WO2003093307A2 - Antigenes mycobacteriels et leurs utilisations - Google Patents

Antigenes mycobacteriels et leurs utilisations Download PDF

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Publication number
WO2003093307A2
WO2003093307A2 PCT/GB2003/001815 GB0301815W WO03093307A2 WO 2003093307 A2 WO2003093307 A2 WO 2003093307A2 GB 0301815 W GB0301815 W GB 0301815W WO 03093307 A2 WO03093307 A2 WO 03093307A2
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Prior art keywords
seq
mycobacterium
polypeptide
diagnostic
bovis
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PCT/GB2003/001815
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English (en)
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WO2003093307A8 (fr
WO2003093307A3 (fr
Inventor
Paul Jason Cockle
Hanns Martin Vordermeier
Stephen Vincent Gordon
Robert Glyn Hewinson
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The Secretary Of State For Environment, Food And Rural Affairs
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Priority claimed from GB0209724A external-priority patent/GB0209724D0/en
Priority claimed from GB0209723A external-priority patent/GB0209723D0/en
Application filed by The Secretary Of State For Environment, Food And Rural Affairs filed Critical The Secretary Of State For Environment, Food And Rural Affairs
Priority to AU2003224313A priority Critical patent/AU2003224313A1/en
Publication of WO2003093307A2 publication Critical patent/WO2003093307A2/fr
Publication of WO2003093307A8 publication Critical patent/WO2003093307A8/fr
Publication of WO2003093307A3 publication Critical patent/WO2003093307A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • 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
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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 to the use of antigens derived from the RDl or RD2 regions of the Mycojbacteriuiii tuberculosis , Mycobacterium bovis or Mycobacterium africanum genomes, and peptides derived therefrom, as diagnostic reagents, in particular in the context of diagnostic kits.
  • certain of these peptides, as well as other antigens and peptides derived from the RD14 region of the genome are suitable for use as vaccines.
  • Novel fusion peptides are also part of the invention.
  • the present invention relates to diagnostic kits comprising such antigens for differentiating between those mammals infected by tuberculosis, those which have been vaccinated against tuberculosis, and those mammals, which have been sensitised by environmental Mycobacteria.
  • the present invention further relates to novel Mycobacterium tuberculosis , Mycobacterium bovis and Mycobacterium africanum peptides derived from such antigens.
  • the present invention also relates to vaccines against Mycobacterium infections, in particular, Mycobacterium tuberculosis, Mycobacterium bovis or Mycobacterium africanum infections, as well as to veterinary and pharmaceutical compositions containing thes'e and their preparations.
  • Bovine tuberculosis is caused by Mycobacterium bovis and shares greater than 99.9% DNA identity with M. tuberculosis, the main cause of human tuberculosis. Moreover, BTB is a zoonotic disease and was responsible during the 1930s and 1940s for approximately 6% of the total human deaths due to TB, and more than 50% of all cervical lymphadentitis cases in children. The introduction of pasteurisation of milk in the 1930s dramatically reduced the transmission from cattle to man. However, it still remains a small but significant cause of human morbidity and mortality especially in developing countries and is seen. as one of the most important infectious diseases of both man and other animals in the world.
  • Mycobacterium bovis causes disease in both cattle and man.
  • TB in cattle can have severe implications for animal welfare, causing reduced productivity and premature death, resulting in substantial economic losses to affected farms.
  • a compulsory eradication programme based upon the slaughter of infected animals, detected by the single intraderrnal comparative tuberculin skin test, began in Great Britain (GB) in 1950 and by 1960 it had been implemented in all of Britain. These measures resulted in the dramatic reduction of bovine tuberculosis in GB from incidence rates of around 40% of cattle infected with M. bovis to 0.41% in 1996.
  • the incidence of BTB in cattle has been steadily rising since 1988, possibly due to a wildlife reservoir of I. -bovis.
  • BCG is an attenuated strain of M. bovis, and is currently the only available vaccine for the prevention of BTB. Encouraging results with BCG have been reported in New Zealand where a significant level of protection in BCG vaccinated cattle against experimental M. bovis infection has been recently demonstrated.
  • Immunity to M. tuberculosis is characterised by three basic features: 1) living bacilli which efficiently induce a protective immune response; 2) specifically sensitised T lymphocytes which mediate this protection, and 3) interferon gamma (IFN- ⁇ ) which is an important mediator molecule.
  • IFN- ⁇ interferon gamma
  • tuberculin PPD has become an integral part of the bovine tuberculosis eradication programme.
  • blood-based diagnostic assays that measure antigen-induced lymphokine production such as the IFN- ⁇ are also under consideration.
  • the cytokine IFN ⁇ appears to be critical in the development of immunity to M. tuberculosis. For example, both mice with a disruptive IFN- ⁇ gene and humans with mutated I N- ⁇ receptor are highly susceptible to mycobacterial in ections .
  • specificity constraints are associated with the use of PPD in such assays. These arise due to the crude mixture of M.
  • jovis proteins that it contains, many that are cross-reactive with other environmental mycobacterial species, e.g., M. avium or M. intracellulare and importantly the vaccine strain M. bovis Bacille Calmette-Guerin (BCG) .
  • BCG Bacille Calmette-Guerin
  • a cattle vaccine would reduce the risk of cattle infection and hence result in lower tuberculin test frequencies and signi icant cost savings . It is believed that the development of an improved cattle vaccine holds the best long-term prospect for BTB control in British herds . In addition, it would be desirable to develop a complimentary diagnostic test to differentiate between vaccinated animals and those infected with M. bovis (differential diagnosis) in parallel with the vaccine to ensure continuation of the test and slaughter-rbased control strategies .
  • diagnostic reagents which distinguish between vaccinated and infected cattle can be developed using specific, defined antigens that are present in virulent M. bovis but absent from the vaccine strain.
  • Genetic analysis of BCG has revealed that several large genomic regions have been deleted during attenuation and subsequent prolonged propagation in culture [Behr, M. A., et al.. 1999. Science 284:1520 - 1523; Gordon, S. V., et al. 1999. Mol. Microbiol. 32: 643-655]. These regions have been characterised and antigens from one of these regions, RDl [Mahairas, G. G., et al. 1996. J. Bacteriol.
  • antigens have not reached that of tuberculin. It would, therefore, be desirable to provide other antigens in order to achieve this desired sensitivity. Such antigens may also be useful in supplementing the ESAT-6 and CFP-10 to achieve even greater sensitivity.
  • Synthetic peptides have the advantages of lower production costs, easier standardisation, improved quality control and carry no risk of infection since they are chemically synthesised.
  • Such synthetic peptide epitopes have been found to have great potential in the study of immune responses in cattle and in the development of diagnostic reagents. For example, formulation of 10 synthetic peptides derived from ESAT-6 and CFP-10 resulted in similar cellular immune responses to those seen with the whole recombinant antigens. When assayed in cattle this cocktail could distinguish between M. bovis infected animals and BCG vaccinated cattle with sensitivity similar to PPD and with a greater specificity [Vordermeier, 2001 supra.].
  • BCG BCG vaccination studies have highlighted the variability with regard to its efficacy. In humans, this ranges from 0 to
  • the present invention seeks to provide an improved diagnostic test to differentiate between vaccinated mammals and those infected with tuberculosis.
  • the test of the present invention differentiates between animals vaccinated against Mycobacterium bovis, Mycobacterium tuberculosis or Mycobacterium africanum and those infected with Mycobacterium bovis, Mycobacterium tuberculosis or Mycobacterium africanum.
  • the present invention also seeks to provide an improved vaccine for control of tuberculosis and in particular to control tuberculosis in cattle.
  • the tuberculosis disease also affects a number of other different animal species such as guinea pigs, badgers, possums and deer.
  • the vaccines of the present invention may therefore be useful in the control of tuberculosis infections in such different animals .
  • Mycobacterium bovis or Mycobacterium africanum genomes, or a variant, ho ologue or fragment of these, other than ESAT-6, CFP- 10, MPT-64 are useful as diagnostic agents, and in particular are a source of diagnostic peptides.
  • the polypeptide is other than polypeptides encoded by the Rvl984c, Rv3871,
  • Rv3872 or at least certain parts of the Rv3873 regions of the Mycobacterium tuberculosis, Mycobacterium bovis or Mycobacterium africanum genomes.
  • polypeptide as used herein includes long chain peptides including proteins and epitopic fragments thereof. Such proteins generally comprise one or more chains of amino acids joined covalently through peptide bonds and are typically greater than 10,000 MW.
  • peptides refers to small proteins (generally less than about 10,000 MW) , and in particular to smaller chains, for example up to 30 amino acids in length, preferably up to 20 amino acids in length. Also included however are small oligopeptides comprising three or more amino acid residues covalently linked through peptide bonds. Peptides will generally comprise two or more amino acid residues linked together covalently through peptide bonds.
  • the polypeptide from which the diagnostic agents are selected are preferably encoded by the Mycobacterium tuberculosis genome and comprise a member of the PE/PPE protein family.
  • derived from means any polypeptide or peptide encoded by an open reading frame from the specified regions of the Mycobacterium tuberculosis, Mycobacterium bovis or Mycobacterium africanum genomes.
  • the applicants utilise peptides encoded by fragments of the open reading frames and variants thereof as long as such peptides are still capable of being used as diagnostic reagents.
  • they will comprise epitopic sequences .
  • a diagnostic reagent comprising a peptide comprising an epitope from at least one polypeptide selected from Rvl986 (SEQ ID NO 1), Rv3878 (SEQ ID NO 3), Rv 1983 (SEQ ID NO 4), Rv3873 (SEQ ID NO 5) or Rv3879 (SEQ ID NO 6).
  • an “epitope” of the sequence comprises those amino acids that are necessary to generate in immune response, and therefore be recognised in a diagnostic test. They may be consecutive amino acids, or they may be spaced apart from one another. In the latter case, the nature of the amino acids between the amino acids of the epitope may not be crucial to the activity and may be varied. Determination of the amino acids which comprise the epitopes can be determined using routine methods, for example by finding antigenic regions or fragments, as illustrated hereinafter, and then carrying out a series of small mutations, for example, point mutations, and then determining whether the immunogenic or diagnostic activity has been retained. Where it has, then the variant retains the epitope. If activity has been lost, then the mutation has disrupted the epitope and so must be reversed.
  • the peptide comprises a series of consecutive amino acids from within the polypeptide sequence.
  • the polypeptide from which the peptide is derived comprises the sequence shown in SEQ ID Nos 1, 3, 4 or 6.
  • polypeptide from which the peptide is derived comprises the sequence shown in SEQ ID Nos 3, 5 or 6.
  • diagnostic reagents comprise peptides which include an epitope from SEQ ID NO 23 as shown in Figure 9 hereinafter, which is a fragment of SEQ ID NO 5, and in particular, an epitope found within SEQ ID NO 25 or SEQ ID NO 7, or within SEQ ID NOs 28 and 29.
  • SEQ ID NO 23, and fragments thereof, such as SEQ ID NOS 7,25, 28 and 29 form particular embodiments of the diagnostic reagents of the invention.
  • Other particular examples of diagnostic reagents comprise peptides which include an epitope from SEQ ID NO 35, shown in Figure 10 hereinafter, which is a fragment of SEQ ID NO 3.
  • SEQ ID NO 35 or fragments thereof form a particular embodiment of the invention.
  • diagnostic reagents comprise peptides which include an epitope from SEQ ID NO 48, shown in Figure 11 hereinafter, which is a fragment of SEQ ID NO 6, and in particular, an epitope found within SEQ ID NO 51.
  • SEQ ID NO 48 or variants thereof, or fragments of these, in particular SEQ ID NO 51 form particular embodiments of the diagnostic reagents of the invention.
  • polypeptides themselves, as well as homologues or variants thereof may also be used as diagnostic agents, but preferably fragments (comprising peptides are employed) .
  • the peptides described above may be used in either specific or differential diagnostic tests.
  • fragment thereof refers to any portion of the given amino acid sequence which has the same activity as the complete amino acid sequence. Fragments will suitably comprise at least 10 and preferably at least 20 consecutive amino acids from the basic sequence. In one embodiment, the fragment sequence comprises 17 amino acids.
  • Fragments of the polypeptide include deletion mutants and polypeptides where small regions of the polypeptides are joined together.
  • the fragments should contain an epitope, and preferably contain at least one antigenic region.
  • the term "homologue” refers to similar genes found in other organisms, such as the Mycobacterium bovis or Mycobacterium africanum genomes,
  • variant thereof as used herein in relation to an amino acid sequence means sequences of amino acids which differ from the base sequence from which they are derived in that one or more amino acids within the sequence are substituted for other amino acids.
  • Amino acid substitutions may be regarded as "conservative” where an amino acid is replaced with a different amino acid with broadly similar properties. Non-conservative substitutions are where amino acids are replaced with amino acids of a different type.
  • conservative substitution is meant the substitution of an amino acid by another one of the same class; the classes being as follows :
  • Nonpolar Ala, Val, Leu, lie, Pro, Met, Phe, Trp
  • Uncharged polar Gly. Ser, Thr, Cys, Tyr, Asn, Gin
  • altering the primary structure of a peptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out. This is so even when the substitution is in a region, which is critical in determining the peptides con ormation.
  • a diagnostic test based upon SEQ ID NOS 1 and 3 or homologues or variants thereof can give a differential diagnostic test, which in particular, can differentiate between tuberculosis-infected and tuberculosis vaccinated mammals .
  • Selection of a diagnostic reagent comprising or derived from these sequences can be made so that they differentiate between Mycobacterium bovis, Mycobacterium tuberculosis or Mycobacterium africanum -infected mammals and mammals vaccinated against Mycobacterium bovis, Mycobacterium tuberculosis or Mycobacterium econum.
  • diagnostic tests based upon SEQ ID Nos 3 or 6, or a homologue or variant thereof can also provide differential diagnostic tests, but in this case they are able to distinguish between mammals, which are either vaccinated against or infected by tuberculosis and mammals, sensitised by environmental mycobacteria.
  • the diagnostic reagent used in a specific diagnostic test preferably differentiates between Mycobacterium bovis -infected and mammals sensitised by environmental mycobacteria.
  • peptides are capable of being used as diagnostic reagents and are preferably synthetic peptides having the advantages discussed above.
  • One such peptide is a peptide derived from SEQ ID NO.5, which is shown in Figure 6 as SEQ ID NO 7. Fragments, homologues and variants of this peptide are also included herein.
  • the peptide as shown in SEQ' ID NO 7 may be used in a specific diagnostic test to differentiate between those mammals, which are either vaccinated against or infected by tuberculosis, and those mammals which have been sensitised by environmental mycobacteria.
  • the peptide is especially useful in differentiating between Mycobacterium bovis- infected mammals, such as cattle or calves, and those animals sensitised by environmental bacteria.
  • Such peptides may be used as diagnostic reagents, either on their own or preferably with one or more other peptides, which may be other peptides according to the present invention, or different peptides, in order to achieve greater sensitivity and specificity of a diagnostic test.
  • protein or peptide cocktails composed of other antigens from the RDl or RD2 regions of the Mycobacterium bovis, Mycobacterium tuberculosis or Mycobacterium africanum genomes may be used in addition to the antigens of the present invention in order to enhance the specificity of the diagnostic test.
  • peptide cocktails may include peptides derived from the antigens, ESAT-6 and CFP-10, as well as those described above.
  • a diagnostic kit comprising at least two diagnostic reagents, at least one of which is a diagnostic reagent as described above.
  • kits of the invention will comprise polypeptides or peptides, at least one of which is selected from a polypeptide derived from the sequences shown as SEQ ID Nos 1, 3, 4, 6 and 7, or a fragment, homologue or variant thereof, and optionally at least one polypeptide derived from the sequences shown as SEQ ID Nos 2 and 5, and optionally one or more reagents.
  • Such kits may be used to differentiate between tuberculosis-infected and tuberculosis-vaccinated mammals.
  • the polypeptide and peptide sequences described herein can provide a means for detecting the recognition of the polypeptides or peptides by the T-cell.
  • diagnostic reagents utilised in the diagnostic kit are able to differentiate between Mycobacterium bovis, Mycobacterium tuberculosis or Mycobacterium africanum -infected mammals and mammals vaccinated against Mycobacterium bovis, Mycobacteri um tuberculosis or Myco-bacteriurr. africanum.
  • the kit will preferably comprise the polypeptides derived from the sequences shown as SEQ ID Nos 1, 2 and 3, or a fragment, homologue or variant thereof.
  • it will comprise diagnostic reagents comprising peptides comprising an epitope from these sequences.
  • the kit will preferably comprise polypeptides or peptides derived from the sequences shown as SEQ ID Nos 4, 5, 6, and optionally, SEQ ID NO. 7 or a fragment, homologue or variant thereof.
  • it will comprise diagnostic reagents comprising peptides comprising an epitope from these sequences .
  • the diagnostic kit may also comprise one or more polypeptides or peptides from the RDl region of the Mycobacterium bovis, Mycobacterium tuberculosis or Mycobacterium africanum genomes. Protein or peptide cocktails composed of such polypeptides may also be used. Especially preferred are peptide cocktails composed of the ESAT-6 and/ or the CFP-10 polypeptides. Such peptide cocktails may be used to enhance the sensitivity of the diagnostic tests of the present invention.
  • polypeptides and peptides described above are encoded by nucleic acids.
  • Novel nucleic acids for example which encode novel peptides or polypeptides as described above form a further aspect of the invention, together with fragments homologues or variants thereof.
  • the nucleic acid may be DNA or RNA, and where it is a DNA molecule, it may comprise a cDNA or genomic DNA. These nucleic acids may themselves be useful as vaccines and such vaccines form a further aspect of the present invention.
  • the nucleic acid comprises the sequence shown in SEQ ID Nos 8, 10, 11 or 13, or a variant or fragment thereof.
  • fragment thereof refers to any portion of the given polynucleotide sequence which exhibits the same activity as the complete polynucleotide sequence. Fragments will suitably comprise at least 15, preferably at least 30 and more preferably at least 60 consecutive bases from the basic sequence.
  • variant thereof in relation to a polynucleotide or nucleic acid sequences means any substitution of, variation of, modification of, replacement of deletion of, or the addition of one or more nucleic acid(s) from or to a polynucleotide sequence providing the resultant protein sequence encoded by the polynucleotide exhibits the same properties as the protein encoded by the basic sequence.
  • the term therefore includes allelic variants and also includes a polynucleotide which substantially hybridises to the polynucleotide sequence of the present invention. Preferably, such hybridisation occurs at, or between low and high stringency conditions.
  • low ' stringency conditions can be defined as 3 x SSC at about ambient temperature to about 55°C and high stringency condition as 0.1 x SSC at about 65°C.
  • SSC is the name of the buffer of 0.15M NaCl. 0.015M tri-sodium citrate. 3 x SSC is three times as strong as SSC and so on.
  • variants have 62% or more of the nucleotides in common with the polynucleotide sequence of the present invention, more typically 65%, preferably 70%, even more preferably 80% or 85% and, especially preferred are 90%, 95%, 98% or 99% or more identity.
  • BESTFIT When comparing nucleic acid sequences for the purposes of determining the degree of identity, programs such as BESTFIT and GAP (both from Wisconsin Genetics Computer Group (GCG) software package) .
  • BESTFIT compares two sequences and produces an optimal alignment of the most similar segments.
  • GAP enables sequences to be aligned along their whole length and fins the optimal alignment by inserting spaces in either sequence as appropriate.
  • the comparison is made by alignment of the sequences along their whole length.
  • diagnosis of infection in a host, or exposure of a host, to a mycobacterium can be carried out by i) contacting a population of cells from the host with a polypeptide derived from an RDl or RD2 region of the Mycobacterium tuberculosis , Mycobacterium bovis or Mycobacterium africanum genomes, or a variant, homologue or fragment of these, which polypeptide may be used as a diagnostic reagent, including those described above, in addition to those derived from ESAT-6, CFP-10, MPT-64; and ii) determining whether the cells of said cell population recognise the polypeptide or fragment or variant thereof.
  • a method of diagnosising infection in a host, or exposure of a host, to a mycobacterium comprising i) contacting a population of cells from the host with a diagnostic reagent according to the invention; and ii) determining whether the cells of said cell population recognise the diagnostic reagent.
  • Suitable diagnostic reagents are as described above.
  • the population of cells used in the method is suitably a population of T-cells.
  • the method preferably diagnoses infection by Mycobacterium tuberculosis , Mycobacterium bovis or Mycobacterium africanum.
  • the diagnostic reagents and peptides described above can be used, in accordance with a further aspect of the invention to produce an antibody specific to the peptide.
  • Polypeptides of the invention may be isolated from strains of M. bovis, M. tuberculosis or M. africanum. Preferably, they are prepared synthetically using conventional peptide synthesisers. Alternatively, they may be prpduced using recombinant DNA technology or isolated from natural sources followed by any chemical modification, if required. In these cases, nucleic acids encoding the polypeptides are incorporated into suitable expression vectors, which are then used to transform a suitable host cell, such as a pro aryotic cell such as E. coli . The transformed cells are cultured and the polypeptide isolated therefrom. Vectors, cells and methods of this type form further aspects of the present invention.
  • a particular diagnostic kit comprising the polypeptides derived from the sequences shown as SEQ ID Nos 1 to 3 and further comprising one or more polypeptides derived from the RDl region of Mycobacterium tuberculosis , Mycobacterium bovis or Mycobacterium africanum, and optionally one or more reagents, for differentiating between cattle infected by M. bovis and cattle which have been vaccinated with BCG or with a vaccine according to the present invention.
  • a specific diagnostic kit comprises the polypeptides and peptides derived from the sequences shown as SEQ ID Nos 4 to 7 and further comprising one or more polypeptides derived from the RDl region of Mycobacterium tuberculosis, Mycobacterium bovis or Mycobacterium africanum, and optionally one or more reagents, for differentiating between cattle which have either been vaccinated against or infected by M. bovis and those cattle which have been sensitised by environmental mycobacteria.
  • a further preferred embodiment of the present invention is a vaccine comprising a peptide having the sequence shown in SEQ ID No 7.
  • An advantage of the present invention is that the level of sensitivity achieved in diagnostic tests with these antigens is higher than the sensitivity achieved with the antigens ESAT-6 and CFP-10.
  • the level of specificity of the antigen of the present invention is higher than that of PPD, which is currently used.
  • PPD has the disadvantage that it cross-reacts with other environmental mycobacterial species and the vaccine strain M. bovis Bacille Calmette-Guerin (BCG) .
  • BCG Bacille Calmette-Guerin
  • a further advantage of the present invention is the provision of a test which can distinguish between those mammals that have been vaccinated against tuberculosis, and in particular M. bovis, and those which have been infected with M. bovis . This allows the selective slaughter of animals which would appear from current tests to all be infected, thereby saving the lives of many animals.
  • the invention further provides a polypeptide comprising any one of SEQ ID NO 1, 2, 3, 4, 5 or 6, or variants thereof, or fragments of any of these, which produce a protective immune response in a mammal to whom they are administered, for use as a vaccine.
  • the present invention provides a polypeptide derived from an RD2 or RD14 region of the Mycobacterium tuberculosis, Mycobacterium bovis or Mycobacterium africanum genomes, or a variant thereof, or a fragment of any of these, for use as a medicament, with the proviso that the polypeptide is not a MPT-64 polypeptide or a polypeptide encoded by the Rvl984c region of the Mycobacterium tuberculosis, Mycobacterium bovis or Mycobacterium africanum genomes.
  • These polypeptides, or variants or fragments are preferably used as a vaccine against tuberculosis caused by Mycobacterium tuberculosis , Mycobacterium bovis or Mycobacterium africanum.
  • polypeptide of the invention is derived from the Mycobacterium tuberculosis genome .
  • polypeptide of the invention comprises the sequence shown in SEQ ID Nos 14 or 15, or a variant thereof or fragment thereof. Most preferably the polypeptide is of SEQ ID NO 14 or 15 or an epitopic fragment thereof.
  • the invention provides a polypeptide comprising a fusion of a region of SEQ ID NO 14 and a region of SEQ ID NO 15, which fusion polypeptide is able to produce a protective immune response in a mammal to which it is administered.
  • fusion polypeptides are illustrated hereinafter, as SEQ ID NOs 18, 20 and 22, and these, together with protective variants and fragments thereof form preferred embodiments of the invention.
  • Polypeptides which are protective are protective against tuberculosis infection and therefore may be used as a prophylactic or therapeutic vaccine, and these form a further aspect of the invention.
  • particular vaccines comprise a polypeptide derived from an RD2 or RDl region of the Mycobacterium tuberculosis , Mycobacterium bovis or Mycobacterium africanum genomes, or a variant thereof or a fragment of any of these, which polypeptide produces a protective immune response against tuberculosis infection in a mammal to which it is administered, with the proviso that the polypeptide is not a MPT-64 polypeptide or a polypeptide encoded by the Rvl98 c region of the McoJbacte.rii-.ffi tuberculosis, Mycobacterium bovis or Mycobacterium africanum genomes.
  • the vaccine is preferably used to vaccinate against tuberculosis. It may be used as a vaccine against tuberculosis in humans, cattle and other mammals including guinea pigs, badgers, possums and deer. It is, however, preferably used as a vaccine in cattle.
  • the vaccine comprises one or more protein subunits.
  • it may comprise a nucleic acid such as a DNA or cDNA encoding for the protein or protein subunits.
  • the nucleic acid When it comprises a nucleic acid, this is suitably incorporated into an expression vector, in such as way that the protein subunit is expressed in the host animal.
  • the nucleic acid may be incorporated into a virus vector such as a vaccinia or adenovirus vector, or a plasmid to form a so-called "naked DNA" vaccine.
  • the vector may contain the usual expression control functions such as promoters, enhancers and signal sequences, as well as selection markers in order to allow detection of successful transformants. The nature of these will depend upon the precise nature of the vector chosen and will be known to or readily determinable by a person skilled in the art.
  • vaccine compositions will further comprise an adjuvant such as in order to enhance the immune response to the biologically active material administered.
  • adjuvants include pharmaceutically acceptable adjuvants such as Freund' s incomplete adjuvant, aluminium compounds and, preferably adjuvants which are known to up-regulate mucosal responses such as CTB, the non-toxic pentameric B subunit of cholera toxin (CT) .
  • nucleic acid encoding a polypeptide of the invention or a fragment or variant thereof.
  • the nucleic acid may be DNA or RNA and where it is a DNA molecule, it may comprise a cDNA or genomic DNA. These nucleic acids may themselves be useful as vaccines .
  • the nucleic acids of the present invention are those shown as SEQ ID Nos 59 and 60, as well as or a variant or fragments thereof.
  • One such variant, which encodes a fusion is SEQ ID NO 17.
  • a pharmaceutical or veterinary composition comprising a protective polypeptide as described above, or a nucleic acid which encodes this, in combination with a pharmaceutically or veterinarily acceptable carrier.
  • the carriers may be solid or liquid as understood in the art. They may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art.
  • compositions of the invention may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs) , for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing or as a suppository for rectal dosing.
  • inhalation for example as a finely divided powder or a liquid aerosol
  • insufflation for example as a finely divided powder
  • parenteral administration for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing or as a suppository for rectal
  • compositions are preferably in the form of a sterile injectable aqueous or oily suspension, • which may be formulated according to known procedures using one or more of the appropriate dispersing or wetting agents and suspending agents, which have been mentioned above.
  • a sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example a solution in 1,3-butanediol.
  • compositions of the invention comprise a nucleic acid
  • they are preferably formulated for parenteral administration and in particular intramuscular injection, although other means of application are possible as described in the pharmaceutical literature, for example administration using a Gene Gun,
  • Such formulations include delivery of the plasmid DNA via a bacterial vector such as species of Salmonella or Listeria (Sizemore et al
  • Dosage unit forms will generally contain about 1 mg to about 500 mg of an active ingredient.
  • the size of the dose for therapeutic or prophylactic purposes of the composition of the invention will naturally vary according to the age and sex of the animal or patient and the nature of the active component and the route of administration, according to well known principles of medicine. Generally speaking however, for administration to a human as a prophylactic vaccine, dosage units of from 0.25 ⁇ g to 2.5mg will be typically employed.
  • the invention provides a method of protecting a mammal against infection against Mycobacterium jbovis, Mycobacterium tuberculosis or Mycobacterium africanum comprising administering to said mammal a polypeptide, a nucleic acid or a composition as described above.
  • the polypeptide is a pharmaceutical or veterinary composition
  • the polypeptide may be administered directly.
  • a nucleic acid encoding the polypeptides is administered to a mammal in a form in which it is expressed in situ.
  • Mycobacterium tuberculosis Mycobacterium bovis or Mycobacterium africanum genomes, or a variant thereof, or a fragment of any of these, which peptide produces a protective immune response against tuberculosis infection in a mammal to which it is administered.
  • peptides derived from the polypeptides of the present invention are also encompassed by the present invention.
  • Such peptides are preferably synthetic peptides.
  • the polypeptide may for example, have the sequence shown in SEQ ID NO. 7 or an epitopic fragment thereof.
  • the vaccine is preferably used to vaccinate against tuberculosis. It may be used as a vaccine against tuberculosis in both humans and cattle. It is, however, preferably used as a vaccine in cattle.
  • the vaccine comprises protein subunits.
  • it may comprise DNA or cDNA encoding for the subunits.
  • a method of protecting a mammal against infection by Mycobacterium bovis, Mycobacterium tuberculosis or Mycobacterium africanum comprising administering to said mammal a polypeptide, peptide or pharmaceutical or veterinary composition according to the present invention which produces an immune response against Mycobacterium bovis, Mycobacterium tuberculosis or Mycobacterium africanum.
  • Figure 1 shows the recognition of RDl products by a M. bovis infected cow (A, C and E) and a PPD-A reactor (B, D and F) .
  • Whole blood was cultured in the presence of peptide pools of between 8-11 peptides representing RDl (A and B), RD2 (C and D) and RD14 (E and F) at 5 ⁇ g each peptide/ml.
  • Dashed horizontal lines indicate positive cut-off (OD 450 values with antigens minus OD450 without antigens > 0.1);
  • Figure 2 shows IFN- ⁇ responses induced by RD region antigens by M. bovis infected (22), BCG vaccinated (6) and PP-A reactor cattle (10) . Only the results of the pool/antigen stimulating the greatest IFN- ⁇ response are shown. Green squares represent M. bovis infected cattle, red triangles represent PP-A reactors and blue circles represent BCG vaccinated cattle. Dashed horizontal line indicate the positive cutOoff (OD45 0 values with antigens minus OD 4 5 0 without antigens >0.1) .
  • Figure 3 shows IFN- ⁇ secretion induced by individual peptides from pool 3 (A) and pool 26 (B) in whole blood cultures from two representative animals.
  • Whole blood was collected from M. bovis experimentally infected cattle and incubated for 48hrs with peptides (25ug/ml each) .
  • Results are expressed as delta mean optical density OD 45 o values with antigens minus OD4 5 0 without antigens) of duplicate determinations, with a positive cut-off of 0.1.
  • Figure 4 shows the antigens selected for evaluation.
  • Figure 5 shows the most frequently recognised antigen.
  • Figure 6 shows the sequence homology between peptide 3.2 from Rv3873 (shown as SEQ ID NO. 7) with other mycobacterial proteins .
  • Figure 7 shows the amino acid sequences which correspond to the open reading frames Rvl979c, Rvl769c, Rvl986, Rv3872, Rv3878, Rvl983, Rv3873 and Rv3879c which are shown as SEQ ID Nos 1 to 6, the amino acid sequences of the antigens which are particular vaccine candidates and in particular the Rv 1979 and Rvl769 antigens which are shown as SEQ ID Nos 14 and 15 respectively.
  • Figure 8 shows the nucleotide sequences of the Rvl979c, Rvl769c, Rvl986, Rv3872, Rv3878, Rvl983, Rv3873 and Rv3879c antigens whose coding sequences are shown as SEQ ID Nos 8 to 13, and whose genomic sequences are illustrated as SEQ ID Nos 61-66 respectively.
  • Figure 9 shows a diagnostic cocktail derived from SEQ ID NO 5.
  • Figure 10 shows a diagnostic cocktail derived from SEQ ID NO 3.
  • Figure 11 shows a diagnostic cocktail derived from SEQ ID NO 6.
  • Figure 12 shows the coding nucleotide sequences of the Rv 1979 and 1769 antigens which are shown as SEQ ID Nos 59 and 60, respectively, together with the genomic sequences 70 and 71 respectively.
  • Figure 13 shows the nucleotide coding and amino acid sequences of a novel vaccine as SEQ ID NOs 17 and 18 respectively.
  • Figure 14 shows the first half of a fusion insert from the ORF of Rv 1979c (SEQ ID NO 15) and the position with the ORF of the segment to be fused in the vaccine (bold) .
  • Figure 15 shows the second half of a fusion insert from the ORF of Rv 1769 (SEQ ID NO 16) and the position with 'the ORF of the segment to be fused in the vaccine (bold) .
  • Cattle Ca. 6 months old calves (Friesian or Friesian crosses) were obtained from herds free of bovine tuberculosis. The following groups of cattle were used in this study:
  • BCG vaccination Calves were vaccinated with BCG Pasteur by subcutaneous injection of 10 6 CFU into the side of the neck followed 8 weeks later by a booster injection using the same route and dose [Buddie, 1995 supra.; Vordermeier, H. M. , et al. 1999. Clin. Diagn. Lab. Immunol. 6:675-682]. Heparinised blood samples were taken between 4-6 weeks after the booster vaccination. Data from 6 calves will be presented in this study.
  • Bovine (PPD-B) and avian (PPD-A) tuberculins were obtained from the Tuberculin Production Units at the Veterinary Laboratories Agency-Weybridge and used in culture at 10 ⁇ g/ml.
  • Peptides A set of five hundred and fifty two synthetic peptides spanning 13 open reading frames (20 residues long with a 12 residue overlap) was prepared by Multi-rod peptide synthesis. These were used in mapping experiments in pools of 10 peptides at 5 ⁇ g each peptide/ml and 25 ⁇ g/ml when used individually. The peptides were purchased from Chiron Mimotopes (Clayton, Australia) . ESAT-6 and CFP-10 derived peptides were synthesised by solid phase peptide synthesis and formulated into a peptide cocktail as described earlier [Vordermeier, H. M. et al. 2001. Clin. Diagn. Lab. Immunol. 8:571-8]. They were also used at 5 ⁇ g each peptide/ml. Peptide purity and sequence fidelity of ESAT-6 and CFP-10 derived peptides was confirmed by analytical reverse-phase HPLC and by electron-spray mass spectrometry, respectively.
  • terferon -gamma LISA In terferon -gamma LISA. Whole blood cultures were performed in 96-well plates in 0.2ml/well aliquots by mixing 0.1 ml of heparinised blood with an equal volume of antigen containing solution [Vordermeier, 1999 supra.]. Supernatants were harvested after 48 h of culture at 37°C/5% C0 2 in a humidified incubator. Interferon-gamma (IFN- ⁇ ) concentration was determined using BOVIGAMTM ELISA kit (Biocore AH, Omaha, NE) . Results were deemed positive when the OD 450 [PPD-B] minus OD 450 values with antigens minus OD 50 value without antigens were > 0.1. For comparative analysis of PPD-B vs. PPD-A responses, a positive result was defined by an OD 450 [PPD-A >0.1, and OD 450 [PPD-B] minus OD 450 [unstimulated] >0.1
  • ORFs from the RDl, RD2 and RD14 regions of M. bovis were selected for screening. These regions are deleted in BCG Pasteur and proteins encoded within these regions hold promise as candidate antigens for the differential diagnosis of M. bovis infected animals from BCG vaccinated cattle and as potential vaccine candidates. Selection criteria were that the ORF should encode a protein that either (i) showed no, or minimal, sequence similarity to other proteins in M. tuberculosis or other organisms, (ii) belonged to the PE or PPE protein family, (iii) had the potential of being induced or upregulated in vivo (e.g. amino acid transporters) , or (iv) had the potential to be secreted.
  • the designations of the antigens encoded by the selected ORF (Rv number), their sizes, and putative functions are listed in Figure .
  • responder frequencies of the 8 most immunogenic antigens are summarised in Figure 5.
  • 21/22 M. bovis infected animals responded to a previously characterised peptide cocktail derived from CFP-10 and ESAT-6 (reference) that had been included for comparison (median responses and range: 1.281 (0.011-2.825)).
  • the peptide pools formulated contain between 8-11 peptides (see Figure 4 for details of peptide pools) .
  • the individual peptides of pool 3 (representing residues 89-188 from Rv3873) and pool 26 (representing residues 161-252 from Rvl983) were tested using blood from 5 M. bovis infected animals . All three animals tested that recognised pool 3 responded exclusively to peptide 3.2 (residues 97-116 - SEQ ID NO 7), whereas both animals tested that responded to pool 26 only recognised peptide 26.2 (residues 169-188) .
  • the results shown in Figure 3 give results from one representative animal responding to pools 3 (Fig. 3A) or 26 (Fig.3B), respectively.
  • the present invention exploits the use of pools of overlapping synthetic peptides derived from the sequences of these proteins.
  • PBMC peripheral monocyte blood cell
  • Peptides as i muno-diagnostic reagents can therefore constitute a practical alternative to recombinant proteins, in addition to substituting them as reagents to assess immunogenicity.
  • the fact that all three animals tested, two from the UK and one from New Zealand, recognised the same peptide within pool 3 (peptide 3.2) is encouraging in this context.
  • peptide 3.2 is a highly immunogenic component of pool 3 derived from the sequence of Rv3873, a member of the PPE family of proteins .
  • the pool consistently produces positive responses when assayed in M. bovis infected cattle with a responder frequency of 82% but was also recognised in BCG vaccinated animals.
  • This is a surprising outcome given that its gene is deleted in BCG and that no homologous proteins were found elsewhere in the BCG genome.
  • the unit of cross-reactivity is the epitope, less than 20 amino acids long, that is recognised by T cells in the context of MHC molecules . Consequently, the molecular nature of cross-reactivity can only be addressed once these epitopes have been identified.
  • sequence of peptide 3.2 (shown as SEQ ID NO.9) to search for similar regions with other genes found within the M. tuberculosis genome.
  • Figure 6 shows the results using the Basic Local Alignment Search Tool (BLAST) program [NCBI. Basic Local Alignment Search Tool (BLAST) . http: //www/ncbi.nlm.nih.gov/BLAST/] to identify similarity between mycobacterial proteins .
  • the table shown in Figure 6 highlights several sequences that contain amino acid identities of greater than 50%. These include five proteins from the M. tuberculosis genome, all of which are also members of the PPE family and several others identified in proteins of various mycobacterial species.
  • the peptide covers an area of the gene that encodes two motifs identified in a number of PPE family members during their annotation [Tekaia, F., et al 1999. Tuber. Lung Dis. 79:329-42. TubercuList. MAST - Motif Alignment and Search Tool http: //genolist.pastuer.fr/TubercuList/3.
  • the responder frequency of 57% in M. bovis infected cattle is due to the recognition of two pools with responder frequencies of 47% and 53% respectively. Whilst one pool is recognised by 50% of the BCG vaccinated animals, the other is not recognised. Therefore, the diagnostic potentials of this antigen can still be realised by using only peptides derived from the second peptide pool.
  • Example 1 The Experiment described in Example 1 above could also be used to demonstrate that two antigens could be considered as potential vaccine candidates because they were highly immunogenic in all groups assayed.
  • a DNA vaccine comprising a fusion of two internal gene sequences was constructed in the vector pvmcLINK (Vordermeier et al. Vaccine 2000 Dec 8;19(9-10) :1246-55) .
  • the two gene sequences derived from a section of the sequence of Rvl979c and Rvl769 respectively, were generated by polymerase chain reaction (PCR) and ligated together via their restriction enzyme (RE) digested termini.
  • the two sections encode for polypeptides that stimulate the production of gamma interferon in blood from cattle inoculated with mycobacterium bovis, mycobacterium bovis BCG and others exposed to environmental mycobacteria (Cockle et al. Infect Immun 2002 Dec;70 (12) : 6996- 7003).
  • the vector itself was then cut using RE's at a specific position and the fused insert sequence ligated within it.
  • the section of the open reading frame (ORF) from Rvl979c that has been fused into the DNA vaccine is shown in Figure 1 .
  • the section is an internal gene sequence that encodes an area of the gene that, when assayed in the translated form as polypeptides, stimulates the production of gamma interferon in blood from cattle inoculated with mycobacterium bovis, mycobacterium bovis BCG and others exposed to environmental mycobacteria (Cockle et al. supra.).
  • the position of the antigenic gene section within the ORF is highlighted in the sequence in bold.
  • Figure 15 shows the section of the open reading frame (ORF) from Rvl769 that has been fused into the DNA vaccine.
  • the section is a gene sequence that encodes an area of the gene that, when assayed in the translated form as polypeptides, stimulates the production of gamma interferon in blood from cattle inoculated with mycobacterium bovis, mycobacterium bovis BCG and others exposed to environmental mycobacteria (Cockle et al. supra) .
  • the position of the antigenic gene section within the ORF is highlighted in the sequence in bold.

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Abstract

L'invention se rapporte à l'utilisation d'antigènes dérivés des régions RD1 ou RD2 des génomes de Mycobacterium tuberculosis, Mycobacterium bovis ou Mycobacterium africanum, et de peptides dérivés de ces antigènes en tant qu'agents réactifs de diagnostic, en particulier dans des trousses de diagnostic. De plus, certains de ces peptides, ainsi que d'autres antigènes et peptides dérivés de la région RD14 du génome, peuvent être utilisés comme vaccins. De nouveaux peptides de fusion sont également décrits.
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WO2005054851A1 (fr) * 2003-11-21 2005-06-16 Institut Pasteur Adenylate-cyclase recombinante de bordetella utilisable a des fins de diagnostic et d'immunosurveillance, methode de diagnostic ou d'immunosurveillance utilisant ladite adenylate-cyclase recombinante, et trousse de diagnostic ou d'immunosurveillance comprenant ladite adenylate-cyclase recombinante
WO2005080990A2 (fr) 2004-02-19 2005-09-01 Istituto Nazionale Delle Malattie Infettive 'lazzaro Spallanzani' Essai de diagnostic immunitaire pour diagnostiquer et pour surveiller une infection de tuberculose
WO2005090988A2 (fr) * 2004-03-19 2005-09-29 Isis Innovation Limited Test de diagnostic
FR2873819A1 (fr) * 2005-07-21 2006-02-03 Pasteur Institut Detection de la tuberculose et de l'infection par mycobacterium tuberculosis a l'aide de hbha
EP1674868A1 (fr) * 2004-12-21 2006-06-28 Chang Gung University (a university of Taiwan) Procédé et dispositif pour la détection d'antigenes de Mycobacterium tuberculosis dans des liquides biologiques
EP1794187A1 (fr) * 2004-08-19 2007-06-13 Proteome Systems Intellectual Property Pty Ltd. Procédés de diagnostic et de traitement d"une infection par m. tuberculosis et réactifs correspondants
CN100393876C (zh) * 2004-05-11 2008-06-11 中国人民解放军第三○九医院 用于结核病诊断的结核分枝杆菌蛋白
US7579141B2 (en) 1999-05-04 2009-08-25 University Of Medicine And Dentistry Of New Jersey Proteins expressed by Mycobacterium tuberculosis and not by BCG and their use as diagnostic reagents and vaccines
CN101661044A (zh) * 2008-08-27 2010-03-03 复旦大学附属华山医院 一种结核病诊断试剂及试剂盒
JP2010524854A (ja) * 2007-04-04 2010-07-22 インフェクティアス ディジーズ リサーチ インスティチュート 結核菌（Ｍｙｃｏｂａｃｔｅｒｉｕｍｔｕｂｅｒｃｕｌｏｓｉｓ）ポリペプチドおよびその融合物を含む免疫原性組成物
CN102260328A (zh) * 2010-07-15 2011-11-30 华中农业大学 结核分枝杆菌的抗原蛋白及应用
CN104628833A (zh) * 2015-01-23 2015-05-20 中国疾病预防控制中心传染病预防控制所 一种结核感染细胞免疫检测抗原组合物及其应用

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