High molecular weight major outer membrane protein of moraxella

This application is a United States National Phase filing pursuant to 35 USC 371 of International Application PCT/CA96/00264 filed Apr. 29, 1996, which is a continuation-in-part of U.S. PAt. application No. 08/621,944 filed Mar. 20, 1996, which itself is a continuation-in-part of U.S. patent application Ser. No. 08/478,370, filed Jun. 7, 1995, (now U.S. Pat. No. 5,808,024) which itself is a continuation-in-part of U.S. patent application Ser. No. 08/431,718 filed May 1, 1995 now U.S. Pat. No. 6,335,018.

The present invention relates to the field of immunology and is particularly concerned with outer membrane proteins from Moraxella, methods of production thereof, genes encoding such proteins and uses thereof.

Otitis media is the most common illness of early childhood with approximately 70% of all children suffering at least one bout of otitis media before the age of seven. Chronic otitis media can lead to hearing, speech and cognitive impairment in children. It is caused by bacterial infection with Streptococcus pneumoniae (approximately 50%), non-typable Haemophilus influenzae (approximately 30%) and Moraxella ( Branhamella ) catarrhalis (approximately 20%). In the United States alone, treatment of otitis media costs between one and two billion dollars per year for antibiotics and surgical procedures, such as tonsillectomies, adenoidectomies and insertion of tympanostomy tubes. Because otitis media occurs at a time in life when language skills are developing at a rapid pace, developmental disabilities specifically related to learning and auditory perception have been documented in youngsters with frequent otitis media.

M. catarrhalis mainly colonizes the respiratory tract and is predominantly a mucosal pathogen. Studies using cultures of middle ear fluid obtained by tympanocentesis have shown that M. catarrhalis causes approximately 20% of cases of otitis media (ref. 1—Throughout this application, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure).

The incidence of otitis media caused by M. catarrhalis is increasing. As ways of preventing otitis media caused by pneumococcus and non-typable H. influenzae are developed, the relative importance of M. catarrhalis as a cause of otitis media can be expected to further increase.

M. catarrhalis is also an important cause of lower respiratory tract infections in adults, particularly in the setting of chronic bronchitis and emphysema (refs. 2, 3, 4, 5, 6, 7, and 8). M. catarrhalis also causes sinusitis in children and adults (refs. 9, 10, 11, 12, and 13) and occasionally causes invasive disease (refs. 14, 15, 16, 17, 18, and 19).

Like other Gram-negative bacteria, the outer membrane of M. catarrhalis consists of phospholipids, lipopolysaccharide (LPS), and outer membrane proteins (OMPs). Eight of the M. catarrhalis OMPs have been identified as major components. These are designated by letters A to H, beginning with OMP A which has a molecular mass of 98 kDa to OMP H which has a molecular mass of 21 kDa (ref. 20).

Recently, a high-molecular-weight outer membrane protein of M. catarrhalis was purified and characterized (ref. 21). The apparent molecular mass of this protein varies from 350 kDa to 720 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This protein appears to be an oligomer of much smaller proteins or subunits thereof of molecular mass 120 to 140 kDa and is antigenically conserved among strains of Moraxella.

A protein molecular mass of about 300 to 400 kDa named UspA was also reported to be present on the surface of Moraxella (ref. 22).

M. catarrhalis infection may lead to serious disease. It would be advantageous to provide other outer membrane proteins for M. catarrhalis and genes encoding such proteins for use as antigens in immunogenic preparations including vaccines, carriers for other antigens and immunogens and the generation of diagnostic reagents.

The present invention is directed towards the provision of a purified and isolated major outer membrane protein of Moraxella catarrhalis and other Moraxella strains, having an apparent molecular mass of about 200 kDa, as well as genes encoding the same.

In accordance with one aspect of the invention, there is provided an isolated and purified, outer membrane protein of a Moraxella strain having a molecular weight of about 200 kDa, as determined by SDS-PAGE, or a fragment or an analog thereof. The outer membrane protein may be substantially in its native conformation (so as to have substantially retained the characteristic immunogenicity of the outer membrane protein in the Moraxella strain) and may be isolated from a M. catarrhalis strain, such as from M. catarrhalis 4223. Such isolated and purified about 200 kDa outer membrane protein is substantially free from non-200 kDa outer membrane proteins, phospholipids and lipopolysaccharide of Moraxella. The about 200 kDa outer membrane protein is at least about 70 wt % pure, preferably at least about 90 wt % pure, and may be in the form of an aqueous solution thereof. Such about 200 kDa outer membrane protein may have substantially the amino acid composition shown in Table III and a deduced amino acid sequence as shown in FIG. 6 (SEQ ID No: 3).

The present invention also provides a purified and isolated nucleic acid molecule encoding an outer membrane protein of a strain of Moraxella having a molecular mass of about 200 kDa, as determined by SDS-PAGE, or a fragment or an analog of the outer membrane protein. The protein encoded by the nucleic acid molecule may comprise a protein containing the amino acid sequence NH 2 -Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-x-Gln-Gly-Ile (SEQ ID No: 5) particularly where X is Lys (SEQ ID No: 18), for Moraxella catarrhalis strain 4223 or containing the corresponding amino acid sequence from other Moraxella strains.

In a further aspect of the present invention, there is provided a purified and isolated nucleic acid molecule having a sequence selected from the group consisting of (a) a DNA sequence as set out in FIG. 6 (SEQ ID Nos: 1 or 2), or the complementary sequence thereto; (b) a DNA sequence encoding an about 200 kDa protein of a strain of Moraxella and containing the amino acid sequence NH 2 -Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-x-Gln-Gly-Ile (SEQ ID No: 5), particularly where x is Lys (SEQ ID No: 18) or the complementary sequence thereto; (c) a DNA sequence encoding the deduced amino acid sequence as set out in FIG. 6 (SEQ ID No: 3) or the complementary sequence thereto; and (d) a nucleotide sequence which hybridizes under stringent conditions to any one of the sequences defined in (a), (b) or (c). The nucleic acid preferably defined in (d) has at least about 90% sequence identity with any one of the sequences defined in (a), (b) or (c).

The nucleic acid molecules provided herein may be included in a vector adapted for transformation of a host. The nucleic acid molecules provided herein also may be included in an expression vector adapted for transformation of a host along with expression means operatively coupled to the nucleic acid molecule for expression by the host of the about 200 kDa outer membrane protein of a strain of Moraxella or the fragment or the analog of the outer membrane protein. A transformed host containing the expression vector is included within the invention, along with a recombinant outer membrane protein or fragment or analog thereof producible by the transformed host.

The expression means may include a nucleic acid portion encoding a leader sequence for secretion from the host of the outer membrane protein or the fragment or the analog of the outer membrane protein. The expression means may include a nucleic acid portion encoding a lipidation signal for expression from the host of a lipidated form of the outer membrane protein or the fragment or analog thereof.

The present invention further includes a live vector for delivery of the outer membrane protein of the invention or a fragment or analog thereof, comprising a vector containing the nucleic acid molecule provided herein. The live vector may be selected from the group consisting of E. coli , Salmonella, BCG, adenovirus, poxvirus, vaccinia and poliovirus.

In accordance with a further aspect of the present invention, there is provided a peptide having no less than six amino acids and no more than 150 amino acids and containing an amino acid sequence corresponding to a portion only of the outer membrane protein of the invention, or a fragment or analog thereof. The peptide may be one having the amino acid sequence NH 2 -Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-Lys-Gln-Gly-Ile (SEQ ID No: 18) for the Moraxella catarrhalis 4223 strain or the amino acid sequence for the corresponding peptide for other strains of Moraxella.

The present invention also provides an immunogenic composition comprising an immunoeffective amount of an active component, which may be the outer membrane protein or fragment or analog thereof, nucleic acid molecules, recombinant outer membrane proteins, fragments or analogs thereof, live vectors, and/or peptides, as provided herein, along with a pharmaceutically acceptable carrier therefor with the active component producing an immune response when administered to a host, which may be a primate, particularly a human.

The immunogenic composition may be formulated as a vaccine for in vivo administration to a host to confer protection against diseases caused by a bacterial pathogen that produces the about 200 kDa outer membrane protein or produces a protein capable of inducing antibodies in the host specifically reactive with the about 200 kDa outer membrane protein. In particular, the bacterial pathogen is a strain of Moraxella, particularly M. catarrhalis.

The immunogenic composition may be formulated as a microparticle capsule, ISCOM or liposome preparation. The immunogenic composition may be used in combination with a targeting molecule for delivery to specific cells of the immune system as to mucosal surfaces. Some targeting molecules include vitamin B12 and fragments of bacterial toxins, as described in WO 92/17167 (Biotech Australia Pty. Ltd.) and monoclonal antibodies, as described in U.S. Pat. No. 5,194,254 (Barber et al). The immunogenic compositions of the invention (including vaccines) may further comprise at least one other immunogenic or immunostimulating material and the immunostimulating material may be at least one adjuvant.

Suitable adjuvants for use in the present invention include, (but are not limited to) aluminum phosphate, aluminum hydroxide, QS21, Quil A, derivatives and components thereof, ISCOM matrix, calcium phosphate, calcium hydroxide, zinc hydroxide, a glycolipid analog, an octadecyl ester of an amino acid, a muramyl dipeptide, polyphosphazene, ISCOPREP, DC-chol, DDBA and a lipoprotein. Advantageous combinations of adjuvants are described in copending U.S. patent application Ser. Nos. 08/261,194 filed Jun. 16, 1994 and 08/483,856, filed Jun. 7, 1995, assigned to the assignee hereof and the disclosures of which is incorporated herein by reference thereto. The invention further includes an antibody specific for the outer membrane protein provided herein producible by immunizing a host with an immunogenic composition as provided herein.

In a further aspect of the invention, there is provided a method of generating an immune response in a host comprising administering thereto an immuno-effective amount of the immunogenic composition as provided herein. The immune response may be a humoral or a cell-mediated immune response. The immune response may provide protection to the host against diseases caused by a bacterial pathogen that produces the about 200 kDa outer membrane protein or produces a protein capable of inducing antibodies in the host specifically reactive with the about 200 kDa outer membrane protein. In particular, the pathogen is a strain of Moraxella, including M. catarrhalis . Hosts in which protection against disease may be conferred include primates, including humans.

The present invention provides, in an additional aspect thereof, a method of producing a vaccine comprising administering the immunogenic composition provided herein to a test host to determine an amount and a frequency of administration of the active component to confer protection against disease caused by a bacterial pathogen that produces the about 200 kDa outer membrane protein or produces a protein capable of inducing antibodies in the host specifically reactive with the about 200 kDa outer membrane protein, and formulating the active component in a form and amount suitable for administration to a treated host in accordance with said determined amount and frequency of administration. In particular, the pathogen is a strain of Moraxella, including M. catarrhalis . The treated host may be a human.

A further aspect of the present invention provides a method of determining the presence of nucleic acid encoding an outer membrane protein of a strain of Moraxella having a molecular mass of about 200 kDa, as determined by SDS-PAGE, or fragment or analog thereof, in a sample, comprising the steps of:

(a) contacting the sample with the nucleic acid molecule provided herein to produce duplexes comprising the nucleic acid molecule and any said nucleic acid molecule encoding the outer membrane protein present in the sample and specifically hybridizable therewith; and

(b) determining the production of the duplexes.

In yet a further aspect of the invention, there is provided a method of determining the presence of antibodies specifically reactive with outer membrane protein of a strain of Moraxella having a molecular mass of about 200 kDa, in a sample, comprising the steps of:

(a) contacting the sample with the outer membrane protein as provided herein to produce complexes comprising the outer membrane protein and any said antibodies present in the sample specifically reactive therewith; and

(b) determining production of the complexes.

In a further aspect of the invention, there is also provided a method of determining the presence of an outer membrane protein of a strain of Moraxella having a molecular mass of about 200 kDa, in a sample comprising the steps of:

(a) immunizing a subject with the immunogenic composition as provided herein, to produce antibodies specific for the outer membrane protein;

(b) contacting the sample with the antibodies to produce complexes comprising any outer membrane protein present in the sample and said outer membrane protein specific antibodies; and

(c) determining production of the complexes.

The outer membrane protein may be part of a Moraxella catarrhalis strain.

The present invention provides, in a yet further aspect, a diagnostic kit for determining the presence of nucleic acid encoding an outer membrane protein of a strain of Moraxella having a molecular mass of about 200 kDa, as determined by SDS-PAGE, or fragment or analog thereof, in a sample, comprising:

(a) the nucleic acid molecule as provided herein;

(b) means for contacting the nucleic acid with the sample to produce duplexes comprising the nucleic acid molecule and any said nucleic acid present in the sample and hybridizable with the nucleic acid molecule; and

(c) means for determining production of the duplexes.

In yet a further aspect of the invention, there is provided a diagnostic kit for determining the presence of antibodies in a sample specifically reactive with the outer membrane protein of a strain of Moraxella having a molecular mass of about 200 kDa, as determined by SDS-PAGE, comprising:

(a) the outer membrane protein as provided herein;

(b) means for contacting the outer membrane protein with the sample to produce complexes comprising the outer membrane protein and any said antibodies present in the sample; and

(c) means for determining production of the complexes.

The invention also provides a diagnostic kit for detecting the presence of an outer membrane protein of a strain of Moraxella having a molecular mass of about 200 kDa, in a sample, comprising:

(a) an antibody specific for the about 200 kDa outer membrane protein as provided herein;

(b) means for contacting the antibody with the sample to produce a complex comprising the outer membrane protein and outer membrane-specific antibody; and

(c) means for determining production of the complex.

In a further aspect of the invention, there is provided a method of producing an isolated and purified outer membrane protein of a strain of Moraxella having a molecular mass of about 200 kDa, as determined by SDS-PAGE, comprising the steps of:

(a) providing a cell mass of the Moraxella strain;

(b) disrupting the cell mass to provide a cell lysate;

(c) fractionating the cell lysate to provide a fraction containing the outer membrane protein substantially free from other cell lysate components, and

(d) recovering said outer membrane protein.

The bacterial strain may be M. catarrhalis . The cell lysate may be fractionated by gel electrophoresis.

In this application, the term “about 200 kDa protein” is used to define a family of outer membrane proteins of Moraxella having a molecular mass of between about 160 and about 230 kDa and includes proteins having variations in their amino acid sequences including those naturally occurring in various strains of Moraxella. The purified and isolated DNA molecules comprising a gene encoding the about 200 kDa protein of the present invention also include those encoding functional analogs of the about 200 kDa protein. In this application, a first protein is a “functional analog” of a second protein if the first protein is immunologically related to and/or has the same function as the second protein. The functional analog may be, for example, a fragment of the protein or a substitution, addition, deletion mutant thereof or a fusion with a second protein.

Advantages of the present invention include:

a method for isolating purified about 200 kDa outer membrane protein of a Moraxella strain that produces the outer membrane protein, including M. catarrhalis;

a gene encoding an about 200 kDa outer membrane protein of M. catarrhalis;

an isolated and purified about 200 kDa outer membrane protein isolatable from a Moraxella strain; and

diagnostic kits and immunological reagents for specific identification of Moraxella and hosts infected thereby.

FIGS. 1A and 1B show an analysis of Moraxella catarrhalis cell proteins by SDS-PAGE. The identification of the lanes and the sources of the proteins are given in Example 2 below;

FIG. 2 shows a comparative analysis of cell proteins from a number of M. catarrhalis strains by SDS-PAGE analysis and shows the variability in the molecular weight of the about 200 kDa protein in different strains of Moraxella. The identification of the lanes and the sources of the proteins are given in Example 4 below;

FIG. 3 shows an analysis of isolated and purified about 200 kDa outer membrane protein of M. catarrhalis by SDS-PAGE;

FIG. 4 shows the specific recognition of about 200 kDa outer membrane protein by anti-peptide antiserum. The identification of the lanes and antiserum are given in Example 8 below;

FIG. 5 shows restriction maps of clones containing a gene encoding the about 200 kDa outer membrane protein of M. catarrhalis . The open reading frame of the about 200 kDa outer membrane protein is indicated by the shaded box. Restriction sites are Sal: SalI, N: NcoI, B: BglII, K: KpnI, Xh: XhoI, RV: EcoRV.

FIGS. 6A to 6 K show the nucleotide sequence (SEQ ID No: 1—entire sequence, SEQ ID No: 2—coding sequence) of the gene encoding the about 200 kDa outer membrane protein of M. catarrhalis and the deduced amino acid sequence (SEQ ID No: 3—identified GTG start codon, SEQ ID No: 4—putative ATG start codon). Peptide 1 (SEQ ID No: 11) and Peptide 2 (SEQ ID No: 12) are identified by underlining;

FIG. 7A is a restriction enzyme map of the gene encoding the about 200 kDa outer membrane protein of M. catarrhalis (SEQ ID No: 1) showing single cutting restriction enzymes;

FIG. 7B is a restriction enzyme map of the gene encoding about 200 kDa outer membrane protein of M. catarrhalis (SEQ ID No: 1) showing double cutting restriction enzymes;

FIG. 8 shows the identification of the GTG initiation codon by expressing the C-terminal truncations of the gene encoding the about 200 kDa outer membrane protein of M. catarrhalis . Restriction sites are N: NcoI, K: KpnI, H: HindIII, Hp: HpaI, RV: EcoRV, Sal: SalI;

FIG. 9 shows the identification of the GTG initiation codon by utilization of anti-sera specific for N-terminal peptides of the about 200 kDa outer membrane protein of M. catarrhalis . Restriction sites are Nco: NcoI, K: KpnI, H: HindIII, RV: EcoRV, Sal: SalI;

FIG. 10 shows the recognition of 200 kDa protein by anti peptide sera;

FIG. 11 shows the construction of vectors for the expression of the about 200 kDa outer membrane protein of M. catarrhalis from E. coli . Nco: NcoI, Pst: PstI, Pvu: PvuII, Sca: ScaI, Sal: SalI;

FIG. 12 shows the expression of N-terminal truncations of the about 200 kDa outer membrane protein of M. catarrhalis in E. coli using the bacteriophage T7 promoter;

FIG. 13 shows the expression of the about 200 kDa outer membrane protein of M. catarrhalis fused with the LacZ-α-peptide in E. coli ; and

FIG. 14 shows the specific identification of M. catarrhalis expressing the about 200 kDa outer membrane protein by guinea pig anti-200 kDa specific antiserum in contrast to other bacteria. Identification of the lanes and bacteria appears below.

Referring to FIGS. 1A and 1B and FIG. 2, there is illustrated the separation of a novel outer membrane protein from a variety of strains of M. catarrhalis having a molecular mass about 200 kDa. The presence of this about 200 kDa protein in a variety of M. catarrhalis strains and, in particular, the almost-universal presence in strains isolated from patients suffering from otitis media is shown in Table I. FIG. 3 shows the isolated and purified outer membrane protein.

Purified protein was eluted from a gel and used to raise antibodies in guinea pigs. The antibodies specifically recognize only strains of M. catarrhalis which produce the outer membrane protein (Table I below).

Referring to FIG. 4, there is shown the recognition of the about 200 kDa outer membrane protein by antibodies raised in guinea pigs to a synthesized peptide corresponding to an internal fragment of the about 200 kDa protein. The synthesized peptide had the amino acid sequence NH 2 -Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys (SEQ ID No: 6).

Referring to FIG. 5, there is shown restriction maps of clones containing a gene encoding the about 200 kDa outer membrane protein. In FIG. 5, the open reading frame of the about 200 kDa gene is shown as a solid box and the GTG start codon is indicated. The nucleotide sequence (SEQ ID No: 1 and 2) of the gene encoding the about 200 kDa outer membrane protein is shown in FIG. 6, along with the deduced amino acid sequence (SEQ ID No: 3) of the protein. Restriction enzyme maps of the gene encoding the about 200 kDa protein are shown in FIGS. 7 (A) and 7 (B). The amino acid composition of the about 200 kDa protein is shown in Table III.

In one embodiment of the present invention, the isolated and purified about 200 kDa outer membrane protein as provided herein is useful for generating antibodies that can be used to specifically distinguish M. catarrhalis from other bacterial pathogens that cause otitis media and other diseases. Thus referring to FIG. 14, there is illustrated an immunoblot showing the specific reactivity of a guinea pig monospecific anti-200 kDa outer membrane protein antiserum produced by immunizing mice with the purified about 200 kDa outer membrane protein as provided herein. The bacterial lysates analyzed were as follows:

The results shown in FIG. 14 clearly show the usefulness of outer membrane-specific antisera as provided herein to distinguish between bacterial pathogens that produce diseases with similar clinical symptoms.

In accordance with another aspect of the present invention, there is provided a vaccine against Moraxella, comprising an immunogenically-effective amount of the outer membrane protein as provided herein and a physiologically-acceptable carrier therefor. The outer membrane protein provided herein also may be used as a carrier protein for hapten, polysaccharides or peptides to make a conjugate vaccine against antigenic determinants unrelated to the about 200 kDa outer membrane protein.

The about 200 kDa outer membrane protein provided herein is useful as a diagnostic reagent, as an antigen for the generation of anti-outer membrane protein antibodies, or as an antigen for vaccination against the diseases caused by species of Moraxella or for detecting infection by Moraxella.

In additional embodiments of the present invention, the about 200 kDa outer membrane protein as provided herein may be used as a carrier molecule to prepare chimeric molecules and conjugate vaccines (including glycoconjugates) against pathogenic bacteria, including encapsulated bacteria. Thus, for example, glycoconjugates of the present invention may be used to confer protection against disease and infection caused by any bacteria having polysaccharide antigens including lipooligosaccharides (LOS) and polyribosylphosphate (PRP). Such bacterial pathogens may include, for example, Haemophilus influenzae, Streptococcus pneumoniae, Escherichia coli, Neisseria meningitidis, Salmonella typhi, Streptococcus mutants, Cryptococcus neoformans , Klebsiella, Staphylococcus aureus and Pseudomonas aeruginosa . Particular antigens which can be conjugated to outer membrane protein and methods to achieve such conjugations are described in published PCT application WO 94/12641, assigned to the assignee hereof and the disclosure of which is hereby incorporated by reference thereto.

In another embodiment, the carrier function of the outer membrane protein may be used, for example, to induce immunity toward abnormal polysaccharides of tumor cells, or to produce anti-tumor antibodies that can be conjugated to chemotherapeutic or bioactive agents.

The present invention extends to the use of the nucleic acid molecules and proteins provided herein as a medicament and in the manufacture of a medicament for the treatment of Moraxella infections.

In a particular embodiment of the invention, there is provided a recombinant about 200 kDa outer membrane protein of Moraxella or fragment or analog thereof or a fusion protein producible by a transformed host containing at least a portion of the gene encoding the about 200 kDa protein. Referring to FIG. 11, there is shown recombinant vectors for the production of such proteins. In FIG. 11, the filled boxes show 1.9 kb and 4.8 kb C-terminal regions of 200 kD protein gene, that were inserted into a vector, pT7—7, under the control of the bacteriophage T7 promoter. The small open boxes show seven N- terminal amino acids from the vector in the same reading frame. The shaded box shows 5.5 kb C-terminal region of 200 kD protein, which contained ATG codon very close to the N-terminus. This gene fragment was fused to lacZ α peptide gene (shown in filled box) under the control of lacZ promoter. The full-length gene, that starts from GTG, is shown in a hatched box.

Referring to FIG. 12, there is shown the expression of N-terminal truncations of the about 200 kDa protein in E. coli. E. coli strain, BL21(DE3)/pLysS, carrying plasmid, pKS94, was grown in LB broth containing 100 μg/ml ampicillin to the early log phase and then IPTG was added. After culturing for 2 more hours, the bacteria were harvested and lysed. The lysates were assayed on Western blot using anti-200 kD protein guinea pig serum as a first antibody. Other procedures were as in FIG. 5 . Lane 1: prestained molecular weight marker, Lane 2: BL21(DE3)/pLysS carrying pT7—7 with an incorrect insert. Lane 3: L21(DE3)/pLysS carrying pKS94.

Referring to FIG. 13, there is shown the expression of fusion protein comprising the β-galactosidase α peptide and a portion of the about 200 kDa protein in E. coli. E. coli strain, DH5α, carried pKS140. The plasmid pKS140 carried the C-terminal 5.5 kb fragment of 200 kD protein gene after a N-terminal portion of LacZ-α-peptide in the same reading frame. The E. coli strain was grown to the stationary phase, harvested and then lysed. The lysate was assayed by Western blotting. Lane 1: prestained molecular weight marker, Lane 2: DH5α carrying pKS140 (total protein, 0.5 μg), Lane 3: sonicate of M. catarrhalis , strain 4223 (total protein, 10 μg).

It is clearly apparent to one skilled in the art, that the various embodiments of the present invention have many applications in the fields of vaccination, diagnosis, treatment of Moraxella infections, and in the generation of immunological reagents. A further non-limiting discussion of such uses is further presented below.

1. Vaccine Preparation and Use

Immunogenic compositions, including those suitable to be used as vaccines, may be prepared from the about 200 kDa outer membrane protein as disclosed herein, as well as immunological fragments and fusions thereof, which may be purified from the bacteria or which may be produced recombinantly. The vaccine elicits an immune response in a subject which produces antibodies, including anti-200 kDa outer membrane protein antibodies and antibodies that are opsonizing or bactericidal. Should the vaccinated subject be challenged by Moraxella or other bacteria that produce proteins capable of producing antibodies that specifically recognize 200 kDa outer membrane protein, the antibodies bind to and inactivate the bacterium. Furthermore, opsonizing or bactericidal anti-200 kDa outer membrane protein antibodies may also provide protection by alternative mechanisms.

Immunogenic compositions including vaccines may be prepared as injectables, as liquid solutions or emulsions. The about 200 kDa outer membrane protein may be mixed with pharmaceutically acceptable excipients which are compatible with the about 200 kDa outer membrane protein. Such excipients may include, water, saline, dextrose, glycerol, ethanol, and combinations thereof. The immunogenic compositions and vaccines may further contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance the effectiveness thereof. Immunogenic compositions and vaccines may be administered parenterally, by injection subcutaneously or intramuscularly. Alternatively, the immunogenic compositions formed according to the present invention, may be formulated and delivered in a manner to evoke an immune response at mucosal surfaces. Thus, the immunogenic composition may be administered to mucosal surfaces by, for example, the nasal or oral (intragastric) routes. Alternatively, other modes of administration including suppositories and oral formulations may be desirable. For suppositories, binders and carriers may include, for example, polyalkalene glycols or triglycerides. Oral formulations may include normally employed incipients such as, for example, pharmaceutical grades of saccharine, cellulose and magnesium carbonate. These compositions can take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 1 to 95% of the about 200 kDa outer membrane protein. The immunogenic preparations and vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective, protective and immunogenic. The quantity to be administered depends on the subject to be treated, including, for example, the capacity of the individual's immune system to synthesize antibodies, and if needed, to produce a cell-mediated immune response. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are readily determinable by one skilled in the art and may be of the order of micrograms of the about 200 kDa outer membrane protein. Suitable regimes for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent administrations. The dosage may also depend on the route of administration and will vary according to the size of the host.

The immunogenic preparations including vaccines may comprise as the immunostimulating material a nucleotide vector comprising at least a portion of the gene encoding the about 200 kDa protein, or the at least a portion of the gene may be used directly for immunization.

The concentration of the about 200 kDa outer membrane antigen in an immunogenic composition according to the invention is in general about 1 to 95%. A vaccine which contains antigenic material of only one pathogen is a monovalent vaccine. Vaccines which contain antigenic material of several pathogens are combined vaccines and also belong to the present invention. Such combined vaccines contain, for example, material from various pathogens or from various strains of the same pathogen, or from combinations of various pathogens.

Immunogenicity can be significantly improved if the antigens are co-administered with adjuvants, commonly used as 0.05 to 0.1 percent solution in phosphate-buffered saline. Adjuvants enhance the immunogenicity of an antigen but are not necessarily immunogenic themselves. Adjuvants may act by retaining the antigen locally near the site of administration to produce a depot effect facilitating a slow, sustained release of antigen to cells of the immune system. Adjuvants can also attract cells of the immune system to an antigen depot and stimulate such cells to elicit immune responses.

Immunostimulatory agents or adjuvants have been used for many years to improve the host immune responses to, for example, vaccines. Intrinsic adjuvants, such as lipopolysaccharides, normally are the components of the killed or attenuated bacteria used as vaccines. Extrinsic adjuvants are immunomodulators which are typically non-covalently linked to antigens and are formulated to enhance the host immune responses. Thus, adjuvants have been identified that enhance the immune response to antigens delivered parenterally. Some of these adjuvants are toxic, however, and can cause undesirable side-effects, making them unsuitable for use in humans and many animals. Indeed, only aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines. The efficacy of alum in increasing antibody responses to diphtheria and tetanus toxoids is well established and a HBsAg vaccine has been adjuvanted with alum. While the usefulness of alum is well established for some applications, it has limitations. For example, alum is ineffective for influenza vaccination and inconsistently elicits a cell mediated immune response.

A wide range of extrinsic adjuvants can provoke potent immune responses to antigens. These include saponins complexed to membrane protein antigens (immune stimulating complexes), pluronic polymers with mineral oil, killed mycobacteria in mineral oil, Freund's complete adjuvant, bacterial products, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A, and liposomes.

To efficiently induce humoral immune responses (HIR) and cell-mediated immunity (CMI), immunogens are typically emulsified in adjuvants. Many adjuvants are toxic, inducing granulomas, acute and chronic inflammations (Freund's complete adjuvant) FCA, cytolysis (saponins and Pluronic polymers) and pyrogenicity, arthritis and anterior uveitis (LPS and MDP). Although FCA is an excellent adjuvant and widely used in research, it is not licensed for use in human or veterinary vaccines because of its toxicity.

Desirable characteristics of ideal adjuvants include:

(1) lack of toxicity;

(2) ability to stimulate a long-lasting immune response;

(3) simplicity of manufacture and stability in long-term storage;

(4) ability to elicit both CMI and HIR to antigens administered by various routes, if required;

(5) synergy with other adjuvants;

(6) capability of selectively interacting with populations of antigen presenting cells (APC);

(7) ability to specifically elicit appropriate T H 1 or T H 2 cell-specific immune responses; and

(8) ability to selectively increase appropriate antibody isotype levels (for example, IgA) against antigens.

U.S. Pat. No. 4,855,283 granted to Lockhoff et al on Aug. 8, 1989 which is incorporated herein by reference thereto, teaches glycolipid analogues including N-glycosylamides, N-glycosylureas and N-glycosylcarbamates, each of which is substituted in the sugar residue by an amino acid, as immuno-modulators or adjuvants. Thus, Lockhoff et al. (U.S. Pat. No. 4,855,283 and ref. 27) reported that N-glycolipid analogs displaying structural similarities to the naturally-occurring glycolipids, such as glycosphospholipids and glycoglycerolipids, are capable of eliciting strong immune responses in both herpes simplex virus vaccine and pseudorabies virus vaccine. Some glycolipids have been synthesized from long chain-alkylamines and fatty acids that are linked directly with the sugars through the anomeric carbon atom, to mimic the functions of the naturally occurring lipid residues.

U.S. Pat. No. 4,258,029 granted to Moloney, assigned to the assignee hereof and incorporated herein by reference thereto, teaches that octadecyl tyrosine hydrochloride (OTH) functioned as an adjuvant when complexed with tetanus toxoid and formalin inactivated type I, II and III poliomyelitis virus vaccine. Also, Nixon-George et al. (ref. 24), reported that octadecyl esters of aromatic amino acids complexed with a recombinant hepatitis B surface antigen, enhanced the host immune responses against hepatitis B virus.

Lipidation of synthetic peptides has also been used to increase their immunogenicity. Thus, Wiesmuller (ref. 25) describes a peptide with a sequence homologous to a foot-and-mouth disease viral protein coupled to an adjuvant tripalmityl-S-glyceryl-cysteinylserylserine, being a synthetic analogue of the N-terminal part of the lipoprotein from Gram negative bacteria. Furthermore, Deres et al. (ref. 26) reported in vivo priming of virus-specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine which comprised of modified synthetic peptides derived from influenza virus nucleoprotein by linkage to a lipopeptide, N-palmityl-S-[2,3-bis(palmitylxy)-(2RS)-propyl-[R]-cysteine (TPC).

2. Immunoassays

The about 200 kDa outer membrane protein of the present invention is useful as an immunogen for the generation of anti-200 kDa outer membrane protein antibodies, as an antigen in immunoassays including enzyme-linked immunosorbent assays (ELISA), RIAs and other non-enzyme linked antibody binding assays or procedures known in the art for the detection of anti-bacterial, anti-Moraxella, and anti-200 kDa outer membrane protein antibodies. In ELISA assays, the about 200 kDa outer membrane protein is immobilized onto a selected surface, for example, a surface capable of binding proteins such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed about 200 kDa outer membrane protein, a nonspecific protein such as a solution of bovine serum albumin (BSA) that is known to be antigenically neutral with regard to the test sample may be bound to the selected surface. This allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific bindings of antisera onto the surface.

The immobilizing surface is then contacted with a sample, such as clinical or biological materials, to be tested in a manner conducive to immune complex (antigen/antibody) formation. This may include diluting the sample with diluents, such as solutions of BSA, bovine gamma globulin (BGG) and/or phosphate buffered saline (PBS)/Tween. The sample is then allowed to incubate for from 2 to 4 hours, at temperatures such as of the order of about 20° to 37° C. Following incubation, the sample-contacted surface is washed to remove non-immunocomplexed material. The washing procedure may include washing with a solution, such as PBS/Tween or a borate buffer. Following formation of specific immunocomplexes between the test sample and the bound about 200 kDa outer membrane protein, and subsequent washing, the occurrence, and even amount, of immunocomplex formation may be determined by subjecting the immunocomplex to a second antibody having specificity for the first antibody. If the test sample is of human origin, the second antibody is an antibody having specificity for human immunoglobulins and in general IgG. To provide detecting means, the second antibody may have an associated activity such as an enzymatic activity that will generate, for example, a colour development upon incubating with an appropriate chromogenic substrate. Quantification may then be achieved by measuring the degree of colour generation using, for example, a visible spectrophotometer.

3. Use of Sequences as Hybridization Probes

The nucleotide sequences of the present invention, comprising the sequence of the about 200 kDa protein gene, now allow for the identification and cloning of the about 200 kDa protein gene from any species of Moraxella.

The nucleotide sequences comprising the sequence of the about 200 kDa protein gene of the present invention are useful for their ability to selectively form duplex molecules with complementary stretches of other about 200 kDa protein genes. Depending on the application, a variety of hybridization conditions may be employed to achieve varying degrees of selectivity of the probe toward the other genes. For a high degree of selectivity, relatively stringent conditions are used to form the duplexes, such as low salt and/or high temperature conditions, such as provided by 0.02 M to 0.15 M NaCl at temperatures of between about 50° C. to 70° C. For some applications, less stringent hybridization conditions are required such as 0.15 M to 0.9 M salt, at temperatures ranging from between about 20° C. to 55° C. Hybridization conditions can also be rendered more stringent by the addition of increasing amounts of formamide, to destabilize the hybrid duplex. Thus, particular hybridization conditions can be readily manipulated, and will generally be a method of choice depending on the desired results. In general, convenient hybridization temperatures in the presence of 50% formamide are: 42° C. for a probe which is 95 to 100% homologous to the target fragment, 37° C. for 90 to 95% homology and 32° C. for 85 to 90% homology.

In a clinical diagnostic embodiment, the nucleic acid sequences of the about 200 kDa protein genes of the present invention may be used in combination with an appropriate means, such as a label, for determining hybridization. A wide variety of appropriate indicator means are known in the art, including radioactive, enzymatic or other ligands, such as avidin/biotin and digoxigenin-labelling, which are capable of providing a detectable signal. In some diagnostic embodiments, an enzyme tag such as urease, alkaline phosphatase or peroxidase, instead of a radioactive tag may be used. In the case of enzyme tags, colorimetric indicator substrates are known which can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridization with samples containing about 200 kDa protein gene sequences.

The nucleic acid sequences of the about 200 kDa protein genes of the present invention are useful as hybridization probes in solution hybridizations and in embodiments employing solid-phase procedures. In embodiments involving solid-phase procedures, the test DNA (or RNA) from samples, such as clinical samples, including exudates, body fluids (e.g., serum, amniotic fluid, middle ear effusion, sputum, bronchoalveolar lavage fluid) or even tissues, is adsorbed or otherwise affixed to a selected matrix or surface. The fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes comprising the nucleic acid sequences of the about 200 kDa protein encoding genes or fragments or analogs thereof of the present invention under desired conditions. The selected conditions will depend on the particular circumstances based on the particular criteria required depending on, for example, the G+C contents, type of target nucleic acid, source of nucleic acid, size of hybridization probe etc. Following washing of the hybridization surface so as to remove non-specifically bound probe molecules, specific hybridization is detected, or even quantified, by means of the label. It is preferred to select nucleic acid sequence portions which are conserved among species of Moraxella. The selected probe may be at least 18 bp and may be in the range of about 30 to 90 bp.

4. Expression of the About 200 kDa Protein Gene

Plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell may be used for the expression of the genes encoding the about 200 kDa protein in expression systems. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli may be transformed using pBR322 which contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The plasmids or phage, must also contain, or be modified to contain, promoters which can be used by the host cell for expression of its own proteins.

In addition, phage vectors containing replicon and control sequences that are compatible with the host can be used as a transforming vector in connection with these hosts. For example, the phage in lambda GEM™-11 may be utilized in making recombinant phage vectors which can be used to transform host cells, such as E. coli LE392.

Promoters commonly used in recombinant DNA construction include the β-lactamase (penicillinase) and lactose promoter systems and other microbial promoters, such as the T7 promoter system as described in U.S. Pat. No. 4,952,496. Details concerning the nucleotide sequences of promoters are known, enabling a skilled worker to ligate them functionally with genes. The particular promoter used will generally be a matter of choice depending upon the desired results. Hosts that are appropriate for expression of the about 200 kDa protein genes, fragments, analogs or variants thereof, may include E. coli , Bacillus species, Haemophilus, fungi, yeast, Bordetella, or the baculovirus expression system may be used.

In accordance with this invention, it is preferred to make the protein by recombinant methods, particularly when the naturally occurring about 200 kDa protein as purified from a culture of a species of Moraxella may include trace amounts of toxic materials or other contaminants. This problem can be avoided by using recombinantly produced protein in heterologous systems which can be isolated from the host in a manner to minimize contaminants in the purified material. Particularly desirable hosts for expression in this regard include Gram positive bacteria which do not have LPS and are, therefore, endotoxin free. Such hosts include species of Bacillus and may be particularly useful for the production of non-pyrogenic about 200 kDa protein, fragments or analogs thereof.

Certain plasmids that contain portions of the gene having the open reading frame of the gene encoding the about 200 kDa outer membrane protein of M. catarrhalis strain 4223 that are described and referred to herein have been deposited with the America Type Culture Collection (ATCC) located at U.S.A., pursuant to the Budapest Treaty and pursuant to 37 CFR 1.808 and prior to the filing of this application. The identifications of the respective portions of the gene present in these plasmids are shown in FIG. 5 .

Samples of the deposited plasmids will become available to the public upon grant of a patent based upon this United States patent application. The invention described and claimed herein is not to be limited in scope by plasmids deposited, since the deposited embodiment is intended only as an illustration of the invention. Any equivalent or similar plasmids that encode similar or equivalent antigens as described in this application are within the scope of the invention.

The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. These Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitations.

Methods of molecular genetics, protein biochemistry, and immunology used but not explicitly described in this disclosure and these Examples are amply reported in the scientific literature and are well within the ability of those skilled in the art.

In summary of the disclosure, the present invention provides an isolated and purified outer membrane protein of a Moraxella strain, particularly M. catarrhalis , having a molecular weight of about 200 kDa as well as isolated and purified DNA molecules encoding the outer membrane protein. The invention also provides analogs, truncations and peptides corresponding to portions of the outer membrane protein. The protein, DNA sequences, recombinant proteins derived therefrom and peptides are useful for diagnosis, immunization and the generation of diagnostic and immunological reagents. Modifications are possible within the scope of this invention.

1. Van Hare, G. F., P. A. Shurin, C. D. Marchant, N. A. Cartelli, C. E. Johnson, D. Fulton, S. Carlin, and C. H. Kim. Acute otitis media caused by Branhamella catarrhalis : biology and therapy. Rev. Infect. Dis. 9:16-27.

2. Chapman, A. J., D. M. Musher, S. Jonsson, J. E. Clarridge, and R. J. Wallace. 1985. Development of bactericidal antibody during Branhamella catarrhalis infection. J. Infect. Dis. 151:878-882.

3. Hager, H., A. Verghese, S. Alvarez, and S. L. Berk. 1987. Branhamella catarrhalis respiratory infections. Rev. Infect. Dis. 9:1140-1149.

4. McLeod, D. T., F. Ahmad, M. J. Croughan, and M. A. Calder. 1986. Bronchopulmonary infection due to M. catarrhalis . Clinical features and therapeutic response. Drugs 31(Suppl.3):109-112.

5. Nicotra, B., M. Rivera, J. I. Luman, and R. J. Wallace. 1986. Branhamella catarrhalis as a lower respiratory tract pathogen in patients with chronic lung disease. Arch.Intern.Med. 146:890-893.

6. Ninane, G., J. Joly, and M. Kraytman. 1978. Bronchopulmonary infection due to Branhamella catarrhalis 11 cases assessed by transtracheal puncture. Br.Med.Jr. 1:276-278.

7. Srinivasan, G., M. J. Raff, W. C. Templeton, S. J. Givens, R. C. Graves, and J. C. Mel. 1981. Branhamella catarrhalis pneumonia. Report of two cases and review of the literature. Am.Rev. Respir. Dis. 123:553-555.

8. West, M., S. L. Berk, and J. K. Smith. 1982. Branhamella catarrhalis pneumonia. South.Med. J. 75:1021-1023.

9. Brorson, J-E., A. Axelsson, and S. E. Holm. 1976. Studies on Branhamella catarrhalis ( Neisseria catarrhalis ) with special reference to maxillary sinusitis. Scan. J. Infect. Dis. 8:151-155.

10. Evans, F. O., Jr., J. B. Sydnor, W. E. C. Moore, G. R. Moore, J. L. Manwaring, A. H. Brill, R. T. Jackson, S. Hanna, J. S. Skaar, L. V. Holdeman, G. S. Fitz-Hugh, M. A. Sande, and J. M. Gwaltney, Jr. 1975. Sinusitis of the maxillary antrum. N.Engl.J.Med. 293:735-739.

11. Tinkelman, D. G., and H. J. Silk. 1989. Clinical and bacteriologic features of chronic sinusitis in children. Am.J.Dis.Child. 143:938-942.

12. Wald, E. R., C. Byers, N. Guerra, M. Casselbrant, and D. Beste. 1989. Subacute sinusitis in children. J.Pediatr. 115:28-32.

13. Wald, E. R., G. J. Milmoe, A. Bowen, J. Ledesma-Medina, N. Salamon, and C. D. Bluestone. 1981. Acute maxillary sinusitis in children. N.Engl.J.Med. 304:749-754.

14. Christensen, J. J., and B. Bruun. 1985. Bacteremia caused by a beta-lactamase producing strain of Branhamella catarrhalis . Acta.Pathol. Microbiol. Immunol. Scand. Sect.B 93:273-275.

15. Craig, D. B., and P. A. Wehrle. 1983. Branhamella catarrhalis septic arthritis. J. Rheumatol. 10:985-986.

16. Gray, L. D., R. E. Van Scoy, J. P. Anhalt, and P. K. W. Yu. 1989. Wound infection caused by Branhamella catarrhalis . J.Clin.Microbiol. 27:818-820.

17. Guthrie, R., K. Bakenhaster, R. Nelson, and R. Woskobnick. 1988. Branhamella catarrhalis sepsis: a case report and review of the literature. J.Infect.Dis. 158:907-908.

18. Hiroshi, S., E. J. Anaissie, N. Khardori, and G. P. Bodey. 1988. Branhamella catarrhalis septicemia in patients with leukemia. Cancer 61:2315-2317.

19. O'Neill, J. H., and P. W. Mathieson. 1987. Meningitis due to Branhamella catarrhalis . Aust. N. Z. J. Med. 17:241-242.

20. Murphy, T. F. 1989. The surface of Branhamella catarrhalis : a systematic approach to the surface antigens of an emerging pathogen. Pediatr. Infect. Dis. J. 8:S75-S77.

21. Klingman, K. L., and T. F. Murphy. 1994. Purification and characterization of a high-molecular-weight outer membrane protein of Moraxella ( Branhamella ) catarrhalis . Infect. Immun. 62:1150-1155.

22. Helminen, M. E., I. Maciver, J. L. Latimer, J. Klesney-Tait, L. D. Cope, M. Paris, G. H. McCracken, Jr., and E. J. Hansen. 1994. A large, antigenically conserved protein on the surface of Moraxella catarrhalis is a target for protective antibodies. J. Infect. Dis. 170:867-872.

23. Panezutti H., O. James, E. J. Hanson, Y. Choi, R. E. Harkness, M. H. Klein and P. Chong, 1993. Identification of surface-exposed B-cell epitopes recognized by Haemophilus influenzae type b P1 specific monoclonal antibodies. Infec. Immun. 61: 1867-1872.

24. Nixon-George et al. (1990), J. Immunology 144:4798-4802.

25. Wiesmuller (1989), Vaccine 8:29-33.

26. Deres et al. (1989), Nature 342:561.

27. Lockhoff, O. Glycolipids as Immmunomodulators: Synthesis and Properties. 1991. Chem. Int. Ed. Engl. 30:1611-1620.

1

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Met Ile Gly

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Ala Thr Leu Ser Gly Ser Ala Tyr Ala Gln Lys Lys Asp Thr Lys His

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Ala Asp Gly Asp Arg Ala Ile Ala Ile Gly Glu Asn Ala Asn Ala Gln

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Gly Gly Gln Ala Ile Ala Ile Gly Ser Ser Asn Lys Thr Val Asn Gly

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Ser Ser Leu Asp Lys Ile Gly Thr Asp Ala Thr Gly Gln Glu Ser Ile

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Ala Ile Gly Gly Asp Val Lys Ala Ser Gly Asp Ala Ser Ile Ala Ile

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Gly Ser Asp Asp Leu His Leu Leu Asp Gln His Gly Asn Pro Lys His

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Thr Ala Ser Gly His Ala Ser Thr Ala Val Gly Ala Met Ser Tyr Ala

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Gln Gly His Phe Ser Asn Ala Phe Gly Thr Arg Ala Thr Ala Lys Ser

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Ala Tyr Ser Leu Ala Val Gly Leu Ala Ala Thr Ala Glu Gly Gln Ser

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Gly Gln Gly Ser Val Val Thr Gln Ser Asp Asn Asn Ser Arg Pro Ala

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Tyr Thr Pro Asn Thr Gln Ala Leu Asp Pro Lys Phe Gln Ala Thr Asn

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Asn Thr Lys Ala Gly Pro Leu Ser Ile Gly Ser Asn Ser Ile Lys Arg

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Lys Ile Ile Asn Val Gly Ala Gly Val Asn Lys Thr Asp Ala Val Asn

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Val Ala Gln Leu Glu Ala Val Val Lys Trp Ala Lys Glu Arg Arg Ile

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Thr Phe Gln Gly Asp Asp Asn Ser Thr Asp Val Lys Ile Gly Leu Asp

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Asn Thr Leu Thr Ile Lys Gly Gly Ala Glu Thr Asn Ala Leu Thr Asp

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Asn Asn Ile Gly Val Val Lys Glu Ala Asp Asn Ser Gly Leu Lys Val

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Arg Asp Lys Ile Gly Phe Ala Arg Asp Gly Asp Val Asp Glu Lys Gln

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Ala Pro Tyr Leu Asp Lys Lys Gln Leu Lys Val Gly Ser Val Ala Ile

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Thr Ile Asp Asn Gly Ile Asp Ala Gly Asn Lys Lys Ile Ser Asn Leu

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Ala Lys Gly Ser Ser Ala Asn Asp Ala Val Thr Ile Glu Gln Leu Lys

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Ala Ala Lys Pro Thr Leu Asn Ala Gly Ala Gly Ile Ser Val Thr Pro

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Thr Glu Ile Ser Val Asp Ala Lys Ser Gly Asn Val Thr Ala Pro Thr

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Asp Lys Phe Ser Val Lys Gly Ser Gly Thr Asn Asn Ser Leu Val Thr

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Ala Glu His Leu Ala Ser Tyr Leu Asn Glu Val Asn Arg Thr Ala Asp

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Ser Ala Leu Gln Ser Phe Thr Val Lys Glu Glu Asp Asp Asp Asp Ala

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Asn Ala Ile Thr Val Ala Lys Asp Thr Thr Lys Asn Ala Gly Ala Val

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Ser Ile Leu Lys Leu Lys Gly Lys Asn Gly Leu Thr Val Ala Thr Lys

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Lys Asp Gly Thr Val Thr Phe Gly Leu Ser Gln Asp Ser Gly Leu Thr

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Ile Gly Lys Ser Thr Leu Asn Asn Asp Gly Leu Thr Val Lys Asp Thr

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Asn Glu Gln Ile Gln Val Gly Ala Asn Gly Ile Lys Phe Thr Asn Val

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Asn Gly Ser Asn Pro Gly Thr Gly Ile Ala Asn Thr Ala Arg Ile Thr

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Arg Asp Lys Ile Gly Phe Ala Gly Ser Asp Gly Ala Val Asp Thr Asn

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Lys Pro Tyr Leu Asp Gln Asp Lys Leu Gln Val Gly Asn Val Lys Ile

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Thr Asn Thr Gly Ile Asn Ala Gly Gly Lys Ala Ile Thr Gly Leu Ser

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Pro Thr Leu Pro Ser Ile Ala Asp Gln Ser Ser Arg Asn Ile Glu Leu

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Gly Asn Thr Ile Gln Asp Lys Asp Lys Ser Asn Ala Ala Ser Ile Asn

760 765 770

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Asp Ile Leu Asn Thr Gly Phe Asn Leu Lys Asn Asn Asn Asn Pro Ile

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Asp Phe Val Ser Thr Tyr Asp Ile Val Asp Phe Ala Asn Gly Asn Ala

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Thr Thr Ala Thr Val Thr His Asp Thr Ala Asn Lys Thr Ser Lys Val

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Val Tyr Asp Val Asn Val Asp Asp Thr Thr Ile His Leu Thr Gly Thr

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Asp Asp Asn Lys Lys Leu Gly Val Lys Thr Thr Lys Leu Asn Lys Thr

840 845 850

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Ser Ala Asn Gly Asn Thr Ala Thr Asn Phe Asn Val Asn Ser Ser Asp

855 860 865

gaa gat gcc ctt gtt aac gcc aaa gac atc gcc gaa aat cta aac acc 3356

Glu Asp Ala Leu Val Asn Ala Lys Asp Ile Ala Glu Asn Leu Asn Thr

870 875 880

cta gcc aag gaa att cac acc acc aaa ggc aca gca gac acc gcc cta 3404

Leu Ala Lys Glu Ile His Thr Thr Lys Gly Thr Ala Asp Thr Ala Leu

885 890 895

caa acc ttt acc gtt aaa aag gta gat gaa aat aat aat gct gat gac 3452

Gln Thr Phe Thr Val Lys Lys Val Asp Glu Asn Asn Asn Ala Asp Asp

900 905 910 915

gcc aac gcc atc acc gtg ggt caa aag aac gca aat aat caa gtc aac 3500

Ala Asn Ala Ile Thr Val Gly Gln Lys Asn Ala Asn Asn Gln Val Asn

920 925 930

acc cta aca ctc aaa ggt gaa aac ggt ctt aat att aaa acc gac aaa 3548

Thr Leu Thr Leu Lys Gly Glu Asn Gly Leu Asn Ile Lys Thr Asp Lys

935 940 945

aat ggt acg gtt acc ttt ggc att aac acc aca agc ggt ctt aaa gcc 3596

Asn Gly Thr Val Thr Phe Gly Ile Asn Thr Thr Ser Gly Leu Lys Ala

950 955 960

ggc aaa agc acc cta aac gac ggt ggc ttg tct att aaa aac ccc act 3644

Gly Lys Ser Thr Leu Asn Asp Gly Gly Leu Ser Ile Lys Asn Pro Thr

965 970 975

ggt agc gaa caa atc caa gtc ggt gct gat ggc gtg aag ttt gcc aag 3692

Gly Ser Glu Gln Ile Gln Val Gly Ala Asp Gly Val Lys Phe Ala Lys

980 985 990 995

gtt aat aat aat ggt gtt gta ggt gct ggc att gat ggc aca act cgc 3740

Val Asn Asn Asn Gly Val Val Gly Ala Gly Ile Asp Gly Thr Thr Arg

1000 1005 1010

att acc aga gat gaa att ggc ttt act ggg act aat ggc tca ctt gat 3788

Ile Thr Arg Asp Glu Ile Gly Phe Thr Gly Thr Asn Gly Ser Leu Asp

1015 1020 1025

aaa agc aaa ccc cac cta agc aaa gac ggc att aac gca ggt ggt aaa 3836

Lys Ser Lys Pro His Leu Ser Lys Asp Gly Ile Asn Ala Gly Gly Lys

1030 1035 1040

aag att acc aac att caa tca ggt gag att gcc caa aac agc cat gat 3884

Lys Ile Thr Asn Ile Gln Ser Gly Glu Ile Ala Gln Asn Ser His Asp

1045 1050 1055

gct gtg aca ggc ggc aag att tat gat tta aaa acc gaa ctt gaa aac 3932

Ala Val Thr Gly Gly Lys Ile Tyr Asp Leu Lys Thr Glu Leu Glu Asn

1060 1065 1070 1075

aaa atc agc agt act gcc aaa aca gca caa aac tca tta cac gaa ttc 3980

Lys Ile Ser Ser Thr Ala Lys Thr Ala Gln Asn Ser Leu His Glu Phe

1080 1085 1090

tca gta gca gat gaa caa ggt aat aac ttt acg gtt agt aac cct tac 4028

Ser Val Ala Asp Glu Gln Gly Asn Asn Phe Thr Val Ser Asn Pro Tyr

1095 1100 1105

tcc agt tat gac acc tca aag acc tct gat gtc atc acc ttt gca ggt 4076

Ser Ser Tyr Asp Thr Ser Lys Thr Ser Asp Val Ile Thr Phe Ala Gly

1110 1115 1120

gaa aac ggc att acc acc aag gta aat aaa ggt gtg gtg cgt gtg ggc 4124

Glu Asn Gly Ile Thr Thr Lys Val Asn Lys Gly Val Val Arg Val Gly

1125 1130 1135

att gac caa acc aaa ggc tta acc acg cct aag ctg acc gtg ggt aat 4172

Ile Asp Gln Thr Lys Gly Leu Thr Thr Pro Lys Leu Thr Val Gly Asn

1140 1145 1150 1155

aat aat ggc aaa ggc att gtc att gac agc caa aat ggt caa aat acc 4220

Asn Asn Gly Lys Gly Ile Val Ile Asp Ser Gln Asn Gly Gln Asn Thr

1160 1165 1170

atc aca gga cta agc aac act cta gct aat gtt acc aat gat aaa ggt 4268

Ile Thr Gly Leu Ser Asn Thr Leu Ala Asn Val Thr Asn Asp Lys Gly

1175 1180 1185

agc gta cgc acc aca gaa cag ggc aat ata atc aaa gac gaa gac aaa 4316

Ser Val Arg Thr Thr Glu Gln Gly Asn Ile Ile Lys Asp Glu Asp Lys

1190 1195 1200

acc cgt gcc gcc agc att gtt gat gtg cta agc gca ggc ttt aac ttg 4364

Thr Arg Ala Ala Ser Ile Val Asp Val Leu Ser Ala Gly Phe Asn Leu

1205 1210 1215

caa ggc aat ggt gaa gcg gtt gac ttt gtc tcc act tat gac acc gtc 4412

Gln Gly Asn Gly Glu Ala Val Asp Phe Val Ser Thr Tyr Asp Thr Val

1220 1225 1230 1235

aac ttt gcc gat ggc aat gcc acc acc gct aag gtg acc tat gat gac 4460

Asn Phe Ala Asp Gly Asn Ala Thr Thr Ala Lys Val Thr Tyr Asp Asp

1240 1245 1250

aca agc aaa acc agt aaa gtg gtc tat gat gtc aat gtg gat gat aca 4508

Thr Ser Lys Thr Ser Lys Val Val Tyr Asp Val Asn Val Asp Asp Thr

1255 1260 1265

acc att gaa gtt aaa gat aaa aaa ctt ggc gta aaa acc acc aca ttg 4556

Thr Ile Glu Val Lys Asp Lys Lys Leu Gly Val Lys Thr Thr Thr Leu

1270 1275 1280

acc agt act ggc aca ggt gct aat aaa ttt gcc cta agc aat caa gct 4604

Thr Ser Thr Gly Thr Gly Ala Asn Lys Phe Ala Leu Ser Asn Gln Ala

1285 1290 1295

act ggc gat gcg ctt gtc aag gcc agt gat atc gtt gct cat cta aac 4652

Thr Gly Asp Ala Leu Val Lys Ala Ser Asp Ile Val Ala His Leu Asn

1300 1305 1310 1315

acc tta tct ggc gac atc caa act gcc aaa ggg gca agc caa gcg aac 4700

Thr Leu Ser Gly Asp Ile Gln Thr Ala Lys Gly Ala Ser Gln Ala Asn

1320 1325 1330

aac tca gca ggc tat gtg gat gct gat ggc aat aag gtc atc tat gac 4748

Asn Ser Ala Gly Tyr Val Asp Ala Asp Gly Asn Lys Val Ile Tyr Asp

1335 1340 1345

agt acc gat aac aag tac tat caa gcc aaa aat gat ggc aca gtt gat 4796

Ser Thr Asp Asn Lys Tyr Tyr Gln Ala Lys Asn Asp Gly Thr Val Asp

1350 1355 1360

aaa acc aaa gaa gtt gcc aaa gac aaa ctg gtc gcc caa gcc caa acc 4844

Lys Thr Lys Glu Val Ala Lys Asp Lys Leu Val Ala Gln Ala Gln Thr

1365 1370 1375

cca gat ggc aca ttg gct caa atg aat gtc aaa tca gtc att aac aaa 4892

Pro Asp Gly Thr Leu Ala Gln Met Asn Val Lys Ser Val Ile Asn Lys

1380 1385 1390 1395

gaa caa gta aat gat gcc aat aaa aag caa ggc atc aat gaa gac aac 4940

Glu Gln Val Asn Asp Ala Asn Lys Lys Gln Gly Ile Asn Glu Asp Asn

1400 1405 1410

gcc ttt gtt aaa gga ctt gaa aaa gcc gct tct gat aac aaa acc aaa 4988

Ala Phe Val Lys Gly Leu Glu Lys Ala Ala Ser Asp Asn Lys Thr Lys

1415 1420 1425

aac gcc gca gta act gtg ggt gat tta aat gcc gtt gcc caa aca ccg 5036

Asn Ala Ala Val Thr Val Gly Asp Leu Asn Ala Val Ala Gln Thr Pro

1430 1435 1440

ctg acc ttt gca ggg gat aca ggc aca acg gct aaa aaa ctg ggc gag 5084

Leu Thr Phe Ala Gly Asp Thr Gly Thr Thr Ala Lys Lys Leu Gly Glu

1445 1450 1455

act ttg acc atc aaa ggt ggg caa aca gac acc aat aag cta acc gat 5132

Thr Leu Thr Ile Lys Gly Gly Gln Thr Asp Thr Asn Lys Leu Thr Asp

1460 1465 1470 1475

aat aac atc ggt gtg gta gca ggt act gat ggc ttc act gtc aaa ctt 5180

Asn Asn Ile Gly Val Val Ala Gly Thr Asp Gly Phe Thr Val Lys Leu

1480 1485 1490

gcc aaa gac cta acc aat ctt aac agc gtt aat gca ggt ggc acc aaa 5228

Ala Lys Asp Leu Thr Asn Leu Asn Ser Val Asn Ala Gly Gly Thr Lys

1495 1500 1505

att gat gac aaa ggc gtg tct ttt gta gac tca agc ggt caa gcc aaa 5276

Ile Asp Asp Lys Gly Val Ser Phe Val Asp Ser Ser Gly Gln Ala Lys

1510 1515 1520

gca aac acc cct gtg cta agt gcc aat ggg ctg gac ctg ggt ggc aag 5324

Ala Asn Thr Pro Val Leu Ser Ala Asn Gly Leu Asp Leu Gly Gly Lys

1525 1530 1535

gtc atc agt aat gtg ggc aaa ggc aca aaa gat acc gac gct gcc aat 5372

Val Ile Ser Asn Val Gly Lys Gly Thr Lys Asp Thr Asp Ala Ala Asn

1540 1545 1550 1555

gta caa cag tta aac gaa gta cgc aac ttg ttg ggt ctt ggt aat gct 5420

Val Gln Gln Leu Asn Glu Val Arg Asn Leu Leu Gly Leu Gly Asn Ala

1560 1565 1570

ggt aat gat aac gct gac ggc aat cag gta aac att gcc gac atc aaa 5468

Gly Asn Asp Asn Ala Asp Gly Asn Gln Val Asn Ile Ala Asp Ile Lys

1575 1580 1585

aaa gac cca aat tca ggt tca tca tct aac cgc act gtc atc aaa gca 5516

Lys Asp Pro Asn Ser Gly Ser Ser Ser Asn Arg Thr Val Ile Lys Ala

1590 1595 1600

ggc acg gta ctt ggc ggt aaa ggt aat aac gat acc gaa aaa ctt gcc 5564

Gly Thr Val Leu Gly Gly Lys Gly Asn Asn Asp Thr Glu Lys Leu Ala

1605 1610 1615

act ggt ggt ata caa gtg ggc gtg gat aaa gac ggc aac gct aac ggc 5612

Thr Gly Gly Ile Gln Val Gly Val Asp Lys Asp Gly Asn Ala Asn Gly

1620 1625 1630 1635

gat tta agc aat gtt tgg gtc aaa acc caa aaa gat ggc agc aaa aaa 5660

Asp Leu Ser Asn Val Trp Val Lys Thr Gln Lys Asp Gly Ser Lys Lys

1640 1645 1650

gcc ctg ctc gcc act tat aac gcc gca ggt cag acc aac tat ttg acc 5708

Ala Leu Leu Ala Thr Tyr Asn Ala Ala Gly Gln Thr Asn Tyr Leu Thr

1655 1660 1665

aac aac ccc gca gaa gcc att gac aga ata aat gaa caa ggt atc cgc 5756

Asn Asn Pro Ala Glu Ala Ile Asp Arg Ile Asn Glu Gln Gly Ile Arg

1670 1675 1680

ttc ttc cat gtc aac gat ggc aat caa gag cct gtg gta caa ggg cgt 5804

Phe Phe His Val Asn Asp Gly Asn Gln Glu Pro Val Val Gln Gly Arg

1685 1690 1695

aac ggc att gac tca agt gcc tca ggc aag cac tca gtg gcg ata ggt 5852

Asn Gly Ile Asp Ser Ser Ala Ser Gly Lys His Ser Val Ala Ile Gly

1700 1705 1710 1715

ttc cag gcc aag gca gat ggt gaa gcc gcc gtt gcc ata ggc aga caa 5900

Phe Gln Ala Lys Ala Asp Gly Glu Ala Ala Val Ala Ile Gly Arg Gln

1720 1725 1730

acc caa gca ggc aac caa tcc atc gcc atc ggt gat aac gca caa gcc 5948

Thr Gln Ala Gly Asn Gln Ser Ile Ala Ile Gly Asp Asn Ala Gln Ala

1735 1740 1745

acg ggc gat caa tcc atc gcc atc ggt aca ggc aat gtg gta gca ggt 5996

Thr Gly Asp Gln Ser Ile Ala Ile Gly Thr Gly Asn Val Val Ala Gly

1750 1755 1760

aag cac tct ggt gcc atc ggc gac cca agc act gtt aag gct gat aac 6044

Lys His Ser Gly Ala Ile Gly Asp Pro Ser Thr Val Lys Ala Asp Asn

1765 1770 1775

agt tac agt gtg ggt aat aac aac cag ttt acc gat gcc act caa acc 6092

Ser Tyr Ser Val Gly Asn Asn Asn Gln Phe Thr Asp Ala Thr Gln Thr

1780 1785 1790 1795

gat gtc ttt ggt gtg ggc aat aac atc acc gtg acc gaa agt aac tcg 6140

Asp Val Phe Gly Val Gly Asn Asn Ile Thr Val Thr Glu Ser Asn Ser

1800 1805 1810

gtt gcc tta ggt tca aac tct gcc atc agt gca ggc aca cac gca ggc 6188

Val Ala Leu Gly Ser Asn Ser Ala Ile Ser Ala Gly Thr His Ala Gly

1815 1820 1825

aca caa gcc aaa aaa tct gac ggc aca gca ggt aca acc acc aca gca 6236

Thr Gln Ala Lys Lys Ser Asp Gly Thr Ala Gly Thr Thr Thr Thr Ala

1830 1835 1840

ggt gca acc ggt acg gtt aaa ggc ttt gct gga caa acg gcg gtt ggt 6284

Gly Ala Thr Gly Thr Val Lys Gly Phe Ala Gly Gln Thr Ala Val Gly

1845 1850 1855

gcg gtc tcc gtg ggt gcc tca ggt gct gaa cgc cgt atc caa aat gtg 6332

Ala Val Ser Val Gly Ala Ser Gly Ala Glu Arg Arg Ile Gln Asn Val

1860 1865 1870 1875

gca gca ggt gag gtc agt gcc acc agc acc gat gcg gtc aat ggt agc 6380

Ala Ala Gly Glu Val Ser Ala Thr Ser Thr Asp Ala Val Asn Gly Ser

1880 1885 1890

cag ttg tac aaa gcc acc caa agc att gcc aac gca acc aat gag ctt 6428

Gln Leu Tyr Lys Ala Thr Gln Ser Ile Ala Asn Ala Thr Asn Glu Leu

1895 1900 1905

gac cat cgt atc cac caa aac gaa aat aag gcc aat gca ggg att tca 6476

Asp His Arg Ile His Gln Asn Glu Asn Lys Ala Asn Ala Gly Ile Ser

1910 1915 1920

tca gcg atg gcg atg gcg tcc atg cca caa gcc tac att cct ggc aga 6524

Ser Ala Met Ala Met Ala Ser Met Pro Gln Ala Tyr Ile Pro Gly Arg

1925 1930 1935

tcc atg gtt acc ggg ggt att gcc acc cac aac ggt caa ggt gcg gtg 6572

Ser Met Val Thr Gly Gly Ile Ala Thr His Asn Gly Gln Gly Ala Val

1940 1945 1950 1955

gca gtg gga ctg tcg aag ctg tcg gat aat ggt caa tgg gta ttt aaa 6620

Ala Val Gly Leu Ser Lys Leu Ser Asp Asn Gly Gln Trp Val Phe Lys

1960 1965 1970

atc aat ggt tca gcc gat acc caa ggc cat gta ggg gcg gca gtt ggt 6668

Ile Asn Gly Ser Ala Asp Thr Gln Gly His Val Gly Ala Ala Val Gly

1975 1980 1985

gca ggt ttt cac ttt taagccataa atcgcaagat tttacttaaa aatcaatctc 6723

Ala Gly Phe His Phe

1990

accatagttg tataaaacag catcagcatc agtcatatta ctgatgctga tgttttttat 6783

cacttaaacc attttaccgc tcaagtgatt ctctttcacc atgaccaaat cgccattgat 6843

cataggtaaa cttattgagt aaattttatc aatgtagttg ttagatatgg ttaaaattgt 6903

gccattgacc aaaaaatgac cgatttatcc cgaaaatttc tgattatgat ccgttgacct 6963

gcaggtcgac 6973

2

5976

Moraxella catarrhalis

2

gtgatcggtg caacgctcag tggcagtgct tatgctcaaa aaaaagatac caaacatatc 60

gcaattggtg aacaaaacca gccaagacgc tcaggcactg ccaaggcgga cggtgatcga 120

gccattgcta ttggtgaaaa tgctaacgca cagggcggtc aagccatcgc catcggtagt 180

agtaataaaa ctgtcaatgg aagcagtttg gataagatag gtaccgatgc tacgggtcaa 240

gagtccatcg ccatcggtgg tgatgtaaag gctagtggtg atgcctcgat tgccatcggt 300

agtgatgact tacatttgct tgatcagcat ggtaatccta aacatccgaa aggtactctg 360

attaacgatc ttattaacgg ccatgcagta ttaaaagaaa tacgaagctc aaaggataat 420

gatgtaaaat atagacgcac aaccgcaagc ggacacgcca gtactgcagt gggagccatg 480

tcatatgcac agggtcattt ttccaacgcc tttggtacac gggcaacagc taaaagtgcc 540

tattccttgg cagtgggtct tgccgccaca gccgagggcc aatctacaat cgctattggt 600

tctgatgcaa catctagctc gttgggagcg atagcccttg gtgcaggtac tcgtgctcag 660

ctacagggca gtattgccct aggtcaaggt tctgttgtca ctcagagtga taataattct 720

agaccggcct atacaccaaa tacccaggca ctagacccca agtttcaagc caccaataat 780

acgaaggcgg gtccactttc cattggtagt aactctatca aacgtaaaat catcaatgtc 840

ggtgcaggtg ttaataaaac cgatgcggtc aatgtggcac agctagaagc ggtggtgaag 900

tgggctaagg agcgtagaat tacttttcag ggtgatgata acagtactga cgtaaaaata 960

ggtttggata atactttaac tattaaaggt ggtgcagaga ccaacgcatt aaccgataat 1020

aatatcggtg tggtaaaaga ggctgataat agtggtctga aagttaaact tgctaaaact 1080

ttaaacaatc ttactgaggt gaatacaact acattaaatg ccacaaccac agttaaggta 1140

ggtagtagta gtagtactac agctgaatta ttgagtgata gtttaacctt tacccagccc 1200

aatacaggca gtcaaagcac aagcaaaacc gtctatggcg ttaatggggt gaagtttact 1260

aataatgcag aaacaacagc agcaatcggc actactcgta ttaccagaga taaaattggc 1320

tttgctcgag atggtgatgt tgatgaaaaa caagcaccat atttggataa aaaacaactt 1380

aaagtgggta gtgttgcaat taccatagac aatggcattg atgcaggtaa taaaaagatc 1440

agtaatcttg ccaaaggtag cagtgctaac gatgcggtta ccatcgaaca gctcaaagcc 1500

gccaagccta ctttaaacgc aggcgctggc atcagtgtca cacctactga aatatcagtt 1560

gatgctaaga gtggcaatgt taccgcccca acttacaaca ttggcgtgaa aaccaccgag 1620

cttaacagtg atggcactag tgataaattt agtgttaagg gtagtggtac gaacaatagc 1680

ttagttaccg ccgaacattt ggcaagctat ctaaatgaag tcaatcgaac ggctgacagt 1740

gctctacaaa gctttaccgt taaagaagaa gacgatgatg acgccaacgc tatcaccgtg 1800

gctaaagata cgacaaaaaa tgccggcgca gtcagcatct taaaactcaa aggtaaaaac 1860

ggtctaacgg ttgctaccaa aaaagatggt acggttacct ttgggcttag ccaagatagc 1920

ggtctgacca ttggcaaaag caccctaaac aacgatggct tgactgttaa agataccaac 1980

gaacaaatcc aagtcggtgc taatggcatt aaatttacta atgtgaatgg tagtaatcca 2040

ggtactggca ttgcaaatac cgctcgcatt accagagata aaattggctt tgctggttct 2100

gatggtgcag ttgatacaaa caaaccttat cttgatcaag acaagctaca agttggcaat 2160

gttaagatta ccaacactgg cattaacgca ggtggtaaag ccatcacagg gctgtcccca 2220

acactgccta gcattgccga tcaaagtagc cgcaacatag aactgggcaa tacaatccaa 2280

gacaaagaca aatccaacgc tgccagcatt aatgatatat taaatacagg ctttaaccta 2340

aaaaataata acaaccccat tgactttgtc tccacttatg acattgttga ctttgccaat 2400

ggcaatgcca ccaccgccac agtaacccat gataccgcta acaaaaccag taaagtggta 2460

tatgatgtga atgtggatga tacaaccatt catctaacag gcactgatga caataaaaaa 2520

cttggcgtca aaaccaccaa actgaacaaa acaagtgcta atggtaatac agcaactaac 2580

tttaatgtta actctagtga tgaagatgcc cttgttaacg ccaaagacat cgccgaaaat 2640

ctaaacaccc tagccaagga aattcacacc accaaaggca cagcagacac cgccctacaa 2700

acctttaccg ttaaaaaggt agatgaaaat aataatgctg atgacgccaa cgccatcacc 2760

gtgggtcaaa agaacgcaaa taatcaagtc aacaccctaa cactcaaagg tgaaaacggt 2820

cttaatatta aaaccgacaa aaatggtacg gttacctttg gcattaacac cacaagcggt 2880

cttaaagccg gcaaaagcac cctaaacgac ggtggcttgt ctattaaaaa ccccactggt 2940

agcgaacaaa tccaagtcgg tgctgatggc gtgaagtttg ccaaggttaa taataatggt 3000

gttgtaggtg ctggcattga tggcacaact cgcattacca gagatgaaat tggctttact 3060

gggactaatg gctcacttga taaaagcaaa ccccacctaa gcaaagacgg cattaacgca 3120

ggtggtaaaa agattaccaa cattcaatca ggtgagattg cccaaaacag ccatgatgct 3180

gtgacaggcg gcaagattta tgatttaaaa accgaacttg aaaacaaaat cagcagtact 3240

gccaaaacag cacaaaactc attacacgaa ttctcagtag cagatgaaca aggtaataac 3300

tttacggtta gtaaccctta ctccagttat gacacctcaa agacctctga tgtcatcacc 3360

tttgcaggtg aaaacggcat taccaccaag gtaaataaag gtgtggtgcg tgtgggcatt 3420

gaccaaacca aaggcttaac cacgcctaag ctgaccgtgg gtaataataa tggcaaaggc 3480

attgtcattg acagccaaaa tggtcaaaat accatcacag gactaagcaa cactctagct 3540

aatgttacca atgataaagg tagcgtacgc accacagaac agggcaatat aatcaaagac 3600

gaagacaaaa cccgtgccgc cagcattgtt gatgtgctaa gcgcaggctt taacttgcaa 3660

ggcaatggtg aagcggttga ctttgtctcc acttatgaca ccgtcaactt tgccgatggc 3720

aatgccacca ccgctaaggt gacctatgat gacacaagca aaaccagtaa agtggtctat 3780

gatgtcaatg tggatgatac aaccattgaa gttaaagata aaaaacttgg cgtaaaaacc 3840

accacattga ccagtactgg cacaggtgct aataaatttg ccctaagcaa tcaagctact 3900

ggcgatgcgc ttgtcaaggc cagtgatatc gttgctcatc taaacacctt atctggcgac 3960

atccaaactg ccaaaggggc aagccaagcg aacaactcag caggctatgt ggatgctgat 4020

ggcaataagg tcatctatga cagtaccgat aacaagtact atcaagccaa aaatgatggc 4080

acagttgata aaaccaaaga agttgccaaa gacaaactgg tcgcccaagc ccaaacccca 4140

gatggcacat tggctcaaat gaatgtcaaa tcagtcatta acaaagaaca agtaaatgat 4200

gccaataaaa agcaaggcat caatgaagac aacgcctttg ttaaaggact tgaaaaagcc 4260

gcttctgata acaaaaccaa aaacgccgca gtaactgtgg gtgatttaaa tgccgttgcc 4320

caaacaccgc tgacctttgc aggggataca ggcacaacgg ctaaaaaact gggcgagact 4380

ttgaccatca aaggtgggca aacagacacc aataagctaa ccgataataa catcggtgtg 4440

gtagcaggta ctgatggctt cactgtcaaa cttgccaaag acctaaccaa tcttaacagc 4500

gttaatgcag gtggcaccaa aattgatgac aaaggcgtgt cttttgtaga ctcaagcggt 4560

caagccaaag caaacacccc tgtgctaagt gccaatgggc tggacctggg tggcaaggtc 4620

atcagtaatg tgggcaaagg cacaaaagat accgacgctg ccaatgtaca acagttaaac 4680

gaagtacgca acttgttggg tcttggtaat gctggtaatg ataacgctga cggcaatcag 4740

gtaaacattg ccgacatcaa aaaagaccca aattcaggtt catcatctaa ccgcactgtc 4800

atcaaagcag gcacggtact tggcggtaaa ggtaataacg ataccgaaaa acttgccact 4860

ggtggtatac aagtgggcgt ggataaagac ggcaacgcta acggcgattt aagcaatgtt 4920

tgggtcaaaa cccaaaaaga tggcagcaaa aaagccctgc tcgccactta taacgccgca 4980

ggtcagacca actatttgac caacaacccc gcagaagcca ttgacagaat aaatgaacaa 5040

ggtatccgct tcttccatgt caacgatggc aatcaagagc ctgtggtaca agggcgtaac 5100

ggcattgact caagtgcctc aggcaagcac tcagtggcga taggtttcca ggccaaggca 5160

gatggtgaag ccgccgttgc cataggcaga caaacccaag caggcaacca atccatcgcc 5220

atcggtgata acgcacaagc cacgggcgat caatccatcg ccatcggtac aggcaatgtg 5280

gtagcaggta agcactctgg tgccatcggc gacccaagca ctgttaaggc tgataacagt 5340

tacagtgtgg gtaataacaa ccagtttacc gatgccactc aaaccgatgt ctttggtgtg 5400

ggcaataaca tcaccgtgac cgaaagtaac tcggttgcct taggttcaaa ctctgccatc 5460

agtgcaggca cacacgcagg cacacaagcc aaaaaatctg acggcacagc aggtacaacc 5520

accacagcag gtgcaaccgg tacggttaaa ggctttgctg gacaaacggc ggttggtgcg 5580

gtctccgtgg gtgcctcagg tgctgaacgc cgtatccaaa atgtggcagc aggtgaggtc 5640

agtgccacca gcaccgatgc ggtcaatggt agccagttgt acaaagccac ccaaagcatt 5700

gccaacgcaa ccaatgagct tgaccatcgt atccaccaaa acgaaaataa ggccaatgca 5760

gggatttcat cagcgatggc gatggcgtcc atgccacaag cctacattcc tggcagatcc 5820

atggttaccg ggggtattgc cacccacaac ggtcaaggtg cggtggcagt gggactgtcg 5880

aagctgtcgg ataatggtca atgggtattt aaaatcaatg gttcagccga tacccaaggc 5940

catgtagggg cggcagttgg tgcaggtttt cacttt 5976

3

1992

Moraxella catarrhalis

3

Met Ile Gly Ala Thr Leu Ser Gly Ser Ala Tyr Ala Gln Lys Lys Asp

1 5 10 15

Thr Lys His Ile Ala Ile Gly Glu Gln Asn Gln Pro Arg Arg Ser Gly

20 25 30

Thr Ala Lys Ala Asp Gly Asp Arg Ala Ile Ala Ile Gly Glu Asn Ala

35 40 45

Asn Ala Gln Gly Gly Gln Ala Ile Ala Ile Gly Ser Ser Asn Lys Thr

50 55 60

Val Asn Gly Ser Ser Leu Asp Lys Ile Gly Thr Asp Ala Thr Gly Gln

65 70 75 80

Glu Ser Ile Ala Ile Gly Gly Asp Val Lys Ala Ser Gly Asp Ala Ser

85 90 95

Ile Ala Ile Gly Ser Asp Asp Leu His Leu Leu Asp Gln His Gly Asn

100 105 110

Pro Lys His Pro Lys Gly Thr Leu Ile Asn Asp Leu Ile Asn Gly His

115 120 125

Ala Val Leu Lys Glu Ile Arg Ser Ser Lys Asp Asn Asp Val Lys Tyr

130 135 140

Arg Arg Thr Thr Ala Ser Gly His Ala Ser Thr Ala Val Gly Ala Met

145 150 155 160

Ser Tyr Ala Gln Gly His Phe Ser Asn Ala Phe Gly Thr Arg Ala Thr

165 170 175

Ala Lys Ser Ala Tyr Ser Leu Ala Val Gly Leu Ala Ala Thr Ala Glu

180 185 190

Gly Gln Ser Thr Ile Ala Ile Gly Ser Asp Ala Thr Ser Ser Ser Leu

195 200 205

Gly Ala Ile Ala Leu Gly Ala Gly Thr Arg Ala Gln Leu Gln Gly Ser

210 215 220

Ile Ala Leu Gly Gln Gly Ser Val Val Thr Gln Ser Asp Asn Asn Ser

225 230 235 240

Arg Pro Ala Tyr Thr Pro Asn Thr Gln Ala Leu Asp Pro Lys Phe Gln

245 250 255

Ala Thr Asn Asn Thr Lys Ala Gly Pro Leu Ser Ile Gly Ser Asn Ser

260 265 270

Ile Lys Arg Lys Ile Ile Asn Val Gly Ala Gly Val Asn Lys Thr Asp

275 280 285

Ala Val Asn Val Ala Gln Leu Glu Ala Val Val Lys Trp Ala Lys Glu

290 295 300

Arg Arg Ile Thr Phe Gln Gly Asp Asp Asn Ser Thr Asp Val Lys Ile

305 310 315 320

Gly Leu Asp Asn Thr Leu Thr Ile Lys Gly Gly Ala Glu Thr Asn Ala

325 330 335

Leu Thr Asp Asn Asn Ile Gly Val Val Lys Glu Ala Asp Asn Ser Gly

340 345 350

Leu Lys Val Lys Leu Ala Lys Thr Leu Asn Asn Leu Thr Glu Val Asn

355 360 365

Thr Thr Thr Leu Asn Ala Thr Thr Thr Val Lys Val Gly Ser Ser Ser

370 375 380

Ser Thr Thr Ala Glu Leu Leu Ser Asp Ser Leu Thr Phe Thr Gln Pro

385 390 395 400

Asn Thr Gly Ser Gln Ser Thr Ser Lys Thr Val Tyr Gly Val Asn Gly

405 410 415

Val Lys Phe Thr Asn Asn Ala Glu Thr Thr Ala Ala Ile Gly Thr Thr

420 425 430

Arg Ile Thr Arg Asp Lys Ile Gly Phe Ala Arg Asp Gly Asp Val Asp

435 440 445

Glu Lys Gln Ala Pro Tyr Leu Asp Lys Lys Gln Leu Lys Val Gly Ser

450 455 460

Val Ala Ile Thr Ile Asp Asn Gly Ile Asp Ala Gly Asn Lys Lys Ile

465 470 475 480

Ser Asn Leu Ala Lys Gly Ser Ser Ala Asn Asp Ala Val Thr Ile Glu

485 490 495

Gln Leu Lys Ala Ala Lys Pro Thr Leu Asn Ala Gly Ala Gly Ile Ser

500 505 510

Val Thr Pro Thr Glu Ile Ser Val Asp Ala Lys Ser Gly Asn Val Thr

515 520 525

Ala Pro Thr Tyr Asn Ile Gly Val Lys Thr Thr Glu Leu Asn Ser Asp

530 535 540

Gly Thr Ser Asp Lys Phe Ser Val Lys Gly Ser Gly Thr Asn Asn Ser

545 550 555 560

Leu Val Thr Ala Glu His Leu Ala Ser Tyr Leu Asn Glu Val Asn Arg

565 570 575

Thr Ala Asp Ser Ala Leu Gln Ser Phe Thr Val Lys Glu Glu Asp Asp

580 585 590

Asp Asp Ala Asn Ala Ile Thr Val Ala Lys Asp Thr Thr Lys Asn Ala

595 600 605

Gly Ala Val Ser Ile Leu Lys Leu Lys Gly Lys Asn Gly Leu Thr Val

610 615 620

Ala Thr Lys Lys Asp Gly Thr Val Thr Phe Gly Leu Ser Gln Asp Ser

625 630 635 640

Gly Leu Thr Ile Gly Lys Ser Thr Leu Asn Asn Asp Gly Leu Thr Val

645 650 655

Lys Asp Thr Asn Glu Gln Ile Gln Val Gly Ala Asn Gly Ile Lys Phe

660 665 670

Thr Asn Val Asn Gly Ser Asn Pro Gly Thr Gly Ile Ala Asn Thr Ala

675 680 685

Arg Ile Thr Arg Asp Lys Ile Gly Phe Ala Gly Ser Asp Gly Ala Val

690 695 700

Asp Thr Asn Lys Pro Tyr Leu Asp Gln Asp Lys Leu Gln Val Gly Asn

705 710 715 720

Val Lys Ile Thr Asn Thr Gly Ile Asn Ala Gly Gly Lys Ala Ile Thr

725 730 735

Gly Leu Ser Pro Thr Leu Pro Ser Ile Ala Asp Gln Ser Ser Arg Asn

740 745 750

Ile Glu Leu Gly Asn Thr Ile Gln Asp Lys Asp Lys Ser Asn Ala Ala

755 760 765

Ser Ile Asn Asp Ile Leu Asn Thr Gly Phe Asn Leu Lys Asn Asn Asn

770 775 780

Asn Pro Ile Asp Phe Val Ser Thr Tyr Asp Ile Val Asp Phe Ala Asn

785 790 795 800

Gly Asn Ala Thr Thr Ala Thr Val Thr His Asp Thr Ala Asn Lys Thr

805 810 815

Ser Lys Val Val Tyr Asp Val Asn Val Asp Asp Thr Thr Ile His Leu

820 825 830

Thr Gly Thr Asp Asp Asn Lys Lys Leu Gly Val Lys Thr Thr Lys Leu

835 840 845

Asn Lys Thr Ser Ala Asn Gly Asn Thr Ala Thr Asn Phe Asn Val Asn

850 855 860

Ser Ser Asp Glu Asp Ala Leu Val Asn Ala Lys Asp Ile Ala Glu Asn

865 870 875 880

Leu Asn Thr Leu Ala Lys Glu Ile His Thr Thr Lys Gly Thr Ala Asp

885 890 895

Thr Ala Leu Gln Thr Phe Thr Val Lys Lys Val Asp Glu Asn Asn Asn

900 905 910

Ala Asp Asp Ala Asn Ala Ile Thr Val Gly Gln Lys Asn Ala Asn Asn

915 920 925

Gln Val Asn Thr Leu Thr Leu Lys Gly Glu Asn Gly Leu Asn Ile Lys

930 935 940

Thr Asp Lys Asn Gly Thr Val Thr Phe Gly Ile Asn Thr Thr Ser Gly

945 950 955 960

Leu Lys Ala Gly Lys Ser Thr Leu Asn Asp Gly Gly Leu Ser Ile Lys

965 970 975

Asn Pro Thr Gly Ser Glu Gln Ile Gln Val Gly Ala Asp Gly Val Lys

980 985 990

Phe Ala Lys Val Asn Asn Asn Gly Val Val Gly Ala Gly Ile Asp Gly

995 1000 1005

Thr Thr Arg Ile Thr Arg Asp Glu Ile Gly Phe Thr Gly Thr Asn Gly

1010 1015 1020

Ser Leu Asp Lys Ser Lys Pro His Leu Ser Lys Asp Gly Ile Asn Ala

1025 1030 1035 1040

Gly Gly Lys Lys Ile Thr Asn Ile Gln Ser Gly Glu Ile Ala Gln Asn

1045 1050 1055

Ser His Asp Ala Val Thr Gly Gly Lys Ile Tyr Asp Leu Lys Thr Glu

1060 1065 1070

Leu Glu Asn Lys Ile Ser Ser Thr Ala Lys Thr Ala Gln Asn Ser Leu

1075 1080 1085

His Glu Phe Ser Val Ala Asp Glu Gln Gly Asn Asn Phe Thr Val Ser

1090 1095 1100

Asn Pro Tyr Ser Ser Tyr Asp Thr Ser Lys Thr Ser Asp Val Ile Thr

1105 1110 1115 1120

Phe Ala Gly Glu Asn Gly Ile Thr Thr Lys Val Asn Lys Gly Val Val

1125 1130 1135

Arg Val Gly Ile Asp Gln Thr Lys Gly Leu Thr Thr Pro Lys Leu Thr

1140 1145 1150

Val Gly Asn Asn Asn Gly Lys Gly Ile Val Ile Asp Ser Gln Asn Gly

1155 1160 1165

Gln Asn Thr Ile Thr Gly Leu Ser Asn Thr Leu Ala Asn Val Thr Asn

1170 1175 1180

Asp Lys Gly Ser Val Arg Thr Thr Glu Gln Gly Asn Ile Ile Lys Asp

1185 1190 1195 1200

Glu Asp Lys Thr Arg Ala Ala Ser Ile Val Asp Val Leu Ser Ala Gly

1205 1210 1215

Phe Asn Leu Gln Gly Asn Gly Glu Ala Val Asp Phe Val Ser Thr Tyr

1220 1225 1230

Asp Thr Val Asn Phe Ala Asp Gly Asn Ala Thr Thr Ala Lys Val Thr

1235 1240 1245

Tyr Asp Asp Thr Ser Lys Thr Ser Lys Val Val Tyr Asp Val Asn Val

1250 1255 1260

Asp Asp Thr Thr Ile Glu Val Lys Asp Lys Lys Leu Gly Val Lys Thr

1265 1270 1275 1280

Thr Thr Leu Thr Ser Thr Gly Thr Gly Ala Asn Lys Phe Ala Leu Ser

1285 1290 1295

Asn Gln Ala Thr Gly Asp Ala Leu Val Lys Ala Ser Asp Ile Val Ala

1300 1305 1310

His Leu Asn Thr Leu Ser Gly Asp Ile Gln Thr Ala Lys Gly Ala Ser

1315 1320 1325

Gln Ala Asn Asn Ser Ala Gly Tyr Val Asp Ala Asp Gly Asn Lys Val

1330 1335 1340

Ile Tyr Asp Ser Thr Asp Asn Lys Tyr Tyr Gln Ala Lys Asn Asp Gly

1345 1350 1355 1360

Thr Val Asp Lys Thr Lys Glu Val Ala Lys Asp Lys Leu Val Ala Gln

1365 1370 1375

Ala Gln Thr Pro Asp Gly Thr Leu Ala Gln Met Asn Val Lys Ser Val

1380 1385 1390

Ile Asn Lys Glu Gln Val Asn Asp Ala Asn Lys Lys Gln Gly Ile Asn

1395 1400 1405

Glu Asp Asn Ala Phe Val Lys Gly Leu Glu Lys Ala Ala Ser Asp Asn

1410 1415 1420

Lys Thr Lys Asn Ala Ala Val Thr Val Gly Asp Leu Asn Ala Val Ala

1425 1430 1435 1440

Gln Thr Pro Leu Thr Phe Ala Gly Asp Thr Gly Thr Thr Ala Lys Lys

1445 1450 1455

Leu Gly Glu Thr Leu Thr Ile Lys Gly Gly Gln Thr Asp Thr Asn Lys

1460 1465 1470

Leu Thr Asp Asn Asn Ile Gly Val Val Ala Gly Thr Asp Gly Phe Thr

1475 1480 1485

Val Lys Leu Ala Lys Asp Leu Thr Asn Leu Asn Ser Val Asn Ala Gly

1490 1495 1500

Gly Thr Lys Ile Asp Asp Lys Gly Val Ser Phe Val Asp Ser Ser Gly

1505 1510 1515 1520

Gln Ala Lys Ala Asn Thr Pro Val Leu Ser Ala Asn Gly Leu Asp Leu

1525 1530 1535

Gly Gly Lys Val Ile Ser Asn Val Gly Lys Gly Thr Lys Asp Thr Asp

1540 1545 1550

Ala Ala Asn Val Gln Gln Leu Asn Glu Val Arg Asn Leu Leu Gly Leu

1555 1560 1565

Gly Asn Ala Gly Asn Asp Asn Ala Asp Gly Asn Gln Val Asn Ile Ala

1570 1575 1580

Asp Ile Lys Lys Asp Pro Asn Ser Gly Ser Ser Ser Asn Arg Thr Val

1585 1590 1595 1600

Ile Lys Ala Gly Thr Val Leu Gly Gly Lys Gly Asn Asn Asp Thr Glu

1605 1610 1615

Lys Leu Ala Thr Gly Gly Ile Gln Val Gly Val Asp Lys Asp Gly Asn

1620 1625 1630

Ala Asn Gly Asp Leu Ser Asn Val Trp Val Lys Thr Gln Lys Asp Gly

1635 1640 1645

Ser Lys Lys Ala Leu Leu Ala Thr Tyr Asn Ala Ala Gly Gln Thr Asn

1650 1655 1660

Tyr Leu Thr Asn Asn Pro Ala Glu Ala Ile Asp Arg Ile Asn Glu Gln

1665 1670 1675 1680

Gly Ile Arg Phe Phe His Val Asn Asp Gly Asn Gln Glu Pro Val Val

1685 1690 1695

Gln Gly Arg Asn Gly Ile Asp Ser Ser Ala Ser Gly Lys His Ser Val

1700 1705 1710

Ala Ile Gly Phe Gln Ala Lys Ala Asp Gly Glu Ala Ala Val Ala Ile

1715 1720 1725

Gly Arg Gln Thr Gln Ala Gly Asn Gln Ser Ile Ala Ile Gly Asp Asn

1730 1735 1740

Ala Gln Ala Thr Gly Asp Gln Ser Ile Ala Ile Gly Thr Gly Asn Val

1745 1750 1755 1760

Val Ala Gly Lys His Ser Gly Ala Ile Gly Asp Pro Ser Thr Val Lys

1765 1770 1775

Ala Asp Asn Ser Tyr Ser Val Gly Asn Asn Asn Gln Phe Thr Asp Ala

1780 1785 1790

Thr Gln Thr Asp Val Phe Gly Val Gly Asn Asn Ile Thr Val Thr Glu

1795 1800 1805

Ser Asn Ser Val Ala Leu Gly Ser Asn Ser Ala Ile Ser Ala Gly Thr

1810 1815 1820

His Ala Gly Thr Gln Ala Lys Lys Ser Asp Gly Thr Ala Gly Thr Thr

1825 1830 1835 1840

Thr Thr Ala Gly Ala Thr Gly Thr Val Lys Gly Phe Ala Gly Gln Thr

1845 1850 1855

Ala Val Gly Ala Val Ser Val Gly Ala Ser Gly Ala Glu Arg Arg Ile

1860 1865 1870

Gln Asn Val Ala Ala Gly Glu Val Ser Ala Thr Ser Thr Asp Ala Val

1875 1880 1885

Asn Gly Ser Gln Leu Tyr Lys Ala Thr Gln Ser Ile Ala Asn Ala Thr

1890 1895 1900

Asn Glu Leu Asp His Arg Ile His Gln Asn Glu Asn Lys Ala Asn Ala

1905 1910 1915 1920

Gly Ile Ser Ser Ala Met Ala Met Ala Ser Met Pro Gln Ala Tyr Ile

1925 1930 1935

Pro Gly Arg Ser Met Val Thr Gly Gly Ile Ala Thr His Asn Gly Gln

1940 1945 1950

Gly Ala Val Ala Val Gly Leu Ser Lys Leu Ser Asp Asn Gly Gln Trp

1955 1960 1965

Val Phe Lys Ile Asn Gly Ser Ala Asp Thr Gln Gly His Val Gly Ala

1970 1975 1980

Ala Val Gly Ala Gly Phe His Phe

1985 1990

4

1833

Moraxella catarrhalis

4

Met Ser Tyr Ala Gln Gly His Phe Ser Asn Ala Phe Gly Thr Arg Ala

1 5 10 15

Thr Ala Lys Ser Ala Tyr Ser Leu Ala Val Gly Leu Ala Ala Thr Ala

20 25 30

Glu Gly Gln Ser Thr Ile Ala Ile Gly Ser Asp Ala Thr Ser Ser Ser

35 40 45

Leu Gly Ala Ile Ala Leu Gly Ala Gly Thr Arg Ala Gln Leu Gln Gly

50 55 60

Ser Ile Ala Leu Gly Gln Gly Ser Val Val Thr Gln Ser Asp Asn Asn

65 70 75 80

Ser Arg Pro Ala Tyr Thr Pro Asn Thr Gln Ala Leu Asp Pro Lys Phe

85 90 95

Gln Ala Thr Asn Asn Thr Lys Ala Gly Pro Leu Ser Ile Gly Ser Asn

100 105 110

Ser Ile Lys Arg Lys Ile Ile Asn Val Gly Ala Gly Val Asn Lys Thr

115 120 125

Asp Ala Val Asn Val Ala Gln Leu Glu Ala Val Val Lys Trp Ala Lys

130 135 140

Glu Arg Arg Ile Thr Phe Gln Gly Asp Asp Asn Ser Thr Asp Val Lys

145 150 155 160

Ile Gly Leu Asp Asn Thr Leu Thr Ile Lys Gly Gly Ala Glu Thr Asn

165 170 175

Ala Leu Thr Asp Asn Asn Ile Gly Val Val Lys Glu Ala Asp Asn Ser

180 185 190

Gly Leu Lys Val Lys Leu Ala Lys Thr Leu Asn Asn Leu Thr Glu Val

195 200 205

Asn Thr Thr Thr Leu Asn Ala Thr Thr Thr Val Lys Val Gly Ser Ser

210 215 220

Ser Ser Thr Thr Ala Glu Leu Leu Ser Asp Ser Leu Thr Phe Thr Gln

225 230 235 240

Pro Asn Thr Gly Ser Gln Ser Thr Ser Lys Thr Val Tyr Gly Val Asn

245 250 255

Gly Val Lys Phe Thr Asn Asn Ala Glu Thr Thr Ala Ala Ile Gly Thr

260 265 270

Thr Arg Ile Thr Arg Asp Lys Ile Gly Phe Ala Arg Asp Gly Asp Val

275 280 285

Asp Glu Lys Gln Ala Pro Tyr Leu Asp Lys Lys Gln Leu Lys Val Gly

290 295 300

Ser Val Ala Ile Thr Ile Asp Asn Gly Ile Asp Ala Gly Asn Lys Lys

305 310 315 320

Ile Ser Asn Leu Ala Lys Gly Ser Ser Ala Asn Asp Ala Val Thr Ile

325 330 335

Glu Gln Leu Lys Ala Ala Lys Pro Thr Leu Asn Ala Gly Ala Gly Ile

340 345 350

Ser Val Thr Pro Thr Glu Ile Ser Val Asp Ala Lys Ser Gly Asn Val

355 360 365

Thr Ala Pro Thr Tyr Asn Ile Gly Val Lys Thr Thr Glu Leu Asn Ser

370 375 380

Asp Gly Thr Ser Asp Lys Phe Ser Val Lys Gly Ser Gly Thr Asn Asn

385 390 395 400

Ser Leu Val Thr Ala Glu His Leu Ala Ser Tyr Leu Asn Glu Val Asn

405 410 415

Arg Thr Ala Asp Ser Ala Leu Gln Ser Phe Thr Val Lys Glu Glu Asp

420 425 430

Asp Asp Asp Ala Asn Ala Ile Thr Val Ala Lys Asp Thr Thr Lys Asn

435 440 445

Ala Gly Ala Val Ser Ile Leu Lys Leu Lys Gly Lys Asn Gly Leu Thr

450 455 460

Val Ala Thr Lys Lys Asp Gly Thr Val Thr Phe Gly Leu Ser Gln Asp

465 470 475 480

Ser Gly Leu Thr Ile Gly Lys Ser Thr Leu Asn Asn Asp Gly Leu Thr

485 490 495

Val Lys Asp Thr Asn Glu Gln Ile Gln Val Gly Ala Asn Gly Ile Lys

500 505 510

Phe Thr Asn Val Asn Gly Ser Asn Pro Gly Thr Gly Ile Ala Asn Thr

515 520 525

Ala Arg Ile Thr Arg Asp Lys Ile Gly Phe Ala Gly Ser Asp Gly Ala

530 535 540

Val Asp Thr Asn Lys Pro Tyr Leu Asp Gln Asp Lys Leu Gln Val Gly

545 550 555 560

Asn Val Lys Ile Thr Asn Thr Gly Ile Asn Ala Gly Gly Lys Ala Ile

565 570 575

Thr Gly Leu Ser Pro Thr Leu Pro Ser Ile Ala Asp Gln Ser Ser Arg

580 585 590

Asn Ile Glu Leu Gly Asn Thr Ile Gln Asp Lys Asp Lys Ser Asn Ala

595 600 605

Ala Ser Ile Asn Asp Ile Leu Asn Thr Gly Phe Asn Leu Lys Asn Asn

610 615 620

Asn Asn Pro Ile Asp Phe Val Ser Thr Tyr Asp Ile Val Asp Phe Ala

625 630 635 640

Asn Gly Asn Ala Thr Thr Ala Thr Val Thr His Asp Thr Ala Asn Lys

645 650 655

Thr Ser Lys Val Val Tyr Asp Val Asn Val Asp Asp Thr Thr Ile His

660 665 670

Leu Thr Gly Thr Asp Asp Asn Lys Lys Leu Gly Val Lys Thr Thr Lys

675 680 685

Leu Asn Lys Thr Ser Ala Asn Gly Asn Thr Ala Thr Asn Phe Asn Val

690 695 700

Asn Ser Ser Asp Glu Asp Ala Leu Val Asn Ala Lys Asp Ile Ala Glu

705 710 715 720

Asn Leu Asn Thr Leu Ala Lys Glu Ile His Thr Thr Lys Gly Thr Ala

725 730 735

Asp Thr Ala Leu Gln Thr Phe Thr Val Lys Lys Val Asp Glu Asn Asn

740 745 750

Asn Ala Asp Asp Ala Asn Ala Ile Thr Val Gly Gln Lys Asn Ala Asn

755 760 765

Asn Gln Val Asn Thr Leu Thr Leu Lys Gly Glu Asn Gly Leu Asn Ile

770 775 780

Lys Thr Asp Lys Asn Gly Thr Val Thr Phe Gly Ile Asn Thr Thr Ser

785 790 795 800

Gly Leu Lys Ala Gly Lys Ser Thr Leu Asn Asp Gly Gly Leu Ser Ile

805 810 815

Lys Asn Pro Thr Gly Ser Glu Gln Ile Gln Val Gly Ala Asp Gly Val

820 825 830

Lys Phe Ala Lys Val Asn Asn Asn Gly Val Val Gly Ala Gly Ile Asp

835 840 845

Gly Thr Thr Arg Ile Thr Arg Asp Glu Ile Gly Phe Thr Gly Thr Asn

850 855 860

Gly Ser Leu Asp Lys Ser Lys Pro His Leu Ser Lys Asp Gly Ile Asn

865 870 875 880

Ala Gly Gly Lys Lys Ile Thr Asn Ile Gln Ser Gly Glu Ile Ala Gln

885 890 895

Asn Ser His Asp Ala Val Thr Gly Gly Lys Ile Tyr Asp Leu Lys Thr

900 905 910

Glu Leu Glu Asn Lys Ile Ser Ser Thr Ala Lys Thr Ala Gln Asn Ser

915 920 925

Leu His Glu Phe Ser Val Ala Asp Glu Gln Gly Asn Asn Phe Thr Val

930 935 940

Ser Asn Pro Tyr Ser Ser Tyr Asp Thr Ser Lys Thr Ser Asp Val Ile

945 950 955 960

Thr Phe Ala Gly Glu Asn Gly Ile Thr Thr Lys Val Asn Lys Gly Val

965 970 975

Val Arg Val Gly Ile Asp Gln Thr Lys Gly Leu Thr Thr Pro Lys Leu

980 985 990

Thr Val Gly Asn Asn Asn Gly Lys Gly Ile Val Ile Asp Ser Gln Asn

995 1000 1005

Gly Gln Asn Thr Ile Thr Gly Leu Ser Asn Thr Leu Ala Asn Val Thr

1010 1015 1020

Asn Asp Lys Gly Ser Val Arg Thr Thr Glu Gln Gly Asn Ile Ile Lys

1025 1030 1035 1040

Asp Glu Asp Lys Thr Arg Ala Ala Ser Ile Val Asp Val Leu Ser Ala

1045 1050 1055

Gly Phe Asn Leu Gln Gly Asn Gly Glu Ala Val Asp Phe Val Ser Thr

1060 1065 1070

Tyr Asp Thr Val Asn Phe Ala Asp Gly Asn Ala Thr Thr Ala Lys Val

1075 1080 1085

Thr Tyr Asp Asp Thr Ser Lys Thr Ser Lys Val Val Tyr Asp Val Asn

1090 1095 1100

Val Asp Asp Thr Thr Ile Glu Val Lys Asp Lys Lys Leu Gly Val Lys

1105 1110 1115 1120

Thr Thr Thr Leu Thr Ser Thr Gly Thr Gly Ala Asn Lys Phe Ala Leu

1125 1130 1135

Ser Asn Gln Ala Thr Gly Asp Ala Leu Val Lys Ala Ser Asp Ile Val

1140 1145 1150

Ala His Leu Asn Thr Leu Ser Gly Asp Ile Gln Thr Ala Lys Gly Ala

1155 1160 1165

Ser Gln Ala Asn Asn Ser Ala Gly Tyr Val Asp Ala Asp Gly Asn Lys

1170 1175 1180

Val Ile Tyr Asp Ser Thr Asp Asn Lys Tyr Tyr Gln Ala Lys Asn Asp

1185 1190 1195 1200

Gly Thr Val Asp Lys Thr Lys Glu Val Ala Lys Asp Lys Leu Val Ala

1205 1210 1215

Gln Ala Gln Thr Pro Asp Gly Thr Leu Ala Gln Met Asn Val Lys Ser

1220 1225 1230

Val Ile Asn Lys Glu Gln Val Asn Asp Ala Asn Lys Lys Gln Gly Ile

1235 1240 1245

Asn Glu Asp Asn Ala Phe Val Lys Gly Leu Glu Lys Ala Ala Ser Asp

1250 1255 1260

Asn Lys Thr Lys Asn Ala Ala Val Thr Val Gly Asp Leu Asn Ala Val

1265 1270 1275 1280

Ala Gln Thr Pro Leu Thr Phe Ala Gly Asp Thr Gly Thr Thr Ala Lys

1285 1290 1295

Lys Leu Gly Glu Thr Leu Thr Ile Lys Gly Gly Gln Thr Asp Thr Asn

1300 1305 1310

Lys Leu Thr Asp Asn Asn Ile Gly Val Val Ala Gly Thr Asp Gly Phe

1315 1320 1325

Thr Val Lys Leu Ala Lys Asp Leu Thr Asn Leu Asn Ser Val Asn Ala

1330 1335 1340

Gly Gly Thr Lys Ile Asp Asp Lys Gly Val Ser Phe Val Asp Ser Ser

1345 1350 1355 1360

Gly Gln Ala Lys Ala Asn Thr Pro Val Leu Ser Ala Asn Gly Leu Asp

1365 1370 1375

Leu Gly Gly Lys Val Ile Ser Asn Val Gly Lys Gly Thr Lys Asp Thr

1380 1385 1390

Asp Ala Ala Asn Val Gln Gln Leu Asn Glu Val Arg Asn Leu Leu Gly

1395 1400 1405

Leu Gly Asn Ala Gly Asn Asp Asn Ala Asp Gly Asn Gln Val Asn Ile

1410 1415 1420

Ala Asp Ile Lys Lys Asp Pro Asn Ser Gly Ser Ser Ser Asn Arg Thr

1425 1430 1435 1440

Val Ile Lys Ala Gly Thr Val Leu Gly Gly Lys Gly Asn Asn Asp Thr

1445 1450 1455

Glu Lys Leu Ala Thr Gly Gly Ile Gln Val Gly Val Asp Lys Asp Gly

1460 1465 1470

Asn Ala Asn Gly Asp Leu Ser Asn Val Trp Val Lys Thr Gln Lys Asp

1475 1480 1485

Gly Ser Lys Lys Ala Leu Leu Ala Thr Tyr Asn Ala Ala Gly Gln Thr

1490 1495 1500

Asn Tyr Leu Thr Asn Asn Pro Ala Glu Ala Ile Asp Arg Ile Asn Glu

1505 1510 1515 1520

Gln Gly Ile Arg Phe Phe His Val Asn Asp Gly Asn Gln Glu Pro Val

1525 1530 1535

Val Gln Gly Arg Asn Gly Ile Asp Ser Ser Ala Ser Gly Lys His Ser

1540 1545 1550

Val Ala Ile Gly Phe Gln Ala Lys Ala Asp Gly Glu Ala Ala Val Ala

1555 1560 1565

Ile Gly Arg Gln Thr Gln Ala Gly Asn Gln Ser Ile Ala Ile Gly Asp

1570 1575 1580

Asn Ala Gln Ala Thr Gly Asp Gln Ser Ile Ala Ile Gly Thr Gly Asn

1585 1590 1595 1600

Val Val Ala Gly Lys His Ser Gly Ala Ile Gly Asp Pro Ser Thr Val

1605 1610 1615

Lys Ala Asp Asn Ser Tyr Ser Val Gly Asn Asn Asn Gln Phe Thr Asp

1620 1625 1630

Ala Thr Gln Thr Asp Val Phe Gly Val Gly Asn Asn Ile Thr Val Thr

1635 1640 1645

Glu Ser Asn Ser Val Ala Leu Gly Ser Asn Ser Ala Ile Ser Ala Gly

1650 1655 1660

Thr His Ala Gly Thr Gln Ala Lys Lys Ser Asp Gly Thr Ala Gly Thr

1665 1670 1675 1680

Thr Thr Thr Ala Gly Ala Thr Gly Thr Val Lys Gly Phe Ala Gly Gln

1685 1690 1695

Thr Ala Val Gly Ala Val Ser Val Gly Ala Ser Gly Ala Glu Arg Arg

1700 1705 1710

Ile Gln Asn Val Ala Ala Gly Glu Val Ser Ala Thr Ser Thr Asp Ala

1715 1720 1725

Val Asn Gly Ser Gln Leu Tyr Lys Ala Thr Gln Ser Ile Ala Asn Ala

1730 1735 1740

Thr Asn Glu Leu Asp His Arg Ile His Gln Asn Glu Asn Lys Ala Asn

1745 1750 1755 1760

Ala Gly Ile Ser Ser Ala Met Ala Met Ala Ser Met Pro Gln Ala Tyr

1765 1770 1775

Ile Pro Gly Arg Ser Met Val Thr Gly Gly Ile Ala Thr His Asn Gly

1780 1785 1790

Gln Gly Ala Val Ala Val Gly Leu Ser Lys Leu Ser Asp Asn Gly Gln

1795 1800 1805

Trp Val Phe Lys Ile Asn Gly Ser Ala Asp Thr Gln Gly His Val Gly

1810 1815 1820

Ala Ala Val Gly Ala Gly Phe His Phe

1825 1830

5

Asn Val Lys Ser Val Ile Asn Lys Glu Gln Val Asn Asp Ala Asn Lys

1 5 10 15

Xaa Gln Gly Ile

20

6

16

Moraxella catarrhalis

6

Asn Val Lys Ser Val Ile Asn Lys Glu Gln Val Asn Asp Ala Asn Lys

1 5 10 15

7

60

Moraxella catarrhalis

7

aatgtcaaat cagtcattaa caaagaacaa gtaaatgatg ccaataaaaa gcaaggcatc 60

8

20

Moraxella catarrhalis

8

Asn Val Lys Ser Val Ile Asn Lys Glu Gln Val Asn Asp Ala Asn Lys

1 5 10 15

Lys Gln Gly Ile

20

9

30

Moraxella catarrhalis

9

Met Ile Gly Ala Thr Leu Ser Gly Ser Ala Tyr Ala Gln Lys Lys Asp

1 5 10 15

Thr Lys His Ile Ala Ile Gly Glu Gln Asn Gln Pro Arg Arg

20 25 30

10

30

Moraxella catarrhalis

10

Ser Gly Thr Ala Lys Ala Asp Gly Asp Arg Ala Ile Ala Ile Gly Glu

1 5 10 15

Asn Ala Asn Ala Gln Gly Gly Gln Ala Ile Ala Ile Gly Ser

20 25 30