WO1997037026A1 - Nouveaux composes - Google Patents

Nouveaux composes Download PDF

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Publication number
WO1997037026A1
WO1997037026A1 PCT/US1997/005306 US9705306W WO9737026A1 WO 1997037026 A1 WO1997037026 A1 WO 1997037026A1 US 9705306 W US9705306 W US 9705306W WO 9737026 A1 WO9737026 A1 WO 9737026A1
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Prior art keywords
seq
polypeptide
sequence
polynucleotide
nucleic acid
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PCT/US1997/005306
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English (en)
Inventor
Michael Terrance Black
John Edward Hodgson
David Justin Charles Knowles
Richard Oakley Nicholas
Robert King Stodola
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Smithkline Beecham Corporation
Smithkline Beecham Plc
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Application filed by Smithkline Beecham Corporation, Smithkline Beecham Plc filed Critical Smithkline Beecham Corporation
Priority to JP09535535A priority Critical patent/JP2000511769A/ja
Priority to EP97920002A priority patent/EP0907738A1/fr
Publication of WO1997037026A1 publication Critical patent/WO1997037026A1/fr

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    • 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
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/02—Local antiseptics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

Definitions

  • This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the uses of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides and recombinant host cells transformed with the polynucleotides.
  • This invention also relates to inhibiting the biosynthesis or action of such polynucleotides or polypeptides and to the use of such inhibitors in therapy.
  • Preferred embodiments of the invention include Streptococcal polypeptides and polynucleotides, particularly those of Streptococcus pneumoniae. Background of the Invention;
  • Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including otitis media, pneumonia and meningitis. Since its isolation more than 100 years ago, Streptococcus pneumoniae has been one of the more intensively studied microbes. For example, much of our early understanding that DNA is, in fact, the genetic material was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe. Despite the vast amount of research with S. pneumoniae, many questions concerning the virulence of this microbe remain.
  • Streptococcal factors associated with pathogenicity e.g., capsule polysaccharides, peptidoglycans, pneumolysins, PspA Complement factor H binding component, autolysin, neuraminidase, peptide permeases, hydrogen peroxide, IgAl protease, the list is certainly not complete. Further very little is known concerning the temporal expression of such genes during infection and disease progression in a mammalian host. Discovering the sets of genes the bacterium is likely to be expressing at the different stages of infection, particularly when an infection is established, provides critical information for the screening and characterization of novel antibacterials which can interrupt pathogenesis. In addition to providing a fuller understanding of known proteins, such an approach will identify previously unrecognised targets. Brief Description of the Invention;
  • This invention provides novel proteins, particularly those from Streptococcus pneumoniae, strain 0100993, characterised in that it comprises the amino acid sequences given herein or fragments, analogues or derivatives thereof.
  • polynucleotides (DNA or RNA) which encode such polypeptides.
  • the invention provides polynucleotides having the DNA sequences given herein.
  • the invention also relates to novel oligonucleotides derived from the sequences given herein which can act as PCR primers in the process herein described to determine whether or not the Streptococcus pneumoniae gene identified herein in whole or in part is expressed in infected tissue. It is recognised that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained.
  • the proteins so identified are also useful as targets in screens designed to identify antimicrobial compounds. It is an object of the invention to provide polypeptides that have been identified as novel polypeptides by homology between the amino acid sequence set out in the Sequence Listing and a known amino acid sequence or sequences of other proteins such as the proteins identified under the heading Identity in Table 1.
  • the polynucleotide comprises a region encoding polypeptides comprising the sequence set out in the Sequence Listing, or a variant thereof.
  • a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization.
  • particularly preferred embodiments of the invention are naturally occurring allelic variants the polynucleotides set forth in the Sequence Listing and polypeptides encoded thereby.
  • inhibitors to such polypeptides useful as antibacterial agents, including, for example, antibodies.
  • products, compositions and methods for assessing expression of the sequences the Sequence Listing treating disease, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid, assaying genetic variation, and administering a polypeptide or polynucleotide of the invention to an organism to raise an immunological response against a bacteria, especially a Streptococcus pneumoniae bacteria.
  • polynucleotides that hybridize to polynucleotide sequences of the invention, particularly under stringent conditions.
  • methods for identifying compounds which bind to or otherwise interact with and inhibit or activate an activity of a polypeptide or polynucleotide of the invention comprising: contacting a polypeptide or polynucleotide of the invention with a compound to be screened under conditions to permit binding to or other interaction between the compound and the polypeptide or polynucleotide to assess the binding to or other interaction with the compound, such binding or interaction being associated with a second component capable of providing a detectable signal in response to the binding or interaction of the polypeptide or polynucleotide with the compound; and determining whether the compound binds to or otherwise interacts with and activates or inhibits an activity of the polypetide or polynucleotide by detecting the presence or absence of a signal generated from the binding or interaction ofthe compound with the polypeptide or polynucleotide.
  • agonists and antagonists of the polynucleotide and/or polypeptides of the invention preferably bacteriostatic or bacteriocidal agonists and antagonists.
  • compositions comprising a polynucleotide or a polypeptide of the invention for administration to a cell or to a multicellular organism.
  • each of the DNA sequences provided herein may be used in the discovery and development of antibacterial compounds. Because each of the sequences contains an open reading frame (ORF) with appropriate initiation and termination codons, the encoded protein upon expression can be used as a target for the screening of antimicrobial drugs. Additionally, the DNA sequences encoding preferably the amino terminal regions of the encoded protein or the Shine-Delgarno region can be used to construct antisense sequences to control the expression of the coding sequence of interest. Furthermore, many of the sequences disclosed herein also provide regions upstream and downstream from the encoding sequence. These sequences are useful as a source of regulatory elements for the control of bacterial gene expression.
  • ORF open reading frame
  • Such sequences are conveniently isolated by restriction enzyme action or synthesized chemically and introduced, for example, into promoter identification strains. These strains contain a reporter structural gene sequence located downstream from a restriction site such that if an active promoter is inserted, the reporter gene will be expressed.
  • this invention also provides several means for identifying particularly useful target genes.
  • the first of these approaches entails searching appropriate databases for sequence matches in related organisms.
  • the Streptococcal-like form of this gene would likely play an analogous role.
  • a Streptococcal protein identified as homologous to a cell surface protein in another organism would be useful as a vaccine candidate.
  • homologies have been identified for the sequences disclosed herein they are reported along with the encoding sequence.
  • the basis of the technique is the random mutagenesis of target organism by various means (e.g., transposons) such that unique DNA sequence tags are inserted in close proximity to the site of mutation.
  • the tags from a mixed population of bacterial mutants and bacteria recovered from an infected hosts are detected by amplification, radiolabeling and hybridisation analysis. Mutants attenuated in virulence are revealed by absence of the tag from the pool of bacteria recovered from infected hosts.
  • Streptococcus pneumoniae because the transposon system is less well developed, a more efficient way of creating the tagged mutants is to use the insertion - duplication mutagenesis technique as described by Morrison et al.. J. Bacteriol. 159:870 (1984) the contents of which is incorporated by reference for background purposes.
  • VERT Vivo Expression Technology
  • random chromosomal fragments of target organism are cloned upstream of a promoter-less recombinase gene in a plasmid vector.
  • This construct is introduced into the target organism which carries an antibiotic resistance gene flanked by. resolvase sites. Growth in the presence of the antibiotic removes from the population those fragments cloned into the plasmid vector capable of supporting transcription of the recombinase gene and therefore have caused loss of antibiotic resistance.
  • the resistant pool is introduced into a host and at various times after infection bacteria may be recovered and assessed for the presence of antibiotic resistance.
  • the chromosomal fragment carried by each antibiotic sensitive bacterium should carry a promoter or portion of a gene normally upregulated during infection. Sequencing upstream of the recombinase gene allows identification of the up regulated gene. 3) Differential display
  • Random insertion of these transposons into target organisms and subsequent isolation of insertion mutants in the presence of inducer of promoter activity ensures that insertions which separate promoter from coding region of a gene whose expression is essential for cell viability will be recovered. Subsequent replica plating in the absence of inducer identifies such insertions, since they fail to survive. Sequencing of the flanking regions of the transposon allows identification of site of insertion and identification of the gene disrupted. Close monitoring of the changes in cellular processes/mo ⁇ hology during growth in the absence of inducer yields information on likely function of the gene.
  • Such monitoring could include flow cytometry (cell division, lysis, redox potential, DNA replication), inco ⁇ oration of radiochemically labeled precursors into DNA, RNA, protein, lipid, peptidoglycan, monitoring reporter enzyme gene fusions which respond to known cellular stresses. 5) Generation of conditional lethal mutants by chemical mutagenesis.
  • RT-PCR Streptococcus pneumoniae messenger RNA is isolated from bacterial infected tissue e.g. 48 hour murine lung infections, and the amount of each mRNA species assessed by reverse transcription of the RNA sample primed with random hexanucleotides followed by PCR with gene specific primer pairs. The determination of the presence and amount of a particular mRNA species by quantification of the resultant PCR product provides information on the bacterial genes which are transcribed in the infected tissue. Analysis of gene transcription can be carried out at different times of infection to gain a detailed knowledge of gene regulation in bacterial pathogenesis allowing for a clearer understanding of which gene products represent targets for screens for novel antibacterials.
  • the bacterial mRNA preparation need not be free of mammalian RNA. This allows the investigator to carry out a simple and quick RNA preparation from infected tissue to obtain bacterial mRNA species which are very short lived in the bacterium (in the order of 2 minute halflives).
  • the bacterial mRNA is prepared from infected murine lung tissue by mechanical disruption in the presence of TRIzole (GIBCO-BRL) for very short periods of time, subsequent processing according to the manufacturers of TRIzole reagent and DNAase treatment to remove contaminating DNA.
  • the process is optimised by finding those conditions which give a maximum amount of S.pneumoniae 16S ribosomal RNA as detected by probing Northerns with a suitably labelled sequence specific oligonucleotide probe.
  • a 5' dye labelled primer is used in each PCR primer pair in a PCR reaction which is terminated optimally between 8 and 25 cycles.
  • the PCR products are separated on 6% polyacrylamide gels with detection and quantification using GeneScanner (manufactured by ABI).
  • nucleotide sequences disclosed herein can be obtained by synthetic chemical techniques known in the art or can be obtained from Streptococcus pneumoniae, strain 0100993by probing a DNA preparation with probes constructed from the particular sequences disclosed herein.
  • oligonucleotides derived from a disclosed sequence can act as PCR primers in a process of PCR-based cloning of the sequence from a bacterial genomic source. It is recognised that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained.
  • a library of clones of chromosomal DNA of Streptococcus pneumoniae, strain 0100993 in E. coli or some other suitable host is probed with a radiolabelled oligonucleotide, preferably a 17mer or longer, derived from the partial sequence.
  • Clones carrying DNA identical to that of the probe can then be distinguished using high stringency washes.
  • sequencing primers designed from the original sequence it is then possible to extend the sequence in both directions to determine the full gene sequence. Conveniently such sequencing is performed using denatured double stranded DNA prepared from a plasmid clone.
  • a polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
  • the DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand.
  • the coding sequence which encodes the polypeptide may be identical to the coding sequence shown or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encoding the same polypeptide.
  • the present invention includes variants of the hereinabove described polynucleotides which encode fragments, analogues and derivatives of the polypeptide characterized by the deduced amino acid sequence given herein.
  • the variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non- naturally occurring variant of the polynucleotide.
  • N is also used.
  • N means that any of the four DNA or RNA bases may appear at such a designated position in the DNA or RNA sequence, except that N cannot be a base 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.
  • the present invention includes polynucleotides encoding the same polypeptide characterized by the deduced amino acid sequence given herein as well as variants of such polynucleotides which variants encode for a fragment, derivative or analogue of the polypeptide.
  • Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
  • the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence characterized by the DNA sequence disclosed herein.
  • an allelic variant is an altemate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.
  • the polynucleotide which encodes for the mature polypeptide may include only the coding sequence for the mature polypeptide or the coding sequence for the mature polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein sequence.
  • polynucleotide encoding a polypeptide encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
  • the present invention therefore includes polynucleotides, wherein the coding sequence for the mature polypeptide may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell.
  • the polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide.
  • the polynucleotides may also encode for a proprotein which is the mature protein plus additional 5' amino acid residues.
  • a mature protein having a prosequence is a proprotein and may be an inactive form of the protein. Once the prosequence is cleaved an active mature protein remains.
  • the polynucleotide of the present invention may code for a mature protein, or for a protein having a prosequence or for a protein having both a prosequence and a presequence (leader sequence).
  • leader sequence the amino acid sequences provided herein show a methionine residue at the NH 2 -terminus. It is appreciated, however, that during post-translational modification of the peptide, this residue may be deleted. Accordingly, this invention contemplates the use of both the methionine-containing and the methionineless amino terminal variants of each protein disclosed herein.
  • the polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence at either the 5' or 3' terminus of the gene which allows for purification of the polypeptide of the present invention.
  • the marker sequence may be a hexa-histidine tag supplied by the pQE series of vectors (supplied commercially by Quiagen Inc.) to provide for purification of the polypeptide fused to the marker in the case of a bacterial host.
  • the present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 50% and preferably at least 70% identity between the sequences.
  • the present invention particularly relates to polynucleotides, particularly Streptococcal polynucleotides, which hybridize under stringent conditions to the hereinabove-described polynucleotides .
  • stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • the polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which retain substantially the same biological function or activity as the polypeptide characterised by the deduced amino acid sequence given herein.
  • An 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 denatured, sheared salmon sperm DNA, followed by washing the hybridization support in 0.1 x 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.
  • the invention also provides a polynucleotide consisting essentially of a polynucleotide sequence obtainable by screening an appropriate library containing the complete gene for a polynucleotide sequence selected from the group consisting of the polynucleotides of the Sequence Listing under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence set forth in said polynucleotide of the Sequence Listing or a fragment thereof; and isolating said DNA sequence.
  • Fragments useful for obtaining such a polynucleotide include, for example, probes and primers described elsewhere herein.
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, 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.
  • Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al., J. Molec. Biol.
  • BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et ai, J. Mol. Biol. 215: 403-410 (1990).
  • a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence selected from the group consisting of the polynucleotide of the Sequence Listing is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • These mutations of the reference sequence may occur at the 5 or 3 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • polypeptide having an amino acid sequence having at least, for example, 95% identity to a reference amino acid sequence of selected from the group consisting of the amino acids of the Sequence Listing is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid.
  • the polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • fragment when referring to the polypeptide characterized by the deduced amino acid sequence herein, means a polypeptide which retains essentially the same biological function or activity as such polypeptide.
  • an analogue includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
  • the fragment, derivative or analogue of the polypeptide characterized by the deduced amino acid sequence herein may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the polypeptid
  • polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • the present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • vectors which include polynucleotides of the present invention
  • host cells which are genetically engineered with vectors of the invention
  • production of polypeptides of the invention by recombinant techniques.
  • the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
  • Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector.
  • the vector may be, for example, in the form of a plasmid, a cosmid, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • Suitable expression vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA.
  • any other vector may be used as long as it is replicable and viable in the host.
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures.
  • the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art.
  • the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
  • promoter for example, LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda Pj ⁇ promoter and other promoters known to control expression of genes in eukaryotic or prokaryotic cells or their viruses.
  • the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • the gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired protein is transcribed into RNA in the host cell transformed by a vector containing this expression construction.
  • the coding sequence may or may not contain a signal peptide or leader sequence.
  • the polypeptides of the present invention can be expressed using, for example, the E. coli tae promoter or the protein A gene (spa) promoter and signal sequence. Leader sequences can be removed by the bacterial host in post-translational processing. See, e.g., U.S. Patent Nos.
  • Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • CAT chloramphenicol transferase
  • Two appropriate vectors are PKK232-8 and PCM7.
  • Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL and t ⁇ .
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • regulatory sequences which allow for regulation of the expression of the protein sequences relative to the growth of the host cell.
  • Regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
  • An expression vector is constructed so that the particular coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the control sequences being such that the coding sequence is transcribed under the "control" of the control sequences (i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence).
  • control i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence.
  • Modification of the coding sequences may be desirable to achieve this end. For example, in some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the reading frame.
  • control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above.
  • a vector such as the cloning vectors described above.
  • the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.
  • recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly- expressed gene to direct transcription of a downstream structural sequence.
  • the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.
  • the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • the vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein. More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example.
  • Bacterial pET-3 vectors (Stratagene), pQE70, pQE60, pQE-9 (Qiagen), pbs, pDIO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).
  • Eukaryotic pBlueBacIII (Invitrogen), pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).
  • any other plasmid or vector may be used as long as they are replicable and viable in the host.
  • Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage 1 (£. coli), pBR322 (E. coli), pACYC177 (E.
  • polypeptides can be expressed in host cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
  • Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al.. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby inco ⁇ orated by reference.
  • the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • appropriate means e.g., temperature shift or chemical induction
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well known to those skilled in the art.
  • the polypeptide of the present invention may be produced by growing host cells transformed by an expression vector described above under conditions whereby the polypeptide of interest is expressed. The polypeptide is then isolated from the host cells and purified. If the expression system secretes the polypeptide into growth media, the polypeptide can be purified directly from the media. If the polypeptide is not secreted, it is isolated from cell lysates or recovered from the cell membrane fraction. Where the polypeptide is localized to the cell surface, whole cells or isolated membranes can be used as an assayable source of the desired gene product. Polypeptide expressed in bacterial hosts such as E. coli may require isolation from inclusion bodies and refolding.
  • the mature protein has a very hydrophobic region which leads to an insoluble product of overexpression
  • the selection of the appropriate growth conditions and recovery methods are within the skill of the art.
  • the polypeptide can be recovered and purified from recombinant cell cultures by methods including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • polypeptides of the present invention may be glycosylated or may be non-glycosylated.
  • Polypeptides of the invention may also include an initial methionine amino acid residue.
  • a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
  • a “vector” is a replicon, such as a plasmid, phage, or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a “double-stranded DNA molecule” refers to the polymeric form of deoxyribonucleotides (bases adenine, guanine, thymine, or cytosine) in a double-stranded helix, both relaxed and supercoiled. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms.
  • this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having the sequence homologous to the mRNA).
  • a DNA "coding sequence of or a "nucleotide sequence encoding" a particular protein is a DNA sequence which is transcribed and translated into a polypeptide when placed under the control of appropriate regulatory sequences.
  • a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bound at the 3' terminus by a translation start codon (e.g., ATG) of a coding sequence and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA” boxes and “CAT” boxes.
  • Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.
  • DNA "control sequences” refers collectively to promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, which collectively provide for the expression (i.e., the transcription and translation) of a coding sequence in a host cell.
  • a control sequence "directs the expression" of a coding sequence in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.
  • a “host cell” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous DNA sequence.
  • a cell has been "transformed” by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane.
  • Exogenous DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the exogenous DNA may be maintained on an episomal element, such as a plasmid.
  • a stably transformed or transfected cell is one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • a "heterologous" region of a DNA construct is an identifiable segment of DNA within or attached to another DNA molecule that is not found in association with the other molecule in nature.
  • This invention is also related to the use of the polynucleotides of the invention for use as diagnostic reagents. Detection of a polynucleotide or polypeptide of the invention in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of a disease. Eukaryotes (herein also "individual(s)"), particularly mammals, and especially humans, infected with an organism comprising a polynucleotide of the invention may be detected at the nucleic acid level by a variety of techniques.
  • Nucleic acids for diagnosis may be obtained from an infected individual's cells and tissues, such as bone, blood, muscle, cartilage, and skin. Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification technique prior to analysis. RNA or cDNA may also be used in the same ways. Using amplification, characterization of the species and strain of prokaryote present in an individual, may be made by an analysis ofthe genotype ofthe prokaryote gene. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the genotype of a reference sequence.
  • Point mutations can be identified by hybridizing amplified DNA to labeled polynucleotide sequences of the invention. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures. DNA sequence differences may also be detected by alterations in the electrophoretic mobility of the DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing. See, e.g., Myers et al., Science, 230: 1242 (1985). Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and SI protection or a chemical cleavage method. See, e.g., Cotton et al., Proc. Natl. Acad. Sci, USA, 85: 4397-4401
  • RNA or cDNA may also be used for the same purpose, PCR or RT-PCR.
  • PCR primers complementary to a nucleic acid encoding a polypeptide of the invention can be used to identify and analyze mutations. These primers may be used for, among othe4r things, amplifying DNA isolated from a sample derived from an individual.
  • the primers may be used to amplify the gene isolated from an infected individual such that the gene may then be subject to various techniques for elucidation of the DNA sequence. In this way, mutations in the DNA sequence may be detected and used to diagnose infection and to serotype and/or classify the infectious agent.
  • the invention further provides a process for diagnosing, disease, preferably bacterial infections, more preferably infections by Streptococcus pneumoniae, comprising determining from a sample derived from an individual a increased level of expression of polynucleotide having a sequence set forth in the Sequence Listing or a sequence of the invention.
  • Increased or decreased expression of a polynucleotide of the invention can be measured using any on of the methods well known in the art for the quantation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
  • a diagnostic assay in accordance with the invention for detecting over ⁇ expression of a polypeptide of the invention compared to normal control tissue samples may be used to detect the presence of an infection, for example.
  • Assay techniques that can be used to determine levels of a polypeptide of the invention, in a sample derived from a host are well- known to those of skill in the art. Such assay methods include radioimmunoassays, competitive- binding assays, Western Blot analysis and ELISA assays.
  • a polypeptide of the invention for therapeutic or prophylactic pu ⁇ oses, for example, as an antibacterial agent or a vaccine.
  • a polynucleotide of the invention for therapeutic or prophylactic pu ⁇ oses, in particular genetic immunisation.
  • inhibitors to such polypeptides useful as antibacterial agents.
  • antibodies against such polypeptides are provided.
  • Polypeptides 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 ai, Current Protocols in Immunology
  • the invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of polypeptides or polynucleotides of the invention, particularly those compounds that are bacteriostatic and/or bacteriocidal.
  • the method of screening may involve high-throughput techniques. For example, to screen for agonists or antagoists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, comprising a polypeptide of the invention and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be an agonist or antagonist of a polypeptide of the invention.
  • the ability of the candidate molecule to agonize or antagonize a polypeptide of the invention 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 a polypeptide of the invention are most likely to be good antagonists.
  • Molecules that bind well and increase the rate of product production from substrate are agonists. Detection ofthe rate or level of production of product from substrate 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 polynucleotide or polypeptide activity, and binding assays known in the art.
  • an assay for antagonists of a polypeptide of the invention is a competitive assay that combines such polypeptide and a potential antagonist with polypeptide- binding molecules, recombinant polypeptide-binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay.
  • Polypeptides of the invention can be labeled, such as by radioactivity or a colorimetric compound, such that the number of such polypeptide molecules bound to a binding molecule or converted to product can be deterrnined accurately to assess the effectiveness of the potential antagonist.
  • Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide or polypeptide of the invention and thereby inhibit or extinguish its activity. 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, without inducing an activities of a polypeptide of the invention, thereby preventing the action of such polypeptide by excluding it 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); OUGODEOXYNUCLEOT1DES 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 the poiypetides of the invention.
  • Each of the DNA 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.
  • the DNA sequences encoding the amino terminal regions of the encoded protein or Shine-Delgamo or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest.
  • the invention also provides the use of the polypeptide, polynucleotide or inhibitor of the invention to interfere with the initial physical interaction between a pathogen and 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 bacteria, to mammalian extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds; to block protein-mediated mammalian cell invasion by, for example, initiating phosphorylation of mammalian tyrosine kinases (Rosenshine et al.. Infect. Immun.
  • the antagonists and agonists of the invention may be employed, for instance, to inhibit and treat otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid.
  • Another aspect of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising the above polypeptide, polynucleotide or inhibitor of the invention and a pharmaceutically acceptable carrier.
  • the invention provides the use of an inhibitor of the invention as an antibacterial agent.
  • the invention further relates to the manufacture of a medicament for such uses.
  • the polypeptide may be used as an antigen for vaccination of a host to produce specific antibodies which have anti-bacterial action.
  • This invention also contemplates the use of the DNA encoding the antigen as a component in a DNA vaccine as discussed more fully below.
  • the polypeptides or cells expressing them can be used as an immunogen to produce antibodies thereto.
  • These antibodies can be, for example, polyclonal or monoclonal antibodies.
  • the term antibodies also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
  • Antibodies generated against the polypeptides of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
  • Polypeptide derivatives include antigenically or immunologically equivalent derivatives which form a particular aspect of this invention.
  • 'antigenically equivalent derivative' encompasses a polypeptide or its equivalent which will be specifically recognised by certain antibodies which, when raised to the protein or polypeptide according to the present invention, interfere with the interaction between pathogen and mammalian host.
  • 'immunologically equivalent derivative' as used herein encompasses a peptide or its equivalent which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the interaction between pathogen and mammalian host.
  • derivatives which are slightly longer or slightly shorter than the native protein or polypeptide fragment of the present invention may be used.
  • polypeptides in which one or more of the amino acid residues are modified may be used.
  • Such peptides may, for example, be prepared by substitution, addition, or rearrangement of amino acids or by chemical modification thereof. All such substitutions and modifications are generally well known to those skilled in the art of peptide chemistry.
  • the polypeptide such as an antigenically or immunologically equivalent derivative or a fusion protein thereof is used as an antigen to immunize a mouse or other animal such as a rat or chicken.
  • the fusion protein may provide stability to the polypeptide.
  • the antigen may be associated, for example by conjugation , with an immunogenic carrier protein for example bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).
  • BSA bovine serum albumin
  • KLH keyhole limpet haemocyanin
  • a multiple antigenic peptide comprising multiple copies of the protein or polypeptide, or an antigenically or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve immunogenicity so as to obviate the use of a carrier.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, Nature. 256:495-497(1975)), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 4:72(1983)), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • antibody-containing cells from the immunised mammal are fused with myeloma cells to create hybridoma cells secreting monoclonal antibodies.
  • the hybridomas are screened to select a cell line with high binding affinity and favorable cross reaction with other Streptococcal species using one or more of the original polypeptide and/or the fusion protein.
  • the selected cell line is cultured to obtain the desired Mab.
  • Hybridoma cell lines secreting the monoclonal antibody are another aspect of this invention.
  • phage display technology could be utilised to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR amplified v- genes of lymphocytes from humans screened for possessing anti-Fbp or from naive libraries (McCafferty, J. et al, Nature 348:552-554(1990), and Marks, J. et al, Biotechnology 10:779-783(1992)).
  • the affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al, Nature 352:624-628(1991)).
  • the antibody should be screened again for high affinity to the polypeptide and/or fusion protein.
  • a fragment of the final antibody may be prepared.
  • the antibody may be either intact antibody of M r approx 150,000 or a derivative of it, for example a Fab fragment or a Fv fragment as described in Skerra, A and Pluckthun, A., Science 240: 1038-1040 (1988). If two antigen binding domains are present each domain may be directed against a different epitope - termed 'bispecific' antibodies.
  • the antibody of the invention may be prepared by conventional means for example by established monoclonal antibody technology (Kohler, G. and Milstein, C. (supra. (1975) or using recombinant means e.g. combinatorial libraries, for example as described in Huse, W.D. _-/ ⁇ /., S_cie_Q£e 246:1275-1281 (1989).
  • the antibody is prepared by expression of a DNA polymer encoding said antibody in an appropriate expression system such as described above for the expression of polypeptides of the invention.
  • the choice of vector for the expression system will be determined in part by the host, which may be a prokaryotic cell, such as E. coli (preferably strain B) or Streptomyces sp. or a eukaryotic cell, such as a mouse C127, mouse myeloma, human HeLa, Chinese hamster ovary, filamentous or unicellular fungi or insect cell.
  • the host may also be a transgenic animal or a transgenic plant (for example, as described in Hiatt, A. et al, Nature 340:76-78(1989).
  • Suitable vectors include plasmids, bacteriophages, cosmids and recombinant viruses, derived from, for example, baculoviruses and vaccinia.
  • the Fab fragment may also be prepared from its parent monoclonal antibody by enzyme treatment, for example using papain to cleave the Fab portion from the Fc portion.
  • the antibody or derivative thereof is modified to make it less immunogenic in the patient.
  • the antibody may most preferably be 'humanised'; where the complimentarity determining region(s) of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody , for example as described in Jones, P. et al, Nature 321 :522-525 (1986), or Tempest et al., Biotechnology 9:266-273 (1991).
  • the modification need not be restricted to one of 'humanisation' ; other primate sequences (for example Newman, R. et al. Biotechnology 10:1455-1460 (1992)) may also be used.
  • the humanised monoclonal antibody, or its fragment having binding activity, form a particular aspect of this invention.
  • This invention provides a method of screening drugs to identify those which interfere with the proteins selected as targets herein, which method comprises measuring the interference of the activity of the protein by a test drug. For example if the protein selected has a catalytic activity, after suitable purification and formulation the activity of the enzyme can be followed by its ability to convert its natural substrates. By inco ⁇ orating different chemically synthesised test compounds or natural products into such an assay of enzymatic activity one is able to detect those additives which compete with the natural substrate or otherwise inhibit enzymatic activity.
  • the invention also relates to inhibitors identified thereby.
  • a polynucleotide of the invention in genetic immunisation will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al., Hum. Mol. Genet. 1:363 (1992); Mantho ⁇ e et al.. Hum. Gene Ther. 4:419 (1963)), delivery of DNA complexed with specific protein carriers ( Wu et al, _ Biol. Chem. 264: 16985 (1989)), coprecipitation of DNA with calcium phosphate (Benvenisty & Reshef, Proc. Nat'l Acad. Sci. USA.
  • Suitable promoters for muscle transfection include CMV, RSV, SRa, actin, MCK, alpha globin, adenovirus and dihydrofolate reductase.
  • the active agent i.e., the polypeptide, polynucleotide or inhibitor of the invention
  • the active agent may be administered to a patient as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.
  • the composition may be formulated for topical application for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams.
  • Such topical formulations may also contain compatible conventional carriers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions.
  • Such carriers may constitute from about 1 % to about 98% by weight of the formulation; more usually they will constitute up to about 80% by weight of the formulation.
  • the daily dosage level of the active agent will be from 0.01 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 patient and will vary with the age, weight and response of the particular patient.
  • 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.
  • 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 ⁇ g/kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks. Within the indicated dosage range, no adverse toxicologicals effects are expected with the compounds of the invention which would preclude their administration to suitable patients.
  • Plasmids are designated by a lower case p preceded and/or followed by capital letters and/or numbers.
  • the starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures.
  • equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
  • Digestion of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA.
  • restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan.
  • analytical pu ⁇ oses typically 1 ⁇ g of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
  • pu ⁇ ose of isolating DNA fragments for plasmid construction typically 5 to 50 ⁇ g of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37° C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
  • Oligonucleotides refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
  • Ligase refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., supra.. p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units to T4 DNA ligase ("ligase”) per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragments to be ligated.
  • ligase T4 DNA ligase
  • each of the DNAs disclosed herein by virtue of the fact that it includes an intact open reading frame is useful to a greater or lesser extent as a screen for identifying antimicrobial compounds.
  • a useful approach for selecting the preferred DNA sequences for screen development is evaluation by insertion-duplication mutagenesis. This system disclosed by Morrison et al., J. Bacteriol. 159:870 (1984), is applied as follows.
  • random fragments of Streptococcus pneumoniae, strain 0100993 DNA are generated enzymatically (by restriction endonuclease digestion) or physically (by sonication based shearing) followed by gel fractionation and end repair employing T4 DNA polymerase. It is preferred that the DNA fragments so produced are in the range of 200-400 base pairs, a size sufficient to ensure homologous recombination and to insure a representative library in E.coli.
  • the fragments are then inserted into appropriately tagged plasmids as described in Hensel el al-, Science 269: 400-403(1995).
  • plasmids can be used for this pu ⁇ ose
  • a particularly useful plasmid is pJDC9 described by Pearce et al., Mol. Microbiol. 9:1037 (1993) which carries the erm gene facilitating erythromycin selection in either E. coli or S. pneumoniae previously modified by inco ⁇ oration of DNA sequence tags into one of the polylinker cloning sites.
  • the tagged plasmids are introduced into the appropriate 5.
  • pneumoniae strain selected, inter alia, on the basis of serotype and virulence in a murine model of pneumococcal pneumonia. It is appreciated that a seventeen amino acid competence factor exists (Havastein et al., Proc. Nat'l.
  • mice eg., Swiss and/or C57B1/6.
  • Preliminary experiments are conducted to establish the optimum complexity of the pools and level of inoculum.
  • a particularly useful model has been described by Veber et al. (J. Antimicrobiol. Chemother.32:432 (1993) in which 10 ⁇ cfu inocula sizes are introduced by mouth to the trachea. Strain differences are observed with respect to onset of disease e.g. ,3-4 days for Swiss mice and 8-10 days for C57B1/6. Infection yields in the lungs approach 10° cfu/lung. IP administration is also possible when genes mediating blood stream infection are evaluated.
  • the mutant bank normally comprising several thousand strains is subjected to the virulence test.
  • Mutants with attenuated virulence are identified by hybridization analysis using the labelled tags from the "input” and “recovered” pools as probes as described in Hensel el al, Science 269: 400-403(1995).
  • S. pneumoniae DNA is colony blotted or dot blotted, DNA flanking the integrated plasmid is cloned by plasmid rescue in E. coli (Morrison et al., J. Bacteriol. 159:870 (1984)) and sequenced.
  • the DNA is compared to the nucleotide sequences given herein and the appropriate ORF is identified and function confirmed for example by knock-out studies.
  • Expression vectors providing the selected protein are prepared and the protein is configured in an appropriate screen for the identification of anti-microbial agents.
  • genomic DNA libraries are probed with restriction fragments flanking the integrated plasmid to isolate full-length cloned virulence genes whose function can be confirmed by "knock-out" studies or other methods, which are then expressed and inco ⁇ orated into a screen as described above.
  • Table 2 where the polynucleotide of SEQ ID NO: 2 encodes the ORFs of SEQ ID NOS: 225 and 226.
  • Table 1 also shows the start ("START” column) and stop (“STOP” column) codons for each ORF and their positions in the encoding polynucleotide sequence.
  • the SEQ ID NOS of the polypeptides of this table are linked to both a deduced identity in this table and a polynucleotide sequence in Table 2 which encodes each polypeptide.
  • the "Direction” column in Table 1 shows the direction of the ORF encoding each poypetide in this table. "Forward” denotes the sense orientation and “Reverse” denotes the antisense orientation of the ORF.
  • 2-Isopropylmalate Synthase (EC 4.1.3.12) 1 -CAT TTA- 31 231 Reverse (Alpha-Isopropylmalate Synthase) (Alph A- IPM Synthetase).
  • Lactococcus Lactis (Subsp. Lactis) (Streptococcus Lactis).
  • PSEGLI NCBI gi 499660 - Pseudomonase 1 -CAT TTA- 50 481 Reverse J putida.
  • Transport ATP-Binding Protein Coma - 1 -CAT TTA- 112 552 Reverse Streptococcus Pneumoniae.
  • Orotate Phosphoribosyltransferase (EC •CAT CTA- 21 311 Reverse 2.4.2.10) (OPRT). - Bacillus Subtilis.
  • prephenate dehydrogenase (EC 1.3.1.12) - ATG TAG 271 492 Forward lactococcus lactis.
  • O-Syaloglycoprotein Endopeptidase (EC CTG TAA 450 Forward 3.4.24.57) (Glycoprotease).
  • - Pasteurella Haemolvtica (Glycoprotease).
  • Beta-Glucosidase A (EC 1 ATG TAA 184 453 Forward
  • Exodeoxyribonuclease Small Subunit (EC 2 -CAT TCA- 470 682 Reverse 3.1.1 1.6) (Exonuclease VII Small Subunit). - Escherichia Coli.
  • Table 2 shows the correlation between the SEQ ID NO of each DNA sequence of the invention with the SEQ ID NO(S) of polypeptide or polypeptides that its open reading frame(s) encodes.
  • the DNA of SEQ ID NO:l encodes one polypeptide, that of SEQ ID NO:224.
  • the DNA of SEQ ID NO:2 encodes two polypeptides, the polypeptides of SEQ ID NO:225 and SEQ ID NO:226.
  • CAGCACGTTC ATGCGCAAAG GTAATATTAC GGTAAATTGA CTTAGCAAAT GGATTGGGGC 300
  • TCTTCTGTGT CAGCATGAAT GGTCTGTCAA AATCCCACCG CATAGCAGGT TTCCCGTGTG 180
  • GCCTCAGCTT CAATATTTCT TACTTTCCAA TAGACTCCCT GCGAAACAAA ATATGGTATA 540
  • GCTGGCATTA TCAATTATGA ACTAGGATTC TAGTTTTGCA GGAAGTCTAT TATTTTCCTT 1380 ATTGTCTGTA AGTCTACTGA CCTTGTTGTT TATCCCAGTC ATGGTTTCTA GTTCGGGCTC 1440
  • TCTCATCTCT TTTATTCTCC TGAAAATTCA CGCGCAGTCT GGGCAACTAC TTCTGTTTCT 960
  • AAAAGCTAAA CCACCCTGAT TTCGATTTTG TCTTCTTTGT ATCTTCGTTC TCCCTACTTT 420
  • GATTAGTTAC CATGATTGTT CCTGCCCCTA GGAAACCCAA
  • TTATAGTTCC AACCTCTTTT CAGTTATTAT TTCCAGTTTA ACATAGCATT CAAGCCATAG 600
  • AACTTTGTCA TTAACTTTGA TAAATGGAAT GTATAGAAGA ACTCCAAGTG CAAAGATGAT 480 GAATTGAACT AGAACTGCTC TCACGTCCCC TGCTGTTGCT AACCATGCAT TTAAGAATAC 540
  • CAAGACGAAT ACCGTAACAC CATCTTGAAG AATGCTGCTC TTGCTCGTCG TTTCAACGAA 840
  • GGTCATGGTC GTGGACTCTG CCAAGGGGAT CGAGGCTCAA ACAAAGAAAT TGTTTGAGGT 420
  • GCATGTTTCC TTAGATGTTN GCATTAAACT CNGATAAGTC TATATGATTG CCTTTTTTCT 1140

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Abstract

Cette invention a trait à des polynucléotides récemment identifiés, à des polypeptides codés par ces polynucléotides, aux utilisations qui sont faites de ces polynucléotides et de ces polypeptides ainsi qu'à leur production et à celle de cellules hôtes de recombinaison transformées par ces polynucléotides. Elle concerne également l'inhibition de la biosynthèse de ces polynucléotides ou polypeptides ou de leur activité ainsi que l'application thérapeutique qui est faite de ces agents d'inhibition.
PCT/US1997/005306 1996-04-02 1997-04-01 Nouveaux composes WO1997037026A1 (fr)

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JP09535535A JP2000511769A (ja) 1996-04-02 1997-04-01 新規化合物
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EP0881292A2 (fr) * 1997-05-29 1998-12-02 Smithkline Beecham Corporation Facteur de dégagement de peptide de Streptococcus pneumoniae
EP0885963A2 (fr) * 1997-06-20 1998-12-23 Smithkline Beecham Corporation Composés contenant des polypeptides et polynucléotides de Streptococcus pneumoniae, ces polypeptides sont apparenté par leur séquence d'acides aminés avec le polypeptide SapR de Streptococcus mutans
EP0889132A2 (fr) * 1997-07-01 1999-01-07 Smithkline Beecham Corporation Streptococcus pneumoniae gidA2 polynucléotides et polypeptides
EP0892059A2 (fr) * 1997-06-20 1999-01-20 Smithkline Beecham Corporation Polypeptides de la kinase de l'histidine
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EP0904105A1 (fr) * 1996-04-18 1999-03-31 Smithkline Beecham Corporation Nouveaux composes
EP0913480A2 (fr) * 1997-11-03 1999-05-06 Smithkline Beecham Corporation Chorismate synthase
EP0913477A2 (fr) * 1997-10-30 1999-05-06 Smithkline Beecham Corporation Gène lacC codant pour une tagatose-6-phosphate kinase
WO2000006738A2 (fr) * 1998-07-27 2000-02-10 Microbial Technics Limited Acides nucleiques et proteines de streptococcus pneumoniae
US6074858A (en) * 1997-04-18 2000-06-13 Smithkline Beecham Corporation Cysteinyl TRNA synthetase from Streptococcus pneumoniae
EP1075841A1 (fr) * 1999-08-13 2001-02-14 Erasmus Universiteit Rotterdam Vaccins pneumococciques
WO2001053334A1 (fr) * 2000-01-19 2001-07-26 Smithkline Beecham Corporation thdF
US6274719B1 (en) * 1998-04-24 2001-08-14 Smithkline Beecham Corporation Gcp
US6589738B1 (en) 1999-11-09 2003-07-08 Elitra Pharmaceuticals, Inc. Genes essential for microbial proliferation and antisense thereto
US6720139B1 (en) 1999-01-27 2004-04-13 Elitra Pharmaceuticals, Inc. Genes identified as required for proliferation in Escherichia coli
EP1624064A3 (fr) * 1998-07-27 2006-05-10 Microbial Technics Limited Acides nucléiques et protéines de streptococcus pneumoniae
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US7632515B2 (en) 1998-07-27 2009-12-15 Sanofi Pasteur Limited Streptococcus pneumoniae proteins and nucleic acid molecules
WO2010079464A1 (fr) 2009-01-12 2010-07-15 Novartis Ag Antigènes à domaines cna_b dans des vaccins contre des bactéries à gram positif
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EP2277595A2 (fr) 2004-06-24 2011-01-26 Novartis Vaccines and Diagnostics, Inc. Composés pour potentialiser l'immunité
US7893238B2 (en) 1997-07-02 2011-02-22 Sanofi Pasteur Limited Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics
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WO2011105891A1 (fr) 2010-02-23 2011-09-01 Stichting Katholieke Universiteit, More Particularly The Radboud University Nijmegen Medical Centre Vaccin combiné pour streptococcus
EP2368569A2 (fr) 2006-01-18 2011-09-28 University Of Chicago Compositions et procédés liés aux protéines des bactéries staphylocoques
WO2011119759A1 (fr) 2010-03-23 2011-09-29 Irm Llc Composés (lipopeptides à base de cystéine) et compositions en tant qu'agonistes des tlr2 utilisés pour traiter des infections, inflammations, maladies respiratoires entre autres
WO2011149564A1 (fr) 2010-05-28 2011-12-01 Tetris Online, Inc. Infrastructure de jeu informatique asynchrone hybride interactif
EP2537857A2 (fr) 2007-12-21 2012-12-26 Novartis AG Formes mutantes de streptolysine O
EP2583678A2 (fr) 2004-06-24 2013-04-24 Novartis Vaccines and Diagnostics, Inc. Immunopotentiateurs de petites molécules et dosages pour leur détection
EP2659912A2 (fr) 2007-07-17 2013-11-06 Novartis AG Purification de conjugué
US8632784B2 (en) 1998-07-27 2014-01-21 Sanofi Pasteur Limited Nucleic acids and proteins from Streptococcus pneumoniae
WO2014053521A2 (fr) 2012-10-02 2014-04-10 Novartis Ag Conjugués saccharidiques non linéaires
WO2014118305A1 (fr) 2013-02-01 2014-08-07 Novartis Ag Administration intradermique de compositions immunologiques comprenant des agonistes des récepteurs de type toll
WO2015082501A1 (fr) 2013-12-03 2015-06-11 Virometix Ag Peptides riches en proline protecteurs contre s. pneumoniae
US9393294B2 (en) 2011-01-20 2016-07-19 Genocea Biosciences, Inc. Vaccines and compositions against Streptococcus pneumoniae
US10105412B2 (en) 2009-06-29 2018-10-23 Genocea Biosciences, Inc. Vaccines and compositions against Streptococcus pneumoniae
WO2019157509A1 (fr) 2018-02-12 2019-08-15 Inimmune Corporation Ligands du récepteur de type toll
WO2022096590A1 (fr) 2020-11-04 2022-05-12 Eligo Bioscience Particules dérivées de phages pour l'administration in situ de charge utile d'adn dans une population de c. acnes

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EP0849362A3 (fr) * 1996-10-24 1999-10-20 Smithkline Beecham Corporation Protéines de Streptococcus de la famille de spoT/relA (spo/rel)
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EP0889132A2 (fr) * 1997-07-01 1999-01-07 Smithkline Beecham Corporation Streptococcus pneumoniae gidA2 polynucléotides et polypeptides
EP0889132A3 (fr) * 1997-07-01 2002-03-20 Smithkline Beecham Corporation Streptococcus pneumoniae gidA2 polynucléotides et polypeptides
US7893238B2 (en) 1997-07-02 2011-02-22 Sanofi Pasteur Limited Nucleic acid and amino acid sequences relating to Streptococcus pneumoniae for diagnostics and therapeutics
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EP0896061A3 (fr) * 1997-08-08 2000-07-26 Smithkline Beecham Corporation Gène rpoA de Staphylococcus aureus
EP0913477A2 (fr) * 1997-10-30 1999-05-06 Smithkline Beecham Corporation Gène lacC codant pour une tagatose-6-phosphate kinase
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EP2368570A2 (fr) 2006-01-18 2011-09-28 University Of Chicago Compositions et procédés liés aux protéines des bactéries staphylocoques
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US8173657B2 (en) 2006-03-23 2012-05-08 Novartis Ag Imidazoquinoxaline compounds as immunomodulators
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WO2010079464A1 (fr) 2009-01-12 2010-07-15 Novartis Ag Antigènes à domaines cna_b dans des vaccins contre des bactéries à gram positif
WO2010136897A2 (fr) 2009-05-28 2010-12-02 Novartis Ag Expression de protéines recombinées
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