CA2426413A1

CA2426413A1 - Basb207 polypeptides and polynucleotides from nontypeable haemophilus influenzae

Info

Publication number: CA2426413A1
Application number: CA002426413A
Authority: CA
Inventors: Joelle Thonnard
Original assignee: Individual
Current assignee: GlaxoSmithKline Biologicals SA
Priority date: 2000-10-17
Filing date: 2001-10-16
Publication date: 2002-04-25
Also published as: WO2002032946A8; WO2002032946A3; WO2002032946A2; GB0025488D0; EP1328541A2; AU2002220635A1

Abstract

The invention provides nontypeable Haemophilus influenzae BASB207 polypeptides and polynucleotides encoding BASB207 polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are diagnostic, prophylactic and therapeutic uses, including vaccines against bacterial infections.

Description

Novel Compounds FIELI3 OF T>EIE INVENTION
This invention relates to polynucleotides, (herein referred to as "BASB207 polynucleotide(s)"), polypeptides encoded by them (referred to herein as °°BASB207" or "BASB207 polypeptide(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.
BACKGROI~ND OF THE INVENTION
Haemophilus influenzae is a non-motile Gram negative bacterium. Man is its only natural host.
H. inflasenzae isolates are usually classified according to their polysaccharide capsule.
Six different capsular types designated a through f have been identif ed.
Isolates that fail to agglutinate with antisera raised against one of these six serotypes are classified as non typeable, and do not express a capsule.
The H. influenzae type b is clearly different from the other types in that it is a major cause of bacterial meningitis and systemic diseases. Non typeable X in, fh~enzae (NTHi) are only occasionally isolated from the blood of patients with systemic disease.
NTHi is a common cause of pneumonia, exacerbation of chronic bronchitis, sinusitis and otitis media.
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 children have experienced at least one episode of otitis before reaching the age of 3 (1). Left untreated, or becoming chronic, this disease may lead to hearing loss that can 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 delayed speech learning.
Three bacterial species are primarily isolated from the middle ear of children with otitis media: Streptococcus pnea~moniae, NTHi and M. catarrhalis. These are present in 60 to 90 % of eases. A review of recent studies shows that S. pnea~moniae and NTHi each represent about 30 °~°, and M. catarrhalis about 15 %
of otitis media cases (2). Other bacteria can be isolated from the middle ear (H. inJluenzae type B, S.
pyogenes, ...) 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 1 S development of an otitis; other factors are however also required to lead to the disease (3-9). 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 other factors are unknown todate. It has been postulated that a transient anomaly of the immune system following a viral infection, for example, could cause an inability to control the colonization of the respiratory tract (5). An alternative explanation is that the exposure to environmental factors allows a more important colonization of some children, who subsequently become susceptible to the development of otitis media because of the sustained presence of middle ear pathogens (2).
Various proteins of H. infla~enzae have been shown to be involved in pathogenesis or have been shown to confer protection upon vaccination in animal models.
Adherence of NTHi to human nasopharygeal epithelial cells has been reported (10).
Apart from fimbriae and pili (11-15), many adhesins have been identified in NTHi.
Among them, two surface exposed high-molecular-weight~proteins designated HMW 1 and HMW2 have been shown to mediate adhesion of NTHi to epithelial cells (16). Another family of high molecular weight proteins has been identified in NTHi strains that lack proteins belonging to HMW IlHMW2 family. The NTHi 115 kDa Hia protein (17) is highly similar to the Hsf adhesin expressed by H.
influenzae type b strains ( 18). Another protein, the Hap protein shows similarity to IgA 1 serine proteases and has been shown to be involved in both adhesion and cell entry ( 19).
Five major outer membrane proteins (OMP) have been identified and numerically numbered.
Original studies using H. influenzae type b strains showed that antibodies specific for P1 and P2 protected infant rats from subsequent challenge (20-21). P2 was found to be able to induce bactericidal and opsonic antibodies, which are directed against the variable regions present within surface exposed loop structures of this integral OMP
(22-23). The lipoprotein P4 also could induce bactericidal antibodies (24).
P6 is a conserved peptidoglycan-associated lipoprotein making up 1-5 % of the outer membrane (25). Later a lipoprotein of about the same mol. wt. was recognized, called PCP (P6 crossreactive protein) (26). A mixture of the conserved lipoproteins P4, P6 and PCP did not reveal protection as measured in a chinchilla otitis-media model (27). P6 alone appears to induce protection in the chinchilla model (28).
P5 has sequence homology to the integral Escherichia coli OmpA (29-30). PS
appears to undergo antigenic drift during persistent infections with NTHi (31).
However, conserved regions of this protein induced protection in the chinchilla model of otitis media.
In line with the observations made with gonococci and meningococci, NTHi expresses a dual human transferrin receptor composed of TbpA and TbpB when grown under iron limitation. Anti-TbpB protected infant rats. (32). Hemoglobin l haptoglobin receptors have also been described for NTHi (33). A receptor for Haem:

Hemopexin has also been identified (34). A lactofernn receptor is also present in NTHi, but is not yet characterized (35).
A 80kDa OMP, the D 15 surface antigen, provides protection against NTHi in a mouse challenge model. (36). A 42kDa outer membrane lipoprotein,LPD is conserved amongst Haemophilus influenzae and induces bactericidal antibodies (37).
A minor 98kDa OMP (38), was found to be a protective antigen, this OMP may very well be one of the Fe-limitation inducible OMPs or high molecular weight adhesins that have been characterized. H. influenzae produces IgAI-protease activity (39).
IgA 1-proteases of NTHi reveals a high degree of antigenic variability (40).
Another OMP of NTHi, OMP26, a 26-kDa protein has been shown to enhance pulmonary clearance in a rat model (41). The NTHi HfrA protein has also been shown to be a protective antigen. Indeed, this protein protected Chinchilla against otitis media and protected infant rats against H. influenzae type b bacteremia (42) Background References 1. Klein, JO ( 1994) Clin.Inf.Dis 19:823 2. Murphy, TF (1996) Microbiol.Rev. 60:267 3. Dickinson, DP et al. (1988) J. Infect.Dis. 158:205 4. Faden, HL et al. (1991) Ann.Otorhinol.Laryngol. 100:612 5. Faden, HL et al (1994) J. Infect.Dis. 169:1312 6. Leach, AJ et al. (1994) Pediatr.Infect.Dis.J. 13:983 7. Prellner, KP et al. (1984) Acta Otolaryngol. 98:343 8. Stenfors, L-E and Raisanen, S. ( 1992) J.Infeet.Dis. 165:1148 9. Stenfors, L-E and Raisanen, S. ( 1994) Acta Otolaryngol. 113:191 10. Read, RC. et al. (1991) J. Infect. Dis. 163:549 11. Brinton, CC. et al. (1989) Pediatr. Infect. Dis. J. 8:S54 12. Kar, S. et al. (1990) Infect. Immun. 58:903 13. Gildorf, JR. et al. (1992) Infect. Immun. 60:374 14. St. Genre, JW et al. (1991) Infect. Immun. 59:3366 15. St. Genre, JW et al. ( 1993) Infect. Immun. 61: 2233 16. St. Genre, JW. et al. (1993) Proc. Natl. Acad. Sci. USA 90:2875 17. Barenkamp, SJ. et JW St Genre ( 1996) Mol. Microbiol. (In press) 18. St. Genre, JW. et al. ( 1996) J. Bact. 178:6281 19. St. Genre, JW. et al. (1994) Mol. Microbiol. 14:217 20. Loeb, MR. et al. ( 1987) Infect. Immun. 55:2612 21. Musson, RS. Jr. et al. (1983) J. Clin. Invest. 72:677 22. Haase, EM. et al. (1994) Infect. Immun. 62:3712 23. Troelstra, A. et al. ( 1994) Infect. Immun. 62:779 24. Green, BA. et al. (1991) Infect.Immun.59:3191 25. Nelson, MB. et al. (1991) Infect. Immun. 59:2658 26. Deich, RM. et al. (1990) Infect. Immun. 58:3388 27. Green, BA. et al. (1993) Infect.immun. 61:1950 28. Demaria, TF. et al. ( 1996) Infect. Immun. 64:5187 29. Miyamoto, N., Bakaletz, LO (1996) Microb. Pathog. 21:343 30. Munson, RS.j.r. et al. (1993) Infect. Immun. 61:1017 31. Duim, B. et a1. (1997) Infect. Immun. 65:1351 32. Loosmore, SM. et a1(1996) Mol.Microbiol. 19:575 33. Maciver, I. et al. (1996) Infect. Immun. 64:3703 34. Cope, LD. et al. (1994) Mol.Microbiol. 13:868 35. Schryvers, AB. et al. (1989) J. Med. Microbiol. 29:121 36. Flack, FS. et al. (1995) Gene 156:97 37. Akkoyunlu, M. et al. (1996) Infect. Immun. 64:4586 38. Kimura, A. et al. (1985) Infect. Immun. 47:253 39. Mulks, MH. et Shoberg, RJ (1994) Meth. Enzymol. 235:543 40. Lomholt, H. Alphen, Lv, Kilian, M. (1993) Infect. Immun. 61:4575 41. Kyd, J.M. and Cripps, A. W. ( 1998) Infect. Immun. 66:2272 42. Loosmore, S.M. et al. (199$) Infect. Immun. 66:899 The frequency of NTHi infections has risen dramatically in the past few decades. This phenomenon has created an unmet medical need for new anti-microbial agents, vaccines, drug screening methods and diagnostic tests for this organism. The present invention aims to meet that need.

SUMMARY OF THE INVENTION
The present invention relates to BASB207, in particular BASB207 polypeptides and BASB207 polynueleotides, 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 BASB207 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 BASB207 polypeptides and polynucleotides as described in greater detail below. In particular, the invention relates to polypeptides and polynucleotides of BASB207 of non typeable H. influenzae. The BASB207 polypeptide has some characteristics of an autotransporter protein. BASB207 has a signal sequence localised from residue 1 to residue 45. This signal sequence is cleaved in the mature protein and it could thus be exposed at the surface of the bacterium. The mature protein has three distinct domains. The hF-terminal domain is located from residue 46 to residue 117 of the BASB207 polypeptide. This domain contains a (3-wrap that has adhesin function in some autotransporter proteins.
This domain is likely to be surface-exposed. The second domain of the mature protein is likely to contain an hydrophobic a-helix located from residue 1175 to the residue 1211 of the BASB207 polypeptide. The third domain is likely to contain four anti-parallel (3-strands located from residue 1237 to the end of the BASB207 polypeptide.
These (3-strands are predicted to span the outer-membrane, forming a pore through which the N-terminal domain is secreted.
The invention relates especially to BASB207 polynucleotides and encoded polypeptides listed in table 1. Those polynucleotides and encoded polypeptides have the nucleotide and amino acid sequences set out in SEQ ID NO:1 to SEQ ID N0:8 as described in table A.
Table A
Strain isolated from Nucleotidic peptidic in sequence sequence 3224A USA Otitis mediaSEQ ID NO:1 SEQ ID N0:2 322~A USA Otitis mediaSEQ ID N0:3 SEQ ID NO:~

32190 USA Otitis mediaSEQ ID NO:S SEQ ID N0:6 A84016~ NL Carrier strainSEQ ID N0:7 SEQ ID N0:8 It is understood 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 ribopolynueleotides.
The sequences of the BASB207 polynucleotides are set out in SEQ ID NO: l, 3, 5, 7.
SEQ Group 1 refers herein to any one of the polynucleotides set out in SEQ ID
NO:1, 3, 5, 7.
The sequences of the BASB207 encoded polypeptides are set out in SEQ ID N0:2, 4, 6, 8. SEQ Group 2 refers herein to any one of the encoded polypeptides set out in SEQ ID
N0:2, 4, 6, 8.
Polypeptides In one aspect of the invention there are provided polypeptides of non typeable H.
influenzae referred to herein as "BASB207" and "BASB207 polypeptides" as well as biologically, diagnostically, prophylaetically, 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 any sequence of SEQ
Group 2;
(b) a polypeptide 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 any sequence of SEQ Group 1 over the entire length of the selected sequence of SEQ Group 1; 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 any sequence of SEQ Group 2.
The BASB207 polypeptides provided in SEQ Group 2 are the BASB207 polypeptides from non typeable H. influenzae strains as described in tableA.
The invention also provides an immunogenic fragment of a BASB207 polypeptide, that is, a contiguous portion of the BASB207 polypeptide which has the same or substantially the same immunogenic activity as the polypeptide comprising the corresponding amino acid sequence selected from SEQ Group 2 ; That is to say, the fragment (if necessary when coupled to a carrier) is capable of raising an immune response which recognises the BASB207 polypeptide. Such an immunogenic fragment may include, for example, the BASB207 polypeptide lacking an N-terminal leader sequence, andlor a transmembrane domain and/or a C-terminal anchor domain.
In a preferred aspect the immunogenic fragment of BASB207 according to the invention comprises substantially all of the extracellular domain of a polypeptide which has at least $5% identity, preferably at least 90% identity, more preferably at least 95% identity, most preferably at least 97-99% identity, to that a sequence selected from SEQ Group 2 over the entire length of said sequence.
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 BASB207 polypeptides, fragments may be "free-standing," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region in a single larger polypeptide.
Preferred fragments include, for example, truncation polypeptides having a portion of an amino acid sequence selected from SEQ Group 2 or of variants thereof, such as a continuous series of residues that includes an amino- 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 structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-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 an amino acid sequence selected from SEQ Group 2 or an isolated polypeptide comprising an amino acid sequence having at least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated or deleted from an amino acid sequence selected from SEQ Group 2 .
BASB207 has a signal sequence localised from residue 1 to residue 45. The mature protein has three distinct domains. The N-terminal domain is located from residue X16 to residue 1174 of the BASB207 polypeptide. This domain contains a j3-wrap that has adhesin function in some autotransporter proteins. This domain is likely to be surface-exposed. The second domain of the mature protein is likely to contain an hydrophobic a-helix located from residue 1175 to the residue 1211 of the BASB207 polypeptide.°'fhe third domain is likely to contain four anti-parallel (3-strands located from residue 1237 to the end of the BASB207 polypeptide. These (3-strands are predicted to span the outer-membrane, forming a pore through which the N-terminal domain is secreted. Each of these peptides/domains (comprised within any of the SEQ Group 2 polypeptides) are preferred fragments of the invention, as are truncates, variants, conjugates and recombinant fusions as described herein. The N-terminal domain is particularly preferred as a vaccine candidate. Furthermore full length polypeptides missing one or more of the above peptides/domains are further peptides/polypeptides of the invention (for instance a polypeptide lacking the signal peptide).
Still further preferred fragments are those which comprise a B-cell or T-helper epitope, for example those fragments/peptides described in Example 13.
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 full-length polypeptides of the invention.
Particularly preferred are variants in which several, 5-10, 1-5, 1-3, 1-2 or 1 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 part of a larger protein such as a precursor or a fusion protein. It is often advantageous to include an additional amina 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 portions 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 IgGI, where fusion takes place at the hinge region. In a particular 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. W094129458 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 partner 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 partner will be both an immunological fusion partner and expression enhancing partner.
Fusion partners include protein D from Haemophilats influenzae and the non-structural protein from influenza virus, NS 1 (hemagglutinin). Another fusion partner is the protein known as Omp26 (WO 97101638). Another fusion partner is the protein known as LytA. Preferably the G terminal portion of the molecule is used.
LytA is derived from Streptococcus pneurnoniae which synthesize an N-acetyl-L-alanine amidase, amidase LytA, (coded by the lvt~t gene tGene, 43 (19$6) page 272 f ) an autolysin that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain of the LytA protein is responsible for the affinity to the choline or to some choline analogues such as DEAF. This property has bee$
exploited for the development ofE.coli 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 Ile; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gln; and among the basic residues Lys and Arg; 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 polypeptide of the invention is derived from non typeable H.
influenzae, 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 invention to provide polynucleotides that encode polypeptides, particularly polynucleotides that encode the polypeptides herein designated BASB207.

In a particularly preferred embodiment of the invention the polynucleotides comprise a region encoding BASB207 polypeptides comprising sequences set out in SEQ
Group° 1 which include full length gene, or a variant thereof.
The BASB207 polynucleotides provided in SEQ Group 1 are the BASB207 polynucleotides from non typeable H. inJlasenzae strains as described in table A.
As a further aspect of the invention there are provided isolated nucleic acid molecules encoding andlor expressing BASB207 polypeptides and polynucleotides, particularly non typeable H. influenzae BASB207 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 polynucleotides, including at least one full length gene, that encodes a BASB207 polypeptide having a deduced amino acid sequence of SEQ Group 2 and polynucleotides closely related thereto and variants thereof.
In another particularly preferred embodiment of the invention relates to polypeptide from non typeable H. inflzienzae comprising or consisting of an amino acid sequence selected from SEQ Group 2 or a variant thereof.
Using the information provided herein, such as a polynucleotide sequences set out in SEQ Group 1 , a polynucleotide of the invention encoding BASB207 polypeptides may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteria using non typeable H
influenzae strain322~A 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 Group l, typically a library of clones of chromosomal DNA of non typeable H. influenzae strain 3224A in E.coli or some other suitable host is probed with a radiolabeled oligonucleotide, 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 LtIBOR~4TORYMANU~1L, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). (see in particular Screening By Hybridization 1.90 and Sequencing Denatured Double-Stranded DNA Templates 13.70). Direct genomic DNA sequencing may also be performed to obtain a full length gene sequence. Illustrative of the invention, each polynucleotide set out in SEQ
Group 1 was discovered in a DNA library derived from non typeable H.
influenzae.
Moreover, each DNA sequence set out in SEQ Group 1 contains an open reading frame encoding a protein having about the number of amino acid residues set forth in SEQ
Group 2 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 polynucleotides of SEQ Group l, between the start codon and the stop codon, encode respectively the polypeptides of SEQ Group 2. The nucleotide number of start codon and first nucleotide of stop codon are listed in table B for each polynucleotide of SEQ Group 1.
Table B
nucleotidic encoded peptidicStart codon 1st nucleotide of sequence sequence stop codon 1 ~1 SEQ ID NO:1 SEQ ID N0:2 1 3895 SEQ ID N0:3 SEQ ID N0:4 1 3892*

SEQ ID N0:5 SEQ iD N0:6 1 3892*

SEQ ID N0:7 SEQ ID N0:8 1 3892**

* first nucleotide of the last codon of encoding sequence ** first nucleotide of the last codon of partial encoding sequence 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 any polynucleotide sequence from SEQ Group 1 over the entire length of the polynucleotide sequence from SEQ Group 1; 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 identity, to any amino acid sequence selected from SEQ Group 2 , over the entire length of the amino acid sequence from SEQ Group 2.
A polynucleotide encoding a polypeptide of the present invention, including homologs and orthologs from species other than non typeable H. infla~enzae, 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°G and an SDS concentration from 0.1 - 1%) with a labeled or detectable probe consisting of or comprising any sequence selected from SEQ Group 1 or a fragment thereof; and isolating a full-length gene andlor genomie clones containing said polynucleotide sequence.
The invention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) set out in SEQ Group 1. 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 secretory 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, Kozak sequences, sequences that stabilize mRNA, introns, and polyadenylation signals. The polynueleotide 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. Natl. Acad. Sci., ZISA 86: 821-824 (1989), or an HA peptide tag (Wilson et al., Cell 37: 767 (1984), both of which may be useful in purifying polypeptide sequence fused to them. Polynucleotides of the invention also include, but are not limited to, polynucleotides comprising a structural gene and its naturally associated sequences that control gene expression.
The nucleotide sequence encoding the BASB207 polypeptide of SEQ Group 2 may be identical to the corresponding polynucleotide encoding sequence of SEQ Group 1. The position of the first and last nucleotides of the encoding sequences of SEQ
Goup 1 are listed in table C. Alternatively it may be any sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes a polypeptide of SEQ
Group 2.
Table C
nueleotidic encoded peptidicStart codon Last nucleotide sequence sequence of encoding sequence SEQ ID NO:1 SEQ ID N0:2 1 3894 SEQ ID N0:3 SEQ ID N0:4 1 3894 SEQ ID NO:S SEQ ID N0:6 1 3894 SEQ ID N0:7 SEQ ID N0:8 1 3893*

last nucleotide of the last codon of partial encoding sequence The term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacterial polypeptide and more particularly a polypeptide of the non typeable H. influenzae BASB207 having an amino acid sequence set out in any of the sequences of SEQ Group 2 . The term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, polynueleotides 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 any of the sequences of SEQ Group 2 . Fragments of polynucleotides of the invention may be used, for example, to synthesize full-length polynucleotides of the invention.
Preferred fragments are those polynucleotides which encode a B-cell or T-helper epitope, for example the fragments/peptides described in Example 13, and recombinant, chimeric genes comprising said polynucleotide fragments.
Further particularly preferred embodiments are polynucleotides encoding variants, that have the amino acid sequence of BASB207 polypeptide of any sequence from SEQ Group 2 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 BASB207 polypeptide.
Further preferred embodiments of the invention are polynucleotides that are at least 85°l0 identical over their entire length to a polynucleotide encoding BASB207 polypeptide having an amino acid sequence set out in any of the sequences of SEQ Group 2 , 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 BASB207 polypeptide and polynucleotides complementary thereto. In this regard, polynucleotides at least 95%
identical over their entire length to the same are particularly 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 sequence selected from SEQ Group 1.
In accordance with certain preferred embodiments of this invention there are provided polynucleotides that hybridize, particularly under stringent conditions, to polynucleotide sequences, such as those polynucleotides of SEQ Group 1.
The invention further relates to 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°l° formamide, 5x SSC (150mM NaGI, lSmM 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 support in 0. lx SSG at about 65°C. Hybridization and wash conditions are well known and exemplified in Sambrook, et al., Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), particularly Chapter 11 therein. 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 obtained by screening an appropriate library containing the complete gene for a polynucleotide sequence set forth in any of the sequences of SEQ
Group 1 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in the corresponding sequence of SEQ
Group 1 or a fragment thereof; and isolating said polynucleotide 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 BASB207 and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to the BASB207 gene. Such probes generally will comprise at least 15 nucleotide residues or base pairs.
Preferably, such probes will have at 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 BASB207 gene may be isolated by screening using a DNA
sequence provided in SEQ Group 1 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 ClS~1 85: 8998-9002, 1988). Recent modifications of the technique, exemplified by the MarathonTM 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 oligonucleotide primers. The PCR reaction is then repeated using "nested" primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 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 directly 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, particularly human diseases, as further discussed herein relating to polynucleotide assays.
The polynucleotides of the invention that are oligonucleotides derived from a sequence of SEQ Group 1 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 every polynueleotide of the invention there is provided a polynucleotide 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, TILT representations for nucleotides, the term "N"
may also be used in describing certain polynucleotides of the invention. "N"
means that any of the four DNA or RNA nucleotides may appear at such a designated position in 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 particu'Iar genetic immunization.
The use of a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet (1992) 1: 363, Manthorpe et al., Hum.
Gene Ther. (1983) 4: 419), delivery of DNA complexed with specific protein carriers (Wu et al., JBiol Chern. (1989) 264: 16985), coprecipitation of DNA with calcium phosphate (Benvenisty & Reshef, PNAS US~1, (1986) 83: 9551), encapsulation of DNA in various forms of liposomes (Kaneda et al., Science (1989) 243: 375), particle bombardment (Tang et al., Nature (1992) 356:152, Eisenbraun et al., DN~1 Cell Biol (1993) 12: 791) and in vivo infection using cloned retroviral vectors (Seeger et al., PN~iS ZIS~1 (1984) 81: 5849).
Vectors, Lost Cells, 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.
Gell-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 in 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 cells 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 engineered to incorporate expression systems or portions 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 al., B~1SICMETHODSINMOLECULAR BIOLOGY, (1986) and Sambrook, et al., MOLECULAR CLONING: ~ LABORATORYMANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, conjugation, transduction, scrape loading, ballistic introduction and infection.
Representative examples of appropriate hosts include bacterial cells, such as cells of streptococci, staphylococci, enterococci, E. coli, streptomyces, cyanobacteria, Bacillus subtilis, Neisseria meningilidis, Haemophilus influenzae and Moraxella catarrhalis;
fungal cells, such as cells of a yeast, Kluveromyces, Saccharomyces, Pichia, a basidiomycete, Candida albicans and ~Ispergillzas; insect cells such as cells of Drosophila S2 and Spodoptera Sf~3; animal cells such as CHO, COS, HeLa, C 127, 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, picornaviruses, retroviruses, and alphaviruses 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 variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., MOLECULAR
CLONING, A LABORATORYMANUtIL, (supra).
In recombinant expression systems in eukaryotes, for secretion of a translated 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 polypeptide 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 canon exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, 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, Venezuelian Equine Encephalitis Virus), adenoviruses, adeno-associated virus, picornaviruses (poliovirus, rhinovirus), herpesviruses (varicella zoster virus, ete), Listeria, Salmonella , Shigella, BGG, streptococci. 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 BASB207 polynucleotides and polypepticf~s of the invention for use as diagnostic reagents. Detection of BASB207 polynucleotides andlor 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, particularly those infected or suspected to be infected with an organism comprising the BASB207 gene or protein, may be detected at the nucleic acid or amino acid level by a variety 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 putatively infected andlor 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 enzyrnatically 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 in 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 BASB207 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. Polynucleotide differences may also be detected by direct DNA or RNA sequencing. See, for example, Myers et al., Science, 230.' 122 (1985). Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase, V 1 and S 1 protection assay or a chemical cleavage method. See, for example, Cotton et al., Proc. Natl. ~lcad. Sci., USA, 85: 4~97-4401 (1985).
In another embodiment, an array of oligonucleotides probes comprising BASB207 nucleotide sequence or fragments thereof can be constructed to conduct efficient screening 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, 274: 610 (1996)).
Thus in another aspect, the present invention relates to a diagnostic kit which compnses:
(a) a polynucleotide of the present invention, preferably any of the nucleotide sequences of SEQ Group 1, or a fragment thereof ;
(b) a nucleotide sequence complementary to that of (a);
(c) a polypeptide of the present invention, preferably any of the polypeptides of SEQ
Group 2 or a fragment thereof; or (d) an antibody to a polypeptide of the present invention, preferably to any of the polypeptides of SEQ Group 2 .
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, preferably any sequence of SEQ Group 1 , which is associated with a disease or pathogenicity will 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 polynucleotide. Organisms, particularly infectious organisms, carrying mutations in such polynueleotide 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 polymorphisms (allelic variations) in a polynucleotide andlor 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 particularly 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 polynucleotide encoding BASB207 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 BASB207 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 serotype and/or classify the infectious agent.
The invention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections caused by non typeable H. influen~ae, comprising determining from a sample derived from an individual, such as a bodily material, an increased level of expression of polynucleotide having a sequence of any of the sequences of SEQ Group 1. Increased or decreased expression of BASB207 polynueleotide 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, spectrometry and other hybridization methods.
In addition, a diagnostic assay in accordance with the invention for detecting over-expression of BASB207 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 BASB207 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 non-typeable H. influenzae, and may be useful in diagnosing and/or prognosing disease or a course of disease. A grid comprising a number of variants of any polynucleotide sequence of SEQ Group 1 is preferred. Also preferred is a number of variants of a polynucleotide sequence encoding any polypeptide sequence of SEQ Group 2 .
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. Alternatively, mimotopes, 3p particularly peptide mimotopes, of epitopes within the polypeptide sequence may also be used as immunogens to produce antibodies immunospecifrc for the polypeptide of the 2$

invention. The term "immunospecific" means that the antibodies have substantially greater affinity for the polypeptides of the invention than their amity for other relate polypeptides in the prior art.
In certain preferred embodiments of the invention there are provided antibodies against BASB207 polypeptides or polynucleotides.
Antibodies generated against the polypeptides or polynueleotides 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 cells expressing either or both, to an animal, preferably a nonhuman, using routine protocols.
For preparation of monoclonal antibodies, 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., Natarre 256:

(1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIES AND G~INCER THERAPY, Alan R. Liss, Inc. (1985).
Techniques for the production of single chain antibodies {LT.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-BASB207 or from naive libraries (McGafferty, et al., (1990), Nature 34$, 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) Natzrre 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 BASB207 polypeptide or BASB207 polynucleotide may be employed to treat infections, particularly 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 human 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 et al. (1986), Natasre 321, 522-525 or Tempest et al., (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 ligands may be natural substrates and ligands or may be structural or functional mimetics.
See, e.g., Coligan et al., Current Protocols in Imrnitnology 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 o~' 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 BASB207 polypeptide and/or polynucleotide activity in the mixture, and comparing the BASB207 polypeptide andlor polynucleotide activity of the mixture to a standard.
Fusion proteins, such as those made from Fc portion and BASB207 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):959-9471 ( 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 mRI~A andlor 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 BASB207 polypeptides or polynucleotides, particularly those compounds that are bacteriostatic andlor 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 BASB207 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 BASB207 agonist or antagonist. The ability of the candidate molecule to agonize or antagonize the BASB207 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 BASB207 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 transduetion, or increase chemical channel activity are agonists. Detection of the rate or level of, as the ease may be, production of product from substrate, signal transduction, or chemical channel activity 1 S 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 BASB207 polynucleotide or polypeptide activity, and binding assays known in the art.
Another example of an assay for BASB207 agonists is a competitive assay that combines BASB207 and a potential agonist with BASB207 binding molecules, recombinant BASB207 binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay.
BASB207 can be labeled, such as by radioactivity or a colorimetric compound, such that the number of BASB207 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, polypeptides 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 BASB207 induced activities, thereby preventing the action or expression of BASB207 polypeptides and/or polynucleotides by excluding BASB207 polypeptides andlor polynueleotides 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. Nez~rochern. 56: 560 (1991);
OLIGODEOXYNUCLEOTIDES ~S ~NTISENSE INHIBITORS OF GENE
EXPRESSION, CRC Press, Boca Raton, FL (1988), for a description of these molecules). Preferred potential antagonists include compounds related to and variants of BASB207.
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 portions 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 IgGI, where fusion takes place at the hinge region. In a particular 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.
W09~129~58 and W094/22914.
Each of the polynucleotide sequences provided herein may be used in the discovery 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-Delgarno 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 andlor 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 BASB207 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 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.
3~

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 G-terminal hydrophobic amidated tail. Alternatively, the addition or substitution of a D-stereoisomer form of one or more of the amino acids (inverso sequences) may be performed to create a beneficial derivative, for example to enhance stability of the peptide. Mimotopes may also be retro sequences of the natural peptide sequences, in that the sequence orientation is reversed. Mimotopes may also be retro-inverso in character. Retro, inverso and retro-inverso peptides are described in WO

and WO 94/05311.
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 B1). 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 BASB207 polynucleotide andlor polypeptide, or a fragment or variant thereof, adequate to produce antibody andl or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly non typeable H. influenzae 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 BASB207 polynucleotide andlor polypeptide, or a fragment or a variant thereof, for expressing BASB207 polynucleotide andlor polypeptide, or a fragment or a variant thereof in vivo in order to induce an immunological response, such as, to produce antibody andl 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 DNAIRNA 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 BASB207 polynucleotide and/or polypeptide encoded therefrom, wherein the composition comprises a recombinant BASB207 polynucleotide andlor polypeptide encoded therefrom and/or comprises DNA and/or RNA which encodes and expresses an antigen of said BASB207 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.
BASB207 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/ar immunogenic properties, and preferably~protective properties.
Thus fused recombinant protein, preferably further comprises an antigenic co-protein, such as lipoprotein D from Haemophilus inJluenzae, Glutathione-S-transferase (GST) off' 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.
In a vaccine composition according to the invention, a BASB207 polypeptide andlor polynucleotide, or a fragment, or a mimotope, or a variant thereof may be present in a vector, such as the live recombinant vectors described above for example live bacterial vectors.
Also suitable are non-live vectors for the BASB207 polypeptide, for example bacterial outer-membrane vesicles or "blebs". OM blebs are derived from the outer membrane of the two-layer membrane of Gram-negative bacteria and have been documented in many Gram-negative bacteria (Zhou, L et al. 1998. FEMS tL~Iicrobiol. Lett.
163:223-228) including C. trachomatis and C. psittaci. A non-exhaustive list of bacterial pathogens reported to produce blebs also includes: Bordetella pertussis, Borrelia burgdorferi, Brucella ntelitensis, Brucella ovas, Esherichia coli, Haemophilus influenzae, Legionella pneumophila, ~Yloraxella catarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis, Psettdomonas aeruginosa and Yersinia enterocolitica.
Blebs have the advantage of providing outer-membrane proteins in their native conformation and are thus particularly useful for vaccines. Blebs can also be improved for vaccine use by engineering the bacterium so as to modify the expression of one or more molecules at the outer membrane. Thus for example the expression of a desired immunogenic protein at the outer membrane, such as the BASB207 polypeptide, can be introduced or upregulated (e.g. by altering the promoter).
Instead or in addition, the expression of outer-membrane molecules which are either not relevant (e.g. unprotective antigens or immunodominant but variable proteins) or detrimental (e.g. toxic molecules such as LPS, or potential inducers of an autoimmune response) can be downregulated. These approaches are discussed in more detail below.
The non-coding flanking regions of the BASB207 gene contain regulatory elements important in the expression of the gene. This regulation takes place both at the transcriptional and translational level. The sequence of these regions, either upstream or downstream of the open reading frame of the gene, can be obtained by DNA sequencing. This sequence information allows the determination of potential regulatory motifs such as the different promoter elements, terminator sequences, inducible sequence elements, repressors, elements responsible for phase variation, the shine-dalgarno sequence, regions with potential secondary structure involved in regulation, as well as other types of regulatory motifs or sequences. This sequence is a further aspect of the invention. Furthermore, SEQ ID NO: 9 is the non typeable Haemophilus influenzae upstream sequence (upstream of the predicted initiation codon of the preferred genes) comprising approximately 1000bp.
This sequence information allows the modulation of the natural expression of the BASB207 gene. The upregulation of the gene expression may be accomplished by altering the promoter, the shine-dalgarno sequence, potential repressor or operator elements, or any other elements involved. Likewise, downregulation of expression can be achieved by similar types of modification. Alternatively, by changing phase variation sequences, the expression of the gene can be put under phase variation control, or it may be uncoupled from this regulation. In another approach, the expression of the gene can be put under the control of one or more inducible elements allowing regulated expression. Examples of such regulation include, but are not limited to, induction by temperature shift, addition of inductor substrates like selected carbohydrates or their derivatives, trace elements, vitamins, co-factors, metal ions, etc.
Such modifications as described above can be introduced by several different means.
The modification of sequences involved in gene expression can be earned out in vivo by random mutagenesis followed by selection for the desired phenotype. Another approach consists in isolating the region of interest and modifying it by random mutagenesis, or site-directed replacement, insertion or deletion mutagenesis.
The modified region can then be reintroduced into the bacterial genome by homologous recombination, and the effect on gene expression can be assessed. In another approach, the sequence knowledge of the region of interest can be used to replace or delete all or part of the natural regulatory sequences. In this ease, the regulatory region targeted is isolated and modified so as to contain the regulatory elements from another gene, a combination of regulatory elements from different genes, a synthetic regulatory region, or any other regulatory region, or to delete selected parts of the wild-type regulatory sequences. These modified sequences can then be reintroduced into the bacterium via homologous recombination into the genome. A non-exhaustive list of preferred promoters that could be used for up-regulation of gene expression includes the promoters porA, porB, lbpB, tbpB, p 110, 1st, hpuAB from N.
meningitides or N. gonorroheae; ompCD, copB, lbpB, ompE, UspAl; UspA2; TbpB from M.
Catarrhalis; p l, p2, p~, p5, p6, lpD, tbpB, D 15, Hia, Hmw 1, Hmw2 from H.
influenzae.
In one example, the expression of the gene can be modulated by exchanging its promoter with a stronger promoter (through isolating the upstream sequence of the gene, in vitro modification of this sequence, and reintroduction into the genome by homologous recombination). Upregulated expression can be obtained in both the bacterium as well as in the outer membrane vesicles shed (or made) from the bacterium.
In other examples, the described approaches can be used to generate recombinant bacterial strains with improved characteristics for vaccine applications.
These can be, but are not limited to, attenuated strains, strains with increased expression of selected antigens, strains with knock-outs (or decreased expression) of genes interfering with the immune response, strains with modulated expression of immunodominant proteins, strains with modulated shedding of outer-membrane vesicles.

Thus, also provided by the invention is a modified upstream region of the BASB2a7 gene, which modified upstream region contains a heterologous regulatory element which alters the expression level of the BASB207 protein located at the outer membrane. The upstream region according to this aspect of the invention includes the sequence upstream of the BASB207 gene. The upstream region starts immediately upstream of the BASB207 gene and continues usually to a position no more than about 1000 by upstream of the gene from the ATG start codon. In the case of a gene located in a polycistronic sequence (operon) the upstream region can start immediately preceding the gene of interest, or preceding the first gene in the operon. Preferably, a modified upstream region according to this aspect of the invention contains a heterologous promotor at a position between 500 and 700 by upstream of the ATG.
The use of the disclosed upstream regions to upregulate the expression of the BASB207 gene, a process for achieving this through homologous recombination (for instance as described in WO 01109350 incorporated by reference herein), a vector comprising upstream sequence suitable for this purpose, and a host cell so altered are all further aspects of this invention.
Thus, the invention provides a BASB207 polypeptide, in a modified bacterial bleb.
The invention further provides modified host cells capable of producing the non-live membrane-based bleb vectors. The invention further provides nucleic acid vectors comprising the BASB207 gene having a modified upstream region containing a heterologous regulatory element.
Further provided by the invention are processes to prepare the host cells and bacterial blebs according to the invention.
Also 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 non typeable H.
influenzae. 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 non typeable H.
inflZrenzae infection, in mammals, particularly humans.
The invention also includes a vaccine formulation which comprises an immunogenic recombinant polypeptide andlor polynucleotide of the invention together with a suitable earner, 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 mufti-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 earner 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 TH 1 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 TH1-type responses (cell-mediated response), and TH2-type immune responses (humoral response).
Extreme TH1-type immune responses may be characterised by the generation of antigen specific, haplotype restricted cytotoxic T lymphocytes, and natural killer cell responses. In mice TH1-type responses are often characterised by the generation of antibodies of the IgG2a subtype, whilst in the human these correspond to IgGI
type antibodies. TH2-type immune responses are characterised by the generation of a broad range of immunoglobulin isotypes including in mice IgGI, 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 TH1-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 TH 1 and TH2-type immune responses is not absolute. In reality an individual will support an immune response which is described as being predominantly TH1 or predominantly TH2. However, it is often convenient to consider the families of cytokines in terms of that described in murine CDR
+ve T
cell clones by Mosmann and Coffman (Mosmann, T.R. and Coffman, R.L. (1989 THl and TH2 cells: different patterns of lyrnphokine secretion lead to different functional properties. ~lnnaral Review oflrnrnasnologv, 7, p145-173).
Traditionally, TH 1-type responses are associated with the production of the INF-y and II,-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 TH 1 or TH2 - type cytokine responses. Traditionally the best indicators of the TH l :TH2 balance of the immune response after a vaccination or infection includes direct measurement of the production of THl or TH2 cytokines by T lymphocytes in vitro after restimulation with antigen, andlor the measurement of the IgGI
: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 TH 1-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 94100153 and WO 95!17209.
3 De-O-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-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains and is manufactured by Ribi Immunochem, Montana. A preferred form of 3 De-O-acylated monophosphoryl lipid A is disclosed in European Patent 0 689 454 B 1 (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 10~g - 100~tg preferably 25-SOpg per dose wherein the antigen will typically be present in a range 2-SOpg 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-O-acylated 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 96133739). Such formulations comprising QS21 and cholesterol have been shown to be successful TH1 stimulating adjuvants when formulated together with an antigen.
Further adjuvants which are preferential stimulators of TH1 cell response include immunomodulatory oligonucleotides, for example unmethylated CpG sequences as disclosed in WO 96/02555.
Combinations of different TH1 stimulating adjuvants, such as those mentioned hereinabove, are also contemplated as providing an adjuvant which is a preferential stimulator of TH1 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 :

to 10 : l; 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: QS21.
Preferably a carrier is also present in the vaccine composition according to the invention. The earner 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 squalene, alpha tocopherol and Tween 80. In a particularly preferred aspect the antigens in the vaccine compositian accarding to the invention are camhined with QS21 and 3D-MPL in such an emulsion. Additionally the oil in water emulsion may contain span 85 andlor lecithin and/or tricaprylin.
Typically for human administration QS21 and 3D-MPL will be present in a vaccine in the range of 1 pg - 200pg, such as 10-100p.g, preferably l Op.g - SOp,g 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, fox example, phosphate buffered saline.
A particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil in water emulsion is described in WO 95117210.
While the invention has been described with reference to certain BASB207 polypeptides and polynucleotides, it is to be understood that this covers fragments of the naturally occurnng 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. Preferred fragments/peptides are described in Example 13.
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 otiti,r media. Such a polyvalent vaccine composition may include a TH-1 inducing adjuvant as hereinbefore described.

In a preferred embodiment, the polypeptides, fragments and immunogens of the invention are formulated with one or more of the following groups of antigens:
a) bne or more pneumococcal capsular polysaccharides (either plain or conjugated to a carrier protein); b) one or more antigens that can protect a host against M.
catarrhalis infection; c) one or more protein antigens that can protect a host against Streptococcus pneurnoniae infection; d) one or more further non typeable Haemophilus inJluenzae protein antigens; e) one or more antigens that can protect a host against RSV;
and f) one or more antigens that can protect a host against influenza virus.
Combinations with: groups a) and b); b) and c); b), d), and a) and/or c); b), d), e), f), and a) andlor c) are preferred. Such vaccines may be advantageously used as global otitis media vaccines.
The pneumococcal capsular polysaccharide antigens axe preferably selected from serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 1 1A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F (most preferably from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F).
Preferred pneumococcal protein antigens are those pneumococcal proteins which are exposed on the outer surface of the pneumococcus (capable of being recognised by a host's immune system during at least part of the life cycle of the pneumococcus), or are proteins which are secreted or released by the pneumococcus. Most preferably, the protein is a toxin, adhesin, 2-component signal tranducer, or lipoprotein of Streptococcus pneZCrnoniae, or fragments thereof. Particularly preferred proteins include, but are not limited to: pneumolysin (preferably detoxified by chemical treatment or mutation) [Mitchell et al. Nucleic Acids Res. 1990 Jul 11;
18(13): 4010 "Comparison of pneumolysin genes and proteins from Streptococcus pneumoniae types 1 and 2.", Mitchell et al. Biochim Biophys Acta 19$9 Jan 23; 1007(1): 67-"Expression of the pneumolysin gene in Escherichia coli: rapid purification and biological properties.", WO 96/05859 {A. Cyanamid), WO 90/06951 {Paton et al), WO 99103884 {NAVA)]; PspA and transmembrane deletion variants thereof {WO
92/14488; WO 99153940; US 5804193 - Briles et al.); PspC and transmembrane deletion variants thereof (WO 99153940; WO 97/09994 - Briles et al); PsaA and transmembrane deletion variants thereof (Berry & Paton, Infect Immun 196 Dec;64(12):5255-62 "Sequence heterogeneity of PsaA, a 37-kilodalton putative adhesin essential for virulence of Streptococcus pneumoniae"); pneumococcal choline binding proteins and transmembrane deletion variants thereof; CbpA and transmembrane deletion variants thereof (WO 97/41151; WO 99/51266);
Glyceraldehyde-3-phosphate - dehydrogenase (Infect. Immun. 1996 64:3544);
HSP70 (WO 96/40928); PcpA (Sanchez-Beato et al. FEMS Rlicrobiol Lett 1998, 164:207-14); M like protein, SB patent application No. EP 0837130; and adhesin 18627, SB Patent application No. EP 0834568. Further preferred pneumococcal protein antigens are those disclosed in WO 98/18931, particularly those selected in WO 98118930 and PGTlUS99/30390.
Preferred Moraxella catarrhalis protein antigens which can be included in a combination vaccine (especially for the prevention of otitis media) are: OMP

[WO 97141731 (Antex) & WO 96/34960 (PMG)]; OMP21; LbpA &/or LbpB [WO
98/55606 (PMC)]; TbpA &/or TbpB [WO 97/13785 & WO 97/32980 (PMC)];
CopB [Helminen ME, et al. (1993) Infect. Immun. 61:2003-2010]; UspAl and/or UspA2 [WO 93/03761 (University of Texas)]; OmpCD; HasR (PCT/EP99/03824);
PiIQ (PCT/EP99/03823); OMP85 (PGT/EP00/01468); lipo06 (GB 9917977.2);
lipol0 (GB 9918208.1); lipoll (GB 9918302.2); lipol8 (GB 9918038.2); P6 (PGT/EP99/03038); D15 (PCT/EP99/03822); OmplAl (PCT/EP99/06781); Hly3 (PCT/EP99/03257); and OmpE.
Preferred further non-typeable Haemophilus inflatenzae protein antigens which can be included in a combination vaccine (especially for the prevention of otitis media) include: Fimbrin protein [(US 5766608 - Ohio State Research Foundation)] and fusions comprising peptides therefrom [cg LB1(f) peptide fusions; US 5843464 (OSU) or WO 99/64067]; OMP26 [WO 97/01638 (Gortecs)]; P6 [EP 281673 (State University of New York)]; protein D (EP 594610); TbpA andlor TbpB; Hia; Hsf;

Hin47; Hif; Hmw l; Hmw2; Hmw3; Hmw4; Hap; D 15 (WO 94/ 12641 ); P2; and PS
(WO 94/26304).
Preferred influenza virus antigens include whole, live or inactivated virus, split influenza virus, grown in eggs or MDCK cells, or Vero cells or whole flu virosomes (as described by R. Gluck, Vaccine, 1992, 10, 915-920) or purified or recombinant proteins thereof, such as HA, NP, NA, or M proteins, or combinations thereof.
Preferred RSV (Respiratory Syncytial Virus) antigens include the F
glycoprotein, the G glycoprotein, the HN protein, or derivatives thereof.
Compositions, kits and administration In a further aspect of the invention there are provided compositions comprising a BASB207 polynucleotide andlor a BASB207 polypeptide for administration to a cell or to a multicellular organism.
The invention also relates to compositions comprising a polynucleotide andlor a polypeptides discussed herein or their agonists or antagonists. The polypeptides and polynucleotides of the invention may be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical earner suitable for administration to an individual. Such compositions comprise, for instance, a media additive or a therapeutically effective amount of a polypeptide andlor 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 andlor 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 salts or fusidic 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 andlor 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 mglkg, typically around 1 mglkg. 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 pg/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 microgramlkg of antigen, and such dose is preferably administered 1-3 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.
3p SO

Sequence Databases, Sequences in a Tangible Medium, and Algorithms Polynucleotide and polypeptide sequences form a valuable information resource wit~l 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 identify 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 wer-a 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 incorporate2l by reference herein in its entirety in the manner described above for publications and references.
DEFINITIONS
"Identity," 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, "identity" 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 IS York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, 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., Na~cleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN (Altschul, S.F. et al., .l. Molec. Biol. 215: 403-410 (1990), and FASTA( Pearson and Lipman Proc. Natl. Acad. Sci. USA 85; 2444-2448 (1988). The BLAST family of programs is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NLH Bethesda, MD 20894;
Altschul, 3p 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. 89:1091 S-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 Biol. 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 WI.
These are the default parameters for nucleic acid comparisons.
A preferred meaning for "identity" for polynucleotides and polypeptides, as the ease 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
NO: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 o~ 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 NO: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
NO: l, or:
nn ~ xn ' ~xn ' Y) wherein nn is the number of nucleotide alterations, xn is the total number of nucleotides in SEQ ID NO:1, y is 0.50 for SO%, 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 polynucleotide sequences encoding the polypeptides 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 sequences of SEQ ID NO: l, 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 polynueleotide 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 NO:1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleic acids irk' SEQ ID NO: l, or:
nn ~ xn ' ~xn ' Y) 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, 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 the 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 amino 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:
na ~ xa' ~xa' Y) 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, 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 xa 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% 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 ~ xa - ~xa ~ Y) 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 essential properties. A typical variant of a polynueleotide 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 an$
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: DNA sequencing of the BASB207 gene from Non typable Haemoplaila~s ira fluenzae strain 3224A.
A: BASB207 in Non typableHaemophilus influenzae strain 322~A
The DNA sequence of the BASB207 polynucleotide from the Non typable Haemophilus Influenzae strain 3224A ( also referred to as strain ATCC PT-1816) is shown in SEQ ID N0:1. The translation of the BASB207 polynucleotidic sequence is showed in SEQ ID N0:2.
B: BASB207 in Non typable Haemophilus in fluenzae strain 3224A
The sequence of the BASB207 polynucleotide was confirmed in Non Typable Haemophilus influenzae strain 322~A. For this purpose, plasmid DNA (see example 3A) containing the gene region encoding BASB207 from Non Typable Haemophilus influenzae strain 3224A was submitted to DNA sequencing using the Big Dyes kit (Applied biosystems) and analyzed on a ABI 373/A DNA sequencer in the conditions described by the supplier M13 Universal Sequence Primer(S'-GTA AAA
CGA CGG CCA GT-3') [SEQ ID NO:10] and M13 Reverse Sequence Primer (5'-CAG GAA ACA GCT ATG AC-3') [SEQ ID NO:11] specific for the vector and additional internal primers specific for the BASB207 polynucleotide. As a result, the polynucleotide and deduced polypeptide sequences, respectively, were obtained.
Using the Clustalx 1.8 program, the polynucleotide sequence SEQ ID NO: 3 was aligned with SEQ ID NO:1; a pairwise comparison of identities showed that the polynucleotide sequence was 99 % identical to SEQ ID NO:1 (fig 1) . Using the same Clustalx 1.8 program, the polypeptide sequence SEQ ID NO: 4 was aligned with SEQ ID N0:2; a pairwise comparison of identities showed that the polypeptide sequence was 99 % identical to SEQ ID N0:2 (FIG 2) Example 2:
Variability analysis of the BASB207 gene among Non Typable Haemophilus influenzae strains.
Genomic DNA was extracted from 2 further NT Haemophilus inflatenzae strains (presented in Table 1) as follows. A SOOmI erlenmeyer flask containing 100 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 4°C. The supernatant was removed. Genomie DNA was extracted from the pellet of the NT Haemophilzts influehzae cells using the QIAGEN genomic DNA
extraction kit (Qiagen Gmbh). leg of this material was submitted to Polymerase Chain Reaction DNA amplification using primers MCM023 (5'- ATG ACA AAA
CAA CTA CAA CCA TC-3') [SEQ ID N0:12] and MCM024 (5'- ATT CAA ATT
TAT AAA GTA AAT CAA ACA-3') [SEQ ID N0:13]. This PCR product was purified using the High Pure PCR Product Purification Kit (Roche) , subjected to DNA sequencing using the Big Dyes kit (Applied biosystems) and analyzed on a ABI PRISM 310 Genetic Analyser by means of the primers MCM023 [SEQ ID
N0:12] and MCM024 [SEQ ID N0:13] and additional internal primers in the conditions described by the supplier. Using the Clustalx 1.8 program, an alignment of the polynucleotide sequences was performed, and is displayed in Figure 3. A
pairwise comparaison of identities showed that the polynucleotidic sequences SEQ
ID NO: 5 and 7 turned out to be between 93 and 95 % identical to SEQ ID N0:3 (Table 2). Using the Clustalx 1.8 program, an alignment of the polypeptidic sequences was performed, and is displayed in Figure 4. A pairwise comparaison of identities showed that the polypeptidic sequences SEQ ID NO: 6 and 8 turned out to be between 94 and 95 °ro identical to SEQ ID N0:4 (Table 3).
'Table 1: Features of the NT Haemophilus influenzae strains used in this study Strain isolated in from nucleotidic peptidic sequence sequence 3224A USA Otitis mediaSEQ ID N0:3 SEQ ID NO:~

3219C USA Otitis mediaSEQ ID NO:S SEQ ID N0:6 A840164 NL Garner strainSEQ ID N0:7 SEQ ID N0:8 Table 2: Pairwaise comparison of polynucleiotidic sequences SEQ ID SEQ ID SEQ ID N0:7 N0:3 NO:S

N0;3 NO:S

SEQ ID
N0;7 i Table 3: Pairwaise comparison of polypeptidic sequences SEQ ID N0:4SEQ ID SEQ ID
N0:6 NO:$

NO;~

SEQ ID
N0:6 SEQ ID
N0:8 Example 3 : Construction of Plasmid to Express Recombinant BASB207 A: Cloning of BASB207.
The BSpHI and BgIII restriction sites engineered into oli 1 nt YtfN (5'-TC ATG
ACA
AAA GAA CTA CAA CC-3') [SEQ ID N0:14] forward and oli 6 nt YtfN (5'-AGA
TGT AAA TTC AAA TTT ATA AAGTAAATG -3') [SEQ ID NO:15] reverse amplification primers, respectively, permitted directional cloning of the PCR
product into the E.coli expression plasmid pQE60 such that BASB207 protein could be expressed as a fusion protein containing a (His)6 affinity chromatography tag at the C-terminus. Due to the large size of the gene, three parallel PCR
experiments were carried out to amplify the entire coding sequence using oli 1 nt YtfN (5'-TC
ATG ACA AAA CAA CTA CAA CC-3') [SEQ ID N0:14] and oli2 nt YtfN (5'-G
GGG TTT GAA GAG AAT GTT CG-3') [SEQ ID N0:16] for one amplification, oli3 nt YtfN (5'iCAG GCG GTT TTG CAG GCTG-3') [SEQ ID N0:17] and oli4 nt YtfN (S'- CAG TTG TTC TTT GCT GTT GTG-3') [SEQ ID N0:18] for the second amplification, and oli5nt YtfN (5'-GCG AGTCAG CGA AGA TGA AGT

G-3') [SEQ ID N0:19]and oli 6 nt YtfN (5'-AGA TCT AAA TTC AAA TTT ATA
AAGTAAATC -3') [SEQ ID NO: I S] for the third amplification . The fragments amplified using oli 1 nt YtfN -oli2 nt YtfN and oli3 nt Ytfn -oli4 nt YtfN
share a common region with a FokI restriction site, while the fragments amplified using oli3 nt YtfN - olio nt YtfN and oli5nt YtfN - oli6 nt YtfN share a common region with a HaeIII restriction site. The three PCR products were first introduced into the pCRIITOPO cloning vector (In vitrogen) using Top 10 bacterial cells, according to the manufacturer's instructions. These intermediate constructs were realized to facilitate further cloning into the expression vector. Transformants containing the fragments were selected by restriction enzyme analysis. Following digestion, a ~20q1 aliquot of each reaction was analyzed by agarose gel electrophoresis (0.8 agarose in a Tris-acetate-EDTA (TAE) buffer). DNA fragments were visualized by LTV illumination after gel electrophoresis and ethidium bromide staining. A
DNA
molecular size standard (1 Kb ladder, Life Technologies) was electrophoresed in parallel with the test samples and was used to estimate the size of the DNA
fragments. Plasmid purified from selected transformants far each cloning were then sequentially digested to completion with either BSpHI and Fokl or Fokl and Haelll or HaeIIII and BgIII restriction enzymes as recommended by the manufacturer (Life Technologies). The digested DNA fragments were then purified using silica gel-based spin columns prior to ligation with the pQE60 plasmid.
B: Production of expression vector.
To prepare the expression plasmid pQE60 for ligation, it was similarly digested to completion with both NcoI and BgIII. An approximately 5-fold molar excess of the digested fragments to the prepared vector was used to program the ligation reaction.
A standard ~20 Itl ligation reaction (~16°C, ~16 hours), using methods well known in the art, was performed using T~ DNA ligase (~2.0 units / reaction, Life Technologies). An aliquot of the ligation (~5 q1) was used to transform M15(pREP4) electro-competent cells according to methods well known in the art.
Following a ~2-3 hour outgrowth period at 37°C in ~l .0 ml of LB
broth, transformed cells were plated on LB agar plates containing ampicillin (100 ~g/ml) and kanamycin (30~.g/m1). Antibiotic was included in the selection. Plates were incubated overnight at 37°G for ~16 hours. Individual ApRIKanR colonies were picked with sterile toothpicks and used to "patch" inoculate fresh LB ApR/KanR
plates as well as a ~1.0 ml LB Apl Kan 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 BASB207 DNA insert. Here, the ~1.0 ml overnight LB Ap/Kan 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 ~200p 1 of sterile water and a ~lOpl aliquot used to program a ~50p1 final volume PCR reaction containing both BASB207 forward and reverse amplification primers. The initial 95°C
denaturation step was increased to 3 minutes to ensure thermal disruption of the bacterial cells and liberation of plasmid DNA. An 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, lmin., were used to amplify the BASB207 fragment from the lysed transformant samples. Following thermal amplification, a ~20p1 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 Technologies) was electrophoresed in parallel with the test samples and was used to estimate the size of the PCR products.
Transformants that produced the expected size PCR product were identified as strains containing a BASB207 expression construct. Expression plasmid containing strains were then analyzed for the inducible expression of recombinant BASB207.
C: Expression Analysis of PCR-Positive Transformants An aliquot of the overnight seed culture (~1.0 ml) was inoculated into a 125 ml erlenmeyer flask containing ~25 ml of LB AplKan broth and grown at 37 °C with shaking 0250 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 012.5 ml) was transferred to a second 125 ml flask and expression of recombinant BASB207 protein induced by the addition of IPTG (1.0 M stock prepared in sterile water, Sigma) to a final concentration of 1.0 mM. Incubation of both the IPTG-induced and non-induced cultures continued for an additional ~4 hours at 37 °C w~'th 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 ~SOp I 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 p1) of both the crude IPTG-induced and the non-induced cell lysates were loaded onto duplicate 12°,~o Tris/glycine polyacrylamide 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 8250 (BioRad) and then destained to visualize novel BASB207 TPTG-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 art. 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 BASB207 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.
Example 4: Production of Recombinant BASB207 Bacterial strain A recombinant expression strain ofE. coli M15(pREP4) containing a plasmid (pQE60) encoding BASB207 from NT haemophilus inJluenzae was used to produce cell mass for purification of recombinant protein. The expression strain was cultivated on LB agar plates containing 100pg/ml ampicillin ("Ap") and 30pg/ml kanamycin ("Km") to ensure that pQE60 and pREP4 were maintained. For eryopreservation 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 growth medium used for the production of recombinant protein consisted of LB
broth (Difco) containing 100pglml Ap and 30 p.glml Km. To induce expression of the BASB207 recombinant protein, IPTG (Isopropyl 13-D-Thiogalactopyranoside) was added to the culture {1 mM, final).
Fermentation A 100-ml erlenmeyer seed flask, containing l Oml working volume, was inoculated with 0.3 ml of rapidly thawed frozen culture, or several colonies from a selective agar plate culture, and incubated for approximately 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 500m1 working volume erlen containing LB
broth and both Ap and Km antibiotics. IPTG ( 1.0 M stock, prepared in sterile water) was added to the erlen when the culture reached mid-log of growth (~0.5 O.D.600 units). Cells were induced for 4 hours then harvested by centrifugation using either a 28RS Heraeus {Sepatech) or RCSC superspeed centrifuge (Sorvall Instruments).
Cell paste was stored at -20 C until processed.
Example 5: Expression and purification of recombinant BASB207 protein in Escherichia coli.
The construction of the pET-BASB207 cloninglexpression vector is described in Example 1. This vector harbours the BASB207 gene isolated from the non typeable Haemophilus influenzae strain 3224A in fusion with a stretch of 6 Histidine residues, placed under the control of the strong bacteriophage T7 gene 10 promoter. For expression study, this vector is introduced into the Escherichia coli strain hFovablue (DE3) (Novagen), in which, the gene for the T7 polymerase is placed under the control of the isopropyl-beta-D thiogalactoside (IPTG)-regulatable lac promoter.
Liquid cultures (100 ml) of the Novablue (DE3) [pET-BASB207] E. coli recombinant strain are grown at 37°C under agitation until the optical density at 600nm (0D600) reached 0.6. At that time-point, IPTG is added at a final concentration of 1 mM and the culture is grown for 4 additional hours. The culture is then centrifuged at 10,000 rpm and the pellet is frozen at -20°C for at least 10 hours. After thawing, the pellet is resuspe$ded during 30 min at 25°C in buffer A (6M guanidine hydrochloride, O.1M
NaH2P04, 0.01 M Tris, pH 8.0), passed three-times through a needle and clarified by centrifugation (20000rpm, 15 min). The sample is then loaded at a flow-rate of lml/min on a Ni2+ -loaded Hitrap column (Pharmacia Biotech). After passsage of the flowthrough, the column is washed succesively with 40m1 of buffer B (8M Urea, O.IMNaH2P0~1, O.OIM Tris, pH 8.0), ~Oml of buffer C (8M Urea, O.IMNaH2P0~1, 0.01 M Tris, pH 6.3). The recombinant protein BASB207/His6 is then eluted from the column with 30m1 of buffer D (8M Urea, O.IMNaH2P04, O.O1M Tris, pH 6.3) containing 500mM of imidazole and 3ml-size fractions are collected. Highly enriched BASB207lHis6 protein can be eluted from the column. This polypeptide is detected by a mouse monoclonal antibody raised against the 5-histidine motif. Moreover, the denatured, recombinant BASB207-His6 protein is solubilized in a solution devoid of urea. For this purpose, denatured BASB207-His6 contained in 8M urea is extensively dialyzed (2 hours) against buffer R (NaCI 150mM, IOmM NaH2P04, Arginine O.SM
pH6.8) containing successively 6M, ~1M, 2M and no urea. Alternatively, this polypeptide is purified under non-denaturing conditions using protocoles described in the Quiexpresssionist booklet (Qiagen Gmbh).
Example 6: Production of Antisera to Recombinant BASB207 Polyvalent antisera directed against the BASB207 protein are generated by vaccinating rabbits with the puribed recombinant BASB207 protein. Polyvalent antisera directed against the BASB207 protein are also generated by vaccinating mice with the purified recombinant BASB207 protein. Animals are bled prior to the first immunization ("pre-bleed") and after the last immunization.
Anti-BASB207 protein titers are measured by an ELISA using purified recombinant BASB207 protein as the coating antigen. The titer is defined as mid-point titers calculated by ~I-parameter logistic model using the JCL Fit so~rivare.The antisera are also used as the first antibody to identify the protein in a western blot as described in example 8 below.
Example 7: Immunological characterization: Surface exposure of BASB207 S Anti-BASB207 protein titres are determined by an ELISA using formalin-killed whole cells of non typable Haemophilus influenzae (NTHi). The titer is defined as mid-point titers calculated by 4-parameter logistic model using the XL Fit software.
Example 8. Immunological Characterisation: Western Blot Analysis Several strains of NTHi, as well as clinical isolates, are grown on Chocolate agar I0 plates for 24 hours at 36°C and S% CO,. Several colonies are used to inoculate Brain Heart Infusion (BHI) broth supplemented by NAD and hemin, each at 10 pg/ml. Cultures are grown until the absorbance at 620nm is approximately 0.4 and cells are collected by centrifugation. Cells are then concentrated and solubilized in PAGE sample buffer. The solubilized cells are then resolved on 4-20%
1 S polyacrylamide gels and the separated proteins are electrophoretically transferred to PVDF membranes. The PVDF membranes are then pretreated with saturation buffer.
All subsequent incubations are carried out using this pretreatment buffer.
PVDF membranes are incubated with preimmune serum or rabbit or mouse immune 20 serum. PVDF membranes are then washed.
PVDF membranes are incubated with biotin-labeled sheep anti-rabbit or mouse Ig.
PVDF membranes are then washed 3 times with wash buffer, and incubated with streptavidin-peroxydase. PVDF membranes are then washed 3 times with wash buffer and developed with 4-chloro-1-naphtol.

Example 9: Immunological characterization' Bactericidal Activity Complement-mediated cytotoxie activity of anti-BASB207 antibodies is examined to determine the vaccine potential of BASB207 protein antiserum that is prepared as described above. The activities of the pre-immune serum and the anti-BASB207 30 antiserum in mediating complement killing of NTI-Ii are examined.

Strains of NTHi are grown on plates. Several colonies are added to liquid medium.
Cultures are grown and collected until the A620 is approximately 0.4. After one wash step, the pellet is suspended and diluted.
Preimmune sera and the anti-BASB207 sera are deposited into the first well of a 96-wells plate and serial dilutions are deposited in the other wells of the same line. Live diluted NTHi is subsequently added and the mixture is incubated. Complement is added into each well at a working dilution defined beforehand in a toxicity assay.
Each test includes a complement control (wells without serum containing active or inactivated complement source), a positive control (wells containing serum with a know titer of bactericidal antibodies), a culture control (wells without serum and complement) and a serum control (wells without complement).
Bactericidal activity of rabbit or mice antiserum (50% killing of homologous strain) is measured.
Example 10: Presence of Antibod to BASB207 in Human Convalescent Sera Western blot analysis of purified recombinant BASB207 is performed as described in Example 5 above, except that a pool of human sera from children infected by NTHi is used as the first antibody preparation.
Example 11: Efficacy of BASB207 vaccine enhancement of lung clearance of NTHi in mice.
This mouse model is based on the analysis of the lung invasion by NTHi following a standard intranasal challenge to vaccinated mice.
Groups of mice are immunized with BASB207 vaccine. After the booster, the mice are challenged by instillation of bacterial suspension into the nostril under anaesthesia. Mice are killed between 30 minutes and 24 hours after challenge and the lungs are removed aseptically and homogenized individually. The 1og10 weighted mean number of CFU/lung is determined by counting the colonies grown on agar plates after plating of dilutions of the homogenate. The arithmetic mean of the 1og10 weighted mean number of CFU/lung and the standard deviations are calculated for each group.
Results are analysed statistically.
In this experiment groups of mice are immunized either with BASB207 or with a killed whole cells (kwc) preparation of NTHi or sham immunized.
Example 12: Inhibition of NTHi adhesion onto cells by anti-BASB207 antiserum.
This assay measures the capacity of anti BASB207 sera to inhibit the adhesion of NTHi bacteria to epithelial cells. This activity could prevent colonization of the nasopharynx by NTHi.
One volume of bacteria is incubated on ice with one volume of pre-immune or anti-BASB207 immune serum dilution. This mixture is subsequently added in the wells of a 24 well plate containing a confluent cells culture that is washed once with culture medium to remove traces of antibiotic. The plate is centrifuged and incubated.
Each well is then gently washed. After the last wash, sodium glycocholate is added to the wells. After incubation, the cell layer is scraped and homogenised.
Dilutions of the homogenate are plated on agar plates and incubated. The number of colonies on each plate is counted and the number of bacteria present in each well calculated.
Example 13: Useful Epitopes The B-cell epitopes of a protein are mainly localized at its surface. To predict B-cell epitopes of BASB207 polypeptide two methods were combined: 2D-structure prediction and antigenic index prediction. The 2D-structure prediction was made using the PSIPRED program (from David Jones, Brunei Bioinformatics Group, Dept.
Biological Sciences, Brunei University, Uxbridge UB8 3PH, UK) (Fig.S). The antigenic index was calculated on the basis of the method described by Jameson and Wolf (CABIOS x:181-186 X1988]). The parameters used in this program are the antigenic index and the minimal length for an antigenic peptide. An antigenic inde~c of 0.9 for a minimum of 5 consecutive amino acids was used as threshold for the program. Peptides comprising good, potential B-cell epitopes are listed in table 4 These can be useful (preferably conjugated or recombinantly joined to a larger protein) in a vaccine composition for the prevention of ntHi infections, as could similar peptides comprising conservative mutations (preferably 70, 80, 95, 99 or 100% identical to the sequences of table 4) or truncates comprising 5 or more (e.g. 6, 7, 8, 9, 10, 1 l, 12, 15 or 20) amino acids therefrom or extensions comprising e. g. l, 2, 3, 5, 10 further amino acids at either or both ends from the native context of BASB207 polypeptide which preserve an effective epitope which can elicit an immune response in a host against the BASB207 polypeptide.
Table 4: Potential B-cell epitopes from SEQ TI3 N0:2 PositionSequence '647 HYGENI

776 NSL,KG

,812 KLDYRTF

845 HNQGR i 10 0 ~ ANpKETrNL

1024 ~ PDTAEPVSE

'1039 GPHKSKEELI

The T-helper cell epitopes are peptides bound to HLA class II molecules and recognized by T-helper cells. The prediction of useful T-helper cell epitopes of BASB207 polypeptide was based on the TEPITOPE method describe by Sturniolo at al. (Nature Biotech. 17: 555-561 [1999]). Peptides comprising good, potential T-cell epitopes are listed in table 5. These can be useful (preferably conjugated to peptides, polypeptides or polysaccharides) for vaccine purposes, as could similar peptides comprising conservative mutations (preferably 70, 80, 95, 99 or 100°~o identical to the sequences below) or truncates comprising 5 or more (e.g. 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 2~1 or 30) amino acids therefrom or extensions comprising e. g. l, 2, 3, 5, 10 further amino acids at either or both ends from the native context of BASB207 polypeptide which preserve an effective T-helper epitope from BASB207 polypeptide.
Table 5: Potential T-helper cell epitopes from SEQ ID N0:2 PositionSequence 100 LQLDFGCLLrSREVCLR

289 LQLQKLAVESSLGNL,SSQGK

349 ~LKKSTALSLKTKGVLDA

I 3 6 LrNGNVQLAQ I

391 YTFVNTMTPLKINDVTLrKLrTGDf~

531 ~f~KGSATLNQ

All identified regions containing epitopes as defined above are in respect of SEQ ID
N0:4. The corresponding regions in SEQ ID NO: 6, 8 as defined by position in table 4&5 with respect to SEQ ID N0:4 and by its corresponding peptide in the alignment of figure 4 for SEQ ID N0:6, 8 are also preferred peptides of the invention as described in this example.

Deposited materials A deposit of strain 3 (strain 3224A) has been deposited with the American Type Culture Collection (ATCC) on May 5 2000 and assigned deposit number PTA-1816.
The non typeable Haemophilus influenzae-strain deposit is referred to herein as "the deposited strain" or as "the DNA of the deposited strain."
The deposited strain contains a full length BASB207 gene.
The sequence of the polynueleotides contained in the deposited strain, 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 strain has 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 strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. The deposited strain is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U.S.C. ~
112. A
license may be required to make, use or sell the deposited strain, and compounds derived therefrom, and no such license is hereby granted.

Applicant's or agent's file NIJLlBM454?4 International application No.
reference number (PCT Rule l3bis) A. The indications made below relate to the microorganism referred to in the description on page 73 ,lines 1-2?

B. IDENTIFICATION OF DEPOSIT
Further deposits are identified on an additional sheet Name of depository institution AMERICAN TYPE CULTURE COLhECTION

Address of depository institution (including postal code and couniry) 10801 UNIVERSITY BLVD, MANASSAS, VIRGINIA 20110-2209, UNITED
STATES OF

AMERICA

Date of deposit 05/0512000 Accession Number C. ADDITIONAL INDICATIONS (leave blank fnot applicable) This information is continued on an additional sheet In respect of those designations where a European Patent is sought, a sample of the deposited microorganisms will be made available until the publication of the mention of the grant of the European Patent or until the date on which the application has been refused or withdrawn, only by issue of such a sample to an expert nominated by the person requesting the sample D. DESIGNATED STATES FOR WHICH
INDICATIONS ARE MADE tifthe indications are not for all designated States) E. SEPARATE FURNISHING OF INDICATIONS
(leave blankifrtot applicable) The indications listad below will be submitted to the International Bureau later (specify the general nahtre of tire indications e.g., "Accession Number of Deposit') For receiving Office use only For International Bureau use only This sheet was received with the ittlernational ~ This sheet was received by the International Bureau application on~
Authorized officer Authorized officer t~
. :~' ~l~ ~tt 3 i"~ t''~ ~ : .... ...~~ ~ : - :,_, a i Form PCTlR01134 (July 199Z) 7.1 SEQUENCE INFORMATION
BASB207 Polynucleotide and Polypeptide Sequences SEQ ID NO:I polynucleotide sequence of BASB207 J ATGACAAAACAACTACAACCATCAGATACTTCACCAAAAGCACCAAAACAAGCAAATAAAAAACACTG
GGTACGCAAAGTTATTTGTATTGGAAGTGCGGTCATTTTTATACCTGTTTTAGGTGTTGCTGGGGCAA
TTTCTTTCGATGCGGGACAAAAAAGTTTAATTCAACTAGCCGACAAAATGCTCGATAGTTTTTCCGTT
GAGCAAATTGAAGGCGGATTACAAAATGGCTTGGTGTTAAAGAATGTTCGTTATCAAACCGCTGGGAT
TGAGACACATATTGCACAAGCACGTTTACAGCTCGATTTCGGTTGTTTGCTTTCACGTGAAGTTTGTT
IO TACGTGACTTTACCTTAAATAAACCGACAATTGCGATTAATACTGCATTATTGCCTCCATCAACGCCT
GATAATTCCAAGTCTAGCCAGATGAAGCGTATTTCCTTGCCAATTAGTATCAATGCAGAAAGTTTGGT
AGTGCAAAATTTATCTGTAAATATTGATCAGACTAATATTACACTTGGAAATTTTAAAAGTGCGGTAA
ATTTAAATAATGAAAAAGGTTTAACTATTGCACCGACTGAAATTAACGATATTTCAGTGATTGCAAAA
AAATTGTCTGAAGTTAAATCAGAGCTGAAAGCCGAACAGCCGAATAAACCTGTAGATTGGGCTACGAT
IS TGAGCAATCTTTAACGCCTGCTTTTTTAGGTAATGTATCAGAAATCATTCTTCCTTTTGATTTACATA
TTCCTGAAATTTCAGGCAAAAACTGGCAGTATCAAGCAGTGAATGAAAAAGGCGAAACGCTTCAATCT
GTTGAAATGTCTAGTTTAATTGCACAGGCGAATACCGTCGATAACCAACTGCAATTACAAAAATTAGC
GGTAGAAAGCTCGTTGGGTAATCTTTCTTCACAAGGTAAATTACAACTCGATGGCGATATGCCTCTCG
ATTTAACTTTAAAATCCCATTTGGAACCGTTGAAATCCGATGGAAAAGAAATTCTACCCGCGAGTGAT
2O GTGGATCTTACGCTTTCAGGATCATTGAAAAAATCTACCGCTCTTTCTTTGAAAACaaaaggCGTATT
AGATGCTGAACTGAATGGCAACGTGCAATTAGCGCAAGACAAAATGCCCTTAAACCTCACATTAAATG
TGGCAAAAGGACAATACACTtttgtaAACACAATGACGCCACTCAAAATTAATGATGTGACACTTAAA
CTCACGGGGGAT'E'TACTAAATTATCACGCCGAACTTAAAGGCGATGTAGCAGGAATGAATTATATTCC
CGCATCTCAAGTGGAACTTAATGCCGACGGTAAACTTTATGAAGTTACCGTCAATAAATTGGGGATAG
ZS CTTCATTAGACGGTAAAAGTGAATTTGTGGGTAATGCGAATTGGAAAAATGGTGCAAACTGGGATATT
CAAGCCGATTTAGAAAAAATGAATATTGGTTTTTTTGTTCCTGTCATGCCTGCAACACTATCTGGTAA
ATTGCATTCACGTGGTTTTGCAGGCTCTCAAGGATGGCAAGTGGAAGTGCCAGTAGCCGATTTAAATG
GAACATTGTCTTCAAACCCCATCAGTTTAAAAGGCTCCGCAACACTTAATCAAAATGTACTTCTAACA
GTACCTGATTTACAGATTAGATATGGCGAAAATTATCTTAAAGCCAGTGGCGTATTAGATGATCATTC
3O TGATTTTGCGCTAGATATAA.ATGCGCCAAATTTACGTGGATTATGGTCAGATTTAAAAGGGCGAGTAA
AAGGTCGAGTAGCGATTTCTGGTCAAATTACGACACCTAATCTTGATCTTGATTTAACTTCATCTAAT
TTACATTTACAGGGTTTTCAGTTAGCCAAAGCAAGTATAAAAGGTCATATAAACAATGCCTCACTCTC
GAGTGGAAAATTGAATATTAAAGCAGAACAACTTCATTATGGCGAAAATATTAAACTCCATTTACTTG
ATCTTGATTTATCAGGCGATGAACAAAATCACAAACTAATCCTAAAATCTCAAGGCGCGCCTGTTGCT

AAAATTTGAAACTCCGATTGGCGATGTAAAATCCAACCAAGCGATTGCAGTGTCCTACGATAACAAAC
AAACACAAGCCAATA'F'TGCCTCACATTGCTGGCAAAACACCGATGTAGAATTATGTTTCCCACAAGCA
TTCAATGCAGGCAAGCAAGGCAATATTCCTTTCCAATTTAAACGTGTTAATTTGGATTTAGTGAATAA
ACTTATCGAACAAAACAGCCTCAAAGGCAATTTACAAGTGCAAGGAAATGTTGCTTGGTTTACGGATA
AACCATTCCAATTTACCGCCAACATAGATGGCAATCATTTAGCCTTCTCTCAAAAATTGGATTACCGC
ACGTTTAAATTAGATATTCCAAAATTAACCCTTAATGCCGACATTCAAAATAATp,ATTTGGTTTTAAA
AACAGACATCAATTTGCATAATCAAGGCAGAATTGTAGGCGATATTCGTTTAAATGATTTTGTTAAAA

CTCGCCAACTTGGCGGAACCCTTGCGATAGAACGATTAAACTTGTCGATCGCAAATCAGCTACTTACT
AGCGGTGAATCAGTGAATGGTAAGGTAGTCTCGAAGTTGAGTTTCGGCGGGAATTTAGAGAAACCATT
a ATTAAATGGTGACTTCAATATTCGTAATATTCGTACCAAATTGAAATCTATGCCAGTCAATATTACAG
ATGGCGATATTGCCCTACGCTTTAATGGTAACCGTTCAACATTGCAAGGGAAAATCGAAACAGTAGAT
S AGTCACTTAAACTTAACTGGACGTGCAAACTGGGCGAATATTGAACATTGGACGACAGAATTGAATGC
ACAAGCAAATAATTTCAACGTGGATATTCCATCTATGGCGAAGCTACGTTTCAGTCCAGATATCACGA
TAAAAGCCAATCCTAAGGAATTGAATTTAAGCGGAACAGTGGATATTCCTTGGGCGCGTATTAAAATT
GACAGTCTGCCAGATACTGCTGAGCCAGTCAGCGAAGATGAAGTGATTTTAAACGGCCCGCACAAGAG
CAAAGAAGAACTGATTAAACGTGAATTTGCCGCAAAAACGAAATCAGGAATGGATATTCGCTCTGATT
IO TACGCATCAATATTGGTAAAGACGTCAGCCTTGATGCCTATGGCTTAAAAACCAATCTTGATGGTTTA
CTTTCTGTGAAACAAGACAAAGGCAATTTAGGTTTATTCGGGCAAATCAATTTAACCAAAGGTCGCTA
TGCTTCTTTCGGACAAGATTTACTCATTCGTAAAGGGCTTATTAGTTTTTCTGGGCAAGCGGCACAAC
CTACGTTGAATATTGAGGCTATTCGTAATCCTGAAACAATGGAAGACAGCAAAATTACTGCTGGAGTG
AGAGTGATTGGCATAGCCGATAGCCCAGAAGTCACTATTTTCAGTGAACCAAGCAAACCACAAGATCA
IS AGCACTTTCTTATTTATTAACAGGTCGTTCGTTAGAAAGTAGTGGAGAAGTGGGTTCAACGGGATCAG
TTGGTGCAGCATTAATTGGTTTAGGCATTTCAAAGAGCGGTAAATTAGTGGGCAGTATTGGCGAAGTT
TTTGGTATTCAAGACTTAAACCTTGGTACATCTGGCGTGGGCGATAAATTTAAGGTTACAGTAAGTGG
CAATATTACCAATCGCTTACAAATAAAATATGGCGTAGGTTTATTTGATGGGTTAGCAGAAGTTACAT
TACGTTATCGCCTAATGCCACAGCTTTATTTTCAATCCGTTTCTAGCACAAACCAAGTGTTTGATTTA
ZO CTTTATAAATTTGAATTTTAG
SEQ ID N0:2 polypeptide sequence of BASB207 MTKQLQPSDTSPKAPKQANKKHWVRKVICIGSAVIFIPVLGVAGAISFDAGQKSLIQLADKMLDSFSV
EQIEGGLQNGLVLKNVRYQTAGIETHIAQARLQLDFGCLLSREVCLRDFTLNKPTIAINTALLPPSTP
~S DNSKSSQMKRTSLPISINAESLWQNLSVNIDQTNITLGNFKSAVNI~NNEKGT~TIAPTEINDTSVIAK
KLSEVKSELKAEQPNKPVDWATIEQSLTPAFLGNVSEIILPFDLHIPEISGKNWQYQAVNEKGETLQS
VEMSSLTAQANTVDNQLQLQKLAVESSLGNLSSQGKLQLDGDMPLDLTLKSHLEPLKSDGKEILPASD
VDLTLSGSLKKSTDLSLKTKGVLDAELNGNVQLAQDKMPLNLTLNVAKGQYTFWTMTPLKINDVTLK
LTGDLLNYHAELKGDVAGMNYIPASQVELNADGKLYEVTWKLGIASIsDGKSEFVGNANWKNGANWDI
3O QADLEKMNIGFFVPVMPATLSGKLHSRGFAGSQGWQVEVPVADLNGTLSSNPISLKGSATLNQNVIrLT
VPDLQIRYGENYLKASGVLDDHSDFALDINAPNLRGLWSDLKGRVKGRVAISGQITTPNLDLDLTSSN
LHLQGFQLrAKASTKGHINNASLSSGKLNIKAEQLHYGENIKLHLLDLDLSGDEQNHKLILKSQGAPVA
ANLQINGHFDRTLEQWKGTISQVKFETPIGDVKSNQAIAVSYDNKQTQANIASHCWQNTDVELCFPQA
FNAGKQGNIPFQFKRVNLDLVNKLIEQNSLrKGNLQVQGNVAWFTDKPFQFTANIDGNHLAFSQKLDYR
3S TFKLDIPKLTLNADIQNNNF~VT~KTDINLHNQGRIVGDIRLNDFVKTRQLGGTLAIERLrNLSIANQLLT
SGESVNGKWSKLSFGGNLEKPLLNGDFNIRNIRTKLKSMPVNTTDGDIALRFNGNRSTLQGKIETVD
SHLNLTGRANWANIEHWTTELNAQANNFNVDIpSMAKLRE'SPDITIKANPKELNLSGTVDIpWARIKI
DSLPDTAEPVSEDEVILNGPHKSKEELIKREFAAKTKSGMDIRSDLRINIGKDVSLDAYGLKTNLDGL
LSVKQDKGNLGLrFGQINLTKGRYASFGQDLLIRKGL,ISFSGQAAQPTLNIEAIRNPETMEDSKITAGV
RVIGIADSPEVTTFSEPSKPQDQALSYLLTGRSLESSGEVGSTGSVGAALIGLGISKSGKLVGSIGEV
FGTQDLNLGTSGVGDKSKVTVSGNITNRLQIKYGVGLFDGLAEVTLRYRLMPQLYFQSVSSTNQVFDL
LYKFEF
SEQ ID N0:3 polynucleotide sequence of BASB207 ATGACAAAACAACTACAACCATCAGATACTTCACCAAAAGCACCAAAACAAGCAAATAAAAAACACTG
GGTACGCAAAGTTATTTGTATTGGAAGTGCGGTCATTTTTATACCTGTTTTAGGTGTTGCTGGGGC~AA
TTTCTTTCGATGCGGGACAAAAAAGTTTAATTCAACTAGCCGACAAAATGCTCGATAGTTTTTCCGTT
GAGCAAATTGAAGGCGGATTACAAAATGGCTTGGTGTTAAAGAATGTTCGTTATCAAACCGCTGGGAT
S TGAGACACATATTGCACAAGCACGTTTACAGCTCGATTTCGGTTGTTTGCTTTCACGTGAAGTTTGTT
TACGTGACTTTACCTTAAA'." ACCGACAATTGCGATTAATACTGCATTATTGCCTCCATCAACGCCT
GATAATTCCAAGTCTAGCCAGATGAAGCGTATTTCCTTGCCAATTAGTATCAATGCAGAAAGTTTGGT
AGTGCAAAATTTATCTGTAAATATTGATCAGACTAATATTACACTTGGAAATTTTAAAAGTGCGGTAA
ATTTAAATAATGAAAAAGGTTTAACTATTGCACCGACTGAAATTAACGATATTTCAGTGATTGCAAAA

TGAGCAATCTTTAACGCCTGCTTTTTTAGGTAATGTATCAGAAATCATTCTTCCTTTTGATTTACATA
TTCCTGAAATTTCAGGCAAAAACTGGCAGTATCAAGCAGTGAATGAAAAAGGCGAAACGCTTCAATCT
GTTGAAATGTCTAGTTTAATTGCACAGGCGAATACCGTCGATAACCAACTGCAATTACAAAAATTAGC
GGTAGAAAGCTCGTTGGGTAATC'.~'TTCTTCACAAGGTAAATTACAACTCGATGGCGATATGCCTCTCG
LS ATTTAACTTTAAAATCCCATTTGGAACCGTTGAAATCCGATGGAAAAGAAATTCTACCCGCGAGTGAT
GTGGATCTTACGCTTTCAGGATCATTGAAAAAATCTACCGCaCTTTCTTTGAAAACaaaaggCGTATT
AGATGCTGAACTGAATGGCAACGTGCAATTAGCGCAAGACAAAATGCCCTTAAACCTCACATTAAATG
TGGCAAAAGGACAATACACTtttgtaAACACAATGACGCCACTCAAAATTAATGATGTGACACTTAAA
CTCACGGGGGATTTACTAAATTATCACGCCGAACTTAAAGGCGATGTAGCAGGAATGAATTATATTCC
ZO CGCATCTCAAGTGGAACTTAATGCCGACGGTAAACTTTATGAAGTTACCGTCAATAAATTGGGGATAG
CTTCATTAGACGGTAAAAGTGAATTTGTGGGTAATGCGAATTGGAAAAATGGTGCAAACTGGGATATT
CAAGCCGATTTAGAAAAAATGAATATTGGTTTTTTTGTTCCTGTCATGCCTGCAACACTATCTGGTAA
ATTGCATTCACGTGGTTTTGCAGGCTCTCAAGGATGGCAAGTGGAAGTGCCAGTAGCCGATTTAAATG
GAACATTGTCTTCAAACCCCATCAGTTTAAAAGGCTCCGCAACACTTAATCAAAATGTACTTCTAACA

TGATTTTGCGCTAGATATAAATGCGCCAAATTTACGTGGATTATGGTCAGATTTAAAAGGGCGAGTAA
AAGGTCGAGTAGCGATTTCTGGTCAAATTACGACACCTAATCTTGATCTTGATTTAACTTCATCTAAT
TTACATTTACAGGGTTTTCAGTTAGCCAAAGCAAGTATAAAAGGTCATATAAACAATGCCTCACTCTC
GAGTGGAAAATTGAATATTAAAGCAGAACAACTTCATTATGGCGAAAATATTAAACTCCATTTACTTG

GCAAATTTACAAATTAACGGACATTTTGACCGCACTTTGGAACAGTGGAAAGGCACAATTTCACAGGT
AAAATTTGAAACTCCGATTGGCGATGTAAAATCCAACCAAGCGATTGCAGTGTCCTACGATAACAAAC
AAACACAAGCCAATATTGCCTCACATTGCTGGCAAAACACCGATGTAGAATTATGTTTCCCACAAGCA
TTCAATGCAGGCAAGCAAGGCAATATTCCTTTCCAAT'h'TAAACGTGTTAATTTGGATTTAGTGAATAA

AACCATTCCAATTTACCGCCAACATAGATGGCAATCATTTAGCCTTCTCTCAA.AAATTGGATTAGCGC
ACGTTTAAATTAGATATTCCAAAATTAACCCTTAATGCCGACATTCAAAATAATAATTTGGTTTTAAA
AACAGACATCAATTTGCATAATCAAGGCAGAATTGTAGGCGATATTCGTTTAAATGATTTTGTTAAAA
CTCGCCAACTTGGCGGAACCCTTGCGATAGAACGATTAAACTTGTCGATCGCAAATCAGCTACTTACT
AGCGGTGAATCAGTGAATGGTAAGGTAGTCTCGAAGTTGAGTTTCGGCGGGAATTTAGAGAAACCATT
ATTAAATGGTGACTTCAATATTCGTAATATTCGTACCAAATTGAAATCTATGCCAGTCAATATTACAG
ATGGCGATATTGCCCTACGCTTTAATGGTAACCGTTCAACATTGCAAGGGAAAATCGAAACAGTAGAT
AGTCACTTAAACTTAACTGGACGTGCAAACTGGGCGAATATTGAACATTGGACGACAGAATTGAATGC
ACAAGCAAATAATTTCAACGTGGATA"~'TCCATCTATGGCGAAGCTACGTTTCAGTCCAGATATCAGGA

TAAAAGCCAATCCTAAGGAATTGAATTTAAGCGGAACAGTGGATATTCCTTGGGCGCGTATTAAAATT
GACAGTCTGCCAGATACTGCTGAGCCAGTCAGCGAAGATGAAGTGATTTTAAACGGCCCGCACAA~G
CAAAGAAGAACTGATTAAACGTGAATTTGCCGCAAAAACGAAATCAGGAATGGATATTCGCTCTGATT
TACGCATCAATATTGGTAAAGACGTCAGCCTTGATGCCTATGGCTTAAAAACCAATCTTGATGGTTTA
S CTTTCTGTGAAACAAGACAAAGGCAATTTAGGTTTATTCGGGCAAATCAATTTAACCAAAGGTCGCTA
TGCTTCTTTCGGACAAGATTTACTCATTCGTAAAGGGCTTATTAGTTTTTCTGGGCAAGCGGCACAAC
CTACGTTGAATATTGAGGCTATTCGTAATCCTGAAACAATGGAAGACAGCAAAATTACTGCTGGAGTG
AGAGTGATTGGCATAGCCGATAGCCCAGAAGTCACTATTTTCAGTGAACCAAGCAAACCACAAGATCA
AGCACTTTCTTATTTATTAACAGGTCGTTCGTTAGAAAGTAGTGGAGAAGTGGGTTCAACGGGATCAG
IO TTGGTGGAGCATTAATTGGTTTAGGCATTTCAAAGAGCGGTAAATTAGTGGGCAGTATTGGCGAAGTT
TTTGGTATTCAAGACTTAAACCTTGGTACATCTGGCGTGGGCGATAAATcTAAGGTTACAGTAAGTGG
CAATATTACCAATCGCTTACAAATAAAATATGGCGTAGGTTTATTTGATGGGTTAGCAGAAGTTACAT
TAGGTTATCGCCTAATGCCACAGCTTTATTTTCAATCCGTTTCTAGCACAAACCAAGTGTTTGATTTA
CTTTATAAATTTGAATTT
15 SEQ ID NO:~ polypeptide sequence of BASB207 MTKQLQPSDTSPKAPKQANKKHWVRKVICTGSAVIFIPVLGVAGAISFDAGQKSLIQLADKMLDSFSV
EQIEGGLQNGLVLKNVRYQTAGIETHIAQARLQLDFGCLLSREVCLRDFTLNKPTIAINTALLPPSTP
DNSKSSQMKRISLPISINAESLVVQNLSVNIDQTNITLGNFKSAVNLNNEKGLTIAPTETNDISVIAK
KLSEVKSELKAEQPNKPVDWATIEQSLTPAFLGNVSEIILPFDLHIPEISGKNWQYQAVNEKGETLQS
ZO VEMSSLIAQANTVDNQLQLQKLAVESSLGNLSSQGKLQLDGDMPLDLTLKSHLEPLKSDGKEILPASD
VDLTLSGSLKKSTALSLKTKGVLDAELNGNVQLAQDKMPLNLTLNVAKGQYTFVNTMTPLKINDVTLK
LTGDLLNYHAELKGDVAGMNYIPASQVELNADGKLYEVTVNKLGIASLDGKSEFVGNANWKNGANWDI
QADLEKMNTGFFVPVMPATLSGKLHSRGFAGSQGWQVEVPVADLNGTLSSNPISLKGSATLNQNVLLT
VPDLQIRYGENYLKASGVLDDHSDFALDINAPNLRGLWSDLKGRVKGRVAISGQITTPNLDLDLTSSN
ZS LHLQGFQLAKASIKGHINNASLSSGKLNIKAEQLHYGENIKLHLLDLDLSGDEQNHKLILKSQGAPVA
ANLQINGHFDRTLEQWKGTISQVKFETPIGDVKSNQAIAVSYDNKQTQANIASHCWQNTDVELCFPQA
FNAGKQGNIPFQFKRVNLDLVNKLIEQNSLKGNLQVQGNVAWFTDKPFQFTANIDGNHLAFSQKLDYR
TFKLDIPKLTLNADIQNNNLVLKTDINLHNQGRIVGDTRLNDFVKTRQLGGTLAIERLNLSIANQLLT
SGESVNGKWSKLSFGGNLEKPLLNGDFNIRNIRTKLKSMPVNITDGDIALRFNGNRSTLQGKIETVD

DSLPDTAEPVSEDEVILNGPHKSKEELIKREFAAKTKSGMDIRSDLRINIGKDVSLDAYGLKTNLDGL
LSVKQDKGNLGLFGQINLTKGRYASFGQDLLIRKGLISFSGQAAQPTLNIEAIRNPETMEDSKITAGV
RVIGIADSPEVTIFSEPSKPQDQALSYLLTGRSLESSGEVGSTGSVGAALIGLGISKSGKLVGSIGEV
FGIQDLNLGTSGVGDKSKVTVSGNITNRLQIKYGVGLFDGLAEVTLRYRLMPQLYFQSVSSTNQVFDL

SEQ ID NO:S polynucleotide sequence of BASB207 ATGACAAAACAACTACAACCATCAGAAACTTCGCCAAAAGCACCAGAGCAACCAAGTAAAAAACATTG
GATGCGTAAAGCTGTTTGTATCGGAAGTGCGGTCATTTTTATACCTGTTTTAGGTGTTGCTGGGGCGC
TTTCTTTCGATGCAGGACAAAAAAGTTTAATTCAACTTGCCGATAAAATGCTTGATAGTTTTTCCGTT
~O GAGCAAATtGAAGGCGGATTaCAAAAtGGCttGGTGTTAAAGAATGTTCAATACCAAACTGCTGGGAT
TGAGACACATATTGCACAAGCACGGT'FACAGATTGATTTCGCTTGTTTGTTTTCACGTGAAGTTTGTT
TACGTGACTTTACCTTAAATAAACCGACGATTGCGATTAATACTGCATTATTGCCTCCATCGACAACT
GATAATTCCAAGTCTAGCCCGATGAAGCGtATTTCCTTGCCAATTGGTATCAATGCAGAAAATTTGGC
7$

GGTGCAAGATTTATCTGTAAACATTGATCAGACTAGTATTACGCTTGGAAATTTTAAAAGTGCGGTAA
GTTTAAATAATGAAAAAGGCTTAACTATTGCACCGACTGAAATTAACGATATTTCAGTGATTGCAAAA
a AAATTGCCCGAAGGTAAACCTGAGCTGAAAGCCGAACAGCCGAATAAGCCTGTAGATTGGGCTGCGAT
TGAGCAATCTTTAACGCCTGCTTTTTTAGGTAATGTATCAGAAATAATTCTTCCTTTTGATTTACATA
S TTCCTGAAATTTCAGGCAAAAACTGGCAGTATCAAGCCGTGAATGAAAAAGGCGAAACGCTTCAATCT
GTTGAAATGTCTAGTTTAATTGCACAGGCGGATACCGTCGATAACCAACTGCAATTACAAAAATTAGC
GGTAGAAAGCTCGTTGGGTAATCTTTATTCGCAAGGTAAATTACAACTTGATGGCGATATGCCGCTAG
ATTTAACTTTAAAATCCCATTTGGAGCCGTTGAAATCCGCTGGAAAAGAAATTCTACCCGCGAGTGAT
GTGGATCTTACGCTTTCGGGATCATTGAAAAAATCCACCGCACTTTCTTTAAAAACAAAAGGCGTATT
IO AGATGCTGAACTGAATGGCGACGTGCAATTAGCACAAGACAAAATGCCCTTAAACCTCACATTAAACG
tGGCAAAAGGACAATACGCTTTTGTAAACFCAATGATGCCACTCAAAATTAATGATGTGACACTTAAA
CTCACGGGGGATTTACTAAATTATCACGCTGAACTTAAGGGCAATGTAGTAGGAATGAATTATATTCC
TGCATCTCAAGTGGAACTTAATGCCGACGGTAAACTTTATGAAGTCACCATAAATAAATTGGGGATAG
CTTCATTAGATGGTAAAAGTGAATTTGTGGGTAATGCGAATTGGAAAAATGGTGCAAACTGGGATATT
IS CAAGCCGaTTTAGAAA.AAATGAATATTGGTTTTTTTGTTCCTGTAATGCCTGCAACACTATCTGgCAA
ATTACATTCGCGTGGTTTTGCAGGCTCTCAAGGATGGCAAGTGGAAGTGCCAGTAGCCGATTTAAACG
GAATACTTTCTGCAAAACC'FATCAGTTTAAAAGGTTCTGCAACGCTCAATCAAAAGGTACTTCTAACA
GTACCTGATTTACAGATTAAATATGGCGAAAATTATCTTAAAGCCAGTGGCGTATTAGATGATCATTC
TGATTTTGCGCTAGATATAAATGCGCCAAATTTACGTGGATTATGGTCAGATTTAAAAGGGCGAGTAA
ZO AAGGTCGAGTAGCGATTTCTGGTCAAATTACGACACCTAATCTTGATCTTGATTTAACTTCATCTGAT
TTACATTTACAGGGTTTTCAGTTAGCTAAAGCAAGTATAAAAGGTCATATAAACAATACCTCACTCTC
AAGTGGAAAATTGAATATTAAAGCAGAACAACTTCATTATGGCGAAAATATTAAACTCCATTTACTTG
ATCTTGATTTATCAGGCGATGAACAAAATCACAAACTAATCCTAAAATCTCAAGGCGAGCCTGTTGCT
GCAAATTTACAAATTAACGGCCATTTTGACCGCACTTTGGAACAGTGGAAAGGCACAATTTCACAGGT
ZS AAAATTTGAAACTCCGATTGGCGATGTAAAATCCAACCAAGCGATTGCGGTGTCCTACGATAACAAAC
AAACACAAGCCAATATTGCCTCACATTGCTGGCAA.AACACCGATGTAGAATTATGTTTCCCACAAGCA
TTCAATGCAGGCAAGCAAGGCAATATTCCTTTCCAATTTAAACGTGTTAATTTGGATTTAGTCAATAA
ACTTATCGAACAAGACAGCCTCAAAGGCAATTTACAAGTGCAAGGAAATGTGGCTTGGTTTACGGATA
AACCATTCCAATTTACCGCCAACGTAGATGGCAATCATTTAGCCTTCTCTCAAAAATTGGATTACCGT
3O aCATTTAAATTGGaTATTCCAAAATTAACCCTTAACGCCGACATTCAAAATAATAATTTGGTTTTAAA
AACAGACATCAATGTGCATAACCAAGGCAGAATTGTAGGCGATATTCGTTTAAACGATCTTGCGAAAA
ATCGCCAACTTAGCGGTAGCCTTGCGATAGAACGATTAAGTTTGAGTATTGCGAATCAACTACTCACT
CAAGGCGAATCCGTGAATGGAGAAGTGGTATCAAAATTGCGTTTCGGTGGGAATTTAGAGAAACCATT
ATTAAATGGTGACTTCAATATTCGTAATATTCGTACCAAATTAAAATCTATGCCAGTCAATATTACAG

AGTCACTTAAACTTAACTGGACGTGCAAACTGGGCTAATATTGAACATTGGCCAACAGAATTAAATGC
GCAAGCAAATAATTTCAACGTGGATATTCCTTCTATGGCGAAGCTACGTTTCAGTCCAAATATCACGA
TAAAAGCCAATCCGAAGGAATTGAATTTAAGCGGAACAGTGGATATTCCTTGGGCACGTATTAAAATT
GACAGTCTGCCAGATACTGCTGAGCCAGTAAGCGAAGATGAAGTTATTTTAAATGGCCCTCATAAGAG
CAAAGAAGAACTGATTAAACGCGAATTTGCGGCAAAAACAAAATCAGGCATGGATATTCGCTCTGATT
TACGCATCAATATTGGTAAAGACGTTAGCCTTGATGCCTATGGCTTGAAAACCAATCTTGATGGTTTA
CTTTCTGTGAAACAAGACAAAGGCAATTTAGGTTTATTCGGTCAAATCAATTTAACCAAAGGTCGCTA
TGCTTCTTTCGGGCAAGATTTACTCATTCGTAAAGGGCTTATTAGCTTTTCTGGGCAAGCGACACAAC
CTACATTGAATATTGAGGCTATTCGTAATCCTGAAACAATGGAAGACAGCAAAATTACTGCTGGCGTG

AGAGTGATTGGCATAGCTGATAGCCCAGAAGTCACTATTTTCAGTGAACCAAGCAAACCACAAGATCA
AGCACTTTCTTATTTATTAACAGGTCGTTCGTTAGAAAGCAGCGGAGAAGTGGGTTTAACGGGATCAG
a TTGGCGCAGCATTGATTGGTTTAGGCATTTCAAAGAGCGGTAAATTAGTGGGCAGTATTGGCGAAGTT
TTTGGTATTCAAGATTTAAACCTTGGTACATCTGGCGTGGGCGATAAATCCAAAGTGACGGTAAGTGG
AAATATTACCAATCGCTTACAAATAAAATATGGCGTAGGTTTATTTGATGGCTTAGCAGAAGTTACAT
TACGTTATCGCCTAATGCCACAGCTTTATTTTCAATCCGTTTCTAGCACAAACCAAGTGTTTGATTTA
CTTTATAAATTTGAATTA
SEQ ID N0:6 polypeptide sequence of BASB207 MTKQLQPSETSPKAPEQPSKKHWMRKAVCIGSAVIFIPVLGVAGALSFDAGQKSLIQLADKMLDSFSV
iO EQIEGGLQNGLVLKNVQYQTAGIETHTAQARLQIDFACT~FSREVCLRDFTLNKPTIAINTALLppSTT
DNSKSSPMKRISLPIGTNAENLAVQDLSVNIDQTSITLGNFKSAVSLNNEKGLTIAPTEINDISVIAK
KLPEGKPELKAEQPNKPVDWAATEQSLTPAFLGNVSEIILPFDLHIPEISGKNWQYQAVNEKGETLQS
VEMSSLIAQADTVDNQLQLQKLAVESSLGNLYSQGKLQLDGDMPLDLTLKSHLEPLKSAGKEILPASD
VDT~TLSGSLKKSTALSLKTKGVT~DAELrNGDVQLAQDKMpLrNLTLNVAKGQYAFVNTMMPDKINDVTLK
IS LTGDI~LNYHAELKGNWGMNYIPASQVELNADGKLYEVTINKLGIASLDGKSEFVGNANWKNGANWDI
QADLEKMNIGFFVPVMPATLSGKLHSRGFAGSQGWQVEVpVADLNGILSAKPISLKGSATLNQKVLrLT
VPDLQIKYGENYLKASGVLDDHSDFALDINAPNLRGLWSDLKGRVKGRVAISGQITTPNLDLDLTSSD
LHT~QGFQLAKASIKGHINNTSLSSGKLNIKAEQLHYGENIKLHLLDLDLSGDEQNHKLrILKSQGEPVA
ANLQINGHFDRTLEQWKGTISQVKFETPIGDVKSNQATAVSYDNKQTQANTASHCWQNTDVELCFPQA
FNAGKQGNIPFQFKRVNT~DLVNKLTEQDSLKGNLQVQGNVAWFTDKPFQFTANVDGNHLAFSQKLDYR
TFKLDIPKLTLNADIQNNNLVLKTDINVHNQGRIVGDIRLNDLAKNRQLSGSLAIERLSLSIANQI~LT
QGESVNGEVVSKLRFGGNLEKPLLNGDFNIRNIRTKLrKSMPVNITDGDIALRFNDNRSTLQGKIETVD
SHLNLTGRANWANIEHWpTELNAQANNFNVDIPSMAKT~RFSPNITIKANPKELNLSGTVDTPWARIKT
DSLrPDTAEPVSEDEVILNGPHKSKEEIiTKREFAAKTKSGMDIRSDLRTNTGKDVSLDAYGLhKTNLDGL
ZS LSVKQDKGNLGLFGQINLTKGRYASFGQDLLIRKGLISFSGQATQPTLNTEAIRNPETMEDSKITAGV
RVTGIADSPEVTIFSEPSKPQDQALSYLIrTGRSLESSGEVGLTGSVGAALIGLGISKSGKE~VGSIGEV
FGIQDLNLGTSGVGDKSKVTVSGNTTNRLQIKYGVGLrFDGLAEVTLRYRLMPQLYFQSVSSTNQVFDL
LYKFEL
SEQ ID N0:7 polynucleotide sequence of BASB207 GGTACGCAAAGTTATTTGTATTGGAAGTGCGGTCATTTTTATACCTGTTTTAGGTGTTGCTGGGGCGA
TTTCTTTCGATGCGGGACAAAAAAGTTTAATTCAACTAGCCGACAAAATGCTCGATAGTTTTTCCGTT
GAGCAAATTGAAGGCGGATTACAAAATGGCTTGGTGTTAAAGAATGTTCGTTATCAAACCGCTGGGAT
TGAGACACATATTGCACAAGCACGGTTACAGCTTGATTTCGGTTGTTTGCTTTCACGTGAAGTTTGTT

GATGATTCAAAGTCTAGCCCGATGAAGCGCATTTCCTTGCCAATTAGTATCAATGCAGAAAATTTAGT
GGTGCAAGATTTATCCGTAAATGTTGATCAGACTAATATTACGCTTGGAAATTTTAAAAGTGCGGTAA
GTTTAAATAATGAAAAAGGCTTAACTATTGCACCGACTGAAATTAACGATATTTCAGTGATTTCAAAA
AAATTGCCTGAAGTTAAATCAGAGGCGAAAGCCGAACAGCCGAATAAGCCTGTAGATTGGGCTGCGAT
O TGAGCAATCTTTAACTCCCGCTTTTTTAGGCAATGTATCAGAAATCATTCTTCCTTTTGATTTACATA
TTCCTGAAATTTCAGGCAAAAACTGGCAGTATCAAGCAGTCAATGAAAAAGGCGAAACGCTTCAATCT
ATTGAAATCCCAAGCCTGATTGCACAGGCGGATACCGTCGATAACCAACTGCAATTACAAAAATTATC
GGTTGAAAGTTCGCTCGGTAATCTTTATTCACAAGGTAAATTACAACTTGATGGCGATATGCCACTAG

ATTTAACCTTAAAATCCCATTTGGAGCCGTTGAAATCCGCTGGAAAAGAAATTCTGCCAGAGAGTGAT
GTGGATCTTACGCTTTCGGGAACATTGAAAAAATCCACCGCACTTTCTTTAAAAACAAAAGGCGT1~TT
AGATGCTGAACTGAATGGCGAAGTGCAATTAGCACAAGACAAAATGCCCTTAAACCTCACATTAAATG
TGGCAAAAGGACAATACGCTTTTGTAAACACAATGGCACCACTCAAAATTAATGATGTGACACTTAAA
S CTCACGGGGGATTTACTGAATTATCACGCCGAATTTAAGGGCAATGTGGCAGGAATGAATTATATTCC
CGCATCTCAAGTAGACTTTAATGCCGACGGCAAACTTTATGAAATAACCGTAAATAAATTGGGGATAG
CTTCATTAGACGGTAAAAGTGAATTTGTGGGTAATGCAAATTGGAAAAATGGTGCAAACTGGGATATT
CAAGCCGATTTAGAAAAAATGAATATTGGTTTTTTTGTTCCAGTTATGCCTGCAATATTATCGGGTAA
ATTACATTCTCGCGGTTTTGCGGACTCTCAAGGTTGGCAAGTGGAAGTGCCTACAGCCGATTTAAACG
IO GAACGCTTTCTGCAAAACCTATCAGTTTAAAAGGTTCTGCAACGCTCAATCAAAAGGTACTTCTAACC
GTGCCTGATTTACAGATTAGATATGGCGAAAATTATCTTAAAGCCAGTGGCGTATTAGATGATCATTC
TGATTTTGTGCTAGATATAGATGCGCCAAAT'FTACGTGGTTTATGGTCAGATTTAAAAGGGCGAGTAA
AAGGTCGTGTATCGATTTCTGGTCAAATTACGACACCTAATCTTGATCTTGATTTAACTTCATCCAAT
TTACATTTACAAGGTTTTCAGTTAGCTAAAGCAAGTATAAAAGGTCATATAAACAATACCCCACTCTC
IS GAGTGGAAAATTGAATATTAAAGCAGAACAACTTCATTATGGCGAAAATATTAAACTCCATTTACTTG
ATCTTGATTTATCAGGCGATGAACAAAATCACAAACTAATCCTAAAATCTCAAGGCGAGCCTGTTGCT
GCAAATTTACAAATTAACGGCCATTTTGACCGCACTTTGGAACAGTGGAAAGGCACACTTTCACAAGC
AAAATTTGAAACCCCGATTGGCGATGTAAAATCCAACCAAGCGATTGCGGTGTCTTACGATAACAAAC
AAACACAAGCCAATATTGCCGCACATTGCTGGCAAAATACAGATGTAGAATTATGTTTCCCACAAGCA
ZO TTCAATGCAGGCAAGCAAGGCAATATTCCTTTCCAATTCAAACGTGTTAATTTGGATTTAATGAATAA
ACTTATCGAACAAGATAGCCTCAAAGGAAACTTGCAAGCGCAAGGTAATGTGGCTTGGTTTACGGATA
AACCATTCCAATTTAGCACAAAAGTAGATGGCAATCATTTAGCTTTCTCTCAAAAATTGGATTACCGC
ACGTTTAAATTAGATA'~'TCCAAAATTAACCCTTAATGCCGACATTCAAAATAATAATTTGGTTTTAAA
AACAGACATCAATTTGCATAATCAAGGCAGAATTGTAGGCGATATTCGTGTAAATGATTTTGTTAAAA

AGCGGTGAATCAGTGAATGGTAAGGTAGTCTCGAAGTTGAGTTTCGGCGGGAATTTAGAGAAACCATT
ATTAAATGGTGACTTCAATATTCGTAATATTCGTACCAAATTGAAATCTATGCCAGTCAATATTACTG
ATGGCGATATTGCCCTACGTTTCAATGGTACCCGTTCAACATTGCAAGGGAAAATCGAAACAGTAGAT
AGTCACTTAAACTTAACTGGACGTGCACACTGGGCTAATATTGAACATTGGACGACAGAATTGAATGC

TAAAAGCCAATCCTAAGGAATTGAATTTAAGCGGAACAGTGGATATTCCTTGGGCGCGTATTAAAATT
GACAGTCTGCCAGATACTGCTGAGCCAGTCAGCGAAGATGAAGTGATTTTAAACGGCCCGCACAAGAG
CAAAGAAGAACTGATTAAACGTGAATTTGCCGCAAAAACGAAATCAGGAATGGATATTCGCTCTGATT
TACGCATCAATATTGGTAAAGACGTCAGCCTTGATGCCTATGGCTTAAAAACCAATCTTGATGGTTTA

TGCTTCTTTCGGACAAGATTTACTCATTCGTAAAGGGCTTATTAGTTTTTCTGGGCAAGCGGCACAAC
CTACGTTGAATATTGAGGCTATTCGTAATCCTGAAACAATGGAAGACAGCAAAATTACTGCTGGAGTG
AGAGTGATTGGCATAGCCGATAGCCCAGAAGTCACTATTTTCAGTGAACCAAGCAAACCACAAGATCA
AGCACTTTCTTATTTATTAACAGGTCGTTCGTTAGAAAGTAGCGGTGAAGCTGGCTCCAGTGGTTCGA
TTGGCGCAGCATTGATTGGTTTAGGCATTTCAAAGAGCGGTAAATTAGTGGGCAGTATTGGCGAAGTT
TTTGGTATTCAAGACTTAAACCTTGGTACATCTGGCGTGGGCGATAAATCTAAGGTTACAGTAAGTGG
AAATATTACCAATCGCTTACAAATAAAATATGGCGTAGGTTTATTTGATGGTTTAGCAGAAGTCACCT
TACGTTATCGCCTAATGCCACAGCTTTATTTTCAATCCGTTTCTAGCACAAACCAAGTGTTTGATTTA
CTTTATATTTTTGAATA
8l SEQ ID N0:8 polypeptide sequence of BASB207 MTKQLQPSDTSPKAPKQANKKHWVRKVTCIGSAVIFIPVLGVAGAISFDAGQKSLIQLADKMLDSF'°5V
EQIEGGLQNGLVLKNVRYQTAGIETHIAQARLQLDFGCLLSREVCLRDFTLNKPTIAINTALLPPSMP
DDSKSSPMKRISLPISINAENLWQDLSVNVDQTNTTLGNFKSAVSLNNEKGLTIAPTETNDISVISK
S KLPEVKSEAKAEQPNKPVDWAAIEQSLTPAFLGNVSEITLPFDLHIPEISGKNWQYQAWEKGETLQS
IEIPSLIAQADTVDNQLQLQKLSVESSLGNLYSQGKLQLDGDMPLDLTLKSHItEPL~KSAGKEILPESD
VDLTLSGTLKKSTALSLKTKGVLDAELNGEVQLAQDKMPLNLTLNVAKGQYAFVNTMAPLKINDVTLK
LTGDLLNYHAEFKGNVAGMNYIPASQVDFNADGKLYEITVNKLGIASLDGKSEFVGNANWKNGANWDI
QADLEKMNIGFFVPWPAILSGKLHSRGFADSQGWQVEVPTADLNGTLSAKPISLKGSATLNQKVLLT
IO VPDl,QIRYGENYLKASGVLDDHSDFVLDIDAPNLRGLWSDLKGRVKGRVSISGQITTPNLDLDLTSSN
LHLQGFQLAKAS I KGHINNTPLSSGKT~NIKAEQLrHYGENIKT~HLLDIsDLSGDEQNHKLTLKSQGEPVA
ANLQTNGHFDRTLEQWKGTLSQAKFETPIGDVKSNQAIAVSYDNKQTQANIAAHCWQNTDVELCFPQA
FNAGKQGNIPFQFKRVNLDLMNKLTEQDSLKGNLQAQGNVAWFTDKPFQFSTKWGNHLAFSQKLDYR
TFKLDIPK1~TLNADTQNNNLVLrKTDINLHNQGRIVGDIRVNDFVKTRQLGGTLAIERLNLSIANQLLT
IS SGESVNGKWSKT~SFGGNLEKPLLNGDFNIRNIRTKLKSMPVNITDGDIALRFNGTRSTLQGKIETVD
SHLNLTGRAHWANIEHWTTELNAQANNFNVDIPSMAKLRFSPDITIKANPKEhNLSGTVDIPWARIKI
DSLPDTAEPVSEDEVILNGPHKSKEELIKREFAAKTKSGMDIRSDLRINIGKDVSLDAYGLKTNLDGIr LSVKQDKGNLGLFGQINLTKGRYASFGQDLLTRKGLISFSGQAAQPTLNIEAIRNPETMEDSKITAGV
RVIGIADSPEVTIFSEPSKPQDQALSYItLTGRSLESSGEAGSSGSIGAALIGLGISKSGKLVGSIGEV
FGIQDLrNT~GTSGVGDKSKVTVSGNITNRLQIKYGVGLFDGLAEVTLRYRLMPQLYFQSVSSTNQVFDL
LYIFE
SEQ ID NO:9 polynucleotide sequence upstream of the predicted initiation codon of polynucleotide BASB207 TGGAACTCGGTGTGGGCTTTGCTACTGATGGCGGCGTTCACGGACAAATAGGCTGGACAAAACCTTGG
ZS ATTAATAGCCGTGGACATAGTTTGCGTTCAAATCTTTATCTCTCTGCACCAAAACAAACTCTAGAGGC
AACTTATCGAATGCCACTGCTTAAAAATCCATTAAATTATTACTATGATTTTGCCGTCGGTTGGGAAG
GGGAAAAAGAGAACGATACCAATACGAGAGCGCTTACGTTGTCAGCGTTACGTTATTGGAATAATGCG
CGTGGTTGGCAATATTTTGGCGGACTTCGAGCAAGATACGATAGTTTTACACAAGCGGATATCACTGA
TAAAACCTTACTTCTTTATCCAACTGTTGGATTTACTCGTACTCGATTACGTGGTGGTTCCTTTGCCA

ATAAAAGTGCAAGCATCAAGCGCGTGGATTCGTACTTATGCAGAAAATCATCGTATTGTTGCTCGTGC
TGAAATCGGGTATTTACATACAAAAGATATTGAAAAAATTCCGCCTACACTGCGTTTCTTTGCTGGTG
GCGATCGCAGTWGCGTGGTTACGGCTATAAAAAAATTGCGCCTAAAAATAAAAATGGGAAATTGGTG
GGTGGCTCACGTTTGCTTACAGGTTCTTTAGAATATCAATATCAAGTTTATCCGAAATGGTGGGTGGC

TGGTGTGCGTTGGGCATCGCCAGTGGGCGCGATTAAATTTGATATTGCCACACCCATTCGCGATAAAG
ATAACAGCAAAAATATTCAATTTTACATTGGACTTGGTACAGAAATTTAAGGTGAATTTA
SEQ ID NO:10 GTA AAA CGA GGG CCA GT
40 SEQ ID NO:11 CAG GAA ACA GGT ATG AC
SEQ ID N0:12 8'_' ATG ACA AAA CAA CTA CAA CCA TC
SEQ ID N0:13 ATT GAA ATT TAT AAA GTA AAT CAA ACA
SEQ ID N0:14 TC ATG AGA AAA CAA CTA CAA GC
SEQ ID NO:15 AGA TCT AAA TTC AAA TTT ATA AAGTAAATC
SEQ ID N0:16 G GGG TTT GAA GAC AAT GTT CC
SEQ ID N0:17 CAC GCG GTT TTG GAG GCTC
SEQ ID N0:18 CAG TTC TTG TTT GCT CTT GTG
SEQ ID N0:19 GCC AGTCAG CGA AGA TGA AGT G
$3

Claims

CLAIMS:

1. An isolated polypeptide comprising an amino acid sequence selected from:
(a) an amino acid sequence which bas 97% identity to an amino acid sequence selected from SEQ ID Nos: 2, 4 and 6, over the entire length of said sequence;
and (b) an amino acid sequence which has at least 95% identity to an amino acid sequence of SEQ ID No: 8, over the entire length of said sequence.

2. An isolated polypeptide as claimed in claim 1 in which the amino acid sequence has at least 97% identity to an amino acid sequence of SEQ ID NO: 8;~over the entire length of said sequence.

3. The polypeptide as claimed in claim 1 comprising an amino acid sequence selected from the group consisting of SEQ Group 2.

4. An isolated polypeptide of SEQ Group 2.

5. An immunogenic fragment of a polypeptide comprising an amino acid sequence which has at least 85% identity to an amino acid sequence selected from the group consisting of SEQ Group 2, over the entire length of said sequence wherein said fragment (if necessary when coupled to a carrier) is capable of raising an immune response which recognises a polypeptide of SEQ Group 2.

6. A polypeptide as claimed in any of claims 1 to 5 wherein said polypeptide is part of a larger fusion protein.

7. An isolated polynucleotide encoding a polypeptide as claimed in any of claims 1 to 6.

8. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide selected from:

(a) an amino acid sequence which has 97% identity to an amino acid sequence selected from SEQ ID Nos: 2, 4 and 6, over the entire length of said sequence; and (b) an amino acid sequence that has at least 95% identity to an amino acid sequence of SEQ ID NO: 8, over the entire length of said sequence; or a nucleotide sequence complementary to said isolated polynucleotide.

9. An isolated polynucleotide comprising a nucleotide sequence that has at least 97%
identity to a nucleotide sequence encoding a polypeptide selected from SEQ
Group 2 over the entire coding region; or a nucleotide sequence complementary to said isolated polynucleotide.

10. An isolated polynucleotide which comprises a nucleotide sequence which has at least 97% identity to a DNA sequene selected from SEQ Group 1 over the entire length of said sequence from SEQ Group 1; or a nucleotide sequence complementary to said isolated polynucleotide.

11. The isolated polynucleotide as claimed in any one of claims 7 to 10 in which the identity is at least 99% to a DNA sequence selected from SEQ Group 1 .

12. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide selected from SEQ Group 2 .

13. An isolated polynucleotide comprising a polynucleotide selected from SEQ
Group 1.

14. An isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide selected from SEQ Group 2 obtainable by screening an appropriate library under stringent hybridization conditions with a labeled probe having the corresponding DNA sequence of SEQ Group 1 or a fragment thereof.

15. An expression vector or a recombinant live microorganism comprising an isolated polynucleotide according to any one of claims 7 - 14.

16. A host cell comprising the expression vector of claim 15 or a subcellular fraction or a membrane of said host cell expressing an isolated polypeptide comprising an amino acid sequence that has at least 85% identity to an amino acid sequence selected from the group consisting of SEQ Group 2.

17. A process for producing a polypeptide of claims 1 to 6 comprising culturing a host cell of claim 16 under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture medium.

18. A process for expressing a polynucleotide of any one of claims 7 -14 comprising transforming a host cell with the expression vector comprising at least one of said polynucleotides and culturing said host cell under conditions sufficient for expression of any one of said polynucleotides.

19. A vaccine composition comprising an effective amount of a polypeptide comprising an amino acid sequence which has at least 85% identity to an amino acid sequence selected from the group consisting of SEQ Group 2, over the entire length of said sequence, and a pharmaceutically acceptable carrier.

20. A vaccine composition comprising an effective amount of a polynucleotide comprising a nucleotide sequence which has at least 85% identity to a DNA
sequence selected from SEQ Group 1 over the entire length of said sequence, and a pharmaceutically effective carrier.

21. The vaccine composition according to either one of claims 19 or 20 wherein said composition comprises at least one other non typeable H. influenzae antigen.

22. An antibody immunospecific for a polypeptide comprising an amino acid sequence which has at least 85% identity to an amino acid sequence selected from the group consisting of SEQ Group 2, over the entire length of said sequence, or an immunological fragment thereof.

23. A method of diagnosing a non typeable H. influenzea infection, comprising identifying a polypeptide comprising an amino acid sequence which has at least 85%
identity to an amigo acid sequence selected from the group consisting of SEQ
Group 2, over the entire length of said sequence, or an antibody that is immunospecific for said polypeptide, present within a biological sample from an animal suspected of having such an infection.

24. Use of a composition comprising an immunologically effective amount of a polypeptide comprising an amino acid sequence which has at least 85% identity to an amino acid sequence selected from the group consisting of SEQ Group 2, over the entire length of said sequence, in the preparation of a medicament far use in generating an immune response in an animal.

25. Use of a composition comprising an immunologically effective amount of a polynucleotide comprising a nucleotide sequence which has at least 85%
identity to a DNA sequence selected from SEQ Group 1, over the entire length of said sequence, in the preparation of a medicament for use in generating an immune response in an animal.

26. A therapeutic composition useful in treating humans with non typeable H.
influenzae disease comprising at least one antibody directed against a polypeptide comprising an amino acid sequence which has at least 85% identity to an amino acid sequence selected from the group consisting of SEQ Group 2, over the entire length of said sequence, and a suitable pharmaceutical carrier.