WO2000069903A1 - LAWSONIA DERIVED GENE AND RELATED SodC POLYPEPTIDES, PEPTIDES AND PROTEINS AND THEIR USES - Google Patents
LAWSONIA DERIVED GENE AND RELATED SodC POLYPEPTIDES, PEPTIDES AND PROTEINS AND THEIR USES Download PDFInfo
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- WO2000069903A1 WO2000069903A1 PCT/AU2000/000436 AU0000436W WO0069903A1 WO 2000069903 A1 WO2000069903 A1 WO 2000069903A1 AU 0000436 W AU0000436 W AU 0000436W WO 0069903 A1 WO0069903 A1 WO 0069903A1
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Classifications
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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
- C07K14/205—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Campylobacter (G)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Definitions
- the present invention relates generally to therapeutic compositions for the treatment and/or prophylaxis of intestinal disease conditions in animals and birds caused or exacerbated by Lawsonia intracellularis or similar or otherwise related microorganism.
- the present invention provides a novel gene derived from Lawsonia intracellularis which encodes an immunogenic peptide, polypeptide or protein.
- the polypeptide described herein, designated as sodC, or a peptide homologue, analogue or derivative thereof is particularly useful as an antigen in vaccine preparation for conferring humoral immunity against Lawsonia intracellularis and related pathogens in animal hosts.
- the present invention is also directed to methods for the treatment and/or prophylaxis of such intestinal disease conditions and to diagnostic agents and procedures for detecting Lawsonia intracellularis or similar or otherwise related microorganisms.
- L. intracellularis includes all microorganisms similar to or otherwise related to this microorganism, as described by Stills (1991) or Jones et a/.(1997) or Lawson et al. (1993) or McOrist et al. (1995).
- the word "sodC”, or the term “sodC gene”, shall be taken to refer to the gene encoding the SodC polypeptide of the present invention.
- the meat-producing sector of the agricultural industry is dependant upon the health of its livestock and there is a need to maintain disease-free livestock for human consumption
- the industry is subject to rapid economic downturn in response to disease conditions adversely affecting livestock and the quality of meat products derived therefrom, including those diseases which may potentially be transmitted to humans It is important, therefore, to have well defined treatments and prophylactic and diagnostic procedures available to deal with infections or potential infections in livestock animals and humans.
- porcine proliferative enteropathy PPE
- PPE proliferative haemorrhagic enteropathy
- PPE has been reported in a number of animal species including pigs (McOrist et al, 1993), hamsters (Stills, 1991), ferrets (Fox er a/, 1989), guinea pigs (Elwell er a/, 20 1981), rabbits (Schodeb and Fox, 1990) as well as avian species (Mason et al, 1998).
- the causative organism of PPE is a Campy/obacter-like organism referred to herein as "Lawsonia intracellularis" (McOrist et al, 1995). The organism has also been previously referred to as Heal symbiont intracellularis (Stills, 1991). PPE-like diseases 25 in pigs may also be caused by other pathogens such as various species of Campylobacter (Gebhart et al, 1983).
- Lawsonia intracellularis is an intracellular, possibly obligate intracellular, bacterium. It can only be cultured in vitro with tissue culture cells (Jones et al., 1997; Lawson et 30 al., 1993; McOrist et al, 1995; International Patent Application No. PCT/US96/09576).
- L. intracellularis is located in the cytoplasm of the villus cells and intestinal crypt cells of infected animals. Pigs suffering from PPE are characterised by irregularities in the villus cells and intestinal crypt structure with epithelial cell dysplasia, wherein crypt abscesses form as the villi and intestinal crypts become branched and fill with inflammatory cells.
- PPE is a significant cost component associated with the pig industry, especially in terms of stock losses, medication costs, reduced growth rates of pigs and increased feed costs. PPE also contributes to downstream indirect costs in, for example, additional labour costs and environmental costs in dealing with antibiotic residue contamination, and in control measures to prevent the organism from being passed on or carried to other animals or humans.
- the most effective vaccine preparations are generally comprised of a highly antigenic component, such as a peptide, polypeptide, protein or other macromolecule which is derived from the pathogenic organism against which the vaccine is directed, wherein said antigenic component produces little or no contraindications when administered to a susceptible host animal, and produces little or no antigenic cross-reactivity with desirable organisms, such as non-pathogenic organisms that are a part of the normal flora of the intestinal tract or other tissues of said host animal.
- a highly antigenic component such as a peptide, polypeptide, protein or other macromolecule which is derived from the pathogenic organism against which the vaccine is directed
- said antigenic component produces little or no contraindications when administered to a susceptible host animal, and produces little or no antigenic cross-reactivity with desirable organisms, such as non-pathogenic organisms that are a part of the normal flora of the intestinal tract or other tissues of said host animal.
- an effective vaccine preparation must be immunogenic, specific and safe.
- intracellularis partial genetic sequences and partial polypeptides encoded thereby.
- polypeptide immunogens produced by the bacterium L. intracellularis and immunogenic peptides derived therefrom including those immunogens which are genus- or species-specific, for use in improved vaccine compositions.
- the presently-described invention provides such immunogens.
- One aspect of the present invention is directed to an isolated or recombinant immunogenic polypeptide which comprises, mimics or cross-reacts with a B-cell or T- cell epitope of the SodC polypeptide derived from Lawsonia spp.
- the isolated or recombinant immunogenic polypeptide is selected from the group consisting of the following:
- a peptide, oligopeptide or polypeptide which comprises an amino acid sequence which has at least about 70% sequence identity overall to the amino acid sequence set forth in SEQ ID NO:1 ;
- a peptide, oligopeptide or polypeptide which comprises an amino acid sequence having at least about 50% sequence identity to about amino acid residues 1 to about 42 of SEQ ID NO:1 ;
- the polypeptide comprises or consists essentially of the amino acid sequence of SEQ ID NO:1 , or about amino acids 1 to 42 thereof.
- a further aspect of the invention extends to an immunologically interactive molecule, such as an antibody or antibody fragment, which is capable of binding to the immunogenic polypeptide of the invention.
- a further aspect of the invention provides a method of diagnosing infection of an animal by Lawsonia intracellularis or a related microorganism, said method comprising the steps of contacting a biological sample derived from said animal with an immunologically interactive molecule of the present invention for a time and under conditions sufficient for a complex, such as an antigen.antibody complex, to form, and then detecting said complex formation.
- a further aspect of the present invention contemplates a method of determining whether or not an animal has suffered from a past infection, or is currently infected, by Lawsonia intracellularis or a related microorganism, said method comprising contacting a tissue or fluid sample, such as blood or serum derived from said animal, with the immunogenic polypeptide of the invention for a time and under conditions sufficient for a complex, such as an antigen.antibody complex, to form, and then detecting said complex formation.
- a tissue or fluid sample such as blood or serum derived from said animal
- a further aspect of the present invention provides an isolated nucleic acid molecule which comprises a sequence of nucleotides that encodes, or is complementary to a nucleic acid molecule that encodes, a peptide, oligopeptide or polypeptide selected from the following:
- a peptide, oligopeptide or polypeptide which comprises an amino acid sequence which has at least about 70% overall sequence identity to the amino acid sequence set forth in SEQ ID NO:1 ;
- a peptide, oligopeptide or polypeptide which comprises an amino acid sequence having at least about 50% overall sequence identity to amino acid residues 1 to 42 of SEQ ID NO:1 ;
- the isolated nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:2, or at least that portion of SEQ ID NO: 2 encoding amino acid residues 1 to 42 of SEQ ID NO: 1 or a degenerate variant thereof, has at least about 50% sequence identity to all or a part thereof.
- a still further aspect of the invention provides a diagnostic method of detecting Lawsonia intracellularis or related microorganism in a biological sample derived from an animal subject, said method comprising the steps of hybridising one or more polynucleotide or r , ⁇ gonucleotide probes or primers derived from the nucleotide sequence set forth in SEQ ID NO:2 or a complementary nucleotide sequence thereof or a homologue, analogue or derivative thereof, to said sample, and then detecting said hybridisation using a detection means.
- the detection means according to this aspect of the invention is any nucleic acid-based hybridisation or amplification reaction.
- a further aspect of the invention provides an isolated probe or primer derived from SEQ ID NO:2 or a complementary nucleotide sequence thereto.
- Figure 1 is a schematic representation of a deduced amino acid sequence alignment of various bacterial [Cu, Zn]-superoxide dismutase (SOD) polypeptides.
- the inventors sought to identify immunogenic proteins of Lawsonia intracellularis for use in vaccines for the prophylaxis and treatment of PPE in animals, including pigs and birds.
- one aspect of the present invention is directed to an isolated or recombinant immunogenic polypeptide which comprises, mimics or cross-reacts with a B-cell or T-cell epitope of the SodC polypeptide derived from Lawsonia spp.
- Epitopes of Lawsonia spp. may be B cell epitopes or T-cell epitopes. It is well-known that antibody-binding sites (B-cell epitopes) involve linear as well as conformational epitopes (van Regenmortel, 1992). B-cell epitopes are predominantly conformational. In contrast, T-cells recognize predominantly linear epitope sequences in combination with MHC class II molecules. A precise identification and careful selection of epitopes of Lawsonia spp. facilitates the development of diagnostic reagents and vaccine compositions for the effective treatment or prophylaxis of Lawsonia infections. Epitope identification and characterization (i.e.
- determination of the molecular weight, amino acid sequence, and structure of epitopes of Lawsonia spp. may be performed using art-recognised techniques.
- degrading and denaturing of the epitope molecule must be avoided in order to conserve the three-dimensional structure, because the antigen-antibody reaction will be diminished if the secondary structure of the epitope is altered significantly.
- the characterisation and isolation of linear non-conformational epitopes is easier, because any immunoreactive regions are contained within a single peptide fragment or single amino acid sequence which is capable of being purified under a range of conditions.
- Both non-conformational and conformational epitopes may be identified by virtue of their ability to bind detectable amounts of antibodies (such as IgM or IgG) from sera of animals immunised against or infected with Lawsonia spp. and, in particular L. intracellularis, or an isolated polypeptide derived therefrom or, alternatively, by virtue of their ability to bind detectable amounts of antibodies in a purified Ig fraction derived from such sera.
- the antibodies may be derived from or contained within pools of polyclonal sera, or may be monoclonal antibodies.
- Antibody fragments or recombinant antibodies, such as those expressed on the surface of a bacteriophage or virus particle, such as in a phage display library, may also be employed.
- T-cell epitopes The determination of T-cell epitopes is performed by analysing the ability of the epitope peptides to induce the proliferation of peripheral blood lymphocytes or T-cell clones.
- the identification of T-cell epitopes is accomplished using a variety of methods as known in the art, including the use of whole and fragmented native or recombinant antigenic protein, as well as the more commonly employed "overlapping peptide" method. In the latter method, overlapping peptides which span the entire sequence of a polypeptide derived from Lawsonia spp. are synthesized and tested for their capacity to stimulate T-cell cytotoxic or proliferative responses in vitro.
- a successful method to recognize non-conformational linear epitopes is the immunoblot and in particular, the Western blot.
- Peptides may be generated from a complete Lawsonia spp. polypeptide by digestion with site-specific proteases, such as trypsin or chymotrypsin, and the peptides generated thereby can be separated using standard electrophoretic or chromatographic procedures.
- AMPHl The AMPHl algorithm (Margalit et a/., 1987), which is based on the periodicity of T cell epitopes, has been widely used for the prediction of T-cell antigenic sites from sequence information alone. Essentially, AMPHl describes a common structural pattern of MHC binding motifs, since MHC binding motifs (i.e., patterns of amino acids that appear to be common to most of the peptides that bind to a specific MHC molecule) appear to exhibit the same periodicity as an alpha helix. Identification of T- cell epitopes by locating MHC binding motifs in an amino acid sequence provides an effective means of identifying immunogenic epitopes in diagnostic assays.
- the EpiMer algorithm (Meister ef a/., 1995; Gabriel er a/., 1995; DeGroot et al., 1995) locates clustered MHC binding motifs in amino acid sequences of proteins, based upon the correlation between MHC binding motif-dense regions and peptides that may have the capacity to bind to a variety of MHC molecules (promiscuous or multi-determinant binders) and to stimulate an immune response in these various MHC contexts as well (promiscuous or multi-determinant epitopes).
- the EpiMer algorithm uses a library of MHC binding motifs for multiple class I and class II HLA alleles to predict antigenic sites within a protein that have the potential to induce an immune response in subjects with a variety of genetic backgrounds.
- EpiMer locates matches to each MHC-binding motif within the primary sequence of a given protein antigen. The relative density of these motif matches is determined along the length of the antigen, resulting in the generation of a motif-density histogram.
- the algorithm identifies protein regions in this histogram with a motif match density above an algorithm-defined cutoff density value, and produces a list of subsequences representing these clustered, or motif-rich regions.
- the regions selected by EpiMer may be more likely to act as multi-determinant binding peptides than randomly chosen peptides from the same antigen, due to their concentration of MHC-binding motif matches.
- the selection of regions that are MHC binding motif-dense increases the likelihood that the predicted peptide contains a "valid" motif, and furthermore, that the reiteration of identical motifs may contribute to peptide binding.
- MHC binding motif-based algorithms have been described by Parker et a/.(1994) and Altuvia et a/. (1994). In these algorithms, binding to a given MHC molecule is predicted by a linear function of the residues at each position, based on empirically defined parameters, and in the case of the Altuvia et a/.(1994) algorithm, known crystallographic structures may also be taken into consideration.
- polypeptide as used herein shall be taken to refer to any polymer consisting of amino acids linked by covalent bonds and includes within its scope full-length proteins and parts or fragments thereof such as, for example, oligopeptides and short peptide sequences consisting of at least about 5 amino acid residues, preferably at least about 10 amino acid residues, more preferably at least about 12 amino acid residues, and even more preferably at least about 15 amino acid residues. Also included within the scope of the definition of a "polypeptide” are amino acid sequence variants, containing one or more preferably conservative amino acid substitutions, '. j -
- a polypeptide may be isolated from a source in nature, or chemically synthesized
- a polypeptide may be derived from a full-length protein by chemical or enzymatic cleavage, using reagents such as CNBr, trypsm, or chymotrypsin, amongst others
- the present invention is not limited by the source of the subject immunogon and clearly extends to isolated and recombinant polypeptides which are derived from a natural or a non-natural occurring source
- Genetic manipulations which may be used in this context will be known to those skilled in the art and include, but are not limited to, nucleic acid isolation, restriction endonuclease digestion, exonuclease digestion, end-filling using the Klenow fragment of E. coli DNA polymerase I or T4 DNA polymerase enzymes, blunt-ending of DNA molecules using T4 DNA polymerase or Exolll enzymes, site-directed mutagenesis, ligation, and amplification reactions.
- nucleic acid hybridisations may also be utilised in the preparation of recombinant polypeptides, in confirming the identity of a nucleic acid molecule encoding a desired recombinant polypeptide and a genetic construct comprising the nucleic acid molecule.
- the polypeptide of the present invention is a recombinant polypeptide
- it may be produced in and, if desirable, isolated from a recombinant viral vector expression system or host cell.
- a cell for production of a recombinant polypeptide is selected on the basis of several parameters including the genetic constructs used to express the polypeptide under consideration, as well as the stability and actr ity of said polypeptide.
- the stability or activity of a recombinant polypeptide may be determined at least in part, by post-translational modifications to the polypeptide such as, for example, glycosylation, acylation or alkylation reactions, amongst others, which may vary between cell lines used to produce the recombinant polypeptide.
- the present invention extends to a recombinant polypeptide or a derivative, homologue or analogue thereof as present in a virus particle, or as produced in prokaryotic or eukaryotic host cell, or in a virus or cell culture thereof.
- the present invention also extends to a recombinant polypeptide according to any of the foregoing embodiments which is produced in a bacterial cell belonging to the genus Lawsonia, in particular a cell of L. intracellularis, or a culture thereof.
- isolated polypeptide refers to a polypeptide of the present invention which has been purified to some extent, preferably to at least about 20% by weight of protein, preferably to at least about 50% by weight of protein, more preferably to at least about 60% by weight of protein, still more preferably to at least about 70% by weight of protein and even more preferably to at least about 80% by weight of protein or greater, from its natural source or, in the case of non-naturally-occurring polypeptides, from the culture medium or cellular environment in which it was produced. Such isolation may be performed to improve the immunogenicity of the polypeptide of the present invention, or to improve the specificity of the immune response against that polypeptide, or to remove toxic or undesirable contaminants therefrom.
- polypeptide preparation will contain no contaminants which would reduce the immunogenicity of the polypeptide when administered to a host animal, in particular a porcine or avian animal being immunized against PPE or, alternatively, which would inhibit immuno-specific binding in an immunoassay for the diagnosis of PPE or a causative agent thereof.
- the purity of an isolated polypeptide of the present invention may e determined by any means known to those skilled in the art, including the degree of homogeneity of a protein preparation as assessed by SDS/polyacrylamide gel electrophoresis, 2- dimensional electrophoresis, or amino acid composition analysis or sequence analysis.
- the polypeptide of the present invention will be substantially homogeneous or substantially free of nonspecific proteins, as assessed by SDS/polyacrylamide gel electrophoresis, 2-dimensional electrophoresis, or amino acid composition analysis or sequence analysis.
- the polypeptide of the present invention can be purified for use as a component of a vaccine composition by any one or a combination of methods known to those of ordinary skill in the art, including, for example, reverse phase chromatography, HPLC, ion-exchange chromatography, and affinity chromatography, among others.
- the isolated or recombinant polypeptide of the invention possesses the enzymatic or biological activity of a SOD polypeptide such as, for example, the L. intracellularis SodC polypeptide, or is at least derived from a SodC polypeptide or, alternatively, is immunologically cross-reactive with the L. intracellularis SodC polypeptide of the present invention.
- a SOD polypeptide such as, for example, the L. intracellularis SodC polypeptide
- the L. intracellularis SodC polypeptide is at least derived from a SodC polypeptide or, alternatively, is immunologically cross-reactive with the L. intracellularis SodC polypeptide of the present invention.
- the isolated or recombinant polypeptide of the invention is derived from Lawsonia spp. or other pathogenic agent associated with the onset and/or development of PPE and more preferably, the subject polypeptide is derived from Lawsonia intracellularis.
- a B-cell or T-cell epitope of a SodC polypeptide or a derivative, homologue or analogue thereof may comprise any combination of the following:
- immunogenic polypeptides or derivatives, homologues or analogues thereof comprising the same, or substantially the same primary amino acid sequence .7 -
- immunogens which comprise a B-cell or T-cell epitope or similar term.
- Immunogenic polypeptides or derivatives, homologues, or analogues thereof comprising different primary amino acid sequences may comprise immunologically identical immunogens, because they possess conformational B-cell or T-cell epitopes that are recognised by the immune system of a host species to be identical.
- immunogenic polypeptides or derivatives, homologues or analogues thereof are hereinafter defined as "immunogens which mimic or cross-react with a B-cell or T-cell epitope", or similar term.
- the present invention extends to an immunogen which comprises, mimics, or cross-reacts with a B-cell or T-cell epitope of an isolated or recombinant polypeptide according to any one of the foregoing embodiments or a derivative, homologue or analogue thereof.
- the present invention provides an immunogen which comprises, mimics, or cross-reacts with a B-cell or T- cell epitope of an isolated or recombinant polypeptide which in its native form is obtainable from a species of Lawsonia such as, but not limited to L. intracellularis, and which polypeptide preferably possesses SodC activity.
- such immunogenic polypeptides will not comprise a primary amino acid sequence which is highly-conserved between L. intracellularis and another non- pathogenic microorganism which is normally resident in the gut or other organ of an animal, in particular a porcine or avian animal.
- a primary amino acid sequence which is highly-conserved between L. intracellularis and another non- pathogenic microorganism which is normally resident in the gut or other organ of an animal, in particular a porcine or avian animal.
- one or more amino acids not corresponding to the original protein sequence can be added to the amino or carboxyl terminus of the polypeptide.
- extra amino acids are useful for coupling the polypeptide to another peptide or polypeptide, to a large carrier protein or to a solid support.
- Amino acids that are useful for these purposes include but are not limited to tyrosine, lysine, glutamic acid, aspartic acid, cysteine and derivatives thereof.
- polypeptide can be immobilised to a polymeric carrier or support material.
- the immunogenicity of a polypeptide of the present invention may be improved using molecular biology techniques to produce a fusion protein containing one or more polypeptides of the present invention fused to a carrier molecules such as a highly immunogenic protein.
- a fusion protein containing a polypeptide of the present invention fused to the highly immunogenic B subunit of cholera toxin can be used to increase the immune response to the polypeptide.
- the present invention also contemplates fusion proteins comprising a cytokine, such as an interleukin, fused to the subject polypeptide of the present invention, and genes encoding same.
- the polypeptide of the present invention when administered to a mammal, induces an immune response in said mammal.
- the polypeptide of the present invention when administered to a mammal, in particular a porcine animal (e.g., a pig) induces a protective immune response against Lawsonia spp., and preferably against L. intracellularis, therein.
- the phrase "induction of a protective immune response”, and the like, refers to the ability of the administered polypeptide of the present invention to prevent or detectably slow the onset, development, or progression of symptoms associated with Lawsonia infection, and preferably, to prevent or detectably slow the onset, development, or progression of symptoms associated with PPE in pigs.
- the immunogenic polypeptide of the invention comprises an amino acid 5 sequence which is substantially the same as the amino acid sequence set forth in SEQ ID NO:1 or is at least about 75% identical overall to SEQ ID NO:1 , or is at least about 75% identical to at least 8 contiguous amino acids of SEQ ID NO:1.
- the immunogenic polypeptide of the present invention consists essentially of the amino acid sequence of SEQ ID NO:1 , or the amino acid sequence 10 encoded by the SodC-encoding nucleotide sequence present in pALK14 (ATCC 207155) or about the first forty two amino acids thereof.
- amino acid sequence set forth in SEQ ID NO:1 represents the partial amino acid sequence of the SodC polypeptide comprising the
- the nucleotide sequence of the 5'-end of the sodC gene is set forth in SEQ ID NO:2.
- the percentage amino acid sequence identity to SEQ ID NO:1 is at least about 80%, more preferably at least about 85%, even more preferably at least about 20 90%, and still even more preferably at least about 95% identical to SEQ ID NO:1.
- amino acid sequence identities or similarities may be calculated using the GAP programme of the Computer Genetics Group, Inc., University Research Park, Madison, Wisconsin, United States of America (Devereaux et al, 1984)
- the GAP programme utilizes the algorithm of Needleman and Wunsch (1970) to maximise the number of identical/similar residues and to minimise the number and/or length of sequence gaps in the alignment
- the ClustalW programme of Thompson er a/ (1994) can be used
- the isolated or recombinant immunogenic polypeptide of the invention comprises at least about 10 contiguous ammo acids derived from SEQ ID NO 1 , more preferably at least about 20 contiguous am o acid residues derived from SEQ ID NO 1 , even more preferably at least about 30 contiguous ammo acid residues derived from SEQ ID NO 1 and still even more preferably at least about 40 contiguous ammo acid residues derived from SEQ ID NO
- the present invention further encompasses homologues, analogues and derivatives of a polypeptide comprising the ammo acid sequence set forth in SEQ ID NO 1
- “Homologues” of a polypeptide are those polypeptides which contain ammo acid substitutions, deletions and/or additions relative to the polypeptide without altering one or more of its properties, such as its immunogenicity, biological activity or catalytic activity
- ammo acids can be replaced by other ammo acids having similar properties such as, for example, hydrophobicity, hydrophilicity, hydrophobic moment, antigenicity, propensity to form or break ⁇ -he cal structures of ⁇ -sheet structures, and so on
- Substitutional variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place
- Ammo acid substitutions are typically of single residues but may be clustered depending upon functional constraints placed upon the polypeptide, insertions will usually be of the order of about 1-10 ammo acid residues and deletions will range from about 1-20 residues
- ammo acid substitutions will comprise conservative ammo acid substitutions, such as those described supra
- Insertional ammo acid sequence variants are those in which one or more ammo acid residues are introduced into a predetermined site in the protein Insertions can comprise amino-terminal and/or carboxyl terminal fusions as well as mtra-sequence insertions of single or multiple ammo acids Generally, insertions within the ammo acid sequence will be smaller than am o or carboxyl terminal fusions, of the order of about 1 to 4 residues
- Ammo acid variants of the polypeptide of the present invention may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations
- peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations
- the manipulation of DNA sequences to produce variant proteins which manifest as substitutional, insertional or deletional variants are well known in the art
- techniques for making substitution mutations at predetermined sites in DNA having known sequence are well known to those skilled in the art, si h as by M13 mutagenesis or other site- directed mutagenesis protocol
- an "analogues” are defined as peptides, o gopeptides and polypeptides which are functionally equivalent to the peptides of the present invention but which contain certain non-naturally occurring or modified ammo acid residues as will be known to those skilled in the art Accordingly, an "analogue” as defined herein need not comprise an ammo acid sequence which is similar to the ammo acid sequence set forth herein such as, for example, peptides, ohgopeptides and polypeptides which are . ⁇ > ⁇ )
- mimotopes polypeptide analogues that cross-react with a B-cell or T-cell epitope of the Lawsonia polypeptide of the invention but, however, comprise a different ammo acid sequence to said epitope
- the antibodies used to identify such mimotopes may be polyclonal or monoclonal or recombinant antibodies, in crude or purified form
- Mimotopes of a T-cell epitope may then be assayed further for their ability to stimulate T-cell cytotoxic or proliferative responses in vitro
- Mimotopes are particularly useful as analogues of nonlinear (i e , conformational) epitopes of the polypeptide of the present invention, because conformational epitopes are generally formed from non-contiguous
- polypeptide analogues can result in polypeptides with increased immunogenic and/or antigenic activity, that are less sensitive to enzymatic degradation, and which are more selective
- a suitable prolme analogue is 2- ammocyclopentane carboxylic acid ( ⁇ AC 5 c) which has been shown to increase the immunogenic activity of a native polypeptide more than 20 times (Mierke et al, 1990, Portoghese et al, 1990, Goodman et al, 1987)
- “Derivatives” of a polypeptide described herein are those peptides, ohgopeptides and polypeptides which comprise at least about five contiguous ammo acid residues of the ammo acid sequence set forth in SEQ ID NO 1
- a “derivative” may further comprise additional naturally-occurring, altered glycosylated, acylated or non-naturally occurring 23
- ammo acid residues compared to the amino acid sequence set forth in SEQ ID NO 1 may comprise one or more non-ammo acid substituents such as for example, a reporter molecule or other ligand, covalently or non-covalently bound to the ammo acid sequence such as, for example, a reporter molecule which is bound thereto to facilitate its detection
- recombinant or synthetic mutants and derivatives of the peptide immunogens of the present invention include those incorporating single or multiple substitutions, deletions and/or additions therein, such as carbohydrates, hpids and/or proteins or polypeptides Naturally occurring or altered glycosylated or acylated forms of the subject peptides are particularly contemplated by the present invention Additionally, homopolymers or heteropolymers comprising one or more copies of the subject peptide listed in SEQ ID NO 1, or one or more derivatives, homologues or analogues thereof, are within the scope of the invention
- homologues, analogues and derivatives of the polypeptide of the invention are "immunogenic", defined hereinafter as the ability of said polypeptide, or a derivative, homologue or analogue thereof, to elicit B cell and/or T cell responses in the host, in response to immunization
- the immunogen of the present invention or a derivative, homologue or analogue thereof is useful in vaccine compositions that protect an individual against infection by L intracellularis and/or as an antigen to elicit polyclonal or monoclonal antibody production and/or in the detection of antibodies against L intracellularis in infected animals, particularly in porcine and avian animals
- N-termmal region of SEQ ID NO 1 is particularly unique, as compared to other immunogenic am o acid sequences, including those of the SodC polypeptides of other animal pathogens. Accordingly, peptides, ohgopeptides and polypeptides which comprise such unique epitope regions of SEQ ID NO 1 , will have improved specificity compared to other regions of the Lawsonia spp SodC molecule The particular advantages of such peptides will be immediately apparent to those skilled in the production of vaccine compositions, where specificity against a pathogen of interest is an important consideration
- ammo acids from about 1 to about 42 of the Lawsonia intracellularis SodC polypeptide is not highly conserved compared to the corresponding region of the Eschenchia coli SodC polypeptide, being only about 15% identical thereto Accordingly, this region of the L intracellularis SodC polypeptide is a promising antigenic peptide for the formulation of awso ⁇ /a-specific vaccines and diagnostics for the specific detection of Lawsonia spp in biological samples
- a second aspect of the present invention provides a vaccine composition for the prophylaxis or treatment of infection in a mammal or bird by L intracellularis or similar or otherwise related microorganism, said vaccine composition comprising
- an immunogenic component which comprises an isolated or recombinant polypeptide having at least about 70% overall ammo acid sequence identity to the ammo acid sequence set forth in SEQ ID NO 1 and/or at least about 50% ammo acid sequence identity to ammo acid residues 1 to about 42 of SEQ ID NO 1 or comprising at least 5 contiguous ammo acids derived from SEQ ID NO 1 or an immunogenic homologue, analogue or derivative thereof which is immunologically cross-reactive with Lawsonia intracellularis, and (II) one or more carriers, diluents and/or adjuvants suitable for veterinary or pharmaceutical use
- the term "immunogenic component” refers to a peptide, polypeptide or a protein encoded by DNA from, or derived from, L intracellularis or a related microorganism thereto which is capable of inducing a protective immune response in an animal, in particular a porcine or avian animal, whether or not said peptide, polypeptide or protein is in an isolated or recombinant form Accordingly, the vaccine composition clearly encompasses those vaccine compositions which comprise attenuated, killed or non-pathogenic isolates or forms of L intracellularis or related microorganisms thereto which comprise or express said peptide, polypeptide or protein
- protective immune response is meant that the immunogenic component elicits an immune response in the animal to which the vaccine composition is administered at the humoral and/or cellular level which is sufficient to prevent infection by Lawsonia intracellularis or a related microorganism thereto and/or which is sufficient to detectably reduce one or more symptoms or conditions, or to detectably slow the onset
- the polypeptide component of the subject vaccine composition comprises an amino acid sequence which is both immunogenic and specific, by virtue of its immunological cross-reactivity with the causative agent of PPE, Lawsonia intracellularis.
- polypeptide components may comprise an amino acid sequence derived from SEQ ID NO:1 or a homologue, analogue or derivative of the amino acid sequence set forth in SEQ ID NO:1 such as, for example, a mimotope of said sequence.
- the immunogenic polypeptide or immunogenic homologue, analogue or derivative may be a naturally-occurring peptide, oligopeptide or polypeptide in isolated or recombinant form according to any of the embodiments described supra or exemplified herein.
- the immunogenic polypeptide or immunogenic homologue, analogue or derivative is derived from Lawsonia spp., in particular L. intracellularis or a microorganism that is related thereto.
- the immunogenic component of the vaccine of the present invention can comprise a single peptide, polypeptide or protein, or a range or combination of different peptides, polypeptides or proteins covering different or similar epitopes.
- a single polypeptide can be provided with multiple epitopes.
- the latter type of vaccine is referred to as a polyvalent vaccine.
- a multiple epitope includes two or more epitopes located within a peptide or polypeptide molecule.
- Attenuated or non-pathogenic host cells include those cells which are not harmful to an animal to which the subject vaccine is administered.
- live vaccines can comprise an attenuated virus vector encoding the immunogenic component or a host cell comprising same, which is capable of replicating in an animal to which it is administered, and using host cell machinery to express the immunogenic component albeit producing no adverse side-effects therein.
- Such vaccine vectors may colonise the gut or other organ of the vaccinated animal.
- live vaccine vectors are efficacious by virtue of their ability to continually express the immunogenic component in the host animal for a time and at a level sufficient to confer protective immunity against a pathogen which expresses an immunogenic equivalent of said immunogenic component.
- the present invention clearly encompasses the use of such attenuated or non-pathogenic vectors and live vaccine preparations.
- the vaccine vector may be a virus, bacterial cell or a eukaryotic cell such as an avian, porcine or other mammalian cell or a yeast cell or a cell line such as COS, VERO, HeLa, mouse C127, Chinese hamster ovary (CHO), WI-38, baby hamster kidney (BHK) or MDCK cell lines.
- Suitable prokaryotic cells include Mycobacterium spp., Corynebacterium spp., Salmonella spp., Escherichia coli, Bacillus spp. and Pseudomonas spp, amongst others.
- Bacterial strains which are suitable for the present purpose are well-known in the relevant art (Ausube 1 et al, 1987; Sambrook et al, 1989).
- Such cells and cell lines are capable of expression of a genetic sequence encoding a SodC peptide, polypeptide or protein of the present invention from L. intracellularis in a manner effective to induce a protective immune response in the animal.
- a non-pathogenic bacterium could be prepared containing a recombinant sequence capable of encoding a peptide, polypeptide or protein from L intracellularis.
- the recombinant sequence would be in the form of an expression vector under the control of a constitutive or inducible promoter.
- the bacterium would then be permitted to colonise suitable locations in a pig's gut and would be permitted to grow and produce the recombinant peptide, polypeptide or protein in amount sufficient to induce a protective immune response against L. intracellularis
- the immunogenic component in a suitable vector system.
- the immunogenic component can be expressed by:
- nucleic acid molecule comprising the coding region of the nucleotide sequence set forth in SEQ ID NO:2 or a protein-encoding homologue, analogue or derivative of SEQ ID NO:2 selected from the group consisting of:
- nucleotide sequences that have at least about 70% sequence identity to SEQ ID NO:2;
- nucleotide sequences that hybridise under at least low stringency hybridisation, preferably under at least moderate stringency conditions, and even more preferably under high stringency conditions, to the complement of SEQ ID NO:2;
- the stringency is increased by reducing the concentration of SSC buffer, and/or increasing the concentration of SDS and/or increasing the temperature of the hybridisation and/or wash
- the conditions for hybridisation and/or wash may vary depending upon the nature of the hybridisation membrane or the type of hybridisation probe used
- Conditions for hybridisations and washes are well understood by one normally skilled in the art
- pages 2 10 8 to 2 10 16 of Ausubel et al (1987) which is herein incorporated by reference is found in pages 2 10 8 to 2 10 16 of Ausubel et al (1987), which is herein incorporated by reference
- nucleic acid molecule in an expressible format is a protem- encoding region of a nucleic acid molecule placed in operable connection with a promoter or other regulatory sequence capable of regulating expression in the vaccine vector system
- promoter includes the transcnptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence and additional regulatory elements (i e , upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner.
- promoter is also used to describe a recombinant, synthetic or fusion molecule, or derivative which confers, activates or enhances the expression of a nucleic acid molecule to which it is operably connected, and which encodes the immunogenic polypeptide.
- Preferred promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and/or to alter the spatial expression and/or temporal expression of the said nucleic acid molecule.
- Placing a nucleic acid molecule under the regulatory control of i.e., "in operable connection with” a promoter sequence means positioning the said molecule such that expression is controlled by the promoter sequence. Promoters are generally, but not necessarily, positioned 5' (upstream) to the genes that they control. In the construction of heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, i.e., the gene from which the promoter is derived. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene.
- the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.
- the prerequisite for producing intact polypeptides in bacteria such as E. coli is the use of a strong promoter with an effective ribosome binding site.
- Typical promoters suitable for expression in bacterial cells such as E. coli include, but are not limited to, the lacz promoter, temperature-sensitive ⁇ L or ⁇ R promoters, T7 promoter or the IPTG- inducible tac promoter.
- a number of other vector systems for expressing the nucleic acid molecule of the invention in E. coli are well-known in the art and are described, for example, in Ausubel et al (1987) or Sambrook et al (1989).
- plasmids with suitable promoter sequences for expression in bacteria and efficient nbosome binding sites have been described, such as for example, pKC30 ( ⁇ L Shimatake and Rosenberg, 1981), pKK173-3 (tac Amann and Brosius 1985), pET-3 (T7 Studier and Moffat, 1986), the pFLEX series of expression vectors (Pfizer Inc , CT, USA) or the pQE series of expression vectors (Qiagen, CA), amongst others
- Typical promoters suitable for expression in viruses of eukaryotic cells and eukaryotic cells include the SV40 late promoter, SV40 early promoter and cytomegalovirus (CMV) promoter, CMV IE (cytomegalovirus immediate early) promoter amongst others
- the immunogenic component of a vaccine composition as contemplated herein exhibits excellent therapeutic activity, for example, in the treatment and/or prophylaxis of PPE when administered in an amount which depends on the particular case
- from about 0 5 ⁇ g to about 20 mg may be administered, preferably from about 1 ⁇ g to about 10 mg, more preferably from about 10 ⁇ g to about 5 mg, and most preferably from about 50 ⁇ g to about 1 mg equivalent of the immunogenic component in a volume of about 1 ml to about 5ml
- a preferred amount is from about 0 1 ⁇ g/ml to about 5 mg/ml in a volume of about 1 to about 5 ml
- the DNA can be present in "naked" form or it can be administered together with an agent facilitating cellular uptake (e g , in liposomes or cationic lipids)
- the important feature is to administer sufficient immunogen to induce a protective immune response
- the above amounts can be administered as stated or calculated per kilogram
- the vaccine of the present invention can further comprise one or more additional immunomodulatory components such as, for example, an adjuvant or cytokme molecule, amongst others, that is capable of increasing the immune response against the immunogenic component
- additional immunomodulatory components such as, for example, an adjuvant or cytokme molecule, amongst others, that is capable of increasing the immune response against the immunogenic component
- adjuvants include the RIBI adjuvant system (Ribi Inc , Hamilton, MT, USA), alum, mineral gels such as aluminium hydroxide gel, oil-in-water emulsions, water-m-oil emulsions such as, for example, Block co-polymer (CytRx, Atlanta GA, USA),QS-21 (Cambridge Biotech Inc , Cambridge MA, USA), SAF-M (Chiron, Emeryville CA, USA), AMPHIGEN ® adjuvant, Freund's complete adjuvant, Freund's incomplete adjuvant, and Saponm, QuilA or other
- the vaccine composition can be administered in a convenient manner such as by oral, intravenous (where water soluble), intramuscular, subcutaneous, mtranasal, mtradermal or suppository routes or by implantation (e g , using slow release techno'ogy)
- the immunogenic component may be required to be coated in a material to protect it from the action of enzymes, acids and other natural conditions which may inactivate it, such as those in the digestive tract
- the vaccine composition may also be administered parenterally or mtraperitoneally Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, or in oils Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms Alternatively, the vaccine composition can be stored in lyophilised form to be rehydrated with an appropriate vehicle or carrier prior to use.
- compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
- the form must be fluid to the extent that easy syringability exists, unless the pharmaceutical form is a solid or semi-solid such as when slow release technology is employed. In any event, it must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms.
- isotonic agents such as, for example, sugars or sodium chloride.
- Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption such as, for example, aluminum monostearate and gelatin.
- Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter-sterilization.
- dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients selected from those enumerated above.
- the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof
- serogroup and "serovar” relate to a classification of microorganisms which is based upon serological typing data, in particular data obtained using agglutination assays such as the microscopic agglutination test (MAT)
- agglutination assays such as the microscopic agglutination test (MAT)
- MAT microscopic agglutination test
- serovar and serogroup antigens are a mosaic on the cell surface and, as a consequence there will be no strict delineation between bacteria belonging to a serovar and/or serogroup
- organisms which belong to different species may be classified into the same serovar or serogroup because they are indistinguishable by antigenic determination
- the term “serovar” means one or more Lawsonia strains which are antigenically-identical with respect to antigenic determinants produced by one or more loci Quantitatively, serovars may be differentiated from one another by cross-agglutination absorption techniques
- the present invention thus clearly extends to vaccine compositions for the treatment and/or prophylaxis of animals, in particular vaccine compositions for the treatment and/or prophylaxis of porcine and/or avian species, against any bacterium belonging to the same serovar or serogroup as Lawsonia intracellularis
- vaccine compositions for the treatment and/or prophylaxis of porcine and/or avian species against any bacterium belonging to the same serovar or serogroup as Lawsonia intracellularis
- such organisms will express a polypeptide having an ammo acid sequence identity of at least about 70% overall with respect to SEQ ID NO 1 and/or at least about 50% with respect to ammo acids 1 to about 42 of SEQ ID NO 1
- the present invention extends further to vaccine compositions capable of conferring protection against a "genetic variant" of Lawsonia intracellularis, the only requirement being that said variant expresses a polypeptide having an overall ammo acid sequence identity of at least about 70% with respect to SEQ ID NO 1 and/or at least about 50% with respect to ammo acids 1 to about 42 of SEQ ID NO 1 or a homologue, analogue or derivative thereof which is immunologically cross-reactive thereto
- Genetic variants of L intracellularis can be developed by mutation, recombination, conjugation or transformation of L intracellularis or may occur naturally It will be known to a person skilled in the art how to produce such derivatives
- the vaccine composition of the invention is intended for or suitable for the prophylaxis and/or treatment of infection in a porcine or avian animal and more preferably, for prophylaxis and/or treatment of a porcine animal for infection by L intracellularis
- the vaccine composition of the invention is intended for or suitable for the prophylaxis and/or treatment of infection in a porcine or avian animal and more preferably, for prophylaxis and/or treatment of a porcine animal for infection by L intracellularis
- the invention further extends to a method of treatment and/or prophylaxis of PPE in an animal such as an avian or porcine animal, said method comprising administering the vaccine composition or the immunogenic polypeptide of the invention as described or exemplified herein to said animal for a time and under conditions sufficient for an immune response to occur thereto.
- the immune response to the immunogen is a protective immune response.
- Animals which may be protected by the vaccine of the present invention include, but are not limited to, humans, primates, companion animals (e.g., cats, dogs), livestock animals (e.g., pigs, sheep, cattle, horses, donkeys, goats), laboratory test animals (e.g., mice, rats, guinea pigs, rabbits) and captive wild animals (e.g., kangaroos, foxes, deer).
- the present invention also extends to the vaccination of birds such as poultry birds, game birds and caged birds.
- the present invention further extends to combination vaccines compripmg an effective amount of a first immunogenic component comprising the polypeptide of the present invention combined with an effective amount of a second immunogenic component comprising one or more other antigens capable of protecting a porcine animal, or bird, against either Lawsonia spp. or another pathogen that infects and causes disease in said animal.
- the second immunogenic component is selected from the group consisting of the L. intracellularis autolysin, hemolysin, FlgE, and OmpH polypeptides and homologues, analogues or derivatives thereof, in particular immunogenic variants or derivatives thereof, and nucleic acid molecules encoding same.
- the isolated or recombinant SodC polypeptide of the invention or an immunologically- equivalent homologue, analogue or derivative thereof is also useful for the preparation of immunologically interactive molecules which are useful in the diagnosis of infection of an animal by Lawsonia spp., in particular by L. intracellularis or a related organism thereto.
- immunologically interactive molecule includes antibodies and antibody derivatives and functional equivalents, such as a Fab, or a SCAB (single- chain antibody), any of which optionally can be conjugated to an enzyme, radioactive or fluorescent tag, amongst others.
- Fab single-chain antibody
- SCAB single- chain antibody
- a further aspect of the invention extends to an immunologically interactive molecule which is capable of binding to any one or more of the following:
- a peptide, oligopeptide or polypeptide which comprises an amino acid sequence which has at least about 70% overall sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 ;
- a peptide, oligopeptide or polypeptide which comprises an amino acid sequence which comprises an amino having at least about 50% overall sequence identity to amino acid residues 1 to 42 of SEQ ID NO:1 ;
- the immunologically interactive molecule is an antibody that binds specifically to a polypeptide consisting of the amino acid of SEQ ID NO:1 , or to the first forty two amino acids thereof.
- Conventional methods can be used to prepare the immunologically interactive molecules.
- polyclonal antisera or monoclonal antibodies can be made using standard methods.
- a mammal e.g., a mouse, hamster, or rabbit
- an immunogenic form of the polypeptide of the present invention which elicits an antibody response in the mammal.
- Techniques for conferring immunogenicity on a polypeptide include conjugation to carriers, or other techniques well known in the art.
- the polypeptide can be administered in the presence of adjuvant or can be coupled to a carrier molecule, as known in the art, that enhances the immunogenicity of the polypeptide.
- the progress of immunization can be monitored by detection of antibody titres in plasma or serum. Standard ELISA or other immunoassay can be used with the immunogen as antigen to assess the levels of antibodies.
- antisera can be obtained and, for example, IgG molecules corresponding to the polyclonal antibodies can be isolated from the antisera.
- antibody producing cells can be harvested from an animal immunised with a peptide of the present invention and fused with myeloma cells by standard somatic cell fusion procedures, thus immortalizing these cells and yielding hybridoma cells.
- Such techniques are well known in the art, and include, for example, the hybridoma technique originally developed by Kohler and Milstein (1975), as well as other techniques such as the human B-celi hybridoma technique (Kozbor ef. al., 1983), the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985), and screening of combinatorial antibody libraries (Huse et al., 1989).
- Hybridoma cells can be isolated and screened immunochemically for production of antibodies that are specifically reactive with the polypeptide and monoclonal antibodies isolated therefrom.
- antibody as used herein, is intended to include fragments thereof which are also specifically reactive with a peptide that mimics or cross-reacts with a B-cell or T- cell epitope of the Lawsonia intracellularis SodC polypeptide set forth in SEQ ID NO 1
- Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies For example, F(ab')2 fragments can be generated by treating antibody with pepsin The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments
- any secondary antibodies (monoclonal, polyclonal or fragments of antibodies), including anti-idiotypic antibodies, directed to the first mentioned antibodies discussed above
- Both the first and second antibodies can be used in detection assays or a first antibody can be used with a commercially available ant ⁇ - ⁇ mmunoglobul ⁇ n antibody
- An antibody as contemplated herein includes any antibody specific to any region of a peptide which mimics, or cross-reacts with a B-cell or T-cell epitope of the Lawsonia intracellularis SodC polypeptide set forth in SEQ ID NO 1 as hereinbefore described
- the polyclonal, monoclonal or chimeric monoclonal antibodies can be used to detect the peptides of the invention and/or any homologues, analogues or derivatives thereof, in various biological materials.
- they can be used in an ELISA, radioimmunoassay, or histochemical test.
- the antibodies can be used to test for binding to a polypeptide of the invention or to a homologue, analogue or derivative thereof, in a biological sample to diagnose the presence of Lawsonia intracellularis therein.
- a further aspect of the invention provides a method of diagnosing infection of an animal by Lawsonia intracellularis or a related microorganism thereto, said method comprising the steps of contacting a biological sample derived from said animal with an immunologically interactive molecule which is capable of binding to a peptide, oligopeptide or polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 or a homologue, analogue or derivative thereof, for a time and under conditions sufficient for an antigen:antibody complex to form, and detecting said complex formation.
- the immunologically interactive molecule is preferably an antibody molecule prepared against the Lawsonia intracellularis SodC polypeptide set forth in SEQ ID NO:1 or an analogue or derivative thereof.
- the biological sample is one which might contain a polypeptide having an amino acid sequence set forth in SEQ ID NO:1 or a homologue, analogue or derivative thereof, in particular a biological sample derived from a porcine or avian host of the pathogen Lawsonia intracellularis or a related microorganism thereto, and can include any appropriate tissue or fluid sample from the animal.
- Preferred biological samples are derived from the ileum, caecum, small intestine, large intestine, whole serum or lymph nodes of the porcine or avian host animal being tested.
- the biological test sample may comprise faeces or a rectal swab derived from the animal.
- the antibodies should not be prepared against highly-conserved epitopes of SodC such as those regions of at least 5 amino acids in length which are conserved between L intracellularis and E.coli as set forth in Figure 1.
- Conventional immunoassays can be used to perform this embodiment of the invention.
- a wide range of immunoassay techniques are available as can be seen by reference to US Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These, of course, include both single-site and two-site or "sandwich” assays of the non-competitive types, as well as the traditional competitive binding assays.
- These assays also include direct binding of a labelled antibody to a target. It will be readily apparent to the skilled technician how to modify or optimise such assays to perform this embodiment of the present invention, and all such modifications and optimisations are encompassed by the present invention.
- the present invention contemplates a method of identifying whether or not an animal has suffered from a past infection, or is currently infected with Lawsonia intracellularis or a related microorganism thereto, said method comprising contacting blood or serum derived from said animal with the immunogenic polypeptide of the invention for a time and under conditions sufficient for an antigen.antibody complex to form, and detecting said complex formation.
- This embodiment differs from the embodiment described supra in that it relies upon the detection of circulating antibodies against Lawsonia intracellularis or related organism in the animals blood or serum which are present as a consequence of a past or present infection by this pathogen.
- the principle of the assay format is the same.
- a further aspect of the present invention provides an isolated nucleic acid molecule which comprises a sequence of nucleotides which encodes, or is complementary to a nucleic acid molecule which encodes, a peptide, oligopeptide or polypeptide selected from the following: (i) a peptide, oligopeptide or polypeptide which comprises an amino acid sequence having at least about 70% identical overall to the amino acid sequence set forth in SEQ ID NO:1 ;
- a peptide, oligopeptide or polypeptide which comprises an amino acid sequence having at least about 50% identity to amino acid residues from about position 1 to about position 42 of SEQ ID NO:1 ;
- polymeric forms of the immunogenic polypeptide described herein such as aggregates of the amino acid sequence set forth in SEQ ID NO:1 or a homologue, analogue or derivative thereof or, alternatively, as polypeptides comprising repeats of the amino acid sequence set forth in SEQ ID NO:1 or a homologue, analogue or derivative thereof.
- the present invention extends further to nucleic acid molecules encoding such polymeric forms. thereof.
- the isolated nucleic acid molecule of the invention further comprises a sequence of nucleotides which has at least about 70% overall sequence identity to the nucleotide sequence set forth in SEQ ID NO:2 or to a complementary nucleotide sequence thereof. More preferably, the percentage sequence identity to SEQ ID NO:2 or to a complementary nucleotide sequence thereto is at least about 80%. Still more preferably, the percentage sequence identity is at least about 90%. Yet still more preferably, the percentage sequence identity is at least about 95%.
- the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:2, or the SodC-encoding nucleotide sequence present in pALK14 (ATCC 207155), or a degenerate variant thereof, and complements thereof.
- nucleotide sequences In determining whether or not two nucleotide sequences fall within these percentage limits, those skilled in the art will be aware that it is necessary to conduct a side-by-side comparison or multiple alignment of sequences. In such comparisons or alignments, differences may arise in the positioning of non-identical residues, depending upon the algorithm used to perform the alignment.
- reference to a percentage identity between two or more nucleotide sequences shall be taken to refer to the number of identical residues between said sequences as determined using any standard algorithm known to those skilled in the art. For example, nucleotide sequences may be aligned and their identity calculated using the BESTFIT programme or other appropriate programme of the Computer Genetics Group, Inc., University Research Park, Madison, Wisconsin, United States of America (Devereaux et al, 1984).
- the isolated nucleic acid molecule of the invention is capable of hybridising under at least low stringency conditions to the nucleotide sequence set forth in SEQ ID NO:2 or to a complementary nucleotide sequence thereto or to a nucleic acid fragment comprising at least about 20 contiguous nucleotides in length derived from the sequence set forth in SEQ ID NO:2 or to a complementary nucleotide sequence thereto.
- said nucleic acid molecule is capable of hybridising under at least moderate stringency conditions, and even more preferably under high stringency conditions.
- a low stringency is defined herein as being a hybridisation and/or a wash carried out in 6xSSC buffer, 0.1% (w/v) SDS at 28°C.
- a moderate stringency is defined herein as being a hybridisation and/or wash carried out in 2xSSC buffer, 0.1% (w/v) SDS at a temperature in the range 45 °C to 65°C.
- a high stringency is defined herein as being a hybridisation and/or wash carried out in O.lxSSC buffer, 0.1% (w/v) SDS at a temperature of at least 65°C.
- the stringency is increased by reducing the concentration of SSC buffer, and/or increasing the concentration of SDS and/or increasing the temperature of the hybridisation and/or wash.
- concentration of SSC buffer and/or increasing the concentration of SDS and/or increasing the temperature of the hybridisation and/or wash.
- the present invention clearly encompasses genetic constructs comprising the subject nucleic acid molecule in an expressible format suitable for the preparation of a recombinant immunogenic polypeptide of the present invention, such as for use in recombinant univalent or polyvalent recombinant vaccines.
- nucleic acid molecule will be operably connected to a promoter sequence which can thereby regulate expression of said nucleic acid molecule in a prokaryotic or eukaryotic cell as described supra.
- the genetic construct optionally further comprises a terminator sequence.
- terminator refers to a DNA sequence at the end of a transcnptional unit which signals termination of transcription.
- a “terminator” is a nucleotide sequence, generally located within the 3'-non-translated region of a gene or mRNA, comprising a polyadenylation signal to facilitate the post-transcriptional addition of a polyadenylate sequence to the 3'-end of a primary mRNA transcript. Terminator sequences may be isolated from the genetic sequences of bacteria, fungi, viruses, animals and/or plants. Terminators active in animal cells are known and described in the literature.
- the genetic construct can be a cloning or expression vector, as known in the art, such as a plasmid, cosmid, or phage, comprising a nucleic acid molecule of the present invention, and host cells transformed or transfected therewith.
- the vector is plasmid pALK14 (ATCC Accession No. 207155).
- the genetic constructs of the present invention are particularly useful for producing the proteinaceous immunogenic component of the vaccine composition described herein or for use in a DNA vaccine.
- probe refers to a nucleic acid molecule which is derived from the nucleotide sequence set forth in SEQ ID NO:2 and which is capable of being used in the detection thereof. Probes may comprise DNA (single-stranded or double- stranded) or RNA (i.e., riboprobes) or analogues thereof.
- primer refers to a probe as hereinbefore defined which is further capable 5 of being used to amplify a nucleotide sequence from Lawsonia intracellularis or a related microorganism thereto, in a PCR.
- Probes and primers comprising the full-length of SEQ ID NO:2 or a complementary nucleotide sequence 0 thereto are also encompassed by the present invention.
- homologues of a nucleotide sequence shall be taken to refer to an isolated nucleic acid mole ule which encodes a polypeptide that is functionally equivalent to the polypeptice encoded by the nucleic acid molecule of the 25 present invention or to a polypeptide which is a homologue, analogue or derivative of SEQ ID NO:1 , notwithstanding the occurrence within said sequence, of one or more nucleotide substitutions, insertions, deletions, or rearrangements.
- nucleic acid molecule which encodes a functionally-equivalent polypeptide to the polypeptide encoded by the nucleic acid molecule of the present invention or a homologue, analogue or derivative of a polypeptide having the amino acid sequence of SEQ ID NO:1 , notwithstanding the occurrence of any non-nucleotide constituents not normally present in said isolated nucleic acid molecule such as, for example, carbohydrates, radiochemicals including radio nucleotides, reporter molecules such as, but not limited to biotin, DIG, alkaline phosphatase or horseradish peroxidase, amongst others.
- Nucleotide insertional derivatives of the nucleotide sequence of the present invention include 5 ' and 3 ' terminal fusions as well as intra-sequence insertions of single or multiple nucleotides or nucleotide analogues.
- Insertional nucleotide sequence variants are those in which one or more nucleotides or nucleotide analogues are introduced into a predetermined site in the nucleotide sequence of said sequence, although random insertion is also possible with suitable screening of the resulting product being performed.
- Deletional nucleotide sequence variants are characterised by the removal of one or more nucleotides from the nucleotide sequence.
- Substitutional nucleotide sequence variants are those in which at least one nucleotide in the sequence has been removed and a different nucleotide or nucleotide analogue inserted in its place.
- such substitutions are selected based on the degeneracy of the genetic code, as known in the art, with the resulting substitutional variant encoding the amino acid sequence of SEQ ID NO:1 or at least about the first 42 amino acids thereof.
- Probes or primers can comprise inosine, adenine, guanine, thymidine, cytidine or uracil residues or functional analogues or derivatives thereof that are capable of being incorporated into a polynucleotide molecule, provided that the resulting probe or primer is capable of hybridising under at least low stringency conditions to SEQ ID NO:2 or to a complementary nucleotide sequence thereof, or is at least about 70% identical to SEQ ID N0:2 or to a complementary nucleotide sequence thereof.
- the biological sample according to this aspect of the invention includes any organ, tissue, cell or exudate which contains or is likely to contain Lawsonia intracellularis or a nucleic acid derived therefrom.
- a biological sample can be prepared in a suitable solution such as, for example, an extraction buffer or suspension buffer.
- the present invention extends to the testing of biological solutions thus prepared, the only requirement being that said solution at least comprises a biological sample as described herein.
- the diagnostic assay of the present invention is useful for the detection of Lawsonia intracellularis or a microorganism which is related thereto which expresses the SodC polypeptide of the present invention or a SodC-like polypeptide.
- the present invention clearly contemplates diagnostic assays which are capable of both genus-specific and species-specific detection.
- the probe or primer, or a homologue, analogue or derivative thereof comprises DNA capable of being used to detect multiple Lawsonia spp.
- the probe or primer or a homologue, analogue or derivative thereof comprises DNA capable of being used to distinguish Lawsonia intracellularis from related microorganisms.
- Less-highly conserved regions within SEQ ID NO:2, such as those encoding about amino acid residues from about position 1 to about position 42 of the Lawsonia intracellularis SodC polypeptide set forth in SEQ ID NO: , are particularly useful as species-specific probes and/or primers for the detection of L. intracellularis and very closely related species.
- diagnostic assays described herein can be adapted to a genus- specific or species-specific assay by varying the stringency of the hybridisation step.
- a low stringency hybridisation can be used to detect several different s ecies of Lawsonia in one or more biolo ical sam les bein assa ed while a hi h stringency hybridisation can be used to distinguish Lawsonia intracellularis from such other species
- the detection means may be any nucleic acid- based detection means such as, for example, nucleic acid hybridisation techniques or paper chromatography hybridisation assay (PACHA), or an amplification reaction such as PCR, or nucleic acid sequence-based amplification (NASBA) system
- the invention further encompasses the use of different assay formats of said nucleic acid-based detection means, including restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), single-strand chain polymorphism (SSCP), amplification and mismatch detection (AMD), interspersed repetitive sequence polymerase chain reaction (IRS-PCR), inverse polymerase chain reaction (iPCR), in situ polymerase chain reaction and reverse transcription polymerase chain reaction (RT-PCR), amongst others
- the probe can be labelled with a reporter molecule capable of producing an identifiable signal (e g , a radioisotope such as 32 P or 35 S, or a biotmylated molecule)
- a reporter molecule capable of producing an identifiable signal
- the detection of said reporter molecule provides for identification of the probe and that, following the hybridisation reaction, the detection of the corresponding nucleotide sequences in the biological sample is facilitated Additional probes can be used to confirm the assay results obtained using a single probe
- a variation of the nucleic acid hybridisation technique contemplated by the present invention is the paper chromatography hybridisation assay (PACHA) described by Reinhartz et al (1993) and equivalents thereof, wherein a target nucleic acid molecule is labelled with a reporter molecule such as biotm, applied to one end of a nitrocellulose or nylon membrane filter strip and subjected to chromatography under the action of capillary or other forces (e g , an electric field) for a time and under conditions sufficient to promote migration of said target nucleic acid along the length of said membrane to a zone at which a DNA probe is immobilised thereto such as, for l
- PACHA paper chromatography hybridisation assay
- target nucleic acid comprising the Lawsonia spp nucleotide sequences complementary to the probe will hybridise thereto and become immobilised in that region of the membrane to which the probe is bound
- Non-complementary sequences to the probe will diffuse past the site at which the probe is bound
- the target nucleic acid may comprise a crude or partially-pure extract of DNA or RNA or, alternatively, an amplified or purified DNA Additional variations of this detection means which utilise the nucleotide sequences described herein are clearly encompassed by the present invention
- the detection means is an amplification reaction such as, for example a polymerase chain reaction or a nucleic acid sequence-based amplification (NASBA) system or a variant thereof same
- amplification reaction such as, for example a polymerase chain reaction or a nucleic acid sequence-based amplification (NASBA) system or a variant thereof same
- one or more nucleic acid primer molecules of at least 15 contiguous nucleotides in length derivable from SEQ ID NO 2 or its complementary nucleotide sequence, or a homologue, analogue or derivative thereof is hybridised to nucleic acid derived from a biological sample, and nucleic acid copies of the SodC-encoding genetic sequences in said sample, or a part or fragment thereof , are enzymically-amplified
- each primer is at least about 95% identical to a region of SEQ ID NO 2 or its complementary nucleotide sequence in the template molecule to which it hybridises
- PCR provides for the hybridisation of non-complementary primers to different strands of the template molecule, such that the hybridised primers are positioned to facilitate the 5 - 3' synthesis of nucleic acid in the intervening region, under the control of a thermostable DNA polymerase enzyme
- PCR provides an advantage over other detection means in so far as the nucleotide sequence in the region between the hybridised primers may be unknown and unrelated to any known nucleotide sequence
- the primers are selected such that, when nucleic acid derived from the biological sample, in particular DNA, is amplified, different length amplification products are produced from different Lawsonia spp
- the amplification products can be subjected to electrophoresis, transferred to a solid support such as, for example, a nylon or nitrocellulose membrane, and hybridised to a probe optionally labelled with a reporter molecule, as hereinbefore described
- a specific pattern of amplified DNA fragments is displayed on the support, said pattern optionally specific for a particular Lawsonia ssp , to enable the user to distinguish between different species of the bacterium in much the same way as for RFLP analysis
- the technique of AMD facilitates, not only the detection of Lawsonia spp DNA in a biological sample, but also the determination of nucleotide sequence variants which differ from the primers and probes used in the assay format
- the detection means is AMD
- the probe is end-labelled with a suitable reporter molecule and mixed with an excess of the amplified template molecule
- the mixtures are subsequently denatured and allowed to renature to form nucleic acid "probe template hybrid molecules" or “hybrids”, such that any nucleotide sequence variation between the probe and the temple molecule to which it is hybridised will disrupt base-pairing in the 33
- cleaved nucleic acid may be analysed using denaturing polyacrylamide gel electrophoresis, followed by standard nucleic acid hybridisation as described supra, to detect the awson/a-de ⁇ ved nucleotide sequences Those skilled in the art will be aware of the means of end-labelling a genetic probe according to the performance of the invention described in this embodiment
- the use of a single end-labelled probe allows unequivocal localisation of the sequence variation
- the distance between the po ⁇ nt(s) of sequence variation and the end-label is represented by the size of the cleavage product
- the probe is labelled at both ends with a reporter molecule, to facilitate the simultaneous analysis of both DNA strands
- the nucleic acid sample comprises an RNA molecule which is a transcription product of Lawson/a-de ⁇ ved DNA or a homologue, analogue or derivative thereof
- this assay format is particularly useful when it is desirable to determine expression of one or more Lawsonia genes
- the RNA sample is reverse-transcribed to produce the complementarv smgle-str? ided DNA which is subsequently amplified using standard procedures
- the present invention clearly extends to the use of any and all detection means referred to supra for the purposes of diagnosing Lawsonia spp and in particular Lawsonia intracellularis infection in animal
- the amplification reaction detection means described supra can be further coupled to a classical hybridisation reaction detection means to further enhance sensitivity and specificity of the inventive method, such as by hybridising the amplified DNA with a probe which is different from any of the primers used in the amplification reaction
- hybridisation reaction detection means described supra can be further coupled to a second hybridisation step employing a probe which is different from the probe used in the first hybridisation reaction
- a further aspect of the invention provides an isolated probe or primer derived from SEQ ID NO 2 or a complementary nucleotide sequence thereto
- Sections of grossly thickened ilea were taken from pigs naturally or experimentally affected by PPE The presence of L intracellularis bacteria in the ilea was confirmed using immunc'lucescent staining with specific monc lonal antibodies (McOrist et al, 1987)
- An example of a suitable antibody is monoclonal antibody IG4 available from the University of Edinburgh, UK
- INFECTED PIG ILEUM Lawsonia tntracellulans bacteria were extracted directly from lesions of PPE in pigs by filtration and further purified over a Percoll (Pharmacia, Uppsala, Sweden) gradient as D3
- Infected ilea were collected from pigs and the presence of L. intracellularis was confirmed histologically before storage at -80°C. Sections of ileum were thawed and approximately 8g of infected mucosa were scraped from the intestinal wall. The mucosa was homogenised with 40 ml sterile phosphate buffered saline (PBS) on half speed for 10 seconds using a Sorvall omnimixer. This suspension was centrifuged at 2000 xg for 4 minutes. The supernatant was discarded and the cell pellet was resuspended in 40 ml PBS and re centrifuged. This washing step was repeated twice. The cell pellet was then resuspended in 20 mi PBS and homogenised at full speed for one minute to release L. intracellularis bacteria.
- PBS sterile phosphate buffered saline
- This homogenate was centrifuged at 1000 xg for 4 minutes giving a pellet containing a crude mixture of homogenised epithelial cells and intestinal bacteria.
- the supernatant was filtered using filters with pore sized 3 ⁇ m, 1.2 ⁇ m and 0.8 ⁇ m (Millipore Corporation, MA, USA).
- the filtrate was centrifuged at 8000 xg for 30 minutes, resulting in a small pellet of L. intracellularis bacteria.
- intracellularis bacteria were further purified using a 45% self forming Percoll gradient as follows: 2 mis of the bacterial preparation was mixed by inversion into 30 mis of a 45% self forming Percoll (Pharmacia LKB, Uppsala, Sweden) gradient (45% v/v of Percoll, 150 mM NaCl). The gradients were centrifuged in a Sorval centrifuge using the SS34 rotor, at 20,000 rpm for 30 minutes at 4°C. Usually a number of bands form within the gradient. The band (usually located approx. 10-20 mm from the base of the tube) containing the L. intracellularis bacteria was collected and the volume made up to 16 mis with PBS. ⁇ he solution was then centrifuged for 15 minutes at 8000rpm. The resultant pellet was washed with PBS before being resuspended in a final volume of approximately one ml.
- a 45% self forming Percoll gradient as follows: 2 mis of the bacterial preparation was
- TE buffer (1 mM Tris-HCl, 0.1 mM EDTA, pH 8.0
- the pellet was then resuspended in 4 ml of TE buffer containing 4 mg/ml lysozyme (Sigma Chemical Co.) and incubated at 37°C for 20 min. SDS and proteinase K (Promega, Wl, USA) were added to final concentrations of 1 % (w/v) and 200 ⁇ g/ml, respectively, and incubation was continued at 45 C C for 4 hours. The lysate was then extracted with an equal volume of phenol, phenol:chloroform (1 :1) and chloroform, respectively, and the nucleic acids were recovered from the supernatant by ethanol precipitation.
- the pellet was gently dissolved in TE, treated with RnaseA (Promega, Wl, USA) at 37°C for 30 min and then digested with proteinase K in the presence of 0.5% (w/v) SDS for 1 h at 50°C. After another round of phenokchloroform (1 :1) and ethanol precipitation, the purified DNA was dissolved in TE. The DNA was then stored at 4°C.
- Example 3 The genomic DNA from Example 3 was partially digested with the restriction endonuclease Sau3A (Promega) and ligated into Lambda ZAP Express (Stratagene,
- the lambda library was plated on a lawn of E. coli XLI-Blue ceils at a density of 1 ,000 phage forming units (pfu) per 150 mm L-broth agar plate.
- the library was screened using the method described in the Protoblot Technical Manual
- the filters were blocked in blocking buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20 and 5% blotto) prior to screening with sera from the pigs Y12 and/or 395.
- the pigs Y12 and 395 had previously been immunised with formalin-killed L. intracellularis and heat-killed L. intracellularis, respectively, as described in International Patent Application No. PCT/AU96/00767. Positive plaques identified in the primary screen were picked, replated at a lower density and rescreened with either or both sera until an individual positive plaque was identified.
- Phagemid DNA from positive ⁇ ZAP Express phage clones was isolated by in vivo excision, by the conditions recommended by the manufacturer (Stratagene).
- the nucleotide sequence of the sodC gene of L. intracellularis was obtained from the clone p98LI50 identified in the preceding Example. DNA sequencing was performed by the Dye-terminator method of automated sequencing (ABI Biosystems, CA, USA). The nucleotide sequence of the complete coding region of the sodC gene is set out in SEQ ID NO: 2.
- the primary sequence of the native SodC protein appears to match the sequence requirements for both a standard secretory signal peptide cleavage site and a prokaryotic lipoprotein cleavage site Both cleavage sites are located at Ala26-Cys27 of the ammo acid sequence set forth in SEQ ID NO 1
- Plasmid p98LI50 was excised from Lambda ZAP Express (Stratagene Cloning Systems, La Jolla, California) and is a pBK- CMV derivative, which was identified by screening a L tntracellulans genomic lambda libraries with ⁇ - intracellularis pig antisera, as described in Example 4
- the PCR amplifications consisted of 0 1 ng of plasmid template, 1 ⁇ M each of the forward primer (RA167 5' GGCCATGGGTACCACCACCACCACCACCTCTCTGTC TGTTACTTCAGAAGTCCATATG 3', SEQ ID NO 4), and the reverse primer (RA175 5' GGCTCTAGAGGTATATAAATATAAAGAGGTATG 3', SEQ ID NO 5), 7 5 units KlenTaql polymerase (Ab Peptides, Inc , St Louis, Missouri), 0.075 units Pfu polymerase (Stratagene Cloning Systems, La Jolla, California) 1 x PC2 (KlenTaql) buffer and 0.2 mM dNTPs in a 50 ⁇ l volume.
- the PCR fragment containing the SodC gene of L. intracellularis was subcloned into pCR2.1-TOPO (Invitrogen Corp., Carlsbad, CA) and designated pALK14.
- the pCR2.1- TOPO intermediate plasmid was digested with ⁇ /col and EcoRI and the 0.6 kb fragment excised therefrom was gel-purified and sub-cloned into ⁇ /col- EcoRI-digested pET28b, to produce the PP-SS(-)SodC expression plasmid, pRL032.
- the ATG start codon of PP-SS(-)SodC is directly downstream from the ribosome- binding site.
- the expression of the PP-SS(-)SodC protein from this plasmid is under control of the T7 promoter, which is inducible by IPTG.
- the plasmid was introduced into E. coli BL21 (DE3) cells for expression of the modified SodC protein.
- the plasmid pALK14 was deposited with the American Type Culture Collection (ATCC) at 10801 University Boulevard, Manassas, VA 20110, USA on 11th March, 1999 and was assigned ATCC Accession No.207155.
- ATCC American Type Culture Collection
Abstract
Description
Claims
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP00924975A EP1177212A4 (en) | 1999-05-13 | 2000-05-11 | Lawsonia derived gene and related sodc polypeptides, peptides and proteins and their uses |
AU43858/00A AU4385800A (en) | 1999-05-13 | 2000-05-11 | Lawsonia derived gene and related sodC polypeptides, peptides and proteins and their uses |
JP2000618319A JP2003501013A (en) | 1999-05-13 | 2000-05-11 | Lawsonia-derived genes and related SodC polypeptides, peptides and proteins and uses thereof |
MXPA01011670A MXPA01011670A (en) | 1999-05-13 | 2000-05-11 | Lawsonia. |
NZ515332A NZ515332A (en) | 1999-05-13 | 2000-05-11 | Lawsonia derived gene and related SodC polypeptides, peptides and proteins and their uses |
BR0011292-5A BR0011292A (en) | 1999-05-13 | 2000-05-11 | Gene derived from lawsonia and related sodc proteins, peptides and polypeptides and their uses |
CA002372095A CA2372095A1 (en) | 1999-05-13 | 2000-05-11 | Lawsonia derived gene and related sodc polypeptides, peptides and proteins and their uses |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13398999P | 1999-05-13 | 1999-05-13 | |
US60/133,989 | 1999-05-13 |
Publications (1)
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WO2000069903A1 true WO2000069903A1 (en) | 2000-11-23 |
Family
ID=22461256
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Application Number | Title | Priority Date | Filing Date |
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PCT/AU2000/000436 WO2000069903A1 (en) | 1999-05-13 | 2000-05-11 | LAWSONIA DERIVED GENE AND RELATED SodC POLYPEPTIDES, PEPTIDES AND PROTEINS AND THEIR USES |
Country Status (10)
Country | Link |
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EP (1) | EP1177212A4 (en) |
JP (1) | JP2003501013A (en) |
AR (1) | AR023975A1 (en) |
AU (1) | AU4385800A (en) |
BR (1) | BR0011292A (en) |
CA (1) | CA2372095A1 (en) |
MX (1) | MXPA01011670A (en) |
NZ (1) | NZ515332A (en) |
PE (1) | PE20010238A1 (en) |
WO (1) | WO2000069903A1 (en) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6605696B1 (en) | 1999-10-22 | 2003-08-12 | Pfizer, Inc. | Lawsonia intracellularis proteins, and related methods and materials |
WO2005026200A2 (en) * | 2003-09-12 | 2005-03-24 | Akzo Nobel N.V. | Lawsonia intracellularis subunit vaccine |
US6921536B2 (en) | 2000-12-20 | 2005-07-26 | Akzo Nobel N.V. | Lawsonia intracellularis vaccine |
WO2005070958A3 (en) * | 2004-01-22 | 2005-11-24 | Akzo Nobel Nv | Lawsonia intracellularis subunit vaccines |
WO2006099561A1 (en) | 2005-03-14 | 2006-09-21 | Boehringer Ingelheim Vetmedica, Inc. | Immunogenic compositions comprising lawsonia intracellularis |
EP2204184A1 (en) | 2005-07-15 | 2010-07-07 | BOEHRINGER INGELHEIM VETMEDICA, Inc. | Lawsonia vaccine and methods of use thereof |
EP2859900A1 (en) | 2006-12-11 | 2015-04-15 | Boehringer Ingelheim Vetmedica, Inc. | Effective method of treatment of porcine circovirus and lawsonia intracellularis infections |
CN111830257A (en) * | 2020-07-17 | 2020-10-27 | 南京农业大学 | Swine lawsonia intracellularis IPMA antigen detection method and application thereof |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996039629A1 (en) * | 1995-06-05 | 1996-12-12 | Boehringer Ingelheim/Nobl Laboratories, Inc. | Lawsonia intracellularis cultivation, anti-lawsonia intracellularis vaccines and diagnostic agents |
WO1997020050A1 (en) * | 1995-11-30 | 1997-06-05 | Daratech Pty. Ltd. | Therapeutic and diagnostic compositions |
-
2000
- 2000-05-11 EP EP00924975A patent/EP1177212A4/en not_active Withdrawn
- 2000-05-11 CA CA002372095A patent/CA2372095A1/en not_active Abandoned
- 2000-05-11 MX MXPA01011670A patent/MXPA01011670A/en unknown
- 2000-05-11 WO PCT/AU2000/000436 patent/WO2000069903A1/en not_active Application Discontinuation
- 2000-05-11 BR BR0011292-5A patent/BR0011292A/en not_active IP Right Cessation
- 2000-05-11 AU AU43858/00A patent/AU4385800A/en not_active Abandoned
- 2000-05-11 JP JP2000618319A patent/JP2003501013A/en active Pending
- 2000-05-11 NZ NZ515332A patent/NZ515332A/en unknown
- 2000-05-12 AR ARP000102294A patent/AR023975A1/en unknown
- 2000-05-12 PE PE2000000445A patent/PE20010238A1/en not_active Application Discontinuation
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996039629A1 (en) * | 1995-06-05 | 1996-12-12 | Boehringer Ingelheim/Nobl Laboratories, Inc. | Lawsonia intracellularis cultivation, anti-lawsonia intracellularis vaccines and diagnostic agents |
WO1997020050A1 (en) * | 1995-11-30 | 1997-06-05 | Daratech Pty. Ltd. | Therapeutic and diagnostic compositions |
Non-Patent Citations (2)
Title |
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BOYE M. ET AL.: "Specific detection of lawsonia intracellularis in porcine proliferative enteropathy inferred from fluorescent tRNA in situ hybridization", VETERINARY PATHOLOGY, vol. 35, no. 2, March 1998 (1998-03-01), pages 153 - 156, XP000984238 * |
See also references of EP1177212A4 * |
Cited By (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6605696B1 (en) | 1999-10-22 | 2003-08-12 | Pfizer, Inc. | Lawsonia intracellularis proteins, and related methods and materials |
US6921536B2 (en) | 2000-12-20 | 2005-07-26 | Akzo Nobel N.V. | Lawsonia intracellularis vaccine |
US7491401B2 (en) | 2000-12-20 | 2009-02-17 | Intervet International B.V. | Lawsonia intracellularis vaccine |
WO2005026200A2 (en) * | 2003-09-12 | 2005-03-24 | Akzo Nobel N.V. | Lawsonia intracellularis subunit vaccine |
WO2005026200A3 (en) * | 2003-09-12 | 2005-06-23 | Akzo Nobel Nv | Lawsonia intracellularis subunit vaccine |
US7662390B2 (en) | 2003-09-12 | 2010-02-16 | Intarvet International B.V. | Lawsonia intracellularis subunit vaccine |
US8025884B2 (en) | 2004-01-22 | 2011-09-27 | Intervet International B.V. | Lawsonia intracellularis subunit vaccines |
WO2005070958A3 (en) * | 2004-01-22 | 2005-11-24 | Akzo Nobel Nv | Lawsonia intracellularis subunit vaccines |
EP2992897A1 (en) | 2005-03-14 | 2016-03-09 | Boehringer Ingelheim Vetmedica, Inc. | Immunogenic compositions comprising lawsonia intercellularis |
US8834891B2 (en) | 2005-03-14 | 2014-09-16 | Boehringer Ingelheim Vetmedica, Inc. | Immunogenic compositions comprising Lawsonia intracellularis |
WO2006099561A1 (en) | 2005-03-14 | 2006-09-21 | Boehringer Ingelheim Vetmedica, Inc. | Immunogenic compositions comprising lawsonia intracellularis |
EP3354279A2 (en) | 2005-03-14 | 2018-08-01 | Boehringer Ingelheim Vetmedica, Inc. | Immunogenic compositions comprising lawsonia intracellularis |
EP3906941A1 (en) | 2005-03-14 | 2021-11-10 | Boehringer Ingelheim Animal Health USA Inc. | Immunogenic compositions comprising lawsonia intercellularis |
EP2204184A1 (en) | 2005-07-15 | 2010-07-07 | BOEHRINGER INGELHEIM VETMEDICA, Inc. | Lawsonia vaccine and methods of use thereof |
EP2859900A1 (en) | 2006-12-11 | 2015-04-15 | Boehringer Ingelheim Vetmedica, Inc. | Effective method of treatment of porcine circovirus and lawsonia intracellularis infections |
CN111830257A (en) * | 2020-07-17 | 2020-10-27 | 南京农业大学 | Swine lawsonia intracellularis IPMA antigen detection method and application thereof |
CN111830257B (en) * | 2020-07-17 | 2023-10-24 | 南京农业大学 | Pig-derived lawsonia intracellularis IPMA antigen detection method and application thereof |
Also Published As
Publication number | Publication date |
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CA2372095A1 (en) | 2000-11-23 |
NZ515332A (en) | 2004-01-30 |
AR023975A1 (en) | 2002-09-04 |
BR0011292A (en) | 2002-02-26 |
MXPA01011670A (en) | 2003-10-14 |
EP1177212A1 (en) | 2002-02-06 |
PE20010238A1 (en) | 2001-03-03 |
AU4385800A (en) | 2000-12-05 |
EP1177212A4 (en) | 2002-08-28 |
JP2003501013A (en) | 2003-01-14 |
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