WO2000037493A2 - Vaccin - Google Patents

Vaccin Download PDF

Info

Publication number
WO2000037493A2
WO2000037493A2 PCT/EP1999/010297 EP9910297W WO0037493A2 WO 2000037493 A2 WO2000037493 A2 WO 2000037493A2 EP 9910297 W EP9910297 W EP 9910297W WO 0037493 A2 WO0037493 A2 WO 0037493A2
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
polynucleotide
genes
seq
sequence
Prior art date
Application number
PCT/EP1999/010297
Other languages
English (en)
Other versions
WO2000037493A3 (fr
Inventor
Alex Bollen
Alain Fauconnier
Edmond Godfroid
Original Assignee
Universite Libre De Bruxelles
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universite Libre De Bruxelles filed Critical Universite Libre De Bruxelles
Priority to EP99969578A priority Critical patent/EP1140996A2/fr
Priority to JP2000589562A priority patent/JP2002534960A/ja
Priority to CA002356764A priority patent/CA2356764A1/fr
Priority to AU29037/00A priority patent/AU2903700A/en
Publication of WO2000037493A2 publication Critical patent/WO2000037493A2/fr
Publication of WO2000037493A3 publication Critical patent/WO2000037493A3/fr
Priority to HK02101214.6A priority patent/HK1041490A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • C07K14/235—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bordetella (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • This invention relates to a general method for detecting pathogenic strains of bacteria that harbour a type III secretion system, and characterising regions of the chromosome of said strain where virulence genes reside. More particularly, this invention relates to the method as applied to the pathogen Bordetella pertussis. Furthermore, the invention relates to newly identified polynucleotides within these regions, virulent polypeptides encoded by them and to the use of such polynucleotides and polypeptides, and to their production.
  • Type III secretion systems Pathogenic bacteria invade many different niches in a broad host range and cause a wide variety of syndromes. It is due to this fact that it was believed previously that each disease might be induced by a distinct molecular mechanism. However, the spectrum of such mechanisms is not as broad as first imagined; rather, bacteria exploit a number of common molecular tools to achieve a range of goals. Among these tools are type III secretion systems, which provide a means for bacteria to target virulence factors directly at host cells. These factors then tamper with host cell functions to the pathogens' benefit.
  • the type III export system is responsible for secretion of Salmonella and Shigella invasion and virulence factors, Enteropathogenic Escherischia coli (EPEC) signal transduction molecules, virulence factors in several plant pathogens (for instance Xanthomonas campestris pv. vesicatoria [Fenselau et al., 1992]) and Yops proteins in Yersinia.
  • Yops export mechanism has been the most intensively investigated type III secretion apparatus (see for instance: Allaoui et al., 1994; Bergman et al., 1994).
  • Ysc/Lcr proteins encoded by the virulence plasmid pYV
  • the pYV plasmid codes for the Yops proteins which are the secreted substrates and appear as the actual effectors of virulence.
  • Pathogenicity islands have emerged as a novel theme in the field of bacterial virulence. Although they can comprise type III secretion systems they do not exlusively do so.
  • virulence genes resided on plasmids. However, numerous virulence genes were also found on the chromosome. Surprisingly, the chromosomal virulence genes are also often clustered in functionally related groups. Such groups of virulence genes gave rise to the concept of pathogenicity islands (Pais) which can be defined as compact, distinct genetic units carrying virulence genes. These units, often flanked by direct repeats, occupy large chromosomal regions (often > 30 kb) and are present in pathogenic strains, whilst being absent or sporadically distributed in less-pathogenic (or non-pathogenic) strains of a bacterial species. These DNA segments are frequently associated with tRNA genes and/or insertion sequence (IS) elements at their boundaries. In addition, their G+C content often differs from that of host bacterial DNA, suggesting a foreign origin.
  • Pathogenicity islands have been discovered in an increasing number of bacterial pathogens, including different categories of E. coli, Salmonella typhimurium, Yersinia spp, Helicobacter pylori, Vibrio cholera etc.
  • the first intensively studied pathogenicity islands were Pai I and Pai II, which encode the haemolysin determinants of uropathogenic E. coli. These two Pais, are flanked by direct repeats and can be deleted from the chromosome at frequencies of 10 "4 , resulting in non- virulent mutant strains.
  • Another pathogenicity island of 35 kb has recently been identified on the chromosome of enteropathogenic E. coli (EPEC) and was found to encode all known determinants involved in the so-called “attaching and effacing" (AE) lesion formation. This region was therefore referred to as "locus of enterocyte effacing" (LEE).
  • EEE locus of enterocyte effacing
  • the pathogenicity islands (PAIs) which code for a type III secretion system encompass genes that divide into two classes, I and II.
  • Class I encompasses the genes coding for the secretion machinery components and their regulators of expression
  • class II encompasses the genes encoding secreted effector proteins.
  • Yersinia IcrD and yscU belong to class I.
  • the precise functions of class I determinants is not well understood.
  • genes of class I can be identified as being present in many different species, and a comparison of their respective gene sequences indicate that equivalent genes share a significant (yscl, yscO) or even high level ⁇ IcrD, yscU, yscN) of sequence similarity (Hueck, 1998).
  • the second class of genes (class II) codes for proteins which constitute the substrate secreted by the translocon. These proteins appear as the actual effectors of virulence and are referred to as target proteins, virulence effector proteins or, simply, effectors. In contrast to the situation prevailing in class I gene products, the effectors share no, or very weak, similarities between species. Effector proteins are those which present the best biological, vaccine and diagnostic potentialities.
  • the inventors have discovered that the clustering of class I and class II genes inside a single pathogenicity island, offers the opportunity of conveniently finding and characterising unknown class II genes by targeting class I genes which can be identified using a known sequence of one of their numerous orthologues.
  • class I type III secretion system virulence genes have recently been shown to exist in B. bronchiseptica and B. pertussis (Yuk et al., 1998), there has been no complete analysis of a pathogenicity island in Bordetella, and the identity and characterisation of effector genes within such a pathogenicity island has been unknown up until the present invention.
  • the invention relates to a method for the identification of new virulence genes in bacterial strains containing a type III secretion system.
  • the invention allows the identification of the effector virulence genes associated within a pathogenicity island containing the genes for the type III secretion system.
  • Another aspect of the invention a method for the identification of pathogenic bacterial strains containing a type III secretion system.
  • Another aspect of the invention relates to Bordetella pertussis BopN, Orfl, Orf2, Or ⁇ , Orf4, Orf5, Orf6, Orf7, Orf8, Orf9, OrflO, Orfl l, Orfl 2, Orfl 3, Orfl 4, Orfl 5 effector proteins, and the respective polynucleotide sequences encoding them.
  • the invention utilises a method that employs ideally- suited primers designed specifically from the sequence of the virulent Yersinia enterocolitica IcrD gene as a target sequence.
  • the presence of a type III secretion system within a pathogenicity island in Bordetella pertussis was discovered, and every gene within the pathogenicity island was characterised.
  • Fig. 1 Nucleotide and deduced amino acid sequences of the cloned 152 bp amplicon.
  • the primers involved in the original amplification, the subsequent nested PCR, and the gene library screening are all derived from this sequence, and listed specifically in Table 1.
  • Fig. 2 PileUp figure from the deduced amino acid sequences homologous to Yersinia LcrD.
  • BbuFlhA Borrelia burgdorferi FlhA
  • TpaFlhA Treponema pallidum FlhA
  • BsuFlhA Bacillus subtilis FlhA
  • CjeFlbA Campylobacter jejuni FlbA
  • HpyFlhA Helicobacter pylori FlhA
  • StyFlhA Salmonella typhimurium FlhA
  • YenFlhA Yersinia enterocolitica FlhA
  • PmiFlhA Proteus mirabilis FlhA
  • CcrFlbF Caulobacter crescentus FlbF
  • EcoFhiA Escherichia coli FhiA
  • EamHrpI Erwin
  • Fig. 3 Organization of the Bordetella pertussis pathogenicity island (Pai).
  • Four house keeping genes hatchched boxes
  • the transposase gene of 1S481 black box
  • the Pai consists of genes coding for determinants involved in the secretory apparatus and its regulation (class I genes, in grey boxes) as well as ORFs which putitively code for effector proteins (class II genes, in white boxes). Letters indicate the respective class I bsc genes whereas numbers correspond to the class II ORFs listed in Table 3.
  • AtuFlhB Agrobacterium tumefaciens FlhB;
  • CcrPodW Caulobacter crescentus PodW;
  • SflSpa40 Shigella flexneri Spa40;
  • StySpaS Salmonella typhimurium SpaS;
  • Fig. 5 The DNA sequence of the Bordatella pertussis genome comprising the type III secretion system pathogenicity island. Reference should be made to tables 2, 3, and 4 and Fig. 3 for information regarding open reading frames.
  • Type III secretion systems identified to date are encoded by either chromosal or plasmidic pathogenicity island genes.
  • the IcrD gene is preferred.
  • the chosen gene will act as a target for detecting unidentified pathogenicity islands in related bacterial species.
  • the IcrD gene from Yersinia is preferred as it codes for the archetype of the recently identified LcrD/FlbF family of proteins. Members of this family are involved in host cell invasion, virulence in several phytopathogenic bacteria or in flagellar assembly. IcrD is preferred because the LcrD protein, and consequently the gene encoding it, is one of the most conserved determinants of the secretion machinary.
  • the preferred method for identifying unknown pathogenicity islands comprising a type III secretion system is by: i) identifying two highly conserved regions of the target protein sequence (preferably of LcrD). Preferably, both regions should contain conserved amino acids which are encoded by the fewest number of codon possibilities e.g. Methionine (ATG being the only possibility) or Tryptophan (TGG being the only possibility).
  • Methionine ATG being the only possibility
  • TGG Tryptophan
  • a degenerate set of primers for both of the chosen regions such that a) the primers are at least 15 bases long, preferably 20-30 bases long, and still more preferably 21-23 bases long, b) they are degenerate at bases that can be more than one type of nucleotide whilst still encoding the same amino acid (due to the degeneracy of codon usage for amino acids), but no more degenerate than is required to cover all permutations for the amino acid region selected, and c) the primer set that encodes the more N-terminal region of the chosen protein should correspond to the coding strand of its corresponding double-stranded DNA sequence, and the set that encodes the more C-terminal region should correspond to the complementary strand of the corresponding double-stranded DNA sequence.
  • iii) synthesising the degenerate primer sets of step ii) using conventional DNA synthesis methods well known in the art.
  • iv) purifying the primer sets of step iii) v) adding both the primer sets and a sample containing nucleic acid from a bacterial strain (preferably a cell sample of the bacterial species itself) together in appropriate quantities and in an appropriate buffer in order to perform a polymerase chain reaction (PCR)
  • PCR polymerase chain reaction
  • PCR polymerase chain reaction
  • PCR polymerase chain reaction
  • PCR polymerase chain reaction
  • a PCR reaction in order to amplify the region of the gene between the two primers (conditions for performing the PCR reaction can be optimised using techniques well known in the art)
  • the preferred method for confirming that the amplified product actually corresponds to a virulence gene is by carrying out steps i)-vii) above (where the target protein is LcrD) and then: viii) optionally separating the product of correct size from any background products of incorrect size by removing the correct band from the gel, purifying the product by conventional means, and amplifying the product once more with the two degenerate primer sets in another PCR reaction (under preferably more stringent PCR conditions) [this step is required should the product of step vii) not be pure enough for direct cloning] ix) inserting the DNA fragment by conventional means into a vector which is capable of being sequenced, and sequencing the fragment x) comparing the deduced amino acid sequence of ix) with that of known members of the LcrD/FlbF family of proteins to associate the amplified product as being part of either a virulence or a flagellar gene.
  • the preferred method for characterising the whole pathogenicity island and defining unidentified virulence effector genes is by carrying out steps i)-xv) above (where the target protein is LcrD) and then: xvi) if the sequence is more homologous with the IcrD gene family, designing primers at either extreme of the gene sequence already ascertained, and scanning and sequencing the genomic library (using a standard chromosome walking strategy - where the insert boundaries of an original clone serves as a probe for screening and cloning adjacent regions) to sequence eventually the whole of the pathogenicity island (both boundaries of which will be defined by the presence of either direct or inverted repeats, or insertion sequences, or the presence of house-keeping genes) xvii)defining unidentified virulence effector genes within the sequenced pathogenicity island xviii)cloning, expressing and characterising the virulence genes of xvii) which encode virulence effector proteins of the organism
  • Bordetella pathogenicity proteins refers generally to polypeptides having the amino acid sequence encoded by the genes defined in tables 2 and 3, or an allelic variant thereof. These proteins are: BcrD, BcrH, BscC, BscD, BscE, BscF, Bscl, BscJ, BscK, BscL, BscN, BscO, BscP, BscQ, BscR, BscS, BscT, BscU, BscV, B ⁇ L, BopN, Orfl, Orf2, OrD, Orf4, Orf5, Orf6, Orf7, Orf , Orf9, OrflO, Orfl l, Orfl2, Orfl3, Orfl4, Orfl5.
  • “Bordetella pathogenicity genes” refers to polynucleotides having the nucleotide sequence defined in tables 2 and 3, or allelic variants thereof and/or their complement
  • genes are: bcrD, bcrH, bscC, bscD, bscE, bscF, bscl, bscJ, bscK, bscL, bscN, bscO, bscP, bscQ, bscR, bscS, bscT, bscU, bscV, brpL, bopN, or ⁇ , orfl, or ⁇ , or ⁇ , or ⁇ , or ⁇ , or ⁇ , or ⁇ , orflO, orfll, or/72, orflS, orfl 4, orflS.
  • Polypeptide refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres.
  • Polypeptide refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
  • Polypeptides include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side- chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslational natural processes or may be made by synthetic methods.
  • Modifications include 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, hydroxy lation, 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.
  • Polynucleotide generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • Polynucleotides include, without limitation 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 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.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
  • Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides.
  • Variant is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties.
  • a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions (preferably conservative), additions, deletions in any combination.
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
  • Variants should retain one or more of the biological activities of the reference polypeptide. For instance, they should have similar (preferably the same) antigenic or immunogenic activities as the reference polypeptide.
  • Antigenicity can be tested using standard immunoblot experiments, preferably using polyclonal sera against the reference polypeptide. The immunogenicity can best be tested by measuring antibody responses (using polyclonal sera generated against the variant polypeptide) against purified reference polypeptide in a standard ELISA test.
  • a variant would retain all of the above biological activities.
  • Identity is a measure of the identity of nucleotide sequences or amino acid sequences. In general, the sequences are aligned so that the highest order match is obtained. “Identity” per se has an art-recognized meaning and can be calculated using published techniques.
  • identity is well known to skilled artisans (Carillo, H., and Lipton, D., SIAM J Applied Math (1988) 48:1073). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., SIAM J Applied Math (1988) 48:1073. 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 (Devereux, J., et al, Nucleic Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S.F. et al, J Molec Biol (1990) 215:403). Most preferably, the program used to determine identity levels was the GCG 9 package, as was used in the Examples below.
  • a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include on average up to five point mutations per each 100 nucleotides of the reference nucleotide sequence.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the present invention relates to Bordetella pathogenicity proteins (or polypeptides).
  • the Bordetella pathogenicity polypeptides include the polypeptides encoded by the genes defined in tables 2 and 3; as well as polypeptides comprising the amino acid sequence encoded by the genes defined in tables 2 and 3; and polypeptides comprising the amino acid sequence which have at least 75% identity to that encoded by the genes defined in tables 2 and 3 over their entire length, and preferably at least 80% identity, and more preferably at least 90% identity. Those with 95-99% identity are highly preferred.
  • the Bordetella pathogenicity polypeptides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein. It may be advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues or Maltose Binding Protein (MBP), or an additional sequence for stability during recombinant production. Furthermore, addition of exogenous polypeptide or lipid tail or polynucleotide sequences to increase the immunogenic potential of the final molecule is also considered.
  • a fragment is a polypeptide having an amino acid sequence that is the same as part, but not all, of the amino acid sequence of the aforementioned Bordetella pathogenicity polypeptides.
  • fragments may be "freestanding," or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region.
  • Representative examples of polypeptide fragments of the invention include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of Bordetella pathogenicity polypeptide.
  • fragments should comprise at least 7 consecutive amino acids from the sequences e.g. 8, 10, 12, 14, 18, 20 or more depending on the particular sequence).
  • fragments comprise an epitope from the sequence.
  • Preferred fragments include, for example, truncation polypeptides having the amino acid sequence of Bordetella pathogenicity polypeptides, except for deletion of a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus and/or transmembrane region or deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus.
  • fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and ttirn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha arnphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
  • Other preferred fragments are biologically active fragments.
  • Biologically active fragments are those that mediate Bordetella pathogenicity protein activity, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those that are antigenic or immunogenic in an animal, especially in a human.
  • all of these polypeptide fragments retain the biological activity (for instance antigenic or immunogenic) of the Bordetella pathogenicity protein, including antigenic activity.
  • Variants of the defined sequence and fragments also form part of the present invention.
  • Preferred variants are those that vary from the referents by conservative amino acid substitutions i.e., those that substitute a residue with another of like characteristics. Typical such substitutions are among Ala, Val, Leu and He; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr.
  • Particularly preferred are variants in which several, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination.
  • Most preferred variants are naturally occurring allelic variants of Bordetella pathogenicity polypeptide present in strains of Bordetella pertussis.
  • the proteins may be chemically conjugated, or expressed as recombinant fusion proteins allowing increased levels to be produced in an expression system as compared to non-fused protein.
  • the fusion partner may assist in providing T helper epitopes (immunological fusion partner), preferably T helper epitopes recognised by humans, or assist in expressing the protein (expression enhancer) at higher yields than the native recombinant protein.
  • the fusion partner will be both an immunological fusion partner and expression enhancing partner.
  • Bordetella pathogenicity polypeptides of the invention can be prepared in any suitable manner.
  • Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
  • a polypeptide of the invention is derived from Bordetella pertussis, however, it may preferably be obtained from other organisms of the same taxonomic genus.
  • a polypeptide of the invention may also be obtained, for example, from organisms of the same taxonomic family or order, such as Bordetella parapertussis or Bordetella bronchiseptica.
  • a further aspect of the invention is substantially purified Bordetella pathogenicity polypeptides of the invention, "substantially purified" when used in reference to a protein or peptide means that the molecule has been largely, but not necessarily wholly, separated an purified from other cellular and non-cellular components.
  • a protein is substantially pure when it is at least about 60 % by weight free from other naturally occurring organic molecules.
  • the purity is at least about 75 %, more preferably at least about 90% , and most preferably at least about 99% by weight pure.
  • Polynucleotides of the invention Another aspect of the invention relates to Bordetella pathogenicity polynucleotides.
  • Bordetella pathogenicity polynucleotides include isolated polynucleotides which encode the Bordetella pathogenicity polypeptides and fragments respectively, and polynucleotides closely related thereto or variants thereof. More specifically, Bordetella pathogenicity polynucleotides of the invention include a polynucleotide comprising the nucleotide sequence of genes defined in table 2 or 3, encoding a Bordetella pathogenicity polypeptide.
  • Bordetella pathogenicity polynucleotides further include a polynucleotide comprising a nucleotide sequence that has at least 75%) identity over its entire length to a nucleotide sequence encoding the Bordetella pathogenicity polypeptide encoded by the genes defined in tables 2 and 3, and a polynucleotide comprising a nucleotide sequence that is at least 75% identical to that of the genes defined in tables 2 and 3.
  • polynucleotides at least 80% identical are particularly preferred, and those with at least 90% are especially preferred.
  • those with at least 95% are highly preferred and those with at least 98-99% are most highly preferred, with at least 99% being the most preferred.
  • Bordetella pathogenicity polynucleotides is a nucleotide sequence which has sufficient identity to a nucleotide sequence of a gene defined in tables 2 and 3 to hybridize under conditions useable for amplification or for use as a probe or marker.
  • the invention also provides polynucleotides which are complementary to such Bordetella pathogenicity polynucleotides.
  • a polynucleotide of the invention encoding a Bordetella pathogenicity polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacteria using Bordetella pertussis cells as starting material, followed by obtaining a full length clone.
  • standard cloning and screening methods such as those for cloning and sequencing chromosomal DNA fragments from bacteria using Bordetella pertussis cells as starting material, followed by obtaining a full length clone.
  • a polynucleotide sequence of the invention typically a library of clones of chromosomal DNA of Bordetella pertussis in E.
  • coli or some other suitable host is probed with a radiolabeled oligonucleotide, preferably a 17-mer or longer, derived from a partial sequence.
  • Clones carrying DNA identical to that of the probe can then be distinguished using stringent hybridization conditions.
  • sequencing primers designed from the original polypeptide or polynucleotide sequence it is then possible to extend the polynucleotide sequence in both directions to determine a full length gene sequence.
  • sequencing is performed, for example, using denatured double stranded DNA prepared from a plasmid clone. Suitable techniques are described by Maniatis, T., Fritsch, E.F.
  • a polynucleotide encoding a polypeptide of the present invention may be obtained by a process which comprises the steps of screening an appropriate library under stringent hybridization conditions (for example, using a temperature in the range of 45 - 65°C and an SDS concentration from 0.1 - 1%) with a labeled or detectable probe consisting of or comprising a sequence defined in table 2 or 3 or a fragment thereof; and isolating a full- length gene and/or genomic clones containing said polynucleotide sequence.
  • the invention also provides a polynucleotide consisting of or comprising a polynucleotide sequence obtained by screening an appropriate library containing the complete gene for a polynucleotide sequence defined in tables 2 and 3 under stringent hybridization conditions with a probe having the sequence of said polynucleotide sequence defined in table 2 or 3 or a fragment thereof; and isolating said polynucleotide sequence.
  • Fragments useful for obtaining such a polynucleotide include, for example, probes and primers are described elsewhere herein.
  • the nucleotide sequence encoding Bordetella pathogenicity polypeptide encoded by the genes defined in tables 2 and 3 may be identical to the polypeptide encoding sequence contained in the genes defined in tables 2 or 3, or it may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide encoded by the genes defined in tables 2 and 3 respectively.
  • the polynucleotide may include the coding sequence for the mature polypeptide or a fragment thereof, by itself; the coding sequence for the mature polypeptide or fragment in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions.
  • a marker sequence which facilitates purification of the fused polypeptide can be encoded.
  • the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al. , Proc NatlAcadSci USA (1989) 86:821-824, or is an HA tag, or is glutathione-s-transferase, or is MBP.
  • the polynucleotide may also contain non-coding 5' and 3' sequences, such as transcribed, non-translated sequences, splicing and polyadenylation signals, ribosome binding sites and sequences that stabilize mRNA.
  • Nucleic acid comprising fragments of the sequences of the invention are also provided. These should comprise at least 10 consecutive nucleotides from the sequences
  • Such fragments can preferably hybridise to the above-mentioned sequences under stringent conditions.
  • the present invention further relates to polynucleotides that hybridize to the herein above-described sequences.
  • the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides.
  • stringent conditions means hybridization will occur only if there is at least 80%, and preferably at least 90%, and more preferably at least 95%, yet even more preferably 97-99% identity between the sequences.
  • Polynucleotides of the invention which are identical or sufficiently identical to a nucleotide sequence of any gene defined in tables 2 and 3 or a fragment thereof, may be used as hybridization probes for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones encoding Bordetella pathogenicity polypeptides respectively and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than Bordetella pertussis) that have a high sequence similarity to the Bordetella pathogenicity genes.
  • Such hybridization techniques are known to those of skill in the art.
  • these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% identical to that of the referent.
  • the probes generally will comprise at least 15 nucleotides. Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotides. Particularly preferred probes will range between 30 and 50 nucleotides.
  • to obtain a polynucleotide encoding Bordetella pathogenicity polypeptide, including homologs and orthologs from species other than Bordetella pertussis comprises the steps of screening an appropriate library under stringent hybridization conditions with a labeled probe having a nucleotide sequence contained in one of the gene sequences defined by tables 2 and 3, or a fragment thereof; and isolating full-length cDNA and genomic clones containing said polynucleotide sequence.
  • Bordetella pathogenicity polynucleotides of the present invention further include a nucleotide sequence comprising a nucleotide sequence that hybridize under stringent condition to a nucleotide sequence having a nucleotide sequence contained in one of the genes defined by table 2 and 3, or a fragment thereof. Also included with Bordetella pathogenicity polypeptides are polypeptides comprising amino acid sequences encoded by nucleotide sequences obtained by the above hybridization conditions. Such hybridization techniques are well known to those of skill in the art.
  • Stringent hybridization conditions are as defined above or, alternatively, conditions under overnight incubation at 42°C in a solution comprising: 50% formamide, 5xSSC (150mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5x Denhardt's solution, 10 % dextran sulfate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65°C.
  • a coding region of a Bordetella pathogenicity gene may be isolated by screening using a DNA sequence defined in table 2 or 3 to synthesize an oligonucleotide probe.
  • a labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
  • RACE Rapid Amplification of cDNA ends
  • MarathonTM technology (Clontech Laboratories Inc.) for example, have significantly simplified the search for longer cDNAs.
  • cDNAs have been prepared from mRNA extracted from a chosen tissue and an 'adaptor' sequence ligated onto each end.
  • Nucleic acid amplification (PCR) is then carried out to amplify the "missing" 5' end of the DNA using a combination of gene specific and adaptor specific oligonucleotide primers.
  • the PCR reaction is then repeated using "nested" primers, that is, primers designed to anneal within the amplified product (typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the selected gene sequence).
  • the products of this reaction can then be analyzed by DNA sequencing and a full-length DNA constructed either by joining the product directly to the existing DNA to give a complete sequence, or carrying out a separate full-length PCR using the new sequence information for the design of the 5' primer.
  • polynucleotides of the invention that are oligonucleotides derived from a sequence defined in table 2 or 3 may be used in the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue. It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained.
  • polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to animal and human disease.
  • This invention also relates to the use of Bordetella pathogenicity polypeptides, or Bordetella pathogenicity polynucleotides, for use as diagnostic reagents. Detection of Bordetella pathogenicity polypeptides will provide a diagnostic tool that can add to or define a diagnosis of B. pertussis disease, among others.
  • Materials for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy.
  • the present invention relates to a diagonostic kit for a disease or suspectability to a disease, particularly B. pertussis disease, which comprises: (a) a Bordetella pathogenicity polynucleotide, preferably the nucleotide sequence of one of the gene sequences defined by tables 2 and 3, or a fragment thereof ; (b) a nucleotide sequence complementary to that of (a); (c) a Bordetella pathogenicity polypeptide, preferably the polypeptide encoded by one of the gene sequences defined in tables 2 and 3, or a fragment thereof;
  • a phage displaying an antibody to a Bordetella pathogenicity polypeptide preferably to the polypeptide encoded by one of the gene sequences defined in tables 2 and 3.
  • Polypeptides and polynucleotides for prognosis, diagnosis or other analysis may be obtained from a putatively infected and/or infected individual's bodily materials.
  • Polynucleotides from any of these sources may be used directly for detection or may be amplified enzymatically by using PCR or any other amplification technique prior to analysis.
  • RNA, particularly mRNA, cDNA and genomic DNA may also be used in the same ways.
  • amplification, characterization of the species and strain of infectious or resident organism present in an individual may be made by an analysis of the genotype of a selected polynucleotide of the organism.
  • Deletions and insertions can be detected by a change in size of the amplified product in comparison to a genotype of a reference sequence selected from a related organism, preferably a different species of the same genus or a different strain of the same species.
  • Point mutations can be identified by hybridizing amplified DNA to labeled Bordetella pathogenicity polynucleotide sequences. Perfectly or significantly matched sequences can be distinguished from imperfectly or more significantly mismatched duplexes by DNase or RNase digestion, for DNA or RNA respectively, or by detecting differences in melting temperatures or renaturation kinetics.
  • Polynucleotide sequence differences may also be detected by alterations in the electrophoretic mobility of polynucleotide fragments in gels as compared to a reference sequence. This may be carried out with or without denaturing agents. Polynucleotide differences may also be detected by direct DNA or RNA sequencing. See, for example, Myers et al, Science, 230: 1242 (1985). Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase, VI and SI protection assay or a chemical cleavage method. See, for example, Cotton et al, Proc. Natl. Acad. Sci, USA, 85: 4397-4401 (1985).
  • This invention also relates to the use of polynucleotides of the present invention as diagnostic reagents. Detection of a mutated form of a polynucleotide of the invention, which is associated with a disease or pathogenicity will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, a prognosis of a course of disease, a determination of a stage of disease, or a susceptibility to a disease, which results from under-expression, over- expression or altered expression of the polynucleotide. Organisms, particularly infectious organisms, carrying mutations in such polynucleotide may be detected at the polynucleotide level by a variety of techniques, such as those described elsewhere herein.
  • the invention further provides a process for diagnosing disease, preferably bacterial (particularly Bordetella) infections, more preferably infections caused by Bordetella pertussis, comprising determining from a sample derived from an individual, such as a bodily material, an increased level of expression of polynucleotide having a sequence defined in table 2 or 3. Increased or decreased expression of a polynucleotide can be measured using any on of the methods well known in the art for the quantitation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and other hybridization methods.
  • the invention also relates to vectors that comprise a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • RNAs derived from the DNA constructs of the invention can also be employed to produce such proteins using RNAs derived from the DNA constructs of the invention.
  • Recombinant polypeptides of the present invention may be prepared by processes well known in those skilled in the art from genetically engineered host cells comprising expression systems. Accordingly, in a further aspect, the present invention relates to expression systems that comprise a polynucleotide or polynucleotides of the present invention, to host cells which are genetically engineered with such expression systems, and to the production of polypeptides of the invention by recombinant techniques.
  • host cells can be genetically engineered to inco ⁇ orate expression systems or portions thereof or polynucleotides of the invention.
  • Introduction of a polynucleotide into the host cell can be effected by methods described in many standard laboratory manuals, such as Davis, et al. , BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
  • bacterial cells such as cells of streptococci, staphylococci, enterococci, E. coli, streptomyces, cyanobacteria, Bacillus subtilis, Moraxella catarrhalis, Haemophilus influenzae and Neisseria meningitidis
  • fungal cells such as cells of a yeast, Kluveromyces, Saccharomyces, a basidiomycete, Candida albicans and Aspergillus
  • insect cells such as cells of Drosophila S2 and Spodoptera Sf9
  • animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells
  • plant cells such as cells of a gymnosperm or angiosperm.
  • vectors include, among others, chromosomal-, episomal- and virus-derived vectors, for example, vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses, retroviruses, and alphaviruses and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • the expression system constructs may contain control regions that regulate as well as engender expression.
  • any system or vector suitable to maintain, propagate or express polynucleotides and/or to express a polypeptide in a host may be used for expression in this regard.
  • the appropriate DNA sequence may be inserted into the expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al, MOLECULAR CLONING, A LABORATORY MANUAL, ⁇ supra).
  • secretion signals may be inco ⁇ orated into the expressed polypeptide. These signals may be endogenous to the polypeptide or they may be heterologous signals.
  • Polypeptides of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, ion metal affinity chromatography (IMAC) is employed for purification.
  • IMAC ion metal affinity chromatography
  • Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during intracellular synthesis, isolation and or purification.
  • the expression system may also be a recombinant live microorganism, such as a virus or bacterium.
  • the gene of interest can be inserted into the genome of a live recombinant virus or bacterium. Inoculation and in vivo infection with this live vector will lead to in vivo expression of the antigen and induction of immune responses.
  • Viruses and bacteria used for this pu ⁇ ose are for instance: poxviruses (e.g; vaccinia, fowlpox, canarypox), alphaviruses (Sindbis virus, Semliki Forest Virus, Dialoguelian Equine Encephalitis Virus), adenoviruses, adeno-associated virus, picornaviruses (poliovirus, rhinovirus), he ⁇ esviruses (varicella zoster virus, etc), Listeria, Salmonella , Shigella, Neisseria, BCG. These viruses and bacteria can be virulent, or attenuated in various ways in order to obtain live vaccines. Such live vaccines also form part of the invention.
  • poxviruses e.g; vaccinia, fowlpox, canarypox
  • alphaviruses Semliki Forest Virus, Kunststoffuelian Equine Encephalitis Virus
  • adenoviruses adeno
  • the invention provides antibodies which bind specifically to the polypeptides of the invention. These may be polyclonal or monoclonal and may be produced by any suitable means well known to a skilled person in the art.
  • a mouse or rat is immunised with a protein (preferably adjuvanted with Freund's complete adjuvant) and injected (doses of 50-200 ⁇ g/injection is typically sufficient).
  • Polyclonal antibodies can be isolated by bleeding the animal to extract serum.
  • monoclonal antibodies can be generated by removing the spleen (or large lymph nodes) and dissociating it into single cells (Kohler and Milstein, (1975) Nature, 256:495-497). These are then induced to fuse with myeloma cells to form hybridoma, and are cultured in a selective medium (eg hypoxanthine, aminopterin, thymidine merium, "HAT").
  • a selective medium eg hypoxanthine, aminopterin, thymidine merium, "HAT"
  • the resulting hybridomas are plated by limiting dilution, and are assayed for the production of antibodies which bind specifically to the immunizing antigen (and which do not bind to unrelated antigens).
  • the selected monoclonal-secreting hybridomas are then cultured either in vitro (eg in tissue culture bottles or hollow fiber reactors), or in vivo (as Ascites in mice).
  • phage display technology may be utilized to select antibody genes with binding activities towards a polypeptide of the invention either from repertoires of PCR amplified v-genes of lymphocytes from humans screened for possessing anti- Bordetella pathogenicity polypeptide or from naive libraries (McCafferty, et al, (1990), Nature 348, 552-554; Marks, et al, (1992) Biotechnology 10, 779-783).
  • the affinity of these antibodies can also be improved by, for example, chain shuffling (Clackson et al, (1991) Nature 352: 628).
  • the above-described antibodies may be employed to isolate or to identify clones expressing the polypeptides or polynucleotides of the invention to purify the polypeptides or polynucleotides by, for example, affinity chromatography.
  • Antibodies against a Bordetella pathogenicity polypeptide or polynucleotide may be employed to treat infections, particularly bacterial infections.
  • Polypeptide variants include antigenically, epitopically or immunologically equivalent variants form a particular aspect of this invention.
  • the antibody or variant thereof is modified to make it less immunogenic in the individual.
  • the antibody may most preferably be "humanized," where the complimentarity determining region or regions of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody, for example as described in Jones et al. (1986), Nature 321, 522-525 or Tempest et al, (1991) Biotechnology 9, 266-273.
  • Another aspect of the invention relates to a method for inducing an immunological response in a mammal which comprises inoculating the mammal with Bordetella pathogenicity polypeptide or epitope-bearing fragments, analogs, outer- membrane vesicles or cells (attenuated or otherwise) adequate to produce antibody and/or T cell immune response to protect said animal from Bordetella (particularly B. pertussis) disease, among others.
  • Bordetella pathogenicity polypeptides encoded by the genes defined in table 3 - the effector proteins.
  • Yet another aspect of the invention relates to a method of inducing immunological response in a mammal which comprises, delivering Bordetella pathogenicity polypeptide via a vector directing expression of Bordetella pathogenicity polynucleotide in vivo in order to induce such an immunological response to produce antibody to protect said animal from diseases.
  • a further aspect of the invention relates to an immunological composition or vaccine formulation which, when introduced into a mammalian host, induces an immunological response in that mammal to a Bordetella pathogenicity polypeptide (particularly one encoded by a gene defined in table 3) wherein the composition comprises a Bordetella pathogenicity gene, or Bordetella pathogenicity polypeptide or epitope-bearing fragments, analogs, outer-membrane vesicles or cells (attenuated or otherwise).
  • the vaccine formulation may further comprise a suitable carrier.
  • the Bordetella pathogenicity polypeptide vaccine composition is preferably administered orally or parenterally (including subcutaneous, intramuscular, intravenous, intradermal etc. injection).
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
  • the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • the vaccine formulations of the invention may also comprise other Bordetella antigens known to be suitable vaccinal agents, for instance: pertussis toxoid, pertactin, agglutinogins 1 and 2, FHA (filamentous haemagglutinin), and adenylate cyclase / haemolysin (AC/HLY), or immunogenic fragments thereof (Locht et al, NAR (1986) 14:3251-3261; Relman et al, PNAS USA (1989) 86:2637-2641; Roberts et al, Mol. Microbiol. (1991) 5:1393-1404; Mooi et al, Microb. Pathog. (1992) 12:127-135; Hewlett and Gordon, In Pathogenesis and Immunity in Pertussis (1988), New York, Wiley & Sons, pp. 193-209.
  • Bordetella antigens known to be suitable vaccinal agents, for instance: per
  • Yet another aspect of the invention relates to an immunological/vaccine formulation which comprises the polynucleotide of the invention.
  • immunological/vaccine formulation which comprises the polynucleotide of the invention.
  • Vaccine compositions can comprise polypeptides, antibodies, or polynucleotides of the invention.
  • the pharmaceutical compositions will comprise a therapeutically effective amount of either polypeptides, antibodies, or polynucleotides of the claimed invention.
  • terapéuticaally effective amount refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition (in this case Bordetella, particularly B. pertussis, disease), or to exhibit a detectable therepeutic or preventative effect.
  • the effect can be detected by, for example, antigen levels.
  • Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature.
  • Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic or immunogenic polypeptides.
  • immunologically effective amount it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention.
  • Example 1 A type III secretion system is present in a pathogenicity island in Bordetella pertussis.
  • the presence of a IcrD homologous gene in the Bordetella pertussis genome was investigated by polymerase chain reaction (PCR).
  • the primers used (oligos 95080 and 95081 shown in Table 1) were degenerate oligonucleotides corresponding to highly conserved regions of the amino acids sequences of the LcrD/FlbF family of proteins. These primers were also designed to favour the amplification of virulence genes instead of their var logu flhA or flbF flagellar genes, present in flagellated bacterial strains.
  • the presence of the 3' triplet CAT in oligonucleotide 95081 is a determinant - indeed when multiple sequence analysis is done using known homologous sequences (database searching was done with either the FASTA and TFASTA programs of the GCG9 package, or with BLASTN, BLASTP and BLASTX programs, and alignments were carried out with the PILEUP program from the GCG9 package) it could be seen that the CAT triplet codes for a methionine which is exclusively present in virulence sequences while absent in the flagellar ones.
  • the PCR product When analysed on agarose gel, the PCR product appeared as a heterogeneous mix of fragments, one of which was presenting the expected size (around 150 bp).
  • a second round of amplification using the approximately 150 bp DNA as template yielded a single amplicon which was cloned in pCRII (obtained from Invitrogen) for further characterisation. It appeared as a 152 bp fragment whose nucleotide sequence (Fig. 1), although similar to all IcrD/flbF homologous genes, shares a higher level of identity with the virulence ⁇ IcrD-like) genes. Table 1.
  • PCR was performed under stringent conditions with serial 10-fold dilutions of DNA from B. pertussis.
  • the optimisation of stringent PCR conditions require a perfect match between template and primers. It was likely, however, that due to the degeneration of the original primers, the 152 bp sequence initially obtained had, at its boundaries, a few base pair differences with the actual B. pertussis IcrD-like (hereafter called bcrD) sequence.
  • a nested PCR approach using internal primers (oligos 95363 and 95364 Table 1) was therefore preferred, as primers known to be the correct B. pertussis sequence are used.
  • the B. pertussis IcrD ⁇ i s, nucleotide sequence crD) has been submitted to EMBL and assigned the accession number Y13383.
  • This technique could be applied to any Gram negative pathogen of medical or agronomic importance such as Neisseria spp, Moraxella catharalis, Vibrio cholerae, any Enterobacteriaceae, Pseudomonas spp, Haemophilus influenzae, Brucella spp, Francisella tularensis, Pasteurella spp, Legionella pneumophila. Even in strains that have been fully sequenced, this technique can be used as a simple method for checking alternate types or strains of the same species. For instance, some types of pathogenic Escherichia coli harbour a type III secretion system whereas others do not.
  • Example 2 Analysis of the B. pertussis bcrD flanking sequences to characterise the pathogenicity island and virulence-related proteins encoded therein
  • YscU like LcrD, is a component of the Yersinia type III secretion machinery involved in the virulence mechanisms of the bacteria. B. pertussis therefore possesses a classical type III secretion system which is most probably involved in pathogenicity. This latter point can be investigated through phentoypic analyses of mutants (see below).
  • the total length of the Pai is approximately 30 to 40 kb.
  • the DNA sequence of the whole region is presented in Figure 5, and is referred to in tables 2, 3, and 4. Restriction analysis on pulsed-field gel electrophoresis allowed the type III locus to be mapped at coordinate position 1 ,590 kb on the Tohama I strain chromosome.
  • a bcrD mutant was engineered by allelic exchange.
  • the bcrD gene was disrupted by an aphA-3 cassette conferring kanamycin resistance. This cassette was inserted in such a way that translation was not interrupted, avoiding any polar effect on expression of putative downstream cistrons.
  • a mutant has been isolated and its associated phenotype is being currently analysed.
  • a 255-bp fragment (codons 363 to 445) was deleted from the bcrD coding sequence and replaced by a cassette containing the aphA-3 gene which confers kanamycin resistance (Menard et al, J. Bacteriol. (1993) 175:5899-5906).
  • the aphA-3 cassette was excised from pUC18K by EcoRI-Rytl digestion and introduced in the bcrD EcoRI-Sse8387l sites. This construct generated an early stop in bcrD translation and allowed in-frame translation of the remaining 3' end of the mutated gene, avoiding possible polar effects on expression of downstream cistrons.
  • pAF214 is a derivative of pAF214 that contained two additional unique Spel and R ⁇ cl sites. These sites, included in a pair of complementary oligonucleotides, were introduced into the BamHl site of pAF214.
  • PCR amplification of a 831 bp fragment covering the 5' region and the 4 first codons of bcrD was generated.
  • This amplicon was further introduced into Bam l-HinOlll linearized pNM480 (Minton, Gene (1984) 31 :269-273), in such a way that the bcrD initiation codon was placed in frame with lacZ, used as a reporter gene.
  • the resulting construct was named pAF245.
  • primers were designed for placing lacZ downstream of a 849 bp fragment that encompassed upstream bscN sequences including its 3 first codons.
  • pAF246 was obtained by cloning this fragment in pNM480.
  • B. pertussis cells from a freshly saturated culture in 10 ml of SS medium, were washed and resuspended in lOO ⁇ l of a cold 10% (v/v) glycerol solution. Up to 10 ⁇ g of supercoiled purified DNA in a maximum of 20 ⁇ l of water were added to 100 ⁇ l of the bacterial suspension. Cells and DNA were transferred to a prechilled 0.2 cm electroporation cuvette (Bio-Rad) and placed in a Gene Pulser apparatus (Bio-Rad). Pulses were achieved with settings of 25 ⁇ F, 2.5 kV, and 600 ⁇ , giving a time constant ranging from 11 to 14 ms.
  • mice model After a two days growing on BG agar plates, wild type and mutant bacteria were recovered and resuspended in PBS at a concentration of 10 8 PFU ml "1 . 25 ⁇ l of the suspension were injected in each nostril of pentobarbital anaesthetized mice. Lungs colonization was assayed after 4 h, 3, 7, 14, 26, 39 and 45 days by treating both lungs of each mouse in an Ultraturax grinder and titrating the resuspended bacteria on BG agar plates. ⁇ -galactosidase assay
  • the Bvg + phase is characterized by the expression of virulence factors and is necessary for colonization of animal models.
  • the bacteria are avirulent in Bvg ' phase which can be induced by nicotinic acid or MgSO 4 .
  • bcrD and bscN Two genes that belonged to distinct unit of transcription, i.e. bcrD and bscN, by using transcriptional fusions of lacZ into these genes.
  • NIVh86 and NIVh87 which integrated pAF245 and pAF246 respectively.
  • ORFs ⁇ or ⁇ to -8) particularly fulfil certain criteria that make them good candidates as effector proteins and vaccine candidates.
  • ORFs bopN, or ⁇ and orflO are also of particular interest as vaccine candidates.
  • the secreted proteins will be analysed using standard techniques to confirm their functional and immunological properties.
  • the immunogenicity of the secreted proteins will be assessed by investigating the presence of antibodies directed against these proteins in the serum of infected patients.
  • their putative recognition as protective antigens will be based on challenge experiments, realized in a mouse model.
  • the biological properties of the effector proteins will be assessed by analysing their catalytic activities. For instance, it is expected that one of the secreted proteins would display a tyrosine phosphatase activity.
  • the function of the effector proteins will be investigated by microinjecting the proteins into the cytoplasm of eukaryotic cells.
  • the IcrB (yscN/U) gene cluster of Yersinia pseudotuberculosis is involved in Yop secretion and shows high homology to the spa gene clusters of Shigella flexneri and Salmonella typhimurium. J Bacteriol 176:2619-2626. Bogdanove, A. J., Wei, Z.-M., Zhao, L., and Beer, S. V.
  • Erwinia amylovora secretes a Ha ⁇ in via a type III pathway and contains a homolog of yopN of Yersinia spp. J Bacteriol 178:1720-1730.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Engineering & Computer Science (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne un procédé permettant de détecter des souches de bactéries pathogènes qui contiennent un système de sécrétion de type III. Plus particulièrement, la présente invention concerne le procédé appliqué au Bordetella pertussis pathogène. En outre, l'invention concerne des polynucléotides qui viennent d'être identifiés dans ces régions, des polypeptides virulents codés par ces derniers, et l'utilisation de ces polynucléotides et polypeptides, et leur production. Plus particulièrement, les polynucléotides et les polypeptides selon l'invention concernent les protéines effectrices virulentes associées au système de sécrétion de type III de Bordetella pertussis, qui sont particulièrement appropriées pour être utilisées dans les vaccins.
PCT/EP1999/010297 1998-12-21 1999-12-21 Vaccin WO2000037493A2 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
EP99969578A EP1140996A2 (fr) 1998-12-21 1999-12-21 Vaccin
JP2000589562A JP2002534960A (ja) 1998-12-21 1999-12-21 ワクチン
CA002356764A CA2356764A1 (fr) 1998-12-21 1999-12-21 Vaccin
AU29037/00A AU2903700A (en) 1998-12-21 1999-12-21 Vaccine
HK02101214.6A HK1041490A1 (zh) 1998-12-21 2002-02-19 來自百日咳杆菌的iii型分泌系統抗原

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9828217.1 1998-12-21
GBGB9828217.1A GB9828217D0 (en) 1998-12-21 1998-12-21 Vacine

Publications (2)

Publication Number Publication Date
WO2000037493A2 true WO2000037493A2 (fr) 2000-06-29
WO2000037493A3 WO2000037493A3 (fr) 2001-02-01

Family

ID=10844715

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1999/010297 WO2000037493A2 (fr) 1998-12-21 1999-12-21 Vaccin

Country Status (8)

Country Link
EP (1) EP1140996A2 (fr)
JP (1) JP2002534960A (fr)
CN (1) CN1371389A (fr)
AU (1) AU2903700A (fr)
CA (1) CA2356764A1 (fr)
GB (1) GB9828217D0 (fr)
HK (1) HK1041490A1 (fr)
WO (1) WO2000037493A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2217614A1 (fr) * 2007-11-07 2010-08-18 Dynamic Microbials Limited Compositions et formulations antimicrobiennes et leurs utilisations
CN113226361A (zh) * 2018-10-15 2021-08-06 代表亚利桑那大学的亚利桑那校董会 疫苗多肽组合物及方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10465252B2 (en) * 2007-10-26 2019-11-05 Quest Diagnostics Investments Incorporated Bordetella detection assay
CN101875974A (zh) * 2010-07-02 2010-11-03 深圳市儿童医院 检测百日咳杆菌的引物组、检测试剂盒以及检测方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999059630A1 (fr) * 1998-05-15 1999-11-25 University Of California Los Angeles Systeme de secretion de bordetella de type iii

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999059630A1 (fr) * 1998-05-15 1999-11-25 University Of California Los Angeles Systeme de secretion de bordetella de type iii

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
FAUCONNIER A ET AL.: "Characterization of a locus encoding a type III secretion system in Bordetella pertussis" ABSTRACTS OF THE GENERAL MEETING OF THE AMERICAN SOCIETY FOR MICROBIOLOGY., vol. 99, 30 May 1999 (1999-05-30), page 31 XP000929802 WASHINGTON US ISSN: 0067-2777 *
GALAN J E ET AL: "TYPE III SECRETION MACHINES: BACTERIAL DEVICES FOR PROTEIN DELIVERYINTO HOST CELLS" SCIENCE,US,AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE,, vol. 284, 21 May 1999 (1999-05-21), pages 1322-1328, XP002922658 ISSN: 0036-8075 *
KERR J.R. ET AL.: "The Bpel locus encodes type III secretion machinery in Bordetella pertussis" MICROBIAL PATHOGENESIS, vol. 27, no. 6, December 1999 (1999-12), pages 349-367, XP000925861 LONDON GB *
LEE C A: "TYPE III SECRETION SYSTEMS: MACHINES TO DELIVER BACTERIAL PROTEINS INTO EUKARYOTIC CELLS?" TRENDS IN MICROBIOLOGY,GB,ELSEVIER SCIENCE LTD., KIDLINGTON, vol. 5, no. 4, April 1997 (1997-04), pages 148-156, XP002922657 ISSN: 0966-842X *
SANO Y. ET AL: "Molecular structure and functions of pyocins S1 and S2 in Pseudomonas aeruginosa" JOURNAL OF BACTERIOLOGY, vol. 175, 1993, pages 2907-2916, XP000926029 WASHINGTON US *
YUK M H ET AL: "THE BVGAS VIRULENCE CONTROL SYSTEM REGULATES TYPE III SECRETION IN BORDETELLA BRONCHISEPTICA" MOLECULAR MICROBIOLOGY,GB,BLACKWELL SCIENTIFIC, OXFORD, vol. 28, no. 5, 1998, pages 945-959, XP002922650 ISSN: 0950-382X *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2217614A1 (fr) * 2007-11-07 2010-08-18 Dynamic Microbials Limited Compositions et formulations antimicrobiennes et leurs utilisations
EP2217614A4 (fr) * 2007-11-07 2011-12-07 Dynamic Microbials Ltd Compositions et formulations antimicrobiennes et leurs utilisations
CN113226361A (zh) * 2018-10-15 2021-08-06 代表亚利桑那大学的亚利桑那校董会 疫苗多肽组合物及方法

Also Published As

Publication number Publication date
HK1041490A1 (zh) 2002-07-12
GB9828217D0 (en) 1999-02-17
JP2002534960A (ja) 2002-10-22
AU2903700A (en) 2000-07-12
EP1140996A2 (fr) 2001-10-10
CN1371389A (zh) 2002-09-25
CA2356764A1 (fr) 2000-06-29
WO2000037493A3 (fr) 2001-02-01

Similar Documents

Publication Publication Date Title
Charles et al. Molecular cloning and characterization of protective outer membrane protein P. 69 from Bordetella pertussis.
CA2347849C (fr) Proteines omp85 de neisseria gonorrhoeae et de neisseria meningitidis, compositions renfermant lesdites proteines et methodes d'utilisation correspondantes
JP2006166919A (ja) 空胞形成毒素欠損Ｈ．ｐｙｌｏｒｉおよび関連する方法
EP0672048A1 (fr) GENE tagA ET PROCEDES PERMETTANT DE DETECTER LA PREDISPOSITION A L'ULCERATION GASTRODUODENALE
TWI328035B (en) Anti-bacterial vaccine compositions
Pedroni et al. Cloning of a novel pilin‐like gene from Bordetella pertussis: homology to the fim2 gene
AU5662190A (en) Compositions and treatments for pneumonia in animals
AU718392B2 (en) Haemophilus adhesion proteins
EP0719155A1 (fr) ANTIGENES DE $i(CAMPYLOBACTER JEJUNI), LEURS PROCEDES DE PRODUCTION ET LEUR UTILISATION
US20020054883A1 (en) Recombinant fusobacterium necrophorum leukotoxin vaccine and preparation thereof
US20040096973A1 (en) Environmentally regulated genes of Streptococcus suis
US6617128B2 (en) Nucleic acid molecules encoding proteins which impart the adhesion of neisseria cells to human cells
EP1140996A2 (fr) Vaccin
US20040219585A1 (en) Nontypeable haemophilus influenzae virulence factors
US20040047871A1 (en) Recombinant fusobacterium necrophorum leukotoxin vaccine and prepaation thereof
WO2000069903A1 (fr) Gene derive de $i(lawsonia) et polypeptides, peptides et proteines sodc associes, et leurs utilisations
WO1994028137A1 (fr) Regulateur de l'hemolysine induite par contact
EP1535928B1 (fr) Vaccin comprenant Omp85 protéines de Neisseria gonorhoeae et Neisseria meningitidis
WO2000071724A2 (fr) Composes nouveaux
Wieles et al. Molecular characterization and T-cell-stimulatory capacity of Mycobacterium leprae antigen T5
JPH11178584A (ja) ｇｉｄＢ
JP2002516333A (ja) ｐｒｉＡ
JPH10313881A (ja) ａｒｏＥ
JPH11225776A (ja) 新規ｓｐｏＩＩＩＥ
WO1992017587A1 (fr) ANTIGENE DE MEMBRANE EXTERIEURE DE $i(BORDETELLA BRONCHISEPTICA)

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 99816279.5

Country of ref document: CN

AK Designated states

Kind code of ref document: A2

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

WWE Wipo information: entry into national phase

Ref document number: 1999969578

Country of ref document: EP

Ref document number: 29037/00

Country of ref document: AU

ENP Entry into the national phase

Ref document number: 2356764

Country of ref document: CA

Ref document number: 2356764

Country of ref document: CA

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 512492

Country of ref document: NZ

ENP Entry into the national phase

Ref document number: 2000 589562

Country of ref document: JP

Kind code of ref document: A

WWP Wipo information: published in national office

Ref document number: 1999969578

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWE Wipo information: entry into national phase

Ref document number: 09868604

Country of ref document: US

WWW Wipo information: withdrawn in national office

Ref document number: 1999969578

Country of ref document: EP