Title of Invention

COMPOUNDS FROM MORAXELLA CATARRHALIS

Abstract The invention provides BASB020 polypeptides and polynucleotides encoding BASB020 polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are diagnostic, prophylactic and therapeutic uses.
Full Text

COMPOUNDS FROM MORAXELLA CATARRHALIS
FIELD OF THE INVENTION
This invention relates to polynucieotides, (herein referred to as 'BASB020 polynucleotide(s)"). polypeptides encoded by them (referred to herein as 'BASB020" or "BASB020 poiypeptide(s), recombinant materials and methods for their production. In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including vaccines against bacterial infections. In a further aspect, the invention relates to diagnostic assays for detecting infection of certain pathogens.
BACKGROUND OF THE INVENTION
.VIoraxelia catarrhalis (also named Branhamella catarrhalis) is a Gram-negaiive bacteria frequently isolated from the human upper respiratory tract. It is responsible for several pathologies the main ones being otitis media in infants and children, and pneumonia in eideriies. It is also responsible of sinusitis, nosocomial infections and less frequently of invasive diseases.
Otitis media is an important childhood disease both by the number of cases and its potential sequelae. More than 3.5 millions cases are recorded every year in the United States, and it is estimated that 80 % of the children have experienced at least one episode of otitis before reaching the age of 3 (Klein. JO (1994) Clin.Inf.Dis 19:823). Left tmtreated, or becoming chronic, this disease may lead to hearing losses that could be temporary (in the case of fluid accumulation in the middle ear) or permanent (if the auditive nerve is damaged). In infants, such hearing losses may be responsible for a delayed speech learning.
Three bacterial species are primarily isolated from the middle ear of children with othis media: Sireptococcus pneumoniae, non tv'peable Haemophilus influenzae (NTHi) and M caiarrhalis. They are present in 60 to 90 % of the cases. A review of recent studies shows

that S. pneumoniae and NTHi represent both about 30 %, and M. catarrhalis about 15 % of the otitis media cases (Murphy, TF (1996) Microbiol.Rev, 60:267). Other bacteria could be isolated from the middle ear (//, influenzae type B. 5". pyogenes etc) but at a much lower frequency (2 % of the cases or less).
Epidemiological data indicate that, for the pathogens found in the middle ear, the colonization of the upper respiratory tract is an absolute prerequisite for the development of an otitis; other are however also required to lead to the disease (Dickinson, DP et al. (1988) J. Infect.Dis. 158:205, Faden, HL et al. (1991) Ann.OtorhinolXaryngoI. 100:612), These are important to trigger the migration of the bacteria into the middle ear via the Eustachian tubes, followed by the initiation of an inflammatory process. These factors are unknown todate. It has been posmiated that a transient anomaly of the immune system following a viral infection, for example, could cause an inability to control the colonization of the respirator}' tract (Faden, HL et al (1994) J. Infect.Dis. 169:1312). An alternative explanation is that the exposure to environmental factors allow a more important colonization of some children, who subsequently become susceptible to the development of odtis media because of the sustained presence of middle ear pathogens (Murphy, TF (1996) Microbiol.Re\'. 60:267).
The immune response to M. catarrhalis is poorly characterized. The analysis of strains isolated sequentially from the nasopharynx of babies followed from 0 to 2 years of age, indicates that they get and eliminate frequently new strains. This indicates that an efficacious immune response against this bacteria is mounted by the colonized children (Faden, HL et al (1994) J. Infect.Dis. 169:1312),
In most adults tested, bactericidal antibodies have been identified (Chapman. AJ et al. (1985) J. Infect.Dis. 151:878). Strains of M catarrhalis present variations in their capacity to resist serum bactericidal activity: in general, isolates from diseased individuals are more

resistant than those who are simply colonized (Hol Cet al, (1993) Lancec 341:1281. Jordan. Kl et al. (1990) Am J.Med. 88 (suppl. 5A):28S). Serum resistance could therfore be considered as a virulence factor of the bacteria. An opsonizing activit)' has been observed in the sera of children recovering from otitis media.
The antigens targened by these different immune responses in humans have not been identified, with the exception of OMP BL a 84 kDa protein which expression is regulated by iron, and that is recognized by the sera of patients with pneumonia (Sethi, S. et al. (1995) Infect.lmmun. 63:1516), and of UspAl and UspA2 (Chen D. et alf 1999), InfecLlmmun. 67:1310).
A few other membrane proteins present on the surface of M catarrhalis have been characterized using biochemical method, or for their potential implication in the induction of a protective immunity (for review, see Murphy, TF (1996) Microbiol.Rev. 60:267). In a mouse pneumonia model the presence of antibodies raised against some of them (UspA. CopB) favors a faster clearance of the pulmonary infection. Another polypeptide (OMP CD) is highly conserved among M caiarrhalis strains, and presents homologies with a porin of Pseudomonas aeruginosa, which has been demonstrated efficacious against this bacteritim in animal models.
The frequency of Moraxella caiarrhalis infections has risen dramatically in the past few decades. This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems. It is no longer uncommon to isolate Moraxella caiarrhalis strains that are resistant to some or all of the standard antibiotics. This phenomenon has created an unmet medical need and demand for new anti-microbial agents, vaccines, drug screening methods, and diagnostic tests for this organism.

SUMMARY OF THE INVENTION
The present invention relates to BASB020, in particular BASB020 polypeptides and B ASB020 polynucleotides, recombinant materials and methods for their production. In another aspect, the invention relates to methods for using such polypeptides and polynucleotides, including prevention and treatment of microbial diseases, amongst others. In a further aspect, the invention relates to diagnostic assays for detecting diseases associated with microbial infections and conditions associated with such infections, such as assays for detecting expression or activity of BASB020 polynucleotides or polypeptides.
Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following descriptions and from reading the other parts of the present disclosure.
DESCRIPTION OF THE INVENTION
The invention relates to BASB020 polypeptides and polynucleotides as described in greater detail below. In panicular. the invention relates to polypeptides and polynucleotides of BASB020 of Moraxella catarrhalis. which is related by amino acid sequence homology to TlyC hemolysin protein of Serpulina hyodysenteriae. The invention relates especially to BASB020 having the nucleotide and amino acid sequences set out in SEQ ID NO;L3,5 or 7 and SEQ ID NO:2,4,6 or 8 respectively. It is imderstood that sequences recited in the Sequence Listing below as "DNA" represent an exemplification of one embodiment of the invention, since those of ordinary skill will recognize that such sequences can be usefully employed in polynucleotides in general, including ribopolynucleotides.

Polypeptides
In one aspect of the invention there are provided polypeptides of Moraxella caiarrhalis referred to herein as "BASB020" and "BASB020 polypeptides" as well as biologically, diagnostically. prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
The present invention further provides for:
(a) an isolated polypeptide which comprises an amino acid sequence which has at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity, most preferably at least 97-99% or exact identity, to that of SEQ ID N0:2, 4, 6 or 8;
(b) a polypepnde encoded by an isolated polynucleotide comprising a polynucleotide sequence which has at least 85% identity preferably at least 90% identity more preferably at least 95% identity, even more preferably at least 97-99% or exact identity' to SEQ IDNO;1. 3, 5 or 7 over the entire length of SEQ ID N0:1, 3. 5 or 7 respectively; or
(c) a polypeptide encoded by an isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide which has at least 85% identity', preferably at least 90% identity, more preferably at least 95% identity, even more preferably at least 97-99% or exact identity, to the amino acid sequence of SEQ ID N0:2, 4, 6 or 8.
The BASBa20 polypeptides provided in SEQ ID NO:2,4.6 or 8 are the BASB020 polypeptides from Moraxella caiarrhalis strains MC2931 (ATCC 43617), MC2912, MC2913 and MC2969.
The invention also provides an immunogenic fragment of a BASB020 polypeptide, that is, a contiguous ponion of the BASB020 polypeptide which has the same or substantially the same immunogenic activity as the polypeptide comprising the amino acid sequence of SEQ ID NO:2,4,6 or 8; That is to say, the fragment (if necessary when coupled to a

carrier) is capable of raising an immune response which recognises the BASB020 polypeptide. Such an immunogenic fragment may include, for example, the BASB020 polypeptide lacking an N-terminal leader sequence, and/or a transmembrane domain and/or a C-terminal anchor domain. In a preferred aspect the immunogenic fragment of BASB020 according to the invention comprises substantially all of the extracellular domam of a polypeptide which has at least 85% identity, preferably at least 90% identit\', more preferably at least 95% identity, most preferably at least 97-99% identity, to that of SEQ ID NO:2.4,6 or 8 over the entire length of SEQ ID N0:2
A fragment is a polypeptide having an amino acid sequence that is entirely the same as part but not all of any amino acid sequence of any polypeptide of the invention. As with BASB020 polypeptides, fragments may be "free-standing," or composed within a larger polypeptide of which they form a part or region, most preferably as a single continuotis region in a single larger polypeptide.
Preferred fragments include, for example, truncation polypeptides having a portion of an amino acid sequence of SEQ ID NO;2.4.6 or 8 or of variants thereof, such as a continuous series of residues that includes an ammo- and/or carboxyl-terminal amino acid sequence. Degradation forms of the polypeptides of the invention produced by or in a host celL are also preferred. Further preferred are fragments characterized by strucmral or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and tum-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
Further preferred fragments include an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids from the

ammo acid sequence of SEQ ID N0:2, 4, 6 or 8. or an isolated polypeptide comprisms an amino acid sequence having at least 15, 20, 30, 40. 50 or 100 contiguous amino acids truncated or deleted from the amino acid sequence of SEQ ID N0:2, 4, 6 or 8,
Fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these fragments may be employed as intermediates for producing the ful-1ength polypeptides of the invention.
Panicularly preferred are variants in which several 5-10. 1-5, lo, 1-2 or I amino acids are substituted. deleted, or added in any combination.
The polypeptides, or immunogenic fragments, of the invention may be in the form of the '"mature" protein or may be a pan of a larger protein such as a prectirsor or a fusion protein. It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production. Furthermore, addition of exogenous polypeptide or lipid tail or polynucleotide sequences to increase the immunogenic potential of the final molecule is also considered.
In one aspect, the invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and various ponions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG. IgM, IgA. IgE). Preferred as an immunoglobulin is the constant pan of the heavy chain of human IgG, panicularly IgGl. where fusion takes place at the hinge region- In a panicular embodiment, the Fc pan can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa.

Furthermore, this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screening, diagnosis and therapy'. A further aspect of the invention also relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology can be found in International Patent Application Nos. W094/29458 and W094/22914.
The proteins may be chemically conjugated, or expressed as recombinant fusion proteins allowing increased levels to be produced in an expression system as compared to non-fused protein. The fusion panner may assist in providing T helper epitopes (immunological fusion partner), preferably T helper epitopes recognised by humans, or assist in expressing the protein (expression enhancer) at higher yields than the native recombinant protein. Preferably the fusion panner will be both an immunological fiision partner and expression enhancing partner.
Fusion panners include protein D from Haemophilus influenzae and the non-structtiral protein from influenza virus, NSl (hemagglutinin). Another fusion panner is the protein known as LytA. Preferably the C terminal portion of the molecule is used. Lyta is derived from Streptococcus pneumoniae which synthesize an N-acetyl-L-alanine amidase LytA, (coded by the lyiA gene {Gene, 43 (1986) page 265-272}) an autolysin thai specifically degrades cenain bonds in the peptidoglycan backbone. The C-terminal domain of the LytA protein is responsible for the affinity to the choline or to some choline analogues such as DEAE. This property has been exploited for the development of E.coIi C- LytA expressing plasmids useful for expression of fusion proteins. Purification of hybrid proteins containing the C- LytA fragment at its amino terminus has been described {Biotechnology: 10, (1992) page 795-798}. It is possible to use the repeat portion of the LytA molecule found in the C terminal end starting at residue 178, for example residues 188 - 305.

The present invention also includes variants of the aforementioned polypeptides, that is polypeptides that vary from the referents by conservative amino acid substitutions, whereby a residue is substituted by another with like characteristics. Typical such substitutions are among Ala, Val Leu and He; among Ser and Thr; among the acidic residues Asp and Glu: amona Asn and Gin; and amonc the basic residues Lvs and Ars; or aromatic residues Phe and Tyr.
Polypeptides of the present invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
It is most preferred that a polypepdde of the invention is derived from Moraxella caiarrhalis. however, it may preferably be obtained from other organisms of the same taxonomic genus. A polypeptide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order.
Polynucleotides
It is an object of the mvention to provide polynucleotides that encode BASB020 polypeptides, panicularly polynucleotides that encode the polypeptide herein designated BASB020,
In a panicularly preferred embodiment of the invention the polynucleotide comprises a region encoding BASB020 polypeptides comprising a sequence set out in SEQ ID NO:L3,5 or 7 which includes a hill length eene, or a variant thereof.

The BASB020 polynucleotides provided in SEQ ID NO;L3.5 or 7 are the BASB020 polynucleotides from Moraxella caiarrhalis strains MC2931 (ATCC 43617), MC2912, MC29I3 and MC2969. .
As a further aspect of the invention there are provided isolated nucleic acid molecules encoding and/or expressing BASB020 polypeptides and polynucleotides, particularly Moraxella caiarrhalis BASB020 polypeptides and polynucleotides, including, for example, unprocessed RNAs, ribozyme RNAs, mRNAs. cDNAs. genomic DNAs, B-and Z-DNAs- Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful polynucleotides and polypeptides, and variants thereof, and compositions comprising the same.
Another aspect of the invention relates to isolated polynucleoddes. including at least one full length gene, that encodes a BASB020 polypeptide having a deduced amino acid sequence of SEQ ID NO:2,4,6 or 8 and polynucleotides closely related thereto and variants thereof
In another particularly preferred embodiment of the invention there is a BASB020 polypeptide from Moraxella caiarrhalis comprising or consisting of an amino acid sequence of SEQ ID NO:2,4,6 or 8 or a variant thereof.
Using the information provided herein, such as a polynucleotide sequence set out in SEQ ID NO; 1, 3, 5 or 7, a polynucleotide of the invention encoding BASB020 polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteria using Moraxella caiarrhalis Catlin cells as starting material, followed by obtaining a full length clone. For example, to obtain a polynucleotide sequence of the invention, such as a polynucleotide sequence given in SEQ ID NO:1. 3. 5 or 7, typically a library of clones of chromosomal DNA oi Moraxella caiarrhalis Catlin in E.coli or some other suitable host is probed with a radiolabeled

oiigonucleotide, preferably a 17-mer or longer, derived from a partial sequence. Clones carrying DNA identical to that of the probe can then be distinguished using stringent hybridization conditions. By sequencing the individual clones thus identified by hybridization with sequencing primers designed from the original polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence in both directions to determine a full length gene sequence. Conveniently, such sequencing is performed, for example, using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are described by Maniatis. T.. Fritsch, E.F. and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spnng Harbor Laboratory Press. Cold Spring Harbor. New York (1989). (see in panicuiar Screening By Hybridization 1.90 and Sequencing Denattired Double-Stranded DNA Templates 13.70). Direct genomic DNA sequencmg may also be performed to obtain a full length gene sequence. Illustrative of the invention, each polynucleotide set out in SEQ ID NO: 1, 3, 5 or 7 was discovered in a DNA librarv derived from Moraxella catarrhalis.
Moreover, each DNA sequence set out in SEQ ID NO:I, 3, 5 or 7 contains an open reading frame encoding a protein having about the number of amino acid residues set forth in SEQ ID N0:2. 4. 6 or 8 with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known to those skilled in the art.
The polynucleotide of SEQ ID NO: 1, between the start codon at nucleotide number 1 and the stop codon which begins at nucleotide number 841 of SEQ ID NO: 1. encodes the polypeptide of SEQ ID N0:2,
The polynucleotide of SEQ ID N0:3, between the start codon at nucleotide number 1 and the stop codon which begins at nucleotide number 841 of SEQ ID N0:3, encodes the polypeptide of SEQ ID N0:4.

The poiynucieotide of SEQ ID N0:5, between the start codon at nucleonde number 1 and the stop codon which begins at nucleotide number 841 of SEQ ID N0:5, encodes the polypeptide of SEQ ID N0:6.
The polynucleotide of SEQ ID N0:7, between the start codon at nucleotide number I and the stop codon which begins at nucleotide number 841 of SEQ ID N0:7, encodes the polypeptide of SEQ ID N0:8.
in a further aspect, the present invention provides for an isolated polynucleotide comprising or consisting of:
(a) a polynucleotide sequence which has at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity, even more preferably at least 97-99% or exact identity to SEQ ID N0:1. 3, 5 or 7 over the entire length of SEQ ID NO:1 3. 5 or 7 respectively; or
(b) a polynucleotide sequence encoding a polypeptide which has at least 85% identity-, preferably at least 90% identity, more preferably at least 95% identity, even more preferably at least 97-99% or 100% exact, to the amino acid sequence of SEQ ID N0:2, 4, 6 or 8. over the entire length of SEQ ID N0;2, 4, 6 or 8 respectively.
A polynucleotide encoding a polypeptide of the present invention, including homologs and orthologs from species other than Moraxella caiarrhalis. may be obtained by a process which comprises the steps of screening an appropriate library under stringent hybridization conditions (for example, using a temperature in the range of 45 - 65°C and an SDS concentration from 0.1 - 1%) with a labeled or detectable probe consisting of or comprising the sequence of SEQ ID NO: 1, 3, 5 or 7 or a fragment thereof; and isolating a full-length gene and/or genomic clones containing said polynucleotide sequence.
The invention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) in SEQ ID N0:1, 3, 5 or 7. Also provided by the invention

is a coding sequence for a mature polypeptide or a fragment thereof, by itself as well as a coding sequence for a mature polypeptide or a fragment in reading frame with another coding sequence, such as a sequence encoding a leader or secretorv- sequence, a pre-, or pro-or prepro-protein sequence. The polynucleotide of the invention may also contain at least one non-coding sequence, including for example, but not limited to at least one non-coding 5' and 3' sequence, such as the transcribed but non-translated sequences, termination signals (such as rho-dependent and rho-independent termination signals), ribosome binding sites. K.ozak sequences, sequences that stabilize mRNA, introns, and polyadenylation signals. The polynucleotide sequence may also comprise additional coding sequence encoding additional amino acids. For example, a marker sequence that facilitates purification of the fused polypeptide can be encoded. In certain embodiments of the invention, the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen. Inc.) and described in Gentz et al.Proc. Nad. Acad. Sci., USA 86: 821-824 (1989), or an HA peptide tag (Wilson ei al. Cell 37:161 (1984), both of which may be useful in puriiying polypeptide sequence fused to them. Polynucleotides of the invention also include, but are not limited to, polynucleotides composing a structural gene and its naturally associated sequences that control gene expression.
The nucleotide sequence encoding BASB020 polypeptide of SEQ ID N0:2, 4, 6 or 8 may be identical to the polypeptide encoding sequence contained in nucleotides 1 to 840 of SEQ ID NO:l.3.5,or 7 respectively. Alternatively it may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ IDN0:2, 4, 6or 8.
The term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, panicuiarly a bacterial polypeptide and more particularly a polypeptide of the Moraxella catarrhal is BASB020 having an amino acid sequence set out in SEQ ID N0:2,4, 6 or 8.

The term also encompasses poiynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by integrated phage, an integrated insertion sequence, an integrated vector sequence, an integrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may contain coding and/or non-coding sequences.
The invention further relates to variants of the polynucleotides described herein that encode variants of a polypeptide having a deduced amino acid sequence of SEQ ID N0:2. 4, 6 or 8. Fragments of polynucleotides of the invention may be used, for example, to synthesize full-length polynucleotides of the invention.
Further particularly preferred embodiments are polynucleotides encoding BASB020 variants, that have the amino acid sequence of BASB020 polypeptide of SEQ ID N0:2. 4. 6 or 8 in which several, a few, 5 to 10, 1 to 5, 1 to 3. 2, 1 or no amino acid residues are substituted, modified, deleted and/or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of BASB020 polypeptide.
Further preferred embodiments of the invention are polynucleotides that are at least 85% identical over their entire length to a polynucleotide encoding BASB020 polypeptide having an amino acid sequence set out in SEQ ID N0;2. 4, 6 or 8, and polynucleotides that are complementary to such polynucleotides. Alternatively, most highly preferred are polynucleotides that comprise a region that is at least 90% identical over its entire length to a polynucleotide encoding BASB020 polypeptide and polynucleotides complementary thereto. In this regard, polynucleotides at least 95% identical over their entire length to the same are paniculariy preferred. Furthermore, those with at least 97% are highly preferred among those with at least 95%. and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred.

Preferred embodiments are polynucleotides encoding polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by a DNA of SEQIDN0:1.3, 5or7.
In accordance with certain preferred embodiments of this invention there are provided polynucleotides that hybridize, panicularly under stringent conditions, to BASB020 polynucleotide sequences, such as those polynucleotides in SEQ ID NO: 1. 3. 5 or 7.
The invention further relates lo polynucleotides that hybridize to the polynucleotide sequences provided herein. In this regard, the invention especially relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein. As herein used, the terms "stringent conditions" and "stringent hybridization conditions" mean hybridization occurring only if there is at least 95% and preferably at least 97% identity between the sequences. A specific example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising: 50% formamide. 5x SSC (ISOmM NaCL ISmM trisodium citrate). 50 mM sodium phosphate (pH7.6). 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml of denatured, sheared salmon sperm DNA. followed by washing the hybridization suppon in 0.1 x SSC at about 65°C, Hybridization and wash conditions are well known and exemplified in Sambrook. ei ai. Molecular Cloning; A Laboratory Manual. Second Ediuon. Cold Spring Harbor, N.Y., (1989), panicularly Chapter 11 therem. Solution hybridization may also be used with the polynucleotide sequences provided by the invention.
The invention also provides a polynucleotide consisting of or comprising a polynucleotide sequence obtamed by screening an appropriate library containing the complete gene for a polynucleotide sequence set forth in SEQ ID N0:1, 3, 5 or 7 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in

SEQ ID NO;1 3. 5 or 7 or a fragment thereof: and isolating said poiynucieotide sequence. Fragments useful for obtaining such a polynucleotide include, for example, probes and primers fully described elsewhere herein.
As discussed elsewhere herein regarding polynucleotide assays of the invention, for instance, the polynucleotides of the invention, may be used as a hybridization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding BASB020 and to isolate cDNA and genomic clones of other genes that have a high identit}', paniculariy high sequence idemity to the BASB020 gene. Such probes generally will comprise at least 15 nucleotide residues or base pairs. Preferably, such probes will have ai least 30 nucleotide residues or base pairs and may have at least 50 nucleotide residues or base pairs. Particularly preferred probes will have at least 20 nucleotide residues or base pairs and will have less than 30 nucleotide residues or base pairs.
A coding region of a BASB020 gene may be isolated by screening using a DNA sequence provided in SEQ ID NO:I, 3, 5 or 7 to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
There are several methods available and well known to those skilled in the art to obtain full-length DNAs. or extend short DNAs, for example those based on the method of Rapid Amplification of cDNA ends (RACE) (see, for example, Frohman, et al.. PNAS USA 85: 8998-9002. 1988). Recent modifications of the technique, exemplified by the Marathon™ technology (Clontech Laboratories Inc.) for example, have significantly simplified the search for longer cDNAs. In the MarathonTM technology, cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence ligated onto each end. Nucleic acid amplification (PCR) is then carried out to amplify the "missing" 5' end

of the DNA using a combination of gene specific and adaptor specific oligonucleoude primers. The PCR reaction is then repeated using "nested" primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific pnmer thai anneals further 3' in the adaptor sequence and a gene specific primer that anneals funher 5' in the selected gene sequence). The products of this reaction can then be analyzed by DNA sequencing and a full-length DNA constructed either by joining the product directi\' to the existing DNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer.
The polynucleotides and polypeptides of the invention may be employed, for example, as research reagents and materials for discovery of treatments of and diagnostics for diseases, paniculariy human diseases, as further discussed herein relating to polynucleotide assays.
The polynucleotides of the invention that are oligonucleotides derived from a sequence of SEQ ID N0S:1 - 8 may be used in the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue. It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained.
The invention also provides polynucleotides that encode a polypeptide that is the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things. As generally is the case in vivo, the additional amino acids may be processed away from the mature protein by cellular enzymes.

For each and everv' polynucleotide of the invention there is provided a polynucieotide complementary to it. It is preferred that these complementary polynucleotides are fully complementary to each polynucleotide with which they are complementary.
A precursor protein, having a mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide. When prosequences are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proproteins.
In addition to the standard A, G, C, T/U representations for nucleotides, the term "N" may also be used in describing cenain polynucleotides of the invention. "N" means that any of the four DNA or RNA nucleotides may appear at such a designated position m the DNA or RNA sequence, except it is preferred that N is not a nucleic acid that when taken in combination with adjacent nucleotide positions, when read in the correct reading frame. would have the effect of generating a premature termination codon in such reading frame.
In sum, a polynucleotide of the invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotein, or a preproprotein, which is a precursor to a proprotein. having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.
In accordance with an aspect of the invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization.

The use of a polynucleotide of the invention in genetic immunization will preferably employ a suitable deliver}' method such as direct injection of piasmid DNA into muscles (Wolff et al, Hum Mol Genet {\992) 1: 363, Manthorpe et al, Hum. Gene Ther. (1983) 4: 419), delivery of DNA complexed with specific protein carriers (Wu et al.J Biol Chem. (1989) 264: 16985), coprecipitation of DNA with calcium phosphate (Benvemsty iS: Reshef. PNAS USA. (1986) 83: 9551), encapsulation of DNA in various forms of liposomes (Kaneda ei ai, Science (1989) 243: 375), panicle bombardment (Tang et ai. Nature (1992) 356:152, Eisenbraun et ai, DNA Cell Biol (199j) 12: 791) and in vivo infection using cloned retroviral vectors (Seeger et ai. PNAS USA (1984) 81: 5849).
Vectors, Host Ceils. Expression Systems
The invention also relates to vectors that comprise a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the invention.
Recombinant polypeptides of the present invention may be prepared by processes well known in those skilled m the art from genetically engineered host cells comprising expression systems. Accordingly, in a further aspect, the present invention relates to expression systems that comprise a polynucleotide or polynucleotides of the present invention, to host ceils which are genetically engineered with such expression systems, and to the production of polypeptides of the invention by recombinant techniques.
For recombinant production of the polypeptides of the invention, host cells can be genetically engmeered to incorporate expression systems or ponions thereof or polynucleotides of the invention. Introduction of a polynucleotide into the host cell can be

effected by methods described in many standard laboratory manuals, such as Davis, et at. BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook. et al. MOLECULAR CLONING: A LABORATORY MANUAL. 2nd Ed.. Cold Spring Harbor Laboratory Press, Cold Spring Harbon N.Y. (1989), such as, calcium phosphate transfection. DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic mtroduction and infection.
Representative examples of appropriate hosts include bacterial cells, such as cells of streptococci, staphyiococci, emerococci. £. coli streptomyces, cyanobacteria. Bacillus subtilis. Neisseria meningitidis and Moraxella caiarrhalis: fungal cells, such as cells of a yeast. Khuveromyces, Saccharomyces. a basidiomycete, Candida albicans and Aspergillus: msect cells such as cells of Drosophila S2 and Spodoptera Sf9: animal cells such as CHO. COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells; and plant cells, such as cells of a gymnosperm or angiosperm.
A great variety of expression systems can be used to produce the polypeptides of the invention. Such vectors include, among others, chromosomal-, episomal- and virus-derived vectors, for example, vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picomaviruses, retroviruses, and aiphaviruses and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. The expression system constructs may contain control regions that regulate as well as engender expression. Generally, any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard. The appropriate DNA sequence may be inserted into the expression system by any

of a vartery of well-known and routine techniques, such as, for example, those set forth in Sambrook et al. MOLECULAR CLONING, A LABORATORY MANUAL, {supra).
In recombinant expression systems in eukaryotes, for secretion of a iranslaled protein into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. These signals may be endogenous to the polypepnde or they may be heterologous signals.
Polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatograph\\ hydroxylapatite chromatography and lectin chromatography. Most preferably, ion metal affinity chromatography (IMAC) is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during intracellular synthesis, isolation and or purification.
The expression system may also be a recombinant live microorganism, such as a virus or bacterium. The gene of interest can be inserted into the genome of a live recombinant virus or bacterium. Inoculation and in vivo infection with this live vector will lead to in VIVO expression of the antigen and induction of immune responses. Viruses and bacteria used for this purpose are for instance: poxviruses (e.g; vaccinia, fowlpox. canarypox), alphaviruses (Sindbis virus, Semliki Forest Virus, Venezueiian Equine Encephalitis Virus), adenoviruses, adeno-associated virus, picomaviruses (poliovirus, rhinovirus), herpesviruses (varicella zoster virus, etc). Listeria. Salmonella . Shigella. Neisseria, BCG. These viruses and bacteria can be virulent, or attenuated in various ways in order to obtain live vaccines. Such live vaccines also form part of the invention.

Diagnostic. Prognostic, Serotyping and Mutation Assays
This invention is also related to the use of BASB020 polynucleotides and polypeptides of the invention for use as diagnostic reagents. Detection of BASB020 polynucleotides and/or polypeptides in a eukaryote. particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious organism to drugs. Eukaryotes, particularly mammals, and especially humans, panicularly those infected or suspected to be infected with an organism comprising the BASB020 gene or protein, may be detected at the nucleic acid or amino acid level by a variet}' of well known techniques as well as by methods provided herein.
Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putativeiy infected and/or infected individual's bodily materials. Polynucleotides from any of these sources, particularly DNA or RNA, may be used directly for detection or may be amplified enzymaticaily by using PCR or any other amplification technique prior to analysis. RNA, particularly mRNA, cDNA and genomic DNA may also be used in the same ways. Using amplification, characterization of the species and strain of infectious or resident organism present in an individual, may be made by an analysis of the genotype of a selected polynucleotide of the organism. Deletions and insertions can be detected by a change m size of the amplified product in comparison to a genotype of a reference sequence selected from a related organism, preferably a different species of the same genus or a different strain of the same species. Point mutations can be identified by hybridizing amplified DNA to labeled BASB020 polynucleotide sequences. Perfectly or significantly matched sequences can be distinguished from imperfectly or more significantly mismatched duplexes by DNase or RNase digestion, for DNA or RNA respectively, or by detecting differences in melting temperatures or renaturation kinetics. Polynucleotide sequence differences may also be detected by alterations in the electrophoretic mobility of polynucleotide fragments in gels as compared to a reference sequence. This may be carried

out with or without denaturing agents. Poiynucieotide differences may also be detected by direct DNA or RNA sequencing. See, for example, Myers et al. Science, 230: 1242 (1985). Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase, V] and S1 protection assay or a chemical cleavage method. See. for example. Couon et al, Proc. Nati Acad Set, USA, 85: 4397-4401 (1985).
In another embodiment, an array of oligonucleotides probes comprising BASB020 nucleotide sequence or fragments thereof can be constructed to conduct efficient screemng of for example, genetic mutations, serotype, taxonomic classification or identification. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see, for example, Chee et al. Science. 2'74: 6/0 (1996)).
Thus in another aspect, the present invention relates to a diagnostic kit which comprises:
(a) a polynucleotide of the present invention, preferably the nucleotide sequence of SEQ ID NO:L 3, 5 or 7, or a fragment thereof;
(b) a nucleotide sequence complementary to that of (a);
(c) a polypeptide of the present invention, preferably the polypeptide of SEQ ID N0:2. 4. 6 or 8 or a fraament thereof; or
(d) an antibody to a polypeptide of the present invention, preferably to the polypeptide of SEQIDNO:2,4, 6or8.
It will be appreciated that in any such kit, (a), (b), (c) or (d) may comprise a substantial component. Such a kit will be of use in diagnosing a disease or susceptibility to a Disease, among others.
This invention also relates to the use of polynucleotides of the present invention as diagnostic reagents. Detection of a mutated form of a polynucleotide of the invention.

preferable- SEQ ID NO: 1,3,5 or 7. which is associated with a disease or pathogenlcm- wili provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, which results from under-expression, over-expression or altered expression of the poiynucieotide. Organisms, panicuiariy infectious organisms, carrying mutations in such polynucleotide may be detected at the polynucleotide level by a variety of techniques, such as those described elsewhere herein.
Cells from an organism carrying mutations or poiymorphisms (allelic variations) in a polynucleotide and/or polypeptide of the invention may also be detected at the polynucleotide or polypeptide level by a variety of techniques, to allow for serotyping. for example. For example. RT-PCR can be used to detect mutations in the RNA. It is panicuiariy preferred to use RT-PCR in conjunction with automated detection systems, such as. for example. GeneScan. RNA, cDNA or genomic DNA may also be used for the same purpose, PCR. As an example. PCR primers complementary to a poiynucieotide encoding BASB020 polypeptide can be used to identify and analyze mutations.
The invention further provides primers with 1, 2, 3 or 4 nucleotides removed from the 5' and/or the 3* end- These primers may be used for, among other things, amplifying BASB020 DNA and/'or RNA isolated from a sample derived from an individual, such as a bodily material. The primers may be used to amplify a polynucleotide isolated from an infected individual, such that the polynucleotide may then be subject to various techniques for elucidation of the polynucleotide sequence. In this way, mutations in the polynucleotide sequence may be detected and used to diagnose and/or prognose the infection or its stage or course, or to serot\'pe and/or classify the infectious agent.
The mvention ftirther provides a process for diagnosing, disease, preferably bacterial mfections, more preferably infections caused by Moraxella caiarrhalis comprising

detennmine from a sample derived from an individual, such as a bodih' matenal, an increased level of expression of polynucleotide having a sequence of SEQ ID N0:1. 3, 5 or 7. Increased or decreased expression of a BASB020 polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides, such as. for example, amplification. PCR, RT-PCR. RNase protection. Northern blotting. spectrometr>' and other hybridization methods.
In addition, a diagnostic assay in accordance with the invention for detecting over-expression of BASB020 polypeptide compared to normal control tissue samples may be used to detect the presence of an infection, for example. Assay techniques that can be used to determine levels of a BASB020 polypeptide, in a sample derived from a host, such as a bodily material are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays. Western Blot analysis, antibody sandwich assays, antibody detection and ELISA assays.
The polynucleotides of the invention may be used as components of polynucleotide arrays, preferably high density arrays or grids. These high density arrays are particularly useful for diagnostic and prognostic purposes. For example, a set of spots each comprising a different gene, and further comprising a polynucleotide or polynucleotides of the invention, may be used for probing, such as using hybridization or nucleic acid amplification, using a probes obtained or derived from a bodily sample. to determine the presence of a particular polynucleotide sequence or related sequence in an individual. Such a presence may indicate the presence of a pathogen, particularly Moraxella catarrhalis. and may be useful in diagnosing and/or prognosing disease or a course of disease. A grid comprising a number of variants of the polynucleotide sequence of SEQ ID NO:1 3. 5 or 7 are preferred. Also preferred is a comprising a number of variants of a polynucleotide sequence encoding the polypeptide sequence of SEQ ID N0;2. 4, 6 or 8.

Antibodies
The polypeptides and polynucleotides of the invention or variants thereof, or cells expressing the same can be used as immunogens to produce antibodies immunospecific for such polypeptides or polynucleotides respectively.
In cenain preferred embodiments of the invention there are provided antibodies agamst BASB020 polypeptides or polynucleotides.
Antibodies generated against the polypeptides or polynucleotides of the invention can be obtained by administering the polypeptides and/or polynucleotides of the invention, or epitope-bearing fragments of either or both, analogues of either or both, or ceils expressing either or both, to an animal preferably a nonhuman. using routine protocols. For preparation of monoclonal annbodies, any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques- such as those in Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor ei ai. Immunology Today 4: 11 (1983); Cole e( al, pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY. Alan R. Liss, Inc. (1985).
Techniques for the production of single chain antibodies (U.S. Patent No. 4,946.778) can be adapted to produce single chain antibodies to polypeptides or polynucleotides of this invention. Also, transgenic mice, or other organisms or animals, such as other mammals, may be used to express humanized antibodies immunospecific to the polypeptides or polynucleotides of the invention.
Alternatively, phage display technology may be utilized to select antibody genes with binding activities towards a polypeptide of the invention either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti-BASB020 or

from naive libraries (McCaffeny. et al, (1990). Nature 348. 552-554: Marks, et al.. (1992) Biotechnology 10. 779-783), The affinity of these antibodies can also be improved by, for example, chain shuffling (Clackson et al. (1991) Nature 352: 628).
The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptides or polynucleotides of the invention to purify the polypeptides or polynucleotides by, for example, affinity chromatography.
Thus, among others, antibodies against BASB020-polypeptide or BASB020-polynucleotide may be employed to treat infections, particuiarly bacterial infections.
Polypeptide variants include antigenically, epitopically or immunologically equivalent variants form a particular aspect of this invention.
Preferably, the antibody or variant thereof is modified to make it less immunogenic in the individual. For example, if the individual is htiman the antibody may most preferably be "humanized," where the complimentarity determining region or regions of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody, for example as described in Jones ei al. (1986). Nature 321 522o25 or Tempest et ai, (1991) Biotechnology 9, 266-273.
Antagonists and Agonists * Assays and Molecules
Polypeptides and polynucleotides of the invention may also be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures. These substrates and Ugands may be natural substrates and lisands or mav be structural or functional mimetics. See. e.g,. Coliaan ei aL. Current Protocols in Immunology 1(2): Chapter 5 (1991).

The screening methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound. Alternatively, the screening method may involve competition with a labeled competitor. Further, these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide or polynucleotide, using detection systems appropriate to the cells comprising the polypeptide or polynucleotide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed. Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of the polypeptide or polynucleotide, as the case may be. Further, the screening methods may simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide or polynucleotide of the present invention, to form a mixture, measuring BASB020 polypeptide and/or polynucleotide activity in the mixture, and comparing the BASB020 polypeptide and/or polynucleotide activity of the mixture to a standard- Fusion proteins, such as those made from Fc portion and BASB020 polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists of the polypeptide of the present invention, as well as of phylogenetically and and/or functionally related polypeptides (see D. Bennett et al, J Mol Recognition, 8:52-58 (1995); and K. Johanson et al, J Biol Chem, 270(16):9459-947I (1995)).
The polynucleotides, polypeptides and antibodies that bind to and/or interact with a polypeptide of the present invention may also be used to configure screening methods for

detecting the effect of added compounds on the production of mRNA and/or polypeptide in cells. For example, an ELISA assay may be constructed for measuring secreted or cell associated levels of polypeptide using monoclonal and polyclonal antibodies by standard methods known in the art. This can be used to discover agents which may inhibit or enhance the production of polypeptide (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues.
The invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of BASB020 polypeptides or polynucleotides, particularly those compounds that are bacteriostatic and/or bactericidal. The method of screening may involve high-throughput techniques. For example, to screen for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, comprising BASB020 polypeptide and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a BASB020 agonist or antagonist. The ability of the candidate molecule to agonize or antagonize the BASB020 polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate. Molecules that bind gratuitously, i.e., without inducing the effects of BASB020 polypeptide are most likely to be good antagonists. Molecules that bind well and, as the case may be. increase the rate of product production from substrate, increase signal transduction, or increase chemical channel activity are agonists. Detection of the rate or level of, as the case may be, production of product from substrate, signal transduction, or chemical channel activity may be enhanced by using a reporter system. Reporter systems that may be useful in this regard include but are not limited to colorimetric, labeled substrate converted into product, a reporter gene that is responsive to changes in BASB020 polynucleotide or polypeptide activity, and binding assays known in the art.

Another example of an assay for BASB020 agonists is a competitive assay that combines BASB020 and a potential agonist with BASB020-binding molecules, recombinant BASB020 binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay. BASB020 can be labeled, such as by radioactivity or a colorimetric compound, such that the number of BASB020 molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist
Potential antagonists include, among others, small organic molecules, peptides, polypepddes and antibodies that bind to a polynucleotide and/or polypeptide of the invention and thereby inhibit or extinguish its activity or expression. Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, without inducing BASB020'induced activities, thereby preventing the action or expression of BASB020 polypeptides and/or polynucleotides by excluding BASB020 polypeptides and/or polynucleotides from binding.
Potential antagonists include a small molecule that binds to and occupies the binding site of the polypeptide thereby preventing binding to cellular binding molecules, such that normal biological activity is prevented. Examples of small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules. Other potential antagonists include antisense molecules (see Okano, J. Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION CRC Press, Boca Raton, FL (1988), for a description of diese molecules). Preferred potential antagonists include compounds related to and variants of BASB020.
In a further aspect, the present invention relates to genetically engineered soluble fusion proteins comprising a polypeptide of the present invention, or a fragment thereof, and

various ponions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is the constant part of the heavy chain of human igG, particularly IgGl, where fusion takes place at the hinge region. In a panicuiar embodiment, the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa. Furthermore, this invention relates to processes for the preparation of these fusion proteins by genetic engineering, and to the use thereof for drug screening, diagnosis and therapy. A further aspect of the invention also relates to polynucleotides encoding such fusion proteins. Examples of fusion protein technology can be found in International Patent Application Nos. W094/29458 and W094/22914.
Each of the polynucleotide sequences provided herein may be used in the discover}' and development of antibacterial compounds. The encoded protein, upon expression, can be used as a target for the screening of antibacterial drugs. Additionally, the polynucleotide sequences encoding the amino terminal regions of the encoded protein or Shine-Delgamo or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest.
The invention also provides the use of the polypeptide, polynucleotide, agonist or antagonist of the invention to interfere with the initial physical interaction between a pathogen or pathogens and a eukaryotic, preferably mammalian, host responsible for sequelae of infection. In particular, the molecules of the invention may be used: in the prevention of adhesion of bacteria, in particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on in¬dwelling devices or to extracellular matrix proteins in wounds; to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial BASB020 proteins that mediate tissue damage and/or; to block the normal progression of

pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques.
In accordance with yet another aspect of the invention, there are provided BASB020 agonists and antagonists, preferably bacteristatic or bactericidal agonists and antagonists.
The antagonists and agonists of the invention may be employed, for instance, to prevent, inhibit and/or treat diseases.
In a further aspect, the present invention relates to mimotopes of the polypeptide of the invention. A mimotope is a peptide sequence, sufficiently similar to the native peptide (sequentially or structurally), which is capable of being recognised by antibodies which recognise the native peptide; or is capable of raising antibodies which recognise the native peptide when coupled to a suitable carrier.
Peptide mimotopes may be designed for a particular purpose by addition, deletion or substitution of elected amino acids. Thus, the peptides may be modified for the purposes of ease of conjugation to a protein carrier. For example, it may be desirable for some chemical conjugation methods to include a terminal cysteine. In addition it may be desirable for peptides conjugated to a protein carrier to include a hydrophobic terminus distal from the conjugated terminus of the peptide, such that the free unconjugated end of the peptide remains associated with the surface of the carrier protein. Thereby presenting the peptide in a conformation which most closely resembles that of the peptide as found in the context of the whole native molecule. For example, the peptides may be altered to have an N-terminal cysteine and a C-terminal hydrophobic amidated tail. Alternatively, the addition or substitution of a D-stereoisomer form of one or more of the amino acids may be performed to create a beneficial derivative, for example to enhance stability of the peptide.

Alternatively, peptide mimotopes may be identified using antibodies which are capable themselves of binding to the polypeptides of the present invention using techniques such as phage display technology (EP 0 552 267 Bl). This technique, generates a large number of peptide sequences which mimic the structure of the native peptides and are, therefore, capable of binding to anti-native peptide antibodies, but may not necessarily themselves share significant sequence homology to the native polypeptide.
Vaccines
Another aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal, preferably humans, which comprises inoculating the individual with BASB020 polynucleotide and/or polypeptide, or a fragment or variant thereof, adequate to produce antibody and/ or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly Morcaella catarrhalis infection. Also provided are methods whereby such immunological response slows bacterial replication. Yet another aspect of the invention relates to a method of inducing immunological response in an individual which comprises delivering to such individual a nucleic acid vector, sequence or ribozyme to direct expression of BASB020 polynucleotide and/or polypeptide, or a fragment or a variant thereof for expressing BASB020 polynucleotide and/or polypeptide, or a fragment or a variant thereof in vivo in order to induce an immunological response, such as, to produce antibody and/ or T cell immune response, including, for example, cytokine-producing T cells or cytotoxic T cells, to protect said individual, preferably a human, from disease, whether that disease is already established within the individual or not. One example of administering the gene is by accelerating it into the desired cells as a coating on particles or otherwise. Such nucleic acid vector may comprise DNA, RNA, a ribozyme, a modified nucleic acid, a DNA/RNA hybrid, a DNA-protein complex or an RNA-protein complex.

A further aspect of the invention relates to an immunological composition that when introduced into an individual, preferably a human, capable of having induced within it an immunological response, induces an immunological response in such individual to a BASB020 polynucleotide and/or polypeptide encoded therefrom, wherein the composition comprises a recombinant BASB020 polynucleotide and/or polypeptide encoded therefrom and/or comprises DNA and/or RNA which encodes and expresses an antigen of said BASB020 polynucleotide, polypeptide encoded therefrom, or other polypeptide of the invention. The immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity and/or cellular immunity, such as cellular immunity arising from CTL or CD4+ T cells.
A BASB020 polypeptide or a fragment thereof may be fused with co-protein or chemical moiety which may or may not by itself produce antibodies, but which is capable of stabilizing the first protein and producing a fused or modified protein which will have antigenic and/or immunogenic properties, and preferably protective properties. Thus fused recombinant protein, preferably further comprises an antigenic co-protein, such as lipoprotein D from Haemophilus influenzae, Glutathione-S-transferase (GST) or beta-galactosidase, or any other relatively large co-protein which solubilizes the protein and facilitates production and purification thereof Moreover, the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system of the organism receiving the protein. The co-protein may be attached to either the amino- or carboxy-terminus of the first protein.
Provided by this invention are compositions, particularly vaccine compositions, and methods comprising the polypeptides and/or polynucleotides of the invention and immunostimulatory DNA sequences, such as those described in Sato, Y. et al. Science
273:352(1996).

Also, provided by this invention are methods using the described polynucleotide or particular fragments thereof, which have been shown to encode non-variable regions of bacterial cell surface proteins, in polynucleotide constructs used in such genetic immunization experiments in animal models of infection with Moraxella catarrhalis. Such experiments will be particularly useful for identifying protein epitopes able to provoke a prophylactic or therapeutic immune response. It is believed that this approach will allow for the subsequent preparation of monoclonal antibodies of particular value, derived from the requisite organ of the animal successfully resisting or clearing infection, for the development of prophylactic agents or therapeutic treatments of bacterial infection, particularly Moraxella catarrhalis infection, in mammals, particularly humans.
The invention also includes a vaccine formulation which comprises an immunogenic recombinant polypeptide and/or polynucleotide of the invention together with a suitable carrier, such as a pharmaceutically acceptable carrier. Since the polypeptides and polynucleotides may be broken down in the stomach, each is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostatic compounds and solutes which render the formulation isotonic with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
The vaccine formulation of the invention may also include adjuvant systems for enhancing the immunogenicity of the formulation. Preferably the adjuvant system raises preferentially a THl type of response-

An immune response may be broadly distinguished into two extreme catagories, being a humoral or cell mediated immune responses (traditionally characterised by antibody and cellular effector mechanisms of protection respectively). These categories of response have been termed THl-type responses (cell-mediated response), and TH2-type immune responses (humoral response).
Extreme THl-type immune responses may be characterised by the generation of antigen specific, haplotype restricted cytotoxic T lymphocytes, and natural killer cell responses. In mice THl-type responses are often characterised by the generation of antibodies of the IgG2a subtype, whilst in the human these correspond to IgGl type antibodies. TH2-type immune responses are characterised by the generation of a broad range of immunoglobulin isotypes including in mice IgGl, IgA, and IgM.
It can be considered that the driving force behind the development of these two types of immune responses are cytokines. High levels of THl-type cytokines tend to favour the induction of cell mediated immune responses to the given antigen, whilst high levels of TH2-type cytokines tend to favour the induction of humoral immune responses to the antigen.
The distinction of THl and TH2-type immune responses is not absolute. In reality an individual will support an immune response which is described as being predominantly THl or predominantly TH2. However, it is often convenient to consider the families of cytokines m terms of that described in murine CD4 +ve T cell clones by Mosmann and Coffman {Mosmann, T.R. and Copnan, R.L (1989) THl and TH2 cells: different patterns oflymphokine secretion lead to different functional properties. Annual Review of Immunology, 7, p145-173). Traditionally, THl-type responses are associated with the production of the INF-y and IL-2 cytokines by T-lymphocytes. Other cytokines

often directly associated with the induction of THl-type immune responses are not produced by T-cells, such as IL-12. In contrast, TH2- type responses are associated with the secretion of IL-4, IL-5, IL-6 and IL-13.
It is known that certain vaccine adjuvants are particularly suited to the stimulation of either TH1 or TH2 - type cytokine responses. Traditionally the best indicators of the THl :TH2 balance of the immune response after a vaccination or mfection includes direct measurement of the production of THl or TH2 cytokines by T lymphocytes in vitro after restimulation with antigen, and/or the measurement of the IgGl :IgG2a ratio of antigen specific antibody responses.
Thus, a THl-type adjuvant is one which preferentially stimulates isolated T-cell populations to produce high levels of THl-type cytokines when re-stimulated with antigen in vitro, and promotes development of both CD8+ cytotoxic T lymphocytes and antigen specific immunoglobulin responses associated with THl-type isotype.
Adjuvants which are capable of preferential stimulation of the THl cell response are described in International Patent Application No. WO 94/00153 and WO 95/17209.
3 De-0-acylated monophosphoryl lipid A (3D-MPL) is one such adjuvant. This is known from GB 2220211 (Ribi). Chemically it is a mixture of 3 De-0-acylated monophosphoryl lipid A with 4, 5 or 6 acyiated chains and is manufactured by Ribi Immunochem, Montana. A preferred form of 3 De-0-acylated monophosphoryl lipid A is disclosed in European Patent 0 689 454 Bl (SmithKline Beecham Biologicals SA).
Preferably, the particles of 3D-MPL are small enough to be sterile filtered through a 0.22micron membrane (European Patent number 0 689 454).

3D-MPL will be present in the range of 10µg - lOOµg preferably 25.50µg per dose wherein the antigen will typically be present in a range 2-50µg per dose.
Another preferred adjuvant comprises QS21, an Hplc purified non-toxic fraction derived from the bark of Quillaja Saponaria Molina. Optionally this may be admixed with 3 De-0-acyiated monophosphoryl lipid A (3D-MPL), optionally together with an carrier.
The method of production of QS21 is disclosed in US patent No, 5,057,540.
Non-reactogenic adjuvant formulations containing QS21 have been described previously (WO 96/33739). Such formulations comprising QS21 and cholesterol have been shown to be successful THl stimulating adjuvants when formulated together with an antigen.
Further adjuvants which are preferential stimulators of THl cell response include immunomodulatory oligonucleotides, for example unmethylated CpG sequences as disclosed in WO 96/02555.
Combinations of different THl stimulating adjuvants, such as those mentioned hereinabove, are also contemplated as providing an adjuvant which is a preferential stimulator of THl cell response. For example, QS21 can be formulated together with 3D-MPL. The ratio of QS21 ; 3D-MPL will typically be in the order of 1 : 10 to 10 : 1; preferably 1:5 to 5 : 1 and often substantially 1:1. The preferred range for optimal synergy is 2.5 : 1 to 1 : 1 3D-MPL: QS2L
Preferably a carrier is also present in the vaccine composition according to the invention. The carrier may be an oil in water emulsion, or an aluminium salt, such as aluminium phosphate or aluminium hydroxide.

A preferred oil-in-water emulsion comprises a metabolisible oil, such as squaiene. alpha tocopherol and Tween 80. In a particularly preferred aspect the antigens in the vaccine composition according to the invention are combined with QS21 and 3D-MPL in such an emulsion. Additionally the oil in water emulsion may contain span 85 and/or lecithin and/or tricaprylin.
Typically for human administration QS21 and 3D-MPL will be present in a vaccine in the range of l)ag - 200)ig, such as 10-100^g, preferably 10|ig - 50(ig per dose. Typically the oil in water will comprise from 2 to 10% squalene, from 2 to 10% alpha tocopherol and from 0.3 to 3% tween 80. Preferably the ratio of squalene: alpha tocopherol is equal to or less than 1 as this provides a more stable emulsion. Span 85 may also be present at a level of 1%. In some cases it may be advantageous that the vaccines of the present invention will further contain a stabiliser.
Non-toxic oil in water emulsions preferably contain a non-toxic oil, e.g. squalane or squalene, an emulsifier. e.g. Tween 80, in an aqueous carrier. The aqueous carrier may be, for example, phosphate buffered saline.
A particularly potent adjuvant formulation involving QS2L 3D*MPL and tocopherol in an oil in water emulsion is described in WO 95/17210.
The present invention also provides a polyvalent vaccine composition comprising a vaccine formulation of the invention in combination with other antigens, in particular antigens useful for treating cancers, autoimmune diseases and related conditions. Such a polyvalent vaccine composition may include a TH-1 inducing adjuvant as hereinbefore described.

While the invention has been described with reference to certain BASB020 polypeptides and polynucleotides, it is to be understood that this covers fragments of the naturally occurring polypeptides and polynucleotides, and similar polypeptides and polynucleotides with additions, deletions or substitutions which do not substantially affect the immunogenic properties of the recombinant polypeptides or polynucleotides.
Compositions, kits and administration
In a further aspect of the invention there are provided compositions comprising a BASB020 polynucleotide and/or a BASB020 polypeptide for administration to a cell or to a multicellular organism.
The invention also relates to compositions comprising a polynucleotide and/or a polypeptides discussed herein or their agonists or antagonists. The polypeptides and polynucleotides of the invention may be employed in combmation with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to an individual. Such compositions comprise, for instance, a media additive or a therapeutically effective amount of a polypeptide and/or polynucleotide of the invention and a pharmaceutically acceptable carrier or excipient. Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. The formulation should suit the mode of administration. The invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
Polypeptides, polynucleotides and other compounds of the invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes among others.
In therapy or as a prophylactic, the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.
In a further aspect, the present invention provides for pharmaceutical compositions comprising a therapeutically effective amount of a polypeptide and/or polynucleotide, such as the soluble form of a polypeptide and/or polynucleotide of the present invention, agonist or antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention. Polypeptides, polynucleotides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
The composition will be adapted to the route of administration, for instance by a systemic or an oral route. Preferred forms of systemic administration include injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, or intraperitoneal, can be used. Alternative means for systemic administration include transmucosal and transdermal administration using penetrants such as bile sahs or fiisidic acids or other detergents. In addition, if a polypeptide or other compounds of the present invention can be formulated in an enteric or an encapsulated formulation, oral administration may also be possible. Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels, solutions, powders and the like.

For administration to mammals, and particularly humans, it is expected that the daily dosage level of the active agent will be from 0.01 mg/kg to 10 mg/kg, typically around 1 mg/kg. The physician in any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular individual. The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
The dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner. Suitable dosages, however, are in the range of 0.1-100 µg/kg of subject.
A vaccine composition is conveniently in injectable form. Conventional adjuvants may be employed to enhance the immune response, A suitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, and such dose is preferably administered 10 times and with an interval of 1-3 weeks. With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals.
Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.

sequence Databases, Sequences in a Tangible Medium, and Algorithms
Polynucleotide and polypeptide sequences form a valuable infonnation resource with which to determine their 2- and 3-dimensional structures as well as to identify further sequences of similar homology. These approaches are most easily facilitated by storing the sequence in a computer readable medium and then using the stored data in a known macromolecular structure program or to search a sequence database using well known searching tools, such as the GCG program package.
Also provided by the invention are methods for the analysis of character sequences or strings, particularly genetic sequences or encoded protein sequences. Preferred methods of sequence analysis include, for example, methods of sequence homology analysis, such as identity and similarity analysis, DNA, RNA and protein structure analysis, sequence assembly, cladistic analysis, sequence motif analysis, open reading frame determination, nucleic acid base calling, codon usage analysis, nucleic acid base trimming, and sequencing chromatogram peak analysis.
A computer based method is provided for performing homology identification. This method comprises the steps of: providing a first polynucleotide sequence comprising the sequence of a polynucleotide of the invention in a computer readable medium; and comparing said first polynucleotide sequence to at least one second polynucleotide or polypeptide sequence to idendfy homology.
A computer based method is also provided for performing homology identification, said method comprising the steps of: providing a first polypeptide sequence comprising the sequence of a polypeptide of the invention in a computer readable medium; and comparing said first polypeptide sequence to at least one second polynucleotide or polypeptide sequence to identify homology.

All publications and references, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference in their entirety as if each individual publication or reference were specifically and individually indicated to be incorporated by reference herein as being fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in its entirety in the manner described above for publications and references.
DEFINITIONS
"ldentit>\" as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences. In the art. "identit>'" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" can be readily calculated by known methods, including but not limited to those described in {Computational Molecular Biology^, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed.. Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M-- and Griffin, H,G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G.. Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, the GAP program in the GCG program package (Devereux, J,, et al.. Nucleic Acids Research 12(1): 387(1984)), BLASTP,BLASTN(AltschuI,S.RetaL, J. Molec Biol. 275:403.410 (1990), and FASTA( Pearson and Lipman Proc. Natl. Acad. Sci. USA 85; 2444-2448

(] 988). The BLAST family of programs is publicly available from NCBI and other sources {BLAST MamaL Altschul, S., et al, NCBI NLM NIH Bethesda, MD 20894; Aitschul, S., et al J Mol Biol. 215: 403-410 (1990). The well known Smith Waterman algorithm may also be used to determine identity.
Parameters for polypeptide sequence comparison include the following:
Algorithm; Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)
Comparison matrix: BLOSSUM62 from Henikoff and Henikoff,
Proc. Natl. Acad. Sci. USA. 89:10915-10919(1992)
Gap Penalty: 8
Gap Length Penalty: 2
A program useful with these parameters is publicly available as the "gap" program from
Genetics Computer Group, Madison WI. The aforementioned parameters are the default
parameters for peptide comparisons (along with no penalty for end gaps).
Parameters for polynucleotide comparison include the following:
Algorithm: Needleman and Wunsch, J. Mol Bioi. 48: 443-453 (1970)
Comparison matrix: matches - +10, mismatch = 0
Gap Penalty: 50
Gap Length Penalty: 3
Available as: The "gap" program from Genetics Computer Group. Madison WL These
are the default parameters for nucleic acid comparisons.
A preferred meaning for "identity" for polynucleotides and polypeptides, as the case may be. are provided in (1) and (2) below.
(1) Polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide sequence having at least a 50, 60, 70, 80, 85, 90, 95, 97 or

100% identity to the reference sequence of SEQ ID NO: 1, wherein said polynucleotide sequence may be identical to the reference sequence of SEQ ID N0:1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID N0:1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleotides in SEQ ID N0:1, or:
nn wherein iin is the number of nucleotide alterations, xn is the total number of nucleotides in SEQ ID NO:K y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0,80 for 80%, 0,85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%, and • is the symbol for the multiplication operator, and wherein any non-integer product of Xn and y is rounded down to the nearest integer prior to subtracting it from xn- Alterations of a polynucleotidesequence encoding the polypeptide of SEQ ID N0:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
By way of example, a polynucleotide sequence of the present invention may be identical to the reference sequence of SEQ ID N0:1, that is it may be 100% identical, or it may include up to a certain integer number of nucleic acid alterations as compared to the reference sequence such that the percent identity is less than 100% identity. Such

alterations are selected from the group consisting of at least one nucleic acid deletion, substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5' or 3' terminal positions of the reference polynucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleic acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of nucleic acids in SEQ ID N0:1 by the integer defining the percent identity divided fay 100 and then subtracting that product from said total number of nucleic acids in SEQ ID N0:1, or:
"n ^ % - (xn • yl
wherein nn is the number of nucleic acid alterations, xn is the total number of nucleic acids in SEQ ID NO: 1, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., • is the symbol for the multiplication operator, and wherein any non-integer product of xn and y is rounded down to the nearest integer prior to subtracting it from xn'
(2) Polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID N0:2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID N0:2 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of

ammo acid alterations is determined by multiplying the total number of amino acids in SEQ ID N0;2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID N0:2, or;

wherein na is the number of amino acid alterations, xa is the total number of amino acids in SEQ ID N0:2, y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or LOO for 100%, and • is the symbol for the multiplication operator, and wherein any non-integer product of x and y is rounded down to the nearest integer prior to subtracting it from xa.
By way of example, a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID N0;2, that is it may be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100%i identity. Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence. The number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in SEQ ID N0:2 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of amino acids in SEQ ID N0:2, or:
na
wherein na is the number of amino acid alterations, xa is the total number of amino acids in SEQ ID N0:2, y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc, and • is the symbol for the multiplication operator, and wherein any non-integer product of xa and y is rounded down to the nearest integer prior to subtracting it from Xa-
"Individual(s)," when used herein with reference to an organism, means a multicellular eukaryote, including, but not limited to a metazoan, a mammal, an ovid, a bovid, a simian, a primate, and a human.
"Isolated" means altered "by the hand of man" from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living organism is not 'isolated." but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. Moreover, a polynucleotide or polypeptide that is introduced into an organism by transformation, genetic manipulation or by any other recombinant method is "isolated" even if it is still present in said organism, which organism may be living or non-living.
"Polynucleotide(s)" generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA including single and double-stranded regions.
"Variant * refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essendal properties. A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in

the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
"Disease(s)" means any disease caused by or related to infection by a bacteria, including, for example, otitis media in infants and children, pneumonia in elderlies, sinusitis, nosocomial infections and invasive diseases, chronic otitis media with hearing loss, fluid accumulation in the middle ear, auditive nerve damage, delayed speech learning, infection of the upper respiratory tract and inflammation of the middle ear.

EXAMPLES:
The examples below are carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. The examples are illustrative, but do not limit the invention.
Example 1:
Discovery and confirmatory DNA sequencing of the BASB020 gene from
Moraxella catarrhalis strain ATCC 43617,
The BASB020 gene of SEQ ID N0:1 was first discovered in the Incyte PathoSeq data base containing unfinished genomic DNA sequences of the Moraxella catarrhalis strain ATCC 43617 (also referred to as strain Mc2931), The translation of the BASB020 polynucleotide sequence, shown in SEQ ID N0:2, showed significant similarity (36 % identity in a 227 amino acids overlap) to the TlyC hemolysin protein of Serpulina hyodysenteriae.
The sequence of the BASB020 gene was further confirmed experimentally. For this purpose, genomic DNA was extracted from 1010 cells of the M catarrhalis cells (strain ATCC 43617) using the QIAGEN genomic DNA extraction kh (Qiagen Gmbh), and l)ig of this material was submitted to Polymerase Chain Reaction DNA amplification using primers E475781a (5'- ACT TGA ATA AAA CCG AGT G -3') [SEQIDN0:9] and E475782a (5'-GAC ATT GGC CGC AAC ATG C-3') [SEQIDNOrlO]. This PCR

product was purified on a Biorobot 9600 (Qiagen Gmbh) apparatus and subjected to DNA sequencing using the Big Dye Cycle Sequencing kit (Perkin-Elmer) and an ABI 377/PRISM DNA sequencer. DNA sequencing was performed on both strands with a redundancy of 2 and the full-length sequence was assembled using the Sequencher'^ software (Applied Biosytems). The resulting DNA sequence turned out to be 100 % identical to SEQ ID NO: 1.
Example 2:
Variability analysis of the BASB020 gene among several Moraxella catarrhalis
strains.
2A: Restriction Fragment Length Analysis (RFLP).
Genomic DNA was extracted from 16 M. catarrhalis strains (presented in Table 1) as described below. M.catarrhalis was streaked for single colonies on BHI agar plates and grown overnight at 37°C, Three or four single colonies were picked and used to inoculate a ---1.5 ml BHI (Brain-heart infusion) broth seed culture which was grown overnight in a shaking incubator. --300 rpm, at 37 °C. A 500ml erlenmeyer flask containing --150 ml of BHI broth was inoculated with the seed culture and grown for --12-16 hours at 37 °C in a shaking incubator, -175 rpm, to generate cell mass for DNA isolation. Cells were collected by centrifugation in a Sorvall GSA rotor at --2000 X g for 15 minutes at room temperature. The supernatant was removed and the cell pellet suspended in --5.0 ml of sterile water. An equal volume of lysis buffer (200 mM NaCl, 20 mM EDTA, 40 mM Tris-Hcl, pH 8.0, 0.5% (w/v) SDS, 0.5% (v/v) 2-mercaptoethanol, and 250 [Ag/ml of proteinase K) was added and the cells suspended by gentle agitation and trituration. The cell suspension was then incubated ~-12 hours at

50°C to lyse the bacteria and liberate chromosomal DNA. Proteinaceous material was precipitated by the addhion of 5.0 ml of saturated NaCl (--6,0 M, in sterile water) and centrifugation at --5,500xg in a Sorvall SS34 rotor at room temperature. Chromosomal DNA was precipitated from the cleared supernatant by the addition of two volumes of 100 % ethanol Aggregated DNA was collected and washed using gentle agitation in a small volume of a 70 % ethanol solution. Purified chromosomal DNA was suspended in sterile water and allowed to dissolve/ disburse overnight at 4 °C by gentle rocking. The concentration of dissolved DNA was determined spectrophotometrically at 260 nm using an extinction coefficient of 1.0 O.D. unit -50 ^ig/ml
This material was next submitted to PCR amplification using the MC-Hly3-BamF (5'-AAG GGC CCA ATT ACQ CAG AGG GGA TCC ATG CGT GOT CTT AGG CGT TGG TTA TCC ACC G -3') [SEQ ID N0:11] and MC-Hly3^SalRC (5'-AAG GGC CCA ATT ACQ CAG AGG GTC GAC TTA TTA TTC AGC ATT CTC AAG CTG TGG TAT CAG -3') [SEQ ID NO: 12] oligonucleotides. The corresponding BASB020 gene ampiicons were then subjected independantly to hydrolysis using restriction enzymes {Acil, AM, HphI, Msel Nlalll, Tsp509I) and restriction products were separated by agarose or polyacrylamide gel electrophoresis using standard molecular biology procedures as described in "Molecular Cloning, a Laboratory Manual, Second Edhion, Eds: Sambrook, Fritsch & Maniatis, Cold Spring Harbor press 1989". The photographs of the resulting electrophoresis gels are displayed in Figure 1. For each strain, RFLP patterns corresponding to the 6 restriction enzymes were scored and combined. Groups of strains sharing identical combination of RFLP patterns were then defined. Using this methodology, the strains tested in this study fell into 6 genomic groups (Group 1: Mc2904, Mc 2905, Mc 2906, Mc2969; Group 2: Mc2907,Mc2913; Group 3: Mc2908, Mc2909, Mc2931, Mc2975; Group 4: Mc2910, Mc2912, Mc2956; Group5: Mc2911; Group 6: Mc2926). These data support that the Moraxella catarrhalis

population used in this study displays some nucleotide sequence diversity for the BASB020 gene.
2B: DNA sequencing in other strains.
The ATCC 43617 (Mc293l) strain used to determine the BASB020 sequence was classified by RFLP in the group number 3. Using the experimental procedure as described in example 1, the sequence of the BASB020 gene was also determined for Moraxella catarrhalis strains representative of three other RFLP genomic groups (Gr.l (Mc2969), Gr2 (Mc29I3) and Gi:4 (Mc2912)). The polynucleotide sequences of the BASB020 gene of the strains Mc2912, Mc2913 and Mc2969 are shown in SEQ ID N0:3, 5 and 7, respectively. These polynucleotide sequences were translated into amino acid sequences, which are shown in SEQ ID N0:4, 6 and 8, respectively. Using the MegAlign program from the DNASTAR Lasergene package, a multiple alignment of the polynucleotide sequences of SEQ ID N0:1, 3, 5 and 7 was performed, and is displayed in Figure 2, A pairwise comparison of identities is summarized in Table 2, showing that the four BASB020 nucleotide gene sequences are all similar at identity level equal to or greater than 99 %. Using the same program, a multiple alignment of the polypeptide sequences of SEQ ID N0:2,4, 6 and 8 was performed, and is displayed in Figure 3. A pairwise comparison of identities is summarized in Table 3, showing that the four BASB020 protein sequences are all similar at a identity level equal to or greater than 99 %.




Example 3: Construction of Plasmid to Express Recombinant BASB020
A: Cloning of BASB02Q.
The BamHl and Sad restriction sites engineered into the forward ([SEQ ID NO; 11 ]) and reverse ([SEQ ID NO: 12]) amplification primers, respectively, permitted directional cloning of the 504 bp PCR product into the commercially available Exoli expression plasmid pQE30 (QiaGen, ampiciUin resistant) such that a mature BASB020 protein could be expressed as a fusion protein containing a (Hts)6 affinity chromatography tag at the N-terminus. The BASB020 PCR product was purified from the amplification

reaction using silica gei-based spin columns (QiaGen) according to the manufacturers instructions. To produce the required BamHl and Sail termini necessary for cloning, purified PCR product was sequentially digested to completion with BamHI and Sail restriction enzymes as recommended by the manufacturer (Life Technologies). Following the first restriction digestion, the PCR product was purified via spin column as above to remove salts and eluted in sterile water prior to the second enzyme digestion. The digested DNA fragment was again purified using silica gel-based spin columns prior to ligation with the pQE30 plasmid.
B: Production of Expression Vector.
To prepare the expression plasmid pQE30 for ligation, it was similarly digested to completion with both BamHI and SaR and then treated with calf intestinal phosphatase (CIP. -0.02 units / pmole of 5' end. Life Technologies) as directed by the manufacturer to prevent self ligation. An approximately 5-fold molar excess of the digested fragment to the prepared vector was used to program the ligation reaction. A standard --20 µl ligation reaction (-•16°C, -16 hours), using methods well known in the art was performed using T4 DNA ligase (-2.0 units / reaction. Life Technologies). An aliquot of the ligation (~5 µl) was used to transform electro-competent M15(pREP4) cells according to methods well known in the art. Following a -2-3 hour outgrowth period at 37°C in -1.0 ml of LB broth, transformed cells were plated on LB agar plates containing kanamycin (50 µg/ml) and ampicillin (100 µg/ml). Both antibiotics were included in the selection media to ensure that all transformed cells carried both the pREP4 plasmid (KnR), which carries the laclq gene necessary for the repression of expression for IPTG-inducible expression of proteins on pQE30, and the pQE30-BASB020 plasmid (ApR). Plates were incubated overnight at 37°C for -16 hours. Individual KnR / ApR colonies were picked with sterile toothpicks and used to "patch" inoculate fresh LB KnR / ApR plates as well as a --1.0 ml LB KnR / ApR broth culture.

Both the patch plates and the broth culture were incubated overnight at 37°C in either a standard incubator (plates) or a shaking water bath.
A whole cell-based PCR analysis was employed to verify that transformants contained the BASB020 DNA insen. Here, the -1,0 ml overnight LB Kn / Ap broth culture was transferred to a 1.5 ml polypropylene tube and the cells collected by centrifugation in a Beckmann microcentrifuge (-3 min., room temperature, -12,000 X g). The cell pellet was suspended in ~200|il of sterile water and a ~10µ1 aliquot used to program a --50µl final volume PCR reaction containing both BASB020 forward and reverse amplification primers. Final concentrations of the PCR reaction components were essentially the same as those specified in example 2 except ~5.0 units of Taq polymerase was used. The initial 95°C denaturation step was increased to 3 minutes to ensure thermal disruption of the bacterial cells and liberation of plasmid DNA. .'n ABI Model 9700 thermal cycler and a 32 cycle, three-step thermal amplification profile, i.e. 95°C, 45sec; 55-58°C, 45sec, 72°C, Imin., were used to amplify the MC-P6 PCR fragment from the lysed transformant samples. Following thermal amplification, a -20µl aliquot of the reaction was analyzed by agarose gel electrophoresis (0.8 % agarose in a Tris-acetate-EDTA (TAE) buffer). DNA fragments were visualized by UV illumination after gel electrophoresis and ethidium bromide staining. A DNA molecular size standard (1 Kb ladder, Life Teclinologies) was electrophoresed in parallel with the test samples and was used to estimate the size of the PCR products. Transformants that produced the expected 504bp PCR product were identified as strains containing a BASB020 expression construct. Expression plasmid containing strains were then analyzed for the inducible expression of recombinant BASB020.
C: Expression Analysis of PCR-Positive Transformants.
For each PCR-positive transformant identified above, -5.0 ml of LB broth containing
kanamycin (50 |ig/ml) and ampicillin (100 |ig/ml) was inoculated with cells from the

patch plate and grown overnight at 37 °C with shaking ('--250 rpm). An aliquot of the overnight seed culture (--1.0 mi) was inoculated into a 125 ml erlemneyer flask containing -25 ml of LB Kn / Ap broth and grown at 37 °C with shaking (-250 rpm) until the culture turbidity reached O.D,600 of-0.5, i,e. mid-log phase (usually about 1.5 - 2.0 hours). At this time approximately half of the culture (-12.5 ml) was transferred to a second 125 ml flask and expression of recombinant BASB020 protein induced by the addition of IPTG (1,0 M stock prepared in sterile water, Sigma) to a fmal concentration of 1.0 mM, Incubation of both the IPTG-induced and non-induced cultures continued for an additional -4 hours at 37 °C with shaking. Samples (-1.0 ml) of both induced and non-induced cultures were removed after the induction period and the cells collected by centrifugation in a microcentrifuge at room temperature for --3 minutes. Individual cell pellets were suspended in 50µl of sterile water, then mixed with an equal volume of 2X Laemelli SDS-PAGE sample buffer containing 2-mercaptoethanol, and placed in boiling water bath for -3 min to denature protein. Equal volumes (-15|il) of both the crude IPTG-induced and the non-induced cell lysates were loaded onto duplicate 12% Tris/glycine poiyacrylamide gel (1 mm thick Mini-gels, Novex). The induced and non-induced lysate samples were electrophoresed together with prestained molecular weight markers (SeeBlue, Novex) under conventional conditions using a standard SDS/Tris/glycine running buffer (BioRad). Following electrophoresis, one gel was stained with commassie brilliant blue R250 (BioRad) and then destained to visualize novel BASB020 IPTG-inducible protein(s). The second gel was electroblotted onto a PVDF membrane (0.45 micron pore size, Novex) for -2 hrs at 4 °C using a BioRad Mini-Protean II blotting apparatus and Towbin's methanol (20 %) transfer buffer. Blocking of the membrane and antibody incubations were performed according to methods well known in the an. A monoclonal anti-RGS (His)3 antibody, followed by a second rabbit anti-mouse antibody conjugated to HRP (QiaGen), was used to confirm the expression and identity of the BASB020 recombinant protein. Visualization of the anti-His antibody reactive pattern was achieved using either an

ABT insoluble substrate or using Hyperfilm with the Amersham ECL chemiluminescence system.
D: Sequence Confirmation.
To further verify that the IPTG-inducible recombinant BASB020 protein being expressed is in the correct open reading frame and not a spurious molecule arising from a cloning artifact (i.e. a frame-shift), the DNA sequence of the cloned insert was determined. The DNA sequence for the M.catarrhalis BASB020 gene was obtained from one strand using conventional asymrnetric PCR cycle sequencing methodologies (ABI Prism Dye-Terminator Cycle Sequencing, Perkin-Elraer), Sequencing reactions were programmed with undigested expression plasmid DNA (-0.5βg/rxn) as a template and appropriate pQE30 vector-specific and ORF-specific sequencing primers (-3.5 pmol/rxn). In addition to the template and sequencing primer, each sequencing reaction (--20β1) contained the four different dNTPs (i.e. A,G,C, and T) and the four corresponding ddNTPs (i.e. ddA, ddG, ddC, and ddT) tenninator nucleotides; with each terminator being conjugated to one of the four fluorescent dyes, Joe, Tarn, Rox, or Fam. Single strand sequencing elongation products were terminated at random positions along the template by the incorporation of the dye-labelled ddNTP terminators. Fluorescent dye-labelled termination products were purified using microcentrifuge size-exclusion chromatography colunms (Princeton Genetics), dried under vacuum, suspended in a Template Resuspension Buffer (Perkin-Elmer) for capillary electrophoresis or deionized formamide for PAGE, denatured at 95°C for --5 min, and analyzed by high resolution capillary electrophoresis (ABI 310 Automated DNA Sequenator. Perkin-Elmer) or high resolution PAGE (ABI 377 Automated DNA Sequenator) as recommended by the manufacturer. DNA sequence data produced from individual reactions were collected and the relative fluorescent peak intensities analyzed automaiically on a PowerMAC computer using ABI Sequence Analysis Software (Perkin-Elmer). Individually autoanalyzed DNA sequences were edited manually for

accuracy before being merged into a consensus single strand sequence "string" using AutoAssembler software (Perkin-Elmer). Sequencing determined that the expression plasmid contained the correct sequence in the correct open reading frame.
Example 4: Production of Recombinant BASB020
Bacterial strain
A recombinant expression strain of E. coli Ml 5 (pREP4) containing a plasmid (pQE30) encoding BASB020 from M. catarrhalis. was used to produce cell mass for purification of recombinant protein. The expression strain was cultivated on LB agar plates containing 50|ig/ml kanamycin ("Kn") and 100|ig/ml arapiciliin ("Ap") to ensure both the pREP4 laclq control plasmid and the pQE30-BASB020 expression construct were both maintained. For cryopreservation at -80 °C, the strain was propagated in LB broth containing the same concentration of antibiotics then mixed with an equal volume of LB broth containing 30% (w/v) glycerol.
Media
The fermentation medium used for the production of recombinant protein consisted of 2X YT broth (Difco) containing 50 µg/ml Kn and lOOµg/ml Ap. Antifoam was added to medium for the fermentor at 0.25 ml/L (Antifoam 204, Sigma). To induce expression of the BASB020 recombinant protein, IPTG (Isopropyl β-D-Thiogalactopyranoside) was added to the fermentor (1 mM, final),
Fermentation
A 500-ml erlenmeyer seed flask, containing 50ml working volume, was inoculated with 03 ml of rapidly thawed frozen culture, or several colonies from a selective agar plate ciihure. and incubated for aDoroximately 12 hours at 37 ± 1°C on a shaking platform at

150rpm (Innova 2100, New Brunswick Scientific). This seed culture was then used to inoculate a 5-L working volume fermentor containing 2X YT broth and both Kn and Ap antibiotics. The fermentor (Bioflo 3000, New Brunswick Scientific) was operated at 37 ± 1 °C, 0.2 - 0.4 VVM air sparge, 250 rpm in Rushton impellers. The pH was not controlled in either the flask seed culture or the fermentor. During fermentation, the pH ranged 6.5 to 7.3 in the fermentor. IPTG (1.0 M stock, prepared in sterile water) was added to the fermentor when the culture reached mid-log of growth (-0.7 O.D.600 units). Cells were induced for 2 - 4 hours then harvested by centrifugation using either a 28RS Heraeus (Sepatech) or RC5C superspeed centrifuge (Sorvall Instruments). Cell paste was stored at -20 C until processed.
Purification
Chemicals and Materials
Imidazole, guanidine hydrochloride, Tris (hydroxymethyl), and EDTA (ethylene-diamine tetraacetic acid) biotechnology grade or better were all obtained from Ameresco Chemical, Solon, Ohio. Triton X-100 (t-Octylphenoxypolyethoxy-ethanol), sodium phosphate, monobasic, and Urea were reagent grade or better and obtained from Sigma Chemical Company, St. Louis, Missouri. Glacial acetic acid and hydrochloric acid were obtained from Mallincrodt Baker Inc., Phillipsburg, New Jersey. Methanol was obtained from Fisher Scientific, Fairlawn, New Jersey. Pefabloc®SC (4-(2-Aminoethyl)-benzenesulfonylfuoride), Complete protease inhibitor cocktail tablets, and PMSF (phenylmethyl-sulfonylfluoride) were obtained from Roche Diagnostics Corporation, Indianapolis, Indiana. Bestatin, Pepstatin A, and E-64 protease inhibitor were obtained from Calbiochem. LaJolla. California. Dulbecco's Phosphate Buffered Saline(lx PBS) was obtained from Quality Biological, Inc., Gaithersburg, Maryland. Dulbecco's Phosphate Buffered Saline (lOx PBS) was obtained from BioWhittaker, Walkersville, Maryland. Penta-His Antibody, BSA free was obtained from QiaGen, Valencia, California. Peroxidase-conjugated AffiniPure Goat Anti-mouse IgG was

obtained from Jackson Immune Research, West Grove, Perm. AEC single solution was obtained from Zymed, South San Francisco, California. All other chemicals were reagent grade or better,
Ni-NTA Superflow resin was obtained from QiaGen Inc., Valencia, California. Precast Tris-Glycine 4-20% and 10-20% polyacrylamide gels, all running buffers and solutions, SeeBlue Pre-Stained Standards, MultiMark Multi-Colored Standards and PVDF transfer membranes were obtained from Novex, San Diego, California. SDS-PAGE Silver Stain kits were obtained from Daiichi Pure Chemicals Company Limited, Tokyo, Japan. Coomassie Stain Solution was obtained from Bio-Rad Laboratories, Hercules, California. Acrodisc® PF 0.2 µm syringe filters were obtained from Pall Gelman Sciences. Ann Arbor. Michigan. GD/X 25mm disposable syringe filters were obtained from Whatman Inc., Clifton, New Jersey. Dialysis tubing 8,000 MWCO was obtained from BioDesign Inc. Od New York, Carmal New York. BCA Protein Assay Reagents and Snake Skin dialysis tubing 3,500 MWCO were obtained from Pierce Chemical Co,, Rockford, Illinois.
Extraction Protocol
Cell paste was thawed at room temperamre for 30 to 60 minutes. Five to six grams of material was weighed out into a 50 ml disposable centrifuge tube. To this five mis/gram of Guanidine hydrochloride (Gu-HCl) buffer was added (6 M Guanidine hydrochloride, 100 mM Sodium phosphate, monobasic, 10 mM Tris and 0.05 % Triton X-100, pH 8.0). Cell paste was resuspended using a PRO300D proscientific homogenizer, at 3/4 power for one minute. The extraction mixture was then placed at room temperature with gentle agitation for 60 to 90 minutes. After 60 to 90 minutes the extraction mixture was centrifuged at 15,800 x g for 15 minutes (Sorvall RC5C centrifuge, 11,500 rpm). The supernatant (SI) was decanted and saved for additional purification. The pellet (PI) was saved for analysis.

Binding of BASB020 to Nickel-NTA Resin
To the SI three to four mis of Ni-NTA resin is added. This is then placed at room temperature with gentle agitation for one hour. After one hour the S 1/Ni-NTA is packed into an XK16 Pharmacia column. The column is then washed with 1 M Gu-HCl buffer (1 M Guanidine hydrochloride, lOOmM Sodium phosphate, monobasic, 10 mM Tris and 0.05% Triton X-lOO, pH 8.0). This is then followed by a wash with phosphate buffer (lOOmM Sodium phosphate, monobasic, 10 mM Tris and 0.05% Triton X-100, pH 6.3). The protein is then eluted from the column with a 250 mM imidazole buffer (250 mM imidazole, lOOmM Sodium phosphate, monobasic, 10 mM Tris and 0.05% Triton X-100, pH 5.9).
Final Formulation
BASB020 was formulated by dialysis overnight against, three changes of 0.1 % Triton X-100 and Ix PBS, pH 7.4, to remove residual Gu-HCl and imidazole. The purified protein vvas characterized and used to produced antibodies as described below.
Biochemical Characterizations SDS-PAGE and Western Blot Analysis
The recombinant purified protein was resolved on 4-20 % polyacryiamide gels and elecirophoretically transferred to PVDF membranes at 100 V for 1 hour as previously described (Thebaine et al. 1979, Proc. Natl. Acad. Sci. USA 76:4350-4354). The PVDF membranes were then pretreated with 25 ml of Dulbecco's phosphate buffered saline containing 5 % non-fat dry milk. All subsequent incubations were carried out using this pretreatment buffer.
PVDF membranes were incubated with 25 ml of a 1:500 dilution of preimmune serum or rabbit anti-His immune serum for 1 hour at room temperature. PVDF membranes were then washed twice with wash buffer (20 mM Tris buffer, pH 7.5, containing 150

mM sodium chloride and 0.05 % Tween-20). PVDF membranes were incubated with 25 ml of a 1:5000 dilution of peroxidase-labeled goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 minutes at room temperature, PVDF membranes were then washed 4 times with wash buffer, and were developed with 3-amino-9-ethylcarbazole and urea peroxide as supplied by Zymed (San Francisco, CA) for ] 0 minutes each.
The results of an SDS-PAGE (Figure 4) show a protein about 32-35 kDa purified to greater than 90 % and that is reactive to an anti-RGS(His) antibody by western blots (Figure 5) of the SDS-PAGE.
Protein Sequencing
Amino terminal amino acid sequencing of the purified protein was performed to confirm the production of the correct recombinant protein using well defined chemical protocols on Hewlett-Packard model G1OOOA sequencer with a model 1090 LC and a Hewlett-Packard model 241 sequencer with a model 1100 LC.
Example 5: Production of Antisera to Recombinant BASBQ20
Polyvalent antisera directed against the BASB020 protein were generated by vaccinating two rabbits with the purified recombinant BASB020 protein. Each animal is given a total of three immunizations intramuscullarly (i.m.) of about 20µg BASB020 protein per injection (beginning with complete Freund's adjuvant and followed with incomplete Freund's adjuvant) at approximately 21 day intervals. Animals were bled prior to the first immunization ("pre-bleed") and on days 35 and 57.

Anti-BASB020 protein litres were measured by an ELISA using purified recombinant BASB020 protein (0.5 (ig/well). The litre is defined as the highest dilution equal or greater than 0.1 as calculated with the following equation: average OD of two test samples of antisera - the average OD of two test samples of buffer. The litres after three immunizations were between 3000 and 8000.
The antisera were used as the first antibody to identify the protein in a western blot as described in example 4 above. The western-blot showed the presence of anti-BASB020 antibody in the sera of immunized animals.
Example 6- Immunological Characterisation
Western Blot Analysis
Several strains of M catarrhalis including ATCC 49143, and ATCC 43617, as well as clinical isolates from various geographic regions, were grown on chocolate agar plates for 48 hours at 35°C in 5% C02. Several colonies were used to inoculate 25ml of Muller Hinton broth in a 250 ml flask. Cultures were grown overnight and collected by centrifugation. Cells were then solubilized by suspending 30µg of cells in 150µ1 of PAGE sample buffer (360 mM Tris buffer, pH 8.8, containing 4% sodium dodecylsulfate and 20% glycerol), and incubating the suspension at 100 C for 5 minutes. The solubilized cells were resolved on 4-20% polyacrylamide gels and the separated proteins were electrophoretically transferred to PVDF membranes at lOOV for 1hour as previously described (Thebaine et al 1979, Proc. Natl. Acad. Sci. USA 76:4350-4354). The PVDF membranes were then pretreated with 25 ml of Dulbecco's phosphate buffered saline containing 5 % non-fat dry milk. All subsequent incubations were carried out using this pretreatmenl buffer.

PVDF membranes were incubated with 25ml of a 1:500 dilution of preimmune serum or rabbit immune serum for 1hour at room temperature. PVDF membranes were then washed twice with wash buffer (20 mM Tris buffer, pH 7.5, containing 150 mM sodium chloride and 0.05% Tween-20). PVDF membranes were incubated with 25ml of a 1 ;5000 dilution of peroxidase-labeied goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30minutes at room temperature. PVDF membranes were then washed 4 times with wash buffer, and were developed with 3-amino-9-ethylcarbazole and urea peroxide as supplied by Zymed (San Francisco, CA) for 10 minutes each.
A protein of about 32-35 kDa (corresponding to BASB020 expected molecular weight) that is reactive with the antisera is detected in all Moraxella strains.
Example 7: Presence of Antibody to BASBQ20 in Human Convalescent Sera
Western blot analysis of purified recombinant BASB020 were performed as described in Example 4 and 6 above, except that a pool of human sera from children infected by M. caiarrhalis is used as the first antibody preparation. Results show that antisera from naturally infected individuals react to the purified recombinant protein, as shown in Fiaure 6.

Example 8. Efficacy of BASB020 vaccine: enhancement of lung clearance of M catarrhaiis in mice.
The protective capacity of purified recombinant BASB020 protein was tested in a mouse model. This mouse model is based on the analysis of the lung invasion by M. catarrhaiis following a standard intranasal challenge to vaccinated mice. Groups of 6 BALB/c mice (females, 6 weeks old) are immunized subcutaneously with lOOµl of vaccine corresponding to a 10µg dose and are boosted 2 weeks later. One week after the booster, the mice are challenged by instillation of 50 µl of bacterial suspension (-H/-106 CFU/50 µl) into the left nostril under anaesthesia (mice are anaesthetised with a combination of ketamine and xylazine anaesthetics, 0.24 mg xyiazine (Rompun) and 0.8 mg ketamine (lmalgene)/100 µl). Mice are killed 4 hours after challenge and the lungs are removed aseptically and homogenized individually. The loglO weighted mean number of CFU/lung is determined by counting the colonies grown on Muelier-Hinton agar plates after plating of 20 \x[ of 5 serial dilutions of the homogenate. The arithmetic mean of the log 10 weighted mean number of CFU/lung and the standard deviations are calculated for each group.
Resuhs are analysed statistically by applying 1-way ANOVA after assuming equality of variance (checked by Brown and Forsythe's test) and normality (checked using the Shapiro-Wilk testl. Differences between groups were analysed using the Dunnet test. Tukey's studentised range test (HSD) and Student-Newman-Keuls test-In this experiment groups of mice were immunized either with BASB020 adsorbed onto AIPO4 (1 Oµg of BASB020 onto lOOµg of A1P04) or with a killed whole cells (kwc) preparation of M catarrhaiis strain ATCC 43617 adsorbed onto AIPO4 (5 108cells onto lOOµg AIPO4) or with lOOµg AIPO4 without antigen. The mice were challenged with 10^ CFU of live M catarrhaiis strain ATCC 43617 bacteria.

The log 10 weighted mean number of CFU/lung and the standard deviation 4 hours after
challenge were calculated for each group. Sham immunized mice had 5.5 (+/- 0.23)
log 10 CFU/lungs 4 hours after challenge
The kwc preparation induced significant lung clearance as compared to the control
group (1.74 log difference). BASB020 vaccine induced a 0.62 log difference in lung
clearance as compared to the control group, which was significantly different ft'om the
control.
A western-blot using purified recombinant BASB020 protein and pooled sera from mice
immunized with BASB020 protein and collected before challenge show the presence of
antibody to BASB020 protem (Figure 7).
Example 9: Production of BASBQ2Q peptides, Antisera and Reactivity Thereof
Two short amino acid BASB020 specific peptides, having the sequences of CNEEAWSQNRRAELSY (SEQ ID N0:13) and YTGVAPLVDNDETV (SEQ ID NO: 14) were produced in the laboratory using generally well known methods. These peptides coupled to KLH were used to produce antibodies in 12 weeks old Specific Pathogen Free New-Zealand female rabbits. Rabbits received 4 injections at approximately 3 weeks intervals of 200 µg of peptide-KLH in complete (1st injection) or incomplete (2nd 3rd and 4th injections) Freund's adjuvant Animals were bled prior to the first immunization and one month after the 4th injection.
Anti-peptide mid-point titres were measured by an ELISA using free peptides. Anti-peptide Mid-point titres one month after the 4thimmunization were superior to 41000. Western blots of purified recombinant BASB020, using anli-peptide antibodies as the first antibody, were prepared as described in Example 4 and 6. The results are presented in Figure 8.

Deposited materials
A deposit containing a Moraxella catarrhalis Catlin strain has been deposited with the American Type Culture Collection (herein "ATCC") on June 21, 1997 and assigned deposit number 43617. The deposit was described as Branhamella catarrhalis (Frosch and Kolle) and is a freeze-dried, 1.5-2.9 kb insert library constructed from M. catarrhalis isolate obtained from a transtracheal aspirate of a coal miner with chronic bronchitits. The deposit is described in Antimicrob. Agents Chemother. 21: 506-508 (1982).
The Moraxella catarrhalis strain deposit is referred to herein as "the deposited strain" or as "the DNA of the deposited strain."
The deposited strain contains a full lengthBASB020 gene.
A deposit of the vector pMC-Hiy3 consisting of Moraxella catarrhalis DNA inserted in pQE30 has been deposited with the American Type Culture Collection (ATCC) on February 12th 1999 and assigned deposit number 207106.
The sequence of the polynucleotides contained in the deposited strain / clone, as well as the amino acid sequence of any polypeptide encoded thereby, are controlling in the event of any conflict with any description of sequences herein.
The deposit of the deposited strains have been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure. The deposited strains will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. The deposited strains are provided merely as convenience to those of skill in the art and are not an admission that a deposit is required for enablement, such as that required under35U.S.C. §112.



WE CLAIM:
1. A polypeptide comprising an amino acid sequence which has at least 85%
identity to the amino acid sequence selected from the group consisting of: SEQ ID
N0:4 and SEQ ID NO:6 over the entire length of SEQ ID N0:4 or SEQ ID N0:6
respectively.
2. The polypeptide as claimed in claim 1 in which the amino acid sequence has at
least 95% identity to the amino acid sequence selected from the group consisting of:
SEQ ID N0:4 and SEQ ID N0:6 over the entire length of SEQ ID N0:4 and SEQ ID
N0:6 respectively.
3. The polypeptide as claimed in claim 1 comprising the amino acid sequence selected from the group consisting of: SEQ ID N0:4 and SEQ ID NO:6.
4. A polypeptide consisting of the amino acid sequence selected from the group consisting of SEQ ID N0:4 and SEQ ID N0;6.
5. An immunogenic fragment of the polypeptide as claimed in any one of claims 1 to 4 in which the immunogenic fragment has at least 15 contiguous amino acids from the amino acid sequence of SEQ ID N0:4 or 6 and which is capable of raising an immune response (if necessary when coupled to a carrier) which recognises the polypeptide of SEQ ID N0:4 or SEQ ID N0:6.
6. The polypeptide as claimed in any of claims 1 to 5 wherein said polypeptide is
part of a larger fusion protein.
7. A polynucleotide encoding the polypeptide or immunogenic fragment as claimed in any of claims 1 to 6.
8. A polynucleotide comprising a nucleotide sequence encoding a polypeptide that has at least 85% identity to the amino acid sequence of SEQ ID N0:4 or SEQ ID N0:6 over the entire length of SEQ ID N0:4 or SEQ ID N0:6 respectively, or a nucleotide sequence complementary to said polynucleotide.

9. A polynucleotide comprising a nucleotide sequence that has at least 85% identity to a nucleotide sequence encoding a polypeptide of SEQ ID N0:4 or SEQ ID N0:6 over the entire coding region; or a nucleotide sequence complementary to said isolated polynucleotide.
10. A polynucleotide which comprises a nucleotide sequence which has at least 85% identity to that of SEQ ID N0:3 or SEQ ID N0:5 over the entire length of SEQ ID N0:3 or SEQ ID N0:5 respectively; or a nucleotide sequence complementary to said isolated polynucleotide.
11. The polynucleotide as claimed in any one of claims 7 to 10 in which the identity is at least 95% to SEQ ID NO:3 or SEQ ID N0:5,
12. A polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID N0:4 or SEQ ID N0;6.
13. A polynucleotide consisting of the polynucleotide of SEQ ID N0:3 or SEQ ID N0:5.
14. A polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID N0;4 or SEQ ID N0:6 obtainable by screening an appropriate library under stringent hybridization conditions with a labeled probe having the sequence of SEQ ID N0;3 or SEQ ID N0;5 or a fragment thereof, wherein the stringent hybridization conditions use a temperature in the range of 45 to 65*^0 and on SDS concentration from 0.1 to 1%.
15. An expression vector comprising a recombinant polynucleotide according to any one ofclaims 7 to 14.
16. A recombinant live microorganism comprising an expression vector according to claim 15.

17 A host cell comprising the expression vector of claim 15, expressing a polypeptide comprising an amino acid sequence that has at least 85% identity to the amino acid sequence selected from the group consisting of: SEQ ID NO:4 or SEQ ID NO: 6, or a membrane of the host cell containing the expressed polypeptide.
18. A process for producing a polypeptide or immunogenic fragment of claims 1 to
6 comprising culturing a host cell as claimed in claim 17 under known conditions sufficient for
production of said polypeptide or immunogenic fragment and recovering the polypeptide from the culture medium.
19. A process for expressing a polynucleotide of any one of claims 7 to 14 comprising transforming a host cell with an expression vector comprising at least one of said polynucleotides and culturing said host cell under known conditions sufficient for expression of any one of said polynucleotides.
20. A vaccine composition comprising an effective amount of the polypeptide or immunogenic fragment of any one of claims 1 to 6 and a pharmaceutically acceptable carrier.
21. A vaccine composition comprising an effective amount of the polynucleotide of any one of claims 7 to 14 and pharmaceutically acceptable carrier.
22. An antibody immunospecific for a polypeptide having an amino acid sequence which has at least 85% identity to the amino acid sequence selected from the group consisting of: SEQ ID N0:4 and SEQ ID N0:6 over the entire length of SEQ ID N0:4 or SEQ ID N0:6 respectively.
23. A therapeutic composition comprising at least one antibody according to claim 22 and a suitable pharmaceutical carrier.


Documents:

in-pct-2000-643-che-abstract.pdf

in-pct-2000-643-che-claims filed.pdf

in-pct-2000-643-che-claims granted.pdf

in-pct-2000-643-che-correspondnece-others.pdf

in-pct-2000-643-che-correspondnece-po.pdf

in-pct-2000-643-che-description(complete)filed.pdf

in-pct-2000-643-che-description(complete)granted.pdf

in-pct-2000-643-che-drawings.pdf

in-pct-2000-643-che-form 1.pdf

in-pct-2000-643-che-form 26.pdf

in-pct-2000-643-che-form 3.pdf

in-pct-2000-643-che-form 5.pdf

in-pct-2000-643-che-other documents.pdf

in-pct-2000-643-che-pct.pdf


Patent Number 213022
Indian Patent Application Number IN/PCT/2000/643/CHE
PG Journal Number 13/2008
Publication Date 28-Mar-2008
Grant Date 19-Dec-2007
Date of Filing 10-Nov-2000
Name of Patentee SMITHKLINE BEECHAM BIOLOGICALS S. A
Applicant Address Rue de l'Institut 89, B-1330 Rixensart,
Inventors:
# Inventor's Name Inventor's Address
1 Joelle THONNARD SmithKline Beecham Biologicals S.A. Rue de L'Institut 89 B-1330 Rixensart,
PCT International Classification Number C07K 14/21
PCT International Application Number PCT/EP1999/003257
PCT International Filing date 1999-05-07
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 9810285.8 1998-05-13 U.K.