US20030170657A1

US20030170657A1 - High molecular weight major outer membrane protein of Moraxella

Info

Publication number: US20030170657A1
Application number: US10/175,282
Authority: US
Inventors: Ken Sasaki; Robin Harkness; Sheena Loosmore; Pele Chong; Michel Klein
Original assignee: Aventis Pasteur Ltd
Current assignee: Sanofi Pasteur Ltd
Priority date: 1995-05-01
Filing date: 2002-06-20
Publication date: 2003-09-11
Also published as: EP0826052A1; CA2219889A1; HK1015412A1; US6448386B2; MX9708438A; JP3771265B2; AU722132B2; US20030171254A1; JPH11502415A; WO1996034960A1; US20020068070A1; US20010014672A1; US6440425B1; BR9608241A; AU5394196A

Abstract

An isolated and purified outer membrane protein of a Moraxella strain, particularly M. catarrhalis, having a molecular mass of about 200 kDa, is provided. The about 200 kDa outer membrane protein as well as nucleic acid molecules encoding the same are useful in diagnostic applications and immunogenic compositions, particularly for in vivo administration to a host 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 a host specifically reactive with the about 200 kDa outer membrane protein.

Description

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of copending U.S. patent application Ser. No. 08/478,370, filed Jun. 7, 1995, which itself is a continuation-in-part of U.S. patent application Ser. No. 08/431,718 filed May 1, 1995.[0001]

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

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 k), 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 (ref s. 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.

SUMMARY OF THE INVENTION

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: 10), 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: 10) 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, poxyirus, 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: 10) 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. No. 08/261,194 filed Jun. 16, 1994 and Ser. No. 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-PACE, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show an analysis of [0060] 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 [0061] 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 [0062] 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; [0063]
FIG. 5 shows restriction maps of clones containing a gene encoding the about 200 kDa outer membrane protein of [0064] 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.
FIG. 6 shows 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 [0065] 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 [0066] 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 [0067] 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 [0068] 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 [0069] 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; [0070]
FIG. 11 shows the construction of vectors for the expression of the about 200 kDa outer membrane protein of [0071] 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 [0072] M. catarrhalis in E. coli using the bacteriophage T7 promoter;
FIG. 13 shows the expression of the about 200 kDa outer membrane protein of [0073] M. catarrhalis fused with the LacZ-α-peptide in E. coli; and
FIG. 14 shows the specific identification of [0074] 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.

GENERAL DESCRIPTION OF THE INVENTION

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 [0075] 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 [0076] 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[0077] 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. [0078] 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:



Lane	Bacterium	Source

1.	Molecular Weight Standard
2.	M. catarrhalis 4223	middle ear fluid
3.	M. catarrhalis RH408	non-clumping variant
		of strain 4223
4.	H. influenzae, MinnA strain	meningitis isolate
5.	non-typable H. influenzae, SB12 strain	otitis media isolate
6.	non-typable H. influenzae, SB33 strain	otitis media isolate
7.	S. pneumoniae type 6	ATCC 6306
8.	S. pneumoniae type 14	ATCC 6314
9.	P. aeruginosa
10.	E. coli DH5α

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. [0080]
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. [0081]
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. [0082]
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, [0083] 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. [0084]
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. [0085]
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 a 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. [0086]
Referring to FIG. 12, there is shown the expression of N-terminal truncations of the about 200 kDa protein in [0087] 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 [0088] E. coli. E. coli strain, DH5a, 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-a-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: DH5a 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. [0089]
1. Vaccine Preparation and Use [0090]
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. [0091]
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. [0092]
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. [0093]
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. [0094]
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. [0095]
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. [0096]
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. [0097]
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. [0098]
Desirable characteristics of ideal adjuvants include: [0099]
(1) lack of toxicity; [0100]
(2) ability to stimulate a long-lasting immune response; [0101]
(3) simplicity of manufacture and stability in long-term storage; [0102]
(4) ability to elicit both CMI and HIR to antigens administered by various routes, if required; [0103]
(5) synergy with other adjuvants; [0104]
(6) capability of selectively interacting with populations of antigen presenting cells (APC); [0105]
(7) ability to specifically elicit [0106] appropriate T _H1 or T _H2 cell-specific immune responses; and
(8) ability to selectively increase appropriate antibody isotype levels (for example, IgA) against antigens. [0107]
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. [0108]
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. [0109]
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). [0110]
2. Immunoassays [0111]
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. [0112]
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 200 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. [0113]
3. Use of Sequences as Hybridization Probes [0114]
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. [0115]
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. [0116]
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, calorimetric 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. [0117]
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. [0118]
4. Expression of the about 200 kDa Protein Gene [0119]
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, [0120] 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 [0121] 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 [0122] 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. [0123]

Biological Deposits

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 [0124] M. catarrhalis strain 4223 that are described and referred to herein have been deposited with the America Type Culture Collection (ATCC) located at 12301 Parklawn Drive, Rockville, Md., 20852, 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. [0125]

Plasmid ATCC Designation Date Deposited

pKS47 97,111 Apr. 7, 1995

pKS5 97,110 Apr. 7, 1995

pKS9 97,114 Apr. 18, 1995

EXAMPLES

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. [0126]
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. [0127]

Example 1

This Example illustrates the generation of a non-clumping strain (RH408) of [0128] M. catarrhalis.
[0129] M. catarrhalis strain 4223, a clumping strain (a common property of Moraxella strains), was inoculated into several flasks containing 20 mL of brain heat infusion (BHI) broth, and the cultures were incubated with shaking (170 rpm) overnight at 37° C. Five mL of each overnight culture were transferred to five individual 1 mL tubes, and were left sitting undisturbed at room temperature for 3 to 8 hours, to allow bacteria to sediment. One hundred AL of the cleared upper phase of each culture were used to inoculate 25 mL of BHI broth and cultures were incubated overnight at 37° C., as described above. This passaging was repeated six times, using 25 μL of cleared culture to inoculate 25 mL of BHI for each overnight culture. Non-clumping bacterial cultures were identified by measuring the absorbency A₅₇₈at intervals over a 3 hour time period, in order to compare the sedimentation rates of the passaged strains to that of the original M. catarrhalis strain 4223 culture. Non-clumping mutants, including M. catarrhalis RH408, did not aggregate during the three hour time period. On BHI agar plates, strain RH408 had a colony morphology typical for all non-clumping strains. Strain RH408 was previously deposited in connection of U.S. application Ser. No. 08/328,589 at the ATCC under the Budapest Treaty on Dec. 13, 1994 with Accession No. 55637.

Example 2

This Example illustrates the identification of the about 200 kDa outer membrane protein of [0130] Moraxella catarrhalis.
[0131] M. catarrhalis strains 4223, RH408, 5191, 8185, M2, M5, ATCC 25240, 3, 56, 135, 585 were grown in brain heart infusion (BHI) broth. The culture was incubated overnight with aeration at 37° C.
[0132] M. catarrhalis cells were sonicated and total protein was determined using the BCA assay system (Pierce, Rockford, Ill.). Ten μg of total protein were mixed with the SDS-PAGE sample buffer containing 0.3M Tris-HCl (pH 8.0), 50% glycerol, 10% SDS, 20% β-mercaptoethanol and 0.01% bromophenol blue, boiled for 5 minutes and loaded on each lane of SDS-PAGE gel (0.75 mm thick, 7.5% acrylamide). The gels were run at 200 V for 1 hour. Proteins were visualized by staining gels with a solution containing 0.13% Coomassie brilliant blue, 10% acetic acid and 45% methanol. Excess stain was removed with a destaining solution of 5% ethanol and 7.5% acetic acid.
The various Moraxella proteins separated by this procedure are shown in FIGS. 1A and 1B. The [0133] M. catarrhalis strains tested were as follows:

FIG. 1A



Lane	Bacterial Strain	Source

1.	Molecular Weight Standards
2.	E. coli
3.	No sample
4.	M. catarrhalis 4223	middle ear fluid
5.	M. catarrhalis RH408	non-clumping variant
		of 4223
6.	M. catarrhalis 5191	middle ear fluid
7.	M. catarrhalis 8185	nasopharynx
8.	M. catarrhalis M2	sputum
9.	M. catarrhalis M5	sputum
10.	M. catarrhalis 25240	ATCC 25240

FIG. 1B



Lane	Bacterial Strain	Source

1.	E. coli
2.	No sample
3.	Molecular Weight Size Markers
4.	M. catarrhalis 4223	middle ear fluid
5.	M. catarrhalis RH408	non-clumping variant
		of 4223
6.	M. catarrhalis 3	sputum
7.	M. catarrhalis 56	sputum
8.	M. catarrhalis 135	middle ear fluid
9.	M. catarrhalis 585	Blood

The about 200 kDa outer membrane protein was clearly seen in all otitis media strains ([0136] M. catarrhalis 4223, 5191, 135), in one strain isolated from the nasopharynx (8185), and in one strain isolated from sputum (M2). However, the about 200 kDa protein was not detected in three isolates from sputum (3, 56 and M5) and in one strain with unknown origin (ATCC 25240). A very narrow band was found in an isolate from blood of a bacteremia patient (585) and this band was recognized by an anti-200 kDa specific guinea pig serum on an immunoblot. Strain RH408 is a non-clumping spontaneous mutant isolated from strain 4223 (see Example 1) and was found to not express the about 200 kDa protein.

When gels were run longer, they showed heterogeneity in the apparent molecular masses of the about 200 kDa outer membrane protein in different strains of M. catarrhalis (FIG. 2). In FIG. 2 the strains analyzed were as follows:



Lane	Strain	Source

1.	Molecular Weight Size Markers
2.	M. catarrhalis H04	middle ear fluid
3.	M. catarrhalis H12	middle ear fluid
4.	M. catarrhalis PO34	middle ear fluid
5.	M. catarrhalis PO51	middle ear fluid
6.	M. catarrhalis E-07	middle ear fluid
7.	M. catarrhalis E-22	middle ear fluid
8.	M. catarrhalis E-23	middle ear fluid
9.	M. catarrhalis RH 4223	middle ear fluid
10.	M. catarrhalis RH 408	Non-clumping variant
		of 4223

The strain H12 (lane 3) was a natural isolate from middle ear fluid, but did not produce the about 200 kDa protein. [0138]
There may be at least three different sizes of protein in the about 200 kDa range. However, antibodies raised against the about 200 kDa outer membrane protein from one strain of [0139] M. catarrhalis (4223) did recognize all about 200 kDa proteins tested, present in different strains of M. catarrhalis. It is possible, however, that in particular immunogenic compositions, for example, as a vaccine and in particular diagnostic embodiments, that the about 200 kDa outer membrane protein from a variety of M. catarrhalis isolates (including immunogenically diverse isolates) may be required.

Example 3

This Example illustrates the detection of antibodies specific for the about 200 kDa outer membrane protein in a serum obtained from a convalescent patient having recovered from otitis media due to [0140] M. catarrhalis.
After separation by SDS-PAGE, bacterial proteins were transferred from polyacrylamide gels to prepared PVDF (polyvinylidene fluoride; Millipore) membranes at a constant voltage of 70 V for 1.5 h in a buffer system consisting of 3 g Tris, 14,4 g glycine and 200 ml methanol per liter at 4° C. Membranes with transferred proteins were blocked with Blocking Reagent (from Boehringer Mannheim) diluted in TBS (0.1M Tris, 0.15M Nacl) at room temperature for 30 min. Blots were exposed to convalescent antiserum diluted 1:500 in Blocking Reagent/TBS with 0.1[0141] % Tween 20 for 2 hours at room temperature. This patient had otitis media and the M. catarrhalis strain isolated from the patient's ear fluid was M. catarrhalis CJ7. Blots were then washed 2 times in Blocking Reagent/TBS with Tween at 15 min per wash. The reporter conjugate, horseradish peroxidase (HRP) conjugated to protein G, was diluted 1:4000 with Blocking Reagent/TBS with Tween and used to immerse the washed membranes for 30 min at room temperature. Blots were washed twice as above, followed by a TBS wash. Bound antibodies were detected using the LumiGlo (Kirkegaard and Perry) chemiluminescent detection system as described by the manufacturer. Treated blots were exposed to X-ray film. Antibodies were detected in this convalescent serum that reacted with the about 200 kDa outer membrane protein of M. catarrhalis CJ7. These results indicate that the about 200 kDa outer membrane protein is an immunogenic protein of M. catarrhalis to which an immune response is elicited during a natural infection by M. catarrhalis.

Example 4

This Example illustrates the isolation and purification of the about 200 kDa outer membrane protein. [0142]
[0143] M. catarrhalis 4223 cells were harvested by centrifugation at 2,000 rpm for 10 min and frozen. The frozen cells were thawed, resuspended in 20 mM sodium phosphate buffer (pH 7.2) and sonicated until the cells were disrupted. The frozen-thawed cells were also lysed in 20 mM Tris buffer (pH 8) containing 4% SDS and 0.2 mM EDTA by boiling for 5 min to produce a cell lysate. The cell sonicates and cell lysates were suspended in a SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, boiled for 5 min and separated by SDS-PAGE on a gel (1.5 mm thick, 7.5% acrylamide). The estimated position of the about 200 kDa protein on the gel was cut out and the protein extracted from the gel by electroelution using the same buffer as the SDS-PAGE running buffer. The isolated about 200 kDa outer membrane protein was shown to be a homogeneous, single band by SDS-PAGE as seen in FIG. 3. The samples analyzed in FIG. 3 are as follows:

Lane Sample

1. Molecular Weight Size Markers

2. Isolated and purified 200 kDa outer membrane

protein
The isolated and purified 200 kDa outer membrane protein of [0144] M. catarrhalis shown in FIG. 3 has a purity of at least 70%. Purified about 200 kDa outer membrane protein preparations of at least 95% could be readily achieved.

Example 5

This Example illustrates the immunization of guinea pigs with purified about 200 kDa protein from [0145] M. catarrhalis.
Approximately 30 to 40 μg of the about 200 kDa protein, which was isolated from [0146] M. catarrhalis strain 4223 by electroelution, were mixed with Preund's complete adjuvant (FCA) and subcutaneously injected into guinea pigs. After two weeks, the animals were boosted with about the same amount of the about 200 kDa protein in incomplete Freund's adjuvant (IFA). Two weeks later, blood was collected from the guinea pigs and antisera were obtained.
One antiserum was examined on Western blot for its reactivity with the about 200 kDa protein present in 54 different strains of [0147] M. catarrhalis, which were isolated in different geographical locations throughout the world (Canada, U.S. and Finland) (see Table 1 below). The about 200 kDa protein was recognized by the antiserum in all strains, in which the presence of the about 200 kDa protein band was detected on SDS-PAGE gels stained with Coomassie Blue. These results indicate that common epitopes of the about 200 kDa protein were present in all M. catarrhalis strains, which possessed this protein. As stated earlier, this protein is not present in all M. catarrhalis strains, but almost all strains, which were isolated from middle ear fluids from otitis media patients, did possess this protein (Table 1).

Example 6

This Example illustrates the specific recognition of [0148] M. catarrhalis strain 4223 with anti-200 kDa protein guinea pig serum by ELISA assay (see Table 2 below).
[0149] M. catarrhalis strains 4223, RH408 (200 kDa protein negative mutant) and H-12 were cultured in 60 mL of BHI broth overnight. E. coli strain BL21 (DE3) was cultured in 60 mL of broth overnight. The cultures were split into three tubes and centrifuged. M. catarrhalis strain 4223 was centrifuged at 1,500 rpm for 10 min., H-12 at 2,000 rpm for 10 min., and RH408 and E. coli BL21 (DE3) at 3,000 rpm for 15 min. The pellet in one tube was suspended in 20 ml of Dulbecco's phosphate buffered saline (D-PBS) and diluted to 1/500 with coating buffer (0.05M carbonate/bicarbonate buffer) pH 9.6. One hundred μL of the bacteria suspension were placed in each well and incubated for 1 hour at room temperature. One hundred μL of 0.2% glutaraldehyde was added to each well and incubated at room temperature for 10 min. to fix the cells on the well. The wells were washed with PBS containing 0.1% Tween 20 and 0.1% BSA (washing buffer), and then blocked with PBS containing 0.1% BSA for 30 min. at room temperature. After washing 5 times for 10 seconds with the washing buffer, serial dilutions of guinea pig antiserum with the washing buffer were added to the wells and incubation at room temperature was continued for 60 min. After washing, goat anti-guinea pig IgG conjugated with horseradish peroxidase was added to each well at the dilution of 1/20,000. After incubation at room temperature for 60 minutes, the wells were washed and then color reaction was developed using 3,3-5,5-tetramethylbenzidene (TMB) and hydrogen peroxide.
The ELISA plate wells were also coated with sonicates containing 10 μg/mL of total proteins in the coating buffer, blocked without the fixation process and then assayed as described above. [0150]
The results shown in Table 2 indicate that the about 200 kDa outer membrane protein specific guinea pig antiserum specifically recognizes strains of [0151] M. catarrhalis which produce the about 200 kDa protein. The ability of the antiserum to recognize whole cells indicates that the protein is present on the surface of the bacterial cells.

Example 7

This Example describes the determination of an internal amino acid sequence of the 200 kDa outer membrane protein. [0152]
The about 200 kDa outer membrane protein was isolated from [0153] M. catarrhalis 4223 by electroelution as described above. The protein was subjected to CNBr degradation, the proteolytic digests subjected to SDS-PAGE and transferred onto PVDF membrane. A peptide band migrating at a position corresponding to approximately 40 kDa was cut out from the membrane and its N-terminal amino acid sequence was determined. In another experiment, the CNBr degradation products of the about 200 kDa protein were subjected to a direct determination of N-terminal amino acid sequencing without separating by SDS-PAGE. Both analyses gave an identical, N-terminal sequence of 20 amino acids with one unidentified amino acid at the 17th position. The internal sequence of the 200 kDa outer membrane protein was:
NH[0154] 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).

Example 8

This Example describes the immunization of guinea pigs with a peptide corresponding to an internal fragment of the about 200 kDa outer membrane protein and the analysis of the antiserum generated. [0155]
Based upon the determination of the amino acid sequence of an internal fragment of the about 200 kDa outer membrane protein as described above, a 16 amino acid long peptide of sequence: [0156]
NH[0157] ₂-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys (SEQ ID No: 6)

was synthesized using standard procedures. This 16-mer peptide was conjugated to KLH using Imject Maleimide Activated KLH (Pierce, Rockford, Ill.) and approximately 500 μg of the conjugate was injected into guinea pigs using the same immunization and boosting schedule as described above. The guinea pig anti-serum raised against the 16-mer internal amino acid sequence (SEQ ID No: 6) was examined by immunoblot analysis and found to specifically recognize 200 kDa outer membrane protein in cell sonicates of M. catarrhalis 4223. The results are shown in FIG. 4 and indicate that the anti-peptide guinea pig antiserum specifically recognizes the 200 kDa protein of M. catarrhalis 4223. The samples analyzed in FIG. 4 were as follows:



Lane	Sample	Antiserum

1.	Molecular Weight Markers
2.	Purified 200 kDa outer membrane	Anti-200 kDa protein
	protein

3.	M. catarrhalis cell sonicate	Anti-peptide 1:5000
4.	M. catarrhalis cell sonicate	Anti-peptide 1:1000
5.	M. catarrhalis cell sonicate	Anti-peptide 1:500
6.	M. catarrhalis cell sonicate	Pre-immune serum

The results obtained confirm that the peptide corresponding to SEQ ID Nos: 5 and 6 are derived from the 200 kDa outer membrane protein. [0159]

Example 9

This Example describes the preparation of a [0160] M. catarrhalis genomic library.
Chromosomal DNA was isolated as follows: [0161]
An [0162] M. catarrhalis cell pellet was resuspended in 20 mL of Tris-EDTA (TE) buffer, pH 7.5. Pronase (final concentration 500 μg/mL) and SDS (final concentration 1%) were added and the suspension was incubated at 37° C. for 2 hours. DNA was isolated by sequential extractions once with phenol, twice with phenol-chloroform (1:1), and once with chloroform-isoamyl alcohol (24:1). Extracted DNA was dialyzed against 1M NaCl at 4° C. for 4 hours. This was followed by dialysis against TE buffer, pH 7.5, at 4° C. for 48 hours (3 buffer changes). DNA was ethanol precipitated from the dialysate. Large-size DNA was collected by spooling on a glass rod, air dried and dissolved in 3 mL water. Small scale Sau3A (New England BioLabs) restriction digests of chromosomal DNA (final volume 10 μl) were done to establish conditions required to obtain maximal amounts of chromosomal DNA with a size range of 15-23 kb. Large scale digests were prepared once the optimal digestion conditions were determined. The large scale digests consisted of 50 μL of chromosomal DNA (290 μg/mL), 33 μL water, 10 μL Sau3A buffer (New England BioLabs), 1 μL BSA (10 mg/ml, New England BioLabs) and 6.3 μL Sau3A (0.04 U/μL), and were incubated at 37° C. for 15 min. Reactions were stopped by the addition of 10 μL 10× loading buffer (100 mM Tris- HCl pH 8, 10 mM EDTA, 0.1% bromophenol blue, 50% glycerol). Digested DNA was applied to 0.5% agarose gels (prepared in Tris-acetate-EDTA (TAE)) and separated according to size at 50 V for 6 hours. The region of the gel encompassing DNA of size 15-23 kb was cut from the gel and placed in dialysis tubing (BRL) with 3 mL of TAE. DNA was electroeluted from the gel-slice overnight at a field strength of 1 V/cm. Electroeluted DNA in TAE was extracted once with phenol, once with phenol-chloroform (1:1), and precipitated with ethanol. The dried DNA pellet was dissolved in 5 μL water. Size-fractionated chromosomal DNA was ligated with BamHI cut EMBL3 arms (Promega) using T4 DNA ligase in a final volume of 9 μL. The entire ligation reaction was packaged into phage λ using a commercial packaging kit (Amersham) following the manufacturer's protocol.
The packaged DNA library was amplified on solid medium. This was accomplished by incubating 0.1 ml [0163] E. coli strain NM539 plating cells suspended in 10 mM MgSO₄with 15-25 μL of the packaged DNA library at 37° C. for 15 minutes. Bacteria with adsorbed phage were plated onto BBL plates (10 g BBL trypticase peptone, 5 g NaCl and 15 g agar per litre) using 3 mL of BBL top-agarose (same as BBL plates except agar replaced with 0.6% agarose) and plates were incubated overnight at 37° C. Phage were eluted from the top-agarose by adding 3 mL SM buffer (50 mM Tris-HCl, pH 7.5, 8 mM MgSO₄, 100 mM NaCl, 0.01% gelatin) to the plates and leaving them at 4° C. for 7 hours. SM buffer containing phage was collected from the plates, transferred to a screwcap tube and stored at 4° C. over chloroform.

Example 10

This Example describes the cloning of a gene encoding the [0164] M. catarrhalis 200 kDa outer membrane protein.
The [0165] M. catarrhalis genomic library in phage lambda EMBL3 was screened using an anti-200 kDa protein guinea pig antiserum. A lambda phage clone 8II, which expressed an about 200 kDa protein, was confirmed by immunoblotting of the phage lysate using the about 200 kDa outer membrane-specific antiserum.
Plate lysate cultures of this recombinant phage were prepared. The DNA was extracted from the plate lysates using a Wizard Lambda Preps DNA Purification System (Promega Corp, Madison, Wis.) according to the manufacturer's instructions. This phage clone carried a DNA insert of about 16 kb in size (the restriction map for which is shown in FIG. 5). The phage DNA was digested with a mixture of the restriction enzymes SalI and XhoI, and separated by agarose gel electrophoresis. Two DNA bands, approximately 5 kb and 11 kb in size, respectively, were cut out from the gel and extracted using a Geneclean kit (BIO 101 Inc., LaJolla, Calif.) according to the manufacturer's direction. [0166]
The smaller 5 kb fragment was ligated into a plasmid vector, pBluescript II SK +/− (Stratagene Cloning Systems, LaJolla, Calif.), which had been previously digested with SalI and XhoI, to produce plasmid pKS5. The larger 11 kb fragment was ligated into a plasmid vector, pSP72 (Promega Corp., Madison, Wis.), to produce plasmid pKS9. Both ligated plasmids were used to transform [0167] E. coli, strain DH5a.
The lambda phage DNA was also digested with a mixture of XhoI and KpnI and the approximately 1.2 kb fragment was isolated after agarose gel separation as described above. This 1.2 kb fragment was ligated into a plasmid vector, pGEM-7Zf(+) (Promega Corp., Madison, Wis.), to produce plasmid pKS47. Restriction maps of the plasmid clones are shown in FIG. 5. [0168]

Example 11

This Example describes the sequencing of the gene encoding the about 200 kDa outer membrane protein of [0169] M. catarrhalis.
The gene encoding the about 200 kDa outer membrane protein was sequenced using an Applied Biosystems sequencer. The one strand of the insert in the plasmid pKS5, was sequenced after construction of a nested set of deletions using a Erase-a-Base system (Promega Corp., Madison, Wis.). The plasmid pKS5 was first digested with XhoI and KpnI, treated with exonuclease III to generate a nested set of deletions in the insert and then recircularized according to the manufacturer's directions. [0170] E. coli DH5a was transformed with a series of plasmids with deletions generated in this way. Plasmids were isolated from the transformants using a Quiagen midi plasmid isolation kit (Qiagen) and the size of plasmids examined by agarose gel electrophoresis after restriction enzyme digestion. The inserts of the plasmids with deletions were sequenced using a bacteriophage T7 promoter sequence as a primer.
Based upon the sequence, nucleotide primers were synthesized. Using the synthetic nucleotide primers, sequence gaps, which were not sequenced by the Erase-a Base system, were determined. [0171]
The sequences of the inserts in plasmids pKS47 and pKS71 were determined from both ends using synthetic nucleotide primers. The nucleotide sequence of the gene has an open reading frame of the gene coding for the about 200 kDa outer membrane protein of [0172] M. catarrhalis as shown in FIG. 6 (SEQ ID No: 2). This sequence included a nucleotide sequence:

(SEQ ID NO: 9)

5′-AATGTCAAATCAGTCATTAACAAAGAACAAGTAAATGATGCCAATAA

AAAGCAAGGCATC-3′
which encodes the internal amino acid sequence of the about 200 kDa outer membrane protein (SEQ ID No: 5) determined above. This result confirms that the cloned gene has an open reading frame of the gene coding for the about 200 kDa outer membrane protein of [0173] M. catarrhalis. The gene encodes a protein having 1992 amino acids, a calculated molecular weight of 204,677 and a calculated amino acid composition as shown in Table III below. The deduced amino acid sequence of the protein is shown in FIG. 6 (SEQ ID No: 3).

Example 12

This Example describes the identification of the start codon of the gene encoding the about 200 kDa gene of [0174] M. catarrhalis.
To identify the translation start codon and the promoter region of the 200 kDa protein gene, a plasmid, pKS80, was constructed from pKS5 and pKS47 (FIG. 5). This construct contained about 250 base pairs of DNA upstream from the ATG. The plasmid, pKS5, was digested with KpnI and XhoI. The digest was separated on 0.8% agarose gel and the about 8 kb DNA fragment was cut out from the gel and extracted. Another plasmid, pKS47, was also digested with the two enzymes and the about 1.1 kb DNA fragment was extracted. The 1.1 kb fragment was ligated to the 8 kb fragment to construct pKS80. Western blots using anti-200 kD protein guinea pig serum failed to detect 200 kD protein in the lysates of the transformants carrying pKS80. [0175]
To examine if the construct was too long to be expressed in [0176] E. coli, three different sizes of C-terminal truncations were constructed, as shown in FIG. 8. First, the whole insert in pKS80 was cut out by digestion with KpnI and BamHI and then inserted into another vector plasmid, pGEM7Zf(+) (Promega, Madison Wis.), which had been previously digested with the same two enzymes. The resulting plasmid, pKS105, was further digested with either one of the following enzymes, (1) HindIII, (2) HpaI and SmaI or (3) EcoRV, gel-purified and then recircularized to produce pKS130, pKS136 and pKS144, respectively. Transformants of E. coli, DH5a, with either one of pKS130, pKS136 or pKS144 did not produce any truncated proteins, when examined on Western blots using anti-200 kD protein guinea pig serum.
Next, to investigate if the start codon was GTG and if the promoter region was further upstream from the GTG, an about 0.9 kb fragment was cut out from pKS71 using ApaI and KpnI, and ligated into pKS130, pKS136 and pKS144, which had been previously digested with ApaI and KpnI. The 0.9 kb fragment from pKS71 carried the NcoI-KpnI fragment, which contained the possible start codon, GTG, and about 700 bp upstream region from the GTG (FIG. 8). The resulting constructs, pKS159, pKS149 and pKS155, produced truncated proteins, which were recognized by anti-200 kDa protein guinea pig serum on Western blots. The ApaI and KpnI fragment was also ligated to pKS105, which had no C-terminal truncation, to produce pKS164. The transformants carrying pKS164 produced a full-[0177] length 200 kDa protein, which was recognized by the same antiserum on Western blot. These results show that the 5′-region of the gene containing the GTG codon and its upstream sequence is necessary for expression of the about 200 kDa protein gene from its own promoter in E. coli, and indicate that a translation start codon of the about 200 kDa protein gene is GTG.
To confirm that the start codon of the gene is GTG, two peptides were synthesized, as shown in FIG. 9, according to the deduced amino acid sequence from the nucleotide sequence in FIG. 6. Peptide 1 (SEQ ID No: 12) encompasses the 30 amino acids from the GTG start codon. Peptide 2 (SEQ ID No: 12) is the next 30 amino acid peptide. The peptides are identified in FIG. 6 by underlining. Antisera were raised against these two peptides in guinea pigs and antisera were obtained. As seen in FIG. 10, antisera raised against these two peptides clearly recognized 200 kDa protein from [0178] M. catarrhalis, strain 4223, by Western blotting. M. catarrhalis, strain 4223, was sonicated. Proteins in the sonicate were separated on a SDS-PAGE gel and transferred to PVDF membrane. The membrane was cut into strips and treated with either anti-peptide 1 or anti-peptide 2 guinea pig serum as a first antibody. The second antibody was goat anti-guinea pig IgG conjugated with horse radish peroxidase (Jackson ImmunoResearch Lab. Inc., West Grove, Pa.). The membrane was finally treated with CN/DAB substrate (Pierce, Rockford, Ill.) for color development. Lane 1: prestained molecular weight marker, Lane 2: anti-200 kD protein serum, Lane 3: anti-peptide I serum from guinea pig No. 1, Lane 4: prebleed serum from guinea pig No. 1, Lane 5: anti-peptide 1 serum from guinea pig No. 2, Lane 6: prebleed serum from guinea pig No. 2, Lane 7: anti-peptide 2 serum from guinea pig No. 3, Lane 8: prebleed serum from guinea pig No. 3, Lane 9: anti-peptide 2 serum from guinea pig No. 4, Lane 10: prebleed serum from guinea pig No. 4. The results shown in FIG. 10 indicate that the GTG is the translation start codon of the gene encoding the about 200 kDa protein.
The coding sequence of the about 200 kDa protein gene, which starts at GTG, is 5976 bp and encodes a protein of 1992 amino acids and a calculated molecular weight of 204,677. The position of the 200 kDa protein gene is shown in FIG. 5. The sequence between NcoI and SalI and its amino acid translation are shown in FIG. 6. The calculated amino acid composition of the about 200 kDa protein is shown in Table III. [0179]
To construct two different sizes of N-terminal truncation genes under the control of the T7 promoter (as shown in FIG. 11), a ScaI-SalI fragment, which carried the about 1.9 [0180] kb 3′-region of the about 200 kDa protein gene, was cut out from pKS5, and the PvuII-SalI fragment, which carried the about 4.8 kb 3′-region, was cut out from pKS80. The two fragments were ligated into a plasmid, pT7-7, previously digested with SmaI and SalI, to produce pKS94 and pKS91, respectively. These ligations resulted in fusions of 1.9 kb and 4.8 kb 3′-regions with seven N-terminal amino acids from the vector. When transformants of an E. coli strain, BL21(DE3)/pLysS, with either pKS94 or pKS91 were induced with IPTG, they produced a large quantity of N-terminally truncated 200 kDa protein. FIG. 12 shows a Western blot showing the expression of the truncated protein by one of transformants carrying the pKS94 plasmid.
A LacZ fusion of the 3′-5.5 kb fragment of the about 200 kDa protein gene, as shown in FIG. 11. The 5.8 kb fragment, which contained the 3′-5.5 kb region of about 200 kDa protein gene, was excised from pKS80 by digestion with PstI, gel-purified, and then ligated to pGEM5Zf(+) (Promega, Madison, Wis.), previously digested with the same enzyme. The [0181] E. coli DH5a clones, which carried the gene in the same direction and reading frame as the LacZ a peptide, were selected by restriction enzyme analyses. These clones constitutively expressed the fusion protein, as shown in FIG. 13.

SUMMARY OF THE DISCLOSURE

In summary of the disclosure, the present invention provides an isolated and purified outer membrane protein of a Moraxella strain, particularly [0182] 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.

TABLE I

Presence of the about 200 kDa outer membrane protein

in various isolates of Moraxella catarrhalis

Number of Number of isolates ^1.

Type of Clinical isolates containing the 200 kDa

Isolate Examined outer membrane protein

Otitis Media 37 36

Sputum/Expectoration/ 13 6

Bronchial

Secretion

Blood

2 2

Nasopharynx 1 1

Unknown 1 0

TABLE II


Detection of about 200 kDa outer membrane protein
of M. catarrhalis by the monospecific anti-200
kDa outer membrane guinea pig antiserum

Strain	Sample	Reciprocal Reactive Titre

4223	Whole cells not fixed	800
RH408	Whole cells not fixed	<200
H12	Whole cells not fixed	<200
E. coli BL21	Whole cells not fixed	<200
4223	Whole cells fixed	3200
RH408	Whole cells fixed	200
H12	Whole cells fixed	<200
E. coli BL21	Whole cells fixed	<200
4223	Sonicate	12,800
RH408	Sonicate		800
H12	Sonicate		800
E. coli BL21	Sonicate		200

TABLE III


Amino acid composition of the about 200 kDa
outer membrane protein of M. catarrhalis

	Residue	Number	Percentage (MW)

N - Asparagine	196	10.9
T - Threonine	221	10.9
K - Lysine	159	10.0
D - Aspartic Acid	147	8.3
A - Alanine	219	7.6
V - Valine	148	7.2
I - Isoleucine	116	6.4
S - Serine	150	6.4
G - Glycine	222	6.2
L - Leucine	111	6.1
Q - Glutamine	83	5.2
E - Glutamic Acid	55	3.5
F - Phenylalanine	40	2.9
R - Arginine	34	2.6
Y - Tyrosine	27	2.2
H - Histidine	24	1.6
P - Proline	30	1.4
M - Methionine	7	.4
W - Tryptophan	3	.3
B - Aspartic Acid Asparagine	0	.0
C - Cysteine	0	.0

REFERENCES

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1 10 1 6973 DNA Moraxella catarrhalis CDS (708)..(6683) 1 ccatggatat gggcaggtgt gctcgcctgc cgtatgatgg cgatgacacc ccatttgccc 60 catatctgta cgatttgaca tgtgatatga tttaacatgt gacatgattt aacattgttt 120 aatactgttg ccatcattac cataatttag taacgcattt agtaacgcat ttgtaaaaat 180 cattgcgccc ctttatgtgt atcatatgaa tagaatatta tgattgtatc tgattattgt 240 atcagaatgg tgatgctata tgatgatgcc tacgagttga tttgggttaa tcactctatg 300 atttgatata ttttgaaact aatctattga cttaaatcac catatggtta taatttagca 360 taatggtagg ctttttgtaa aaatcacatc gcaatattgt tctactgtta ctaccatgct 420 tgaatgacga tcccaatcac cagattcatt caagtgatgt gtttgtatac gcaccattta 480 ccctaattat ttcaatcaaa tgcctatgtc agcatgtatc atttttttaa ggtaaaccac 540 catgaatcac atctataaag tcatctttaa caaagccaca ggcacattta tggcagtggc 600 agagtacgcc aaatcccaca gcacgggggg ggggtagctg tgctacaggg caagttggca 660 gtgtatgcac tctgagcttt gcccgtattg ccgcgctcgc tgtcctc gtg atc ggt 716 Met Ile Gly 1 gca acg ctc agt ggc agt gct tat gct caa aaa aaa gat acc aaa cat 764 Ala Thr Leu Ser Gly Ser Ala Tyr Ala Gln Lys Lys Asp Thr Lys His 5 10 15 atc gca att ggt gaa caa aac cag cca aga cgc tca ggc act gcc aag 812 Ile Ala Ile Gly Glu Gln Asn Gln Pro Arg Arg Ser Gly Thr Ala Lys 20 25 30 35 gcg gac ggt gat cga gcc att gct att ggt gaa aat gct aac gca cag 860 Ala Asp Gly Asp Arg Ala Ile Ala Ile Gly Glu Asn Ala Asn Ala Gln 40 45 50 ggc ggt caa gcc atc gcc atc ggt agt agt aat aaa act gtc aat gga 908 Gly Gly Gln Ala Ile Ala Ile Gly Ser Ser Asn Lys Thr Val Asn Gly 55 60 65 agc agt ttg gat aag ata ggt acc gat gct acg ggt caa gag tcc atc 956 Ser Ser Leu Asp Lys Ile Gly Thr Asp Ala Thr Gly Gln Glu Ser Ile 70 75 80 gcc atc ggt ggt gat gta aag gct agt ggt gat gcc tcg att gcc atc 1004 Ala Ile Gly Gly Asp Val Lys Ala Ser Gly Asp Ala Ser Ile Ala Ile 85 90 95 ggt agt gat gac tta cat ttg ctt gat cag cat ggt aat cct aaa cat 1052 Gly Ser Asp Asp Leu His Leu Leu Asp Gln His Gly Asn Pro Lys His 100 105 110 115 ccg aaa ggt act ctg att aac gat ctt att aac ggc cat gca gta tta 1100 Pro Lys Gly Thr Leu Ile Asn Asp Leu Ile Asn Gly His Ala Val Leu 120 125 130 aaa gaa ata cga agc tca aag gat aat gat gta aaa tat aga cgc aca 1148 Lys Glu Ile Arg Ser Ser Lys Asp Asn Asp Val Lys Tyr Arg Arg Thr 135 140 145 acc gca agc gga cac gcc agt act gca gtg gga gcc atg tca tat gca 1196 Thr Ala Ser Gly His Ala Ser Thr Ala Val Gly Ala Met Ser Tyr Ala 150 155 160 cag ggt cat ttt tcc aac gcc ttt ggt aca cgg gca aca gct aaa agt 1244 Gln Gly His Phe Ser Asn Ala Phe Gly Thr Arg Ala Thr Ala Lys Ser 165 170 175 gcc tat tcc ttg gca gtg ggt ctt gcc gcc aca gcc gag ggc caa tct 1292 Ala Tyr Ser Leu Ala Val Gly Leu Ala Ala Thr Ala Glu Gly Gln Ser 180 185 190 195 aca atc gct att ggt tct gat gca aca tct agc tcg ttg gga gcg ata 1340 Thr Ile Ala Ile Gly Ser Asp Ala Thr Ser Ser Ser Leu Gly Ala Ile 200 205 210 gcc ctt ggt gca ggt act cgt gct cag cta cag ggc agt att gcc cta 1388 Ala Leu Gly Ala Gly Thr Arg Ala Gln Leu Gln Gly Ser Ile Ala Leu 215 220 225 ggt caa ggt tct gtt gtc act cag agt gat aat aat tct aga ccg gcc 1436 Gly Gln Gly Ser Val Val Thr Gln Ser Asp Asn Asn Ser Arg Pro Ala 230 235 240 tat aca cca aat acc cag gca cta gac ccc aag ttt caa gcc acc aat 1484 Tyr Thr Pro Asn Thr Gln Ala Leu Asp Pro Lys Phe Gln Ala Thr Asn 245 250 255 aat acg aag gcg ggt cca ctt tcc att ggt agt aac tct atc aaa cgt 1532 Asn Thr Lys Ala Gly Pro Leu Ser Ile Gly Ser Asn Ser Ile Lys Arg 260 265 270 275 aaa atc atc aat gtc ggt gca ggt gtt aat aaa acc gat gcg gtc aat 1580 Lys Ile Ile Asn Val Gly Ala Gly Val Asn Lys Thr Asp Ala Val Asn 280 285 290 gtg gca cag cta gaa gcg gtg gtg aag tgg gct aag gag cgt aga att 1628 Val Ala Gln Leu Glu Ala Val Val Lys Trp Ala Lys Glu Arg Arg Ile 295 300 305 act ttt cag ggt gat gat aac agt act gac gta aaa ata ggt ttg gat 1676 Thr Phe Gln Gly Asp Asp Asn Ser Thr Asp Val Lys Ile Gly Leu Asp 310 315 320 aat act tta act att aaa ggt ggt gca gag acc aac gca tta acc gat 1724 Asn Thr Leu Thr Ile Lys Gly Gly Ala Glu Thr Asn Ala Leu Thr Asp 325 330 335 aat aat atc ggt gtg gta aaa gag gct gat aat agt ggt ctg aaa gtt 1772 Asn Asn Ile Gly Val Val Lys Glu Ala Asp Asn Ser Gly Leu Lys Val 340 345 350 355 aaa ctt gct aaa act tta aac aat ctt act gag gtg aat aca act aca 1820 Lys Leu Ala Lys Thr Leu Asn Asn Leu Thr Glu Val Asn Thr Thr Thr 360 365 370 tta aat gcc aca acc aca gtt aag gta ggt agt agt agt agt act aca 1868 Leu Asn Ala Thr Thr Thr Val Lys Val Gly Ser Ser Ser Ser Thr Thr 375 380 385 gct gaa tta ttg agt gat agt tta acc ttt acc cag ccc aat aca ggc 1916 Ala Glu Leu Leu Ser Asp Ser Leu Thr Phe Thr Gln Pro Asn Thr Gly 390 395 400 agt caa agc aca agc aaa acc gtc tat ggc gtt aat ggg gtg aag ttt 1964 Ser Gln Ser Thr Ser Lys Thr Val Tyr Gly Val Asn Gly Val Lys Phe 405 410 415 act aat aat gca gaa aca aca gca gca atc ggc act act cgt att acc 2012 Thr Asn Asn Ala Glu Thr Thr Ala Ala Ile Gly Thr Thr Arg Ile Thr 420 425 430 435 aga gat aaa att ggc ttt gct cga gat ggt gat gtt gat gaa aaa caa 2060 Arg Asp Lys Ile Gly Phe Ala Arg Asp Gly Asp Val Asp Glu Lys Gln 440 445 450 gca cca tat ttg gat aaa aaa caa ctt aaa gtg ggt agt gtt gca att 2108 Ala Pro Tyr Leu Asp Lys Lys Gln Leu Lys Val Gly Ser Val Ala Ile 455 460 465 acc ata gac aat ggc att gat gca ggt aat aaa aag atc agt aat ctt 2156 Thr Ile Asp Asn Gly Ile Asp Ala Gly Asn Lys Lys Ile Ser Asn Leu 470 475 480 gcc aaa ggt agc agt gct aac gat gcg gtt acc atc gaa cag ctc aaa 2204 Ala Lys Gly Ser Ser Ala Asn Asp Ala Val Thr Ile Glu Gln Leu Lys 485 490 495 gcc gcc aag cct act tta aac gca ggc gct ggc atc agt gtc aca cct 2252 Ala Ala Lys Pro Thr Leu Asn Ala Gly Ala Gly Ile Ser Val Thr Pro 500 505 510 515 act gaa ata tca gtt gat gct aag agt ggc aat gtt acc gcc cca act 2300 Thr Glu Ile Ser Val Asp Ala Lys Ser Gly Asn Val Thr Ala Pro Thr 520 525 530 tac aac att ggc gtg aaa acc acc gag ctt aac agt gat ggc act agt 2348 Tyr Asn Ile Gly Val Lys Thr Thr Glu Leu Asn Ser Asp Gly Thr Ser 535 540 545 gat aaa ttt agt gtt aag ggt agt ggt acg aac aat agc tta gtt acc 2396 Asp Lys Phe Ser Val Lys Gly Ser Gly Thr Asn Asn Ser Leu Val Thr 550 555 560 gcc gaa cat ttg gca agc tat cta aat gaa gtc aat cga acg gct gac 2444 Ala Glu His Leu Ala Ser Tyr Leu Asn Glu Val Asn Arg Thr Ala Asp 565 570 575 agt gct cta caa agc ttt acc gtt aaa gaa gaa gac gat gat gac gcc 2492 Ser Ala Leu Gln Ser Phe Thr Val Lys Glu Glu Asp Asp Asp Asp Ala 580 585 590 595 aac gct atc acc gtg gct aaa gat acg aca aaa aat gcc ggc gca gtc 2540 Asn Ala Ile Thr Val Ala Lys Asp Thr Thr Lys Asn Ala Gly Ala Val 600 605 610 agc atc tta aaa ctc aaa ggt aaa aac ggt cta acg gtt gct acc aaa 2588 Ser Ile Leu Lys Leu Lys Gly Lys Asn Gly Leu Thr Val Ala Thr Lys 615 620 625 aaa gat ggt acg gtt acc ttt ggg ctt agc caa gat agc ggt ctg acc 2636 Lys Asp Gly Thr Val Thr Phe Gly Leu Ser Gln Asp Ser Gly Leu Thr 630 635 640 att ggc aaa agc acc cta aac aac gat ggc ttg act gtt aaa gat acc 2684 Ile Gly Lys Ser Thr Leu Asn Asn Asp Gly Leu Thr Val Lys Asp Thr 645 650 655 aac gaa caa atc caa gtc ggt gct aat ggc att aaa ttt act aat gtg 2732 Asn Glu Gln Ile Gln Val Gly Ala Asn Gly Ile Lys Phe Thr Asn Val 660 665 670 675 aat ggt agt aat cca ggt act ggc att gca aat acc gct cgc att acc 2780 Asn Gly Ser Asn Pro Gly Thr Gly Ile Ala Asn Thr Ala Arg Ile Thr 680 685 690 aga gat aaa att ggc ttt gct ggt tct gat ggt gca gtt gat aca aac 2828 Arg Asp Lys Ile Gly Phe Ala Gly Ser Asp Gly Ala Val Asp Thr Asn 695 700 705 aaa cct tat ctt gat caa gac aag cta caa gtt ggc aat gtt aag att 2876 Lys Pro Tyr Leu Asp Gln Asp Lys Leu Gln Val Gly Asn Val Lys Ile 710 715 720 acc aac act ggc att aac gca ggt ggt aaa gcc atc aca ggg ctg tcc 2924 Thr Asn Thr Gly Ile Asn Ala Gly Gly Lys Ala Ile Thr Gly Leu Ser 725 730 735 cca aca ctg cct agc att gcc gat caa agt agc cgc aac ata gaa ctg 2972 Pro Thr Leu Pro Ser Ile Ala Asp Gln Ser Ser Arg Asn Ile Glu Leu 740 745 750 755 ggc aat aca atc caa gac aaa gac aaa tcc aac gct gcc agc att aat 3020 Gly Asn Thr Ile Gln Asp Lys Asp Lys Ser Asn Ala Ala Ser Ile Asn 760 765 770 gat ata tta aat aca ggc ttt aac cta aaa aat aat aac aac ccc att 3068 Asp Ile Leu Asn Thr Gly Phe Asn Leu Lys Asn Asn Asn Asn Pro Ile 775 780 785 gac ttt gtc tcc act tat gac att gtt gac ttt gcc aat ggc aat gcc 3116 Asp Phe Val Ser Thr Tyr Asp Ile Val Asp Phe Ala Asn Gly Asn Ala 790 795 800 acc acc gcc aca gta acc cat gat acc gct aac aaa acc agt aaa gtg 3164 Thr Thr Ala Thr Val Thr His Asp Thr Ala Asn Lys Thr Ser Lys Val 805 810 815 gta tat gat gtg aat gtg gat gat aca acc att cat cta aca ggc act 3212 Val Tyr Asp Val Asn Val Asp Asp Thr Thr Ile His Leu Thr Gly Thr 820 825 830 835 gat gac aat aaa aaa ctt ggc gtc aaa acc acc aaa ctg aac aaa aca 3260 Asp Asp Asn Lys Lys Leu Gly Val Lys Thr Thr Lys Leu Asn Lys Thr 840 845 850 agt gct aat ggt aat aca gca act aac ttt aat gtt aac tct agt gat 3308 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 DNA 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 PRT 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 PRT 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 20 PRT Moraxella catarrhalis UNSURE (17) 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 PRT 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 DNA Moraxella catarrhalis 7 aatgtcaaat cagtcattaa caaagaacaa gtaaatgatg ccaataaaaa gcaaggcatc 60 8 20 PRT 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 PRT 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 PRT 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

Claims

What we claim is:

1. An isolated and purified outer membrane protein of a Moraxella strain having an apparent molecular mass of about 200 kDa, as determined by SDS-PAGE or a fragment or an analog thereof.

2. The protein of claim 1 wherein the Moraxella strain is Moraxella catarrhalis.

3. The protein of claim 2 wherein the strain is Moraxella catarrhalis 4223.

4. The protein of claim 1 containing the amino acid sequence NH₂-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-Lys-Gln-Gly-Ile (SEQ ID No: 10) for Moraxella catarrhalis strain 4223 or containing the corresponding amino acid sequence from other Moraxella strains.

5. The protein of claim 1 which is at least about 70 wt % pure.

6. The protein of claim 5 which is at least about 95 wt % pure.

7. The protein of claim 1 in the form of an aqueous solution thereof.

8. The protein of claim 1 recognizable by an antibody preparation specific for a peptide having the amino acid sequence of NH₂-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-Lys-Gln-Gly-Ile (SEQ ID No: 10).

9. The protein of claim 1 having substantially the amino acid composition as shown in Table III.

10. 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.

11. The nucleic acid molecule of claim 10, wherein the strain of Moraxella is a strain of Moraxella catarrhalis.

12. The nucleic acid molecule of claim 11, wherein the strain is Moraxella catarrhalis 4223.

13. The nucleic acid molecule of claim 10, wherein the encoded protein contains the amino acid sequence NH₂-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-Lys-Gln-Gly-Ile (SEQ ID No: 10) for Moraxella catarrhalis strain 4223 or contains the corresponding amino acid sequence from other Moraxella strains.

14. 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 and 2), or the complementary sequence thereto;

(b) a DNA sequence encoding a 200 kDa protein of a strain of Moraxella and containing the amino acid sequence NH₂-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-Lys-Gln-Gly-Ile (SEQ ID No: 10), or the complementary sequence thereto;

(c) a DNA sequence encoding a deduced amino acid sequence as set out in FIG. 6 (SEQ ID No: 3), or the complimentary sequence thereto; and

(d) a nucleotide sequence which hybridizes under stringent conditions to any one of the sequences defined in (a), (b) or (c).

15. The nucleic acid molecule of claim 14, wherein the nucleotide sequence defined in (d) has at least about 90% sequence identity with any one of the sequences defined in (a), (b) or (c).

16. A vector adapted for transformation of a host comprising the nucleic acid molecule of claim 10 or 14.

17. An expression vector adapted for transformation of a host comprising the nucleic acid molecule of claim 10 or 14 and expression means operatively coupled to the nucleic acid molecule for expression by the host of said outer membrane protein of a strain of Moraxella or the fragment or the analog of the outer membrane protein.

18. The expression vector of claim 17, wherein the expression means includes 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.

19. The expression vector of claim 17, wherein the expression means includes 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 the analog of the outer membrane protein.

20. A transformed host containing an expression vector as claimed in claim 17.

21. A recombinant outer membrane protein or fragment or analog thereof producible by the transformed host of claim 20.

22. A live vector for delivery of an outer membrane protein of a strain of Moraxella having a molecular weight of about 200 kDa or a fragment or analog thereof to a host, comprising a vector containing the nucleic acid molecule of claim 10 or 14.

23. The live vector of claim 21, wherein the vector is selected from the group consisting of E. coli, Salmonella, Mycobacteria, adenovirus, poxyirus, vaccinia and poliovirus.

24. 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 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 of an analog of the outer membrane protein.

25. The peptide of claim 24, wherein the Moraxella strain is a Moraxella catarrhalls strain.

26. The peptide of claim 25, wherein the strain is Moraxella catarrhalis 4223.

27. The peptide of claim 24 having the amino acid sequence NH₂-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-x-Gln-Gly-Ile (SEQ ID No: 5) or NH₂-Asn-Val-Lys-Ser-Val-lle-Asn-Lys-GlU-Gln-Val-Asn-Asp-Ala-Asn-Lys (SEQ ID No: 6) for the Moraxella catarrhalis 4223 strain or the amino acid sequence for the corresponding peptide for other strains of Moraxella.

28. An immunogenic composition, comprising at least one active component selected from the group consisting of:

(A) an isolated and purified outer membrane protein of a Moraxella strain having a molecular mass of about 200 kDa, as determined by SDS-PAGE, or a fragment or an analog thereof;

(B) 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 thereof;

(C) a purified and isolated nucleic acid molecule having a sequence selected from the group consisting of:

(a) a DNA sequence set out in FIG. 6 (SEQ ID No: 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 NH₂-Asn-Val-Lys-Ser-Val-Ile-Asn-Lys-Glu-Gln-Val-Asn-Asp-Ala-Asn-Lys-Lys-Gln-Gly-Ile (SEQ ID No: 10), or the complementary sequence thereto;

(c) a DNA sequence encoding an amino acid sequence as set forth in FIG. 6 (SEQ ID No: 3), or the complimentary sequence thereto; and

(d) a nucleotide sequence which hybridizes under stringent conditions to any one of the sequences defined in (a), (b) or (c);

(D) a recombinant outer membrane protein or fragment or analog thereof producible in a transformed host containing an expression vector comprising a nucleic acid molecule as defined in (B) or (C) and expression means operatively coupled to the nucleic acid molecule for expression by the host of the recombinant outer membrane protein or fragment or analog thereof;

(E) a live vector, comprising a vector containing a purified and isolated nucleic acid molecule encoding a protein of a strain of Moraxella having a molecular mass of about 200 kDa, as determined by SDS-PAGE, or a fragment or analog thereof;

(F) a live vector, comprising a vector containing 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 No: 1), or the complementary sequence thereto;

(d) a nucleotide sequence which hybridizes under stringent conditions to any one of the sequences defined in (a), (b) or (c); and

(G) 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 an outer membrane protein of a strain of Moraxella having a molecular mass of about 200 kDa, as determined by SDS-PAGE, or of an analog of the outer membrane protein; and

a pharmaceutically acceptable carrier therefor, said at least one active component producing an immune response when administered to a host.

29. The immunogenic composition of claim 28 formulated as a vaccine for in vivo administration to a host to confer protection against disease caused by a strain of Moraxella.

30. The immunogenic composition of claim 29 wherein the strain is Moraxella catarrhalis.

31. The immunogenic composition of claim 29 formulated as a microparticle, capsule, ISCOM, or liposome preparation.

32. The immunogenic composition of claim 29 in combination with a targeting molecule for delivery to specific cells of the immune system or to mucosal surfaces.

33. The immunogenic composition of claim 29 further comprising at least one other immunogenic or immunostimulating material.

34. The immunogenic composition of claim 33 wherein the at least one other immunostimulating material is at least one adjuvant.

35. The immunogenic composition of claim 34 wherein the at least one adjuvant is selected from the group consisting of aluminum phosphate, aluminum hydroxide, QS21, Quil A, derivatives and components thereof, ISCOM matrix, calcium phosphate, calcium hydroxide, zinc hydroxide, a glycolipid analog, an octodecyl ester of an amino acid, a muramyl dipeptide, polyphosphazene, ISCOPREP, DC-chol, DDBA and a lipoprotein.

36. The immunogenic composition of claim 35 wherein the host is a primate.

37. The immunogenic composition of claim 36 wherein the primate is a human.