WO2000061165A1 - Motif d'adhesine conserve et ses procedes d'utilisation - Google Patents

Motif d'adhesine conserve et ses procedes d'utilisation Download PDF

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WO2000061165A1
WO2000061165A1 PCT/US2000/009866 US0009866W WO0061165A1 WO 2000061165 A1 WO2000061165 A1 WO 2000061165A1 US 0009866 W US0009866 W US 0009866W WO 0061165 A1 WO0061165 A1 WO 0061165A1
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ala
val
asp
gly
asn
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Andrei Nicolae Lupas
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Smithkline Beecham Corporation
Smithkline Beecham Plc
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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
    • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
    • 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
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • C07K14/212—Moraxellaceae, e.g. Acinetobacter, Moraxella, Oligella, Psychrobacter
    • 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
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/285—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides.
  • the polypeptides of the present invention have been putatively identified as conserved binding domains of bacterial adhesins.
  • Adhesins are multifunctional, proteinaceous structures formed on the surface of pathogenic bacteria. They can mediate attachment or can assist the invading bacteria in avoiding the immune responses of the infected host, which are intended to protect the host against the infection. Adhesins can interaction with host cell receptors, in adherence to extracellular matrix proteins (ECM), in activation or inactivation of host proteases (e.g. activation of plasminogen or inhibition of complement).
  • ECM extracellular matrix proteins
  • the virulence plasmid (pYV)-encoded non-fimbrial surface protein YadA (Yersinia adhesin, formerly known as Yopl or PI) of the enteropathogenic Yersinia enterocolitica and Y. pseudotuberculosis is an important virulence determinant of the bacterial enteropathogenic Yersinia species [A. Roggenkamp et al, Infec. Immun., 64(7):2506 (July 1996)].
  • the adhesin has several functions.
  • YadA is also involved in auto-agglutination, a phenomenon occurring after growth in tissue culture medium at 37EC [Skurnik et al, J. BacJerioL, 158: 1033-1036 (1984)].
  • compositions could be developed to interfere with the functions of the adhesin, however, such compositions and methods of use thereof would prove useful in the therapeutic and prophylactic treatment of the bacterial infections mediated by these pathogen.
  • proteins, antagonists and agonists of these bacterial adhesins as well as compositions and methods for their use in the vaccine and diagnostic fields.
  • the present invention provides isolated polypeptides of about 20 amino acids in length, which are conserved in proteobacterial extracellular proteins and which bind to a protein or proteinaceous ligand expressed by a mammalian cell.
  • the polypeptides of this invention include the sequences of the invention set forth herein, which are found in Neisseria, Actinobacillus, Haemophilus, Moraxella and Yersinia pathogens. Biologically active and diagnostically or therapeutically useful fragments, variants, analogs and derivatives of these sequences are provided, as well as variants and derivatives of the fragments, and analogs of the foregoing.
  • These polypeptides, which are free from association with other contaminating or proteinaceous materials with which they are found in nature may be produced synthetically or by recombinant means.
  • non-naturally occurring synthetic, isolated and/or recombinant polypeptides, fragments, consensus fragments and/or sequences having conservative amino acid substitutions of the conserved proteobacterial sequences of the present invention may bind proteobacterial adhesin ligands, or may also modulate, quantitatively or qualitatively, adhesin ligand binding.
  • the present invention provides synthetic, isolated or recombinant polypeptides which are designed to inhibit or mimic various conserved proteobacterial adhesin sequences or fragments thereof.
  • the invention provides a fusion protein which comprises at least one of the polypeptides of this invention fused in frame to a second protein.
  • the invention provides an isolated or synthetic polynucleotide sequence free from association with other materials with which it is found in nature, which encodes a polypeptide or fusion protein described herein, including nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to nucleic acid sequences of the present invention.
  • the invention provides a nucleic acid molecule, e.g., a vector or plasmid or recombinant virus, comprising a polynucleotide sequence encoding a polynucleotide sequence or fusion protein of this invention under the control of regulatory sequences which direct the expression of the polypeptide or fusion protein in a host cell.
  • a nucleic acid molecule e.g., a vector or plasmid or recombinant virus, comprising a polynucleotide sequence encoding a polynucleotide sequence or fusion protein of this invention under the control of regulatory sequences which direct the expression of the polypeptide or fusion protein in a host cell.
  • the invention provides a host cell comprising the nucleic acid molecule described above.
  • the invention provides a composition which inhibits or retards the binding of a proteobacterial adhesin to its ligand or to a cell expressing its ligand.
  • antibodies which bind to the conserved polypeptides including humanized antibodies, anti-antibodies, monoclonal and polyclonal antibodies, among others.
  • the invention provides an anti-idiotype of the antibody described above.
  • the invention provides an immunogenic composition useful as a vaccine to prevent infection by a proteobacterial species comprising in a pharmaceutically acceptable carrier a polypeptide or fusion protein of this invention.
  • this composition contains a conserved proteobacterial polypeptide or fusion protein of this invention, or an immunogenic fragment thereof.
  • the composition contains an amino acid sequence at least 70% identical to the aforementioned sequences as determined by a sequence comparison algorithm, which sequence binds the adhesin ligand.
  • the composition contains a small molecule which binds the adhesin ligand.
  • the composition contains an antibody which binds the adhesin ligand, or an anti-idiotype antibody of that antibody.
  • These compositions may also contain one or more adjuvants or
  • the invention provides a process for producing the aforementioned polypeptides, polypeptide fragments, variants and derivatives, fragments of the variants and derivatives, and analogs of the foregoing.
  • the invention provides methods for producing the aforementioned polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing (i.e., having expressibly inco ⁇ orated therein) a nucleic acid sequence encoding a polypeptide of the present invention under conditions for expression of the polypeptide in the host and then recovering or isolating the expressed polypeptide from the cell or cell lysate.
  • the polypeptides of this invention are produced by conventional synthesis methods.
  • the invention provides a method for vaccinating a mammalian subject against infection by a proteobacteria which includes administering to the subject a prophylactically effective amount of the immunogenic composition described above.
  • the invention provides a method of making an immunogenic composition for use as a vaccine component against proteobacterial infection comprising fusing a polypeptide of this invention to a second protein capable of resisting degradation in vivo, wherein said polypeptide elicits antibodies in vivo which interfere with the binding of proteobacterial adhesin molecules to their ligands.
  • the invention provides diagnostic assays for detecting diseases related to expression of the adhesin polypeptides of the present invention in infected host cells.
  • a process for diagnosing a proteobacterial infection comprises contacting a biological sample from a possibly infected subject with a labeled antibody which binds to the conserved polypeptide described herein; and measuring the signal generated by the label with a suitable assay. Detection of said signal indicates the presence of an adhesin molecule from the proteobacteria.
  • the invention provides a diagnostic reagent which comprises a composition capable of binding to a conserved proteobacterial polypeptide of the invention, the composition associated with a detectable label.
  • Antagonists of adhesin binding activity can be used in the treatment of proteobacterial infection.
  • the invention provides a method for utilizing the polypeptides of the present invention for the screening of chemical or natural compounds or ligands thereof which inhibit or retard interaction of proteobacterial adhesins with other proteins, including their ligands expressed on the cells of their infected hosts.
  • the invention provides a method for identifying compounds which antagonize the binding of a proteobacterial adhesin to its ligand comprising the steps of providing a sample of the ligand or a cell which expresses the ligand immobilized on a support; contacting the sample with a known amount of a polypeptide of this invention and a known amount of a test compound; washing unbound materials from said sample; contacting the sample with a labeled reagent which binds to said polypeptide; washing unbound reagent from said sample; and measuring the amount of signal generated by said label.
  • the amount of signal generated is inversely proportional to the ability of the test compound to disrupt or inhibit binding between said polypeptide and its ligand.
  • the method involves identifying those test compounds as antagonists which are associated with a low signal.
  • the invention provides a method for generating a small molecule which antagonizes the binding between a proteobacterial adhesin and its ligand comprising analyzing an antibody to a polypeptide of this invention in a computer modelling program.
  • the invention provides products, compositions, processes and methods that utilize the aforementioned polypeptides and polynucleotides, antibodies and small molecules, as well as other antagonists of bacterial adhesins, for scientific research, biological, clinical and therapeutic purposes, synthesis of DNA and manufacture of DNA vectors, inter alia.
  • the invention provides products, compositions and methods, inter alia, for, among other things, assessing proteobacterial infection or the expression of the bacterial adhesin in an infected host by determining the presence of the adhesins with antibodies of this invention or with nucleotide probes of this invention.
  • Figure 1 A shows a graphical illustration of the coiled-coiled probability of YadA, UspAl and UspA2.
  • Figure 1 B shows a graphical illustration of normalized FFT intensity for head sequence and stalk sequence.
  • FIG. 1 Figure 2 A, B and C show sequence comparisons of proteins of the invention.
  • the present invention meets the needs in the art by providing immunogenic sequences which are derived from the conserved, sequences in adhesins of proteobacteria, as well as fusion proteins, pharmaceutical compositions and methods of utilizing these sequences and compositions for diagnostic, therapeutic and vaccine methods and compositions.
  • the present invention relates to novel polypeptide sequences, which are isolated, highly conserved sequences of extracellular domains of certain pathogens of the family Proteobacteria, consensus sequences thereof, and other variants and analogs which share the immunogenic function of the native isolated sequences.
  • the inventor identified significantly similar proteins in the beta branch of Proteobacteria, e.g., the species Neisseria, and in the gamma branch of Proteobacteria, e.g., the species Actinobacillus, Heomophilus, Moraxella and Yersinia.
  • the inventor surprisingly discovered and isolated a highly conserved sequence of approximately 20 to about 24 amino acid residues, with approximately 14-18 residues centered around a conserved block of four hydrophobic residues, ending with an invariant glycine, e.g., Lys-Ala-Ala-Gly, which has not previously been identified in any eubacterial extracellular domain.
  • This sequence which in the non-fimbrial adhesin YadA of the Yersinia species, is found at the end of the head domain, partly overlapping the tetradecad repeat, was found in many putative open reading frames from Proteobacterial genomes.
  • the sequence was not readily detectable by BLASTr) program homology searches, and it is significantly misaligned by automated alignment programs, such as the CLUSTAL ⁇ program and the PILEUPr) program.
  • An isolated highly conserved Proteobacterial sequence of this invention comprises about 20-24 amino acids. Presented herein are several generic formulae which are conserved or partially conserved sequences of the present inventions.
  • the N-terminal R in each sequence below may represent hydrogen (i.e., the hydrogen on the unmodified N terminal amino acid), or a lower alkyl, or a lower alkanoyl having 1 to 10 carbon atoms. R may also include a sequence of between 1 to about 25 amino acids, optionally substituted with a lower alkyl or lower alkanoyl.
  • the C-terminal R " can be the hydroxyl group on the C terminal amino acid or an amide, optionally substituted with a lower alkyl or a lower alkanoyl having from 1 to 25 amino acids. It should be understood that R and R " will be completely omitted or defined differently, where the polypeptide or fragment of this invention is employed as part of a fusion protein with other proteins, as discussed below.
  • polypeptides of this invention may be preceded at the N terminus (e.g., R) by a selected signal peptide and followed at the C terminus by an optional spacer sequence (e.g., R" ).
  • R N terminus
  • R optional spacer sequence
  • an isolated consensus sequence of this invention comprises a sequence of the formula:
  • X 1 can be Gin, Lys, Thr, Val, or Arg
  • X 2 and the hydrophobic residue X 4 are independently Leu, De, or Val
  • X 3 can be His, Gly, Ser, Asn, or Gin
  • the hydrophobic residue X 5 can be Ala, Lys, Val, Asp, Pro, Asn, Gly, or Glu
  • X 6 can be Thr, Val, Ser, Arg, Leu, Gin, Asp, Glu, Lys, or Asn
  • X 7 can be Lys, Glu, Ala, Gin, He, Asn, or Val
  • X 8 can be Asp, Asn, Gly, Ala, Ser, or Pro
  • X 9 can be Val, Leu, Phe, Gly, Lys, Met, or lie
  • X 10 can be Ala, Gly, Ser, Asp, Arg, or Lys.
  • R-Arg Gin lie Thr X 1 Val Lys X 2 Gly Val X 3 X 4 Thr Asp X 5 X 6 Asn Val X 7 Gin Leu - R 2 [SEQ ED NO: 5].
  • X 1 and X 7 are independently Gly or Ser; X " is Ala or Lys; X 3 is Ala or Glu; X 4 is Asp or Asn; X is Ala or Thr; and X 6 is Ala or lie.
  • R and R 2 are as defined above. For example, specifically desirable sequences of this formula are
  • R and R " are as defined above.
  • R and R" are as defined above.
  • specifically desirable sequences of this formula isolated from the raw genomic sequence (Sanger) of Yersinia pestis are:
  • SEQ ID NOS: 9 and 10 were isolated as multiple copies of the conserved sequence from the same proteins.
  • R and R 2 are as defined above.
  • specifically desirable sequences of this formula which were isolated from the raw genomic sequences of Yersinia pestis include:
  • polypeptide sequences of this invention include the following:
  • Polypeptides of the present invention also include the polypeptide of the
  • sequences of the invention set forth herein, as well as other polypeptides which share at least 50% identity to the consensus sequences described above and/or to the highly conserved sequences in proteobacterial extracellular domains of Proteobacterial species: Neisseria, Actinobacillus, Haemophilus, Moraxella and Yersinia, according to the algorithm BESTFEP from the GCG program package [J. Devereux et al., Nucl. Acids Res., JL2(1):387 (1984)].
  • Other polypeptide sequences of this invention share at least 70% identity to the consensus sequences described above and/or to the highly conserved sequences in proteobacterial extracellular domains.
  • polypeptide sequences of this invention share at least 90% identity to the consensus sequences described above and/or to the highly conserved sequences in proteobacterial extracellular domains. These polypeptides are also anticipated to be useful in the compositions and methods for which the above-identified polypeptides are useful.
  • similarity between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
  • identity also known in the art is “identity” which means the degree of sequence relatedness between two polypeptide or two polynucleotide sequences as determined by the identity of the match between two lengths of such sequences.
  • Preferred methods to determine identity are designed to give the largest match between the two sequences tested. Methods to determine identity and similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package [J. Devereux et al.. Nucl. Acids Res., 1_2(1):387 (1984)], BLAST [S. F. Atschul et al., J. Mol. Biol.. 215:403 (1990)] and FASTA (Pearson) programs. For instance, searches for sequence similarities in databases with other proteobacterial species are likely to detect other similar highly conserved sequences.
  • polypeptide ⁇ encompasses the above- identified polypeptides, and all modifications, particularly those that are present in polypeptides synthesized by expressing a polynucleotide in a host cell.
  • the polypeptides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity. "Isolated” means altered “by the hand of man” from its natural state; i.e., that, if it occurs in nature, it has been changed or removed from its original environment, or both.
  • a naturally occurring polypeptide naturally present in a living animal in its natural state is not “isolated”, but the same polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
  • the polypeptides may occur in a composition, such as a media, formulations, solutions for introduction of polypeptides, for example, into cells, compositions or solutions for chemical or enzymatic reactions, for instance, which are not naturally occurring compositions, and, therein remain isolated polypeptides within the meaning of that term as it is employed herein.
  • polypeptide of the present invention may be a recombinant polypeptide expressed from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells.
  • a polypeptide of the invention may be a synthetic polypeptide.
  • the invention also relates to variants, analogs, derivatives and fragments of these isolated or consensus polypeptides, and variants, analogs and derivatives of the fragments.
  • "Variant(s)" of polypeptides are polypeptides that differ in amino acid sequence from the above-identified polypeptides, which serve as reference polypeptides. Generally, differences are limited so that the sequences of the
  • variant and reference polypeptide are closely similar overall and, in many regions, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination.
  • preferred variants are those that vary from a reference by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics.
  • conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and He; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe and Tyr.
  • fragment when referring to the polypeptide of the sequences of the invention set forth herein, means a polypeptide which retains essentially the same biological function or activity as such polypeptide, i.e., functions as an immunogen or retains the ability to bind its ligand expressed on a host cell.
  • the fragment, derivative or analog of the polypeptide the sequences of the invention set forth 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; (ii) one in which one or more of the amino acid residues includes a substituent group; (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 polypeptide.
  • a conserved or non-conserved amino acid residue preferably a conserved amino acid residue
  • substituted amino acid residue may or may not be one encoded by the genetic code
  • silent substitutions, additions and fragments are especially preferred among these.
  • polypeptides having the amino acid sequence of the sequences of the invention set forth herein without substitutions.
  • shorter versions of the above-identified sequences and formulae may also be useful where these fragments retain immunogenicity. It is anticipated that for the specific polypeptide identified above, an optional truncation of 1 amino acid at the C terminus is likely to produce a useful fragment. Additionally, a truncation of up to about 4 or 5 amino acids from the N terminus of the polypeptides of this invention is also likely to produce a useful peptide fragment.
  • a polypeptide has a sequence similarity or identity of at least about 80% to the sequence of the sequences of the invention set forth herein, and more preferably at least 90% similarity (more preferably at least 95% identity) to a polypeptide of the invention, and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of the sequences of the invention set forth herein and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 14 amino acids.
  • Other embodiments of fragments of this invention contain at least 18 amino acids of the highly conserved or consensus sequences; and still others contain at least 20 amino acids.
  • Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention. Fragments may be "free-standing," i.e., not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region. When comprised within a larger polypeptide, the presently discussed fragments most preferably form a single continuous region.
  • fragments may be comprised within a single larger polypeptide.
  • certain preferred embodiments relate to a fragment of a polypeptide of the present invention comprised within a precursor polypeptide designed for expression in a host cell and having heterologous pre- and pro-polypeptide regions fused to the amino terminus of the polypeptide or fragment and an additional
  • fragments in one aspect of the meaning intended herein, refers to the portion or portions of a fusion polypeptide or fusion protein derived from the highly conserved Proteobacterial sequences or the consensus sequence therefrom.
  • polypeptide fragments of the invention there may be mentioned those which have from about 5-15, 10-20, and 24 amino acids in length.
  • “about” includes the particularly recited range and ranges larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid residues.
  • about 20 amino acids in this context means a polypeptide fragment of 20 plus or minus several, a few, 5, 4, 3, 2 or 1 amino acid residues.
  • Further preferred fragments are those that have a chemical, biological or other activity of a conserved polypeptide of the invention, including those with a similar activity or an improved activity, or with a decreased undesirable activity.
  • Highly preferred in this regard are the recited ranges plus or minus as many as 5 amino acids at either or at both extremes.
  • Polypeptides of this invention may also contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids, and that these amino acids, including the terminal amino acids, may be modified, either by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques which are well known to the art.
  • amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids, and that these amino acids, including the terminal amino acids, may be modified, either by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques which are well known to the art.
  • the numerous common modifications that occur naturally in polypeptides are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and are well known to those of skill in the art.
  • polypeptides of the present invention include, without limitation, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of
  • cystine formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation. oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation. sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • polypeptides of this invention may be linear, or branched as a result of ubiquitination, or they may be circular, with or without branching, generally as a result of posttranslational events, including natural processing event and events brought about by human manipulation which do not occur naturally.
  • Circular, branched and branched circular polypeptides may be synthesized by non-translation natural processes and by entirely synthetic methods, as well.
  • Modifications can occur anywhere in a polypeptide of this invention, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
  • blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification is common in naturally occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention.
  • the amino terminal residue of polypeptides made in E. coli, prior to processing almost invariably will be N-formylmethionine.
  • the modifications that occur in a polypeptide often will be a function of how it is produced. For polypeptides produced by expression in a host, for instance, the nature and extent of the modifications in large part will be
  • glycosylation often does not occur in bacterial hosts such as E. coli. Accordingly, when glycosylation is desired, a polypeptide should be expressed in a glycosylating host, generally a eukaryotic cell. Insect cells often carry out the same posttranslational glycosylations as mammalian cells and, for this reason, insect cell expression systems have been developed to express efficiently mammalian proteins having the native patterns of glycosylation, inter alia. Similar considerations apply to other modifications.
  • the highly conserved sequence of the present invention acts as a binding motif, and is likely involved in host cell recognition, auto-agglutination, or cell defense
  • polypeptides of this invention are useful in the development of antibodies and the design of diagnostic probes, screening methods for the development of vaccine agents to prevent bacterial infection, and the like discussed below.
  • a "fusion protein” as the term is used herein, is a protein encoded by a polynucleotide sequence encoding a polypeptide, variant, or fragment of this invention to another, often unrelated, gene or fragments thereof.
  • the Aother ⁇ protein to which the polypeptide of this invention is fused or coupled may be selected from among any proteins or peptides which are at least 90% likely to form a coiled coil, as defined by the COILS algorithm [A. Lupas et al, Science, 252: 1 162-1 164 (1991), inco ⁇ orated by reference herein]. See, also, European Patent Application No. EP-A-0464 533 [Canadian counte ⁇ art Patent Application No.
  • fusion proteins comprising various portions of constant regions of immunoglobulin molecules together with another human protein or part thereof.
  • employing an immunoglobulin Fc region as a part of a fusion protein is advantageous for use in therapy and diagnosis resulting in, for example, improved pharmacokinetic properties [See, e.g., European Patent Application No. EP-A 0 232 262].
  • Fc portion proves to be a hindrance to use in therapy and diagnosis, for example, when the fusion protein is to be used as an antigen for immunizations.
  • human proteins have been fused with Fc portions for use in high-throughput screening assays to identify antagonists of those proteins. See, D. Bennett et al., J. Mol. Recog., 8:52-58 (1995); and K. Johanson et al.. J. Biol. Chem.. 270(16):9459-9471 (1995).
  • this invention also relates to genetically engineered fusion proteins comprised of one of the conserved proteobacterial sequences or a variant, derivative or fragment thereof, and of various portions of the constant regions of heavy or light chains of immunoglobulins of various subclasses (IgG, IgM, IgA, IgE).
  • immunoglobulin is the constant part of the heavy chain of human IgG, particularly IgGl, where fusion takes place at the hinge region.
  • the Fc part can be removed simply by inco ⁇ oration of a cleavage sequence which can be cleaved with blood clotting factor Xa.
  • Membrane-bound receptors are particularly useful in the formation of fusion proteins. Such receptors are generally characterized as possessing three distinct structural regions: an extracellular domain, a transmembrane domain and a cytoplasmic domain. This invention contemplates the use of one or more of these regions as components of a fusion protein. Examples of such fusion protein technology can be found in International Patent Application Nos. WO94/29458 and WO94/22914.
  • fusion proteins of the present invention may be prepared and used in a variety of forms, for example, chemically synthesized or as recombinant peptides, polypeptides, proteins, fusion proteins or fused peptides.
  • a composition of the present invention may be a synthetic peptide, containing single or multiple copies of the same or different polypeptide of this invention, coupled to a selected carrier protein.
  • one or more polypeptides or fragments thereof as described above may be coupled or fused to a carrier protein, or several may be admixed to create a immunogenic composition.
  • the carrier protein is desirably a protein or other molecule which can enhance the immunogenicity of the selected immunogen.
  • a carrier may be a larger molecule which has an adjuvanting effect.
  • Exemplary conventional protein carriers include, without limitation, E. coli DnaK protein, galactokinase (galK, which catalyzes the first step of galactose metabolism in bacteria), ubiquitin, ⁇ -mating factor, ⁇ -galactosidase, and influenza NS-1 protein.
  • Toxoids i.e., the sequence which encodes the naturally occurring toxin, with sufficient modifications to eliminate its toxic activity
  • diphtheria toxoid and tetanus toxoid may also be employed as carriers.
  • bacterial heat shock proteins e.g., mycobacterial hsp-70 may be used.
  • Glutathione reductase (GST) is another useful carrier.
  • Glutathione reductase is another useful carrier.
  • a polypeptide or fusion protein of the present invention may also be modified to increase its immunogenicity.
  • the polypeptide or fusion protein may be coupled to chemical compounds or immunogenic carriers, provided that the coupling does not interfere with the desired biological activity of either the polypeptide or the carrier.
  • useful immunogenic carriers known in the art include, without limitation, keyhole limpet hemocyanin (KLH); bovine serum albumin (BSA).
  • ovalbumin PPD (purified protein derivative of tuberculin); red blood cells; tetanus toxoid; cholera toxoid; agarose beads; activated carbon; or bentonite.
  • Useful chemical compounds for coupling include, without limitation, dinitrophenol groups and arsonilic acid.
  • the polypeptide or fusion protein antigen may also be modified by other techniques, such as denaturation with heat and/or SDS.
  • one or more polypeptides or fragments of this invention may be covalently linked to a mycobacterial or E. coli heat shock protein 70 (hsp70) [K. Suzue et al, J. Immunol.. 156:873 (1996)].
  • the composition is formed by covalently linking the polypeptide sequence(s) to diphtheria toxoid.
  • polypeptides are assembled as multi-antigenic peptide (MAP) complexes [see, e.g., European Patent Application 0339695, published November 2, 1989] or as simple mixtures of antigenic proteins/peptides and employed to elicit high titer antibodies capable of binding the selected antigen(s) as it appears in the biological fluids of an infected animal or human.
  • MAP multi-antigenic peptide
  • each amino acid sequence may be optionally separated by optional amino acid sequences called "spacers".
  • Spacers are sequences of between 1 to about 4 amino acids which are inte ⁇ osed between two sequences to permit linkage therebetween without adversely effecting the three dimensional structure of the fusion protein. Spacers may also contain restriction endonuclease cleavage sites to enable separation of the sequences, where desired. Suitable spacers or linkers are known and may be readily designed and selected by one of skill in the art. This invention also relates to processes for the preparation of
  • the present invention provides an isolated nucleic acid (polynucleotide) which encodes the highly conserved isolated polypeptide sequences having the amino acid sequences defined above [the sequences of the invention set forth herein].
  • isolated means that it is separated from the chromosome and cell in which it naturally occurs.
  • such polynucleotides can be joined to other polynucleotides, such as DNAs, for mutagenesis, to form fusion proteins, and for propagation or expression in a host, for instance.
  • the isolated polynucleotides. alone or joined to other polynucleotides such as vectors, can be introduced into host cells, in culture or in whole organisms. Introduced into host cells in culture or in whole organisms, such DNAs still would be isolated, because they would not be in their naturally occurring form or environment.
  • Apolynucleotide(s) generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA which encodes a polypeptide of the present invention.
  • the term includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
  • the term also encompasses polynucleotides that include a single continuous region encoding the polypeptide together with additional regions, that also may contain coding and/or non-coding sequences.
  • sequences include mRNAs, DNAs, cDNAs, genomic DNAs and fragments thereof.
  • the polynucleotides may be single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
  • polynucleotide as used herein refers to triple-stranded regions comprising 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 triple-helical region often is an oligonucleotide.
  • polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases.
  • DNAs or RNAs with backbones modified for stability or for other reasons are polynucleotides as that term is intended herein.
  • DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful pu ⁇ oses known to those of skill in the art.
  • polynucleotide as it is employed herein, embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including ter alia simple and complex cells.
  • the invention also relates to, among others, polynucleotides encoding the aforementioned polypeptide fragments, polynucleotides that hybridize to polynucleotides encoding the fragments, particularly those that hybridize under stringent conditions, and polynucleotides, such as PCR primers, for amplifying polynucleotides that encode the fragments.
  • preferred polynucleotides are those that correspond to the preferred fragments, as discussed above.
  • sequences which encode the desired highly conserved or consensus polypeptide as defined above include polynucleotides with a different coding sequence, which, as a result of the redundancy (degeneracy) of the genetic code, encode the same polypeptide or desired fragment thereof of any of the sequences of the invention set forth herein.
  • the particularly preferred embodiments of this aspect of the invention are naturally occurring alleles of the bacterial adhesins which contain the conserved sequences described herein as well as analogs and biologically active and diagnostically or therapeutically useful variants, derivatives, and fragments thereof.
  • Polynucleotides of the present invention which encode the polypeptide of this invention may include, but are not limited to, the coding sequence for the polypeptide,
  • the coding sequence for the polypeptide and additional coding sequences such as transcribed, non-translated sequences that play a role in transcription, and mRNA processing, including splicing and polyadenylation signals, for example, for ribosome binding and stability of mRNA. Coding sequences which provide additional functionalities may also be inco ⁇ orated into the polypeptide.
  • Variant(s) of polynucleotides are polynucleotides that differ in nucleotide sequence from a reference polynucleotide. Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical. Changes in the nucleotide sequence of the variant may be silent. That is, they may not alter the amino acids encoded by the polynucleotide. Where alterations are limited to silent changes of this type, a variant will encode a polypeptide with the same amino acid sequence as the reference.
  • changes in the nucleotide sequence of the variant may alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide.
  • Such nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • the present invention further relates to variants of the herein above-described polynucleotides which encode for fragments, analogs and derivatives of the polypeptides of this invention.
  • a variant of the polynucleotide may be a naturally occurring polynucleotide that encodes the sequences of the invention set forth herein, such as a naturally occurring allelic variant, a variant which occurs in another Proteobacterial species, or a variant that is not known to occur naturally.
  • an allelic variant is an alternate 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.
  • Non-naturally occurring variants of the polynucleotide may be prepared by mutagenesis techniques, including those applied to polynucleotides, cells or organisms.
  • variants in this regard are variants that differ from the aforementioned polynucleotides by nucleotide substitutions, deletions or additions.
  • the substitutions, deletions or additions may involve one or more nucleotides.
  • the variants may be altered in coding or non-coding regions or both. Alterations in the coding regions
  • polypeptides having the amino acid sequence of the sequences of the invention set forth herein, variants, analogs, derivatives and fragments thereof, and fragments of the variants, analogs and derivatives as described above.
  • a polynucleotide of the present invention encoding a highly conserved Proteobacterial sequence may be obtained using standard cloning and screening procedures.
  • the polynucleotide sequences of this invention may be produced by conventional synthetic means or a combination of both techniques.
  • the present invention also includes polynucleotides, wherein the coding sequence for the isolated 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 or which aids in the stability of the polypeptide in a cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell.
  • the polypeptide may be fused in frame to a marker sequence, such as a peptide, which facilitates purification of the fused polypeptide.
  • the marker sequence is a hexa-histidine peptide, such as the tag provided in the pQE-9 vector (Qiagen, Inc.) to provide for purification of the polypeptide fused to the marker in the case of a bacterial host.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • the marker sequence is a hemagglutinin (HA) tag, particularly when a mammalian host, e.g. COS-7 cells, is used.
  • the HA tag corresponds to an epitope derived from influenza hemagglutinin protein, which has been described by Wilson et al, Cell, 1984, 37:767, for instance. Many other such tags are commercially available.
  • polynucleotides of the invention may be used as hybridization probes for isolating other highly conserved sequences from other Proteobacterial species, or to isolate
  • probes generally will comprise at least 15 nucleotides.
  • An example of such a screen comprises labeling an oligonucleotides having a sequence complementary to that of the sequence of the present invention and using it as a probe for hybridize to sequences in a library of Proteobacterial species DNA, or mRNA to determine novel conserved sequences.
  • the polynucleotides which hybridize to the herein above-described polynucleotides in a preferred embodiment encode polypeptides which either retain substantially the same biological function or activity as the isolated or consensus polypeptide encoded by the sequences of the invention set forth herein.
  • polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of compositions, diagnostic methods, treatments and vaccine methods for the diagnosis, treatment or prevention of Proteobacterial infections.
  • 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.
  • Host cells can be genetically engineered to inco ⁇ orate polynucleotides and express polypeptides of the present invention.
  • polynucleotides may be introduced into host cells using well known techniques of infection, transduction, transfection, transvection and transformation. Unless otherwise stated, transformation was performed as described in the method of Graham, F. and Van der Bb, A., Virology, 52:456-457 (1973).
  • polynucleotides may be introduced alone or with other polynucleotides. Such other polynucleotides may be introduced independently, co-introduced or introduced joined to the polynucleotides of the invention. Thus, for instance, polynucleotides of the invention may be transfected into host cells with another, separate polynucleotide encoding a selectable marker, using standard techniques for co-transfection and selection
  • polynucleotides generally will be stably inco ⁇ orated into the host cell genome.
  • the polynucleotides may be joined to a vector or plasmid containing a selectable marker for propagation in a host.
  • the vector construct may be introduced into host cells by the aforementioned techniques.
  • “Plasmids” are genetic elements that are stably inherited without being a part of the chromosome of their host cell. They may be comprised of DNA or RNA and may be linear or circular. They can also encode genes that confer resistance to antibiotics. Plasmids are widely used in molecular biology as vectors used to clone and express recombinant genes.
  • Plasmids generally are designated herein by a lower case p preceded and/or followed by capital letters and/or numbers, in accordance with standard naming conventions that are familiar to those of skill in the art.
  • Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known and readily available to those of skill in the art.
  • those of skill readily may construct any number of other plasmids suitable for use in the invention. The properties, construction and use of such plasmids, as well as other vectors, in the present invention will be readily apparent to those of skill from the present disclosure.
  • a plasmid vector is introduced as DNA in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. Electroporation may also be used to introduce polynucleotides into a host. If the vector is a virus, it may be packaged in vitro or introduced into a packaging cell and the packaged virus may be transduced into cells.
  • a wide variety of techniques suitable for making polynucleotides and for introducing polynucleotides into cells in accordance with this aspect of the invention are well known and routine to those of skill in the art.
  • the vector may be, for example, a plasmid vector, a single or double-stranded phage vector, or a single or double-stranded RNA or DNA viral vector.
  • a plasmid vector for example, a single or double-stranded phage vector, or a single or double-stranded RNA or DNA viral vector.
  • Such vectors may be introduced into cells as
  • 26 polynucleotides, preferably DNA. by well known techniques for introducing DNA and RNA into cells.
  • the vectors in the case of phage and viral vectors may also be and preferably are introduced into cells as packaged or encapsidated virus by well known techniques for infection and transduction.
  • Viral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
  • vectors are those for expression of polynucleotides and polypeptides of the present invention.
  • such vectors comprise cis-acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed.
  • Appropriate trans-acting factors are either supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
  • the vectors provide for specific expression.
  • Such specific expression may be inducible expression or expression only in certain types of cells or both inducible and cell-specific expression.
  • Particularly preferred among inducible vectors are vectors that can be induced for expression by environmental factors that are easy to manipulate, such as temperature and nutrient additives.
  • a variety of vectors suitable to this aspect of the invention, including constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, are well known and employed routinely by those of skill in the art. Presently prokaryotic expression systems are preferred.
  • the engineered host cells can be cultured in conventional nutrient media, which may be modified as appropriate for, inter alia, activating promoters, selecting transformants or amplifying genes.
  • Culture conditions such as temperature, pH and the like, previously used with the host cell selected for expression, generally will be suitable for expression of polypeptides of the present invention as will be apparent to those of skill in the art.
  • a great variety of expression vectors can be used to express a polypeptide of the invention.
  • Such vectors include chromosomal, episomal and virus-derived vectors e.g., vectors derived from bacterial plasmids, bacteriophages, yeast episomes, yeast chromosomal elements, and viruses such as baculoviruses, papova viruses, SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors
  • any vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide in a host may be used for expression in this regard.
  • the appropriate DNA sequence may be inserted into the vector by any of a variety of well-known and routine techniques.
  • a DNA sequence for expression is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction endonucleases and then joining the restriction fragments together using T4 DNA ligase.
  • Procedures for restriction and ligation that can be used to this end are well known and routine to those of skill. Suitable procedures in this regard, and for constructing expression vectors using alternative techniques, which also are well known and routine to those skilled in the art, are set forth in great detail in Sambrook et al.
  • the DNA sequence in the expression vector is operatively linked to appropriate expression control sequence(s), including, for instance, a promoter to direct mRNA transcription.
  • appropriate expression control sequence(s) including, for instance, a promoter to direct mRNA transcription.
  • promoters include the phage lambda PL promoter, the E. coli lac, t ⁇ and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name just a few of the well-known promoters. It will be understood that numerous other promoters useful in this aspect of the invention are well known and may be routinely employed by those of skill in the manner illustrated by the discussion and the examples herein.
  • expression constructs will contain sites for transcription initiation and termination, and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.
  • constructs may contain control regions that regulate as well as engender expression. Generally, in accordance with many commonly practiced procedures, such regions will operate by controlling transcription. Examples include repressor binding sites and enhancers, among others.
  • Vectors for propagation and expression generally will include selectable markers. Selectable marker genes provide a phenotypic trait for selection of transformed host cells. Preferred markers include, but are
  • the vectors may contain additional markers for this pu ⁇ ose.
  • the vector containing the appropriate DNA sequence as described elsewhere herein, as well as an appropriate promoter, and other appropriate control sequences, may be introduced into an appropriate host using a variety of well known techniques suitable for expression therein of a desired polypeptide.
  • appropriate hosts include bacterial cells, such as E. coli, Streptomyces and Salmonella typhitnurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells.
  • Host cells for a great variety of expression constructs are well known, and those of skill will be enabled by the present disclosure to routinely select a host for expressing a polypeptide in accordance with this aspect of the present invention.
  • prokaryotic expression systems and host cells are preferred.
  • the present invention also includes recombinant constructs, such as expression constructs, comprising one or more of the sequences described above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which such a sequence of the invention has been inserted.
  • the sequence may be 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 there are many commercially available vectors suitable for use in the present invention.
  • vectors which are commercially available, are provided by way of example.
  • vectors preferred for use in bacteria are pQ ⁇ 70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNHl ⁇ a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRlT5 available from Pharmacia.
  • eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. These vectors are listed
  • Promoter regions can be selected from any desired gene using vectors that contain a reporter transcription unit lacking a promoter region, such as a chloramphenicol acetyl transferase ("CAT") transcription unit, downstream of a restriction site or sites for introducing a candidate promoter fragment; i.e., a fragment that may contain a promoter.
  • CAT chloramphenicol acetyl transferase
  • introduction into the vector of a promoter-containing fragment at the restriction site upstream of the CAT gene engenders production of CAT activity, which can be detected by standard CAT assays.
  • Vectors suitable to this end are well known and readily available. Two examples of such vectors include pKK232-8 and pCM7.
  • promoters for expression of polynucleotides of the present invention include not only well known and readily available promoters, but also promoters that may be readily obtained by the foregoing technique, using a reporter gene.
  • bacterial promoters suitable for expression of polynucleotides and polypeptides in accordance with the present invention are the E. coli lad and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, PL promoters and the t ⁇ promoter.
  • known eukaryotic promoters suitable in this regard are the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous Sarcoma Virus ("RSV"), and metallothionein promoters, such as the mouse metallothionein-I promoter.
  • Selection of appropriate vectors and promoters for expression in a host cell is a well known procedure and the requisite techniques for construction of expression vectors, introduction of the vector into the host and expression in the host are routine skills in the art.
  • the present invention also relates to host cells containing the above-described constructs.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, a lower eukaryotic cell, such as a yeast cell, or a prokaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the host cell can be effected by calcium
  • Constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
  • Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other 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.
  • recombinant expression vectors will include origins of replication, a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence, and a selectable marker to permit isolation of vector containing cells following exposure to the vector.
  • suitable promoters are those derived from the genes that encode glycolytic enzymes such as 3-phosphoglycerate kinase ("PGK”), -factor, acid phosphatase, and heat shock proteins, among others.
  • PGK 3-phosphoglycerate kinase
  • Selectable markers include the ampicillin resistance gene of E. coli and the t ⁇ l gene of S. cerevisiae.
  • Enhancers are cis-acting elements of DNA, usually from about 10 to 300 bp, that act to increase transcriptional activity of a promoter in a given host cell-type.
  • enhancers include the S V40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • a polynucleotide of the invention encoding the heterologous structural sequence of a polypeptide of the invention generally will be inserted into the vector using standard techniques so that it is operably linked to the promoter for expression.
  • the polynucleotide will be positioned so that the transcription start site is located appropriately
  • the ribosome binding site will be 5' to the AUG that initiates translation of the polypeptide to be expressed.
  • the ribosome binding site will be 5' to the AUG that initiates translation of the polypeptide to be expressed.
  • Appropriate secretion signals may be inco ⁇ orated into the expressed polypeptide for secretion of the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment.
  • the signals may be endogenous to the polypeptide or heterologous.
  • the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions.
  • a region of additional amino acids, particularly charged amino acids may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell during purification or subsequent handling and storage.
  • a region may also be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide.
  • the addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
  • Suitable prokaryotic hosts for propagation, maintenance or expression of polynucleotides and polypeptides in accordance with the invention include Escherichia coli, Bacillus subtilis and Salmonella typhimurium. Various species of Pseudomonas, Streptomyces, and Staphylococcus are also suitable hosts in this regard. Moreover, many other hosts also known to those of skill may be employed in this regard.
  • useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pB22 (ATCC 37017).
  • commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, ison, WI, USA).
  • the pB22 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
  • the host strain Following transformation of a suitable host strain, the host strain is grown to an appropriate cell density. Where the selected promoter is inducible. it is induced by appropriate means (e.g., temperature shift or exposure to chemical inducer) and cells are cultured for an additional period. Cells typically then are harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • appropriate means e.g., temperature shift or exposure to chemical inducer
  • 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.
  • mammalian cell culture systems can be employed for expression, as well.
  • mammalian expression systems include, without limitation, the C127, 3T3, CHO, HeLa, human kidney 293 and BHK cell lines, and the COS-7 line of monkey kidney fibroblasts, described by Gluzman et al., Cell, 1981, 23: 175.
  • Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non-transcribed sequences that are necessary for expression.
  • DNA sequences derived from the SV40 splice sites and the SV40 polyadenylation sites are used for required non-transcribed genetic elements.
  • polypeptide or fusion protein of this invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.
  • HPLC high performance liquid chromatography
  • polynucleotides and polypeptides of the present invention may be used in accordance with the present invention for a variety of applications, particularly in the
  • polypeptides of this invention are preferably used as fusion proteins.
  • the polynucleotides and polypeptides may occur in a composition, such as a media, formulations, solutions for introduction of polynucleotides or polypeptides, for example, into cells, compositions or solutions for chemical or enzymatic reactions, for instance, which are not naturally occurring compositions, and, therein remain isolated polynucleotides or polypeptides within the meaning of that term as it is employed herein.
  • the polypeptides are preferably used as individual peptides, or peptides coupled to a polylysine core (so-called multiple antigenic peptides), and/or labeled peptides.
  • polynucleotides and polypeptides may also be employed in the development of binding molecules that interfere with the binding of the bacterial adhesin to its ligand, and in the use thereof as pharmaceutical agents. Additional applications relate to diagnosis and to treatment of disorders of cells, tissues and organisms. These aspects of the invention are illustrated further by the following discussion.
  • This invention is also related to the use of the polynucleotides described above to detect complementary polynucleotides for use, for example, as a diagnostic reagent. Detection of one of the conserved sequences of a bacterial adhesin identified above provides a diagnostic tool that can add to or define diagnosis of an infection with a proteobacterial species.
  • Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using polymerase chain reaction (PCR) [Saiki et al., Nature, 324: 163-166 (1986)] prior to analysis.
  • RNA or cDNA may also be used in similar fashion.
  • PCR primers complementary to the nucleic acid encoding a polypeptide of this invention can be used to identify and analyze expression of a bacterial adhesin.
  • DNA segments may be employed as probes to detect specific DNA segments.
  • the sensitivity of such methods can be greatly enhanced by appropriate use of PCR or other amplification methods.
  • a sequencing primer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures with fluorescent-tags.
  • Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method [e.g., Cotton et al., Proc. Natl. Acad. Sci., USA, 85:4397-4401 (1985)].
  • the detection of a specific bacterial adhesin DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g., restriction fragment length polymo ⁇ hisms ("RFLP"), PCR, RT-PCR, Northern blotting and Southern blotting, and in situ analysis.
  • restriction enzymes e.g., restriction fragment length polymo ⁇ hisms ("RFLP"), PCR, RT-PCR, Northern blotting and Southern blotting, and in situ analysis.
  • the present invention also relates to diagnostic assays for detecting, qualitatively or quantitatively, the presence of proteobacterial adhesin protein in infected cells and tissues.
  • Assay techniques that can be used to determine levels of a protein, such as conserved polypeptide of the present 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. Among these, ELISAs are frequently preferred.
  • An ELISA assay initially comprises preparing an antibody specific to a polypeptide of this invention or , preferably a monoclonal antibody.
  • a reporter antibody generally is prepared which binds to the monoclonal antibody.
  • the reporter antibody is attached to a detectable reagent such as a radioactive, fluorescent or enzymatic reagent, e.g., horseradish peroxidase enzyme.
  • a sample is removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin.
  • a non-specific protein such as bovine serum albumin.
  • the monoclonal antibody is then incubated in the dish during which time the monoclonal antibodies attach to any
  • a competition assay may also be employed to determine levels of the polypeptide of the present invention in a sample derived from the infected hosts.
  • Such an assay comprises isolating cells which express the polypeptide of the present invention.
  • a test sample containing the polypeptides of the present invention which have been labeled are then added to the purified cells and then incubated for a set period of time.
  • Also added to the reaction mixture is a sample derived from a host which is suspected of containing the polypeptide of the present invention.
  • the reaction mixtures are then passed through a filter which is rapidly washed and the bound radioactivity is then measured to determine the amount of competition for the polypeptides and therefore the amount of the polypeptides of the present invention in the sample.
  • Another competition assay may involve antibodies specific to a polypeptide of the invention, which are attached to a solid support and labeled or and a sample derived from the host are passed over the solid support.
  • the amount of detected label attached to the solid support can be correlated to a quantity of a bacterial adhesin sequence in the sample.
  • Binding molecules refer to molecules, including ligands, that specifically bind to or interact with polypeptides of the present invention. Such binding molecules are a part of the present invention. Binding molecules may also be non-naturally occurring, such as antibodies and antibody-derived reagents that bind specifically to polypeptides of the invention.
  • polypeptides of this invention can also be used as immunogens to produce antibodies thereto.
  • These antibodies can be, for example, polyclonal or monoclonal antibodies.
  • the present invention 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 corresponding to a sequence 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 polypeptide itself. In this manner, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies binding the whole native polypeptide. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique [G. Kohler and C. 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 [Cole et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY, pg. 77-96, Alan R. Liss, Inc., (1985)].
  • antibodies may be employed to isolate or to identify clones expressing the polypeptide or purify the polypeptide of the present invention by attachment of the antibody to a solid support for isolation and/or purification by affinity chromatography.
  • polypeptides of this invention may also be employed to treat or prohibit proteobacterial infections.
  • a polypeptide of this invention can be used to isolate proteins which interact with it, or to identify its ligand when expressed on an infected host cell; and this interaction can be a target for interference.
  • Inhibitors of protein-protein interactions between the conserved Proteobacterial polypeptide of the and other factors could lead to the development of pharmaceutical agents for the treatment of infection by such bacterial species.
  • this invention also provides a method for identification of binding molecules to the conserved polypeptides of this invention.
  • Polynucleotide sequences encoding proteins for binding molecules to the polypeptides of this invention can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting. Such methods are described in many laboratory manuals such as, for instance, Coligan et al., CURRENT PROTOCOLS EN IMMUNOLOGY 1, Chapter 5 (1991).
  • the yeast two-hybrid system provides methods for detecting the interaction between a first test protein and a second test protein, in vivo, using reconstitution of the activity of a transcriptional activator.
  • the method is disclosed in U.S. Patent No. 5,283,173; reagents are available from Clontech and Stratagene. Briefly, a polynucleotide sequence encoding a conserved polypeptide of this invention is fused to a Gal4 transcription factor DNA binding domain and expressed in yeast cells. cDNA library members obtained from cells of interest are fused to a transactivation domain of Gal4. cDNA clones which express proteins which can interact with the conserved polypeptide sequence will lead to reconstitution of Gal4 activity and transactivation of expression of a reporter gene such as Gall-lacZ.
  • An alternative method involves screening of lambda gtl 1 or lambda ZAP (Stratagene) or equivalent cDNA expression libraries with recombinant polypeptides of this invention.
  • Recombinant polypeptides of this invention are fused to small peptide tags such as FLAG, HSV or GST.
  • the peptide tags can possess convenient phosphorylation sites for a kinase such as heart muscle creatine kinase or they can be
  • Recombinant polypeptides of this invention can be phosphorylated with ⁇ ⁇ [P] or used unlabeled and detected with streptavidin or antibodies against the tags.
  • Lambda gtl 1 cDNA expression libraries are made from cells of interest and are incubated with the recombinant polypeptide of this invention, washed and cDNA clones which interact with the polypeptide are isolated. Such methods are routinely used by skilled artisans. See, e.g., Sambrook et al, cited above.
  • Another method for obtaining molecules that bind the polypeptides of the present invention involves the screening of a mammalian expression library.
  • cDNAs are cloned into a vector between a mammalian promoter and polyadenylation site and transiently transfected in COS or 293 cells. Forty-eight hours later, the binding protein is detected by incubation of fixed and washed cells with labeled polypeptide.
  • the polypeptide of this invention is iodinated, and any bound polypeptide is detected by autoradiography. See Sims et al, Science, 1988, 24 . :585-589 and McMahan et al, EMBO J., 1991, 10:2821-2832.
  • pools of cDNAs containing the cDNA encoding the binding protein of interest can be selected and the cDNA of interest can be isolated by further subdivision of each pool followed by cycles of transient transfection, binding and autoradiography.
  • the cDNA of interest can be isolated by transfecting the entire cDNA library into mammalian cells and panning the cells on a dish containing a polypeptide of this invention bound to the plate. Cells which attach after washing are lysed and the plasmid DNA isolated, amplified in bacteria, and the cycle of transfection and panning repeated until a single cDNA clone is obtained. See Seed et al, Proc. Natl. Acad. Sci.
  • binding protein If the binding protein is secreted, its cDNA can be obtained by a similar pooling strategy once a binding or neutralizing assay has been established for assaying supernatants from transiently transfected cells. General methods for screening supernatants are disclosed in Wong et al, Science, 1985, 228:810-815.
  • Another method of identifying a binding molecule involves isolation of proteins interacting with a polypeptides of this invention directly from cells infected with a proteobacterial species. Fusion proteins of a polypeptide of this invention with GST or small peptide tags are made and immobilized on beads. Biosynthetically labeled polypeptide of this invention unlabeled protein extracts from the cells of interest
  • the cells can be treated with agents that induce a functional response such as tyrosine phosphorylation of cellular proteins.
  • agents that induce a functional response such as tyrosine phosphorylation of cellular proteins.
  • An example of such an agent would be a growth factor or cytokine such as interleukin-2.
  • Another method for identifying binding molecules is immunoaffinity purification.
  • a recombinant polypeptide of this invention is incubated with a labeled polypeptide of this invention, unlabeled cell extracts and immunoprecipitated with antibodies to the polypeptide of this invention.
  • the immunoprecipitate is recovered with protein A-Sepharose and analyzed by SDS-PAGE. Unlabelled proteins are labeled by biotinylation and detected on SDS gels with streptavidin. Binding partner proteins are analyzed by microsequencing. Further, standard biochemical purification steps known to those skilled in the art may be used prior to microsequencing.
  • Yet another alternative method involves screening of peptide libraries for binding partners.
  • Recombinant tagged or labeled polypeptides of this invention are used to select peptides from a peptide or phosphopeptide library which interact with the polypeptides of the invention. Sequencing of the peptides leads to identification of consensus peptide sequences which might be found in interacting proteins.
  • Another method for identifying compounds which antagonize the binding of a bacterial adhesin to its ligand comprise the steps of providing a sample of the ligand or a cell which expresses the ligand immobilized on a support; contacting the sample with a known amount of a polypeptide of this invention and a known amount of a test compound; washing unbound materials from the sample; contacting the sample with a labeled reagent which binds to the polypeptide; washing unbound reagent from the sample; measuring the amount of signal generated by the label.
  • the amount of signal generated is inversely proportional to the ability of the test compound to disrupt or inhibit binding between the polypeptide and the ligand; and identifying those test compounds as antagonists which are associated with a low signal.
  • binding partners or antagonists identified by any of these methods or other methods can be used in the assay method of the invention.
  • Assaying for the presence of the native conserved polypeptide/binding partner complex is accomplished by, for example, the yeast two-hybrid system, ELISA or immunoassays using antibodies specific for the complex.
  • test substances which interrupt or inhibit formation of a conserved Proteobacterial sequence/binding partner interaction, a decreased amount of complex will be determined relative to a control lacking the test substance.
  • Assays for free polypeptide or binding partner are accomplished by, for example, ELISA or immunoassay using specific antibodies or by incubation of radiolabeled or with cells or cell membranes followed by centrifugation or filter separation steps. In the presence of test substances which interrupt or inhibit formation of the interaction between a Proteobacterial conserved sequence and a binding partner, an increased amount of free conserved polypeptide or free binding partner will be determined relative to a control lacking the test substance.
  • Polypeptides of the invention also can be used to assess or binding capacity of the polypeptides of this invention or their binding molecules in cells or in cell-free preparations.
  • Other methods for detecting ligands (agonists or antagonists) for the polypeptides of this invention include the yeast based technology as described in U. S. Patent No. 5,482,835.
  • Examples of potential ligands include antibodies or, in some cases, oligonucleotides which bind to the polypeptide.
  • Potential antagonists also include proteins which are closely related to a ligand of the polypeptide of this invention, i.e., a fragment of the ligand.
  • a potential antagonist also includes an antisense construct prepared through the use of antisense technology.
  • Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both methods of which are based on binding of a polynucleotide to DNA or RNA.
  • the 5 ' coding portion of the polynucleotide sequence which encodes for the polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 24 base pairs in length [see, I_ee et al., Nucl. Acids Res., 3:173
  • oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA is expressed in vivo to inhibit the binding of the conserved sequence to its ligand in infected cells.
  • Another potential antagonist is a small molecule which binds to a conserved Proteobacterial sequence of this invention, making it inaccessible to ligands such that normal biological activity is prevented.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules.
  • the small molecules may also bind the interaction protein to the sequence.
  • One exemplary method for generating a small molecule which antagonizes the binding between a proteobacterial adhesin and its ligand involves analyzing an antibody to a polypeptide as described above in a computer modelling program.
  • Potential antagonists also include fragments of the polypeptides of the invention, which bind to the ligand and prevent the ligand from interacting with the conserved Proteobacterial sequence in infected cells. It is desirous to find compounds and drugs which can inhibit the function of the conserved Proteobacteral sequence.
  • agonists or antagonists for the polypeptides of this invention are employed for diagnostic, therapeutic and prophylactic pmposes for the diagnosis or treatment of infection by the Proteobacterial species identified herein, among others.
  • a process for diagnosing a bacterial infection comprises contacting a biological sample from a possibly infected subject with a labeled antibody which binds to the conserved polypeptide of the sequences of the invention set forth herein; and measuring the signal generated by the label with a suitable assay, wherein detection of the signal indicates the presence of an adhesin molecule from the bacteria.
  • a diagnostic reagent of this invention comprises a composition capable of binding to one of the polypeptides of the sequences of the invention set forth herein, the composition associated with a detectable label.
  • polypeptides, fusion proteins and/or other reagents of the invention identified above are associated with conventional labels which are capable, alone or in concert with other compositions or
  • the labels may be interactive to produce a detectable signal. Most desirably, the label is detectable visually, e.g. colorimetrically.
  • a variety of enzyme systems have been described in the art which will operate to reveal a colorimetric signal in an assay. As one example, glucose oxidase (which uses glucose as a substrate) releases peroxide as a product. Peroxidase, which reacts with peroxide and a hydrogen donor such as tetramethyl benzidine (TMB) produces an oxidized TMB that is seen as a blue color.
  • TMB tetramethyl benzidine
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • hexokinase in conjunction with glucose-6-phosphate dehydrogenase which reacts with ATP, glucose, and NAD+ to yield, among other products, NADH that is detected as increased absorbance at 340 nm wavelength.
  • Other label systems that may be utilized in the methods of this invention are detectable by other means, e.g., colored latex microparticles [Bangs Laboratories, Indiana] in which a dye is embedded may be used in place of enzymes to form conjugates with the antibodies and provide a visual signal indicative of the presence of the resulting complex in applicable assays.
  • Still other labels include fluorescent compounds, radioactive compounds or elements.
  • Detectable labels for attachment to polypeptides, proteins, and antibodies useful in diagnostic assays of this invention may be easily selected from among numerous compositions known and readily available to one skilled in the art of diagnostic assays.
  • the methods and antibodies of this invention are not limited by the particular detectable label or label system employed.
  • any number of conventional protein assay formats may be designed to utilize the isolated polypeptides, fusion proteins, antibodies, binding moleucles or their nucleic acid sequences or anti-sense sequences of this invention for the detection of Proteobacterial infection in animals and humans.
  • This invention is thus not limited by the selection of the particular assay format, and is believed to encompass assay formats which are known to those of skill in the art.
  • kits are useful for diagnosing infection with Proteobacterial species in a human or an animal sample.
  • kits are useful for diagnosing infection with Proteobacterial species in a human or an animal sample.
  • a diagnostic kit contains an antigen of this invention and/or at least one polypeptide, fusion
  • kits may contain a simple mixture of such antigens or sequences, or means for preparing a simple mixture.
  • kits can include microtiter plates to which the Proteobacterial antigen proteins or antibodies or nucleic acid sequences of the invention have been pre-adsorbed, various diluents and buffers, labeled conjugates for the detection of specifically bound antigens or antibodies, or nucleic acids and other signal-generating reagents, such as enzyme substrates, cofactors and chromogens.
  • Other components of these kits can easily be determined by one of skill in the art.
  • Such components may include polyclonal or monoclonal capture antibodies, antigen of this invention, or a cocktail of two or more of the antibodies, purified or semi-purified extracts of these antigens as standards, MAb detector antibodies, an anti-mouse or anti-human antibody with indicator molecule conjugated thereto, an ELISA plate prepared for abso ⁇ tion, indicator charts for colorimetric comparisons, disposable gloves, decontamination instructions, applicator sticks or containers, and a sample preparator cup.
  • Such kits provide a convenient, efficient way for a clinical laboratory to diagnose Proteobacterial infection.
  • this invention provides an immunogenic composition useful as a vaccine to prevent infection by a proteobacterial species comprising in a pharmaceutically acceptable carrier, at least one component described above, such as, a polypeptide derived from a conserved sequence of a Proteobacterial species, e.g., the sequences of the invention set forth herein; an amino acid sequence at least 50% identical to the polypeptide sequence as determined by a sequence comparison algorithm, which sequence binds the ligand of the polypeptide sequence.
  • these polypeptides of the present invention are employed as fusion proteins comprising the Proteobacterial polypeptide described above fused in frame to a second protein for vaccine use. Such fusion proteins are described in detail above.
  • the polypeptides of this invention are administered, desirably as fusion proteins, to develop in a mammalian subject in vivo, antibodies to the conserved polypeptides sequences of the infecting Proteobacterial
  • polypeptides of this invention are useful as vaccine components. These polypeptides are administered in an amount effective to induce a humoral or cellular immune response against the invading bacteria in a manner so as to inhibit the infection by blocking binding of ligands to the conversed polypeptide of the invention.
  • a small molecule which binds the ligand of the polypeptide sequence; an antibody which binds the polypeptide sequence; or an anti- idiotype antibody of the aforementioned antibody may be employed in vaccine compositions or in therapeutic compositions.
  • an optional adjuvant may be included of which many types are available for selection by one of skill in the pharmaceutical arts.
  • this invention additionally provides a method of treating an infection by a Proteobacteral species bacteria which comprises administering to a subject an inhibitor compound (antagonist) as herein above-described along with a pharmaceutically acceptable carrier in an amount effective to inhibit the spread of infection by blocking binding of ligands to the conversed polypeptide of the invention.
  • a binding molecule preferably an antibody, developed as described above, may be employed as a passive vaccine to prevent infection by a Proteobacteral species.
  • a mammalian subject is administered an antibody or cocktail of antibodies in a suitable pharmaceutical carrier and with optional adjuvants to the polypeptides of this invention prior to infection to provide passive prophylaxsis whenever exposure to such bacterial species is contemplated.
  • These antibodies are administered in an amount effective to inhibit the spread of infection by blocking binding of ligands to the conversed polypeptide of the invention.
  • polypeptides of the invention and compounds which bind or inhibit interaction between the Proteobacterial conserved sequences and their ligands, may be employed in combination with a suitable pharmaceutical, physiologically acceptable carrier.
  • a suitable pharmaceutical, physiologically acceptable carrier for example, one such vaccine composition may be formulated to contain a carrier or diluent and one or more of the polypeptide/fusion protein or multimeric
  • Suitable pharmaceutically acceptable carriers facilitate administration of the proteins but are physiologically inert and/or nonharmful.
  • Carriers may be selected by one of skill in the art. Such carriers include but are not limited to, sterile saline, phosphate, buffered saline, dextrose, sterilized water, glycerol, ethanol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil. olive oil, sesame oil, and water and combinations thereof.
  • the carrier or diluent may include a time delay material, such as glycerol monostearate or glycerol distearate alone or with a wax.
  • slow release polymer formulations can be used. The formulation should suit the mode of administration. Selection of an appropriate carrier in accordance with the mode of administration is routinely performed by those skilled in the art.
  • the vaccine composition may further contain adjuvants, preservatives, chemical stabilizers, or other antigenic proteins.
  • stabilizers, adjuvants, and preservatives are optimized to determine the best formulation for efficacy in the target human or animal.
  • Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallade, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.
  • Suitable stabilizing ingredients which may be used include, for example, casamino acids, sucrose, gelatin, phenol red, N-Z amine, monopotassium diphosphate, lactose, lactalbumin hydrolysate, and dried milk.
  • One or more of the above described vaccine components may be admixed or adsorbed with a conventional adjuvant.
  • the adjuvant is used to attract leukocytes or enhance an immune response.
  • adjuvants include, among others, Ribi, mineral oil and water, aluminum hydroxide, Amphigen, Avridine, L121/squalene, D- lactide-polylactide/glycoside, pluronic plyois, muramyl dipeptide, killed Bordetella, and saponins, such as Quil A.
  • Other vaccinal antigens originating from other bacterial species may also be included in these compositions.
  • Polypeptides and other compounds of the present invention which inhibit the interaction between the conserved sequence of the Proteobacterial species and its ligand may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
  • other agents useful in treating a Proteobacterial infection e.g., antibiotics or immunostimulatory agents and cytokine regulation elements, are expected to be useful in preventing, reducing
  • compositions of this invention may operate in concert with the compositions of this invention.
  • the invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
  • Infection by the Proteobacterial species may be partially or completely ameliorated by the systemic clinical administration of the polypeptides/antibodies of this invention, or by administration as a vaccine and again as multiple boosters, as required.
  • This administration can be through the administration of peptides agonists or antagonists synthesized from recombinant constructs of polynucleotides encoding the polypeptide of this invention or from peptide chemical synthesis [see, e.g., Woo et al, Protein Engineering, 3:29-37 (1989)].
  • the pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes, among others.
  • a human or an animal may be vaccinated against Proteobacterial infection by administering an effective amount of such a composition.
  • An "effective amount" is defined as an amount of antigen that is effective in a route of administration to provide a vaccinal benefit, i.e., protective immunity. Such an amount may be between about 1 ng to 1000 mg protein, and more preferably, 0.05 ⁇ g to 1 mg per mL of protein; or 0.05 to about 1000 ⁇ g/mL of a polypeptide or fusion protein of the invention.
  • a suitable dosage may be about 1.0-5.0 mL of a vaccine composition.
  • Suitable dosage adjustments and the need for any boosters may be made by the attending physician or veterinarian depending upon the age, sex, weight and general health of the human or animal patient, as well as the level of immune response desired.
  • the vaccine may be administered by any suitable route.
  • such a composition is administered parenterally, preferably intramuscularly or subcutaneously.
  • it may also be formulated to be administered by any other suitable route, including orally or topically. Routes of administration may be combined, if desired, or
  • the vaccine may be a DNA vaccine, which includes a nucleotide sequence encoding one or more of the polypeptides or fusion proteins of this inveniton, optionally under the control of regulatory sequences.
  • the antigen- encoding DNA may be carried in a vector, e.g., a viral vector.
  • a suitable vector- based treatment contains between 1x10 " " pfu to lxlO 1" pfu per dose.
  • the dose, timing and mode of administration of these compositions may be determined by one of skill in the art. Such factors as the age, and physical condition of the vaccinate may be taken into account in determining the dose, timing and mode of administration of the immunogenic or vaccine composition of the invention.
  • the pharmaceutical compositions generally are administered in an amount effective for treatment or prophylaxis of infection by a bacterial species described herein.
  • the amount employed of the subject polypeptide or binding compound will vary with the manner of administration, the employment of other active compounds, and the like. Another conventional general range is about 1 ⁇ g to 100 ⁇ g.
  • the amount of compound employed will be determined empirically, based on the response of cells in vitro and response of experimental animals to the subject polypeptides or formulations containing the subject polypeptides.
  • the compositions are administered in an amount of at least about 10 ⁇ g/kg body weight. In most cases they will be administered in an amount not in excess of about 8 mg/kg body weight per day.
  • the administered dose is from about 10 ⁇ g/kg to about 1 mg/kg body weight, daily. It will be appreciated that optimum dosage will be determined by standard methods for each treatment modality and indication, taking into account the indication, its severity, route of administration, complicating conditions and the like.
  • size separation of fragments in the examples below is carried out using standard techniques of agarose and polyacrylamide gel electrophoresis ("PAGE") as described in Sambrook et al and numerous other references, such as D. Goeddel et al, Nucleic Acids Res., 8: 4057 (1980) (i.e., using 8 percent polyacrylamide gel).
  • ligations are accomplished using standard buffers, incubation temperatures and times, e.g., approximately 10 units of T4 DNA ligase (“ligase”) per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragments to be ligated.
  • a polypeptide corresponding to the sequences of the invention set forth herein is synthesized as described below.
  • the amino acid sequence of this immunogen is synthesized by solid phase methodology on polypropylene pegs according to the methods of H. M. Geysen et al, J. Immunol. Meth., 102:259 (1987), with an N-terminal cysteinyl being inco ⁇ orated to facilitate coupling to a carrier protein.
  • the N-terminus is left as a free amine and the C-terminus was amidated in the immunizing polypeptides.
  • Immunizing polypeptides are generally purified to greater than 95% purity by reverse phase HPLC, and purity is further confirmed by mass spectometry (MS).
  • immunizing polypeptides are covalently coupled to diphtheria toxoid (DT) carrier protein via the cysteinyl side chain by the method of A. C. J. Lee et al, Molec. Immunol., 1_7:749 (1980), using a ratio of 6-8 moles peptide per mole of diphtheria toxoid.
  • DT diphtheria toxoid
  • the polypeptide conjugates are taken up in purified water and emulsified 1 : 1 with complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (EFA)
  • Total volume per immunized rabbit is 1 ml. and this contains 100 ⁇ g of peptide coupled to DT.
  • the DNA sequence encoding a polypeptide or fusion protein of this invention is cut from a Bluescript plasmid using the restriction enzyme sites corresponding to the restriction enzyme sites on the bacterial expression vector pBluescript SK (+/-) phagemid (Stratagene, Inc.).
  • pBluescript SK (+/-) phagemid encodes antibiotic resistance (Ampr), a bacterial origin of replication (ori), an ⁇ -galactosidase promoter operator, and other regulatory sequences [GENBANK 52325].
  • Plasmid GEX-tl [Pharmacea, Uppsala, Sweden] is then digested with EcoRI and Xhol and the polynucleotide sequence encoding a polypeptide sequence of this invention is ligated into the digested plasmid.
  • the polypeptide-encoding sequence is inserted in frame with the sequence encoding for the glutathione S transferase gene in this commercially available plasmid.
  • This plasmid is designed to generate fusion of the inserted polypeptide-encoding sequence, with GST.
  • the ligation mixture is then used to transform E. coli strain SOLR (Stratagene) by conventional techniques.
  • polypeptide-GST is purified using GST Sephadex (Pharmacea) according to manufacturer's instructions.
  • Enzyme assay procedures for identifying agonists and antagonists of a polypeptide of this invention include assays which use the FLASHPLATE system (DuPont), as follows.
  • the phosphorylation reaction was performed in the plate using a total volume of 60 ⁇ L per well containing 33 mM Tris-HCl (pH 7.4), 17 mM MgCl 2 , 33 ⁇ M ATP, 0.7 mM DTT, 0.25 ⁇ Ci of [ ⁇ PJ-ATP (DuPont NEG-302H), 20 ⁇ g of and varying amounts of purified polypeptide or fusion protein.
  • the plate is incubated overnight at 30EC. Following aspiration of the solution, the wells are rinsed lx with 250 ⁇ L per well of 10 mM sodium pyrophosphate/PBS which reduces non-specific binding. The plate is counted on a Packard TopCount.
  • the protein immobilized directly onto FlashPlate serves as a functional substrate for the conserved Proteobacterial sequence.
  • the reaction only require a single pyrophosphate rinse to remove unreacted [ ⁇ 3 P]-ATP and cell lysate from the wells.
  • Background counts in wells containing no Proteobacterial sequence has a characteristic count and a signal to noise ratio. This ratio increases as the amount of interaction between the antibody on the plate and the polypeptide in the cell lysate increases.
  • Immobilized substrate at 750 ng/well can be phosphorylated in a dose dependent fashion, thus allowing quantitation of binding activity.
  • Coating the plate with an antibody against the polypeptide is also efficient in enabling the bound substrate to be phosphorylated by the Proteobacteral
  • reaction is dose dependent with respect to the amount of Proteobacterial sequences added.
  • an enzyme assay Such assays enable one to insert into the system an unknown compound, which can inhibit the binding reaction by interacting with the polypeptide of the invention or with its ligand expressed by an infected cell.
  • the choice of format depends upon the sensitivity required and the pu ⁇ ose of the assay. Regardless of format, such enzyme assays are advantageous both for automation and for high throughput screening.
  • a Proteobacterial species e.g., Yersinia pestis
  • the rabbits are randomized into two groups.
  • Each rabbit of group 1 (control group) is immunized with 0.4 mg diphtheria toxoid (Commonwealth Scrum Laboratories, Victoria, Australia) with 0.25 mg threonyl muramyl dipeptide (T-MDP) in 0.5 ml water, this being emulsified with 0.5 ml MF75 adjuvant (Chiron Co ⁇ , Emeryville CA).
  • Each rabbit of group 2 (test group) is immunized with 0.1 mg of the synthetic polypeptide of the invention (e.g., the sequences of the invention set forth herein) in which R is H and R " is an amide coupled to 0.4 mg diphtheria toxoid [A. C. Lee et al, Mol. Immunol., 17:749 (1980)].
  • the conjugate is dissolved in 0.5 ml water containing 0.25 mg T-MDP and emulsified with 0.5 ml MF75 adj
  • Each rabbit is immunized at day 0 and day 28 (week 4) with two 0.5 ml intramuscular injections at two distinct sites.
  • day 42 week 6
  • serums are drawn and tested by ELISA for binding to a sequence of the invention set forth herein, as described above in Example 1. This assay indicates the background titers of the control rabbits and the titers of the test group.
  • Plasma is drawn in EDTA at weeks 2, 4 and 8, and copies of viral RNA per ml of plasma were measured by RT-PCR.
  • Yersinia pestis in control animals causes a characteristic infection.
  • Rabbits immunized with a synthetic polypeptide according to this invention that induce antibodies to the conserved sequence of the Yersinia pestis YadA of the challenge bacteria are anticipated to show, by comparison with control immunized rabbits, a reduction in bacterial levels in plasma after challenge, with inhibition being still detectable in the plasma bacterial levels thereafter. This shows that bacterial infection was inhibited in the presence of antibodies to the YadA protein and suggests that a similar effect would prevail in other infected mammals.
  • Fig. 2C All YadA-like sequences have a conserved C-terminal region, which presumably anchors these proteins to the outer membrane (Fig. 2C). It consists of a short coiled-coil segment (Fig. 1A) and four transmembrane ⁇ -strands, as judged from secondary structure prediction and comparisons to a profile of porin ⁇ -strands (Baldermann et al, 1998; A. Lupas and H. Engelhardt, unpublished). The ⁇ -strands are most similar to the equivalent C-terminal strands of eight-stranded porins (Baldermann et al, 1998) and autotransporters (Loveless and Saier, 1997), which include many adhesins
  • Yersinia Ail such as Yersinia Ail, neisserial opacity proteins, Escherichia coli AidA. and Bordetella pertactin (Fig. 2C).
  • the similarity includes a nearly invariant glycine, but the reasons for its conservation are unclear to us and do not appear to be illuminated by the recently published structure of the OmpA transmembrane domain (Pautsch and Schulz, 1998).
  • the amphipathic nature of the ⁇ -strands suggests that they form a solvent- accessible pore in the YadA oligomer. If so, one may envisage that - like autotransporters - YadA-like proteins mediate their own passage through the outer membrane. This possibility has several interesting implications. For example, a pore of sufficient size to translocate a polypeptide chain would require at least a trimer, probably a tetramer.
  • the polypeptide segments connecting the transmembrane ⁇ - strands to the conserved coiled-coil domain would be running through the pore, presumably in extended conformation, with the coiled-coil domain plugging the outer opening of the pore. It is interesting to note in this context that the C-terminal end of the
  • 55 coiled-coil domain is hydrophobic and mainly composed of a residue with a minimal side- chain - alanine (Fig. 2C).
  • Fig. 2C The conserved nature of the entire C-terminal region would imply that all proteins of the YadA family have the same membrane-bound structure and therefore also the same number of subunits per oligomer.
  • Sinorhizobium meliloti 1021 Sme genomic Stanford ( putative fragments ol the same gene
  • Salmonella cntci iltdis LK Sen genomic U ol Illinois S 1 same gene in all loin Salmonella species, contains 10 copies ol the neck scq
  • Yersinia enterocolitica Ye GenBank 2 from plasmids pYV647 l/76 and pYV808 l
  • Yersinia pcslis CO-92 biovar O ⁇ entalis Ypc genomic Sanger 5 YadA gene is Irameshilted two YadA like genes contiguous on contig 653
  • Hi3 GenBank 2 Hia gene is frameshifted; incomplete gene resembles YadA Pasteurella multocida PM70 Pmu genomic U of Minnesota ' 2 Pmu 70b contains a unique repeat motif leudomonas group
  • Moraxella catarrhalis 035E Mca GenBank 2 UspA2 has a unique head sequence
  • Salmonella paratyphi A ATCC 9150 19646 1 ITNLAAGTLAADSTDAVNGSQL 2 LTNLAAGTLAADSTDAVNGSQL 3 ITNLAAGTLTADSTDAVNGSQL 4 ITNVAAGELSEESTDAVNGSQL
  • Yersinia enterocolitica plasmid pYV6471/76 YadA LTHLAAG— T DTDAVNVAQL plasmid pYV8081 YadA LTHLAAG— TKDTDAVNVAQL
  • VSNVADG LQATDAVNLRQL en 4 ITNVAPA— TQGTDAVNFDQL o Actinobacillus act ⁇ nomycetcmcom ⁇ tans(Aac) HK 165 I contig 220 1 ITQVADG— VNDKDAVNKSQL
  • Pasteurella multocida Pmu

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  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne des polypeptides isolés qui son conservés dans des domaines extracellulaires d'eubactéries identifiés dans cinq pathogènes des branches bêta et gamma de probactéries. Ces polypeptides, seuls ou en tant que protéines hybrides avec une deuxième protéine, sont utiles dans la génération d'anticorps et d'autres antagonistes. Les peptides, protéines hybrides, et anticorps sont utiles an tant que constituants de vaccin ou en tant qu'agents thérapeutiques contre les infections bactériennes ou en tant que réactifs de diagnostic. Lesdits polypeptides sont également utiles dans des procédés de criblage pour d'autres agonistes et antagonistes utilisables en diagnostic, en thérapie et en tant que vaccins.
PCT/US2000/009866 1999-04-13 2000-04-13 Motif d'adhesine conserve et ses procedes d'utilisation WO2000061165A1 (fr)

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US60/129,073 1999-04-13

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1252182A1 (fr) * 2000-01-25 2002-10-30 The University Of Queensland PROTEINES COMPRENANT DES REGIONS CONSERVEES DE L'ANTIGENE DE SURFACE NEISSERIA MENINGITIDIS NhhA
US20100190710A1 (en) * 2007-07-06 2010-07-29 Valorisation Hsj, Societe En Commandite Il-23 receptor antagonists and uses thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998028333A2 (fr) * 1996-12-20 1998-07-02 The Board Of Regents, The University Of Texas System ANTIGENES USPA1 ET USPA2 DE $i(MORAXELLA CATARRHALIS)

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998028333A2 (fr) * 1996-12-20 1998-07-02 The Board Of Regents, The University Of Texas System ANTIGENES USPA1 ET USPA2 DE $i(MORAXELLA CATARRHALIS)

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Title
SKURNIK ET AL.: "Analysis of the yopA gene encoding the Yop1 virulence determinants of Yersinia spp", MOLECULAR MICROBIOLOGY,, vol. 3, 1989, pages 517 - 529, XP002930961 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1252182A1 (fr) * 2000-01-25 2002-10-30 The University Of Queensland PROTEINES COMPRENANT DES REGIONS CONSERVEES DE L'ANTIGENE DE SURFACE NEISSERIA MENINGITIDIS NhhA
EP1252182A4 (fr) * 2000-01-25 2005-03-02 Univ Queensland PROTEINES COMPRENANT DES REGIONS CONSERVEES DE L'ANTIGENE DE SURFACE NEISSERIA MENINGITIDIS NhhA
US7947291B2 (en) 2000-01-25 2011-05-24 The University Of Queensland Modified surface antigen
EP2395013A3 (fr) * 2000-01-25 2012-03-21 The University of Queensland Protéines comprenant des régions conservées d'antigène de surface NhhA de Neisseria meningitidis
US8367070B2 (en) 2000-01-25 2013-02-05 The University Of Queensland Modified surface antigen
US8383790B2 (en) 2000-01-25 2013-02-26 The University Of Queensland Modified surface antigen
US20100190710A1 (en) * 2007-07-06 2010-07-29 Valorisation Hsj, Societe En Commandite Il-23 receptor antagonists and uses thereof

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