WO1999023212A1 - Novel streptococcal ers - Google Patents

Novel streptococcal ers Download PDF

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Publication number
WO1999023212A1
WO1999023212A1 PCT/US1997/020005 US9720005W WO9923212A1 WO 1999023212 A1 WO1999023212 A1 WO 1999023212A1 US 9720005 W US9720005 W US 9720005W WO 9923212 A1 WO9923212 A1 WO 9923212A1
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Prior art keywords
poiypeptide
polynucleotide
glus
comp
seq
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PCT/US1997/020005
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English (en)
French (fr)
Inventor
Deborah D. Jaworski
Elizabeth J. Lawlor
Min Wang
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Smithkline Beecham Corporation
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Publication date
Application filed by Smithkline Beecham Corporation filed Critical Smithkline Beecham Corporation
Priority to JP52614999A priority Critical patent/JP2001509682A/ja
Priority to PCT/US1997/020005 priority patent/WO1999023212A1/en
Priority to EP97947329A priority patent/EP0964921A4/en
Publication of WO1999023212A1 publication Critical patent/WO1999023212A1/en

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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • 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
    • 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 and polypeptides, and their production and uses, as well as their variants, agonists and antagonists, and their uses.
  • the invention relates to novel polynucleotides and polypeptides of the glutamyl tRNA synthetase family, hereinafter referred to as "gluS”.
  • Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including, 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.
  • Streptococcus pneumoniae 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.
  • Streptococcal genes and gene products as targets for the development of antibiotics.
  • Streptococcus pneumoniae infections has risen dramatically in the past 20 years. This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems. It is no longer uncommon to isolate Streptococcus pneumoniae strains which are resistant to some or all of the standard antibiotics. This has created a demand for both new anti-microbial agents and diagnostic tests for this organism.
  • t-RNA synthetases have a primary role in protein synthesis according to the following scheme:
  • Enzyme +ATP + AA ⁇ t > Enzyme.AA-AMP + PPi Enzyme.AA-AMP + t-RNA Enzyme + AMP + AA-t-RNA in which AA is an amino acid.
  • RNA synthetase have potential as antibacterial agents.
  • One example of such is mupirocin which is a selective inhibitor of isoleucyl t-RNA synthetase.
  • Other t-RNA synthetases are now being examined as possible anti-bactenal targets, this process being greativ assisted bv the isolation of the synthetase
  • the polynucleotide comprises a region encoding gluS polypeptides comp ⁇ sing the sequence set out in Table 1 [SEQ ID NO 1], or a va ⁇ ant thereof
  • gluS protein from Streptococcus pneumoniae comp ⁇ sing the amino acid sequence of Table 1 [SEQ ID NO 2] or a va ⁇ ant thereof
  • an isolated nucleic acid molecule encoding a mature poiypeptide expressible by the Streptococcus pneumoniae 0100993 strain contained in the deposited strain
  • a further aspect of the invention there are provided isolated nucleic acid molecules encoding gluS, particularly Streptococcus pneumoniae gluS, including mRNAs cDNAs, ge ⁇ omic DNAs Further embodiments of the invention include biologicalK. diagnostically, prophylacticalK , chnicallv or therapeuticalK useful . a ⁇ ants thereof, and compositions comp ⁇ sing the same
  • gluS novel polypeptides of Streptococcus pneumoniae referred to herein as gluS as well as biologicallv. diagnostically, prophylactically. clinically or therapeutically useful va ⁇ ants thereof, and compositions comp ⁇ sing the same
  • products, compositions and methods for assessing gluS expression 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 va ⁇ ation, and administe ⁇ ng a gluS poiypeptide or polynucleotide to an organism to raise an immunological response against a bacte ⁇ a, especialK a Streptococcus pneumoniae bacte ⁇ a.
  • polynucleotides that hvb ⁇ dize to gluS polynucleotide sequences, particularly under st ⁇ ngent conditions
  • compositions comp ⁇ sing a gluS polynucleotide or a gluS poiypeptide for administration to a cell or to a multicellular organism Va ⁇ ous changes and modifications within the spi ⁇ t and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following desc ⁇ ptions and from reading the other parts of the present disclosure GLOSSARY
  • “Host cell” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence
  • Identit ⁇ is a relationship between two or more poiypeptide sequences or two or more polynucleotide sequences, as determined b ⁇ compa ⁇ ng the sequences In the art. identity also means the degree of sequence relatedness between poiypeptide or polynucleotide sequences, as the case may be, as determined by the match between st ⁇ ngs of such sequences "Identity' and "simila ⁇ ty” can be readily calculated bv 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 .
  • 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 publicK available computer programs
  • Preferred computer program methods to determine identity and similantv 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 f Atschul. S F et al . J Molec Bwl 215 403-410 ( 1990)
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual. Altschul. S . et al .
  • nucleotide having a nucleotide sequence having at least, for example, 95 "identity' to a reference nucleotide sequence of SEQ ID NO 1 it 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 of SEQ ID NO 1
  • 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
  • a poiypeptide having an amino acid sequence having at least, for example, 95% identity to a reference amino acid sequence of SEQ ID NO 2 is intended that the amino acid sequence of the poiypeptide is identical to the reference sequence except that the poiypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference am o acid of SEQ ID NO 2
  • 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 mav be inserted into the reference sequence
  • PolynucleotidefsV include, without limitation, single- and double-stranded DNA.
  • polynucleotide refers to t ⁇ ple-stranded regions comp ⁇ sing RNA or DNA or both RNA and DNA
  • the strands in such regions may be from the same molecule or from different molecules
  • the regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules
  • One of the molecules of a t ⁇ ple-hehcal region often is an o gonucleotide
  • the term "polynucleotide.s)" also includes DNAs or RNAs as desc ⁇ bed above that contain one or more modified bases
  • DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleot ⁇ de(s)” as that term is intended herein
  • DNAs or RNAs comp ⁇ sing unusual bases such as mosine, or modified
  • Polynucleot ⁇ de(s) also embraces short polynucleotides often referred to as ohgonucleot ⁇ de(s)
  • Polypept ⁇ de(s) refers to any peptide or protein comp ⁇ sing two or more amino acids joined to each other by peptide bonds or modified peptide bonds
  • Polypept ⁇ de(s) refers to both short chains, commonly referred to as peptides.
  • Polypeptides mav contain amino acids other than the 20 gene encoded amino acids "Polypept ⁇ de(s)" include those modified either by natural processes, such _5 processing and other post-translational modifications, but also by chemical modification techniques Such modifications are well desc ⁇ bed in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given poiypeptide Also, a given poiypeptide may contain many types of modifications Modifications can occur anywhere in a poiypeptide.
  • Modifications include, for example, acetylation. acvlation. ADP- ⁇ bosylation. amidation. covalent attachment of flavin, covalent attachment of a heme moietv . covalent attachment of a nucleotide or nucleotide de ⁇ vative. covalent attachment of a lipid or lipid de ⁇ vative. covalent attachment of phosphotidy nositol. cross-linking, cvc zation. disulfide bond formation, demethvlation. tormauon of covalent cross-links, tormauon of cysteine. formation of pyroglutamate.
  • Polypeptides may be branched or cyclic, with or without branching Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well
  • Va ⁇ ant(s) is a polynucleotide or poiypeptide that differs from a reference polynucleotide or poiypeptide respectively, but retains essential properties
  • a typical va ⁇ ant 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 poiypeptide encoded by the reference polynucleotide Nucleotide changes mav result in amino acid substitutions, additions, deletions, fusions and truncations in the poiypeptide encoded bv the reference sequence, as discussed below
  • a typical va ⁇ ant ot a poiypeptide differs in amino acid sequence from another, reference poiypeptide Generally, differences are limited so that the sequences of the reference poiypeptide and the va ⁇ ant are closely similar overall and, in many regions, identical.
  • a va ⁇ ant and reference poiypeptide 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 bv the genetic code
  • a ariant of a polynucleotide or poiypeptide may be a naturally occur ⁇ ng such as an allelic va ⁇ ant.
  • Non-naturally occur ⁇ ng variants of polynucleotides and polypeptides may be made bv mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans DESCRIPTION OF THE INVENTION
  • the invention relates to novel gluS polypeptides and polynucleotides as described in greater detail below.
  • the invention relates to polypeptides and polynucleotides of a novel gluS of Streptococcus pneumoniae. which is related by amino acid sequence homology to Bacillus subtilis glutamyl tRNA synthetase poiypeptide.
  • the invention relates especially to gluS having the nucleotide and amino acid sequences set out in Table 1 [SEQ ID NO: 1 ] and Table 1 [SEQ ID NO: 2] respectively, and to the gluS nucleotide sequences of the DNA in the deposited strain and amino acid sequences encoded thereby.
  • a deposit containing a Streptococcus pneumoniae 0100993 strain has been deposited with the National Collections of Indust ⁇ al and Ma ⁇ ne Bacte ⁇ a Ltd ( herein 'NCIMB"), 23 St Machar D ⁇ ve, Aberdeen AB2 IRY. Scotland on 1 1 Ap ⁇ l 1996 and assigned deposit number 40794
  • the deposit was desc ⁇ bed as Streptococcus pneumoniae 0100993 on deposit On 17 Ap ⁇ l 1996 a Streptococcus pneumoniae 0100993 DNA library in E. coli was similarly depositedwith the NCIMB and assigned deposit number 40800
  • the Streptococcus pneumoniae strain deposit is referred to herein as "the deposited strain” or as "the DNA of the deposited strain "
  • the deposited strain contains the full length gluS gene
  • the sequence of the polynucleotides contained in the deposited strain, as well as the amino acid sequence of the poiypeptide encoded thereby, are controlling in the event of any conflict with any desc ⁇ ption of sequences herein
  • the deposit of the deposited strain has been made under the terms of the Budapest
  • polypeptides of the invention include a poiypeptide of Table 1 [SEQ ID NO 2] (in particular the mamre poiypeptide) as well as poK peptides and fragments, particularly those which have the biological activity of gluS.
  • the invention also includes polypeptides of the formula set forth in Table 1 (D) wherein, at the amino terminus.
  • X is hydrogen, and at the carboxyl terminus.
  • Y is hydrogen or a metal
  • R j and Ro is amino acid residue
  • m is an integer between 1 and 1000 or zero
  • n is an integer between 1 and 1000 or zero
  • Any stretch of amino acid residues denoted by either R group, where n and/or m is an integer greater than 1 may be either a heteropolymer or a homopolymer. preferably a heteropolymer
  • Other preferred embodiments provide polypeptides where n is zero and m is the integer 1, 2. 3, 4, 5, 6, 7, 8, 9, or 10 In such preferred embodiments it is most preferred that the amino terminal amino acid residue is a methionyl residue
  • a fragment is a va ⁇ ant poiypeptide having an amino acid sequence that entirely is the same as part but not all of the amino acid sequence of the aforementioned polypeptides
  • fragments may be free-standing, or comp ⁇ sed within a larger poiypeptide ot which thev form a part or region, most preferably as a single continuous region, a single larger poiypeptide
  • Preferred fragments include, for example, truncation polypeptides having a portion of the amino acid sequence of Table 1 [SEQ ID NO.2], or of va ⁇ ants thereof, such as a continuous se ⁇ es of residues that includes the amino terminus, or a continuous se ⁇ es of residues that includes the carboxyl terminus Degradation forms of the polypeptides of the invention in a host cell, particularly a Streptococcus pneumoniae.
  • aie fragments characte ⁇ zed bv structural or functional att ⁇ butes such as fragments that comp ⁇ se alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and tum- forming regions, coil and coil-forming regions, hvdrophi c regions, hv dropnooic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions surface-forming regions. substrate binding region, and high antigenic index regions
  • biologically active fragments which are those fragments that mediate activities of gluS. including those with a similar activity or an improved activity, or with a decreased undesirable activity
  • fragments that are antigenic or lmmunogenic in an animal especially in a human
  • Particularlv preferred are fragments comp ⁇ sing receptors or domains of enzymes that confer a runction essential for viabilitv of Streptococcus pneumoniae or the ability to initiate, or maintain cause disease in an individual, particularly a human
  • Va ⁇ ants that are fragments of the polypeptides of the invention may be employed for producing the corresponding full-length poiypeptide bv peptide sv nthesis. therefore, these vanants may be employed as intermediates for producing the full-length polypeptides of the invention
  • Another aspect of the invention relates to isolated polynucleotides that encode the gluS poiypeptide having the deduced amino acid sequence of Table 1 [SEQ ED NO.2] and polynucleotides closely related thereto and va ⁇ ants thereof
  • a polynucleotide of the invention encoding gluS poiypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacte ⁇ a using Streptococcus pneumoniae 0100993 cells as starting mate ⁇ al, followed by obtaining a full length clone
  • a polynucleotide sequence of the invention such as the full length gene sequence given in Table 1 [SEQ ID NO 1 ]
  • typically a library of clones of chromosomal DNA of Streptococcus pneumoniae 0100993 in E coli or some other suitable host is probed with a radiolabeled ohgonucleotide, preferably a 17-mer or longer, de ⁇ ved from a partial sequence Clones carrying DNA identical to that of the probe can then be distinguished
  • the DNA sequence set out in Table 1 [ SEQ ID NO 1 ] contains an open reading frame encoding a protein having about the number of amino acid residues set forth in Table 1 [SEQ ID NO.2 respectively] with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known in the art
  • gluS of the invention is structurally related to other proteins of the glutamyl tRNA synthetase family, as shown by the results of sequencing the DNA encoding gluS of the deposited strain
  • the protein exhibits greatest homology to Bacillus subtilis glutamyl tRNA synthetase protein among kno n proteins
  • the invention provides a polynucleotide sequence identical over its entire length to the coding sequence in Table 1 [SEQ ED NO 1 ]
  • the invention is the coding sequence for the mature poiypeptide or a fragment thereof, by itself as well as the coding sequence for the mature poiypeptide or a fragment in reading frame with other coding sequence
  • the marker sequence is a hexa-histidine peptide. as provided in the pQE vector (Qiagen, Inc ) and desc ⁇ bed in Gentz et al . Proc Natl Acad Sci USA 86 821-824 ( 1989).
  • Polvnucleotides ot the invention also include, but are not limited to. polynucleotides comp ⁇ sing a structural gene and its naturally associated sequences that control gene expression
  • a preferred embodiment of the invention is the polynucleotide comp ⁇ sing a nucleotide sequence set forth in SEQ ED NO. l of Table 1 which encodes the gluS poiypeptide
  • the invention also includes polynucleotides of the formula set forth in Table 1 (C) wherein, at the 5' end of the molecule, X is hydrogen, and at the 3' end of the molecule.
  • Y is hydrogen or a metal
  • R [ and R is anv nucleic acid residue
  • m is an integer between 1 and 1000 or zero
  • n is an integer between 1 and 1000 or zero
  • Other preferred embodiments provide polynucleotides where n is zero and m is the integer 3 6, 9, 12. 15, 18, 21, 24. 27 or 30 In such preferred embodiments it is most preferred that the 5 terminal nucleic acid residues be ATG or GTG or encode a methionine
  • polynucleotide encoding a poiypeptide encompasses polynucleotides that include a sequence encoding a poiypeptide of the invention, particularly a bacte ⁇ al poiypeptide and more particularlv a poiypeptide of the Streptococcus pneumoniae gluS having the amino acid sequence set out in Table 1 [SEQ ED NO 2]
  • the term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the poiypeptide (for example, interrupted bv integrated phage or an insemon sequence or editing) together with additional regions, that also may contain coding and/or non-coding sequences
  • the invention further relates to va ⁇ ants of the polynucleotides desc ⁇ bed herein that encode for va ⁇ ants of the poiypeptide having the deduced amino acid sequence of Table 1 [SEQ ED NO.2] Va ⁇ ants that are fragments of the polynucle
  • polynucleotides that are at least 707c identical over their entire length to a polynucleotide encoding gluS poiypeptide having an ammo acid sequence set out in Table 1 [SEQ ID NO.2], and polynucleotides that are complementary to such polynucleotides Alternatively, most highly preferred are polynucleotides that comp ⁇ se a region that is at least 807c identical over its entire length to a polynucleotide encoding gluS poiypeptide of the deposited strain and polynucleotides complementary thereto In this regard, polynucleotides that are at least 707c identical over their entire length to a polynucleotide encoding gluS poiypeptide having an ammo acid sequence set out in Table 1 [SEQ ID NO.2], and polynucleotides that are complementary to such polynucleotides Alternatively, most highly preferred are polynucleotides that comp ⁇ se
  • Preferred embodiments are polynucleotides that encode polypeptides that retain substantially the same biological function or activ ity as the mature poiypeptide encoded by a DNA of Table 1 [SEQ ED NO 1 ]
  • the invention further relates to polynucleotides that hyb ⁇ dize to the herein above- desc ⁇ bed sequences
  • the invention especially relates to polynucleotides that hyb ⁇ dize under st ⁇ ngent conditions to the herein above-desc ⁇ bed polynucleotides
  • st ⁇ ngent conditions' and st ⁇ ngent hyb ⁇ dization conditions' mean hyb ⁇ dization will occur only if there is at least 957c and preferably at least 977c identity between the sequences
  • An example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising 507c formamide. 5x SSC ( 150mM NaCl.
  • the invention also provides a polynucleotide consisting essentially of a polynucleotide sequence obtainable by screening an approp ⁇ ate library containing the complete gene for a polynucleotide sequence set forth in SEQ ID NO.1 under stringent hyb ⁇ dization conditions with a probe having the sequence of said polynucleotide sequence set forth in SEQ ID NO.
  • polynucleotide assays of the invention may be used as a hyb ⁇ dization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding gluS and to isolate cDNA and genomic clones of other genes that ha e a high sequence similanty to the gluS gene
  • probes generally will compnse at least 15 bases Preferably. such probes will have at least 30 bases and may have at least 50 bases Particularly preferred probes will have at least 30 bases and will have 50 bases or less
  • the coding region of the gluS gene may be isolated by screening using a DNA sequence provided in SEQ ED NO 1 to synthesize an o gonucleotide probe A labeled o gonucleotide 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 hyb ⁇ dizes to
  • polynucleotides and polypeptides of the invention mav be employed, for example as research reagents and mate ⁇ ais for discovery ot treatments of and diagnostics for disease particularly human disease, as further discussed herein relating to polynucleotide assays
  • Polynucleotides of the invention that are o gonucleotides derived from the sequences of SEQ ID NOS.
  • 1 and/or 2 and/or 3 and/or 4 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 ot infection the pathogen has attained
  • the invention also provides polv nucleotides that mav encode a poiypeptide that is the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids inte ⁇ or to the mamre poiypeptide (when the mature form has more than one poiypeptide 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 A precursor protem.
  • prosequences having the mature form of the poiypeptide fused to one or more prosequences may be an inactive form of the poiypeptide
  • inactive precursors generally are activated
  • proproteins Some or all of the prosequences may be removed before activation Generally, such precursors are called proproteins
  • a polynucleotide of the invention may encode a mamre protein, a mamre protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mamre 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 du ⁇ ng processing steps that produce active and mamre forms of the poiypeptide Vectors, host cells, expression
  • the invention also relates to vectors that compnse 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 de ⁇ ved from the DNA constructs of the invention
  • host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention
  • Introduction ot a polynucleotide into the host cell can be effected bv methods descnbed in many standard laboratory manuals, such as Davis et al , BASIC METHODS IN MOLECL LAR BIOLOGY, ( 1986) and Sambrook et al., MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spnng Harbor Laboratory Press, Cold Sp ⁇ ng Harbor, N Y (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection. transvection. microinjection. cationic pid-mediated transfection.
  • approp ⁇ ate hosts include bactenal cells, such as streptococci, staphylococci enterococci E toll, streptomvces and Bacillus suotihs cells, fungal cells, such as east cells and Aspergdlus cells, insect cells such as Drosophda S2 and Spodoptera Sf9 cells, animal cells such as CHO. COS. HeLa, C 127. 3T3. BHK. 293 and Bowes melanoma cells, and plant cells A great va ⁇ ety of expression systems can be used to produce the polypeptides of the invention
  • Such vectors include, among others, cnromosomal.
  • episomal and virus-de ⁇ ved vectors e g., vectors de ⁇ ved from bacte ⁇ al plasmids. from bacte ⁇ ophage, 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 and retroviruses.
  • 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 poiypeptide in a host may be used for expression in this regard
  • the approp ⁇ ate DNA sequence may be inserted into the expression system by any of a va ⁇ ety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al . MOLECULAR CLONING. A LABORATORY MANUAL, (supra)
  • approp ⁇ ate secretion signals may be incorporated into the expressed poiypeptide These signals may be endogenous to the poiypeptide or they may be heterologous signals
  • Polypeptides of the invention can be recovered and pu ⁇ fied 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 chromatographv . hydroxylapatite chromatographv and lectin chromatography Most preferabK high pertormance liquid chromatographv is employed for punfication Well known techniques tor refolding protein mav Iz employed to regenerate active conformation w hen the poiypeptide is denatured du ⁇ ng isolation and or punfication
  • This invention is also related to the use of the gluS polynucleotides of the invention for use as diagnostic reagents Detection of gluS in a eukaryote. particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of a disease Eukaryotes (herein also ⁇ nd ⁇ v ⁇ dual(s)"), particularly mammals, and especially humans, infected with an organism compnsing the gluS gene may De detected at the nucleic acid lev el by a va ⁇ ety of techniques
  • Nucleic acids for diagnosis may be obtained from an infected individual's cells and tissues, such as bone, blood, muscle, cartiiaee. and skin Genomic DNA mav be used directly for detection or may be amplified enzymatically by using PCR or other amplification technique p ⁇ or to analysis. RNA or cDNA may also be used in the same ways Using amplification, characte ⁇ zation of the species and strain of prokaryote present in an individual, may be made by an analysis of the genotype of the prokaryote gene.
  • Deletions and insertions can be detected by a change in size of the amplified product in compa ⁇ son to the genotype of a reference sequence
  • Point mutations can be identified by hyb ⁇ dizmg amplified DNA to labeled gluS polynucleotide sequences
  • 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 denatu ⁇ ng agents, or by direct DNA sequencmg See. e.g., Myers et al..
  • Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and S I protection or a chemical cleavage method See. e.g . Cotton et al . Proc Nad Acad Set . USA. 85 4397-4401 ( 1985)
  • Cells carrying mutations or polymorphisms in tne gene of the inv ention may also be detected at the DNA level by a va ⁇ ety of techniques, to allow for serotyping.
  • RT-PCR can be used to detect mutations It is particularly preferred to used RT-PCR in conjunction with automated detection systems, such as. for example.
  • GeneScan RNA or cDNA may also be used for the same purpose, PCR or RT-PCR
  • PCR pnmers complementary to a nucleic acid encoding gluS can be used to identify and analyze mutations
  • the invention further provides these pnmers with 1, 2, 3 or 4 nucleotides removed from the 5' and or the 3' end These pnmers may be used for.
  • amplifying gluS DNA isolated from a sample denved from an individual The pnmers may be used to amplify the gene isolated from an infected individual such that the gene may then be subject to v anous 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, and most preferably otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid, comp ⁇ sing determining from a sample derived from an individual a increased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID NO 1 ] Increased or decreased expression of gluS polynucleotide 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 hyb ⁇ dization methods
  • a diagnostic assay in accordance with the invention for detecting over- expression of gluS protein 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 gluS protein, in a sample de ⁇ ved 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 Antibodies The polypeptides of the invention or va ⁇ ants thereof, or cells expressing them can be used as an immunogen to produce antibodies immunospecific for such polypeptides "Antibodies" as used herein includes monoclonal and polyclonal antibodies, chime ⁇ c.
  • Antibodies generated against the polypeptides ot the invention can be obtained by administe ⁇ ng the polypeptides or epitope-bea ⁇ ng fragments, analogues or cells 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 va ⁇ ous techniques, such as those in Kohler, G and Milstein, C . Nature 256 495-497 ( 1975), Kozbor et al . Toda ⁇ 4 72 ( 1983).
  • phage display technology may be utilized to select antibody genes with binding activities towards the poiypeptide either from repertoires of PCR amplified v- genes of lymphocytes from humans screened for possessing anti-gluS or from naive libraries (McCafferty. J et al.. ( 1990). Nature 348. 552-554. Marks. J et al . ( 1992) Biotechnology 10. 779-783).
  • the affinity of these antibodies can also be improved by chain shuffling (Clackson. T et al , ( 1991 . Nature 352. 624-628)
  • each domain mav be directed against a different epitope - termed bispecific antibodies
  • the above-desc ⁇ bed antibodies may be employed to isolate or to identify clones expressing the polypeptides to punfy the polypeptides by affinity chromatography
  • antibodies against gluS- poiypeptide may be employed to treat infections, particularly bacte ⁇ al infections and especially otitis media, conjunctivitis, pneumonia. bacteremia. meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid
  • Poiypeptide va ⁇ ants include antigenically. epitopically or immunologically equivalent va ⁇ ants that form a particular aspect of this invention
  • the term ' antigenically equivalent de ⁇ vative encompasses a poiypeptide or its equivalent which will be specifically recognized by certain antibodies which, when raised to the protein or poiypeptide according to the invention, interfere with the immediate physical interaction between pathogen and mammalian host
  • the term immunologically equivalent de ⁇ vative 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 immediate physical interaction between pathogen and mammalian host
  • the poiypeptide 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 poiypeptide
  • the antigen may be associated, for example by conjugation, with an lmmunogemc carrier protein for example bovine serum albumin (BSA) or keyhole limpet haemocvamn (KLH) AlternativeK a multiple antigenic peptide comprising multiple copies of the protein or poiypeptide. or an antigemcalK or immunologicallv equivalent poiypeptide thereof may be sufficiently antigenic to improv e immunogenicity so as to obviate the use of a earner
  • BSA bovine serum albumin
  • KLH keyhole limpet haemocvamn
  • the antibody or va ⁇ ant thereof is modified to make it less lmmunogemc in the individual
  • the antibody may most preferably be "humanized", where the comphmenta ⁇ ty determining reg ⁇ on(s) of the hyb ⁇ doma-de ⁇ ved antibody has been transplanted into a human monoclonal antibody , for example as described in Jones, P et al ( 1986), Nature 321 , 522-525 or Tempest et al.,( 1991 ) Biotechnology 9, 266-273
  • the use of a polynucleotide of the inv ention in genetic immunization will preferably employ a suitable delivery method such as direct miection of plasmid DNA into muscles (Wolff et al , Hum Mol Genet 1992.
  • Polypeptides of the invention may also be used to assess the binding of small molecule substrates and gands in. for example, cells, cell-free preparations, chemical hbranes. and natural product mixtures
  • substrates and hgands may be natural substrates and gands or may be structural or functional mimetics. See, e.g., Cohgan et al.. Current Protocols in Immunology 7(2): Chapter 5 (1991).
  • the invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of gluS polypeptides or polynucleotides, particularly those compounds that are bacte ⁇ ostatic and/or bacte ⁇ ocidal
  • the method of screening mav involve high-throughput techniques
  • a synthetic reaction mix to screen for agonists or antagoists.
  • a cellular compartment such as a membrane, cell envelope or cell wall, or a preparation of any thereof, compnsing gluS poiypeptide and a labeled substrate or hgand of such poiypeptide is incubated in the absence or the presence of a candidate molecule that may be a gluS agonist or antagonist.
  • the ability of the candidate molecule to agonize or antagonize the gluS poiypeptide is reflected in decreased binding of the labeled gand or decreased production of product from such substrate Molecules that bind gratuitously, ; e .
  • Molecules that bind well and increase the rate ot product production from substrate are agonists
  • Detection of the rate or level of production of product from substrate may be enhanced by using a importer system Reporter systems that may be useful in this regard include but are not limited to colo ⁇ met ⁇ c labeled substrate converted into product, a reporter gene that is responsive to changes in gluS polynucleotide or poiypeptide activit , and binding assays known in the art.
  • an assay for gluS antagonists is a competitive assay that combines gluS and a potential antagonist with gluS-binding molecules, recombinant gluS binding molecules, natural substrates or hgands. or substrate or hgand mimetics.
  • gluS can be labeled, such as by radioactivity or a colo ⁇ met ⁇ c compound, such that the number of gluS 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 small organic molecules, peptides. polypeptides and antibodies that bind to a polynucleotide or poiypeptide of the invention and thereby inhibit or extinguish its activity
  • Potential antagonists also may be small organic molecules, a peptide. a poiypeptide such as a closely related protein or antibody that binds the same sues on a binding molecule, such as a binding molecule, without inducing gluS-induced activities, thereby preventing the action of gluS by excluding gluS from binding
  • Potential antagonists include a small molecule that binds to and occupies the binding site of the poiypeptide 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).
  • Preferred potential antagonists include compounds related to and va ⁇ ants ot gluS
  • Preferred potential antagonists include compounds related to and va ⁇ ants ot gluS
  • the encoded protein upon expression, can be used as a target for the screening of antibacte ⁇ al drugs
  • the DNA 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 poiypeptide, 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 gluS protein-mediated mammalian cell invasion by, for example, initiating phosphorylation of mammalian tyrosine kinases (Rosenshine et al , Infect Immun 60 221 1 ( 1992) to block bacte ⁇ al adhesion between mammalian extracellular matrix proteins and bacte ⁇ al gluS proteins that mediate tissue damage and. to block the normal progression of pathogenesis in infections initiated other than by the implantation of in-dwelling devices or by other surgical techniques
  • the antagonists and agonists of the invention mav 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 Vaccines
  • Another aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal which comprises inoculating the individual with gluS. or a fragment or variant thereof, adequate to produce antibodv and/ or T cell immune response to protect said individual from infection, particularly bacte ⁇ al infection and most particularly Streptococcus pneumoniae infection Also provided are methods whereby such immunological response slows bacte ⁇ al replication Yet another aspect of the invention relates to a method of inducing immunological response in an individual which comp ⁇ ses dehve ⁇ ng to such individual a nucleic acid vector to direct expression of gluS, or a fragment or a variant thereof, for expressing gluS.
  • nucleic acid vector may compnse DNA, RNA.
  • a further aspect of the inv ention relates to an immunological composition which, when introduced into an individual capable or having induced within it an immunological response induces an immunological response in such individual to a gluS or protein coded therefrom wherein the composition comprises a recombinant gluS or protein coded therefrom comprising DNA which codes for and expresses an antigen of said gluS or protein coded therefrom
  • the immunological response may be used therapeutically or prophvlactically and may take the form of antibody immunity or cellular immunity such as that a ⁇ sing from CTL or CD4+ T cells
  • a gluS poiypeptide or a fragment thereot mav be fused with co-protein which may not bv itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will hav e immunogenic and protectiv e properties
  • fused recombinant protein preferabK further comprises an antigenic co-protein such as poprotein D from Hemophilus mfluenzae.
  • co-protein mav act as an adjuvant in the sense of providing a generalized stimulation of the immune system
  • the co-protein may be attached to either the amino or carboxy terminus of the first protein
  • compositions particularly vaccine compositions, and methods comp ⁇ sing the polypeptides or polynucleotides of the invention and immunostimulatory DNA sequences, such as those desc ⁇ bed in Sato, Y et al Science 273 352 (1996)
  • the poiypeptide may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacte ⁇ a, for example by blocking adherence of bacteria to damaged tissue
  • tissue damage include wounds in skin or connective tissue caused, e g., by mechanical, chemical or thermal damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, mammary glands, urethra or vagina
  • the invention also includes a vaccine formulation which comprises an immunogenic recombinant protein of the invention together with a suitable carrier Since the protein may be broken down in the stomach, it 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, bacte ⁇ ostats and solutes w hich render the formulation lnsotomc 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 ampules and v ials and may be stored in a treeze-d ⁇ ed condition requi ⁇ ng only the addition of the sterile liquid earner immediatelv p ⁇ or to use
  • compositions for purposes of compositions, kits and administration
  • the invention also relates to compositions comp ⁇ sing the polynucleotide or the polypeptides discussed above or their agonists or antagonists
  • the polypeptides of the invention may be employed in combination with a non-ste ⁇ le or ste ⁇ le earner or earners for use with cells, tissues or organisms, such as a pharmaceutical earner suitable for administration to a subject
  • Such compositions compnse for instance, a media additive or a therapeuticallv effective amount of a poiypeptide of the invention and a pharmaceutically acceptable earner or excipient
  • Such earners 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 comp ⁇ sing one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention
  • Polypeptides and other compounds of the invention may be employed alone or in conjunction
  • the pnarmaceutical compositions may be administered in anv effective, convenient manner including, for instance, administration bv topical, oral, anal, vaginal, intravenous liitrape ⁇ toneal. 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
  • compositions 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 c to about 987c by weight of the formulation, more usually thev will constitute up to about 80% by weight of the formulation
  • 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 ary with the age, weight and response of the particular individual
  • the above dosages are exemplary of the average case There can.
  • In-dwelling devices include surgical implants, prosthetic devices and catheters, i.e , devices that are introduced to the body of an individual and remain in position for an extended time
  • Such devices include, for example, artificial joints, heart valves, pacemakers, v ascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, continuous ambulatory pentoneal dialysis (CARD) catheters
  • the composition of the invention may be administered by injection to achieve a systemic effect against relevant bacte ⁇ a shortly before insertion of an in-dwelling device Treatment may be continued after surgery during the m-body time of the device
  • the composition could also be used to broaden pe ⁇ operative cover for any surgical technique to prevent bacterial wound infections, especially Streptococcus pneumoniae wound infections
  • compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to mat ⁇ x proteins exposed in wound tissue and for prophylactic use in dental treatment as an alternative to. or in conjunction with, antibiotic prophylaxis
  • composition of the invention may be used to bathe an indwelling device immediately before insertion
  • the active agent will preferably be present at a concentration of l ⁇ g/ml to lOmg/ml for bathing of wounds or indwelling devices
  • 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
  • Libra ⁇ es may be prepared by routine methods, for example Methods 1 and 2 below
  • Total cellular DNA is isolated from Streptococcus pneumoniae 0100993 according to standard procedures and size-fractionated by either of two methods
  • Total cellular DNA is mechanically sheared by passage through a needle in order to size-fractionate according to standard procedures.
  • DNA fragments of up to 1 lkbp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added. Fragments are gated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E.coh infected with the packaged library The library is amplified by standard procedures.
  • Total cellular DNA is partially hydrolyzed with a one or a combination of rest ⁇ ction enzymes appropriate to generate a series of fragments for cloning into library vectors (e g , Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated according to standard procedures.
  • EcoRI linkers are hgated to the DNA and the fragments then hgated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E.coli infected with the packaged library
  • the library is amplified by standard procedures.
  • Example 2 gluS Characterization
  • the enzyme mediated incorporation of radiolabelled ammo acid into tRNA may be measured by the aminoacylation method which measures amino acid-tRNA as trichloroacetic acid-precipitable radioactivity from radiolabelled amino acid in the presence of tRNA and ATP (Hughes J. Mellows G and Soughton S. 1980, FEBS Letters, 122.322- 324).
  • inhibitors of glutamyl tRNA synthetase can be detected by a reduction in the trichloroacetic acid precipitable radioactivity relative to the control
  • the tRNA synthetase catalysed partial PPi/ATP exchange reaction which measures the formation of radiolabelled ATP from PPi can be used to detect glutamyl tRNA synthetase inhibitors (Calender R & Berg P, 1966, Biochemistr , 5, 1681 - 1690)
  • ATCGAAGATT ACCGAAAGAA AGGTTACCTT CCAGAAGCAG TCTTTAACTT TATTGCTCTT 340
  • GAAGCATTCA AAGCAAAACT TGAAGCGATG ACAGATGATG AATTTGTGAC AGAAAATATC 1260

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EP2275124A2 (en) * 2002-04-02 2011-01-19 Ben Gurion University Of The Negev Research And Development Authority Protein-based streptococcus pneumoniae vaccines
US8691243B2 (en) 2002-04-02 2014-04-08 Ben-Gurion University Of The Negev Research And Development Authority Protein-based Streptococcus pneumoniae vaccine

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2275124A2 (en) * 2002-04-02 2011-01-19 Ben Gurion University Of The Negev Research And Development Authority Protein-based streptococcus pneumoniae vaccines
EP2275125A2 (en) * 2002-04-02 2011-01-19 Ben Gurion University Of The Negev Research And Development Authority Protein-based streptococcus pneumoniae vaccines
EP2275120A3 (en) * 2002-04-02 2011-06-08 Ben-Gurion University Of The Negev Research And Development Authority Protein-based streptococcus pneumoniae vaccines
US8691243B2 (en) 2002-04-02 2014-04-08 Ben-Gurion University Of The Negev Research And Development Authority Protein-based Streptococcus pneumoniae vaccine

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