment of said surface exposed protein. DNA segments, vaccines, plasmids and phages, non human hosts, recombinant DNA molecules and plants, fusion proteins and polypeptides and fusion products are also described. A method of detecting IgD, a method of separating IgD, a method of isolation of a surface exposed protein of Moraxella catarrhalis and a method for treatment of an autoimmune disease are also disclosed.

Description

[0001] This application is a divisional application of U.S. patent application Ser. No. 12/124,404, filed on May 21, 2008, now U.S. Pat. No. ______, which is a Continuation Application of U.S. patent application Ser. No. 10/480,456, filed on Jul. 13, 2004, now U.S. Pat. No. 7,470,432, issued on Dec. 30, 2008, which is a national stage filing under 35 U.S.C. §371 of International Patent Application No. PCT/SE02/01299, filed on Jul. 1, 2002, which claims the benefit of Swedish Patent Application No. 0102410-8, filed on Jul. 4, 2001, the entire contents of which are hereby incorporated by reference for all purposes.

FIELD OF THE INVENTION

[0002] The present invention relates to a surface exposed protein, which can be detected in Moraxella catarrhalis, having an amino acid sequence as described in SEQ ID NO: 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, and to an immunogenic or IgD-binding fragment of said surface exposed protein, and to an immunogenic and adhesive fragment of said surface exposed protein.

BACKGROUND OF THE INVENTION

[0003] Moraxella catarrhalis is a Gram-negative diplococcus that for a long time was considered a relatively harmless commensal in the respiratory tract. At present, it is the third most frequent cause of otitis media and also a significant agent in sinusitis and lower respiratory tract infections in adults with pulmonary disease. M. catarrhalis is also one of the most common inhabitants of the pharynx of healthy children.

[0004] Two decades ago, Haemophilus influenzae and M. catarrhalis were shown to display a strong affinity for both soluble and surface-bound human IgD (1). The IgD-binding seems to be paralleled by a similar interaction with surface-bound IgD at the cellular level, a phenomenon that explains the strong mitogenic effects on human lymphocytes by H. influenzae and M. catarrhalis (2-4). An IgD-binding outer membrane protein from H. influenzae (protein D) was isolated and cloned, and shown to be an important pathogenicity factor (5). However, protein D does not bind to the majority of IgD myelomas tested, and it was suggested that encapsulated H. influenzae of serotype b expresses an additional IgD receptor (6).

[0005] Early studies demonstrated that the outer membrane proteins (OMPs) from a diverse collection of Moraxella isolates exhibit a high degree of similarity (7). Investigators have primarily focused their research efforts on a selected group of proteins. Recent studies have demonstrated that the high-molecular-weight surface antigen, termed UspA or HMW-OMP, is actually comprised of two different proteins. These proteins are named UspA1 and UspA2 (8,9,10). The apparent molecular masses of these OMPs are greater than 250 kDa as determined by SDS-PAGE analysis. Reduction with formic acid yields bands of approximately 120 to 140 kDa, suggesting that the UspA proteins form an oligomeric complex composed of several monomeric subunits (11). The predicted mass of each protein, as deduced from the cloned genes, is 88 kDa and 62 kDa for UspA1 and UspA2, respectively. It is thought that the difference in the deduced mass and the mass determined using SDS-PAGE is due to a predicted coiled coil structure (9).

[0006] In a recent patent publication, an outer membrane protein of M. catarrhalis with a molecular mass of approximately 200 kDa was isolated (12). A sequence encoding a protein of approximately 200 kDa was also provided. The protein was shown to be immunogenic, but no further biological functions were presented. In addition, a 200 kDa protein is associated with hemagglutinating M. catarrhalis (13,14).

[0007] CopB is an 80 kDa surface exposed major OMP that shows a moderate antigenic conservation. In addition, OMP CD is a 46 kDa highly conserved protein with numerous surface exposed epitopes and OMP E a 47 kDa protein detected on a variety of heterologous strains. The lactoferrin-binding (LbpA and B) and transferrin-binding (TbpA and B) proteins have molecular sizes of 99-111 and 74-105 kDa, respectively.

[0008] Certain strains of Staphylococcus aureus produce immunostimulatory exotoxins such as toxic shock syndrome toxin-1 (TSST-1), staphylococcal enterotoxin A (SEA), SEB and SEC, all of which are associated with food poisoning and toxic shock syndrome (TSS). These exotoxins have been denominated as superantigens (SAg) due to their ability to activate a high frequency of T lymphocytes. SAg bind as unprocessed proteins to HLA class II molecules on APC and oligoclonally activate T cells expressing particular TCR Vβ chains. In vivo exposure to excessive amounts of SAg results in a strong cytokine production and includes IL-2, TNF-α and IFN-γ, which are associated with a toxic shock like syndrome.

[0009] Since the discovery of the first immunoglobulin-binding bacterial protein, S. aureus protein A (SpA) in 1966, this protein has been extensively characterized. The ability of SpA to bind the Fc part of IgG is well known, but SpA also binds a fraction of Ig-molecules of all classes due to the so called `alternative` binding, which represents an interaction with the variable region of certain heavy chains. All IgG-binding capacity of S. aureus has been considered to be mediated by SpA. However, the existence of a second gene in S. aureus encoding an Ig-binding protein has also been reported.

[0010] Streptococcus pyogenes and Peptostreptococcus magnus are other examples of Ig-binding bacteria. S. pyogenes produces protein H belonging to the M family of proteins, and has strong affinity for the Fc region of IgG. Proteins expressed by some strains bind IgA instead of IgG or both IgG and IgA. Protein Bac or the B-antigen is an IgA-binding protein expressed by certain strains of group B streptococci. Finally, P. magnus expresses protein L that shows high and specific affinity for Ig light chains, especially k light chains, and thereby interacts with all classes of Ig.

[0011] IgD is a unique immunoglobulin that exists in both a soluble and a surface-bound form. Both forms are encoded by the same gene and are splicing products. All mature B lymphocytes have B cell receptors (BCR) consisting of membrane-bound IgD and IgM. Soluble IgD comprises approximately 0.25% of the total amount of serum-Ig. The main function of IgD seems to be as an antigen-receptor on the B cell surface in order to optimize B cell recruitment and accelerated affinity maturation. Antigen is taken up through IgD by endocytosis followed by intracellular degradation and presentation on MHC class II for T cells, which in turn are activated and produce cytokines. Hereby, T cell help is obtained including numerous cytokines (e.g. interleukin-4) and co-stimulatory molecules such as CD28.

[0012] Despite macrophages, dendritic cells, and B cells all can present antigens to T lymphocytes, the B cells are 100-fold more efficient due to the importance of the antigen-presenting immunoglobulin on the surface. An attractive strategy in order to potentiate immunization is to directly target an antigen to the B cell receptor. It was early shown that the mouse antibody-response against bovine serum albumin (BSA) conjugated to anti-IgD monoclonal antibodies was 100-fold stronger compared to BSA administration without any antibody. In parallel, it has been demonstrated that a mouse myeloma antigen incorporated into the constant region of anti-IgD-antibodies targeted to the surface-bound IgD results in an up to 1000-fold more efficient antigen presentation on MHC class II (15).

[0013] Tolerance induction can be achieved experimentally by B cell activation through the IgD BCR without any additional T cell help. It would also be possible to treat autoimmune diseases by inducing B cell anergy and thus inhibit the production of auto-antibodies. In fact, SLE-prone mice administered dextran-conjugated anti-IgD antibodies exhibit a delayed development of autoimmunity. In yet another study it was shown that B cell activation via IgD decreases a T helper 2-induced IgE response suggesting a therapy for diminishing the IgE production in severely allergic individuals by displacing the antibody response from a Th2- to a Th1-response. By targeting antigens to the B cell receptor IgD, stimulation, tolerance, and a switch from IgE-production can be achieved. In addition, polyclonal activation has been reported. The outcome is depending on the experimental model used. With different constructs including various repeating IgD-binding segments, it is possible to tailor the response.

[0014] The T cell is a significant player in the anti-tumor response since it recognizes tumor-specific antigens. However, the important T cells display commonly depressed activity in the cancer patient due to a general immunosuppression. A triggering of T helper cells would therefore be very beneficial. Vaccination against tumors using antigen presenting cells (APC) has recently been acknowledged (17). Immunization protocols with APC pulsed ex vivo with tumor antigens (peptides) have been shown to induce effective MHC class I presentation for cytotoxic T cells. It has also been demonstrated that EBV-transformed B cells are able to present melanoma antigens for tumor-infiltrating lymphocytes (TIL). In experimental models, it has also been shown that tumor cells transfected with MHC class II and B7 surface molecules, receptors that are abundant on B cells, would be a feasible approach for tumor vaccination. Interestingly, B16 melanoma bearing mice that were injected with B cells pulsed with a tumor lysate from the corresponding cell line showed a prolonged survival due to an increase in IFN-γ producing T cells. It was also demonstrated that the induced T helper cells evoked a stronger cytotoxic response against the solid tumors. Since myeloma antigen targeted to IgD induces a T cell response, the suggested approach using IgD-binding bacterial proteins conjugated to specific tumor antigens would be feasible.

[0015] To target an antigen (e.g. peptide derived from a microbe or a specific tumor) to IgD-bearing B cells in order to trigger both humoral and cellular immune responses a IgD-binding protein or a shorter IgD-binding peptide would be a very feasible vector. Several examples of successful strategies with a similar angle of approach exist. The humoral immune response in mice against bovine serum albumin (BSA) conjugated to anti-IgD monoclonal antibodies is 100-fold stronger compared to when BSA is administered alone. A recent publication by Lunde et al. (15) describes that when a myeloma-derived peptide is integrated in the constant region of anti-IgD Fab' fragments and injected into mice, a 1,000-fold more efficient antigen presentation is achieved against the antigen in question (15). In parallel, the Ig-binding fragment of S. aureus protein A fused with cholera toxin significantly increases both systemic and mucosal immune responses 10- to 100-fold against the cholera toxin (16). Finally, in a mouse tumor model consisting of the experimentally well defined B16 melanoma, activated B lymphocytes that are pulsed ex vivo with peptides derived from the tumor tissue can evoke a stronger anti-tumor response in vivo and consequently a prolonged survival (17).

SUMMARY OF THE INVENTION

[0016] In one aspect the present invention relates to a surface exposed protein, which can be detected in Moraxella catarrhalis, having an amino acid sequence as described in SEQ ID NO: 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, or an immunogenic or IgD-binding fragment of said protein or variants, or an immunogenic and adhesive fragment of said surface exposed protein.

[0017] In another aspect the present invention relates to an immunogenic or IgD-binding fragment of a surface exposed protein as defined above, which fragment can be detected in Moraxella catarrhalis, having a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof.

[0018] In a further aspect the present invention relates to an immunogenic or IgD-binding fragment as described above, having an amino acid sequence as described in SEQ ID NO:10.

[0019] In still a further aspect the present invention relates to an immunogenic and adhesive fragment of a surface exposed protein as defined above, which fragment can be detected in Moraxella catarrhalis, having a capacity of binding erythrocytes and epithelial cells.

[0020] In still another aspect the present invention relates to an immunogenic and adhesive fragment as defined above, having an amino acid sequence as described in SEQ ID NO: 8.

[0021] In one aspect the present invention relates to a DNA segment comprising a DNA sequence, as shown in SEQ ID NO: 2, which DNA sequence codes for a surface exposed protein of Moraxella catarrhalis as defined above, or naturally occurring or artificially modified variants of said DNA sequence.

[0022] In yet another aspect the present invention relates to a DNA segment comprising a DNA sequence which codes for an immunogenic or IgD-binding fragment as defined above.

[0023] In a further aspect the present invention relates to a DNA segment as defined above, comprising a DNA sequence, as shown in SEQ ID NO: 11, which DNA sequence codes for an immunogenic or IgD-binding fragment as defined above.

[0024] In still a further aspect the present invention relates to a DNA segment comprising a DNA sequence, which codes for an immunogenic and adhesive fragment of a surface exposed protein as defined above.

[0025] In another aspect the present invention relates to a DNA segment as above, comprising a DNA sequence, as shown in SEQ ID NO: 9, which DNA sequence codes for an immunogenic and adhesive fragment as defined above.

[0026] In a further aspect the present invention relates to a vaccine containing a surface exposed protein of Moraxella catarrhalis, said protein having an amino acid sequence as shown in SEQ ID NO: 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said protein, or an immunogenic or IgD-binding fragment of said protein or variants, or an immunogenic and adhesive fragment of said surface exposed protein.

[0027] In another aspect the present invention relates to a vaccine containing an immunogenic or IgD-binding fragment of a surface exposed protein of Moraxella catarrhalis, which has a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said fragment, preferably a vaccine containing an immunogenic or IgD-binding fragment having an amino acid sequence as described in SEQ ID NO: 10.

[0028] In still another aspect the present invention relates to a vaccine containing an immunogenic and adhesive fragment of a surface exposed protein of Moraxella catarrhalis as defined above, preferably a vaccine containing an immunogenic and adhesive fragment having an amino acid sequence as described in SEQ ID NO: 8.

[0029] In one preferred embodiment said vaccines are combined with another vaccine and in another preferred embodiment said vaccines are combined with an immunogenic portion of another molecule.

[0030] In one aspect the present invention relates to a plasmid or phage comprising a DNA sequence, which codes for a surface exposed protein of Moraxella catarrhalis, said protein having an amino acid sequence as shown in SEQ ID NO: 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, or an immunogenic or IgD-binding fragment of said protein or variants.

[0031] In another aspect the present invention relates to a plasmid or phage comprising a DNA sequence, which codes for a an immunogenic or IgD-binding fragment of a surface exposed protein, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said fragment, preferably a plasmid or phage comprising a DNA sequence, which codes for a an immunogenic or IgD-binding fragment having an amino acid sequence as described in SEQ ID NO: 10.

[0032] In still another aspect the present invention relates to a plasmid or phage comprising a DNA sequence, which codes for an immunogenic and adhesive fragment of a surface exposed protein as defined above, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding erythrocytes and epithelial cells or naturally occurring or artificially modified variants of said fragment, preferably a plasmid or phage comprising a DNA sequence, which codes for a an immunogenic and adhesive fragment having an amino acid sequence as described in SEQ ID NO: 8.

[0033] In yet another aspect the present invention relates to a non human host comprising at least one plasmid or phage as defined above, and capable of producing said protein or variants, or said immunogenic or IgD-binding fragment of said protein or variants, or said immunogenic and adhesive fragment of said protein, which host is chosen among bacteria, yeast and plants. In one embodiment the host is E. coli.

[0034] In one aspect the present invention relates to a recombinant DNA molecule comprising a DNA sequence coding for a surface exposed protein of Moraxella catarrhalis, said protein having an amino acid sequence as shown in SEQ ID NO: 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, or for an immunogenic or IgD-binding fragment of said protein, or variants, which DNA sequence is combined with another gene.

[0035] In another aspect the present invention relates to a recombinant DNA molecule comprising a DNA sequence coding for an immunogenic or IgD-binding fragment of a surface exposed protein, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, which DNA sequence is combined with another gene, preferably a recombinant DNA molecule comprising a DNA sequence coding for an immunogenic or IgD-binding fragment having an amino acid sequence as described in SEQ ID NO: 10.

[0036] In still another aspect the present invention relates to a recombinant DNA molecule comprising a DNA sequence coding for an immunogenic and adhesive fragment of a surface exposed protein as above, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding erythrocytes and epithelial cells, or naturally occurring or artificially modified variants of said fragment, which DNA sequence is combined with another gene, preferably a recombinant DNA molecule comprising a DNA sequence coding for an immunogenic and adhesive fragment having an amino acid sequence as described in SEQ ID NO: 8.

[0037] In yet another aspect the present invention relates to a plasmid or phage comprising said fused DNA sequence as defined above.

[0038] In a further aspect the present invention relates to a non-human host comprising at least one plasmid or phage as defined above, which host is chosen among bacteria, yeast and plants. In one embodiment the host is E. coli.

[0039] In one aspect the present invention relates to a fusion protein or polypeptide, in which a surface exposed protein of Moraxella catarrhalis, said protein having an amino acid sequence as shown in SEQ ID NO: 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, or an immunogenic or IgD-binding fragment of said protein or variants, is combined with another protein by the use of a recombinant DNA molecule as defined above.

[0040] In another aspect the present invention relates to a fusion protein or polypeptide, in which an immunogenic or IgD-binding fragment of a surface exposed protein, which fragment can be detected in Moraxella catarrhalis, which has a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, is combined with another protein by the use of a recombinant DNA molecule as defined above.

[0041] In still another aspect the present invention relates to a fusion protein or polypeptide in which an immunogenic and adhesive fragment of a surface exposed protein as defined above, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding erythrocytes and epithelial cells, or naturally occurring or artificially modified variants of said fragment, is combined with another protein by the use of a recombinant DNA molecule as defined in above.

[0042] In yet another aspect the present invention relates to a fusion product, in which a surface exposed protein of Moraxella catarrhalis, said protein having an amino acid sequence as shown in SEQ ID NO: 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said protein, or an immunogenic or IgD-binding fragment of said protein or variants, is covalently or by any other means bound to a protein, carbohydrate or matrix.

[0043] In a further aspect the present invention relates to a fusion product in which an immunogenic or IgD-binding fragment of a surface exposed protein, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said fragment, is covalently or by any other means bound to a protein, carbohydrate or matrix.

[0044] In still another aspect the present invention relates to a fusion product in which an immunogenic and adhesive fragment of a surface exposed protein as defined above, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding erythrocytes and epithelial cells, or naturally occurring or artificially modified variants of said fragment, is covalently, or by any other means, bound to a protein, carbohydrate or matrix. Preferably, a fusion product in which an immunogenic or IgD-binding fragment, having an amino acid sequence described in SEQ ID NO: 10, is covalently, or by any other means, bound to a protein, carbohydrate or matrix. Preferably, a fusion product in which an immunogenic and adhesive fragment, having an amino acid sequence described in SEQ ID NO: 8, is covalently, or by any other means, bound to a protein, carbohydrate or matrix.

[0045] In one aspect the present invention relates to a method of detecting IgD using a surface exposed protein of Moraxella catarrhalis, said protein having an amino acid sequence as shown in SEQ ID NO: 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said protein, or an immunogenic or IgD-binding fragment of said protein or variants, optionally labeled and/or bound to a matrix.

[0046] In a further aspect the present invention relates to a method of detecting IgD using an immunogenic or IgD-binding fragment of a surface exposed protein, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said fragment, optionally labeled and/or bound to a matrix.

[0047] In another aspect the present invention relates to a method of detecting IgD using an immunogenic or IgD-binding fragment of a surface exposed protein of Moraxella catarrhalis, having an amino acid sequence as described in SEQ ID NO: 10, and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said fragment, optionally labeled and/or bound to a matrix.

[0048] In a further aspect the present invention relates to a method of separating IgD using a surface exposed protein of Moraxella catarrhalis, said protein an amino acid sequence as shown in SEQ ID NO: 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said protein, or an immunogenic or IgD-binding fragment of said protein or variants, optionally bound to a matrix.

[0049] In yet another aspect the present invention relates to method of separating IgD using an immunogenic or IgD-binding fragment of a surface exposed protein, which fragment can be detected in Moraxella catarrhalis and has a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said fragment, optionally bound to a matrix.

[0050] In another aspect the present invention relates to a method of separating IgD using an immunogenic or IgD-binding fragment of a surface exposed protein of Moraxella catarrhalis, having an amino acid sequence as described in SEQ ID NO: 10, and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said fragment, optionally labeled and/or bound to a matrix.

[0051] In one aspect the present invention relates to a method of isolation of a surface exposed protein of Moraxella catarrhalis, said protein having an amino acid sequence as shown in SEQ ID NO: 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said protein, or an immunogenic or IgD-binding fragment of said protein or variants. Said method comprises the steps:

[0052] a) subjecting a suspension of Moraxella catarrhalis to an extraction process by adding a zwitterionic or non-ionic detergent, optionally in the presence of EDTA;

[0053] b) applying the extract comprising the IgD-binding protein of Moraxella catarrhalis from step a) to an adsorption column;

[0054] c) eluting the IgD-binding protein; and

[0055] d) separating the IgD-binding protein.

[0056] In another embodiment the concentration of the detergent in step a) of the method is within the range 0.1-5%, preferably 3%.

[0057] In yet another aspect the present invention relates to a method for treatment of an autoimmune disease comprising extra corporal circulation of the blood trough a material comprising a surface exposed protein as defined above, or a fragment thereof as defined above, for removal of IgD from the blood.

[0058] In one aspect the present invention relates to a purified antibody which is specific to an immunogenic portion of a surface exposed protein Moraxella catarrhalis, said protein having an amino acid sequence as described in SEQ ID NO: 1, an apparent molecular weight of 200 kDa and a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants thereof, or an immunogenic or IgD-binding fragment of said protein or variants.

[0059] In another aspect the present-invention relates to a purified antibody as described above, which is specific to an immunogenic or IgD-binding fragment as defined above, having a capacity of selectively binding membrane bound or soluble IgD, or naturally occurring or artificially modified variants of said fragment.

[0060] In still another aspect the present invention relates to a purified antibody as described above, which is specific to an immunogenic or adhesive fragment as defined above, having a capacity of binding erythrocytes and epithelial cells.

DESCRIPTION OF THE FIGURES

[0061] FIG. 1. Chromatography and rechromatography on a SEPHACRYL® S-400 column of EMPIGEN® soluble extract from M. catarrhalis after ion exchange chromatograpy. The solid line indicates protein content of the first chromatography and the broken line rechromatography of the first peak. Vo specifies the void volume.

[0062] FIG. 2. Analysis on SDS-PAGE of fractions representing different purification steps of MID. The fractions are shown for crude extract in 3% EMPIGEN®, after an ion-exchange chromatography on Q-SEPHAROSE® column, and after the 1st and 2nd gelfiltrations on a SEPHACRYL® S-400 column. Two gels were run simultaneously, one was stained with Coomassie blue (Stain) and one was blotted onto Immobilon-P membranes, probed with human IgD(κ) myeloma protein (IgD), anti-UspA (αUsp), or anti-CopB ((βB) monoclonal antibodies followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies. Molecular weights of marker proteins are indicated to the left.

[0063] FIG. 3. Binding of MID to human myeloma sera representing different immunoglobulin classes. All sera were diluted in two-fold steps (4 to 0.3 μg) and applied to a nitrocellulose membrane. After saturation, washing and blocking, an [125I]-MID-labeled probe was added. After overnight incubation and additional washings, specific MID-IgD binding was visualized by autoradiography.

[0064] FIG. 4. IgD-bearing B cells specifically bound FITC-conjugated MID. PBLs stained with RPE-conjugated mAbs against CD19+ (A) or CD3+ (D) followed by incubation with MID-FITC were compared to PBLs incubated with anti-CD19 mAb in addition to an anti-IgD mAb (B). Double staining with CD3+ and anti-IgD mAb is demonstrated in (E). In (C), a panel with PBLs pre-incubated with a rabbit immunoglobulin fraction against human IgD followed by addition of anti-CD19 mAb and MID-FITC is shown. A control sample with no antibodies or MID-FITC is also included (F). PBLs were isolated from heparinized human blood using Lymphoprep one-step gradients. Lymphocytes (2.5×105) were incubated with the appropriate anti-bodies, washed and further incubated with MID-FITC (10 μg/ml). All incubations were performed on ice and after final washings, PBLs were analyzed by flow cytometry. In this particular experiment, 68% of the total lymphocyte population was gated and analyzed. Less than 2% of the cells were labeled when isomatched mAbs were included as negative controls. A pre-immune rabbit serum did not significantly block MID-FITC binding to the IgD BCR (not shown). An experiment with a typical donor out of three separate ones analyzed is shown.

[0065] FIG. 5. Schematic map of the mid gene showing the cloning strategy. Oligonucleotide primers used for DNA amplification are indicated by arrows placed above (PCR) and below (inverse PCR [IPCR]) the relevant sequences. Degenerated primers based upon the amino acid sequences outlined in Table II and specific primers are shown by broken and solid lines, respectively.

[0066] FIG. 6. Nucleotide sequence (nucleotides 106-6889 of SEQ ID NO: 2) of the mid gene from M. catarrhalis Bc5 together with the deduced amino acid sequence (SEQ ID NO: 1). Putative -35, -10 regions, a possible ribosome binding site (RBS), inverted repeat, the predicted signal peptide, and two alternative start-codons at amino acid positions 1 and 17 are indicated. The stop-codon and the inverted repeat is also shown.

[0067] FIG. 7. The degrees of identity and similarity between MID isolated from five M. catarrahlis strains and UspA1 and A2 from ATCC 25238 are demonstrated. The identity and similarity were calculated using the software Needle.

[0068] FIG. 8. Comparison of the amino acid sequence of MID (SEQ ID NO: 1) with the protein presented in U.S. Pat. No. 5,808,024 (SEQ ID NO: 16).

[0069] FIG. 9. Recombinantly expressed MID retained its IgD-binding capacity. The left panel shows a Coomassie brilliant blue stained gel and the right panel a Western blot probed with human IgD. Native MID protein (MID) was run and compared to cytoplasmic (C), periplasmic (P), and membrane (M) fractions. Numbers on the left indicate a molecular weight standard. E. coli BL21DE3 containing pET16-MID were induced for 4 h by IPTG. Cellular fractions were collected and proteins were separated by two SDS-PAGE that was run in parallel and either stained with Coomassie brilliant blue or blotted onto an Immobilon-P membrane. The membrane was probed with human IgD followed by incubation with a horseradish peroxidase-conjugated secondary antibody.

[0070] FIG. 10. MID764-913 (fragment E) and MID902-1200 (fragment F) is responsible for erythrocyte hemagglutination and IgD-binding, respectively. A series of truncated MID proteins (designated A to I) were manufactured. Recombinant proteins containing histidine tags in their C-terminals (A to H) or fused with maltose binding protein (I) were produced in E. coli and purified on nickel and amylose resin columns, respectively.

[0071] FIG. 11. MID962-1200 (fragment F2) has a conserved IgD-binding capacity compared with full length MID1-2139. Equimolar concentrations (range 240 to 0.06 nmol) of purified full length MID1-2139 and 8 truncated MID fragments (F1 to F8) were analyzed for IgD-binding by dot blots. The proteins MID902-1130 (F8), MID985-1130 (F7), and MID1000-1200 (F4) did not attract IgD, whereas all other fragments bound IgD. DNA encoding for the various truncated MID proteins were cloned into the expression vector pET26b(+) and produced in E. coli. The recombinant proteins containing His-tags were purified and dot blotted onto a nitrocellulose membrane. The membrane was probed with human IgD followed by secondary HRP-conjugated polyclonal antibodies that were used for detection.

[0072] FIG. 12. A tetrameric structure of MID962-1200 (F2) is a prerequisite for optimal IgD-binding. (A), SDS-PAGE of MID962-1200 after treatment at 60° C. separates monomers and tetramers. After heat treatment at 100° C. monomers only can be detected. (B), Corresponding Western blot with IgD as probe reveals weak IgD-binding to monomers. (C), Mean IgD-binding to tetramers and monomers, respectively in 6 different experiments. IgD-binding is shown as arbitrary units/μg protein. MID962-1200 was treated in SDS-sample buffer at 60° C. or 100° C. for 10 min, and subjected to SDS-PAGE and Western blot analysis. The resulting Coomassie-stained gel and Western blot were analyzed by densitometry. The percentage of protein migrating as tetramer or monomer was calculated and compared with the IgD-binding capacity.

[0073] FIG. 13. [125I]-labeled recombinant MID764-913 (fragment E) is specifically attracted to erythrocytes and epithelial cells. [125I]-labeled MID and a series of truncated [125I]-MID fragments (C, E, F, G, and I) were added to human erythrocytes (A). The recombinant [125I]-labeled MID fragments were also added to epithelial cells (B). All truncated MID proteins (except fragment I) were produced in E. coli followed by purification on nickel resins. Fragment I was a fusion protein with MBP and consequently purified on an amylose resin. The recombinant proteins were labeled with [125I] and added to erythrocytes or the epithelial cell line A549. After several washings, bound radioactivity was measured in a γ-counter. Data are presented as mean values of 2 experiments with duplicates. Error bars indicate SD.

[0074] FIG. 14. Adhesion of MID-expressing M. catarrhalis to epithelial cells depends on the amino acid residues MID764-913 (fragment E). A decreased adhesion to epithelial cells was observed with MID-expressing bacteria coated with rabbit anti-MID1-2139 or anti-MID764-913 polyclonal antibodies compared to a pre-immune serum or anti-MID1011-1446 (fragment G) pAb. Bacteria were preincubated with the pre-immune serum or specific antisera for 1 h at 4° C. Bacteria were added to the epithelial cells followed by centrifugation and incubation for 30 min at 37° C. After washings, cells were treated with trypsin-EDTA and the suspensions were plated on blood agar plates. Colony forming units were counted after an overnight incubation. The adherence ratio (cfu added/cfu adhered) was calculated. Results are shown as mean values of 4 separate experiments with duplicates. Error bars indicate SD. ***P≦0.001, **P≦0.01 and *P≦0.05.

DESCRIPTION OF THE INVENTION

[0075] MID is not identical to previously well characterized outer membrane proteins of M. catarrhalis. It is not recognized by monoclonal antibodies derived against the UspA or CopB outer membrane antigens. MID also has a different migration pattern in SDS-PAGE and a different composition as shown by amino acid and DNA sequence analysis. MID appeares as a 200 kDa band in accordance with the Mw from the deduced amino acid sequence, but also as an extra band with an estimated molecular mass of more than 1,000 kDa. The extra band indicates that native MID is an oligomeric complex in a similar fashion as UspA (11). This is further supported by the fact that MID was eluted immediately after the void volume from a SEPHACRYL®. S-400 column with a fractionation range of up to -8,000 kDa. The amino acid sequences for MID shows 11.1 and 6.7% identity, respectively, with the USPA1 and USPA2 outer membrane proteins from M. catarrhalis (FIG. 7).

[0076] In a recent patent publication, an outer membrane protein of M. catarrhalis with a molecular mass of approximately 200 kDa was isolated (12). A sequence encoding a protein of approximately 200 kDa was also provided. However, that protein sequence is not identical to the sequence provided by us and shows only 45.9 to 54.4% identity with MID (FIG. 7). The protein was shown to be immunogenic, but no further biological functions were presented. In addition, a 200 kDa protein is associated with hemagglutinating M. catarrhalis (13,14).

Experimental Part

[0077] The present investigation describes the isolation, purification, characterization, cloning and expression of the novel Ig-binding protein named MID of M. catarrhalis, which has affinity for human IgD, of an immunogenic or IgD-binding fragment of said surface exposed protein, and of an immunogenic and adhesive fragment of said surface exposed protein.

Materials and Methods

Bacteria and Plasmids

[0078] M. catarrhalis, strain Bc5, was a clinical isolate from a nasopharyngeal swab culture at our Department. 118 strains isolated from blood, nasopharynx, and sputum were obtained from Sweden, Denmark, Finland, Hungary, Japan, and USA. Sequenced strains and plasmids used for expression are shown in Table I.

TABLE-US-00001 TABLE I Bacterial strains and plasmids used in this study Description Strains or Plasmid (site of isolation) Reference or source Strains DH5α E. coli Novagen BL21DE3 E. coli Novagen BBH17 M. cararrhalis (sputum) Christensen (Denmark) Bc5 M. cararrhalis Dept. Clinical (nasopharynx) Microbiology, (Malmo, Sweden) NCTC 4103 M. cararrhalis CCUG (Gothenburg, (nasopharynx) Sweden) RH1 M. cararrhalis (blood) Christensen (Denmark) RH4 M. cararrhalis (blood) Christensen (Denmark) Plasmid pET16(b) Expression vector Novagen pET16-MID PET16(b) with the ORF of This study mid

Bacteria were grown overnight in Nutrient Broth (Oxoid, Basingstoke Hampshire, England), harvested and washed in phosphate-balanced saline (PBS), pH 7.2 by centrifugation.

Immunoglobulins, Sera and Other Proteins

[0079] The Ig preparations IgG1(κ), IgG1(λ), IgG2(κ), IgG2(λ), IgG3(κ), IgG3(λ), IgG4(κ), IgG4(λ), IgA1(κ) IgA1(λ), IgA2(κ), IgA2(λ), IgM(κ), IgD(λ), IgD(κ), IgD(λ) and IgE(κ) were all of human origin and purchased from The Binding Site (Birmingham, England). IgD myeloma sera IgD(κ) and IgD(λ) were from the same company and IgD-standard serum OTRD 02/b3 was from Behringwerke AG (Marburg, Germany). Myeloma sera IgD(λ)A, IgD(λ) B, IgG A, IgG B, IgG C, IgM, IgA A and IgA B were obtained from the Department of Clinical Chemistry, Malmo, Sweden. The concentration of respective immunoglobulins was according to the suppliers.

Antibodies

[0080] Horseradish peroxidase (HRP)-conjugated goat anti-human IgD was from Biosource (Camarillo, Calif.). Fluoresceinisothiocyanate (FITC)-conjugated mouse anti-human IgD, unlabeled rabbit anti-human IgD, and HRP-labeled rabbit anti-mouse Ig were purchased from Dakopatts (Gentofte, Denmark). Goat anti-human IgD and HRP-conjugated rabbit anti-human polyvalent immunoglobulins was from Sigma (St. Louis, Mo.). Phycoerythrin (RPE)-conjugated mouse anti-human CD3 and CD19 were from Becton Dickinson (San Jos, Calif.). Mouse monoclonal antibodies 17C7 (UspA) and 10F3 (CopB) were kindly provided by Dr. Eric J. Hansen, Department of Microbiology, University of Texas (Dallas, Tex.).

Antisera

[0081] Rabbits were immunized intramuscularly with 200 μg of purified MID (Forsgren et al., 2001), recombinant MID fragments, or recombinant UspA1 emulsified in complete Freunds adjuvans (Difco, Becton Dickinson, Heidelberg, Germany) and boosted on days 18 and 36 with the same dose of protein in incomplete Freunds adjuvans. Blood was drawn 2 to 3 weeks later. The anti-UspA1 polyclonal antibodies reacted with both recombinant UspA1 and UspA2 as examined by Western blots.

SDS-PAGE and Detection of Proteins on Membranes (Western Blot)

[0082] SDS-PAGE was run using a commercial electrophoresis system consisting of 10% Bis-Tris gels with running (MES), sample (LDS), and transfer buffer as well as a blotting instrument (Novex, San Diego, Calif.). Briefly, samples were boiled for 10 min followed by electrophoresis at room temperature using Protein II vertical slab electrophoresis cells (Novex) at 150 constant voltage. Gels were stained with Coomassie Brilliant Blue R-250 (Bio-Rad, Sundbyberg, Sweden). In addition, electrophoretical transfer of protein bands from the gel to an immobilon-P membrane (Millipore, Bedford, Mass.) was carried out at 30 V for 2-3 h. After transfer, the immobilon-P membrane was blocked in PBS with 0.05% Tween 20 (PBS-Tween) containing 5% milk powder. After several washings in PBS-Tween, the membrane was incubated for 1 h in room temperature with purified IgD myeloma protein (0.5 μg/ml, hu IgD(κ) myeloma; The Bindingsite, Birmingham, UK) in PBS-Tween including 2% milk powder. HRP-conjugated goat anti-human IgD diluted 1/1000 in the same buffer was added after several washings in PBS-Tween. In some experiments, IgD myeloma protein was replaced by myeloma protein of other immunoglobulin classes and HRP-labeled anti-human polyvalent immunoglobulins (Sigma) was used as secondary layer. Mouse mAbs 17C7 and 10F3 were used to detect Moraxella outer membrane proteins UspA1, 2 and Cop B, respectively (7,8). In these experiments, HRP-labeled rabbit anti-mouse immunoglobulins were used as a secondary layer. After incubation for 40 min at room temperature and several additional washings in PBS-Tween, developement was performed with ECL Western blotting detection reagents (Amersham Pharmacia Biotech, Uppsala, Sweden). Western blots were analyzed by a Personal Molecular Imager FX (Bio-Rad).

Enzyme Linked Immunosorbent Assay (ELISA)

[0083] ELISA was used to quantitate the immunoglobulin D-binding protein. Extracts of M. catarrhalis diluted in five-fold steps in 0.1 M Tris-HCl, pH 9.0 were added in 100 μl volumes to microtiter plates (F96 Maxisorb, Nunc-Immuno module, Roskilde, Denmark), which were sealed and incubated at 4° C. overnight. After washing the plate four times in PBS-Tween, blocking buffer PBS-Tween containing 1.5% ovalbumin, was added. The plate was incubated for 1 h at room temperature and further washed four times with PBS-Tween. IgD(κ) myeloma protein, 0.05 μg in 100 μl PBS-Tween containing 1.5% ovalbumin was added to each well and after incubation for 1 h at room temperature the plate was washed four times with PES-Tween. After 1 h incubation with HRP-conjugated goat anti-human IgD diluted 1/1000 in the same buffer and subsequent washing with PBS-Tween, tetramethylbenzidine (20 mM) in 0.1 M potassium citrate solution, pH 4.25, mixed with hydrogen peroxide (final concentration 0.002%) was added. After 30 min, the enzymatic reaction was stopped by adding 2 M sulphuric acid. The optical density (OD) was then measured at 450 nm in an automated ELISA reader (Multiskan Plus, Labsystems, Finland)

Dot Blot Assay

[0084] Purified MID (0.0005-0.2 μg) in a volume of 100 μl in 0.1 M Tris-HCl, pH 9.0 were manually applied to nitrocellulose membranes (Schleicher & Schuell, Dessel, Germany) by using a dot blot apparatus (Schleicher & Schuell). After saturation, the membranes were incubated for 2 h at room temperature in PBS-Tween containing 1% ovalbumin and 5% milk powder and washed four times with PBS-Tween. Human myeloma protein 0.5 μg in 100 μl PBS-Tween was added and after 2 h of incubation, followed by several washings in PBS-Tween, HRP-labeled anti-human light chains (κ and λ) (Dakopatts) in dilution 1/200 was used as a secondary antibody. Development was performed as described above for the Western blots. In another set of experiments, dilutions of human myeloma sera in a volume of 100 μl in 0.1 M Tris-HCl, pH 9.0 was first applied to the membranes. After saturation, incubations, blocking, and washing steps were performed as described above. Thereafter, [125I]-labeled protein MID probe (5 to 10×10⁵ cpm/ml) in PBS-Tween was added. After overnight incubation, the membrane was washed four times with PBS-Tween, air dried, and exposed to Kodak CEA.C x-ray films at -70° C. using Kodak X-Omat regular intensifying screen (Eastman Kodak, Rochester, N.Y.).

Extraction of IgD-Binding Protein

[0085] M. catarrhalis bacteria (1-5×1011 colony forming units (cfu)/ml) were suspended in 0.05 M Tris-HCl-buffer (pH 8.8) containing 0.1-5% EMPIGEN® (Calbiochem Novabiochem, Bedford, Mass.). In some experiments EMPIGEN® was replaced by CHAPS (Sigma), n-Octyl-p-D-glucoside (Bachem, Budendorf, Switzerland) or Triton X-100 (Sigma). All these detergents at a concentration of 0.1-5% were tested with or without 0.01 M EDTA. The bacterial suspensions were mixed by magnetic stirring for 2 h at 37° C. After centrifugation at 8000×g for 20 min at 4° C., the supernatants were filtrated with sterile filters (0.45 μm; Sterivex-HV, Millipore).

Purification of IgD-Binding Protein

[0086] M. catarrhalis extract in 3% EMPIGEN® was applied to a Q-SEPHAROSE® column (Amersham Pharmacia Biotech) equilibrated with 0.05 M Tris-HCl (pH 8.8) containing 0.1% EMPIGEN®. The column was eluted using a 0 to 1 M NaCl linear gradient in the same buffer. Fractions showing most IgD-binding activity as detected by ELISA and Western Blot were pooled, dialyzed in Spectraphor membrane tubes (molecular weight cut off 25,000; Spectrum, Laguna hills, Calif.) against 0.05 M Tris-HCl, pH 8.8, concentrated on YM100 disc membranes (molecular weight cut off 100,000; Amicon, Beverly, Mass.) and then applied to gel-chromatography. The gel-filtration of IgD-binding protein was done on a SEPHACRYL® S-400 high resolution column (20 by 900 mm; Amersham Pharmacia Biotech), equilibrated with 0.05 M Tris-HCl, pH 8.8 containing 0.1% EMPIGEN®. Fractions containing the strongest IgD-binding activity were concentrated and re-chromatographed as described above.

Peptide Cleavage and Amino Acid Sequence Analysis

[0087] Purified MID in 0.05 M Tris-HCl (pH 8.8) containing 0.1% EMPIGEN® was treated with trypsin or chymotrypsin in an enzyme-protein-ratio of 1:10 at 37° C. overnight. The cleavage mixtures were subjected to SDS-PAGE and peptide bands transferred to Immobilon membranes were automatically sequenced or exposed to Western blot analysis as described above. In order to get an N-terminal sequence of the protein, deblocking of intact MID from a possible pyroglutamate group was attempted. Two different protocols were used to deblock both soluble and membrane-bound protein. Automated amino acid sequence analysis was performed with an Applied Biosystems (Foster City, Calif.) 470A gas-liquid solid phase sequenator with on-line detection of the released aminoacid phenylthiohydantoin derivatives by Applied Biosystems model 120A PTH analyzer.

Labeling of Protein MID

[0088] Purified MID was radioiodinated ([125I]; Amersham, Buckinghamshire, England) to high specific activity with lactoperoxidase. The preparations contained approximately 0.05 mol iodine per mol protein. FITC (Sigma) was conjugated to purified MID using a standard protocol. Briefly, MID (2 mg/ml) in 0.1 M carbonate buffer, pH 9.5, was incubated with 0.15 μg/ml FITC solubilized in DMSO. After 45 min at room temperature and constant stirring, the sample was diluted and subjected to a PD10 column (Pharmacia Biotech) pre-equilibrated with PBS, pH 7.4. The resulting MID-FITC was used for binding studies.

DNA Isolation and Sequencing

[0089] Genomic DNA was extracted from five M. catarrhalis strains (see Table I) using a genomic DNA preparation kit (Qiagen, Hilden, Germany) and was subsequently used as template for amplification of the MID gene by PCR. Degenerate primers were synthesized according to the amino terminal sequences of the four peptide fragments (Table II).

TABLE-US-00002 TABLE II Amino acid sequences derived from highly purified MID after protease digestions (SEQ ID NOS: 12-15, respectively in order of appearance). Peptide sequence Protease TAQANTESSIAVG Trypsin GNTATNFSVNSGDDNALIN Trypsin QGINEDNAFVKGLEK Trypsin PSTVKADN Chymotrypsin

[0090] In some of the PCR reactions (High Fidelity PCR System; Roche, Bromma, Sweden), specific primers were used in combination with the degenerate ones. DNA sequences flanking the central region of the gene, where the peptide fragments originated from, were isolated using inverse PCR (IPCR). Briefly, genomic DNA was cleaved with the following restriction enzymes used separately; EcORV, SphI and PstI for the isolation of the start codon, and AccI, AsuI and finally HincII for the isolation of the stop codon sequences. The resulting fragments were religated upon themselves (Rapid DNA Ligation Kit; Roche) and the DNA was used in IPCR. To amplify the start and stop codon areas of the gene, specific primers were designed and used in a long template PCR (LTPCR) (Expand Long Template PCR System; Roche). All PCR products were cloned into pPCR-Script-Amp (Stratagene, La Jolla, Calif.) and sequenced using the Big Dye Cycle Sequencing Ready Reaction kit (Applied Biosystems, Warrington, England). Primers for amplification of genomic DNA were designed using the Oligo Primer Analysis software (Molecular Biology Insights, Cascade, Colo.). The signal peptide was deduced using the SignalP V1.1 World Wide Web Prediction Server Center for Biological Sequence Analysis (http://www.cbs.dtu.dk/services/SignalP/)

PCR Amplification of the Mid Gene

[0091] The complete 6.4 kb open reading frame of the mid gene was amplified by PCR using M. catarrhalis BcS strain genomic DNA as template. The oligonucleotide primers containing BamHI restriction enzyme recognition sequences were 5'-cgggatccgatggccgtggcggaatatgcc-3' (primer A, SEQ ID NO: 3) and 5'-cgcggatccgaaaagtgaaaacctgcaccaactgctgc-3' (primer B, SEQ ID NO: 4) generating a PCR product of 6391 base pairs. BamHI-digested insert was ligated into pET16(b) and the resulting plasmid pET16-MID was transformed into DH5α. Both strands of the cloned PCR product were sequenced.

[0092] To examine the full length mid gene in other M. catarrhalis strains, the primers A and B were used. In addition, primers used for narrowing down the sequence encoding the signal peptide were either primer A or 5'-tgtcagcatgtatcatttttttaaggtaaaccaccatg-3' (primer C; detecting the upper start codon, SEQ ID NO: 5) in combination with 5'-catcaattgcgatatgtctgggatcttg-31 (primer D; located at a conserved region just outside the signal peptide, SEQ ID NO: 6) generating 192- and 266-base pair long PCR products (using Bc5 genomic DNA as template), respectively. Furthermore, primer A or C in combination with 5'-cttcaccccatcagtgccatagacc-3' (primer E, SEQ ID NO: 7) were used for confirming the existence of the mid gene resulting in 1355- and 1429-base pair long fragments, respectively. The expand long template PCR system was used in all reactions and conditions were as recommended by the manufacturer (Roche, Bromma, Sweden).

Expression of the Mid Gene Product in E. coli and Cell Fraction

[0093] To express the mid gene product, pET16-MID was transformed into the expression host BL21DE3, containing a chromosomal copy of the T7 RNA polymerase gene under lacUV5 control. The recombinant bacteria were grown in LB medium supplemented with 2% of glucose and ampicillin. Overexpression was achieved by growing cells to logarithmic-growth phase at OD₆₀₀ of 0.6 followed by addition of 1 mM IPTG. After 4 h of induction, bacteria were sonicated according to a standard protocol and the resulting proteins were analyzed by SDS-PAGE.

[0094] Localisation of recombinant protein from pET16-MID was carried out by osmotic shock as described. Briefly, broth cultures of induced and uninduced cells were harvested and resuspended in 30 mM Tris-HCl, pH 8, containing 20% sucrose. EDTA was added to a final concentration of 1 mM and the solution was slowly stirred at room temperature for 10 min. After centrifugation at 10,000 g for 10 min at 4° C., cells were resuspended in ice-cold 5 mM MgSO₄ and stirred for 10 min on ice. During this step, the periplasmic proteins were released into the buffer. The supernatant containing the periplasmic fraction was collected by centrifugation. Bacteria were completely lysed by lysozyme at a final concentration of 100 mg/ml followed by sonication. Finally, the soluble cytoplasmic and insoluble membrane fractions were collected.

Truncated MID-Derived Recombinant Proteins

[0095] The different truncated MID fragments designated A to I with their specific sizes and primers for generating the proteins are shown in FIG. 10. The open reading frame of the mid gene from M. catarrhalis Bc5 (in pET26-MID) (Forsgren et al., 2001) was used as template. All MID constructs, except for MID367-590 (C), were amplified by PCR using specific primers introducing BamHI and HindIII restriction enzyme sites. Due to an internal HindIII restriction enzyme site in fragment C, an XhoI site was used instead of HindIII at the 3' end. All PCR products, except for MID1616-2139 (I), were cloned into pET26 (Novagen, Madison, Wis.). The PCR product encoding for the I fragment was cloned into pMAL-c2 (New England Biolabs, Beverly, Mass.). To avoid presumptive toxicity, the resulting plasmids were first transformed into the non-expressing host E. coli DH5α. Thereafter, plasmids encoding for fragments A to D, G and H were transformed into E. coli BL21(DE3), whereas the host BL21(DE3)-pLysS was used for vectors containing fragments E and F. Both E. coli strains were incubated in the presence of kanamycin, whereas chloramphenicol also was supplemented when BL21(DE3)-pLysS transformants were used. Fragment I was expressed in DH5α. Bacteria were grown to mid-log phase followed by induction with 1 mM isopropyl-1-thio-.β.-D-g-alactoside (IPTG). After 3.5 h, transformants were sonicated and the overexpressed proteins were purified according to the manufacturers instructions. Resulting recombinant proteins having a histidine tag or combined with maltose binding protein were purified on resins containing nickel amylose, respectively. The concentrations of the eluted proteins were determined using the BCA Protein Assay Kit (Pierce). Thereafter, recombinant proteins were analyzed by SDS-PAGE and Western blots.

Hemagglutination

[0096] Human erythrocytes were obtained from freshly drawn heparinized human blood. The erythrocytes were washed twice in PBS (pH 7.2) and suspended in PBS at a final concentration of 1%. Bacteria cultured in Nutrient Broth were harvested by centrifugation, washed and suspended to 1-2×109/ml in PBS. Bacteria and erythrocyte suspension (50 μl of each) were mixed in round bottom microtiter plates (Sarstedt, Newton, N.C.). In some experiments, erythrocytes were mixed with MID-SEPHAROSE® or BSA-SEPHAROSE® in 150 μl PBS. Agglutination was read by the naked eye.

Cell Line and Adherence Assay

[0097] The lung carcinoma cell line A549 (type II alveolar epithelial cells; CCL-185) was obtained from ATCC. The cells were cultured in RPMI 1640 medium (Gibco BRL, Life Technologies, Paisley, Scotland) supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 12 μg/ml gentamicin (referred to as culture medium). On the day before adherence experiments, cells were harvested, washed twice in gentamicin-free RPMI 1640 and added to 12-well tissue culture plates (Nunc, Roskilde, Denmark) at a concentration of 1×104 cells/well in 2.0 ml gentamicin free culture medium. Cells were thereafter incubated overnight at 37° C. in 5% CO₂. On the day of experiments, M. catarrhalis (-2×108) in PBS, 0.15% gelatin (Sigma) was inoculated onto the monolayers. In neutralization experiments with specific antisera, bacteria were preincubated with polyclonal antibodies (dilution 1/250). After 1 h at 4° C., bacteria were added to the epithelial cells. In all experiments, tissue culture plates were centrifuged at 3,000 g for 5 min and incubated at 37° C., 5% CO₂. After 30 min, the infected monolayers were rinsed twice with PBS, 0.15% gelatin with gentle rocking to remove nonadherant bacteria and then treated with trypsin-EDTA (0.05% trypsin, 0.5 mM EDTA) to release them from the plastic support. Thereafter, the resulting cell/bacteria suspension was seeded to agar plates containing 1.1% isovitalex, 7.8% human blood, and finally 0.9% proteose peptone. Data was calculated from duplicate cultures.

Flow Cytometry Analysis

[0098] Human peripheral blood lymphocytes (PBLs) were isolated from heparinized blood from healthy donors by centrifugation on a step gradient of Ficoll-Isopaque (Lymphoprep; Pharmacia, Uppsala, Sweden). For flow cytometry analyses, a standard staining protocol was used with 0.5% BSA (w/v) in PBS as buffer. PBLs (2.5×105 in 100 μl) were labeled with anti-CD3 or anti-CD19 mAbs with or without FITC-conjugated anti-IgD mAb on ice for 30 min according to the manufacturer's instructions. In blocking experiments, lymphocytes were also pre-incubated with anti-IgD immunoglobulins for 30 min. After two washes, 10 μg/ml of purified FITC-conjugated MID was supplemented to the cells followed by incubation for 45 min on ice. After 4 final washes with excess PBS 0.5% BSA, 105 cells for each sample were analyzed in an EPICS® XL-MCL flow cytometer (Coulter, Hialeah, Fla.). Where appropriate, rabbit and goat pre-immune sera and mouse IgG1 and IgG2a were included as negative controls (Dakopatts).

Results

Extraction and Purification of MID

[0099] Solubilization of MID was a major obstacle in the process of purification. Amongst several detergents tested, only EMPIGEN® and n-Octyl-b-D glucoside alone at a final concentration of 3% solubilized MID from a suspension of M. catarrhalis efficiently as estimated by ELISA and Western blot. The two detergents were equally efficient. Triton X-100 alone did not solubilize MID, but Triton X-100 plus 0.01 M EDTA solubilized MID efficiently. C HAPS alone or CHAPS with EDTA or EDTA alone did not solubilize MID. In the following experiments, EMPIGEN® extraction was used for solubilization and subsequent purification of MID. When the EMPIGEN® extract of M. catarrhalis was applied to a Q-SEPHAROSE® column, all IgD-binding material was eluted from the column with 0.1% EMPIGEN® in 0.05 M Tris HCl, pH 8.8. No additional IgD-binding material could be eluted when a NaCl-gradient up to 1 M was applied to the same column. After concentration of the IgD-binding material obtained after separation on Q-SEPHAROSE®, fractionation of the extract was achieved by gel filtration in the presence of 0.1% EMPIGEN® on a SEPHACRYL® S-400 column (FIG. 1). Most IgD-binding material was eluted in this first peak immediately after the void volume. MID was further purified by rechromatography of the first peak under the same conditions.

[0100] FIG. 2 shows that after purification MID appeared as two bands, one 200 kDa-band and a second band with an apparent molecular mass of more than 1,000 kDa. Western blot experiments were performed to confirm that MID was not identical to the previously described outer membrane proteins UspA1 and 2 with an apparent molecular mass varying from 350 to 720 kD (8-10) or CopB with a molecular weight of 80 kDa. The crude EMPIGEN® extract of M. catarrhalis or partly purified preparations of MID were subjected to SDS-PAGE, transferred to Immobilon filters and blotted with antibodies to those Moraxella proteins and also with human IgD. As can be seen in FIG. 2, MID (as revealed by IgD-binding) is not identical with the outer membrane proteins UspA and Cop B.

[0101] Three attempts were made to determine the amino-terminal amino acid sequence of purified MID. Approximately 1000 pmol of MID was applied each time in an automated amino acid sequencer. Inasmuch as no amino acid phenylthiohydantoin derivatives were obtained, the amino-terminal end of the single MID polypeptide chain was probably blocked. It was recently determined that the moraxella UspA1 and UspA2 proteins, which are also resistant to Edman degradation, contained a pyroglutamyl residue that was removed by the treatment with pyroglutamate aminopeptidase. However, when MID purified from M. catarrhalis or recombinant MID was treated with this enzyme according to two different protocols (twice for each method) and then subjected to Edman degradation, no N-terminal amino acid sequence was obtained.

IgD-Binding Properties of MID

[0102] Crude EMPIGEN® extracts of M. catarrhalis and highly purified MID subjected to SDS-PAGE and transferred to filters were exposed to highly purified commercially available Ig-preparations representing all human Ig-classes and subclasses (Table III).

TABLE-US-00003 TABLE III Summary of Western Blot and dot blot analyses showing the binding specificity of highly purified commercially available myeloma immunoglobulin D preparations against a crude EMPIGEN ® extract of M. catarrhalis and highly purified MID. 200 kDa-protein Immunoglobulin in crude extract Purified MID 200 kDa-protein in Immunoglobulin crude extract Purified MID IgD(κ), IgD(λ) + + IgG1(κ), IgG1(λ) - - IgG2(κ), IgG2(λ) - - IgG3(κ), IgG3(λ) - - IgG4(κ), IgG4(λ) - - IgA1(κ), IgA1(λ) - - IgA2(κ), IgA2(λ) - - IgM; (κ), IgM(λ) - - IgE(κ) - -

[0103] Only the two IgD preparations interacted with the MID-band in the 200 kDa-position in a similar fashion as shown for IgD in FIG. 2. When dot blot experiments were performed and purified MID in dilutions was first added to membranes and purified human myeloma proteins and secondary antibodies were subsequently applied, only the two IgD myelomas interacted with MID. One of the two myelomas detected as little as 0.001 μg of MID on the membrane. The specificity of the interaction between MID and IgD was further verified by using radiolabeled MID in other dot blot experiments In FIG. 3, it is demonstrated that MID effectively bound four IgD myeloma sera. A distinct reaction could be detected in the range 0.03-4 μg of IgD. For the IgD standard serum (B.W.) reactivity was seen at even lower concentrations (not shown). In contrast, 6 different Ig myeloma sera representing IgG, IgA and IgM showed no visible reaction with MID at 4 μg.

[0104] Purified MID specifically attracted human soluble IgD as revealed in dot and Western blots (FIGS. 2 and 3, Table III). To test whether MID bound to the surface-expressed B cell receptor (BCR) IgD, human peripheral blood lymphocytes (PBLs) were isolated. FITC was conjugated to MID followed by incubation with PBLs for 45 min on ice. In parallel, PBLs were labeled with RPE-conjugated mAbs directed against the T cell marker CD3 or the B cell specific surface antigen CD19 and subsequently analyzed by flow cytometry (FIG. 4). Interestingly, a large fraction of CD19.sup.+ lymphocytes bound significant amounts of MID-FITC (FIG. 4A), whereas T cells (CD3.sup.+ lymphocytes) only displayed a non-specific background binding (FIG. 4D). The MID-FITC signal corresponded well with CD19.sup.+ cells incubated with anti-IgD mAbs revealing IgD-positive B cells (FIG. 4B). To further elucidate the specificity of MID-FITC binding to IgD bearing CD19.sup.+ lymphocytes, PBLs were preincubated with a rabbit anti-human IgD immunoglobulin fraction. After incubation and washings, MID-FITC binding was analyzed by flow cytometry according to the standard procedure. The antiserum almost completely inhibited specific MID-FITC binding to the IgD BCR when compared to cells incubated with the pre-immune serum. Mean fluorescence intensity decreased from 79.2 to 14.6 arbitrary units. Similar results were obtained with goat immunoglobulins raised against IgD (not shown). Thus, IgD-expressing B cells promoted specific MID-FITC binding to the surface-expressed BCR IgD.

Cloning of the Gene Encoding MID and DNA Sequence Analysis

[0105] Degenerate primers were designed according to the obtained amino terminal sequences of four peptide fragments originating from MID (Table II) and were used in PCRs in all possible combinations. The specific primers 2982+ and 3692- (FIG. 5) were synthesized using the deduced sequence of a distinctive PCR product generated with the degenerate primer pair 2629+/3693-. A PCR reaction using the specific primers in combination with the degenerate ones (718+ and 5772-) resulted in totally 5054 by of the gene coding for MID. Flanking sequences surrounding the core of the mid gene were obtained by inverse PCR (IPCR). IPCR on EcORV- and AsuI/AccI-digested M. catarrhalis genomic DNA with the primer-pairs 2982+/945- and 3668+/120-, respectively, provided the sequence for the start-codon area. In addition, IPCR on HincII-digested moraxella genomic DNA with the primer-pair 5898+/5511- generated the 3' sequence including the stop-codon. The complete nucleotide sequence of the gene encoding MID in M. catarrhalis Bc5 is shown in SEQ ID NO: 2 and the resulting amino acid sequence is shown in SEQ ID NO: 1. Two alternative open reading frames were revealed and are 20' indicated at amino acid positions 1 and 17, see FIG. 6). Consequently, the length of the mid gene product was either 2123 or 2139 amino acids. In addition to a putative ribosome-binding site (AAGG), -10 (TAATTA) and -35 (TTGAAT) consensus sequence boxes were identified. Furthermore, 62 bases downstream of the TAA stop-codon an inverted repeat was found with the potential of stem-loop formation that is necessary for transcriptional termination. To get an overview of the similarity and identity between different mid genes, the sequences of the five ORF MID proteins were analyzed. For 4 strains, the degree of identity and similarity was ≧75.8% and ≧78.3%, respectively (FIG. 7). In contrast, slightly lower values, ≧65.3% and ≧71.2%, respectively, were obtained for the fifth isolate (RH4). Identity and similarity with UspA1 was 5.5-11.1% and 8.3-17.9%, respectively, and with UspA2 6.5-7.5% respectively 11.1-12.4%.

The Mid Gene Can Be Detected in All M. catarrhalis Strains

[0106] By PCR analyses, the mid-I gene was detected in all 118 M. catarrhalis strains, whereas the Moraxella (nesseria)-related controls were negative. In addition, the size of the mid-1 gene was confirmed using primers spanning the whole gene including the start and stop codons. Analysis of the deduced amino acid sequence of MID differs from UspA1, UspA2 and the protein described in U.S. Pat. No. 5,808,024

[0107] The open reading frame defined a protein with a calculated molecular mass of just below 220 kDa that readily corresponded to the empirical value of approximately 200 kDa found by SDS-PAGE. The N-terminal amino acid sequence showed the typical characteristics of a signal peptide with a potential cleavage site between amino acids 66 and 67. Despite that the first amino acid after the signal peptidase cleavage site most likely was a glutamine residue, any sequence could not be determined by Edman degradation. Furthermore, no amino acid sequence was obtained after pyroglutamate aminopeptidase treatment. The predicted amino acid sequence was also subjected to a hydrophobicity profile analysis by the method of Kyte and Doolittle and showed mainly hydrophilic properties except for the putative signal peptide that was strongly hydrophobic. The deduced amino acid sequence for MID differs significantly from those for the protein described in U.S. Pat. No. 5,808,024 and also from the UspA-proteins (FIGS. 7 and 8).

[0108] The mid gene is distributed in all M. catarrhalis strains. To investigate whether or not the mid gene existed in all M. catarrhalis strains, primers were chosen based upon a conserved area upstream of the open reading frame (ORF) and a conserved area downstream including the stop codon sequence (Forsgren et al., 2001). The mid gene was detected in all 86 clinical isolates and 7 type strains analyzed, and the length of the genomic mid DNA was approximately 6,000 base pairs. The existence was further verified by Southern blots using a probe containing a sequence selected from the 3'-end of the gene. Southern blot experiments revealed that the moraxella strains contained only one mid gene.

Expression of Recombinant MID in E. Coli

[0109] To confirm that the cloned mid gene corresponded to the purified IgD-binding protein, the gene including the predicted signal sequence and start codon was subcloned into the expression vector pET16(b) and thereby under the control of a T7 promoter. The resulting pET16-MID was subsequently transformed into E. coli BL21DE3 followed by induction with IPTG. Bacterial cells were lysed and subfractionated, and recombinant MID was localized by Western blots using human IgD as a probe. Important verifying characteristics of MID were provided from the expression experiments (FIG. 9). Firstly, following induction, cells containing pET16-MID were able to produce recombinant MID confirming the correct reading frame of the gene. Secondly, recombinant MID (as shown by SDS-PAGE) displayed a molecular mass of approximately 200 kDa, corresponding to the 217 kDa calculated value from the amino acid sequence. Thirdly, the recombinant protein was indeed the mid gene product in E. coli as its IgD-binding phenotype was confirmed by Western blot analysis. Total protein from E. coli containing induced pET16(b) vector without insert did not display any IgD-binding capacity (data not shown). Fourthly, the subcellular localization of the recombinant protein showed that MID was equally located in the cytoplasmic and the membrane fractions, but not in the periplasmic space. The localization of MID's in the membrane fraction correlated very well with the known outer membrane localization in M. catarrhalis. IgD-binding is preserved in 238 amino acids of MID

[0110] To in detail determine the MID IgD-binding region, 9 sequences derived from the full length MID were cloned into pET26b(+) and expressed in E. coli. The recombinant proteins covered the entire MID sequence and their individual lengths and positions were as demonstrated in FIG. 10. The recombinant proteins comprising amino acid residues 69-1111 or 1011-2139 of MID did not bind IgD as revealed in Western and dot blots. In contrast, the protein MID902-1200 (protein fragment F1) attracted IgD-, strongly suggesting that the single IgD-binding region of MID was within that particular sequence.

[0111] To pinpoint the sequence responsible for the IgD-binding, the truncated MID902-1200 was systematically shortened at the N- and C-terminal ends (FIG. 11). Equimolar concentrations of the various recombinant proteins were compared to native full length MID1-2139 isolated from M. catarrhalis. The different recombinant proteins were diluted in four-fold steps, added to membranes and incubated with human IgD. On a molar basis, an essentially preserved IgD-binding capacity was detected for the truncated MID protein stretching from amino acid residue 962 to 1200. The shortest truncated protein still interacting with IgD-was localized between MID985 and MID1142 (fragment F6). The IgD-binding property was lost when the N-terminus was reduced to the MID1000 residue (fragment F4) or when the C-terminal was shortened to MID1130 (fragment F7). Finally, a fragment (MID902-1130; F8) with a longer N-terminal and a shorter C-terminal (compared to MID985-1200; F3) was also manufactured and analyzed. However, this truncated MID did not interact with IgD, suggesting that the binding capacity was depending on a longer-C-terminal.

[0112] To further characterize the specific MID-dependent IgD-binding, an IgD ELISA was constructed using human IgD as bate. All the recombinant truncated MID fragments were subjected to ELISA followed by incubation with a specific rabbit anti-serum directed against MID902-1200. The ELISA was developed using HRP-conjugated goat anti-rabbit polyclonal antibodies. The same pattern as with the dot blot (FIG. 11) was observed, i.e. fragments F4, F7, and F8 was not attracted to the solid phase IgD, whereas the other fragments bound to a variable degree compared to full length MID (not shown).

Optimal MID962-1200--IgD Interaction is Depending on a Tetramer Structure

[0113] To shed light upon the need for a tetramer structure in order to obtain an optimal IgD-binding, MID962-1200 (F2, SEQ ID NO: 10) was incubated at 60 or 100° C. followed by analysis on SDS-PAGE and Western blots. MID962-1200 formed both a monomer and a tetramer after pre-treatment at 60° C. (FIG. 12A). The tetrameric structure was, however, disrupted at 100° C. and resulted in a monomeric form, which displayed a considerably weaker binding to IgD when examined in Western blots (FIGS. 12A and B). To investigate the capability of the tetramer to bind IgD in comparison with the monomeric form, the MID962-1200 fragment, SEQ ID NO: 10, was subjected to analysis at 60° C. in 6 different experiments. The heat treated protein was subjected to SDS-PAGE and the IgD-binding activity was analyzed by Western blots. Resulting gels and filters were analyzed by densitometry and the protein concentration (density) of the monomer was divided with the corresponding tetramer concentration. The obtained value (%) was related to the concentration (μg) of total protein loaded on the gels. Interestingly, when IgD-binding to the tetrameric respectively monomeric forms were compared, a 23-fold more efficient binding to IgD was found with the tetrameric MID962-1200 (FIG. 12C). M. catarrhalis IgD-binding protein (MID) hemagglutinates human erythrocytes

[0114] To investigate a putative involvement of MID in hemagglutination, a series of clinical isolates that either expressed MID or by phase variation had shut off the mid gene was selected. Interestingly, all out of 21 isolates expressing MID hemagglutinated human erythrocytes, whereas only four out of the MID-negative strains (n=21) hemagglutinated the red blood cells. An almost full correlation between hemagglutinating capacity and MID expression was observed. UspA1/2 expression was similar and irrespective of the MID expression.

[0115] These initial experiments prompted us to examine whether or not purified MID protein from the model strain M. catarrhalis Bc5 (Forsgren et al., 2001) hemagglutinates erythrocytes. To mimic the bacterial surface, MID was conjugated to SEPHAROSE® beads and incubated with the human erythrocytes. Bovine serum albumin (BSA) linked to SEPHAROSE® was included as a negative control. Interestingly, the human erythrocytes were hemagglutinated in the presence of MID-SEPHAROSE®, whereas BSA-SEPHAROSE® did not interfere with the erythrocytes (data not shown). The hemagglutinating domain of MID is located between amino acid residues Alanine764 and Serine913

[0116] To dissect the molecule and pin-point the specific site of the molecule that was responsible for the hemagglutination, a series of truncated DNA fragments of the mid gene was cloned and recombinantly expressed in E. coli (FIG. 10). Polyclonal antibodies against the truncated MID proteins were raised in rabbits and used in an ELISA. In preparatory experiments, antibodies to MID and the MID-derived proteins were titrated to give similar values when tested in ELISA against respective antigens. The capacity of the truncated MID proteins to bind to lysed erythrocytes was then measured in ELISA using the specific antibodies at appropriate concentrations. MID or MID764-913 (fragment E) gave higher ELISA values (4 to 16 times) as compared to the other truncated MID proteins. Thus, the hemagglutinating structure of MID seemed to be located within amino acid residues 764-913 of MID (SEQ ID NO: 8).

MID764-913 (Fragment E, SEQ ID NO: 8) Binds Directly to Both Erythrocytes and Type II Alveolar Epithelial Cells

[0117] To further confirm the importance of MID764-913 as an adhesin, MID and a selection of the truncated MID-derived proteins were radiolabeled and tested in direct binding experiments with human erythrocytes and alveolar epithelial cells (FIG. 13). Both [125I]-MID and [125I]-MID764-913 strongly bound to erythrocytes, whereas the truncated MID fragments MID367-590 (fragment C), MID902-1200 (F), MID1011-1446 (G), and MID1616-2139 (I) did not bind above background levels (FIG. 13A). In parallel, the alveolar epithelial cell line A549 also attracted both the full length [125I]-labeled MID and the truncated MID764-913 (FIG. 13B). All the other fragments did not bind to the epithelial cells. Taken together, the fragment MID764-913 (SEQ ID NO: 8) was the crucial part of the adhesin MID that mediated the attachment to mammalian cells.

Antibodies to Full Length MID1-2139 and MID764-913 Inhibit Adherence of M. catarrhalis to Type II Alveolar Epithelial Cells

[0118] To further analyze the influence of full length MID and MID764-963 on M. catarrhalis adherence to type II alveolar epithelial cells, a MID-expressing and a MID-deficient M. catarrhalis strain were preincubated with antibodies to MID and subsequently added to alveolar epithelial cells for adherence. As demonstrated in FIG. 14, polyclonal antibodies directed against full length MID1-2139 and MID763-913 (fragment E, SEQ ID NO: 8) effectively inhibited adherence for the MID-expressing isolate. In contrast, pre-immune serum and a pAb directed against MID1011-1466 (fragment G) did not significantly interfere with adhesion.

REFERENCES

[0119] 1. Forsgren, A. and Grubb, A. (1979) Many bacterial species bind human IgD. J. Immunol. 122, 1468-1472. [0120] 2. Banck, G. and Forsgren, A. (1978) Many bacterial species are mitogenic for human blood lymphocytes. Scand. J. Immunol. 8, 347-354. [0121] 3. Calvert, J. E. and Calogeres, A. (1986) Characteristics of human B cells responsive to the T-independent mitogen Branhamella catarrhalis. Immunology 58, 37-41. [0122] 4. Forsgren, A., Penta, A., Schlossman, S. F. and Tedder, T. F. (1988) Branhamella catarrhalis activates human B lymphocytes following interactions with surface IgD and class I major histocompatibility complex antigens. Cell. Immunol. 112, 78-88. [0123] 5. Janson, H., Carin, B., Cervin, A., Forsgren, A., Bjork-Magnusdottir, A., Lindberg, S. and Runer, T. (1999) Effects on the ciliated epithelium of protein D-producing and -nonproducing nontypeable Haemophilus influenzae in nasopharyngeal tissue cultures. J. Infect. Dis. 180, 737-746. [0124] 6. Sasaki, K. and Munson Jr., R. S. (1993) Protein D of Haemophilus influenzae is not a universal immunoglobulin D-binding protein. Infect. Immun. 61, 3026-3031. [0125] 7. Helminen, M. E., Beach, R., Maciver, I., Jarosik, G., Hansen, E. J. and Leinonen, M. (1995) Human immune response against outer membrane proteins of Moraxella (Branhamella) catarrhalis determined by immunoblotting and enzyme immunoassay. Clin. Diagn. Lab. lmmunol. 2, 35-39. [0126] 8. Aebi, C., Maciver, I., Latimer, J. L., Cope, L. D., Stevens, M. K., Thomas, S. E., McCracken, G. H. and Hansen, E. J. (1997) A protective epitope of Moraxella catarrhalis is encoded by two different genes. Infect. Immun. 65, 4367-4377. [0127] 9. Cope, L. D., Lafontaine, E. R., Slaughter, C. A., Hasemann, C. A. Jr., Aebi, C., Henderson, F. W., McCracken, G. H. Jr and Hansen, E. J. (1999) Characterization of Moraxella catarrhalis uspA1 and uspA2 genes and their encoded products. J Bacteriol 181, 4026-4034. [0128] 10. Klingman, K. L. and Murphy, T. F. (1994) Purification and characterization of a high-molecular-weight outer membrane protein of Moraxella (Branhamella) catarrhalis. Infect. Immun. 62,1150-1155. [0129] 11. WO 98/28333. [0130] 12. Sasaki, K., Harkness, R. E., Loosmoore, S. M. and Klein, M. H. (1998) U.S. Pat. No. 5,808,024. [0131] 13. Fitzgerald, M., Mulcahy, R., Murphy, S., Keane, C., Coakley, D. and Scott, T. (1997) A 200 kDa protein is associated with haemagglutinating isolates of Moraxella (Branhamella) catarrhalis. FEMS Immun. Med. Microbiol. 18, 209-216. [0132] 14. Tucker, K., Plosila, L., and Samuel, J. (1994) Correlation between hemagglutination and globotetraosyl-ceramide binding by Branhamella catarrhalis. Abstract 117 of the 94th General meeting of the American Society for Microbiology. [0133] 15. Lunde E, Munthe L A, Vabo A, Sandlie I, Bogen B. (1999) Antibodies engineered with IgD specificity efficiently deliver integrated, T-cell epitopes for antigen presentation by B cells. Nat Biotechnol. 17, 670-675. [0134] 16. Lycke N. (2001) The B-cell targeted CTA1-DD vaccine adjuvant is highly effective at enhancing anti-body as well as CTL responses. Curr. Opin. Mol. Ther. 3, 37-44. [0135] 17. Ito O, Harada M, Takenoyama M, Tamada K, Li T, Abe K, Fujie H, Nomoto K. 1998 Vaccination with activated B cells pulsed with tumor-lysates can induce tumor-specific CD4+ T cells in vivo. Immunobiol. 199, 133-147.

Sequence CWU 1

1612139PRTMoraxella catarrhalis 1Met Asn His Ile Tyr Lys Val Ile Phe Asn Lys Ala Thr Gly Thr Phe1 5 10 15Met Ala Val Ala Glu Tyr Ala Lys Ser His Ser Thr Gly Gly Ser Cys 20 25 30Ala Thr Gly Gln Val Gly Ser Val Cys Thr Leu Ser Phe Ala Arg Val 35 40 45Ala Ala Leu Ala Val Leu Val Ile Gly Ala Thr Leu Asn Gly Ser Ala 50 55 60Tyr Ala Gln Gln Asp Pro Arg His Ile Ala Ile Asp Gly Asn Ser Ser65 70 75 80Asn Thr Ser Ser Gly Thr Ala Arg Ala Thr Gly Asp Arg Ala Ile Ala 85 90 95Leu Gly Glu Asn Ala Asn Ala Glu Gly Gly Gln Ala Ile Ala Ile Gly 100 105 110Ser Ser Asn Lys Thr Gly Gly Arg Asn Ala Leu Asn Ile Gly Thr Asp 115 120 125Ala Lys Gly Glu Glu Ser Ile Ala Ile Gly Gly Asp Val Val Ala Glu 130 135 140Gly Thr Ala Ser Ile Ala Ile Gly Gly Asp Asp Leu His Leu Trp Asp145 150 155 160Glu Pro Asn Lys Gln Lys Phe Leu Asp Pro Lys Val Lys Glu Leu Ile 165 170 175Leu Lys His Gln Glu Leu Asn Asn Ile Tyr Lys Leu Pro Asp Gly Ser 180 185 190Pro Gln Arg Tyr Phe Arg Thr Tyr Ala Lys Gly His Ala Ser Ile Ala 195 200 205Leu Gly Thr Met Thr Gln Ala Glu Gly His Phe Ala Asn Ala Phe Gly 210 215 220Thr Arg Ala Thr Ala Lys Gly Asn Tyr Ser Leu Ala Val Gly Leu Thr225 230 235 240Ala Gln Ala Asn Thr Glu Ser Ser Ile Ala Val Gly Ser Asn Ala Gln 245 250 255Ala Asn Gly Phe Ala Ala Thr Ala Ile Gly Gly Gly Thr Lys Ala Asp 260 265 270Leu Gly Arg Ser Ile Ala Leu Gly Phe Gly Ser Gln Ile Leu Thr Lys 275 280 285Glu Lys Asp Ser His Asn Asn Thr Asn Val Tyr Val Pro Gln Gly Glu 290 295 300Ile Leu Glu Glu Arg Tyr Lys Ala Thr Glu Asn Gly Gln Ser Pro Asn305 310 315 320Lys Val Val Asp Ile Phe Ser Ile Gly Ser Ser Ser Ile Lys Arg Lys 325 330 335Ile Ile Asn Val Gly Ala Gly Ser Gln Glu Thr Asp Ala Val Asn Val 340 345 350Ala Gln Leu Lys Leu Val Glu Arg Val Ala Lys Arg Gln Ile Thr Phe 355 360 365Gln Gly Asp Asp Ser Asn Asn Ser Val Lys Lys Gly Leu Gly Gln Thr 370 375 380Leu Thr Ile Lys Gly Gly Lys Thr Glu Thr Gly Glu Leu Thr Glu Asn385 390 395 400Asn Ile Gly Val Val Gln Asp Asp Asn Gly Asn Gly Leu Lys Val Lys 405 410 415Leu Ala Lys Asp Leu Thr Gly Leu Thr Lys Val Ala Val Asn Gly Asn 420 425 430Gly Ala Asn Asn Ala Glu Leu Leu Asn Gly Gly Leu Thr Phe Ser Thr 435 440 445Ser Gly Ala Asn Ala Gly Lys Thr Val Tyr Gly Thr Asp Gly Val Lys 450 455 460Phe Thr Asn Asn Thr Gly Thr Gly Thr Gly Thr Ala Ile Pro Asp Thr465 470 475 480Thr Arg Ile Thr Lys Asn Lys Ile Gly Phe Ala Gly Ala Asp Glu Gln 485 490 495Val Asp Glu Ser Lys Pro Tyr Leu Asp Asn Glu Lys Leu Lys Val Gly 500 505 510Thr Val Glu Ile Lys Lys Thr Gly Ile Asn Ala Gly Asn Gln Glu Ile 515 520 525Thr Lys Val Lys Ser Ala Ile Val Asp Ala Val Asn Gly Gln Ala Asn 530 535 540Gln Ser Phe Val Asn Leu Leu Glu Thr Ala Gly Thr Asn Thr Asn Lys545 550 555 560Gln Asn Ser Ala Ala Thr Val Lys Asp Leu Tyr Asp Leu Ser Gln Ser 565 570 575Pro Leu Thr Phe Thr Gly Asp Ser Gly Asn Val Lys Arg Lys Leu Gly 580 585 590Gln Thr Leu Thr Ile Thr Gly Gly Gln Thr Lys Thr Asp Gln Leu Thr 595 600 605Asp Asn Asn Ile Gly Val Val Ala Gly Thr Asn Gly Leu Thr Val Lys 610 615 620Leu Ala Lys Thr Leu Asn Ser Leu Thr Glu Val Asn Thr Ala Thr Leu625 630 635 640Asn Ala Thr Asn Lys Val Lys Val Asp Asn Ser Thr Gly Asn Thr Ala 645 650 655Glu Leu Leu Asn Asn Gly Leu Thr Phe Thr Gln Thr Thr Gly Ala Asn 660 665 670Ser Gly Lys Thr Val Tyr Gly Asn Asp Gly Leu Lys Phe Thr Asn Ser 675 680 685Ala Asn Lys Ala Leu Gly Gly Thr Thr Tyr Ile Thr Lys Asp Gln Val 690 695 700Gly Phe Ser Asn Gln Asp Gly Leu Leu Asp Glu Ser Lys Pro Tyr Leu705 710 715 720Asn Arg Glu Lys Leu Lys Val Gly Lys Ile Glu Ile Lys Asp Ser Gly 725 730 735Ile Asn Ala Gly Gly Lys Ala Ile Thr Gly Leu Pro Ser Thr Leu Pro 740 745 750Asn Thr Thr Tyr Thr Ala Pro Gly Val His Thr Ala Leu His Gly Ser 755 760 765Thr Ile Ser Asn Asp Asp Lys Thr Arg Ala Ala Ser Ile Ala Asp Val 770 775 780Leu Asn Ala Gly Phe Asn Leu Glu Gly Asn Gly Glu Ala Val Asp Phe785 790 795 800Val Ser Thr Tyr Asp Thr Val Asn Phe Ala Asp Gly Asn Ala Thr Thr 805 810 815Ala Lys Val Thr Tyr Asp Asn Lys Thr Ser Lys Val Ala Tyr Asp Val 820 825 830Asn Val Asp Gly Thr Thr Ile His Leu Thr Gly Thr Asn Gly Lys Lys 835 840 845Asn Gln Ile Gly Val Lys Thr Thr Thr Leu Thr Thr Lys Arg Ala Lys 850 855 860Gly Asn Thr Ala Thr Asn Phe Ser Val Asn Ser Gly Asp Asp Asn Ala865 870 875 880Leu Ile Asn Ala Lys Asp Ile Ala Asp Asn Leu Asn Thr Leu Ala Gly 885 890 895Glu Ile Arg Thr Ala Lys Gly Thr Ala Ser Thr Ala Leu Gln Thr Phe 900 905 910Ser Ile Ile Asp Glu Gln Gly Asn Asn Phe Met Val Gly Asn Leu Tyr 915 920 925Ser Gly Tyr Asp Thr Ser Asn Thr Ser Glu Thr Val Thr Phe Val Gly 930 935 940Glu Asn Gly Ile Thr Thr Lys Val Asn Lys Gly Lys Val Lys Val Gly945 950 955 960Ile Asp Gln Thr Lys Gly Leu Thr Thr Pro Lys Leu Thr Val Gly Ser 965 970 975Ser Asn Gly Lys Gly Ile Val Ile Asp Ser Lys Asp Gly Gln Asn Thr 980 985 990Ile Thr Gly Leu Ser Asn Thr Leu Thr Asp Ala Thr Asn Ala Thr Thr 995 1000 1005Gly His Val Ser Glu Ile Gln Gly Leu Ala Gln Gly Ala Asn Lys Thr 1010 1015 1020Arg Ala Ala Ser Ile Gly Asp Val Leu Asn Ala Gly Phe Asn Leu Gln1025 1030 1035 1040Gly Asn Gly Glu Ala Lys Asp Phe Val Ser Thr Tyr Asp Thr Val Asn 1045 1050 1055Phe Ile Asp Gly Asn Ala Thr Thr Ala Lys Val Thr Tyr Asp Asp Thr 1060 1065 1070Lys Gln Thr Ser Thr Val Thr Tyr Asp Val Asn Val Asp Asn Lys Thr 1075 1080 1085Leu Glu Val Thr Gly Asp Lys Lys Leu Gly Val Lys Thr Thr Thr Leu 1090 1095 1100Thr Lys Thr Ser Ala Asn Gly Asn Ala Thr Lys Phe Ser Ala Ala Asp1105 1110 1115 1120Gly Asp Ala Leu Val Lys Ala Ser Asp Ile Ala Thr His Leu Asn Thr 1125 1130 1135Leu Ala Gly Asp Ile Gln Thr Ala Lys Gly Ala Ser Gln Ala Ser Ser 1140 1145 1150Ser Ala Ser Tyr Val Asp Ala Asp Gly Asn Lys Val Ile Tyr Asp Ser 1155 1160 1165Thr Asp Lys Lys Tyr Tyr Gln Ala Lys Asn Asp Gly Thr Val Asp Lys 1170 1175 1180Thr Lys Glu Val Ala Lys Asp Lys Leu Val Ala Gln Ala Gln Thr Pro1185 1190 1195 1200Asp Gly Thr Leu Ala Arg Met Asn Val Lys Ser Val Ile Asn Lys Glu 1205 1210 1215Gln Val Asn Asp Ala Asn Lys Lys Gln Gly Ile Asn Glu Asp Asn Ala 1220 1225 1230Phe Val Lys Gly Leu Glu Lys Ala Ala Ser Asp Asn Lys Thr Lys Asn 1235 1240 1245Ala Ala Val Thr Val Gly Asp Leu Asn Ala Val Ala Gln Thr Pro Leu 1250 1255 1260Thr Phe Ala Gly Asp Thr Gly Thr Thr Ala Lys Lys Leu Gly Glu Thr1265 1270 1275 1280Leu Thr Ile Lys Gly Gly Gln Thr Asp Thr Asn Lys Leu Thr Asp Asn 1285 1290 1295Asn Ile Gly Val Val Ala Gly Thr Asp Gly Phe Thr Val Lys Leu Ala 1300 1305 1310Lys Asp Leu Thr Asn Leu Asn Ser Val Asn Ala Gly Gly Thr Lys Ile 1315 1320 1325Asp Asp Lys Gly Val Ser Phe Val Asp Ala Asn Gly Gln Ala Lys Ala 1330 1335 1340Asn Thr Pro Val Leu Ser Ala Asn Gly Leu Asp Leu Gly Gly Lys Arg1345 1350 1355 1360Ile Ser Asn Ile Gly Ala Ala Val Asp Asp Asn Asp Ala Val Asn Phe 1365 1370 1375Lys Gln Phe Asn Glu Val Ala Lys Thr Val Asn Asn Leu Asn Asn Gln 1380 1385 1390Ser Asn Ser Gly Ala Ser Leu Pro Phe Val Val Thr Asp Ala Asn Gly 1395 1400 1405Lys Pro Ile Asn Gly Thr Asp Asp Lys Pro Gln Lys Ala Ile Lys Gly 1410 1415 1420Ala Asp Gly Lys Tyr Tyr His Ala Asn Ala Asn Gly Val Pro Val Asp1425 1430 1435 1440Lys Asp Gly Asn Pro Ile Thr Asp Ala Asp Lys Leu Ala Asn Leu Ala 1445 1450 1455Ala His Gly Lys Pro Leu Asp Ala Gly His Gln Val Val Ala Ser Leu 1460 1465 1470Gly Gly Asn Ser Asp Ala Ile Thr Leu Thr Asn Ile Lys Ser Thr Leu 1475 1480 1485Pro Gln Ile Asp Thr Pro Asn Thr Gly Asn Ala Asn Ala Gly Gln Ala 1490 1495 1500Gln Ser Leu Pro Ser Leu Ser Ala Ala Gln Gln Ser Asn Ala Ala Ser1505 1510 1515 1520Val Lys Asp Val Leu Asn Val Gly Phe Asn Leu Gln Thr Asn His Asn 1525 1530 1535Gln Val Asp Phe Val Lys Ala Tyr Asp Thr Val Asn Phe Val Asn Gly 1540 1545 1550Thr Gly Ala Asp Ile Thr Ser Val Arg Ser Ala Asp Gly Thr Met Ser 1555 1560 1565Asn Ile Thr Val Asn Thr Ala Leu Ala Ala Thr Asp Asp Asp Gly Asn 1570 1575 1580Val Leu Ile Lys Ala Lys Asp Gly Lys Phe Tyr Lys Ala Asp Asp Leu1585 1590 1595 1600Met Pro Asn Gly Ser Leu Lys Ala Gly Lys Ser Ala Ser Asp Ala Lys 1605 1610 1615Thr Pro Thr Gly Leu Ser Leu Val Asn Pro Asn Ala Gly Lys Gly Ser 1620 1625 1630Thr Gly Asp Ala Val Ala Leu Asn Asn Leu Ser Lys Ala Val Phe Lys 1635 1640 1645Ser Lys Asp Gly Thr Thr Thr Thr Thr Val Ser Ser Asp Gly Ile Ser 1650 1655 1660Ile Gln Gly Lys Asp Asn Ser Asn Ile Thr Leu Ser Lys Asp Gly Leu1665 1670 1675 1680Asn Val Gly Gly Lys Val Ile Ser Asn Val Gly Lys Gly Thr Lys Asp 1685 1690 1695Thr Asp Ala Ala Asn Val Gln Gln Leu Asn Arg Ser Thr Gln Leu Val 1700 1705 1710Gly Ser Trp Val Met Ala Gly Asn Asp Asn Ala Asp Gly Asn Gln Val 1715 1720 1725Asn Ile Ala Asp Ile Lys Lys Asp Pro Asn Ser Gly Ser Ser Ser Asn 1730 1735 1740Arg Thr Val Ile Lys Ala Gly Thr Val Leu Gly Gly Lys Gly Asn Asn1745 1750 1755 1760Asp Thr Glu Lys Leu Ala Thr Gly Gly Val Gln Val Gly Val Asp Lys 1765 1770 1775Asp Gly Asn Ala Asn Gly Asp Leu Ser Asn Val Trp Val Lys Thr Gln 1780 1785 1790Lys Asp Gly Ser Lys Lys Ala Leu Leu Ala Thr Tyr Asn Ala Ala Gly 1795 1800 1805Gln Thr Asn Tyr Leu Thr Asn Asn Pro Ala Glu Ala Ile Asp Arg Ile 1810 1815 1820Asn Glu Gln Gly Ile Arg Phe Phe His Val Asn Asp Gly Asn Gln Glu1825 1830 1835 1840Pro Val Val Gln Gly Arg Asn Gly Ile Asp Ser Ser Ala Ser Gly Lys 1845 1850 1855His Ser Val Ala Val Gly Tyr Lys Ala Lys Ala Asp Gly Glu Ala Ala 1860 1865 1870Val Ala Ile Gly Arg Gln Thr Gln Ala Gly Asn Gln Ser Ile Ala Ile 1875 1880 1885Gly Asp Asn Ala Gln Ala Thr Gly Asp Gln Ser Ile Ala Ile Gly Thr 1890 1895 1900Gly Asn Val Val Ala Gly Lys His Ser Gly Ala Ile Gly Asp Pro Ser1905 1910 1915 1920Thr Val Lys Ala Asp Asn Ser Tyr Ser Val Gly Asn Asn Asn Gln Phe 1925 1930 1935Thr Asp Ala Thr Gln Thr Asp Val Phe Gly Val Gly Asn Asn Ile Thr 1940 1945 1950Val Thr Glu Ser Asn Ser Val Ala Leu Gly Ser Asn Ser Ala Ile Ser 1955 1960 1965Ala Gly Thr His Ala Gly Thr Gln Ala Lys Lys Ser Asp Gly Thr Ala 1970 1975 1980Gly Thr Thr Thr Thr Ala Gly Ala Thr Gly Thr Val Lys Gly Phe Ala1985 1990 1995 2000Gly Gln Thr Ala Val Gly Ala Val Ser Val Gly Ala Ser Gly Ala Glu 2005 2010 2015Arg Arg Ile Gln Asn Val Ala Ala Gly Glu Val Ser Ala Thr Ser Thr 2020 2025 2030Asp Ala Val Asn Gly Ser Gln Leu Tyr Lys Ala Thr Gln Ser Ile Ala 2035 2040 2045Asn Ala Thr Asn Glu Leu Asp His Arg Ile His Gln Asn Glu Asn Lys 2050 2055 2060Ala Asn Ala Gly Ile Ser Ser Ala Met Ala Met Ala Ser Met Pro Gln2065 2070 2075 2080Ala Tyr Ile Pro Gly Arg Ser Met Val Thr Gly Gly Ile Ala Thr His 2085 2090 2095Asn Gly Gln Gly Ala Val Ala Val Gly Leu Ser Lys Leu Ser Asp Asn 2100 2105 2110Gly Gln Trp Val Phe Lys Ile Asn Gly Ser Ala Asp Thr Gln Gly His 2115 2120 2125Val Gly Ala Ala Val Gly Ala Gly Phe His Phe 2130 213526889DNAMoraxella catarrhalisCDS(356)..(6772) 2aatcattacc ccccccttta tgggggatca tatgaataga atattatgat tgtatctgat 60tattgtatca gaatggtgat gcctacgagt tgatttgggt gaatcactct attatttgat 120atgttttgaa actaatctat tgacttaaat caccatatgg ttataattta gcataatggt 180aggctttttg taaaaatcac atcgcaatat tgttctactg ttactaccat gcttgaatga 240cgatcccaat catcagattc attcaagtga tgtgtttgta tacgcatcat ttaccctaat 300tatttcaatc gaaatgccta tgtcagcatg tatcattttt ttaaggtaaa ccacc atg 358 Met 1aat cac atc tat aaa gtc atc ttt aac aaa gcc aca ggc aca ttt atg 406Asn His Ile Tyr Lys Val Ile Phe Asn Lys Ala Thr Gly Thr Phe Met 5 10 15gcc gtg gcg gaa tat gcc aaa tcc cac agc acg ggg ggt agc tgt gct 454Ala Val Ala Glu Tyr Ala Lys Ser His Ser Thr Gly Gly Ser Cys Ala 20 25 30aca ggg caa gtt ggc agt gta tgc act ctg agc ttt gcc cgt gtt gcc 502Thr Gly Gln Val Gly Ser Val Cys Thr Leu Ser Phe Ala Arg Val Ala 35 40 45gcg ctc gct gtc ctc gtg atc ggt gcg acg ctc aat ggc agt gct tat 550Ala Leu Ala Val Leu Val Ile Gly Ala Thr Leu Asn Gly Ser Ala Tyr50 55 60 65gct caa caa gat ccc aga cat atc gca att gat ggc aac agc tcg aac 598Ala Gln Gln Asp Pro Arg His Ile Ala Ile Asp Gly Asn Ser Ser Asn 70 75 80aca tcc tca ggc act gcc cgt gcg aca ggt gat cga gcc att gct ctt 646Thr Ser Ser Gly Thr Ala Arg Ala Thr Gly Asp Arg Ala Ile Ala Leu 85 90 95ggt gaa aat gct aat gca gag ggc ggt caa gcc atc gcc atc ggt agt 694Gly Glu Asn Ala Asn Ala Glu Gly Gly Gln Ala Ile Ala Ile Gly Ser 100 105 110agc aat aaa aca ggt ggt aga aac gcg ctg aat ata ggt acc gat gcc 742Ser Asn Lys Thr Gly Gly Arg Asn Ala Leu Asn Ile Gly Thr Asp Ala 115 120 125aaa ggt gag gag tcc atc gcc atc ggt ggt gat gta gtg gct gag ggt 790Lys Gly Glu Glu Ser Ile Ala Ile Gly Gly Asp Val Val Ala Glu Gly130 135 140 145act gcc tcg att gcc atc ggt ggt gat gac tta cat ttg tgg gat gaa 838Thr Ala Ser Ile

Ala Ile Gly Gly Asp Asp Leu His Leu Trp Asp Glu 150 155 160cca aat aag caa aag ttc ctc gac cca aaa gtt aaa gaa ttg att tta 886Pro Asn Lys Gln Lys Phe Leu Asp Pro Lys Val Lys Glu Leu Ile Leu 165 170 175aaa cat caa gaa tta aac aac ata tac aaa ctg cct gat ggt agt cct 934Lys His Gln Glu Leu Asn Asn Ile Tyr Lys Leu Pro Asp Gly Ser Pro 180 185 190caa aga tat ttt cgc aca tac gca aag gga cac gcc agt att gca cta 982Gln Arg Tyr Phe Arg Thr Tyr Ala Lys Gly His Ala Ser Ile Ala Leu 195 200 205gga acc atg aca cag gca gag ggt cat ttt gcc aac gcc ttt ggt aca 1030Gly Thr Met Thr Gln Ala Glu Gly His Phe Ala Asn Ala Phe Gly Thr210 215 220 225cgg gca aca gct aaa ggc aac tat tcc ttg gca gtg ggt ctt acc gcc 1078Arg Ala Thr Ala Lys Gly Asn Tyr Ser Leu Ala Val Gly Leu Thr Ala 230 235 240caa gcc aac aca gaa tct tca atc gct gtt ggt tct aat gca caa gct 1126Gln Ala Asn Thr Glu Ser Ser Ile Ala Val Gly Ser Asn Ala Gln Ala 245 250 255aac ggg ttt gca gcg aca gcc att ggt gga ggt act aaa gct gat ttg 1174Asn Gly Phe Ala Ala Thr Ala Ile Gly Gly Gly Thr Lys Ala Asp Leu 260 265 270ggt cga agc ata gcc cta ggt ttt ggt tct cag atc ctt act aag gag 1222Gly Arg Ser Ile Ala Leu Gly Phe Gly Ser Gln Ile Leu Thr Lys Glu 275 280 285aag gat agt cat aac aat acc aat gtc tat gta cca caa ggt gaa ata 1270Lys Asp Ser His Asn Asn Thr Asn Val Tyr Val Pro Gln Gly Glu Ile290 295 300 305tta gaa gag cgg tat aaa gcc acc gaa aac ggt cag tcg cct aat aag 1318Leu Glu Glu Arg Tyr Lys Ala Thr Glu Asn Gly Gln Ser Pro Asn Lys 310 315 320gta gtg gat ata ttt tcc att ggt agt agc tct atc aaa cgt aaa atc 1366Val Val Asp Ile Phe Ser Ile Gly Ser Ser Ser Ile Lys Arg Lys Ile 325 330 335atc aat gtc ggt gcg ggt tct cag gag acc gat gcg gtc aat gtg gca 1414Ile Asn Val Gly Ala Gly Ser Gln Glu Thr Asp Ala Val Asn Val Ala 340 345 350cag ctt aaa ttg gtg gag cgg gtg gct aag cgt caa att act ttt cag 1462Gln Leu Lys Leu Val Glu Arg Val Ala Lys Arg Gln Ile Thr Phe Gln 355 360 365ggt gat gat agc aat aat agc gta aaa aaa ggt ttg ggt cag act tta 1510Gly Asp Asp Ser Asn Asn Ser Val Lys Lys Gly Leu Gly Gln Thr Leu370 375 380 385act att aaa ggt ggt aaa aca gag acc ggt gaa cta acc gaa aat aac 1558Thr Ile Lys Gly Gly Lys Thr Glu Thr Gly Glu Leu Thr Glu Asn Asn 390 395 400atc ggt gtg gta caa gat gat aat ggt aat ggt ctg aaa gtt aaa ctt 1606Ile Gly Val Val Gln Asp Asp Asn Gly Asn Gly Leu Lys Val Lys Leu 405 410 415gct aaa gat ctg act ggt ttg acc aag gtt gca gta aat ggt aat ggt 1654Ala Lys Asp Leu Thr Gly Leu Thr Lys Val Ala Val Asn Gly Asn Gly 420 425 430gct aac aac gcc gag cta cta aac ggt ggt ctg acc ttt tcg aca tca 1702Ala Asn Asn Ala Glu Leu Leu Asn Gly Gly Leu Thr Phe Ser Thr Ser 435 440 445ggt gcc aat gca ggc aaa acg gtc tat ggc act gat ggg gtg aag ttt 1750Gly Ala Asn Ala Gly Lys Thr Val Tyr Gly Thr Asp Gly Val Lys Phe450 455 460 465act aat aat aca gga aca gga aca gga acg gca ata ccc gac act act 1798Thr Asn Asn Thr Gly Thr Gly Thr Gly Thr Ala Ile Pro Asp Thr Thr 470 475 480cgt att acc aaa aat aaa att ggc ttt gct ggg gct gat gaa caa gtt 1846Arg Ile Thr Lys Asn Lys Ile Gly Phe Ala Gly Ala Asp Glu Gln Val 485 490 495gat gaa agc aaa cct tat ctt gac aac gaa aag cta aaa gtt ggc aca 1894Asp Glu Ser Lys Pro Tyr Leu Asp Asn Glu Lys Leu Lys Val Gly Thr 500 505 510gtt gag att aaa aaa act ggc atc aat gca ggt aat caa gaa att acc 1942Val Glu Ile Lys Lys Thr Gly Ile Asn Ala Gly Asn Gln Glu Ile Thr 515 520 525aag gtc aaa tct gcc att gtt gat gca gtt aat gga caa gca aat caa 1990Lys Val Lys Ser Ala Ile Val Asp Ala Val Asn Gly Gln Ala Asn Gln530 535 540 545agc ttt gtg aac ctt cta gaa aca gca ggc aca aac acc aac aaa caa 2038Ser Phe Val Asn Leu Leu Glu Thr Ala Gly Thr Asn Thr Asn Lys Gln 550 555 560aac tct gcc gcc acg gtt aaa gac tta tac gac cta tca caa tca ccg 2086Asn Ser Ala Ala Thr Val Lys Asp Leu Tyr Asp Leu Ser Gln Ser Pro 565 570 575ctg acc ttt aca ggt gat agc ggt aac gtt aag aga aaa ctg ggt cag 2134Leu Thr Phe Thr Gly Asp Ser Gly Asn Val Lys Arg Lys Leu Gly Gln 580 585 590act tta acc atc aca ggc gga caa aca aag acc gat caa tta acc gac 2182Thr Leu Thr Ile Thr Gly Gly Gln Thr Lys Thr Asp Gln Leu Thr Asp 595 600 605aat aac atc ggt gtg gta gca ggt act aat ggc tta acc gtt aaa ctt 2230Asn Asn Ile Gly Val Val Ala Gly Thr Asn Gly Leu Thr Val Lys Leu610 615 620 625gct aaa act tta aac agt ctt act gaa gtt aat acg gct aca tta aac 2278Ala Lys Thr Leu Asn Ser Leu Thr Glu Val Asn Thr Ala Thr Leu Asn 630 635 640gcc acc aat aaa gtt aag gta gat aat agt act ggt aat acg gct gaa 2326Ala Thr Asn Lys Val Lys Val Asp Asn Ser Thr Gly Asn Thr Ala Glu 645 650 655tta tta aac aat ggt tta acc ttt acc caa aca aca ggt gca aat tca 2374Leu Leu Asn Asn Gly Leu Thr Phe Thr Gln Thr Thr Gly Ala Asn Ser 660 665 670ggt aaa acc gtc tat ggc aat gat ggc ttg aag ttt act aat agt gct 2422Gly Lys Thr Val Tyr Gly Asn Asp Gly Leu Lys Phe Thr Asn Ser Ala 675 680 685aat aaa gca ctt ggc ggc aca act tac atc acc aaa gat caa gtt ggt 2470Asn Lys Ala Leu Gly Gly Thr Thr Tyr Ile Thr Lys Asp Gln Val Gly690 695 700 705ttt agc aat caa gat ggc tta ctt gat gaa agc aaa ccg tat ctt aac 2518Phe Ser Asn Gln Asp Gly Leu Leu Asp Glu Ser Lys Pro Tyr Leu Asn 710 715 720cga gaa aag cta aaa gtt ggt aaa att gag att aaa gac agt ggc att 2566Arg Glu Lys Leu Lys Val Gly Lys Ile Glu Ile Lys Asp Ser Gly Ile 725 730 735aat gca ggt ggt aaa gcc atc aca gga ctg ccc tca aca ctg ccc aac 2614Asn Ala Gly Gly Lys Ala Ile Thr Gly Leu Pro Ser Thr Leu Pro Asn 740 745 750act acc tat act gca cct ggc gtg cat act gca cta cat ggc agt aca 2662Thr Thr Tyr Thr Ala Pro Gly Val His Thr Ala Leu His Gly Ser Thr 755 760 765att tct aac gac gac aaa acc cgt gcc gcc agt atc gcc gat gtg cta 2710Ile Ser Asn Asp Asp Lys Thr Arg Ala Ala Ser Ile Ala Asp Val Leu770 775 780 785aac gca ggc ttt aac ttg gaa ggt aat ggt gaa gcg gtt gac ttt gtc 2758Asn Ala Gly Phe Asn Leu Glu Gly Asn Gly Glu Ala Val Asp Phe Val 790 795 800tcc act tat gac acc gtc aac ttt gcc gat ggc aat gcc acc acc gct 2806Ser Thr Tyr Asp Thr Val Asn Phe Ala Asp Gly Asn Ala Thr Thr Ala 805 810 815aag gta act tat gat aac aaa acc agt aaa gtg gcg tat gat gtc aat 2854Lys Val Thr Tyr Asp Asn Lys Thr Ser Lys Val Ala Tyr Asp Val Asn 820 825 830gtg gat ggt aca acc att cat cta aca ggc act aat ggc aag aaa aac 2902Val Asp Gly Thr Thr Ile His Leu Thr Gly Thr Asn Gly Lys Lys Asn 835 840 845caa att ggc gta aaa acc acc aca ctg acc aca aaa cgt gct aaa ggt 2950Gln Ile Gly Val Lys Thr Thr Thr Leu Thr Thr Lys Arg Ala Lys Gly850 855 860 865aat aca gca act aat ttt agt gtt aac tct ggt gat gac aat gcc ctt 2998Asn Thr Ala Thr Asn Phe Ser Val Asn Ser Gly Asp Asp Asn Ala Leu 870 875 880att aac gcc aaa gac atc gcc gac aat cta aac acc cta gct ggt gaa 3046Ile Asn Ala Lys Asp Ile Ala Asp Asn Leu Asn Thr Leu Ala Gly Glu 885 890 895att cgc acc gcc aaa ggc aca gca agc acc gcc cta caa acc ttc tct 3094Ile Arg Thr Ala Lys Gly Thr Ala Ser Thr Ala Leu Gln Thr Phe Ser 900 905 910att att gat gaa caa ggt aat aac ttt atg gtc ggt aac ctt tac tct 3142Ile Ile Asp Glu Gln Gly Asn Asn Phe Met Val Gly Asn Leu Tyr Ser 915 920 925ggt tat gac acc tca aat acc tct gag acc gtc acc ttt gta ggt gaa 3190Gly Tyr Asp Thr Ser Asn Thr Ser Glu Thr Val Thr Phe Val Gly Glu930 935 940 945aac ggc att acc acc aag gta aat aaa ggt aaa gtc aaa gtt ggt att 3238Asn Gly Ile Thr Thr Lys Val Asn Lys Gly Lys Val Lys Val Gly Ile 950 955 960gac caa acc aaa ggc tta acc acg cct aag ctg acc gtg ggt agt agt 3286Asp Gln Thr Lys Gly Leu Thr Thr Pro Lys Leu Thr Val Gly Ser Ser 965 970 975aat ggc aaa ggc att gtc att gac agt aaa gat ggt caa aat acc atc 3334Asn Gly Lys Gly Ile Val Ile Asp Ser Lys Asp Gly Gln Asn Thr Ile 980 985 990aca gga cta agc aac act cta acc gat gcc acc aac gca aca aca ggg 3382Thr Gly Leu Ser Asn Thr Leu Thr Asp Ala Thr Asn Ala Thr Thr Gly 995 1000 1005cat gtc agt gaa atc cag ggc ttg gca caa ggt gca aac aaa acc cgt 3430His Val Ser Glu Ile Gln Gly Leu Ala Gln Gly Ala Asn Lys Thr Arg1010 1015 1020 1025gcc gcc agc att ggt gat gta cta aac gca ggc ttt aac ttg caa ggc 3478Ala Ala Ser Ile Gly Asp Val Leu Asn Ala Gly Phe Asn Leu Gln Gly 1030 1035 1040aat ggt gaa gcc aaa gac ttt gtc tcc act tat gac acc gtc aac ttt 3526Asn Gly Glu Ala Lys Asp Phe Val Ser Thr Tyr Asp Thr Val Asn Phe 1045 1050 1055atc gat ggc aat gcc acc acc gct aag gtg acc tat gat gac acg aaa 3574Ile Asp Gly Asn Ala Thr Thr Ala Lys Val Thr Tyr Asp Asp Thr Lys 1060 1065 1070cag acc agc aca gta act tat gat gtc aat gtg gat aat aaa acc ctt 3622Gln Thr Ser Thr Val Thr Tyr Asp Val Asn Val Asp Asn Lys Thr Leu 1075 1080 1085gaa gtg aca ggc gat aaa aaa ctt ggc gtc aaa acc acc aca ctg acc 3670Glu Val Thr Gly Asp Lys Lys Leu Gly Val Lys Thr Thr Thr Leu Thr1090 1095 1100 1105aaa aca agt gct aat ggt aat gca acc aaa ttt agt gcc gcc gat ggc 3718Lys Thr Ser Ala Asn Gly Asn Ala Thr Lys Phe Ser Ala Ala Asp Gly 1110 1115 1120gat gcc ctt gtt aaa gcc agt gat atc gcc acc cat cta aat acc ttg 3766Asp Ala Leu Val Lys Ala Ser Asp Ile Ala Thr His Leu Asn Thr Leu 1125 1130 1135gct ggc gac atc caa acc gcc aaa gga gca agc caa gca agc agc tca 3814Ala Gly Asp Ile Gln Thr Ala Lys Gly Ala Ser Gln Ala Ser Ser Ser 1140 1145 1150gca agc tat gtg gat gct gat ggc aac aag gtc atc tat gac agt acc 3862Ala Ser Tyr Val Asp Ala Asp Gly Asn Lys Val Ile Tyr Asp Ser Thr 1155 1160 1165gat aag aag tac tat caa gcc aaa aat gat ggc aca gtt gat aaa acc 3910Asp Lys Lys Tyr Tyr Gln Ala Lys Asn Asp Gly Thr Val Asp Lys Thr1170 1175 1180 1185aaa gaa gtt gcc aaa gac aaa ctg gtc gcc caa gcc caa acc cca gat 3958Lys Glu Val Ala Lys Asp Lys Leu Val Ala Gln Ala Gln Thr Pro Asp 1190 1195 1200ggc aca ttg gct cga atg aat gtc aaa tca gtc att aac aaa gaa caa 4006Gly Thr Leu Ala Arg Met Asn Val Lys Ser Val Ile Asn Lys Glu Gln 1205 1210 1215gta aat gat gcc aat aaa aag caa ggc atc aac gaa gac aac gcc ttt 4054Val Asn Asp Ala Asn Lys Lys Gln Gly Ile Asn Glu Asp Asn Ala Phe 1220 1225 1230gtt aaa gga ctt gaa aaa gcc gct tct gat aac aaa acc aaa aac gcc 4102Val Lys Gly Leu Glu Lys Ala Ala Ser Asp Asn Lys Thr Lys Asn Ala 1235 1240 1245gca gta act gtg ggt gat tta aat gcc gtt gcc caa aca ccg ctg acc 4150Ala Val Thr Val Gly Asp Leu Asn Ala Val Ala Gln Thr Pro Leu Thr1250 1255 1260 1265ttt gca ggg gat aca ggc aca acg gct aaa aaa ctg ggc gag act ttg 4198Phe Ala Gly Asp Thr Gly Thr Thr Ala Lys Lys Leu Gly Glu Thr Leu 1270 1275 1280acc atc aaa ggt ggg caa aca gac acc aat aag cta acc gat aat aac 4246Thr Ile Lys Gly Gly Gln Thr Asp Thr Asn Lys Leu Thr Asp Asn Asn 1285 1290 1295atc ggt gtg gta gca ggt act gat ggc ttc act gtc aaa ctt gcc aaa 4294Ile Gly Val Val Ala Gly Thr Asp Gly Phe Thr Val Lys Leu Ala Lys 1300 1305 1310gac cta acc aat ctt aac agc gtt aat gca ggt ggc acc aaa att gat 4342Asp Leu Thr Asn Leu Asn Ser Val Asn Ala Gly Gly Thr Lys Ile Asp 1315 1320 1325gac aaa ggc gtg tct ttt gta gac gca aac ggt caa gcc aaa gca aac 4390Asp Lys Gly Val Ser Phe Val Asp Ala Asn Gly Gln Ala Lys Ala Asn1330 1335 1340 1345acc cct gtg cta agt gcc aat ggg ctg gac ctg ggt ggc aaa cgc atc 4438Thr Pro Val Leu Ser Ala Asn Gly Leu Asp Leu Gly Gly Lys Arg Ile 1350 1355 1360agt aac atc ggt gca gct gtt gat gat aac gat gcg gtg aac ttt aag 4486Ser Asn Ile Gly Ala Ala Val Asp Asp Asn Asp Ala Val Asn Phe Lys 1365 1370 1375cag ttt aat gaa gtt gcc aaa acg gtc aac aac cta aac aac caa agt 4534Gln Phe Asn Glu Val Ala Lys Thr Val Asn Asn Leu Asn Asn Gln Ser 1380 1385 1390aac tca ggt gcg tca ttg ccc ttt gta gta acc gat gcc aat ggc aag 4582Asn Ser Gly Ala Ser Leu Pro Phe Val Val Thr Asp Ala Asn Gly Lys 1395 1400 1405ccc atc aat ggc acc gat gac aag ccc caa aaa gcc atc aag ggc gcc 4630Pro Ile Asn Gly Thr Asp Asp Lys Pro Gln Lys Ala Ile Lys Gly Ala1410 1415 1420 1425gat ggt aaa tac tat cac gcc aac gcc aac ggc gta cct gtg gac aaa 4678Asp Gly Lys Tyr Tyr His Ala Asn Ala Asn Gly Val Pro Val Asp Lys 1430 1435 1440gat ggc aac ccc atc acc gat gcg gac aaa ctt gcc aat ctg gca gct 4726Asp Gly Asn Pro Ile Thr Asp Ala Asp Lys Leu Ala Asn Leu Ala Ala 1445 1450 1455cat ggc aaa ccc ctt gat gca ggt cat caa gtg gtg gca agc cta ggc 4774His Gly Lys Pro Leu Asp Ala Gly His Gln Val Val Ala Ser Leu Gly 1460 1465 1470ggc aac tca gat gcc atc acc cta acc aac atc aag tcc act ttg cca 4822Gly Asn Ser Asp Ala Ile Thr Leu Thr Asn Ile Lys Ser Thr Leu Pro 1475 1480 1485caa att gac aca cca aac aca ggt aat gcc aat gca ggg caa gcc caa 4870Gln Ile Asp Thr Pro Asn Thr Gly Asn Ala Asn Ala Gly Gln Ala Gln1490 1495 1500 1505agt ctg ccc agc cta tca gca gca cag caa agt aat gct gcc agt gtc 4918Ser Leu Pro Ser Leu Ser Ala Ala Gln Gln Ser Asn Ala Ala Ser Val 1510 1515 1520aaa gat gtg cta aat gta ggc ttt aac ttg cag acc aat cac aat caa 4966Lys Asp Val Leu Asn Val Gly Phe Asn Leu Gln Thr Asn His Asn Gln 1525 1530 1535gtg gac ttt gtc aaa gcc tat gat acc gtc aac ttt gtc aat ggt aca 5014Val Asp Phe Val Lys Ala Tyr Asp Thr Val Asn Phe Val Asn Gly Thr 1540 1545 1550ggt gcc gac atc aca agc gtg cgt agt gct gat ggc acg atg agt aac 5062Gly Ala Asp Ile Thr Ser Val Arg Ser Ala Asp Gly Thr Met Ser Asn 1555 1560 1565atc acc gtc aac acc gcc tta gca gcg acc gat gat gat ggc aat gtg 5110Ile Thr Val Asn Thr Ala Leu Ala Ala Thr Asp Asp Asp Gly Asn Val1570 1575 1580 1585ctt atc aaa gcc aaa gat ggt aag ttc tac aaa gca gac gac ctc atg 5158Leu Ile Lys Ala Lys Asp Gly Lys Phe Tyr Lys Ala Asp Asp Leu Met 1590 1595 1600cca aac ggc tca cta aaa gca ggc aaa tca gcc agt gat gcc aaa act 5206Pro Asn Gly Ser Leu Lys Ala Gly Lys Ser Ala Ser Asp Ala Lys Thr 1605 1610 1615cca act ggt cta agc ctt gtc aac ccc aat gct ggt aaa ggc agt aca 5254Pro Thr Gly Leu Ser Leu Val Asn Pro Asn Ala Gly Lys Gly Ser Thr 1620 1625 1630ggc gat gca gtg gct ctt aat aac tta tca aaa gcg gta ttt aaa tcc 5302Gly Asp Ala Val Ala Leu Asn Asn Leu Ser Lys Ala Val Phe Lys Ser 1635 1640 1645aaa gat ggt aca act act acc aca gta agc tct gat ggc atc agt atc 5350Lys Asp Gly Thr Thr Thr Thr Thr Val Ser Ser Asp Gly Ile Ser Ile1650 1655 1660 1665caa ggc aaa gat aac agc aac atc acc cta agc aaa gat ggg ctg aat 5398Gln Gly Lys Asp Asn Ser Asn Ile Thr Leu Ser Lys Asp Gly Leu Asn 1670 1675 1680gta ggc ggt aag gtc atc agc aat gtg ggt aaa ggc aca aaa gac acc 5446Val Gly Gly Lys Val

Ile Ser Asn Val Gly Lys Gly Thr Lys Asp Thr 1685 1690 1695gac gct gcc aat gta caa cag tta aac cga agt acg caa ctt gtt ggg 5494Asp Ala Ala Asn Val Gln Gln Leu Asn Arg Ser Thr Gln Leu Val Gly 1700 1705 1710tct tgg gta atg gct ggt aat gat aac gct gac ggc aat cag gta aac 5542Ser Trp Val Met Ala Gly Asn Asp Asn Ala Asp Gly Asn Gln Val Asn 1715 1720 1725att gcc gac atc aaa aaa gac cca aat tca ggt tca tca tct aac cgc 5590Ile Ala Asp Ile Lys Lys Asp Pro Asn Ser Gly Ser Ser Ser Asn Arg1730 1735 1740 1745act gtc atc aaa gca ggc acg gta ctt ggc ggt aaa ggt aat aac gat 5638Thr Val Ile Lys Ala Gly Thr Val Leu Gly Gly Lys Gly Asn Asn Asp 1750 1755 1760acc gaa aaa ctt gcc act ggt ggt gta caa gtg ggc gtg gat aaa gac 5686Thr Glu Lys Leu Ala Thr Gly Gly Val Gln Val Gly Val Asp Lys Asp 1765 1770 1775ggc aac gct aac ggc gat tta agc aat gtt tgg gtc aaa acc caa aaa 5734Gly Asn Ala Asn Gly Asp Leu Ser Asn Val Trp Val Lys Thr Gln Lys 1780 1785 1790gat ggc agc aaa aaa gcc ctg ctc gcc act tat aac gcc gca ggt cag 5782Asp Gly Ser Lys Lys Ala Leu Leu Ala Thr Tyr Asn Ala Ala Gly Gln 1795 1800 1805acc aac tat ttg acc aac aac ccc gca gaa gcc att gac aga ata aat 5830Thr Asn Tyr Leu Thr Asn Asn Pro Ala Glu Ala Ile Asp Arg Ile Asn1810 1815 1820 1825gaa caa ggt atc cgc ttc ttc cat gtc aac gat ggc aat caa gag cct 5878Glu Gln Gly Ile Arg Phe Phe His Val Asn Asp Gly Asn Gln Glu Pro 1830 1835 1840gtg gta caa ggg cgt aac ggc att gac tca agt gcc tca ggc aag cac 5926Val Val Gln Gly Arg Asn Gly Ile Asp Ser Ser Ala Ser Gly Lys His 1845 1850 1855tca gtg gcg gtc ggt tat aag gcc aag gca gat ggt gaa gcc gcc gtt 5974Ser Val Ala Val Gly Tyr Lys Ala Lys Ala Asp Gly Glu Ala Ala Val 1860 1865 1870gcc ata ggc aga caa acc caa gca ggc aac caa tcc atc gcc atc ggt 6022Ala Ile Gly Arg Gln Thr Gln Ala Gly Asn Gln Ser Ile Ala Ile Gly 1875 1880 1885gat aac gca caa gcc aca ggc gat caa tcc atc gcc atc ggt aca ggc 6070Asp Asn Ala Gln Ala Thr Gly Asp Gln Ser Ile Ala Ile Gly Thr Gly1890 1895 1900 1905aat gtg gta gca ggt aag cac tct ggt gcc atc ggc gac cca agc act 6118Asn Val Val Ala Gly Lys His Ser Gly Ala Ile Gly Asp Pro Ser Thr 1910 1915 1920gtt aag gct gat aac agt tac agt gtg ggt aat aac aac cag ttt acc 6166Val Lys Ala Asp Asn Ser Tyr Ser Val Gly Asn Asn Asn Gln Phe Thr 1925 1930 1935gat gcc act cag acc gat gtc ttt ggt gtg ggc aat aac atc acc gtg 6214Asp Ala Thr Gln Thr Asp Val Phe Gly Val Gly Asn Asn Ile Thr Val 1940 1945 1950acc gaa agt aac tcg gtt gcc tta ggt tca aac tct gcc atc agt gca 6262Thr Glu Ser Asn Ser Val Ala Leu Gly Ser Asn Ser Ala Ile Ser Ala 1955 1960 1965ggc aca cac gca ggc aca caa gcc aaa aaa tct gac ggc aca gca ggt 6310Gly Thr His Ala Gly Thr Gln Ala Lys Lys Ser Asp Gly Thr Ala Gly1970 1975 1980 1985aca acc acc aca gca ggt gca aca ggt acg gtt aaa ggc ttt gct gga 6358Thr Thr Thr Thr Ala Gly Ala Thr Gly Thr Val Lys Gly Phe Ala Gly 1990 1995 2000caa acg gcg gtt ggt gcg gtc tcc gtg ggt gcc tca ggt gct gaa cgc 6406Gln Thr Ala Val Gly Ala Val Ser Val Gly Ala Ser Gly Ala Glu Arg 2005 2010 2015cgt atc caa aat gtg gca gca ggt gag gtc agt gcc acc agc acc gat 6454Arg Ile Gln Asn Val Ala Ala Gly Glu Val Ser Ala Thr Ser Thr Asp 2020 2025 2030gcg gtc aat ggt agc cag ttg tac aaa gcc acc caa agc att gcc aac 6502Ala Val Asn Gly Ser Gln Leu Tyr Lys Ala Thr Gln Ser Ile Ala Asn 2035 2040 2045gca acc aat gag ctt gac cat cgt atc cac caa aac gaa aat aaa gcc 6550Ala Thr Asn Glu Leu Asp His Arg Ile His Gln Asn Glu Asn Lys Ala2050 2055 2060 2065aat gca ggg att tca tca gcg atg gcg atg gcg tcc atg cca caa gcc 6598Asn Ala Gly Ile Ser Ser Ala Met Ala Met Ala Ser Met Pro Gln Ala 2070 2075 2080tac att cct ggc aga tcc atg gtt acc ggg ggt att gcc acc cac aac 6646Tyr Ile Pro Gly Arg Ser Met Val Thr Gly Gly Ile Ala Thr His Asn 2085 2090 2095ggt caa ggt gcg gtg gca gtg gga ctg tcg aag ctg tcg gat aat ggt 6694Gly Gln Gly Ala Val Ala Val Gly Leu Ser Lys Leu Ser Asp Asn Gly 2100 2105 2110caa tgg gta ttt aaa atc aat ggt tca gcc gat acc caa ggc cat gta 6742Gln Trp Val Phe Lys Ile Asn Gly Ser Ala Asp Thr Gln Gly His Val 2115 2120 2125ggg gca gca gtt ggt gca ggt ttt cac ttt taagccataa atcgcaagat 6792Gly Ala Ala Val Gly Ala Gly Phe His Phe2130 2135tttacttaaa aatcaatctc accatagttg tataaaacag catcagcatc agtcatatta 6852ctgatgcttg atggttttta ttacttaaac catttta 6889330DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 3cgggatccga tggccgtggc ggaatatgcc 30438DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 4cgcggatccg aaaagtgaaa acctgcacca actgctgc 38538DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 5tgtcagcatg tatcattttt ttaaggtaaa ccaccatg 38628DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 6catcaattgc gatatgtctg ggatcttg 28725DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 7cttcacccca tcagtgccat agacc 258150PRTMoraxella catarrhalis 8Ala Leu His Gly Ser Thr Ile Ser Asn Asp Asp Lys Thr Arg Ala Ala1 5 10 15Ser Ile Ala Asp Val Leu Asn Ala Gly Phe Asn Leu Glu Gly Asn Gly 20 25 30Glu Ala Val Asp Phe Val Ser Thr Tyr Asp Thr Val Asn Phe Ala Asp 35 40 45Gly Asn Ala Thr Thr Ala Lys Val Thr Tyr Asp Asn Lys Thr Ser Lys 50 55 60Val Ala Tyr Asp Val Asn Val Asp Gly Thr Thr Ile His Leu Thr Gly65 70 75 80Thr Asn Gly Lys Lys Asn Gln Ile Gly Val Lys Thr Thr Thr Leu Thr 85 90 95Thr Lys Arg Ala Lys Gly Asn Thr Ala Thr Asn Phe Ser Val Asn Ser 100 105 110Gly Asp Asp Asn Ala Leu Ile Asn Ala Lys Asp Ile Ala Asp Asn Leu 115 120 125Asn Thr Leu Ala Gly Glu Ile Arg Thr Ala Lys Gly Thr Ala Ser Thr 130 135 140Ala Leu Gln Thr Phe Ser145 1509450DNAMoraxella catarrhalis 9gcactacatg gcagtacaat ttctaacgac gacaaaaccc gtgccgccag tatcgccgat 60gtgctaaacg caggctttaa cttggaaggt aatggtgaag cggttgactt tgtctccact 120tatgacaccg tcaactttgc cgatggcaat gccaccaccg ctaaggtaac ttatgataac 180aaaaccagta aagtggcgta tgatgtcaat gtggatggta caaccattca tctaacaggc 240actaatggca agaaaaacca aattggcgta aaaaccacca cactgaccac aaaacgtgct 300aaaggtaata cagcaactaa ttttagtgtt aactctggtg atgacaatgc ccttattaac 360gccaaagaca tcgccgacaa tctaaacacc ctagctggtg aaattcgcac cgccaaaggc 420acagcaagca ccgccctaca aaccttctct 45010239PRTMoraxella catarrhalis 10Asp Gln Thr Lys Gly Leu Thr Thr Pro Lys Leu Thr Val Gly Ser Ser1 5 10 15Asn Gly Lys Gly Ile Val Ile Asp Ser Lys Asp Gly Gln Asn Thr Ile 20 25 30Thr Gly Leu Ser Asn Thr Leu Thr Asp Ala Thr Asn Ala Thr Thr Gly 35 40 45His Val Ser Glu Ile Gln Gly Leu Ala Gln Gly Ala Asn Lys Thr Arg 50 55 60Ala Ala Ser Ile Gly Asp Val Leu Asn Ala Gly Phe Asn Leu Gln Gly65 70 75 80Asn Gly Glu Ala Lys Asp Phe Val Ser Thr Tyr Asp Thr Val Asn Phe 85 90 95Ile Asp Gly Asn Ala Thr Thr Ala Lys Val Thr Tyr Asp Asp Thr Lys 100 105 110Gln Thr Ser Thr Val Thr Tyr Asp Val Asn Val Asp Asn Lys Thr Leu 115 120 125Glu Val Thr Gly Asp Lys Lys Leu Gly Val Lys Thr Thr Thr Leu Thr 130 135 140Lys Thr Ser Ala Asn Gly Asn Ala Thr Lys Phe Ser Ala Ala Asp Gly145 150 155 160Asp Ala Leu Val Lys Ala Ser Asp Ile Ala Thr His Leu Asn Thr Leu 165 170 175Ala Gly Asp Ile Gln Thr Ala Lys Gly Ala Ser Gln Ala Ser Ser Ser 180 185 190Ala Ser Tyr Val Asp Ala Asp Gly Asn Lys Val Ile Tyr Asp Ser Thr 195 200 205Asp Lys Lys Tyr Tyr Gln Ala Lys Asn Asp Gly Thr Val Asp Lys Thr 210 215 220Lys Glu Val Ala Lys Asp Lys Leu Val Ala Gln Ala Gln Thr Pro225 230 23511717DNAMoraxella catarrhalis 11gaccaaacca aaggcttaac cacgcctaag ctgaccgtgg gtagtagtaa tggcaaaggc 60attgtcattg acagtaaaga tggtcaaaat accatcacag gactaagcaa cactctaacc 120gatgccacca acgcaacaac agggcatgtc agtgaaatcc agggcttggc acaaggtgca 180aacaaaaccc gtgccgccag cattggtgat gtactaaacg caggctttaa cttgcaaggc 240aatggtgaag ccaaagactt tgtctccact tatgacaccg tcaactttat cgatggcaat 300gccaccaccg ctaaggtgac ctatgatgac acgaaacaga ccagcacagt aacttatgat 360gtcaatgtgg ataataaaac ccttgaagtg acaggcgata aaaaacttgg cgtcaaaacc 420accacactga ccaaaacaag tgctaatggt aatgcaacca aatttagtgc cgccgatggc 480gatgcccttg ttaaagccag tgatatcgcc acccatctaa ataccttggc tggcgacatc 540caaaccgcca aaggagcaag ccaagcaagc agctcagcaa gctatgtgga tgctgatggc 600aacaaggtca tctatgacag taccgataag aagtactatc aagccaaaaa tgatggcaca 660gttgataaaa ccaaagaagt tgccaaagac aaactggtcg cccaagccca aacccca 7171213PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 12Thr Ala Gln Ala Asn Thr Glu Ser Ser Ile Ala Val Gly1 5 101319PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 13Gly Asn Thr Ala Thr Asn Phe Ser Val Asn Ser Gly Asp Asp Asn Ala1 5 10 15Leu Ile Asn1415PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 14Gln Gly Ile Asn Glu Asp Asn Ala Phe Val Lys Gly Leu Glu Lys1 5 10 15158PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 15Pro Ser Thr Val Lys Ala Asp Asn1 5161833PRTMoraxella catarrhalis 16Met Ser Tyr Ala Gln Gly His Phe Ser Asn Ala Phe Gly Thr Arg Ala1 5 10 15Thr Ala Lys Ser Ala Tyr Ser Leu Ala Val Gly Leu Ala Ala Thr Ala 20 25 30Glu Gly Gln Ser Thr Ile Ala Ile Gly Ser Asp Ala Thr Ser Ser Ser 35 40 45Leu Gly Ala Ile Ala Leu Gly Ala Gly Thr Arg Ala Gln Leu Gln Gly 50 55 60Ser Ile Ala Leu Gly Gln Gly Ser Val Val Thr Gln Ser Asp Asn Asn65 70 75 80Ser Arg Pro Ala Tyr Thr Pro Asn Thr Gln Ala Leu Asp Pro Lys Phe 85 90 95Gln Ala Thr Asn Asn Thr Lys Ala Gly Pro Leu Ser Ile Gly Ser Asn 100 105 110Ser Ile Lys Arg Lys Ile Ile Asn Val Gly Ala Gly Val Asn Lys Thr 115 120 125Asp Ala Val Asn Val Ala Gln Leu Glu Ala Val Val Lys Trp Ala Lys 130 135 140Glu Arg Arg Ile Thr Phe Gln Gly Asp Asp Asn Ser Thr Asp Val Lys145 150 155 160Ile Gly Leu Asp Asn Thr Leu Thr Ile Lys Gly Gly Ala Glu Thr Asn 165 170 175Ala Leu Thr Asp Asn Asn Ile Gly Val Val Lys Glu Ala Asp Asn Ser 180 185 190Gly Leu Lys Val Lys Leu Ala Lys Thr Leu Asn Asn Leu Thr Glu Val 195 200 205Asn Thr Thr Thr Leu Asn Ala Thr Thr Thr Val Lys Val Gly Ser Ser 210 215 220Ser Ser Thr Thr Ala Glu Leu Leu Ser Asp Ser Leu Thr Phe Thr Gln225 230 235 240Pro Asn Thr Gly Ser Gln Ser Thr Ser Lys Thr Val Tyr Gly Val Asn 245 250 255Gly Val Lys Phe Thr Asn Asn Ala Glu Thr Thr Ala Ala Ile Gly Thr 260 265 270Thr Arg Ile Thr Arg Asp Lys Ile Gly Phe Ala Arg Asp Gly Asp Val 275 280 285Asp Glu Lys Gln Ala Pro Tyr Leu Asp Lys Lys Gln Leu Lys Val Gly 290 295 300Ser Val Ala Ile Thr Ile Asp Asn Gly Ile Asp Ala Gly Asn Lys Lys305 310 315 320Ile Ser Asn Leu Ala Lys Gly Ser Ser Ala Asn Asp Ala Val Thr Ile 325 330 335Glu Gln Leu Lys Ala Ala Lys Pro Thr Leu Asn Ala Gly Ala Gly Ile 340 345 350Ser Val Thr Pro Thr Glu Ile Ser Val Asp Ala Lys Ser Gly Asn Val 355 360 365Thr Ala Pro Thr Tyr Asn Ile Gly Val Lys Thr Thr Glu Leu Asn Ser 370 375 380Asp Gly Thr Ser Asp Lys Phe Ser Val Lys Gly Ser Gly Thr Asn Asn385 390 395 400Ser Leu Val Thr Ala Glu His Leu Ala Ser Tyr Leu Asn Glu Val Asn 405 410 415Arg Thr Ala Asp Ser Ala Leu Gln Ser Phe Thr Val Lys Glu Glu Asp 420 425 430Asp Asp Asp Ala Asn Ala Ile Thr Val Ala Lys Asp Thr Thr Lys Asn 435 440 445Ala Gly Ala Val Ser Ile Leu Lys Leu Lys Gly Lys Asn Gly Leu Thr 450 455 460Val Ala Thr Lys Lys Asp Gly Thr Val Thr Phe Gly Leu Ser Gln Asp465 470 475 480Ser Gly Leu Thr Ile Gly Lys Ser Thr Leu Asn Asn Asp Gly Leu Thr 485 490 495Val Lys Asp Thr Asn Glu Gln Ile Gln Val Gly Ala Asn Gly Ile Lys 500 505 510Phe Thr Asn Val Asn Gly Ser Asn Pro Gly Thr Gly Ile Ala Asn Thr 515 520 525Ala Arg Ile Thr Arg Asp Lys Ile Gly Phe Ala Gly Ser Asp Gly Ala 530 535 540Val Asp Thr Asn Lys Pro Tyr Leu Asp Gln Asp Lys Leu Gln Val Gly545 550 555 560Asn Val Lys Ile Thr Asn Thr Gly Ile Asn Ala Gly Gly Lys Ala Ile 565 570 575Thr Gly Leu Ser Pro Thr Leu Pro Ser Ile Ala Asp Gln Ser Ser Arg 580 585 590Asn Ile Glu Leu Gly Asn Thr Ile Gln Asp Lys Asp Lys Ser Asn Ala 595 600 605Ala Ser Ile Asn Asp Ile Leu Asn Thr Gly Phe Asn Leu Lys Asn Asn 610 615 620Asn Asn Pro Ile Asp Phe Val Ser Thr Tyr Asp Ile Val Asp Phe Ala625 630 635 640Asn Gly Asn Ala Thr Thr Ala Thr Val Thr His Asp Thr Ala Asn Lys 645 650 655Thr Ser Lys Val Val Tyr Asp Val Asn Val Asp Asp Thr Thr Ile His 660 665 670Leu Thr Gly Thr Asp Asp Asn Lys Lys Leu Gly Val Lys Thr Thr Lys 675 680 685Leu Asn Lys Thr Ser Ala Asn Gly Asn Thr Ala Thr Asn Phe Asn Val 690 695 700Asn Ser Ser Asp Glu Asp Ala Leu Val Asn Ala Lys Asp Ile Ala Glu705 710 715 720Asn Leu Asn Thr Leu Ala Lys Glu Ile His Thr Thr Lys Gly Thr Ala 725 730 735Asp Thr Ala Leu Gln Thr Phe Thr Val Lys Lys Val Asp Glu Asn Asn 740 745 750Asn Ala Asp Asp Ala Asn Ala Ile Thr Val Gly Gln Lys Asn Ala Asn 755 760 765Asn Gln Val Asn Thr Leu Thr Leu Lys Gly Glu Asn Gly Leu Asn Ile 770 775 780Lys Thr Asp Lys Asn Gly Thr Val Thr Phe Gly Ile Asn Thr Thr Ser785 790 795 800Gly Leu Lys Ala Gly Lys Ser Thr Leu Asn Asp Gly Gly Leu Ser Ile 805 810 815Lys Asn Pro Thr Gly Ser Glu Gln Ile Gln Val Gly Ala Asp Gly Val 820 825 830Lys Phe Ala Lys Val Asn Asn Asn Gly Val Val Gly Ala Gly Ile Asp 835 840 845Gly Thr Thr Arg Ile Thr Arg Asp Glu Ile Gly Phe Thr Gly Thr Asn 850 855 860Gly Ser Leu Asp Lys Ser Lys Pro His Leu Ser Lys Asp Gly Ile Asn865 870 875 880Ala Gly Gly Lys Lys Ile Thr Asn Ile Gln Ser Gly Glu Ile Ala Gln 885 890 895Asn Ser His Asp Ala Val Thr Gly Gly Lys Ile Tyr Asp Leu Lys Thr

900 905 910Glu Leu Glu Asn Lys Ile Ser Ser Thr Ala Lys Thr Ala Gln Asn Ser 915 920 925Leu His Glu Phe Ser Val Ala Asp Glu Gln Gly Asn Asn Phe Thr Val 930 935 940Ser Asn Pro Tyr Ser Ser Tyr Asp Thr Ser Lys Thr Ser Asp Val Ile945 950 955 960Thr Phe Ala Gly Glu Asn Gly Ile Thr Thr Lys Val Asn Lys Gly Val 965 970 975Val Arg Val Gly Ile Asp Gln Thr Lys Gly Leu Thr Thr Pro Lys Leu 980 985 990Thr Val Gly Asn Asn Asn Gly Lys Gly Ile Val Ile Asp Ser Gln Asn 995 1000 1005Gly Gln Asn Thr Ile Thr Gly Leu Ser Asn Thr Leu Ala Asn Val Thr 1010 1015 1020Asn Asp Lys Gly Ser Val Arg Thr Thr Glu Gln Gly Asn Ile Ile Lys1025 1030 1035 1040Asp Glu Asp Lys Thr Arg Ala Ala Ser Ile Val Asp Val Leu Ser Ala 1045 1050 1055Gly Phe Asn Leu Gln Gly Asn Gly Glu Ala Val Asp Phe Val Ser Thr 1060 1065 1070Tyr Asp Thr Val Asn Phe Ala Asp Gly Asn Ala Thr Thr Ala Lys Val 1075 1080 1085Thr Tyr Asp Asp Thr Ser Lys Thr Ser Lys Val Val Tyr Asp Val Asn 1090 1095 1100Val Asp Asp Thr Thr Ile Glu Val Lys Asp Lys Lys Leu Gly Val Lys1105 1110 1115 1120Thr Thr Thr Leu Thr Ser Thr Gly Thr Gly Ala Asn Lys Phe Ala Leu 1125 1130 1135Ser Asn Gln Ala Thr Gly Asp Ala Leu Val Lys Ala Ser Asp Ile Val 1140 1145 1150Ala His Leu Asn Thr Leu Ser Gly Asp Ile Gln Thr Ala Lys Gly Ala 1155 1160 1165Ser Gln Ala Asn Asn Ser Ala Gly Tyr Val Asp Ala Asp Gly Asn Lys 1170 1175 1180Val Ile Tyr Asp Ser Thr Asp Asn Lys Tyr Tyr Gln Ala Lys Asn Asp1185 1190 1195 1200Gly Thr Val Asp Lys Thr Lys Glu Val Ala Lys Asp Lys Leu Val Ala 1205 1210 1215Gln Ala Gln Thr Pro Asp Gly Thr Leu Ala Gln Met Asn Val Lys Ser 1220 1225 1230Val Ile Asn Lys Glu Gln Val Asn Asp Ala Asn Lys Lys Gln Gly Ile 1235 1240 1245Asn Glu Asp Asn Ala Phe Val Lys Gly Leu Glu Lys Ala Ala Ser Asp 1250 1255 1260Asn Lys Thr Lys Asn Ala Ala Val Thr Val Gly Asp Leu Asn Ala Val1265 1270 1275 1280Ala Gln Thr Pro Leu Thr Phe Ala Gly Asp Thr Gly Thr Thr Ala Lys 1285 1290 1295Lys Leu Gly Glu Thr Leu Thr Ile Lys Gly Gly Gln Thr Asp Thr Asn 1300 1305 1310Lys Leu Thr Asp Asn Asn Ile Gly Val Val Ala Gly Thr Asp Gly Phe 1315 1320 1325Thr Val Lys Leu Ala Lys Asp Leu Thr Asn Leu Asn Ser Val Asn Ala 1330 1335 1340Gly Gly Thr Lys Ile Asp Asp Lys Gly Val Ser Phe Val Asp Ser Ser1345 1350 1355 1360Gly Gln Ala Lys Ala Asn Thr Pro Val Leu Ser Ala Asn Gly Leu Asp 1365 1370 1375Leu Gly Gly Lys Val Ile Ser Asn Val Gly Lys Gly Thr Lys Asp Thr 1380 1385 1390Asp Ala Ala Asn Val Gln Gln Leu Asn Glu Val Arg Asn Leu Leu Gly 1395 1400 1405Leu Gly Asn Ala Gly Asn Asp Asn Ala Asp Gly Asn Gln Val Asn Ile 1410 1415 1420Ala Asp Ile Lys Lys Asp Pro Asn Ser Gly Ser Ser Ser Asn Arg Thr1425 1430 1435 1440Val Ile Lys Ala Gly Thr Val Leu Gly Gly Lys Gly Asn Asn Asp Thr 1445 1450 1455Glu Lys Leu Ala Thr Gly Gly Ile Gln Val Gly Val Asp Lys Asp Gly 1460 1465 1470Asn Ala Asn Gly Asp Leu Ser Asn Val Trp Val Lys Thr Gln Lys Asp 1475 1480 1485Gly Ser Lys Lys Ala Leu Leu Ala Thr Tyr Asn Ala Ala Gly Gln Thr 1490 1495 1500Asn Tyr Leu Thr Asn Asn Pro Ala Glu Ala Ile Asp Arg Ile Asn Glu1505 1510 1515 1520Gln Gly Ile Arg Phe Phe His Val Asn Asp Gly Asn Gln Glu Pro Val 1525 1530 1535Val Gln Gly Arg Asn Gly Ile Asp Ser Ser Ala Ser Gly Lys His Ser 1540 1545 1550Val Ala Ile Gly Phe Gln Ala Lys Ala Asp Gly Glu Ala Ala Val Ala 1555 1560 1565Ile Gly Arg Gln Thr Gln Ala Gly Asn Gln Ser Ile Ala Ile Gly Asp 1570 1575 1580Asn Ala Gln Ala Thr Gly Asp Gln Ser Ile Ala Ile Gly Thr Gly Asn1585 1590 1595 1600Val Val Ala Gly Lys His Ser Gly Ala Ile Gly Asp Pro Ser Thr Val 1605 1610 1615Lys Ala Asp Asn Ser Tyr Ser Val Gly Asn Asn Asn Gln Phe Thr Asp 1620 1625 1630Ala Thr Gln Thr Asp Val Phe Gly Val Gly Asn Asn Ile Thr Val Thr 1635 1640 1645Glu Ser Asn Ser Val Ala Leu Gly Ser Asn Ser Ala Ile Ser Ala Gly 1650 1655 1660Thr His Ala Gly Thr Gln Ala Lys Lys Ser Asp Gly Thr Ala Gly Thr1665 1670 1675 1680Thr Thr Thr Ala Gly Ala Thr Gly Thr Val Lys Gly Phe Ala Gly Gln 1685 1690 1695Thr Ala Val Gly Ala Val Ser Val Gly Ala Ser Gly Ala Glu Arg Arg 1700 1705 1710Ile Gln Asn Val Ala Ala Gly Glu Val Ser Ala Thr Ser Thr Asp Ala 1715 1720 1725Val Asn Gly Ser Gln Leu Tyr Lys Ala Thr Gln Ser Ile Ala Asn Ala 1730 1735 1740Thr Asn Glu Leu Asp His Arg Ile His Gln Asn Glu Asn Lys Ala Asn1745 1750 1755 1760Ala Gly Ile Ser Ser Ala Met Ala Met Ala Ser Met Pro Gln Ala Tyr 1765 1770 1775Ile Pro Gly Arg Ser Met Val Thr Gly Gly Ile Ala Thr His Asn Gly 1780 1785 1790Gln Gly Ala Val Ala Val Gly Leu Ser Lys Leu Ser Asp Asn Gly Gln 1795 1800 1805Trp Val Phe Lys Ile Asn Gly Ser Ala Asp Thr Gln Gly His Val Gly 1810 1815 1820Ala Ala Val Gly Ala Gly Phe His Phe1825 1830

Claims

1. A vaccine comprising the amino acid sequence of SEQ ID NO:8,

2. An isolated fusion product comprising an amino acid sequence comprising SEQ ID NO:8 bound to a protein, carbohydrate, or matrix.

3. A method of detecting IgD using an amino acid sequence comprising SEQ ID NO:8, optionally labeled and/or bound to a matrix.

4. A method of detecting IgD using an amino acid sequence comprising SEQ ID NO:1, optionally labeled and/or bound to a matrix.

5. A method of detecting IgD using an isolated IgD-binding fragment of a surface exposed protein, optionally labeled and/or bound to a matrix, wherein: said surface exposed protein comprises the amino acid sequence of SEQ ID NO:1, or a naturally-occurring or artificially-modified varient thereof; and said isolated IgD-binding fragment selectively binds membrane bound or soluble IgD.

6. The method according to claim 5, wherein said isolated IgD-binding fragment comprises the amino acid sequence of SEQ ID NO:10.

7. The method according to claim 5, wherein said isolated IgD-binding fragment further selectively binds erythrocytes and epithelial cells.