Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/285—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/53—DNA (RNA) vaccination
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• BASB221 polynucleotide(s) polynucleotide(s)
• BASB221 polypeptide(s) polypeptides encoded by them
• BASB221 polypeptides encoded by them
• BASB221 polypeptides encoded by them
• reeombinant materials and methods for their production referred to herein as "BASB221” or “BASB221 polypeptide(s)”
• the invention relates to methods for using such polypeptides and polynucleotides, including vaccines against bacterial infections.
• the invention relates to diagnostic assays for detecting infection of certain pathogens.
• Haemophilus influenzae is a non-motile Gram negative bacterium. Man is its only natural host.
• H. influenzae isolates are usually classified according to their polysaccharide capsule. Six different capsular types designated a through f have been identified. 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.
• 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 H. influenzae (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.
• Streptococcus pneumoniae Three bacterial species are primarily isolated from the middle ear of children with otitis media: Streptococcus pneumoniae, NTHi and catarrhalis. These are present in 60 to 90 % of cases. A review of recent studies shows that S. pneumoniae and NTHi each represent about 30 %, andM catarrhalis about 15 % of otitis media cases (2). Other bacteria can be isolated from the middle ear (H. influenzae 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 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).
• H. influenzae Various proteins of H. influenzae have been shown to be involved in pathogenesis or have been shown to confer protection upon vaccination in animal models.
• NTHi 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 HMW1 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 HM 1/HMW2 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 IgAl serine proteases and has been shown to be involved in both adhesion and cell entry (19).
• OMP outer membrane proteins
• 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). P5 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.
• NTHi expresses a dual human transferrin receptor composed of TbpA and TbpB when grown under iron limitation. Anti-TbpB protected infant rats. (32). Hemoglobin / haptoglobin receptors have also been described for NTHi (33). A receptor for Haem: Hemopexin has also been identified (34). A lactofemn receptor is also present in NTHi, but is not yet characterized (35).
• a 80kDa OMP the D15 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- li itation inducible OMPs or high molecular weight adhesins that have been characterized.
• H. influenzae produces IgAl-protease activity (39). IgAl -proteases of NTHi reveals a high degree of antigenic variability (40).
• NTHi, OMP26, a 26-kDa protein has been shown to enhance pulmonary clearance in a rat model (41).
• the NTHi HtrA 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)
• the present invention relates to BASB221, in particular BASB221 polypeptides and BASB221 polynucleotides, recombinant materials and methods for their production.
• the invention relates to methods for using such polypeptides and polynucleotides, including prevention and treatment of microbial diseases, amongst others.
• 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 BASB221 polynucleotides or polypeptides.
• the invention relates to BASB221 polypeptides and polynucleotides as described in greater detail below.
• the invention relates to polypeptides and polynucleotides ofBASB221 of non typeable H. influenzae.
• the BASB221 polypeptide has a signal sequence characteristic of a lipoprotein, and could thus be exposed at the surface of the bacteria. This signal sequence is located from residue 1 to the residue 21 of the BASB221 polypeptide.
• the invention relates especially to BASB221 polynucleotides and encoded polypeptides listed in table A. Those polynucleotides and encoded polypeptides have the nucleotide and amino acid sequences set out in SEQ ID NO: 1 to SEQ ID NO: 10 as described in table A. Table A
• sequences recited in the Sequence Listing below as "DNA” represent an exemplification of one embodiment of the invention, since those of ordinary skill will recognize that such sequences can be usefully employed in polynucleotides in general, including ribopolynucleotides.
• sequences of the BASB221 polynucleotides are set out in SEQ ID NO:l, 3, 5, 7, 9.
• SEQ Group 1 refers herein to any one of the polynucleotides set out in SEQ ID NO:l, 3,
• sequences of the BASB221 encoded polypeptides are set out in SEQ ID NO:2, 4, 6, 8,
• SEQ Group 2 refers herein to any one of the encoded polypeptides set out in SEQ ID
• polypeptides of non typeable H. influenzae referred to herein as "BASB221” and “BASB221 polypeptides” as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
• the present invention further provides for:
• 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;
• 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
• 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 BASB221 polypeptides provided in SEQ Group 2 are the BASB221 polypeptides from non typeable H. influenzae strains as described in table A.
• the invention also provides an immunogenic fragment of a BASB221 polypeptide, that is, a contiguous portion of the BASB221 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 BASB221 polypeptide.
• an immunogenic fragment may include, for example, the BASB221 polypeptide lacking an N-terminal leader sequence (residues 1-21), and/or a transmembrane domain and/or a C-terminal anchor domain.
• the immunogenic fragment of BASB221 comprises substantially all of the extracellular domain of a polypeptide which has at least 85% 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.
• 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.
• 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 arnphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
• 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 .
• 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.
• 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 amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production. Furthermore, addition of exogenous polypeptide or lipid tail or polynucleotide sequences to increase the immunogenic potential of the final molecule is also considered.
• 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).
• immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hinge region.
• the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa.
• 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. WO 94/29458 and WO 94/22914.
• 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.
• the fusion partner will be both an immunological fusion partner and expression enhancing partner.
• Fusion partners include protein D from Haemophilus influenzae and the non-structural protein from influenza virus, NS 1 (hemagglutinin). Another fusion partner is the protein known as Omp26 (WO 97/01638). Another fusion partner is the protein known as LytA. Preferably the C terminal portion of the molecule is used. LytA is derived from Streptococcus pneumoniae which synthesize an N-acetyl-L-alanine amidase, amidase LytA, (coded by the lytA gene ⁇ Gene, 43 (1986) page 265-272 ⁇ ) 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 DEAE. This property has been exploited for the development of E. 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 He; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; 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 comprise a region encoding BASB221 polypeptides comprising sequences set out in SEQ Group 1 which include full length gene, or a variant thereof.
• the BASB221 polynucleotides provided in SEQ Group 1 are the BASB221 polynucleotides from non typeable H. influenzae strains as described in table A.
• isolated nucleic acid molecules encoding and/or expressing BASB221 polypeptides and polynucleotides, particularly non typeable H. influenzae BASB221 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 BASB221 polypeptide having a deduced amino acid sequence of SEQ Group 2 and polynucleotides closely related thereto and variants thereof.
• BASB221 polypeptide from non typeable H. influenzae comprising or consisting of an amino acid sequence selected from SEQ Group 2 or a variant thereof.
• a polynucleotide of the invention encoding BASB221 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 strain3224A cells as starting material, followed by obtaining a full length clone.
• standard cloning and screening methods such as those for cloning and sequencing chromosomal DNA fragments from bacteria using non typeable H. influenzae strain3224A cells as starting material, followed by obtaining a full length clone.
• a polynucleotide sequence of the invention such as a polynucleotide sequence given in SEQ Group 1, 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.
• 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.
• 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.
• 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 nucleotide number of start codon and first nucleotide of stop codon are listed in table B for each polynucleotide of SEQ Group 1.
• the present invention provides for an isolated polynucleotide comprising or consisting of:
• a polynucleotide encoding a polypeptide of the present invention may be obtained by a process which comprises the steps of screening an appropriate library under stringent hybridization conditions (for example, using a temperature in the range of 45 - 65°C and an SDS concentration from 0.1 - 1%) with a labeled or detectable probe consisting of or comprising any sequence selected from SEQ Group 1 or a fragment thereof; and isolating a full-length gene and/or genomic clones containing said polynucleotide sequence.
• the invention provides a polynucleotide sequence identical over its entire length to a coding sequence (open reading frame) 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 polynucleotide sequence may also comprise additional coding sequence encoding additional amino acids. For example, a marker sequence that facilitates purification of the fused polypeptide can be encoded.
• 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., USA 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 BASB221 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.
• 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.
• polynucleotide encoding a polypeptide 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 BASB221 haying an amino acid sequence set out in any of the sequences of SEQ Group 2 .
• polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, polynucleotides interrupted by integrated phage, an integrated insertion sequence, an integrated vector sequence, an integrated transposon sequence, or due to RNA editing or genomic DNA reorganization) together with additional regions, that also may contain coding and/or non-coding sequences.
• the invention further relates to variants of the polynucleotides described herein that encode variants of a polypeptide having a deduced amino acid sequence of 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, chirneric genes comprising said polynucleotide fragments.
• Further particularly preferred embodiments are polynucleotides encoding BASB221 variants, that have the amino acid sequence of BASB221 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 BASB221 polypeptide.
• polynucleotides that are at least 85% identical over their entire length to a polynucleotide encoding BASB221 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.
• polynucleotides that comprise a region that is at least 90% identical over its entire length to a polynucleotide encoding BASB221 polypeptide and polynucleotides complementary thereto.
• polynucleotides at least 95% identical over their entire length to the same are particularly preferred.
• 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.
• polynucleotides that hybridize, particularly under stringent conditions, to BASB221 polynucleotide sequences, such as those polynucleotides of SEQ Group 1.
• the invention further relates to polynucleotides that hybridize to the polynucleotide sequences provided herein.
• the invention especially relates to polynucleotides that hybridize under stringent conditions to the polynucleotides described herein.
• stringent conditions and “stringent hybridization conditions” mean hybridization occurring only if there is at least 95% and preferably at least 97% identity between the sequences.
• a specific example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising: 50% formamide, 5x SSC (150mM NaCl, 15mM 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 O.lx SSC 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.
• 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 BASB221 and to isolate cDNA and genomic clones of other genes that have a high identity, particularly high sequence identity, to the BASB221 gene.
• Such probes generally will comprise at least 15 nucleotide residues or base pairs.
• 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 BASB221 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.
• PCR Nucleic acid amplification
• PCR Nucleic acid amplification
• 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.
• 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.
• 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.
• the additional amino acids may be processed away from the mature protein by cellular enzymes.
• polynucleotide 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.
• inactive precursors 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.
• 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.
• 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.
• 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.
• a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization.
• 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
• the invention also relates to vectors that comprise a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
• Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the invention.
• Recombinant polypeptides of the present invention may be prepared by processes well known in those skilled 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.
• 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, BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, ⁇ t al, MOLECULAR CLONING: A LABORATORY MANUAL, 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.
• bacterial cells such as cells of streptococci, staphylococci, enterococci, E. coli, streptomyces, cyanobacteria, Bacillus subtilis, Neisseria meningitidis, Haemophilus influenzae and Moraxella catarrhalis
• fungal cells such as cells of a yeast, Kluveromyces, Saccharomyces, Pichia, a basidiomycete, Candida albicans and Aspergillus
• insect cells such as cells of Drosophila S2 and Spodoptera Sf9
• animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells
• plant cells such as cells of a gymnosperm or angiosperm.
• 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.
• 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 LABORATORY MANUAL, (supra).
• 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 cation 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.
• IMAC ion metal affinity chromatography
• 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, Dialoguelian Equine Encephalitis Virus), adenoviruses, adeno-associated virus, picornaviruses (poliovirus, rhinovirus), herpesviruses (varicella zoster virus, etc), Listeria, Salmonella , Shigella, BCG, 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.
• poxviruses e.g; vaccinia, fowlpox, canarypox
• alphaviruses Semliki Forest Virus, Kunststoffuelian Equine Encephalitis Virus
• adenoviruses adeno-associated virus
• This invention is also related to the use of BASB221 polynucleotides and polypeptides of the invention for use as diagnostic reagents.
• Detection of BASB221 polynucleotides and/or polypeptides in a eukaryote, particularly a mammal, and especially a human will provide a diagnostic method for diagnosis of disease, staging of disease or response of an infectious organism to drugs.
• Eukaryotes, particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism comprising the BASB221 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 and/or infected individual's bodily materials.
• Polynucleotides from any of these sources may be used directly for detection or may be amplified enzymatically 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.
• 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 BASB221 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: 1242 (1985). Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase, VI and S 1 protection assay or a chemical cleavage method. See, for example, Cotton et al, Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985).
• an array of oligonucleotides probes comprising BASB221 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)).
• the present invention relates to a diagnostic kit which comprises:
• a polynucleotide of the present invention preferably any of the nucleotide sequences of SEQ Group 1 , or a fragment thereof ;
• kits 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 polynucleotide may be detected at the polynucleotide level by a variety of techniques, such as those described elsewhere herein.
• Cells from an organism carrying mutations or polymorphisms (allelic variations) in a polynucleotide and/or polypeptide of the invention may also be detected at the polynucleotide or polypeptide level by a variety of techniques, to allow for serotyping, for example.
• 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.
• PCR primers complementary to a polynucleotide encoding BASB221 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 BASB221 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. influenzae, 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 BASB221 polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and other hybridization methods.
• a diagnostic assay in accordance with the invention for detecting over-expression of BASB221 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 BASB221 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.
• 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 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 .
• 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.
• mimotopes, particularly peptide mimotopes, of epitopes within the polypeptide sequence may also be used as immunogens to produce antibodies immunospecific for the polypeptide of the invention.
• immunospecific means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.
• antibodies against BASB221 polypeptides or polynucleotides there are provided antibodies against BASB221 polypeptides or polynucleotides.
• Antibodies generated against the polypeptides or polynucleotides of the invention can be obtained by administering the polypeptides and/or polynucleotides of the invention, or epitope-bearing fragments of either or both, analogues of either or both, or cells expressing either or both, to an animal, preferably a nonhuman, using routine protocols.
• any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques, such as those in Kohler, G. and Milstein, C, Nature 256: 495-497 (1975); Kozbor et al. ,
• 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-BASB221 or from naive libraries (McCafferty, et al, (1990), Nature 348, 552-554; Marks, et al, (1992) Biotechnology 10, 779-783).
• the affinity of these antibodies can also be improved by, for example, chain shuffling (Clackson et al, (1991) Nature 352: 628).
• the above-described antibodies may be employed to isolate or to identify clones expressing the polypeptides or polynucleotides of the invention to purify the polypeptides or polynucleotides by, for example, affinity chromatography.
• antibodies against BASB221 polypeptide or BASB221 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.
• the antibody or variant thereof is modified to make it less immunogenic in the individual.
• 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), Nature 321, 522-525 or Tempest et al, (1991) Biotechnology 9, 266-273.
• 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.
• 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 Immunology 1(2): Chapter 5 (1991).
• the screening methods may simply measure the binding of a candidate compound to the polypeptide or polynucleotide, or to cells or membranes bearing the polypeptide or polynucleotide, or a fusion protein of the polypeptide by means of a label directly or indirectly associated with the candidate compound.
• the screening method may involve competition with a labeled competitor.
• these screening methods may test whether the candidate compound results in a signal generated by activation or inhibition of the polypeptide or polynucleotide, using detection systems appropriate to the cells comprising the polypeptide or polynucleotide. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed.
• Constitutively active polypeptide and/or constitutively expressed polypeptides and polynucleotides may be employed in screening methods for inverse agonists or inhibitors, in the absence of an agonist or inhibitor, by testing whether the candidate compound results in inhibition of activation of the polypeptide or polynucleotide, as the case may be.
• 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 BASB221 polypeptide and or polynucleotide activity in the mixture, and comparing the BASB221 polypeptide and/or polynucleotide activity of the mixture to a standard.
• Fusion proteins such as those made from Fc portion and BASB221 polypeptide, as hereinbefore described, can also be used for high-throughput screening assays to identify antagonists of the polypeptide of the present invention, as well as of phylogenetically and and/or functionally related polypeptides (see D. Bennett et al., J Mol Recognition, 8:52-58 (1995); and K. Johanson et al, J Biol Chem, 270(16):9459-9471 (1995)).
• polypeptides and antibodies that bind to and/or interact with a polypeptide of the present invention may also be used to configure screening methods for detecting the effect of added compounds on the production of mRNA and/or polypeptide in cells.
• 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 BASB221 polypeptides or polynucleotides, particularly those compounds that are bacteriostatic and/or bactericidal.
• the method of screening may involve high-throughput techniques. For example, to screen for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, comprising BASB221 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 BASB221 agonist or antagonist.
• the ability of the candidate molecule to agonize or antagonize the BASB221 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 BASB221 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 fransduction, or increase chemical channel activity are agonists. Detection of the rate or level of, as the case may be, production of product from substrate, signal fransduction, or chemical channel activity may be enhanced by using a reporter system.
• Reporter systems that may be useful in this regard include but are not limited to colorimetric, labeled substrate converted into product, a reporter gene that is responsive to changes in BASB221 polynucleotide or polypeptide activity, and binding assays known in the art.
• an assay for BASB221 agonists is a competitive assay that combines BASB221 and a potential agonist with BASB221 binding molecules, recombinant BASB221 binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay.
• BASB221 can be labeled, such as by radioactivity or a colorimetric compound, such that the number of BASB221 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 BASB221 induced activities, thereby preventing the action or expression of BASB221 polypeptides and/or polynucleotides by excluding B ASB221 polypeptides and/or polynucleotides from binding.
• Potential antagonists include a small molecule that binds to and occupies the binding site of the polypeptide thereby preventing binding to cellular binding molecules, such that normal biological activity is prevented.
• small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules.
• Other potential antagonists include antisense molecules (see Okano,J. Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988), for a description of these molecules).
• Preferred potential antagonists include compounds related to and variants of BASB221.
• 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).
• immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hinge region.
• the Fc part can be removed simply by incorporation of a cleavage sequence which can be cleaved with blood clotting factor Xa.
• 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. WO94/29458 and WO94/22914.
• 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.
• 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.
• the molecules of the invention may be used: in the prevention of adhesion of bacteria, in particular gram positive and/or gram negative bacteria, to eukaryotic, preferably mammalian, extracellular matrix proteins on indwelling devices or to extracellular matrix proteins in wounds; to block bacterial adhesion between eukaryotic, preferably mammalian, extracellular matrix proteins and bacterial BASB221 proteins that mediate tissue damage and/or; to block the normal progression of pathogenesis in infections initiated other than by the implantation of indwelling devices or by other surgical techniques.
• BASB221 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.
• the present invention relates to mimotopes of the polypeptide of the invention.
• a mimotope is a peptide sequence, sufficiently similar to the native peptide (sequentially or structurally), which is capable of being recognised by antibodies which recognise the native peptide; or is capable of raising antibodies which recognise the native peptide when coupled to a suitable carrier.
• Peptide mimotopes may be designed for a particular purpose by addition, deletion or substitution of elected amino acids.
• 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.
• peptides conjugated to a protein carrier may 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.
• the peptides may be altered to have an N-terminal cysteine and a C-terminal hydrophobic amidated tail.
• 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 95/24916 and WO 94/05311.
• peptide mimotopes may be identified using antibodies which are capable themselves of binding to the polypeptides of the present invention using techniques such as phage display technology (EP 0 552 267 Bl). This technique, generates a large number of peptide sequences which mimic the structure of the native peptides and are, therefore, capable of binding to anti-native peptide antibodies, but may not necessarily themselves share significant sequence homology to the native polypeptide.
• Vaccines 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.
• 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 BASB221 polynucleotide and/or polypeptide, or a fragment or variant thereof, adequate to produce antibody and/ or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly 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 BASB221 polynucleotide and/or polypeptide, or a fragment or a variant thereof, for expressing BASB221 polynucleotide and/or polypeptide, or a fragment or a variant thereof in vivo in order to induce an immunological response, such as, to produce antibody and/ or T cell immune response, including, for example, cytokine- producing T cells or cytotoxic T cells, to protect said individual, preferably a human, from disease, whether that disease is already established within the individual or not.
• an immunological response such as, to produce antibody and/ or T cell immune response, including, for example, cytokine- producing T cells or cytotoxic T cells, to protect said individual, preferably a human, from disease, whether that disease is already established within the individual or not.
• nucleic acid vector may comprise DNA, RNA, a ribozyme, a modified nucleic acid, a DNA/RNA hybrid, a DNA-protein complex or an RNA-protein complex.
• a further aspect of the invention relates to an immunological composition that when introduced into an individual, preferably a human, capable of having induced within it an immunological response, induces an immunological response in such individual to a BASB221 polynucleotide and/or polypeptide encoded therefrom, wherein the composition comprises a recombinant BASB221 polynucleotide and/or polypeptide encoded therefrom and/or comprises DNA and/or RNA which encodes and expresses an antigen of said BASB221 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.
• BASB221 polypeptide or a fragment thereof may be fused with co-protein or chemical moiety which may or may not by itself produce antibodies, but which is capable of stabilizing the first protein and producing a fused or modified protein which will have antigenic and/or immunogenic properties, and preferably protective properties.
• fused recombinant protein preferably further comprises an antigenic co-protein, such as lipoprotein D from Haemophilus influenzae, Glutathione-S-transferase (GST) or beta- galactosidase, or any other relatively large co-protein which solubilizes the protein and facilitates production and purification thereof.
• 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.
• a B ASB221 polypeptide and/or 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.
• non-live vectors for the BASB221 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 Microbiol. 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 melitensis, Brucella ovis, Esherichia coli, Haemophilus influenzae, Legionella pneumophila, Moraxella catarrhalis, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas 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.
• a desired immunogenic protein at the outer membrane such as the BASB221 polypeptide
• 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.
• the non-coding flanking regions of the BASB221 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 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.
• SEQ ID NO: 11 is the non typeable Haemophilus influenzae upstream sequence (upstream of the predicted initiation codon of the preferred genes) comprising approximately lOOObp.
• This sequence information allows the modulation of the natural expression of the BASB221 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.
• downregulation of expression can be achieved by similar types of modification.
• the expression of the gene can be put under phase variation control, or it may be uncoupled from this regulation.
• 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.
• modifications as described above can be introduced by several different means.
• the modification of sequences involved in gene expression can be carried 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.
• the sequence knowledge of the region of interest can be used to replace or delete all or part of the natural regulatory sequences.
• 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, IbpB, tbpB, pi 10, 1st, hpuAB from N. meningitidis orN.
• gonorroheae ompCD, copB, IbpB, ompE, UspAl; UspA2; TbpB from M. Catarrhalis; pi, p2, p4, p5, p6, lpD, tbpB, D15, Hia, Hmwl, Hmw2 from H. influenzae.
• 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.
• 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.
• a modified upstream region of the BASB221 gene which modified upstream region contains a heterologous regulatory element which alters the expression level of the BASB221 protein located at the outer membrane.
• the upstream region according to this aspect of the invention includes the sequence upstream of the BASB221 gene.
• the upstream region starts immediately upstream of the BASB221 gene and continues usually to a position no more than about 1000 bp upstream of the gene from the ATG start codon.
• the upstream region can start immediately preceding the gene of interest, or preceding the first gene in the operon.
• a modified upstream region according to this aspect of the invention contains a heterologous promotor at a position between 500 and 700 bp upstream of the ATG.
• upstream regions to upregulate the expression of the BASB221 gene, a process for achieving this through homologous recombination (for instance as described in WO 01/09350 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.
• the invention provides a BASB221 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 B ASB221 gene having a modified upstream region containing a heterologous regulatory element.
• 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).
• 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. influenzae infection, in mammals, particularly humans.
• the invention also includes a vaccine formulation which comprises an immunogenic recombinant polypeptide and/or polynucleotide of the invention together with a suitable carrier, such as a pharmaceutically acceptable carrier. Since the polypeptides and polynucleotides may be broken down in the stomach, each is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal.
• Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostatic compounds and solutes which render the formulation isotonic with the bodily fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
• the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
• the vaccine formulation of the invention may also include adjuvant systems for enhancing the immunogenicity of the formulation. Preferably the adjuvant system raises preferentially a TH1 type of response.
• An immune response may be broadly distinguished into two extreme catagories, being a humoral or cell mediated immune responses (traditionally characterised by antibody and cellular effector mechanisms of protection respectively). These categories of response have been termed THl-type responses (cell-mediated response), and TH2-type immune responses (humoral response).
• Extreme THl-type immune responses may be characterised by the generation of antigen specific, haplotype restricted cytotoxic T lymphocytes, and natural killer cell responses.
• mice THl-type responses are often characterised by the generation of antibodies of the IgG2a subtype, whilst in the human these correspond to IgGl type antibodies.
• TH2- type immune responses are characterised by the generation of a broad range of immunoglobulin isotypes including in mice IgGl, IgA, and IgM.
• cytokines the driving force behind the development of these two types of immune responses.
• High levels of THl-type cytokines tend to favour the induction of cell mediated immune responses to the given antigen, whilst high levels of TH2-type cytokines tend to favour the induction of humoral immune responses to the antigen.
• TH1 and TH2-type immune responses are 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 CD4 +ve T cell clones by Mosmann and Coffman (Mosmann, TR. and Coffman, R.L. (1989) TH1 and TH2 cells: different patterns oflymphokine secretion lead to different functional properties. Annual Review of Immunology, 7, pi 45-173). Traditionally, THl-type responses are associated with the production of the INF- ⁇ and IL-2 cytokines by T-lymphocytes.
• cytokines often directly associated with the induction of THl-type immune responses are not produced by T-cells, such as IL-12.
• TH2- type responses are associated with the secretion of IL-4, IL-5 , IL-6 and IL- 13.
• the best indicators of the TH1: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, and/or the measurement of the IgGl:IgG2a ratio of antigen specific antibody responses.
• a THl-type adjuvant is one which preferentially stimulates isolated T-cell populations to produce high levels of THl-type cytokines when re-stimulated with antigen in vitro, and promotes development of both CD8+ cytotoxic T lymphocytes and antigen specific immunoglobulin responses associated with THl-type isotype.
• Adjuvants which are capable of preferential stimulation of the THl cell response are described in International Patent Application No. WO 94/00153 and WO 95/17209.
• 3 De-O-acylated monophosphoryl lipid A 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 Bl (SmithKline Beecham Biologicals SA).
• the particles of 3D-MPL are small enough to be sterile filtered through a 0.22micron membrane (European Patent number 0 689454).
• 3D-MPL will be present in the range of lO ⁇ g - lOO ⁇ g preferably 25-50 ⁇ g per dose wherein the antigen will typically be present in a range 2-50 ⁇ g per dose.
• Another preferred adjuvant comprises QS21, an Hplc purified non-toxic fraction derived from the bark of Quillaja Saponaria Molina.
• this may be admixed with 3 De-O-acylated monophosphoryl lipid A (3D-MPL), optionally together with an carrier.
• 3D-MPL 3 De-O-acylated monophosphoryl lipid A
• Non-reactogenic adjuvant formulations containing QS21 have been described previously (WO 96/33739). Such formulations comprising QS21 and cholesterol have been shown to be successful THl stimulating adjuvants when formulated together with an antigen.
• Further adjuvants which are preferential stimulators of THl cell response include immunomodulatory oligonucleotides, for example unmethylated CpG sequences as disclosed in WO 96/02555.
• THl stimulating adjuvants such as those mentioned hereinabove, are also contemplated as providing an adjuvant which is a preferential stimulator of THl cell response.
• QS21 can be formulated together with 3D-MPL.
• the ratio of QS21 : 3D-MPL will typically be in the order of 1 : 10 to 10 : 1; preferably 1:5 to 5 : 1 and often substantially 1 : 1.
• the preferred range for optimal synergy is 2.5 : 1 to 1 : 1 3D-MPL: QS21.
• a carrier is also present in the vaccine composition according to the invention.
• the carrier may be an oil in water emulsion, or an aluminium salt, such as aluminium phosphate or aluminium hydroxide.
• a preferred oil-in- water emulsion comprises a metabolisible oil, such as squalene, alpha tocopherol and Tween 80.
• the antigens in the vaccine composition according to the invention are combined with QS21 and 3D-MPL in such an emulsion.
• the oil in water emulsion may contain span 85 and/or lecithin and/or tricaprylin.
• QS21 and 3D-MPL will be present in a vaccine in the range of l ⁇ g - 200 ⁇ g, such as 10-100 ⁇ g, preferably lO ⁇ g - 50 ⁇ g per dose.
• 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.
• 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.
• a non-toxic oil e.g. squalane or squalene
• an emulsifier e.g. Tween 80
• the aqueous carrier may be, for example, phosphate buffered saline.
• the present invention also provides a polyvalent vaccine composition
• a polyvalent vaccine composition comprising a vaccine formulation of the invention in combination with other antigens, in particular antigens useful for treating otitis media.
• a polyvalent vaccine composition may include a TH-1 inducing adjuvant as hereinbefore described.
• the polypeptides, fragments and immunogens of the invention are formulated with one or more of the following groups of antigens: a) one 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 pneumoniae infection; d) one or more further non typeable Haemophilus influenzae protein antigens; e) one or more antigens that can protect a host against RSN; and f) one or more antigens that can protect a host against influenza virus.
• antigens a) one 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
• Combinations with: groups a) and b); b) and c); b), d), and a) and/or c); b), d), e), f), and a) and/or c) are preferred.
• Such vaccines may be advantageously used as global otitis media vaccines.
• the pneumococcal capsular polysaccharide antigens are preferably selected from serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9 ⁇ , 9V, 10A, 11 A, 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.
• the protein is a toxin, adhesin, 2-component signal tranducer, or lipoprotein of Streptococcus pneumoniae, 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.
• pneumococcal protein antigens are those disclosed in WO 98/18931, particularly those selected in WO 98/18930 and PCT/US99/30390.
• Preferred Moraxella catarrhalis protein antigens which can be included in a combination vaccine (especially for the prevention of otitis media) are: OMP 106 [WO 97/41731 (Antex) & WO 96/34960 (PMC)]; 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.
• Preferred further non-typeable Haemophilus influenzae 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 [eg LB 1(f) peptide fusions; US 5843464 (OSU) or WO 99/64067]; OMP26 [WO 97/01638 (Cortecs)]; P6 [EP 281673 (State University of New York)]; protein D (EP 594610); TbpA and or TbpB; Hia; Hsf; Hin47; Hif; Hmwl; Hmw2; Hmw3; Hmw4; Hap; D15 (WO 94/12641); P2; and P5 (WO 94/26304).
• Fimbrin protein (US 5766608 - Ohio State Research Foundation)] and fusions comprising peptides therefrom [e
• 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 for purposes of compositions, kits and administration
• compositions comprising a BASB221 polynucleotide and/or a BASB221 polypeptide for administration to a cell or to a multicellular organism.
• compositions comprising a polynucleotide and/or a polypeptides discussed herein or their agonists or antagonists.
• the polypeptides and polynucleotides of the invention may be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to an individual.
• a pharmaceutical carrier suitable for administration to an individual.
• Such compositions comprise, for instance, a media additive or a therapeutically effective amount of a polypeptide and/or polynucleotide of the invention and a pharmaceutically acceptable carrier or excipient.
• Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof.
• the formulation should suit the mode of administration.
• the invention further relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
• Polypeptides, polynucleotides and other compounds of the invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
• 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.
• the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.
• the present invention provides for pharmaceutical compositions comprising a therapeutically effective amount of a polypeptide and/or polynucleotide, such as the soluble form of a polypeptide and/or polynucleotide of the present invention, agonist or antagonist peptide or small molecule compound, in combination with a pharmaceutically acceptable carrier or excipient.
• a pharmaceutically acceptable carrier or excipient 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.
• 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.
• penetrants such as bile salts or fusidic acids or other detergents.
• oral administration may also be possible. Aclministration of these compounds may also be topical and or localized, in the form of salves, pastes, gels, solutions, powders and the like.
• the daily dosage level of the active agent will be from 0.01 mg/kg to 10 mg/kg, typically around 1 mg/kg.
• the physician in any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular individual.
• the above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
• Suitable dosages are in the range of 0.1-100 ⁇ g/kg of subject.
• a vaccine composition is conveniently in injectable form. Conventional adjuvants may be employed to enhance the immune response.
• a suitable unit dose for vaccination is 0.5-5 microgram kg of antigen, and such dose is preferably administered 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.
• Polynucleotide and polypeptide sequences form a valuable information resource with which to determine their 2- and 3 -dimensional structures as well as to identify further sequences of similar homology. These approaches are most easily facilitated by storing the sequence in a computer readable medium and then using the stored data in a known macromolecular structure program or to search a sequence database using well known searching tools, such as the GCG program package.
• sequence analysis includes, 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 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 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.
• Identity 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.
• 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 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.
• Methods to determine identity are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available computer programs. Computer program methods to determine identity between two sequences include, but are not limited to, the GAP program in the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN (Altschul, S.F. et al., J. Molec. Biol.
• 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:l, 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 or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO: 1 by the integer
• n n is the number of nucleotide alterations
• x n is the total number of nucleotides in SEQ ID NO:l
• y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%
• • is the symbol for the multiplication operator, and wherein any non-integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n .
• Alterations of polynucleotide sequences encoding the polypeptides of SEQ ID NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
• 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 polynucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleic acids in the reference sequence or in one or more contiguous groups within the reference sequence.
• the number of nucleic acid alterations for a given percent identity is determined by multiplying the total number of nucleic acids in SEQ ID NO: 1 by the integer defining the percent identity divided by 100 and then subtracting that product from said total number of nucleic acids in SEQ ID NO:l, or:
• n n is the number of nucleic acid alterations
• x n is the total number of nucleic acids in SEQ ID NO:l
• y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc.
• « is the symbol for the multiplication operator, and wherein any non-integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n .
• 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 NO:2, wherein said polypeptide sequence may be identical to the reference sequence of SEQ ID NO: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 NO: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 NO
• n a is the number of amino acid alterations
• x a is the total number of amino acids in SEQ ID NO:2
• y is 0.50 for 50%, 0.60 for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%, 0.97 for 97% or 1.00 for 100%
• • is the symbol for the multiplication operator, and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a .
• a polypeptide sequence of the present invention may be identical to the reference sequence of SEQ ID NO: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 NO: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 NO:2, or:
• n a is the number of amino acid alterations
• x a is the total number of amino acids in SEQ ID NO:2
• y is, for instance 0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc.
• • is the symbol for the multiplication operator, and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x a .
• 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.
• 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.
• 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 polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
• a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide.
• a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
• a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
• a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non- naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
• Disease(s) means any disease caused by or related to infection by a bacteria, including, for example, otitis media in infants and children, pneumonia in elderlies, sinusitis, nosocomial infections and invasive diseases, chronic otitis media with hearing loss, fluid accumulation in the middle ear, auditive nerve damage, delayed speech learning, infection of the upper respiratory tract and inflammation of the middle ear.
• Example 1 DNA sequencing of the BASB221 gene from Non typable Haemophilus influenzae strain 3224A.
• the DNA sequence of the BASB221 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 BASB221 polynucleotidic sequence is showed in SEQ ID N0:2.
• plasmid D ⁇ A (see example 3 A) containing the gene region encoding BASB221 fromNo « Typable Haemophilus influenzae strain 3224A was submitted to D ⁇ A sequencing using the Big Dyes kit (Applied biosystems) and analyzed on a ABI 373/A D ⁇ A sequencer in the conditions described by the supplier using primers ⁇ LPB olil (5'-TC ATG AAA AAA ATA ATA TTG AGT TTA ACA ACG GC -3') [SEQ ID NO: 12] and NLPB oli2 (5'-AGA TCT TTG AAT TGA AGT GCT TGA TAA ATC C -3') [SEQ ID NO: 13] specific for the BASB221 polynucleotide and M13 Universal Sequence Primer(5'-GTA AAA CGA CGG CCA GT-3') [SEQ ID NO:
• Genomic DNA was extracted from 4 NT Haemophilus influenzae strains (presented in Table 1) as follows. A 500ml 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 ⁇ m, 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. Genomic DNA was extracted from the pellet of the NT Haemophilus influenzae cells using the QIAGEN genomic DNA extraction kit (Qiagen Gmbh).
• the BspHI and BgRl restriction sites engineered into NLPB olil (5'-TC ATG AAA AAA ATA ATA TTG AGT TTA ACA ACG GC-3') [SEQ ID NO: 12] forward and NLPB oli2 (5'-AGA TCT TTG AAT TGA AGT GCT TGA TAA ATC C -3') [SEQ . ID NO: 13] reverse amplification primers, respectively, permitted directional cloning of the PCR product into the E.coli expression plasmid pQE60 such that BASB221 protein could be expressed as a fusion protein containing a (His)6 affinity chromatography tag at the C-terminus.
• the BASB221 PCR product was first introduced into the pCRIITOPO cloning vector (In vitrogen) using Top 10 bacterial cells, according to the manufacturer's instructions. This intermediate construct was realized to facilitate further cloning into an expression vector. Transformants containing the BASB221 DNA insert were selected by restriction enzyme analysis. Following digestion, a ⁇ 20 ⁇ l 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 UN illumination after gel electrophoresis and ethidium bromide staining.
• TAE Tris-acetate-EDTA
• B Production of expression vector. To prepare the expression plasmid pQE60 for ligation, it was similarly digested to completion with both Ncol and Bgtll. An approximately 5-fold molar excess of the digested fragments to the prepared vector was used to program the ligation reaction.
• a standard -20 ⁇ l ligation reaction ( ⁇ 16°C, -16 hours), using methods well known in the art, was performed using T4 D ⁇ A ligase (-2.0 units / reaction, Life Technologies). An aliquot of the ligation (-5 ⁇ l) 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 -1.0 ml of LB broth, transformed cells were plated on LB agar plates containing ampicillin (100 ⁇ g/ml) and kanamycin (30 ⁇ g/ml). Antibiotic was included in the selection. Plates were incubated overnight at 37°C for -16 hours.
• the cell pellet was suspended in ⁇ 200 ⁇ l of sterile water and a ⁇ 10 ⁇ l aliquot used to program a ⁇ 50 ⁇ l final volume PCR reaction containing both BASB221 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.
• one gel was stained with commassie brilliant blue R250 (BioRad) and then destained to visualize novel B ASB221 IPTG-inducible protein.
• the second gel was electroblotted onto a PNDF membrane (0.45 micron pore size, ⁇ ovex) 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 recombinant expression strain of E. coli M15(pREP4) containing a plasmid (pQE60) encoding BASB221 from NT haemophilus influenzae was used to produce cell mass for purification of recombinant protein.
• the expression strain was cultivated on LB agar plates containing lOO ⁇ g/ml ampicillin. ("Ap") and 30 ⁇ g/ml kanamycin (“Km”) to ensure that pQE60 and pREP4 were maintained.
• Ap lOO ⁇ g/ml ampicillin.
• Km 30 ⁇ g/ml kanamycin
• 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.
• the growth medium used for the production of recombinant protein consisted of LB broth (Difco) containing lOO ⁇ g/ml Ap and 30 ⁇ g/ml Km.
• IPTG Isopropyl ⁇ -D-Thiogalactopyranoside
• Example 5 Expression and purification of recombinant BASB221 protein in Escherichia coli.
• the construction of the pET-BASB221 cloning/expression vector is described in Example 1.
• This vector harbours the BASB221 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.
• this vector is introduced into the Escherichia coli strain Novablue (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-BASB221] E. coli recombinant strain are grown at 37°C under agitation until the optical density at 600nm (OD600) reached 0.6. At that time- point, IPTG is added at a final concentration of ImM and the culture is grown for 4 additional hours.
• the culture is then centrifuged at 10,000 ⁇ m and the pellet is frozen at - 20°C for at least 10 hours. After thawing, the pellet is resuspended during 30 min at 25°C in buffer A (6M guanidine hydrochloride, 0.1M NaH2PO4, 0.01M Tris, pH 8.0), passed three-times through a needle and clarified by centrifugation (20000 ⁇ m, 15 min). The sample is then loaded at a flow-rate of lml/min on a Ni2+ -loaded Hitrap column (Pharmacia Biotech).
• the column is washed successiveively with 40ml of buffer B (8M Urea, 0.1MNaH2PO4, 0.01M Tris, pH 8.0), 40ml of buffer C (8M Urea, 0.1MNaH2PO4, 0.0 IM Tris, pH 6.3).
• buffer B 8M Urea, 0.1MNaH2PO4, 0.01M Tris, pH 8.0
• buffer C 8M Urea, 0.1MNaH2PO4, 0.0 IM Tris, pH 6.3
• the recombinant protein BASB221/His6 is then eluted from the column with 30ml of buffer D (8M Urea, 0.1MNaH2PO4, 0.0 IM Tris, pH 6.3) containing 500mM of imidazole and 3ml-size fractions are collected. Highly enriched BASB221/His6 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 BASB221-His6 protein is solubilized in a solution devoid of urea. For this pu ⁇ ose, denatured BASB221-His6 contained in 8M urea is extensively dialyzed (2 hours) against buffer R (NaCl 150mM, lOmM NaH2PO4, Arginine 0.5M pH6.8) containing successively 6M, 4M, 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 BASB221
• Polyvalent antisera directed against the BASB221 protein are generated by vaccinating rabbits with the purified recombinant BASB221 protein. Polyvalent antisera directed against the BASB221 protein are also generated by vaccinating mice with the purified recombinant BASB221 protein. Animals are bled prior to the first immunization ("pre- bleed") and after the last immunization.
• Anti-BASB221 protein titers are measured by an ELISA using purified recombinant BASB221 protein as the coating antigen.
• the titer is defined as mid-point titers calculated by 4-parameter logistic model using the XL Fit software.
• 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 BASB221 Anti-BASB221 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.
• PNDF membranes are incubated with preimmune serum or rabbit or mouse immune serum. PNDF 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-l-naphtol.
• Example 9 Immunological characterization: Bactericidal Activity Complement-mediated cytotoxic activity of anti-BASB221 antibodies is examined to determine the vaccine potential of BASB221 protein antiserum that is prepared as described above. The activities of the pre-immune serum and the anti-BASB221 antiserum in mediating complement killing of NTHi are examined.
• Preimmune sera and the anti-BASB221 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).
• Example 5 except that a pool of human sera from children infected by NTHi is used as the first antibody preparation.
• Example 11 Efficacy of BASB221 vaccine: enhancement of lung clearance of
• This mouse model is based on the analysis of the lung invasion by NTHi following a standard intranasal challenge to vaccinated mice.
• mice are immunized with BASB221 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 log 10 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 log 10 weighted mean number of CFU/lung and the standard deviations are calculated for each group.
• mice are immunized either with BASB221 or with a killed whole cells (kwe) preparation of NTHi or sham immunized.
• Example 12 Inhibition of NTHi adhesion onto cells by anti-BASB221 antiserum.
• This assay measures the capacity of anti BASB221 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- BASB221 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.
• 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.3).
• the antigenic index was calculated on the basis of the method described by Jameson and Wolf (CABIOS 4:181- 186 [1988]).
• the parameters used in this program are the antigenic index and the minimal length for an antigenic peptide.
• 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 nfHi 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, 11, 12 or 15) amino acids therefrom or extensions comprising e. g. 1, 2, 3, 5, 10 further amino acids at either or both ends from the native context of BASB221 polypeptide which preserve an effective epitope which can elicit an immune response in a host against the BASB221 polypeptide.
• 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 BASB221 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 pu ⁇ oses, as could similar peptides comprising conservative mutations (preferably 70, 80, 95, 99 or 100% identical to the sequences below) or truncates comprising 5 or more (e.g.
• SEQ ID NO:2 All identified regions containing epitopes as defined above are in respect of SEQ ID NO:2.
• the corresponding regions in SEQ ID NO:4, 6, 8, 10 as defined by position in table 4&5 with respect to SEQ ID NO:2 and by its corresponding peptide in the alignment of figure 2 for SEQ ID NO:4, 6, 8, 10 are also preferred peptides of the invention as described in this example.
• strain 3224A 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 influe ⁇ zae ⁇ tr ⁇ n deposit is referred to herein as "the deposited strain” or as "the DNA of the deposited strain.”
• the deposited strain contains a full length BASB221 gene.
• sequence of the polynucleotides 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 Pu ⁇ oses 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.
• SEQ ID NO:6 polypeptide sequence of BASB221 MKKIILSLTTAIILVGCSSNPETLKASNDSFQKSEASIPHFSPLATGGVQLPKADDAYSLPNIEVKKRGD IDIRPPLIPLAIIQNSITKFDGERSLIVYPKQEA LYNLQQVERLLKEEGISSTTNGSILTTDWTKTERI GDKPIEIKYQIEQVMTPDVSAITVSILHMRRDGIIFTPNVSDKQYYTSERLNRIVLTLTTAYNKQLQDLS STSIQ

Landscapes

• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Medicinal Chemistry (AREA)
• Gastroenterology & Hepatology (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Life Sciences & Earth Sciences (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Communicable Diseases (AREA)
• Peptides Or Proteins (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Applications Claiming Priority (5)

Publications (1)

Family

ID=26245409

Family Applications (1)

Country Status (4)

Family Cites Families (1)

• 2001
  • 2001-12-07 CA CA002436306A patent/CA2436306A1/en not_active Abandoned
  • 2001-12-07 AU AU2002229625A patent/AU2002229625A1/en not_active Abandoned
  • 2001-12-07 EP EP01990521A patent/EP1339851A2/de not_active Withdrawn
  • 2001-12-07 WO PCT/EP2001/014336 patent/WO2002046215A2/en not_active Application Discontinuation

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events