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WO2003018053A1 - Vaccin contre des pathologies induites par mycobacteries - Google Patents

Vaccin contre des pathologies induites par mycobacteries Download PDF

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Publication number
WO2003018053A1
WO2003018053A1 PCT/EP2002/009345 EP0209345W WO03018053A1 WO 2003018053 A1 WO2003018053 A1 WO 2003018053A1 EP 0209345 W EP0209345 W EP 0209345W WO 03018053 A1 WO03018053 A1 WO 03018053A1
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WIPO (PCT)
Prior art keywords
polypeptide
peptide
polynucleotide
pharmaceutical composition
eliciting
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PCT/EP2002/009345
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English (en)
Inventor
Leander Grode
Peter R. Jungblut
Stefan H. E. Kaufmann
Jens Mattow
Hans-Joachim Mollenkopf
Ulrich Schaible
Original Assignee
MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Publication of WO2003018053A1 publication Critical patent/WO2003018053A1/fr

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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
    • C07K14/35—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
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one polypeptide selected from the group consisting of (a) a polypeptide encoded by a polynucleotide comprising a nucleic acid sequence as defined herein; (b) a polypeptide comprising an amino acid sequence as defined herein; (c) a polypeptide which is or which comprises a functional domain, an antigenic fragment and/or a fragment capable of eliciting and/or triggering an immune response in a mammal of the polypeptide of (a) or (b); (d) a polypeptide which is encoded by a polynucleotide which is at least 80% identical to the polynucleotide as defined in (a) and which is capable of eliciting and/or triggering an immune response in a mammal; and (e) a polypeptide which is encoded by a polynucleotide which hybridizes under stringent conditions with the polynucleotide as
  • the invention provides for pharmaceutical compositions comprising fusion proteins, polynucleotides, vector(s), host cell(s) or antibodies as described herein.
  • the invention relates to recombinant (bacterial) host cells and methods for the production of a vaccine.
  • Tuberculosis caused by mycobacteria of the tuberculosis complex, mostly M. tuberculosis, is a major infectious disease. According to the World Health Organization each year about 8 million new cases of TB are notified globaly, 2 million of which prove fatal [1].
  • the currently available vaccine against TB Mycobacterium bovis Bacillus Calmette-Guerin, was originally developed by Albert Calmette and Camille Guerin. It is a viable vaccine that was attenuated by 230 passages in vitro between 1908 and 1921 [2-3].
  • the necessity to develop a novel vaccine against TB has been recognized for many years. It results from the observation, that BCG prevents miliary and meningeal TB in children, but rather fails to protect adults effectively against pulmonary TB, the most prevalent form of the disease [5-6].
  • M. tuberculosis strain H37Rv with a total of 3924 ORFs (according to the gene classification of the Sanger Center) was published [7].
  • M. tuberculosis clinical isolate CDC1551 and Mycobacterium leprae strain TN have been completely sequenced and the sequencing projects of 4 additional mycobacterial strains including M. bovis strain AF2122/97 are nearing completion [8-10].
  • M. tuberculosis shares > 99.9% identity with wild-type M. bovis and the various BCG substrains. Although highly related, M. tuberculosis and M.
  • bovis can be distinguished on the basis of their host range, virulence and physiological properties [11]. To date our knowledge of the genetic basis for these phenotypic differences, as well as for the virulence of M. tuberculosis and for the attenuation of BCG is still fragmentary. Comparative genomic hybridization experiments have revealed that M. bovis and the different BCG substrains lack distinct regions of the genome of M. tuberculosis H37Rv [4, 12-16]. For example, the BCG substrains Chicago and Copenhagen examined in this study lack 13 regions of the genome of M. tuberculosis H37Rv comprising 111 ORFs.
  • a complementary approach towards comparative analysis of M. tuberculosis and M. bovis BCG strains employs proteomics, e.g. protein separation by 2-DE (or liquid chromatography) in combination with protein identification and characterization by MS and other protein analytical methods.
  • 2-DE allowed us to separate mycobacterial whole cell preparations into approximately 1 800 distinct protein species.
  • a protein species is defined by its chemical structure [17].
  • BCG Bacillus Calmette-Guerin
  • M. bovis BCG has failed to protect against mycobacterial induced diseases, in particular TB, in several trials (WHO, Tech. Rep. Ser. (1980), 651 , 1- 15) for reasons that are not entirely clear (Fine, Tubercle 65 (1984), 137-153). Additionally, it has been shown that the vaccine strain of M. bovis BCG only confers protection against the severe form of miliary tuberculosis in children (Fine, Lancet 346 (1995), 1339-1345). In contrast, its protective capacity against the most common form, pulmonary tuberculosis in adults, is low and highly variable (Colditz (1994), JAMA 271 , 698).
  • Diagnosis of TB is commonly achieved using a skin test, which involves intradermal exposure to tuberculin PPD (protein-purified derivative). Antigen-specific T cell responses result in measurable induration at the injection site by 48-72 hours after injection, which indicates exposure to Mycobacterial antigens. Sensitivity and specificity have, however, been a problem with this test, and individuals vaccinated with BCG cannot be distinguished from infected individuals. Therefore, it is of major concern that effective and safe vaccines and therapies for the immunization and the treatment of mycobacterial-induced diseases/disorders.
  • the technical problem of the present invention was thus to provide compositions useful for effective immunization against pathogenic organisms, in particular mycobacteria, for effective therapy of infected humans and animals that can be reliably used in low doses and with substantially no side effects.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one polypeptide selected from the group consisting of
  • polypeptide which is or which comprises a functional domain, an antigenic fragment and/or a fragment capable of eliciting and/or triggering an immune response in a mammal of the polypeptide of (a) or (b);
  • polypeptide which is encoded by a polynucleotide which is at least 80% identical to the polynucleotide as defined in (a) and which is capable of eliciting and/or triggering an immune response in a mammal;
  • polypeptide which is encoded by a polynucleotide which hybridizes under stringent conditions with the polynucleotide as defined in (a) or (d) and is capable of eliciting and/or triggering an immune response in a mammal.
  • a gene product encoded by Rv1511 is a potent vaccination candidate against diseases caused or elicidated by bacterial strains, in particular virulent Mycobacteria (e.g. M. tuberculosis).
  • Said gene product is not limited to full length gene products but also to fragments thereof as descibed herein below. Proteins present in virulent M. tuberculosis but absent in the attenuated M. bovis BCG vaccine represent putative virulence factors. It has been proposed that these gene products are also interesting antigen candidates for rational vaccine design against TB. M.
  • bovis is the causative agent of cattle TB, and in rare cases it can also cause human TB. Therefore, those proteins of M. tuberculosis which are encoded by genes that are missing in M. bovis BCG but not in wild-type M. bovis (e.g. proteins encoded by ORFs from the deletion regions RD1-2; RD8; RD14; and RD16 of the genome of M. tuberculosis H37Rv) are of particular interest. The best known of these are: the early secretory antigen target (ESAT-6; encoded by Rv3875 from RD1 ) and the secreted immunogenic protein Mpb64/Mpt64 (MPT64; encoded by Rv1980c from RD2).
  • ESAT-6 early secretory antigen target
  • Mpb64/Mpt64 MPT64; encoded by Rv1980c from RD2
  • M. tuberculosis specific proteins encoded by genes deleted in both M. bovis BCG and wild-type M. bovis e.g. proteins encoded by ORFs from the deletion regions RD3-RD7; RD9-13; and RD15 of the genome of M. tuberculosis H37Rv
  • putative GDP-D-mannose dehydratase encoded by Rv1511 located in the deletion region RD6 of the genome of M. tuberculosis H37Rv may provide constant protection against TB-challenge over all time points examined.
  • encoded RV1511 and its fragments have highly homologous counterparts in mammalian species, like, inter alia, in mouse and human. Accordingly, it is surprising that a putative, bacterial GDP-D-mannose dehydratase (and fragments thereof ) is capable of eliciting and/or triggering an immune response in mammals.
  • Rv1511 relates to a deduced open reading frame as described in Cole (1998), Nature 393, 537.
  • Rv1511 (RD6) was further described by Behr, Science 284 (1999), 1520 and described as differentially expressed in M. tuberculosis and M. bovis in WO 00/44392. Yet, Rv1511 had never been proposed as a potential vaccine candidate since said (expressed) ORF appears to be missing in virulent and attenuated M. bovis strains.
  • polypeptide means, in accordance with the present invention, (a) peptide(s) or (a) (poly)peptide(s) which encompass amino acid chains of any length, wherein the amino acid residues are linked by covalent peptide bonds.
  • peptidomimetics of such proteins wherein amino acid(s) and/or peptide bond(s) have been replaced by functional analogs are also encompassed by the invention.
  • a protein may comprise different protein species.
  • a protein species is (furthermore and not limiting) defined by its chemical composition and modifications of said peptide(s)/(poly)peptide(s) by, inter alia, glycosylations, acetylations, phosphorylations, lipidations or by amino acid exchanges, the term describes a chemically clearly-defined molecule and corresponds, inter alia, to one spot on a high-performance 2-DE pattern (Jungblut, Electorphoresis 17 (1996), 839-847).
  • the term protein species is therefore defined as the smallest unit of a protein classification, defined by its chemical structure.
  • polypeptide encoded by a nucleic acid sequence as shown in SEQ ID NO: 1 is to be employed in the pharmaceutical composition of this invention.
  • Said polypeptide has preferably the amino acid sequence as shown in SEQ ID NO: 2.
  • homologous polypeptides may be employed which are capable of elicidating an immunobiological response in a mammal, preferably in a human.
  • said polypeptides are at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 99% identical to the polypeptide as shown in SEQ ID NO: 2.
  • the polypeptide to be employed in the pharmaceutical composition of this invention preferably is a putative GDP-D-mannose-dehydratase, preferably of M. tuberculosis, more preferably a putative GDP-D-mannose-dehydratase as deposited with NCBI as B70714 or CAB02025.
  • Corresponding polynucleotides/nucleic acid molecules, encoding for the Rv1511 gene product can be found in NCBI under the accession number Z79701 (M. tuberculosis H37Rv complete genome; segment 65/162; strand: plus, nudeotides: 30672-316949) and AD000001 (M.
  • the polynucleotide encoding for Rv1511 is at least 80%, more preferably at least 90%, and most preferably at least 95% homologous to the nucleic acid sequence as shown in SEQ ID NO: 1.
  • composition comprises at least one protein, an antigenic fragment of said protein, a fusion protein, a nucleic acid molecule and/or an antibody as defined herein and, optionally, further molecules, either alone or in combination, like e.g. molecules which are capable of optimizing antigen processing, cytokines, immunoglobulins, lymphokines or CpG-containing DNA stretches.
  • the composition in form of a vaccine may include an adjuvant.
  • Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1 ) aluminium slats (alum), such as aluminium hydroxide, aluminium phosphate, aluminium sulfate, etc.; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example, (a) MF59TM (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds.
  • aluminium slats alum
  • oil-in-water emulsion formulations with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components
  • MF59TM WO 90/14837
  • RibiTM adjuvant system Ribi Immunochem, Hamilton, MT
  • MPL monophosphorylipid A
  • TDM trehalose dimycolate
  • CWS cell wall skeleton
  • saponin adjuvants such as QS21 or Stimulon
  • WO 00/07621 (4) Complete Freund's Adjuvant (CFA) in Incomplete Freund's Adjuvant (IFA); (5) cytokines, such as interleukins (e.g. IL-1 , IL-2, IL-4, IL- 5, IL-6, IL-7, IL-12 (WO 99/44636), etc.), interferons (e.g. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (6) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL) e.g.
  • MPL monophosphoryl lipid A
  • 3dMPL 3-O-deacylated MPL
  • EP-A-0 689 545 optionally in the substantial absence of alum when used with pneumococcal saccharides e.g. WO 00/56358; (7) combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions e.g. EP-A-0 835 318, EP-A-0 735 898, EP-A-0 761 231 ; (8) oligonucleotides comprising CpG motifs (Roma, Nat. Med. 3 (1997), 849-854; Weiner, PNAS USA 94 (1997), 10833-10837; Davis, J. Immunol.
  • a polyoxyethylene ether or a polyoxyethylene ester e.g. WO 99/52549
  • a polyoxytheylene sorbitan ester surfactant in combination with an oxtoxynol WO 00/21207
  • a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol (WO 00/21152)
  • a saponin and an immunostimulatory oligonudeotide e.g.
  • a CpG oligonudeotide (WO 00/62800); (11 ) an immunostimulant and a particle of metal salt e.g. WO 00/23105; (12) a saponin and an oil-in-water emulsion e.g. WO 99/11241 ; (13) a saponin (e.g. QS21 ) + 3dMPL + IL-12 (optionally + a sterol) e.g. WO 98/57659; (14) other substances that act as immunostimulating agents to enhance the effectiveness of the composition.
  • Alum especially aluminium phosphate and/or hydroxide
  • MF59 are preferred for use with the saccharide antigens of the present invention.
  • the composition may be in solid, liquid or gaseous form and may be, inter alia, in form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an) aerosol(s).
  • said composition comprises one protein (Rv1511 ) combined with at least one, preferably two, preferably three, more preferably four, most preferably five differentially expressed proteins.
  • Differentially expressed proteins, in particular of mycobacteria are known in the art and, inter alia, described in WO 00/44392.
  • polypeptides/proteins to be employed in the pharmaceutical composition of the invention may be produced by, for example, recombinant techniques or by biochemical or synthetic techniques which are known to the skilled artisan (Sambrook et al., "Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Laboratory, N.Y. (1989); Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N.Y. (1989)).
  • differential protein expression of the gene product of Rv1511 can be detected by preparation of microorganisms or, less preferred, compartments/fragments thereof, 2-DE, subtractive analysis and identification of proteins by peptide mass fingerprinting (PMF) with or without confirmation by further methods.
  • PMF peptide mass fingerprinting
  • virulent strain in accordance with the present invention, denotes the capacity of a pathogenic strain of Mycobacteria to infect a host and/or to cause disease - defined broadly in terms of severity of symptoms in a host.
  • a "virulent strain” might cause symptoms in a susceptible host, whereas another host might be unaffected by this strain, which can be therefore considered as being an "avirulent strain” in this second host.
  • the term "avirulent strain” denotes strains of a Mycobacteria which is not capable of inducing infection and/or causing disease in a specific host or in a host species.
  • avirulent strains denotes furthermore attenuated strains of microorganisms.
  • antigenic fragment refers to the ability of said fragment to elicit an immune response (e.g. humoral or cellular) in a subject, such as a human, and/or in a biological sample.
  • immunogenic fragments may consist entirely of the antigenic and/or immunogenic portion of the protein or may contain additional sequences.
  • the additional sequences may be derived from said protein or may be heterologous, and such additional sequences may (but need not) be antigenic and/or immunogenic.
  • antigenic peptides/fragments of expressed Rv1511 may be deduced by computer-assisted methods.
  • Preferred "antigenic fragments" are shown, but not limited to, the peptide- stretches as shown in Table 1 of appended example 3.
  • Antigenic fragments may be produced recombinantly using a polynucleotide sequence that encodes the antigenic fragment or may be produced by biochemical or synthetic techniques. Those methods are known to those of ordinary skill in the art (see, e.g. Sambrook et al., loc. cit.; Harlow and Lane “Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, NY (1988); Merrifield, J. Am. Chem. Soc.
  • antigenic fragment also relates to the polynucleotide sequence encoding the antigenic fragment as, inter alia, depicted in Table 1 of appended example 3.
  • fragment capable of eliciting and/or triggering an immune response in a mammal relates, in accordance with the invention to fragments of Rv1511 as defined herein which are capable of eliciting said immunological responses in a mammal, in particular in mouse, cattle (bovine) and most preferably in humans.
  • said fragments comprise at least 8, preferably at least 9, more preferably at least 10, more preferably at least 12, more preferably at least 14, more preferably at least 16, more preferably at least 18 and more preferably at least 20 amino acids, in particular when B-cell stimulation is desired.
  • fragments of Rv1511 may be employed which are considerably longer or which are comprised in fusion proteins or peptides as described herein.
  • fragments of RV1511 to be employed in context of this invention may comprise the fragments as depicted in Table 1 or as shown in SEQ ID NOs: 3 to 48.
  • Particular preferred fragments are fragments as shown in SEQ ID NOs: 13, 20, 23, 24, 29, 35 and 42.
  • the appended examples furthermore illustrate and document that such fragments are particularly useful in the preparation of a composition, preferably of a pharmaceutical composition, for the treatment as well as for the prevention of a disease related to a mycobacterial infection.
  • polynucleotides/nucleic acid molecules encoding fragments of Rv1511 as described herein are to be employed in pharmaceutical compositions described herein.
  • the person skilled in the art can deduce antigenic fragments and/or fragments of Rv1511 capable of eliciting and/or triggering immune responses in mammals by methods known in the art, which comprise not only computer-assisted predictions (as documented in appended examples) but also in vitro and in vivo experiments. Said in vivo experiments also comprise the use of test animals, like mice or rats. Furthermore, clinical trials are envisaged after safety and efficacy has been established in pre-clinical experiments.
  • cytokine enzyme-linked ImmunoSPOT ELISPOT
  • FACS flow cytometric cell analysis and cell sorting
  • the fusion protein as described herein and to be employed in accordance with this invention may also comprise (besides Rv1511 -related antigens and/or fragments thereof) antigens and/or immunogenic domains which are not restricted to Mycobacterium antigens and can be selected from autoantigens, tumor antigens and pathogen antigens such as virus antigens, parasite antigens, bacterial antigens in general and immunogenic fragments thereof.
  • suitable tumor antigens for broad spectrum anti-tumor therapy are human antitumor antigens such as members of the MAGE multigene family, such as MAGE-A to MAGE-L, BAGE and GAGE and melanocyte differentiation antigens, such as MELAN-A/MART; see Valmori, J. Immunol. 168 (2002), 4231-4240, Jager, Int. J. Cancer 98 (2002), 376- 388, or Chomez, Cancer Res. 61 (2001 ), 5544-5551.
  • suitable virus antigens are human tumor virus antigens such as human papilloma virus antigens, e.g. antigens E6 and E7, influenza virus antigens, e.g.
  • influenza virus nucleoprotein or retroviral antigens such as HIV antigens, e.g. the HIV antigens p17, p24, RT and Env.
  • suitable parasite antigens are Plasmodium antigens such as liver stage antigen (LSA-1 ), circumsporozoite protein (CS or allelic variants cp26 or cp29), thrombospondin related amonymous protein (TRAP), sporozoite threonine and asparagine rich protein (STARP) from Plasmodium falciparum and Toxoplasma antigens such as p30 from Toxoplasma gondii.
  • suitable bacterial antigens are Legionella antigens such as Major Secretary Protein (MSP) from Legionella pneumophila.
  • MSP Major Secretary Protein
  • the invention relates to a (pharmaceutical) composition
  • a (pharmaceutical) composition comprising a polynucleotide encoding for a fusionprotein/polypeptide as defined herein or comprising a fusion protein comprising a polypeptide and/or an antigenic fragment as defined in the above.
  • the protein and/or the antigenic fragment to be employed herein and being (expressed) Rv1511 or a fragment thereof can comprise a further domain, said domain being linked by covalent or non-covalent bonds. Further domains are illustratively described herein.
  • the linkage can be based on genetic fusion according to the methods known in the art (Sambrook et al., loc. cit.; Ausubel, loc. cit.) or can be performed by, e.g., chemical cross-linking as described in, e.g., WO 94/04686.
  • the additional domain present in the fusion protein comprising the protein of the invention may be joined directly (i.e.
  • fusion protein may further comprise a cleavable linker or cleavage site, which, for example, is specifically recognized and cleaved by proteinases or chemical agents.
  • Cleavable linker sequences include, but are not limited to, Factor XA or enterokinase (Invitrogen, San Diego, CA, USA).
  • said further domain may be of a predefined specificity or function.
  • the protein to be employed in the pharmaceutical composition of the invention may be further modified by conventional methods known in the art. This allows for the construction of fusion proteins comprising the protein defined herein (Rv1511 ) or a functional fragment/antigenic fragment thereof and other functional amino acid sequences, e.g., immunologically relevant proteins like cytokines, lymphocytes, interferones, or protein tags (GST, GFP, h-myc peptide, FLAG, HA peptide) which may be derived from heterologous proteins.
  • the Rv1511 or the functional fragment thereof is capable of elucidating an immunoresponse in animals or humans and/or may confer protective immunity in animals or humans.
  • the invention relates to a pharmaceutical composition comp ⁇ sing a fusion protein comprising at least two proteins as defined herein and/or (an) antigenic fragment(s) as defined herein.
  • the fusion protein to be employed in the present invention for the preparation of a pharmaceutical composition comprises an immunostimulatory molecule.
  • a pharmaceutical composition comprising a polypeptide as defined herein above or a fragment thereof (i.e. Rv1511 or a fragment thereof) further comprises, optionally, an immunostimulatory molecule.
  • immunological molecule denotes in accordance with the present invention molecules or fragments thereof which, inter alia, activate and/or stimulate the humoral and cellular response of an immune system. They might, e.g. activate antigen-presenting cells, stimulate natural killer cells, enhance the production of antibodies directed against an antigen and/or a pathogen or induce the proliferation of cells of the immune system.
  • These molecules are known in the art and comprise, inter alia, cytokines, lymphokines, immunoglobulins, interleukins and/or complement factors (see, e.g. Paul, "Fundamental Immunology", Raven Press (1989); Schaible, Adv. In Immunology 71 (1999), 261-377).
  • said fusion protein comprises a molecule capable of optimizing antigen processing or comprises a peptide/polypeptide which is capable of eliciting and/or triggering an immune response in a mammal, preferably in a human.
  • pharmaceutical compositions comprising RV1511 or (a) fragment(s) thereof or comprising a polynucleotide encoding the same, may also further comprise, optionally, (a) molecule(s) capable of optimizing antigen processing.
  • T- cells Cellular immune recognition is mediated by a special class of lymphoid cells, T- cells. These cells do not recognize whole antigens but instead they respond to degraded peptide fragments thereof which appear on the surface of the target cell bound to proteins called major histocompatibility complex (MHC) molecules (antigen processing). Essentially all nucleated cells have MHC class I molecules, whereas MHC II are restricted to immune cells with special presenting qualities. Molecules which are capable of optimizing antigen processing are known in the art and comprise, inter alia, listeriolysin, which improves MHC class I restricted immune responses (see, e.g., Hess, PNAS 95 (1998), 5299-5304).
  • MHC major histocompatibility complex
  • the immunogenic domain of Rv1511 or of the peptide/polypeptide capable of eliciting and/or triggering an immune response is capable of eliciting a T-cell mediated immune response, more preferably a MHC class l-restricted CD8 T-cell response.
  • Peptides/polypeptides capable of eliciting and/or triggering an immune response in a mammal, preferably in a human are preferably selected from immunogenic peptides or polypeptides from M. bovis or M. tuberculosis or from immunogenic fragments thereof.
  • suitable antigens are Ag85B (p30) from M. tuberculosis, Ag85B ( ⁇ -antigen) from M. bovis BCG, Ag85A from M. tuberculosis and ESAT-6 from M. tuberculosis.
  • the immunogenic domain is derived from antigens as disclosed in WO 99/10496.
  • the invention also provides for pharmaceutical compositions, in particular to vaccines to be employed before and/or after an infectious mycobacterial challenge of an individual, wherein said at least one polypeptide being or comprising a functional domain, a antigenic fragment and/or a fragment capable of eliciting and/or triggering an immune response in a mammal is a polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequences as depicted in any one of SEQ ID NOs: 3 to 48.
  • Said fragments are fragments derived from the amino acid sequence of Rv1511 disclosed in SEQ ID NO: 2 or as encoded by the nucleic acid sequence shown in SEQ ID NO: 1.
  • said functional domain, said antigenic fragment and/or said fragment capable of eliciting and/or triggering an immune response in a mammal is the peptide shown in SEQ ID NO: 13, 20, 23, 24, 29, 35 or 42.
  • nucleic acid molecules coding for any one of the amino acid sequences as depicted in any one of SEQ ID NOs: 3 to 48 are employed in context of this invention, i.e. in the prepation of a pharmaceutical composition.
  • Nucleic acid molecules encoding amino acid molecules/fragments as shown in SEQ ID NOs : 3 to 48 are easily obtainable by the skilled artisan, and may be produced, in accordance with the genetic code, by, inter alia, recombinant techniques, by chemical synthesis or by isolation from natural sources.
  • the invention is not limited to the use of (antigenic) fragments shown in SEQ ID NOS: 3 to 48, but also relates to peptides which are highly homologous to the peptides shown in any one of said SEQ ID NOs. Most preferably, said homologous peptides are at least 80% more preferably at least 90% homologous to the corresponding peptide shown in any one of SEQ ID NO: 3 to 48. Accordingly in a peptide comprising 10 amino acids and shown in table 1 appended hereto, an exchange of one or two amino acids are envisaged to be still useful in accordance with this invention.
  • the person skilled in the art is provided with means and methods to test whether modified and or homologous peptides to the peptides shown in any one of SEQ ID NOs: 3 to 48 are useful in the preparation of a pharmaceutical composition, in particular of a vaccine and/or as an immunogenic composition.
  • the invention further relates to a pharmaceutical composition comprising at least one nucleic acid molecule/polynucleotide coding for any one of the polypeptides as defined herein, the antigenic fragment as defined herein and/or a fusion protein as defined herein. Said nucleic acid molecules/polypeptides are particularly useful in DNA-vaccination approaches as documented in the appended examples.
  • the nucleic acid molecule of the invention may be DNA such as cDNA or RNA such as mRNA. Additionally, the nucleic acid molecule of the invention may be PNA. Its origin may be natural, synthetic or semisynthetic or it may be a derivative, such as said peptide nucleic acid (Nielsen, Science 254 (1991), 1497-1500). Furthermore, said nucleic acid molecule may be a recombinantly produced chimeric nucleic acid molecule comprising any of the aforementioned nucleic acid molecules either alone or in combination. Preferably, said nucleic acid molecule is part of a vector. Said vector may also be compared in the pharmaceutical composition of the invention.
  • Such vectors may be, e.g., a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering, and may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.
  • the vectors may, in addition to the nucleic acid sequences to be employed in the invention, comprise expression control elements, allowing proper expression of the coding regions in suitable hosts.
  • expression control elements are known to the artisan and may include a promoter, translation initiation codon, translation and insertion site for introducing an insert into the vector.
  • the nucleic acid molecule of the invention is operatively linked to said expression control sequences allowing expression in eukaryotic or prokaryotic cells.
  • Control elements ensuring expression in eukaryotic and prokaryotic cells are well known to those skilled in the art. As mentioned above, they usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions. Possible regulatory elements permitting expression in for example mammalian host cells comprise the CMV- HSV thymiakine kinase promoter, SV40, RSV-promoter (Rous sarcoma virus), human elongation factor 1 ⁇ -promoter, CMV enhancer or SV40- enhancer.
  • promoters including, for example, the tac-lac-promoter or the trp promoter, has been described.
  • Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide.
  • suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNAI , pcDNA3 (In- vitrogene), pSPORTI (GIBCO BRL), or prokaryotic expression vectors, such as lambda gt11.
  • the vector may further comprise nucleic acid sequences encoding for secretion signals. Such sequences are well known to the person skilled in the art.
  • leader sequences capable of directing the protein/(poly)peptide to a cellular compartment may be added to the coding sequence of the nucleic acid molecules of the invention and are well known in the art.
  • the leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a protein thereof, into the periplasmic space or extracellular medium.
  • the heterologous sequence can encode a fusion protein including a C- or N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • the vector can also comprise regulatory regions from pathogenic and eukaryotic organisms. Transcriptional targeting of genes and transcriptional regulatory sequences like CD molecules [Immunology. 103 (2001 ), 351] are an important way to control gene expression and can direct high level expression in vivo and in vitro. Expression can additionally be enhanced by non-coding sequences that modify cis-acting regulatory domains, including the Kozak consensus element, the gene-end signal, and the mRNA 5'- untranslated sequence.
  • an internal ribosome entry site (IRES) can be used for co-expression instead of a second subgenomic promoter to ensure plasmid stability and integrity by elimination of homologues regions.
  • a growth factor-like domain may be used to stabilize and target antigen expression.
  • said vector may also be a gene transfer or targeting vector.
  • Gene therapy which is based on introducing therapeutic genes (for example for vaccination) into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer.
  • Suitable vectors, vector systems and methods for in- vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813, Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res.
  • nucleic acid molecules and vectors to be employed in the present invention in particular for the preparation of a pharmaceutical composition as described herein above may be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g. adenoviral, retroviral) into the cell. Additionally, a baculoviral system can be used as eukaryotic expression system for the nucleic acid molecules of the invention.
  • fragments of the protein, the fusion protein or antigenic fragments to be employed in accordance with this invention may be produced by direct peptide synthesis using solid-phase techniques (cf Stewart et al. (1969) ' Solid Phase Peptide Synthesis, WH Freeman Co, San Francisco; Merrifield, J. Am. Chem. Soc. 85 (1963), 2149-2154).
  • In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer, Foster City CA) in accordance with the instructions provided by the manufacturer.
  • Various fragments may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.
  • the invention relates to a pharmaceutical composition
  • a host cell which comprises the polynucleotide or the vector as defined herein.
  • Said host cell may be, inter alia, a dendritic cell; see also Tascon, Immunology 99 (2000), 473-480.
  • eukaryotic cells are envisaged as host cells, but also prokaryotic cells, like a Mycobacterium cell, Salmonella-cells, Listria-cells, Lactobacillus-cells, Lactococcus-cells, Yersinia-cells or Shigella-cells.
  • the vector comprised in the pharmaceutical composition of the invention may also be a viral vector. Such vectors, e.g.
  • vectors for gene transfer into antigen presenting cells are, inter alia, described in Monahan, Curr. Opin. Mol. Ther. 5 (1999), 558-564.
  • further vectors are envisaged which comprise but are not limited to, Vaccinia-, Semliki Forest Virus-, Sendai Virus-, Rubella Virus (RUB)- or Influenza-, Poliovirus Replicon-Vectors.
  • the invention in addition relates to a composition
  • a composition comprising at least one nucleic acid molecule/polynudeotide as defined herein.
  • Said composition is useful, inter alia, for medical and diagnostic purposes, in particular, for pharmaceutic and vaccination purposes.
  • the invention relates to pharmaceutical compositions comprising an antibody or a fragment or a derivative thereof directed against the polypeptide, the antigenic fragment, the nucleic acid molecule or the fusion protein as defined herein.
  • Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric or single chain antibodies or fragments or derivatives of such antibodies.
  • Rv1511 or fragments thereof is surprisingly capable of eliciting a potent immune response. Accordingly, in context of this invention, it is envisaged that antibodies directed against the antigenic fragment, the nucleic acid molecule or the fusion protein as defined herein are employed in medical settings, for example in preventive or therapeutic immunization approaches through passive immunizations.
  • antibodies generated in non-human animals like, mouse, rat, or swine.
  • particularly preferred is the generation of antibodies for passive immunization in horse, camel and goat.
  • the general methodology for producing antibodies is well-known and has been described in, for example, Kohler and Milstein, Nature 256 (1975), 494 and reviewed in J.G.R. Hurrel, ed., "Monoclonal Hybridoma Antibodies: Techniques and Applications", CRC Press Inc., Boco Raron, FL (1982), as well as that taught by L. T. Mimms et al., Virology 176 (1990), 604-619.
  • the generation of polyclonal antibodies/antisera is well known in the art.
  • antibody relates to monoclonal or polyclonal antibodies.
  • Antibody fragments or derivatives comprise F(ab') 2 , Fab, Fv, scFv fragments, chimeric antibodies, humanized antibodies, CDR-grafted antibodies and the like; see, for example, Harlow and Lane, “Antibodies, A Laboratory Manual", CSH Press 1988, Cold Spring Harbor, NY or in Riechmann (1988), Nature 332, 323- 327, Whittle (1989), J. Cell. Biochem 13A, p.96, Queen (1989), PNAS 86, 10029- 10033, WO 86/01533, WO 90/07861 , .
  • the derivatives of antibodies can be produced by peptidomimetics. It is also envisaged that antibodies (or fragments thereof) are further optimized. Optimization protocols for antibodies are known in the art. These optimization protocols comprise, inter alia, CDR walking mutagenesis as disclosed and illustrated herein and described in Yang (1995), J. Mol. Biol. 25, 392-403; Schier (1996), J. Mol. Biol. 263, 551-567; Barbas (1996), Trends. Biotech 14, 230-34 or Wu (1998), PNAS 95, 6037-6042; Schier (1996), Human Antibodies Hybridomas 7, 97; Moore (1997), J. Mol. Biol. 272, 336.
  • the invention also relates to a method for the production of an antibody or a fragment thereof directed against a polypeptide as defined in herin above, i.e. to Rv1511 or (a) fragment(s) thereof comprising the steps of (a) administering to an non-human animal a peptide as shown in any one SEQ ID NOs. 3 to 48, an isolated or recombinantly produced RV1511 or a fragment thereof or a nucleic acid molecule encoding a peptide as shown in any one SEQ ID NOs.
  • the invention relates to a method for the production of an antibody or fragments thereof directed against a polypeptide as herein, i.e. Rv1511 , comprising the steps of (a) administering to an non-human animal a peptide as shown in any one SEQ ID NOs. 3 to , an isolated or recombinantly produced RV1511 or a fragment thereof or a nucleic acid molecule encoding a peptide as shown in any one SEQ ID NOs.
  • the method of production of antbodies as disclosed herein may also further comprise the modification of the isolated antibody and/or the production of a modified antibody molecule or an antibody derivative.
  • Said modifiactions may comprise the isolation and determination of the corresponding CDR regions of the antibodies as well as the generation of humanized or chimeric antibodies by techniques known in the art.
  • antibodies or fragment(s) thereof may be labeled, preferably detectably labeled. Labeled antibodies or fragments thereof are particularly useful for diagnostic purposes.
  • the invention relates to a composition
  • a composition comprising at least one antibody, a fragment or a derivative thereof as defined above.
  • Such antibodies, fragments or derivatives can be used for diagnostic or for pharmaceutical purposes, i.e. for the treatment of Mycobacteria-induced diseases, the vaccination against these pathogens or for the detection of a mycobacterial infection or the detection of RV1511 or (a) fragment(s) thereof.
  • the composition of the invention comprising the above defined antibodies, derivatives or fragments thereof is particularly useful in passive immunization approaches.
  • the invention also relates to a pharmaceutical composition further comprising, optionally, a pharmaceutically acceptable carrier.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a recombinant bacterial host cell of an avirulent strain or a vaccine strain which comprises at least one polynucleotide as defined herein above.
  • Said recombinant bacterial host cell of an avirulent strain may be, but is not limited to, M. bovis, M. bovis BCG.
  • recombinant host cells in particular bacterial host cells are described herein above and comprise, but are not limited to, Mycobacterium-cells, Salmonella-cells, Listeria-cells, Lactobacillus-cells, Lactococcus-cells, Yersinia-cells and Shigella-cells.
  • the invention provides for a pharmaceutical composition as defined herein above, wherein said recombinant cell comprises at least one further nucleic acid molecule/polynudeotide encoding a peptide or polypeptide capable of eliciting and/or triggering an immune response in a mammal, preferably in a human.
  • the invention additionally relates to a pharmaceutical composition, wherein the peptide or polypeptide capable of eliciting and/or triggering an immune response is selected from the group consisting of auto-antigens, tumor-antigens, virus-antigens, parasite-antigens, bacterial-antigens and/or immunogenic fragments thereof.
  • antigens are described herein above.
  • the pharmaceutical composition of the invention comprises a host cell which is capable of expressing at least one recombinant nucleic acid molecule as defined herein.
  • said host cell may be a bacterial host cell.
  • said bacterial host cell is selected from the group consisting of Salmonella ssp., M. bovis, M. bovis BCG, Lactobacillus ssp., Lactococcus ssp., or may also comprise' host cells like further Mycobacterium-cells, Listeria-cells, Yersinia-cells as well as Shigella-cells.
  • the pharmaceutical composition as defined herein is a vaccine and/or said pharmaceutical composition has use as an immunogenic composition.
  • Vaccines may be prepared, inter alia, from one or more proteins, derivatives of the proteins, nucleic acid molecules, fusion proteins, antigenic fragments or antibodies, fragments of said antibodies or derivatives of the antibodies as defined herein.
  • nucleic acid molecules/polynucleotides may be used for gene vaccination or as DNA vaccines the appended examples illustrate how DNA vaccines may be prepared and employed.
  • Routes for administration of gene/DNA vaccines are well known in the art and DNA vaccination has been successfully used to elicit alloimmune, anti-tumor and antiidiotype immune responses (Tighe M. et al., Immunology Today 19 (1998), 89-97).
  • DNA vaccines encode eukaryotic vectors expressing antigens in the vaccinated host (59) and therefore elicit humoral and cellular immune responses.
  • DNA is injected as an aqueous solution intramuscular (i. m.) (72) or is administered by particle bombardment of skin with DNA coated onto gold particles using a gene gun (g. g.). Vaccination via the i. m.
  • Th1 immune response characterized by interferon gamma (IFN- ⁇ ) secreting T-cells and the presence of cytotoxic T-lymphocytes (CTL) (69).
  • CTL cytotoxic T-lymphocytes
  • IL4 interleukin-4
  • DNA vaccines may have a special role in preventing bacterial (70) and viral infections (68), and in tumor therapy (69).
  • the World Health Organization has started a global project for designing effective control strategies and to determine treatment levels for eradication of tuberculosis (58). Results were already published for i. m.
  • the polynucleotide to be employed as DNA-vaccine comprises the coding region for at least one, preferably at least two, more preferably at least three antigenic fragments of Rv1511 or of homologous proteins capable of, inter alia, eliciting an immune response preferably in a mammal, most preferably in a human.
  • the proteins, nucleic acid molecules, fusion proteins, antigenic fragments or antibodies, fragments or derivatives of said antibodies to be used in the pharmaceutical composition of the invention as a vaccine may be formulated e.g. as neutral or salt forms.
  • Pharmaceutically acceptable salts, such as acid addition salts, and others, are known in the art.
  • Vaccines can be, inter alia, used for the treatment and/or the prevention of an infection with pathogens and are administered in dosages compatible with the method of formulation, and in such amounts that will be pharmacologically effective for prophylactic or therapeutic treatments.
  • Proteins, protein fragments and/or protein derivatives used as vaccines are well known in the art (see, e.g. Cryz, "Immunotherapy and Vaccines", VCH Weinheim (1991 ); Paul (1989), loc. cit.). Furthermore, it has been shown that even intracellular enzymes of bacterial pathogens can act as antigenic entities which provide immunological protection (Michetti, Gastroenterology 107 (1994), 1002; Radcliff, Infect. Immun. 65 (1997), 4668; Lowrie, Springer Semin. Immunopathol.
  • a vaccination protocol can comprise active or passive immunization, whereby active immunization entails the administration of an antigen or antigens (like the compositions of the present invention or proteins, nucleic acid molecules, fusion proteins, antigenic fragments or antibodies, fragments of said antibodies or derivatives of the antibodies of the present invention) to the host/patient in an attempt to elicit a protective immune response.
  • Active immunization entails the transfer of preformed immunoglobulins or derivatives or fragments thereof (e.g., the antibodies, the derivatives or fragments thereof of the present invention) to a host/patient.
  • vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in or suspension in liquid prior to injection also may be prepared.
  • the preparation may be emulsified or the protein may be encapsulated in liposomes.
  • the active immunogenic ingredients often are mixed with pharmacologically acceptable excipients which are compatible with the active ingredient.
  • Suitable excipients include but are not limited to water, saline, dextrose, glycerol, ethanol and the like; combinations of these excipients in various amounts also may be used.
  • the vaccine also may contain small amounts of auxiliary substances such as wetting or emulsifying reagents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine.
  • such adjuvants can include aluminum compositions, like aluminumhydroxide, aluminumphosphate or aluminumphosphohydroxide (as used in "Gen H-B-Vax®” or “DPT-lmpfstoff Behring”), N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to as nor- MDP), N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'2'-dipalmitoyl-sn- glycero-3-hydroxphaosphoryloxy)-ethylamine (CGP 19835A, also referred to as MTP-PE), MF59 and RIBI (MPL + TDM + CWS) in a 2% squalene/T
  • Further adjuvants may comprise DNA or oligonucleotides, like, inter alia, CpG-containing motifs (CpG-oligonucleotides; Krieg, Nature 374 (1995), 546-549; Pisetsky, An. Internal. Med. 126 (1997), 169-171 ).
  • the vaccines usually are administered by intravenous or intramuscular injection.
  • the vaccines described herein are administered by mucosal (e.g. intranasal, oral, subcutaneaous) or transdermal routes.
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • binders and carriers may include but are not limited to polyalkylene glycols or triglycerides.
  • Oral formulation include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions may take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 10% to about 95% of active ingredient, preferably about 25% to about 70%.
  • Vaccines are administered in a way compatible with the dosage formulation, and in such amounts as will be prophylactically and/or therapeutically effective.
  • the quantity to be adminstered generally is in the range of about 5 micrograms to about 250 micrograms of antigen per dose, and depends upon the subject to be dosed, the capacity of the subject's immune system to synthesize antibodies, and the degree of protection sought. Precise amounts of active ingredient required to be administered also may depend upon the judgment of the practitioner and may be unique to each subject.
  • the vaccine may be given in a single or multiple dose schedule.
  • a multiple dose is one in which a primary course of vaccination may be with one to ten separate doses, followed by other doses given at subsequent time intervals required to maintain and/or to reinforce the immune response, for example, at one to four months for a second dose, and if required by the individual, a subsequent dose(s) after several months.
  • the dosage regimen also will be determined, at least in part, by the need of the individual, and be dependent upon the practitioner's judgment. It is contemplated that the vaccine containing the immunogenic compounds of the invention may be administered in conjunction with other immunoregulatory agents, for example, with immunoglobulins, with cytokines or with molecules which optimize antigen processing, like listeriolysin.
  • the vaccines described herein may be used for prophylactic as well as for therapeutic purposes.
  • the invention accordingly, also relates to a pharmaceutical composition as defined herein which is a living vaccine suitable for administration to a mucosal surface via the parenteral route.
  • the pharmaceutical composition may comprise the herein defined proteins, the fusion proteins, antigenic fragments and/or antibodies (or their fragments or derivatives) of the invention, either alone or in combination.
  • the pharmaceutical composition of the present invention may be used for effective therapy of infected humans and animals for vaccination purposes and/or prevention.
  • composition of the present invention may, additionally, comprise further antigenic determinants or determinants capable of eliciting an immune response or reaction.
  • Said determinants may comprise other differentially expressed polypeptides, like differentially expressed polypeptides as described in WO 00/44392.
  • These further determinants may comprise, but are not limited to proteins/polypeptides which are differentially expressed in M. tuberculosis H37Rv/Erdman as compared to M. bovis BCG.
  • Such proteins comprise Rv3710, Rv1392, Rv0952, Rv2971 , Rv0068, Rv0120c, Rv2883c, Rv1463, Rv1856c, Rv2579, Rv3275c, Rv2557, Rv3407, Rv3881c, Rv2449c, Rv0036c, Rv2005c or Rv3676.
  • Functional fragments of said polypeptides capable of eliciting an immunologic reaction or being antigenic fragments are also envisaged to be comprised, preferably additionally comprised in the (pharmaceutical) composition of this invention. It is understood that also polynucleotides/nucleic acid molecules encoding said differentially expressed polypeptides or functional fragments thereof be comprised in said (pharmaceutical) composition.
  • the above-mentioned differentially expressed polypeptides may also be linked to the polypeptides being Rv1511 or being derived from Rv1511.
  • Such linkage may comprise fusion proteins as well as polynucleotide constructs comprising coding- sequences for Rv1511 or fragments thereof in combination with coding sequences of the above-mentioned additional antigenic fragments.
  • Such polynucleotides are particularly useful in DNA-vaccination approaches.
  • the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient and/or diluent.
  • suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
  • Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. The dosage regimen will be determined by the attending physician and clinical factors.
  • dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • Proteinaceous pharmaceutically active matter may be present in amounts between 1 ng and 10 mg per dose; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.
  • Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. If the regimen is a continuous infusion, it should also be in the range of 1 ⁇ g to 10 mg units per kilogram of body weight per minute, respectively.
  • compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously.
  • the compositions of the invention may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the pharmaceutical composition of the invention may comprise further agents such as interleukins, interferons and/or CpG-containing DNA stretches, depending on the intended use of the pharmaceutical composition.
  • the invention also provides for a recombinant bacterial host cell of an avirulent strain or a vaccine strain which comprises at least one polynucleotide as defined herein.
  • Said recombinant (bacterial) host cell is particularly useful as a vaccine strain against mycobacterial-induced diseases, in particular against TB.
  • said recombinant cell comprises at least one further nucleic acid molecule encoding a peptide or polypeptide capable of eliciting and/or trigger an immune response in a mammal.
  • Said peptide or polypeptide capable of eliciting and/or trigger an immune response is preferably selected from the group consisting of autoantigens, tumor-antigens, virus-antigens, parasite-antigens, bacterial- antigens and/or immunogenic fragments thereof.
  • said host cell is capable of expressing said polynucleotide and said at least one further recombinant nucleic acid molecule.
  • This invention also relates to a fusion protein as defined herein above.
  • Said fusion protein is particularly useful in vaccination protocols against bacteria-induced diseases, in particular mycobacteria-induced disease.
  • the invention also relates to a polynucleotide encoding for the fusion protein as defined herein.
  • Said polynucleotide is preferably comprised in a vector, e.g. an expression vector as defined herein.
  • the invention provides for a method for the production of a vaccine against a virulent strain of the genus Mycobacterium comprising the step of combining a recombinant polypeptide as defined herein with a pharmaceutically acceptable carrier.
  • Said polypeptide may not comprise expressed Rv1511 and/or fragments thereof but may also be a fusionprotein/polypeptide as defined herein.
  • said method additionally comprises the recombinant expression of a polynucleotide as defined herein for recombinantly producing said polypeptide.
  • the polypeptide to be used in said method may be recombinantly produced.
  • the invention also provides for a method for the production of a vaccine against a virulent strain of the genus Mycobacterium comprising the step of combining a peptide as shown in any one of SEQ ID NOs: 3 to 48 (or as depicted in the appended table 1 )with a pharmaceutically acceptable carrier.
  • the invention also provides for a method for the production of a vaccine against a virulent strain of the genus Mycobacterium comprising the step of combining a polynucleotide or a vector as defined herein with a biologically and/or pharmaceutically acceptable carrier, wherein said polynucleotide or said polynucleotid comprised in said vector is placed under the control of an expression control sequence.
  • Useful expression control sequences are defined herein above but further, specific expression control sequences comprise tissue plasminogen activator (tpa), epidermal growth hormone (EGF), human growth hormone (HGH).
  • tissue plasminogen activator tpa
  • EGF epidermal growth hormone
  • HGH human growth hormone
  • the invention relates to the use of a polynucleotide or a vector as defined herein above for the preparation of a vaccine for vaccination against a virulent strain of the genus Mycobacterium or against a mycobacterium-induced disease.
  • a peptide as depicted in any one of SEQ ID NOs: 3 to 48 is usedfor the preparation of a vaccine for the vaccination against a virulent strain of the genus Mycobacterium or against a mycobacterium-induced disease.
  • the peptide to be employed for the preparation of said vaccine is selected from the group consisting of SEQ ID NO: 13, 20, 23, 24, 29, 35 or 42.
  • Said vaccination may be employed to prevent as well as to treat such a disease.
  • said mycobacterium-induced disease is selected from the group consisting of tuberculosis, tropical skin ulcer, ulceration, absess, granulomatous
  • mycobacteria-induced diseases in humans but also in the prevention and/or treatment of animal diseases, like bovine tuberculosis.
  • the present invention also provides for specific, isolated peptides derived from Rv1511 and shown in table 1. Therefore, the invention relates in a further embodiment to an isolated peptide selected from the group consisting of a peptide as depicted in table 1 or in any one of SEQ ID NOs: 3 to 48 (in particular peptides as shown in SEQ ID NO: 13, 20, 23, 24, 29, 35 or 42 or an isolated peptide which is at least 80% homologous to a peptide as shown in SEQ ID NOs: 3 to 48.
  • the invention also provides for nucleic acid molecules encoding the peptides disclosed herein, in particular peptides SEQ ID NO: 13, 20, 23, 24, 29, 35 or 42
  • Figure 1 Sectors from two-dimensional electrophoretic patterns of total cell proteins of attenuated (A: M. bovis BCG Chicago; B: M. bovis BCG Copenhagen) and virulent (C: M. tuberculosis H37Rv; D: M. tuberculosis Erdman) mycobacterial strains.
  • the spots 2_25 of M. tuberculosis H37Rv and 4_25 of M. tuberculosis Erdman had no counterparts in the protein patterns of the attenuated M. bovis BCG strains and were identified as probable GDP-D-mannose dehydratase (encoded by the ORF Rv1511 ) of M. tuberculosis H37Rv.
  • FIG. 2 Plasmid map of the DNA vector pCMVtpaRvl 511.
  • the DNA vector pCMVtpaRv1511 is a constitutive mammalian expression plasmid optimized for heterologous expression of antigens. Expression of Rv1511 is driven by the human cytomegalovirus (CMV) immediate early gene promoter downstream of the mycobacterial antigen Rv1511.
  • CMV human cytomegalovirus
  • the antigen is proximally fused to the human tissue plasminogen activator (tpa) leader sequence following an EcoRI restriction endonuclease site and translation begins at the first start codon (AUG) distal an untranslated sequence derived from tpa.
  • tpa tissue plasminogen activator
  • CFU colony forming unit
  • FIG. 4-5 Further vaccination results and Protection against tuberculosis by DNA vaccination Protection against tuberculosis by DNA vaccination.
  • BALB/c mice were immunised three times with 100 ⁇ g of the DNA construct. Mice were challenged with TB (200 bacteria per lung) 21 days after the last boost by aerosol. Bars represent the mean +/- SD of the CFU counts of 7 mice per group.
  • FIG. 6-9 IFN- ⁇ ELISpot assay on day 14 after the last boost.
  • BALB/c mice were immunised three times with 100 ⁇ g of the DNA constructs. Potential peptide epitopes were selected using a new MHC class I prediction program. For Rv 1511 seven peptides were generated (n.b. for the others two peptides). Bars represent the mean +/- SD of the spot counts of 3 mice per group.
  • FIGS 7-9 IFN- ⁇ ELISpot assay on days 7, 14, 28 and 60 after the last boost.
  • BALB/c mice were immunised three times with 100 ⁇ g of the DNA constructs. Potential peptide epitopes were selected using a new MHC class I prediction program. For Rv 1511 seven peptides were generated (n.b. for the others two peptides). Bars represent the mean +/- SD of the spot counts of 3 mice per group.
  • Figure 10 Western-blot of a 10% SDS-PAGE.
  • IgG isotypes were measured from mice vaccinated with Rv1511 or control vector. For additional control blood was taken from naive mice. IgGl and lgG2a isotypes were only determined from mice vaccinated with Rv1511. The figure shows ELISA-resultes with sera from Rv1511 immunised mice. Plates were coated with lysate from M.tb or recombinant Rv1511 from E.coli. Sera dilution of 1/50.
  • the Examples illustrate the invention:
  • Cellular proteins of M. tuberculosis H37Rv were prepared from mycobacterial whole cell lysates as described [26].
  • mycobacteria were grown in Middlebrook medium for 6-8 days to a cell density of 1-2 x 10 8 cells per ml, cells were washed and sonicated in the presence of proteinase inhibitors, and proteins were treated with 9 M urea, 70 mM dithiothreitol and 2% Triton X-100 to obtain completely denatured and reduced proteins.
  • the separation of proteins by 2-DE was performed as described [31], using a combination of carrier ampholyte IEF and SDS-PAGE. The gel size was 22 x 30 cm.
  • IEF was performed in rod gels containing 9 M urea, 3.5% acrylamide, 0.3% piperazine diacrylamide and a total of 4% carrier ampholytes pH 2-11. Protein samples were applied at the anodic side of the IEF gels and focused under non-equilibrium pH gradient electrophoresis conditions (8 870 Vh). For analytical and preparative investigations 0.75 or 1.5 mm thick gels were used, respectively. For analytical investigations 100 ⁇ g of protein sample were applied. For preparative experiments we used up to 900 ⁇ g of protein sample. SDS-PAGE was performed in gels containing 15% acrylamide using the IEF gels as stacking gels. Following electrophoresis the gels were stained. For analytical and preparative investigations proteins were visualized as described by either silver staining or Coomassie Brilliant Blue G250 (CBB G250) staining [32-33].
  • CBB G250 Coomassie Brilliant Blue G250
  • sequence support was required, to confirm the identity of a proposed protein or to establish protein identity. Sequence support was obtained by either PSD MALDI-MS [42] or ESI-MS/MS [43] performed with a Q-Tof mass spectrometer (Micromass, Manchester, UK) as described [34]. Here the sequence tag method [44] was used to search the proteins in the NCBI-database.
  • Silver stained 2-DE protein patterns of whole cell preparations of mycobacteria encompassed about 1 800 distinct protein species. The exact number of spots depended on the amount of sample applied to the gels and the staining conditions. Silver stained 2-DE protein patterns of whole cell preparations of all mycobacterial strains examined in this study are presented in our 2-DE database [29]. The genomes of members of the M. tuberculosis complex, including the four mycobacterial strains investigated, are highly conserved [45]. Consistent with this, the vast majority of protein spots had counterparts with identical electrophoretic mobility in all mycobacterial strains investigated.
  • the vaccine candidate Rv1511 was derived from proteome analysis and was found to be differentially expressed in virulent M. tuberculosis strains compared to M. bovis BCG; see Example I herein above. As will be demonstrated here, the DNA vaccine encoding for Rv1511 resulted in an constant protection over all observed/examined time points.
  • mice were bred at the central animal facilities of the Max-Planck-lnstitute for Infection Biology at the Bundesinstitut fur Surgicallichen Medeau für Veterinarytechnik (BGVV, Berlin, Germany). Animals were kept under specific pathogen-free conditions and fed autoclaved food and water ad libitum. In given experiments, female mice were used at 8 weeks of age. Groups of at least 5 mice were used in all experiments.
  • M. tuberculosis H37Rv and M. bovis Calmette-Guerin bacillus (BCG) Danish 1331 (Statens Serum Institute, Copenhagen, Denmark) were cultivated on Middlebrook 7H11 agar supplemented with OADC enrichment w/WR 1339 (Difco, Detroit, U.S.A.). Tuberculosis stocks for infection were grown to an cell density of approximately 10 8 cells per ml in Middlebrook 7H9 medium supplemented with Middlebrook ADC enrichment (Difco, Detroit, U.S.A.). All bacterial infection stocks were washed once with PBS w/o Ca and were maintained as glycerol stocks at - 70°C.
  • PCR primers Second, the amplification product is subcloned into a PCR cloning vector and its sequence identity is verified. Third the confirmed sequence is excised and ligated with a specific eukaryotic expression vector comprising the DNA vaccine.
  • the M. tuberculosis gene Rv1511 was cloned into the vaccine DNA plasmid vector pCMVtpa (Chiron-Behring, Emeryville, USA) under the control of the CMV promoter fused with the tissue plasminogen activator leader sequence, resulting in plasmid pCMVtpaRv1511.
  • the Mycobacterium tuberculosis gene Rv1511 was amplified from chromosomal DNA by gene specific oligonucleotides ( primers pCMVtpaRv1511 5' 5' AGA TCT GTG AAG CGA GCG CTC ATC 3" (SEQ ID NO: 49) and pCMVtpaRvl 511 3' 5' AGA TCT TGT CCG GCC GGC GAT 3' (SEQ ID NO: 50)) containing at their 5' ends an additional Bglll cloning site (underlined sequence) to facilitate subsequent ligation into the DNA vaccine vector pCMVtpa.
  • gene specific oligonucleotides primers pCMVtpaRv1511 5' 5' AGA TCT GTG AAG CGA GCG CTC ATC 3" (SEQ ID NO: 49) and pCMVtpaRvl 511 3' 5' AGA TCT TGT CCG GCC GGC GAT 3
  • the amplification product encoding the terminal Bglll cloning site and the complete M. tuberculosis gene Rv1511 was purified by agarose gel electrophoresis and subcloned into the Smal restriction site of the commercial PCR cloning vector pUC18.
  • the resulting plasmid carrying the subcloned PCR product was verified by restriction endonuclease digestion and by DNA sequencing.
  • the inserted sequence was cleaved from the PCR cloning vector by Bglll restriction endonuclease, specific for the incorporated 5' sites of the PCR product.
  • the excised fragment was subsequently ligated with the BamHI predigested DNA vaccine vector pCMVtpa resulting in the final DNA vaccine construct pCMVtpaRv1511.
  • the inserted sequence in the DNA vaccine encoding the M. tuberculosis antigen was confirmed by restriction endonuclease digestion and DNA sequencing.
  • the DNA vaccine was purified as endotoxin free plasmid DNA from E. coli.
  • mice per group were immunized at the age of 6-8 weeks by intramuscular injection (i. m.). Before immunization mice were narcotized and their hind legs were shaved. The DNA vaccine was injected into the tibialis anterior muscles. A dose of 50 ⁇ g naked DNA in 50 ⁇ l PBS was injected into each tibialis anterior muscle. Mice were initially immunized at day -90 and were boosted twice in 30 day intervals. At day 0 vaccinated and control mice were challenged with M. tuberculosis.
  • mice were vaccinated with 5 x 10 5 live M. bovis BCG at day -90 by i. v. injection into the tail vain. Mice were cured with rifampicin (100 mg/ml) and isoniazide (200 mg/ml) in the drinking water starting at day -60 over 21 days. Curing was verified by enumeration of bacterial titer at day -30 in spleen, liver and lung.
  • mice were challenged with 200 M. tuberculosis H37Rv bacilli by the use of an aerosol exposure system model 099C A4212 (Glas-Cole - Sch ⁇ tt Labortechnik, G ⁇ ttingen, Germany). At day 1 after the challenge the infection dose was verified as CFU per lung. Mice were sacrificed at days 28 and 60 post challenge. Spleens and lungs were aseptically removed and organ homogenates were prepared. Bacterial counts were enumerated by plating serial dilutions of organ homogenates on Middlebrook 7H11 agar.
  • the M. tuberculosis DNA vaccine testing comprises three steps.
  • the initial step of DNA vaccination is most often performed by intra muscular (i. m.) injection of naked DNA or by biolistic bombardment of DNA coated onto gold particles.
  • vaccinated and control animals are challenged by infection with virulent M. tuberculosis.
  • the degree of protection is subsequently measured by enumeration of the bacterial load in different organs at various time points after the challenge. Seven female mice per group were immunized at the age of 8 weeks by intramuscular (i. m.) injection. Before immunization, mice were anesthesized and their hind legs were shaved.
  • the DNA vaccine was injected into the tibialis anterior muscles at a dose of 50 ⁇ g DNA in 50 ⁇ l PBS leading to a final concentration of 100 ⁇ g DNA per mouse for each immunization.
  • Mice were boosted twice in 30 day intervals. Thirty days after the last boost immunization animals were challenged with 250 bacilli of virulent M. tuberculosis bacilli via the aerosol route using an inhalation exposure system. Groups of mice were sacrificed at various time points (days 14, 28 and 60) after challenge. Organs (spleen and lung) were aseptically removed and homogenates were prepared by grinding them in sealed bags.
  • bovis BCG Copenhagen were cured with rifampicin / isoniazide prior to challenge with M. tuberculosis. They were free of the vaccine strain as determined by CFU. Although no sterile protection was achieved, leading to complete eradication of M. tuberculosis bacilli after challenge in the model of murine tuberculosis, the live attenuated vaccine strain M. bovis BCG Copenhagen induced a high level of protection throughout the examined course of challenge infection. Additionally, the DNA vaccine pCMVtpaRv1511 caused a statistically significant protection with P values ⁇ 0.05 as determined by the Mann- Whitney test as compared to the plasmid vector control over the examined time course after challenge.
  • the protection induced by the DNA vaccine pCMVtpaRv1511 showed a constant CFU reduction of challenge tubercle bacilli with no decrease in protection at later time points after challenge. There was no difference between naive mice and the plasmid DNA control vector pCMVtpa treated mice, indicating that the protection induced by the DNA vaccine pCMVtpaRv1511 was not due to an unspecific effect of the DNA vector itself. Exacerbation of challenge infection with tubercle bacilli were determined for all groups, but only M. bovis BCG and pCMVtpaRv1511 lead to a significant reduction in CFU of lung.
  • Example III Antigenic peptides of a M. tuberculosis DNA vaccine
  • MHC I major histocompatibility complex
  • CTL cytotoxic T lymphocytes
  • Most of these antigenic peptides are generated during protein degradation in the cytoplasm and are then transported into the endoplasmic reticulum where they are loaded onto MHC I molecules.
  • CTL antigenic peptide fragments derived from the M. tuberculosis DNA vaccine pCMVtpaRv1511 by a combination of different predictions.
  • MAPPP MHC-I Antigenic Peptide Processing Prediction
  • MAPPP first generates a probability for the cleavage of each possible peptide from a protein by proteasome activity. This probability is based on a statistic-empirical method. Peptides with the highest probabilities of proteasome processing are then given an additional score reflecting their ability of binding to MHC I molecules. This binding score employs coefficient tables. A combination of two independent predictions for proteasome processing and MHC I presentation increases the possibility to retrieve more precise predictions on MHC I presented immunodominant T- cell epitopes.
  • the probability of a cut after each of the residues within the sequence is determined first.
  • the cleavage probability for all possible fragments between two cut-sites within the right length was calculated.
  • the flanking regions to the C-terminal and the N-terminal end of a fragment and the probabilities of their residues were also considered within the calculation.
  • the program used the algorithms first implemented in FRAGPREDICT, a computer program for the prediction of proteasomal cleavage sites and proteolytic fragments [46] or alternatively the prediciton algorithms of PaProC [47].
  • the prediction of antigens derived from pCMVtpaRv1511 presented by MHC class I molecules is based on a score table calculation for each subsequence generated by proteasomal prediction.
  • An additional score was derived from a comparison with different matrices specific for individual MHC haplotypes (e.g. HLA-A201 , H2Kb, H2Dd, H2Kd, H2Dd, H2Ld etc.) and different species, in particular, mouse, cattle (bovine) and most preferably in human.
  • Each specific amino acid at a specific position within a subsequence was given a value.
  • the values for potential epitopes of the correct length were then multiplied for BIMAS calculation [48] or added in case of SYFPEITHI [49] to determine the score for the subsequence.
  • These values for different species and their distinct haplotypes used for calculation were pre- calculated and stored in static matrices. Yet, even if certain peptides in the appended table are linked to distinct haplotypes of certain species, i.e mouse, cattle or human, it is of note that said peptide/fragment of Rv1511 may also be employed in context of this invention in any other species.
  • Each time-point consists of 7 animals per group. Naked DNA (100 ⁇ g) constructs encoding for Rv1511. The DNA vaccine candidate was administered a total of three injections via the intramuscular route at 21 day intervals. 21 days after the last injection the mice were aerosol challenged with M. tuberculosis H37Rv (200 organisms per mouse). At early (30 days) and late (60 days) time points post- challenge the bacterial load in the lung was determined as a measurement of levels of protection.
  • Antigen Rv1511 and Rv3407 provided protection at both time points (compared to BCG) whereas, antigen Rv2520 provided protection only at the early time point. These antigens were also administered in pair-wise combinations. Splenocytes were used in ELIspot assays to determine the IFN- ⁇ immune response to the antigens used for DNA vaccination. The increase in IFN ⁇ production correlated well with the antigen induced in the antigen protection experiments.
  • Example V Frequency of IFN- ⁇ secretion T lymphocytes specific for Rv1511
  • Cytokine ELISPOT Assay The frequency of IFN ⁇ -secreting T lymphocytes specific for the Rv1511 H-2K d epitope and stated in example III (Table 1 ) P1-P7 was determined by a slightly modified ELISPOT technique (Fensterle, J., Grade, L., Hess, J. & Kaufmann, S.H.E. (1999) J. Immunol. 163, 4510-4518). Briefly, 96-well nitrocellulose plates (Millititer HA; Millipore, Bedford, MA) were coated with 5 mg/ml of the anti-mouse IFN ⁇ -mAb R4 (PharMingen) in 100 ⁇ l of carbonate buffer, pH 9.6.
  • Coated or uncoated P815 cells (10 5 ) were added to splenocytes in 100 ⁇ l of RP10, and after 20 h incubation at 37°C, 5% C0 2 in the presence of 30 U/ml IL-2, the plates were washed 10 times with 0.05% Tween 20 in PBS (washing buffer).
  • PBS washing buffer
  • 0.25 ⁇ g/ml biotinylated anti-mouse IFN ⁇ mAb XMG1.2 (PharMingen) in 100 ⁇ l washing buffer was added and incubated at 37°C for 2 h.
  • Rv1511 -specific MHC class ll-restricted T cells To detect Rv1511 -specific MHC class ll-restricted T cells, a slightly modified protocol was used. To induce specific cytokine secretion by Rv1511 -specific CD4 T cells, 10 5 splenocytes per well were stimulated with 10 ⁇ g/ml heat-denatured protein lysat of H37Rv 100 ⁇ l RP10 for 3 d. J774A.1 macrophage-like cells were pulsed with 10 ⁇ g/ml heat-denatured, protein lysat of H37Rv in RP10 at 37°C for 1 h and subsequently washed twice with RP10.
  • Coated or uncoated J774A.1 cells (10 5 ) were added to splenocytes in 100 ⁇ l RP10 and after 20 h incubation at 37°C, 5% C0 2 in the presence of 30 U/ml IL-2, plates were washed 10 times with 0.05% Tween 20 in PBS (washing buffer). Plates for IFN ⁇ -detection were prepared as described above. Incubation times and the detection followed the ELISPOT protocol for MHC class I- restricted CD8 T cells.
  • These results underline the capacity of Rv1511 to induce an acquired cellular immune response, needed to control intracellular pathogens such as M. tuberculosis.
  • the MHC class I restricted immune response observed in this study identifies the course of protection against tuberculosis.
  • Example VI Antibody titers contribute to Th1 type of immune response in Rv1511 immunized mice
  • Antibody response was measured by enzyme-linked immunosorbent assay (ELISA) of sera pooled from groups of vaccinated mice. Briefly, 96-well microtiter plates were coated overnight with 1 ⁇ g of purified recombinant Rv1511 protein in 0.1 M carbonate buffer. Blood sera was collected from Rv1511 immunized mice. The sera were diluted and the endp ⁇ int titer was determined. Serum samples and peroxidase conjugated rabbit anti-mouse IgG (1:2500) were diluted and incubated sequentially in the plates for 2h at 37°C. For determination of antibody isotypes, antibodies specific for murine lgG1 and lgG2a were used. Sera from unimmunised mice was used as the controls.
  • ELISA enzyme-linked immunosorbent assay
  • SYFPEITHI database for MHC ligands and peptide motifs.

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Abstract

L'invention concerne une composition pharmaceutique comprenant au moins un polypeptide sélectionné dans le groupe comprenant (a) un polypeptide codé par un polynucléotide comprenant une séquence d'acide nucléique, comme défini ici ; (b) un polypeptide comprenant une séquence d'acide aminé, comme défini ici ; (c) un polypeptide qui est ou qui comprend un domaine fonctionnel, un fragment antigénique et/ou un fragment apte à provoquer et/ou à déclencher une réponse immunitaire du polypeptide de (a) ou de (b) chez un mammifère ; (d) un polypeptide codé par un polynucléotide identique au moins à 80 % au polynucléotide, comme défini en (a) et qui est apte à provoquer et/ou à déclencher une réponse immunitaire chez un mammifère ; et (e) un polypeptide codé par un polynucléotide qui s'hybride dans des conditions strictes avec le polynucléotide, comme défini en (a) ou en (d) et est apte à provoquer et/ou à déclencher une réponse immunitaire chez un mammifère. L'invention concerne en outre des compositions pharmaceutiques comprenant des protéines de fusion, des polynucléotides, des vecteur(s), des cellule(s) hôte(s) ou des anticorps, comme décrit ici. L'invention concerne par ailleurs des cellules hôtes (bactériennes) recombinées et des procédés pour produire un vaccin.
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WO2005021790A3 (fr) * 2003-08-29 2005-05-06 Consiglio Nazionale Ricerche Utilisation de sequences geniques propres au bacille de koch et proteines associees pour le diagnostic et la prevention des infections tuberculeuses
CN100350048C (zh) * 2005-07-21 2007-11-21 复旦大学 一种重组卡介苗及其制备方法
CN100457912C (zh) * 2005-07-21 2009-02-04 复旦大学 重组Ag85B卡介苗
US8246944B2 (en) 2003-10-31 2012-08-21 Archivel Farma, S.L. Immunotherapeutic agent for the combined treatment of tuberculosis in association with other drugs
CN103884847A (zh) * 2014-03-14 2014-06-25 中国科学院生物物理研究所 一种结核分枝杆菌全蛋白质芯片及应用
US9289483B2 (en) 2006-10-30 2016-03-22 Archivel Farma, S.L. Prophylactic tuberculosis vaccine
CN107304231A (zh) * 2016-04-18 2017-10-31 华中农业大学 一种结核分枝杆菌融合蛋白及应用

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005021790A3 (fr) * 2003-08-29 2005-05-06 Consiglio Nazionale Ricerche Utilisation de sequences geniques propres au bacille de koch et proteines associees pour le diagnostic et la prevention des infections tuberculeuses
US8246944B2 (en) 2003-10-31 2012-08-21 Archivel Farma, S.L. Immunotherapeutic agent for the combined treatment of tuberculosis in association with other drugs
CN100350048C (zh) * 2005-07-21 2007-11-21 复旦大学 一种重组卡介苗及其制备方法
CN100457912C (zh) * 2005-07-21 2009-02-04 复旦大学 重组Ag85B卡介苗
US9289483B2 (en) 2006-10-30 2016-03-22 Archivel Farma, S.L. Prophylactic tuberculosis vaccine
CN103884847A (zh) * 2014-03-14 2014-06-25 中国科学院生物物理研究所 一种结核分枝杆菌全蛋白质芯片及应用
CN107304231A (zh) * 2016-04-18 2017-10-31 华中农业大学 一种结核分枝杆菌融合蛋白及应用
CN107304231B (zh) * 2016-04-18 2021-01-01 华中农业大学 一种结核分枝杆菌融合蛋白及应用

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