# US 7,744,905 B2 · Immunogenic polypeptide isolated from mycobacterium avium subspecies paratuberculosis and uses thereof > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,744,905 B2 - Document ID: US7744905B2 - Status: Granted - Kind: B2 - Application number: 10/518,432 - Filed: 2003-06-19 - Published: 2006-09-21 - Granted: 2010-06-29 - Earliest claimed: 2002-06-19 - Assignees: Massey University; Institut Pasteur - Inventors: Alan Murray; Christine Dupont - Web page: https://patentgrep.com/patent/US7744905B2 ## CPC - C07K 14/35: from Mycobacteriaceae (F) - A61P 1/00: Drugs for disorders of the alimentary tract or the digestive system - A61P 31/04: Antibacterial agents - A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract The present invention relates to an immunogenic polypeptide isolated from Mycobacterium avium subspecies paratuberculosis and variants of the polypeptide. The polypeptide and variants may be used in vaccines against Johne's disease and in methods for detection of the disease. Antibodies against the polypeptide or variants may be used in diagnostic tests for Johne's disease. Also included are polynucleotides encoding the polypeptide and variants, and methods for preparing these. ## Claims ### Claim 1 A recombinant, purified, or isolated polypeptide comprising an amino acid sequence selected from (a) the sequence of SEQ ID NO: 1; (b) amino acids 20 to 235 of SEQ ID NO: 1; (c) a sequence which has greater than 95% amino acid sequence identity with SEQ ID NO: 1; or (d) a sequence which has greater than 95% amino acid sequence identity with the sequence of amino acids 20 to 235 of SEQ ID NO: 1. ### Claim 2 (depends on 1) The polypeptide as claimed in claim 1 wherein the amino acid sequence of the polypeptide has greater than 97% amino acid sequence identity with SEQ ID NO:1. ### Claim 3 (depends on 1) The polypeptide as claimed in claim 1 wherein the amino acid sequence of the polypeptide has greater than 99% amino acid sequence identity with SEQ ID NO: 1. ### Claim 4 (depends on 1) The polypeptide as claimed in claim 1 wherein the amino acid sequence of the polypeptide has greater than 99% amino acid sequence identity with the sequence of amino acids 20 to 235 of SEQ ID NO: 1. ### Claim 5 (depends on 1) The polypeptide as claimed in claim 1 wherein the polypeptide consists of an amino acid sequence selected from (a) the sequence of SEQ ID NO: 1; (b) amino acids 20 to 235 of SEQ ID NO: 1; (c) a sequence which has greater than 95% amino acid sequence identity with SEQ ID NO: 1; or (d) a sequence which has greater than 95% amino acid sequence identity with the sequence of amino acids 20 to 235 of SEQ ID NO: 1. ### Claim 6 (depends on 5) A kit for use in detecting the presence of Mycobacterium avium subspecies paratuberculosis comprising at least two of the following: a polypeptide as claimed in claim 5; an antibody that binds a polypeptide consisting of an amino acid sequence selected from (a) the sequence of SEQ ID NO:1 or (b) amino acids 20 to 235 of SEQ ID NO:1; or a reagent for determining antigen-antibody binding. ### Claim 7 (depends on 1) The polypeptide as claimed in claim 1 obtained from a bacterium. ### Claim 8 (depends on 1) The polypeptide as claimed in claim 1 obtained from Mycobacterium avium subspecies paratuberculosis. ### Claim 9 (depends on 1) The polypeptide as claimed in claim 1 obtained from a heterologous host transformed with a polynucleotide which encodes the polypeptide, wherein said host is capable of expressing said polypeptide. ### Claim 10 (depends on 9) The polypeptide as claimed in claim 9 wherein the host is E coli. ### Claim 11 (depends on 1) A genetic construct comprising (a) a promoter sequence; (b) an open reading frame polynucleotide encoding a polypeptide as claimed in claim 1; and (c) a termination sequence. ### Claim 12 (depends on 11) A host cell incorporating a construct of claim 11. ### Claim 13 (depends on 11) A vector comprising the construct as claimed in claim 11. ### Claim 14 (depends on 13) A host cell incorporating a vector as claimed in claim 13. ### Claim 15 (depends on 14) The host cell according to claim 14 wherein said vector exists within the host cell as a plasmid. ### Claim 16 (depends on 14) The host cell according to claim 14 wherein said vector is integrated into the genome of the host cell. ### Claim 17 (depends on 1) A vaccine composition comprising a polypeptide as claimed in claim 1 and an acceptable diluent, carrier, excipient, or adjuvant, said polypeptide being present in an amount sufficient to generate a protective immune response to Mycobacterium avium subspecies paratuberculosis infection. ### Claim 18 (depends on 17) A method of vaccinating against Johne's disease which comprises administering to an animal a vaccine composition as claimed in claim 17 in an amount sufficient to produce a protective response. ### Claim 19 (depends on 18) The method according to claim 18 wherein said administration is performed on a single occasion. ### Claim 20 (depends on 18) The method according to claim 18 wherein said administration is performed on more than one occasion. ### Claim 21 (depends on 18) The method as claimed in claim 18 wherein 0.1-1000 μg/Kg is administered of a recombinant, purified, or isolated polypeptide comprising an amino acid sequence selected from (a) the sequence of SEQ ID NO: 1; (b) amino acids 20 to 235 of SEQ ID NO:1; (c) a sequence which has greater than 95% amino acid sequence identity with SEQ ID NO:1; or (d) a sequence which has greater than 95% amino acid sequence identity with the sequence of amino acids 20 to 235 of SEQ ID NO:1. ### Claim 22 (depends on 21) The method as claimed in claim 21 wherein 5-500 μg/Kg of the polypeptide is administered. ### Claim 23 (depends on 1) A diagnostic composition for use in detecting the presence of Mycobacterium avium subspecies paratuberculosis, wherein said composition comprises a polypeptide as claimed in claim 1 together with one or more acceptable diluents, carriers, excipients, or adjuvants. ### Claim 24 (depends on 1) A method of prophylactically or therapeutically treating an animal against Johne's disease which comprises administering to an animal a polypeptide as claimed in claim 1 to produce a protective immunological response in the animal. ### Claim 25 (depends on 24) The method according to claim 24 which is a therapeutic method. ### Claim 26 (depends on 24) The method according to claim 24 which is a prophylactic method. ### Claim 27 (depends on 24) The method as claimed in claim 24 wherein the animal is a ruminant. ### Claim 28 (depends on 27) The method as claimed in claim 27 wherein the ruminant is a sheep. ### Claim 29 A recombinant, purified, or isolated polynucleotide comprising the sequence of SEQ ID NO: 2 or a variant thereof encoding a polypeptide comprising an amino acid sequence selected from (a) the sequence of SEQ ID No:1; (b) amino acids 20 to 235 of SEQ ID NO:1; (c) a sequence which has greater than 95% amino acid sequence identity with SEQ ID NO:1; or (d) a sequence which has greater than 95% amino acid sequence identity with the sequence of amino acids 20 to 235 of SEQ ID NO:1. ### Claim 30 (depends on 29) A recombinant, purified or isolated polynucleotide with a nucleotide sequence complementary to the polynucleotide of claim 29. ### Claim 31 (depends on 29) A diagnostic composition for detecting the presence of Mycobacterium avium subspecies paratuberculosis, wherein said composition comprises a polynucleotide according to claim 29 together with one or more acceptable diluents, carriers, excipients, or adjuvants. ### Claim 32 (depends on 29) A host cell transformed with a polynucleotide of claim 29. ### Claim 33 A purified or isolated antibody capable of binding a polypeptide consisting of an amino acid sequence selected from (a) the sequence of SEQ ID NO:1 or (b) amino acids 20 to 235 of SEQ ID NO:1. ### Claim 34 (depends on 33) A diagnostic composition for detecting the presence of Mycobacterium avium subspecies paratuberculosis comprising an antibody according to claim 33 together with one or more acceptable diluents, carriers, excipients, or adjuvants. ### Claim 35 A method of detecting Johne's disease including preclinical Johne's disease in an animal, the method comprising contacting a sample from the animal either with a purified or isolated antibody capable of binding a polypeptide consisting of an amino acid sequence selected from (a) the sequence of SEQ ID NO:1 or (b) amino acids 20 to 235 of SEQ ID NO:1 or with a composition comprising an antibody specific to a polypeptide consisting of an amino acid sequence selected from (a) the sequence of SEQ ID NO:1 or (b) amino acids 20 to 235 of SEQ ID NO:1, and one or more acceptable diluents, carriers, excipients, or adjuvants, and detecting a polypeptide which binds to the antibody. ### Claim 36 (depends on 35) The method according to claim 35 wherein the presence of bound antibody is determined by ELISA, radioimmunoassay or Western blotting. ### Claim 37 (depends on 35) The method according to claim 35 for detecting the presence of Mycobacterium avium subspecies paratuberculosis at a preclinical phase of Johne's disease. ### Claim 38 A method of detecting Johne's disease including preclinical Johne's disease in an animal comprising contacting either the animal or a sample from the animal with a polypeptide and detecting an immune response indicative of the presence of Mycobacterium avium subspecies paratuberculosis, the polypeptide consists of an amino acid sequence selected from (a) the sequence of SEQ ID NO:1 or (b) amino acids 20 to 235 of SEQ ID NO:1. ### Claim 39 (depends on 38) The method according to claim 38 wherein the response is a delayed-type hypersensitivity response. ### Claim 40 (depends on 38) The method according to claim 38 wherein said detecting comprises detecting the presence of antibodies that bind the polypeptide. ### Claim 41 (depends on 40) The method according to claim 40 wherein the detection of the presence of antibodies is by ELISA, radioimmunoassay or Western blotting. ### Claim 42 (depends on 38) The method as claimed in claim 38 wherein the animal is a ruminant. ### Claim 43 (depends on 42) The method as claimed in claim 42 wherein the animal is a sheep.