WO2006102767A1 - Vaccin contre la tuberculose et sa methode de fabrication - Google Patents

Vaccin contre la tuberculose et sa methode de fabrication Download PDF

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Publication number
WO2006102767A1
WO2006102767A1 PCT/CA2006/000503 CA2006000503W WO2006102767A1 WO 2006102767 A1 WO2006102767 A1 WO 2006102767A1 CA 2006000503 W CA2006000503 W CA 2006000503W WO 2006102767 A1 WO2006102767 A1 WO 2006102767A1
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Prior art keywords
bcg
seq
nucleic acid
tuberculosis
mpb70
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PCT/CA2006/000503
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English (en)
Inventor
Marcel Behr
Serge Mostowy
Danielle Charlet
David Alexander
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Mcgill University
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Priority to CA002603298A priority Critical patent/CA2603298A1/fr
Priority to EP06721759A priority patent/EP1863914A1/fr
Publication of WO2006102767A1 publication Critical patent/WO2006102767A1/fr

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    • 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
    • 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
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins

Definitions

  • the present invention relates to an improved tuberculosis (TB) vaccine and includes a method for making this vaccine. Vaccination against mycobacterial infection occurs through peptides and proteins expressed in response to sigma K stimulation.
  • the invention further includes a method for determining the potency of tuberculosis (TB) strains which is based on the detection of an anti-sigma K cell response.
  • BCG Mycobacterium bovis Bacille Calmette-Guerin
  • MPB70 represents the most abundant protein in the culture filtrate (Nagai et al., 1981). In other BCG strains, such as BCG Pasteur, production of MPB70 is markedly reduced, leading to the division of BCG strains into high-producers or low-producers (Miura et al., 1983; Harboe and Nagai, 1984). The same pattern of antigen production across BCG strains has also been observed for MPB83, although the differences have generally not been as dichotomous (Wiker et al., 1996).
  • Mycobacterium bovis Bacille Calmette-Guerin (BCG) strains are genetically and phenotypically heterogeneous. Expression of the antigenic proteins MPB70 and MPB83 is known to vary considerably across BCG strains; however, the reason for this phenotypic difference has remained unknown.
  • BCG strains were separated into high- and low-producing strains.
  • RT-PCR quantitative reverse transcription polymerase chain reaction
  • Transcriptome comparison of the same BCG strains by DNA microarray revealed two gene regions consistently downregulated in low-producing strains compared with high-producing strains, one including mpb70 (Rv2875) and mpb83 (Rv2873) and a second that includes the predicted sigma factor, sigK.
  • DNA sequence analysis revealed a point mutation in the start codon of sigK in all low-producing BCG strains. Complementation of a low-producing strain, BCG Pasteur, with wild-type sigK fully restored MPB70 and MPB83 production.
  • Microarray-based analysis and confirmatory RT-PCR of the complemented strains revealed an upregulation in gene transcription limited to the sigK and the mpb83/mpb70 gene regions.
  • the present invention relates to an improved tuberculosis (TB) vaccine and includes a method for making this vaccine.
  • This vaccine is based on the complementation of a defective sigma K protein with a wild- type sigma K protein.
  • the nucleotide sequence for wild- type sigma K protein is introduced via a nucleotide vector.
  • Host organisms to make the tuberculosis vaccine include but are not limited to the following: M. tuberculosis, M. bovis, M. caprae, M. microti, M. africanum, M. canettii, M. pinnipedii.
  • the tuberculosis vaccine is BCG and wild-type sigma K is introduced into a host cell to stimulate the production of the immunogens mpb70 and mpb83 (or mpt70 an mpt83).
  • Possible BCG strains are chosen from the following non-limited group: BCG Russia, BCG Moreau, BCG Japan, BCG Sweden, BCG Birkhaug, BCGska, BCG Glaxo, BCG Denmark, BCG Tice, BCG Connaught, BCG Frappier, BCG Phipps and BCG Pasteur.
  • the vaccines produced in accordance with the present invention are useful for the immunization of mammals against tuberculosis.
  • Mammals include but are not limited to man, sheep, goats, pigs, deer, elk, bison, cows, steers, bulls and oxen.
  • the invention relates to a method for determining the potency of tuberculosis (TB) strains.
  • this method is based on the detection of an anti-sigma K cell response reflected in the overall production of mpb70, mpb83 or both of these antigenic proteins.
  • the antigenic proteins may be analogous proteins, such as mpt70 and mpt83.
  • the method is based on microarray hybridization and analysis. This method relies on the use of the newly identified nucleotide sequence described in the present specification for a mutant form of sigma K wherein the G at position 3 is replaced by A.
  • the nucleotide sequence of this mutant form of sigma K, as well as its peptidic sequence are part of the present invention, as are nucleotide and amino acid fragments comprising the point mutation described here.
  • FIG 1 SDS-PAGE and immunoblotting of culture filtrate proteins (A and C) and cell extracts (B and D) from BCG strains, using monoclonal antibodies 1- 5c for MPB70 (A and B) and MBS43 for MPB83 (C and D).
  • the strains used were as follows: Ja, BCG Japan; Pa, BCG Pasteur; Ru, BCG Russia; Sw, BCG Sweden; Bi, BCG Birkhaug; Ph, BCG Phipps; Gl, BCG Glaxo; Mo, BCG Moreau; Pr, BCGska; Fr, BCG Frappier; Co, BCG Connaught; Ti, BCG Tice; De, BCG Denmark.
  • Ratio of expression is to that of BCG Pasteur. All values were normalized to the levels of sigA mRNA.
  • Figure 3 A and B. Expression of sigK (A) and mpblO (white) and mpb83 (grey) (B) upon complementation of BCG Pasteur with sigK from M. tuberculosis H37Rv, BCG Russia, BCG Birkhaug and BCG Pasteur. Values are expressed as a ratio of mRNA copies in complemented strains compared with BCG Pasteur::pMV306. All values were normalized to the levels of sigA mRNA and error bars represent the standard error of the mean. C and D.
  • Figure 4 A. Expression analysis of the genes RvO441c to Rv0450c.
  • B Expression analysis for the genes Rv2870c to Rv2881c. Levels presented represent the ratio of mRNA copies in s/gK-complemented strain to the control strain BCG Pasteur::pMV306 (empty vector). All values were normalized to the levels of sigA mRNA and the ratio presented represents the mean of results from different clones, specifically BCG Pasteur::pH37Rv, BCG Pasteur::pRUSS and BCG Pasteur::pBIRK.
  • Figure 5 Protective efficacy against M. tuberculosis low-dose aerosol challenge in a guinea pig model.
  • Figure 7 Immunogenicity, as measured by MPB70-induced gamma- interferon production by splenocytes in mice.
  • a target polynucleotide includes a plurality of target polynucleotides.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), "including” (and any form of including, such as “include” and “includes”) or “containing” (and any form of containing, such as “contain” and “contains”), are inclusive or open-ended and do not exclude additional, unrecited elements or process steps.
  • polynucleotide oligonucleotide
  • nucleic acid nucleic acid molecule
  • nucleic acid molecule polymeric form of nucleotides of any length, and may comprise ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. These terms refer only to the primary structure of the molecule. Thus, the terms include triple-, double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”). They also include modified (for example, by alkylation and/or by capping) and unmodified forms of the polynucleotide.
  • DNA triple-, double- and single-stranded deoxyribonucleic acid
  • RNA triple-, double- and single-stranded ribonucleic acid
  • polynucleotide examples include polydeoxyribonucleotides (containing 2- deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing a phosphate or other polyanionic backbone, and other synthetic sequence-specific nucleic acid polymers provided that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA.
  • polynucleotide oligonucleotide
  • nucleic acid nucleic acid molecule
  • these terms include, for example, 3'-deoxy-2',5'-DNA, oligodeoxyribonucleotide N3' P5' phosphoramidates, 2'-0-alkyl-substituted RNA, double- and single-stranded DNA, as well as double- and single-stranded RNA, and hybrids thereof, including, for example, hybrids between DNA and RNA, and also include known types of modifications, for example, labels, alkylation, "caps," substitution of one or more of the nucleotides with an analog, internucleotide modifications such as, for example, those with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example
  • nucleases nucleases
  • toxins antibodies
  • signal peptides poly-L-lysine, etc.
  • intercalators e.g., acridine, psoralen, etc.
  • chelates of, e.g., metals, radioactive metals, boron, oxidative metals, etc.
  • alkylators those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide or oligonucleotide.
  • Standard A-T and G-C base pairs form under conditions which allow the formation of hydrogen bonds between the N3-H and C4-oxy of thymidine and the N1 and C6-NH2, respectively, of adenosine and between the C2-oxy, N3 and C4-NH2, of cytidine and the C2-NH2, N'-H and C6-oxy, respectively, of guanosine.
  • guanosine (2-amino-6-oxy-9-.beta.-D-ribofuranosyl-purine) may be modified to form isoguanosine (2-oxy-6-amino-9-.beta.-D-ribofuranosyl-purine).
  • Hybridization conditions will typically include salt concentrations of less than about 1 M, more usually less than about 500 mM and preferably less than about 200 mM.
  • Hybridization temperatures can be as low as 5 0 C, but are typically greater than 22 0 C, more typically greater than about 3O 0 C, and preferably in excess of about 37 0 C Longer fragments may require higher hybridization temperatures for specific hybridization.
  • Other factors may affect the stringency of hybridization, including base composition and length of the complementary strands, presence of organic solvents and extent of base mismatching, and the combination of parameters used is more important than the absolute measure of any one alone.
  • Suitable hybridization conditions for a given assay format can be determined by one of skill in the art; nonlimiting parameters which may be adjusted include concentrations of assay components, salts used and their concentration, ionic strength, temperature, buffer type and concentration, solution pH, presence and concentration of blocking reagents to decrease background binding such as repeat sequences or blocking protein solutions, detergent type(s) and concentrations, molecules such as polymers which increase the relative concentration of the polynucleotides, metal ion(s) and their concentration(s), chelator(s) and their concentrations, and other conditions known in the art.
  • the target polynucleotide can be single-stranded, double-stranded, or higher order, and can be linear or circular.
  • Exemplary single-stranded target polynucleotides include MRNA, rRNA, tRNA, hnRNA, ssRNA or ssDNA viral genomes, although these polynucleotides may contain internally complementary sequences and significant secondary structure.
  • Exemplary double-stranded target polynucleotides include genomic DNA, mitochondrial DNA, chloroplast DNA, dsRNA or dsDNA viral genomes, plasmids, phage, and viroids.
  • the target polynucleotide can be prepared synthetically or purified from a biological source.
  • the target polynucleotide may be purified to remove or diminish one or more undesired components of the sample or to concentrate the target polynucleotide. Conversely, where the target polynucleotide is too concentrated for the particular assay, the target polynucleotide may be diluted.
  • Oligonucleotide probes or primers of the present invention may be of any suitable length, depending on the particular assay format and the particular needs and targeted genomes employed. In general, the oligonucleotide probes or primers are at least 12 nucleotides in length, preferably between 15 and 24 molecules, and they may be adapted to be especially suited to a chosen nucleic acid amplification system.
  • the oligonucleotide probes and primers can be designed by taking into consideration the melting point of hybridization thereof with its targeted sequence (see below and in Sambrook et al., 1989, Molecular Cloning - A Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel et al., 1989, in Current Protocols in Molecular Biology, John Wiley & Sons Inc., N. Y.).
  • Probes of the invention can be utilized with naturally occurring sugar-phosphate backbones as well as modified backbones including phosphorothioates, dithionates, alkyl phosphonates and ⁇ -nucleotides and the like. Modified sugar-phosphate backbones are generally taught by Miller, 1988, Ann. Reports Med. Chem. 23:295 and Moran et al., 1987, Nucleic Acids Res., 14:5019. Probes of the invention can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and preferably of DNA.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • probes can be used.
  • Southern blots DNA detection
  • dot or slot blots DNA, RNA
  • Northern blots RNA detection
  • labeled proteins could also be used to detect a particular nucleic acid sequence to which it binds.
  • Other detection methods include kits containing probes on a dipstick setup and the like.
  • Probes can be labeled according to numerous well known methods (Sambrook et al., 1989, supra). Non-limiting examples of labels include 3 H, 14 C, 32 P, and 35 S. Non-limiting examples of detectable markers include ligands, fluorophores, chemiluminescent agents, enzymes, and antibodies. Other detectable markers for use with probes, which can enable an increase in sensitivity of the method of the invention, include biotin and radionucleotides. It will become evident to the person of ordinary skill that the choice of a particular label dictates the manner in which it is bound to the probe.
  • radioactive nucleotides can be incorporated into probes of the invention by several methods.
  • Non-limiting examples thereof include kinasing the 5' ends of the probes using gamma 32 P ATP and polynucleotide kinase, using the Klenow fragment of Pol I of E. coli in the presence of radioactive dNTP (e.g. uniformly labeled DNA probe using random oligonucleotide primers in low-melt gels), using the SP6/T7 system to transcribe a DNA segment in the presence of one or more radioactive NTP, and the like.
  • radioactive dNTP e.g. uniformly labeled DNA probe using random oligonucleotide primers in low-melt gels
  • oligonucleotides or “oligos” define a molecule having two or more nucleotides (ribo or deoxyribonucleotides). The size of the oligo will be dictated by the particular situation and ultimately on the particular use thereof and adapted accordingly by the person of ordinary skill.
  • An oligonucleotide can be synthesized chemically or derived by cloning according to well known methods. While they are usually in a single-stranded form, they can be in a double-stranded form and even contain a "regulatory region".
  • a "primer” defines an oligonucleotide which is capable of annealing to a target sequence, thereby creating a double stranded region which can serve as an initiation point for DNA synthesis under suitable conditions.
  • Primers can be, for example, designed to be specific for certain alleles so as to be used in an allele-specific amplification system.
  • Amplification of a selected, or target, nucleic acid sequence may be carried out by a number of suitable methods. See generally Kwoh et al., 1990, Am.
  • amplification techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), transcription-based amplification, the Q ⁇ replicase system and NASBA (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177; Lizardi et al., 1988, BioTechnology 6:1197-1202; Malek et al., 1994, Methods MoI. Biol., 28:253-260; and Sambrook et al., 1989, supra).
  • amplification will be carried out using PCR.
  • PCR Polymerase chain reaction
  • U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188 the disclosures of all three U.S. Patent are incorporated herein by reference.
  • PCR involves, a treatment of a nucleic acid sample (e.g., in the presence of a heat stable DNA polymerase) under hybridizing conditions, with one oligonucleotide primer for each strand of the specific sequence to be detected.
  • An extension product of each primer which is synthesized is complementary to each of the two nucleic acid strands, with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith.
  • the extension product synthesized from each primer can also serve as a template for further synthesis of extension products using the same primers.
  • the sample is analyzed to assess whether the sequence or sequences to be detected are present. Detection of the amplified sequence may be carried out by visualization following EtBr staining of the DNA following gel electrophores, or using a detectable label in accordance with known techniques, and the like.
  • EtBr staining of the DNA following gel electrophores, or using a detectable label in accordance with known techniques, and the like.
  • the term "gene” is well known in the art and relates to a nucleic acid sequence defining a single protein or polypeptide.
  • a "structural gene” defines a DNA sequence which is transcribed into RNA and translated into a protein having a specific amino acid sequence thereby giving rise to a specific polypeptide or protein. It will be readily recognized by the person of ordinary skill, that the nucleic acid sequence of the present invention can be incorporated into anyone of numerous established kit formats which are well known in the art.
  • heterologous e.g. a heterologous gene region of a DNA molecule is a subsegment of DNA within a larger segment that is not found in association therewith in nature.
  • heterologous can be similarly used to define two polypeptidic segments not joined together in nature.
  • Non-limiting examples of heterologous genes include reporter genes such as luciferase, chloramphenicol acetyl transferase, ⁇ -galactosidase, and the like which can be juxtaposed or joined to heterologous control regions or to heterologous polypeptides.
  • vector is commonly known in the art and defines a plasmid DNA, phage DNA, viral DNA and the like, which can serve as a DNA vehicle into which DNA of the present invention can be cloned. Numerous types of vectors exist and are well known in the art.
  • expression defines the process by which a gene is transcribed into mRNA (transcription), the mRNA is then being translated (translation) into one polypeptide (or protein) or more.
  • expression vector defines a vector or vehicle as described above but designed to enable the expression of an inserted sequence following transformation into a host.
  • the cloned gene (inserted sequence) is usually placed under the control of control element sequences such as promoter sequences.
  • control element sequences such as promoter sequences.
  • the placing of a cloned gene under such control sequences is often referred to as being operably linked to control elements or sequences.
  • Operably linked sequences may also include two segments that are transcribed onto the same RNA transcript.
  • two sequences such as a promoter and a "reporter sequence” are operably linked if transcription commencing in the promoter will produce an RNA transcript of the reporter sequence.
  • a promoter and a reporter sequence are operably linked if transcription commencing in the promoter will produce an RNA transcript of the reporter sequence.
  • Expression control sequences will vary depending on whether the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host or both (shuttle vectors) and can additionally contain transcriptional elements such as enhancer elements, termination sequences, tissue-specificity elements, and/or translational initiation and termination sites.
  • Prokaryotic expressions are useful for the preparation of large quantities of the protein encoded by the DNA sequence of interest.
  • This protein can be purified according to standard protocols that take advantage of the intrinsic properties thereof, such as size and charge (e.g. SDS gel electrophoresis, gel filtration, centrifugation, ion exchange chromatography).
  • the protein of interest can be purified via affinity chromatography using polyclonal or monoclonal antibodies. The purified protein can be used for therapeutic applications.
  • the DNA construct can be a vector comprising a promoter that is operably linked to an oligonucleotide sequence of the present invention, which is in turn, operably linked to a heterologous gene, such as the gene for the luciferase reporter molecule.
  • Promoter refers to a DNA regulatory region capable of binding directly or indirectly to RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter is bound at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • RNA polymerase a transcription initiation site (conveniently defined by mapping with S1 nuclease), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA” boxes and "CCAT” boxes.
  • Prokaryotic promoters contain -10 and -35 consensus sequences, which serve to initiate transcription and the transcript products contain Shine-Dalgarno sequences, which serve as ribosome binding sequences during translation initiation.
  • the designation "functional derivative” denotes, in the context of a functional derivative of a sequence whether a nucleic acid or amino acid sequence, a molecule that retains a biological activity (either function or structural) that is substantially similar to that of the original sequence.
  • This functional derivative or equivalent may be a natural derivative or may be prepared synthetically.
  • Such derivatives include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of the protein is conserved.
  • derivatives of nucleic acid sequences which can have substitutions, deletions, or additions of one or more nucleotides, provided that the biological activity of the sequence is generally maintained.
  • the substituting amino acid When relating to a protein sequence, the substituting amino acid generally has chemico- physical properties which are similar to that of the substituted amino acid.
  • the similar chemico-physical properties include, similarities in charge, bulkiness, hydrophobicity, hydrophylicity and the like.
  • the term “functional derivatives” is intended to include “fragments”, “segments”, “variants”, “analogs” or “chemical derivatives” of the subject matter of the present invention.
  • variant refers herein to a protein or nucleic acid molecule which is substantially similar in structure and biological activity to the protein or nucleic acid of the present invention.
  • the functional derivatives of the present invention can be synthesized chemically or produced through recombinant DNA technology. All these methods are well known in the art.
  • allele defines an alternative form of a gene which occupies a given locus on a chromosome.
  • a “mutation” is a detectable change in the genetic material which can be transmitted to a daughter cell.
  • a mutation can be, for example, a detectable change in one or more deoxyribonucleotide.
  • nucleotides can be added, deleted, substituted for, inverted, or transposed to a new position. Spontaneous mutations and experimentally induced mutations exist.
  • a mutant polypeptide can be encoded from this mutant nucleic acid molecule.
  • purified refers to a molecule having been separated from a cellular component.
  • a purified protein has been purified to a level not found in nature.
  • a “substantially pure” molecule is a molecule that is lacking in most other cellular components.
  • molecule As used herein, the terms “molecule”, “compound”, “agent” or “ligand” are used interchangeably and broadly to refer to natural, synthetic or semi-synthetic molecules or compounds.
  • the term “molecule” therefore denotes for example chemicals, macromolecules, cell or tissue extracts (from plants or animals) and the like.
  • Non limiting examples of molecules include nucleic acid molecules, peptides, antibodies, carbohydrates and pharmaceutical agents.
  • the agents can be selected and screened by a variety of means including random screening, rational selection and by rational design using for example protein or ligand modeling methods such as computer modeling.
  • the terms “rationally selected” or “rationally designed” are meant to define compounds which have been chosen based on the configuration of interacting domains of the present invention.
  • molecules having non-naturally occurring modifications are also within the scope of the term "molecule".
  • peptidomimetics well known in the pharmaceutical industry and generally referred to as peptide analogs can be generated by modeling as mentioned above.
  • the polypeptides of the present invention are modified to enhance their stability. It should be understood that in most cases this modification should not alter the biological activity of the interaction domain.
  • sequences and polypeptides useful to practice the invention include without being limited thereto mutants, homologs, subtypes, alleles and the like. It shall be understood that generally, the sequences of the present invention should encode a functional (albeit defective) interaction domain. It will be clear to the person of ordinary skill that whether an interaction domain of the present invention, variant, derivative, or fragment thereof retains its function in binding to its partner can be readily determined by using the teachings and assays of the present invention and the general teachings of the art.
  • a host cell or indicator cell has been "transfected" by exogenous or heterologous DNA (e.g. a DNA construct) when such DNA has been introduced inside the cell.
  • the transfecting DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the transfecting DNA may be maintained on a episomal element such as a plasmid.
  • a stably transfected cell is one in which the transfecting DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication.
  • the term therapeutic agent should be taken in a broad sense so as to also include a combination of at least two such therapeutic agents.
  • the DNA segments or proteins according to the present invention can be introduced into individuals in a number of ways. For example, erythropoietic cells can be isolated from the afflicted individual, transformed with a DNA construct according to the invention and reintroduced to the afflicted individual in a number of ways, including intravenous injection. Alternatively, the DNA construct can be administered directly to the afflicted individual, for example, by injection in the bone marrow. The DNA construct can also be delivered through a vehicle such as a liposome, which can be designed to be targeted to a specific cell type, and engineered to be administered through different routes.
  • a vehicle such as a liposome
  • the prescribing medical professional will ultimately determine the appropriate form and dosage for a given patient, and this can be expected to vary according to the chosen therapeutic regimen (e.g. DNA construct, protein, cells), the response and condition of the patient as well as the severity of the disease.
  • the chosen therapeutic regimen e.g. DNA construct, protein, cells
  • composition within the scope of the present invention should contain the active agent (e.g. fusion protein, nucleic acid, and molecule) in an amount effective to achieve the desired therapeutic effect while avoiding adverse side effects.
  • the nucleic acids in accordance with the present invention can be administered to mammals (e.g. humans) in doses ranging from 0.005 to 1 mg per kg of body weight per day of the mammal which is treated.
  • Pharmaceutically acceptable preparations and salts of the active agent are within the scope of the present invention and are well known in the art (Remington's Pharmaceutical Science, 16th Ed., Mack Ed.).
  • the amount administered should be chosen so as to avoid adverse side effects.
  • the dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient. Typically, 0.001 to 50 mg/kg/day will be administered to the mammal.
  • kits for determining the characteristics (such as the potency) of a tuberculosis vaccine comprising a nucleic acid, a protein or a ligand in accordance with the present invention.
  • a compartmentalized kit in accordance with the present invention includes any kit in which reagents are contained in separate containers.
  • Such containers include small glass containers, plastic containers or strips of plastic or paper.
  • Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.
  • Such containers will include a container which will accept the test sample (DNA protein or cells), a container which contains the primers used in the assay, containers which contain enzymes, containers which contain wash reagents, and containers which contain the reagents used to detect the extension products.
  • Genomic DNA contamination was removed by RNAeasy on-column digestion, following the manufacturer's protocol (Qiagen, Mississauga, Canada). The quality of RNA was confirmed by denaturing gel electrophoresis (formaldehyde).
  • Targeted gene expression levels were determined using RTPCR with molecular beacons or sybr green, according to established protocols (Manganelli et al., 1999; Mostowy et al., 2004). To provide a normalization standard for mRNA expression, expression of sigA was also determined, and the level of expression of a gene of interest divided by that of sigA to normalize for differences in total mRNA extracted (Manganelli et al., 1999). Sequences of the primers used for molecular beacon and sybr green analysis and the sequences of the molecular beacons used are as listed in Table 4.
  • Microarray hybridization and analysis were performed as previously described (Mostowy et al., 2004).
  • mRNA from BCG strains and complemented strains was extracted during log-phase in vitro growth and labelled with Cy3 or Cy5 dUTP by reverse-transcriptase (Amersham Biosciences).
  • Labelled cDNA was hybridized to microarrays composed of oligonucleotide probes from the TB Array-Ready Oligo SetTM (Operon) that had been printed onto SigmascreenTM microarray slides (Sigma).
  • Initial comparisons were BCG Russia versus BCG Pasteur, and BCG Birkhaug versus Denmark.
  • the sigK region (including the complete gene and 288 bp upstream) was amplified via PCR from M. tuberculosis H37Rv, and BCG strains Russia and Birkhaug.
  • the sigK from BCG Pasteur was also complemented to determine the effect of having a second copy of the mutated gene.
  • PCR was performed using the following primers RvO445cl_ and sigKR (left primer: 5'-agctcgagcagctcaaaatc-3'; right primer: 5'- acgcgtcaccccaactact-3') and amplified products were cloned into the T-vector, pDRIVE (Qiagen).
  • the sigK region was then removed by digestion with H/ndlll and Kpn ⁇ and ligated to the integrative mycobacterial vector pMV306 (de Stover et al., 1991 ) cut with the same restriction endonucleases.
  • Integrity of the cloned genes was confirmed by DNA sequencing, then the resulting plasmids (pH37Rv, pRUSS, pBIRK and pPAST) were electroporated into M. bovis BCG Pasteur cells, using previously described methods (Belley et al., 2004).
  • the empty pMV306 vector was also included as a control.
  • Transformants were grown at 37 0 C on Middlebrook 7H10 agar supplemented with 10% ADC [albumin (bovine fraction V), dextrose and catalase; BD/BBL media] and kanamycin (25 mg ml-1 ). Complementation was PCR-confirmed by amplifying the sigK gene with primers specific for the regions of pMV306 flanking the sigK insert and these amplicons were sequence-confirmed for all transformants.
  • BCG strains were cultivated as surface pellicles on liquid synthetic Sauton medium for 3 weeks at 37 0 C.
  • the bacteria were washed and disrupted by a bead beater to yield a cellular extract and the culture medium was filtered to remove residual bacteria and concentrated by ammonium sulphate precipitation at 80% saturation.
  • the antigens were separated under reducing conditions by horizontal SDSPAGE in precast 8-18% gradient Excel gel using a Multiphor Il unit 2117 (Amersham Pharmacia). After separation, the proteins were transferred to a nitrocellulose membrane (pore size, 0.2 mm) by diffusion blotting (Olsen and Wiker, 1998) and the gel was stained with CBB. The membranes were blocked with PBS containing 2% bovine serum albumin (BSA) and 1% gelatin and incubated with antibodies overnight.
  • BSA bovine serum albumin
  • Bound antibodies were recognized by horseradish peroxidase (HRP)-labelled anti- rabbit or anti-mouse Ig.
  • HRP horseradish peroxidase
  • 3,3-diaminobenzidine was added to visualize the bound antibodies.
  • Cultures were grown at 37°C in 7H9 with 10% ADC, supplemented with kanamycin (25 mg ml-1 ) for 7 days. The cultures were then centrifuged and the supernatant was filtered with a 0.22 mm membrane filter and concentrated with an Amicon Ultra-15 Centrifugal Filter Unit, 10 000 MWCO. Cell pellets were frozen and whole-cell lysates were prepared by resuspending the cell pellet in 100 ml of PBS and boiling for 20 min. The culture filtrate proteins (CFP) were precipitated by the following protocol: 1 volume of sample was mixed with 3 volumes of methanol, 1 volume of chloroform, 4 volumes of water. Samples were centrifuged at max speed (-13 200 rpm) for 1 min.
  • Mouse monoclonal antibodies 1-5C (anti-MPB70) and MBS43 (anti-MPB83) (Wiker et a/., 1998) were used at a dilution of 1/500 and the HRP-conjugated anti-mouse antibody was used at a dilution of 1/10 000. Protein bands were detected using ECL PlusTM Western Blotting Detection Reagents (Amersham).
  • RvO445c-RvO449c A second region that showed consistent downregulation in the low-producing strains was RvO445c-RvO449c.
  • RvO445c sigK
  • tuberculosis strains H37Rv, 210 and CDC1551 have the adenine residue.
  • the start codon of sigK is the predominant start codon sequence AUG, while in BCG Pasteur there is a G ⁇ A mutation at the third nucleotide, resulting in an altered AUA start codon.
  • the AUG was observed in all members of the M. tuberculosis complex except for the eight low-producing BCG strains obtained after 1927 in which the altered AUA start codon was observed (Table 2).
  • codon AUA has been identified as a functional start codon in Escherichia coli, Bacillus subtilis and Salmonella spp.
  • levels of translation are substantially reduced with this codon compared with the conventional start codon AUG (Romero and Garcia, 1991 ; Sussman et al., 1996). Because the start codon mutation correlated precisely with the BCG strains having decreased sigK and mpb83/mpb 70 expression, the functional consequence of the sigK mutation was examined.
  • BCG Pasteur::pPAST and BCG Pasteur::pMV306 levels of mRNA were comparable to those previously demonstrated in low-producing strains of BCG.
  • BCG Pasteur::pH37Rv, BCG Pasteur::pRUSS and BCG Pasteur::pBIRK manifested highly increased transcription levels for mpb70 and mpb83, comparable to the levels observed with high-producing strains of BCG ( Figure 3B).
  • the same results were obtained with a second clone of the same strains (data not shown).
  • MPB70 and MPB83 exhibit striking amino acid sequence homology (Hewinson et al., 1996) and both are exported, but localize differently.
  • the single form of MPB70 is secreted into culture media while MPB83 is present in two forms, a 26 kDa lipoprotein which remains associated with the mycobacterial cell wall and a 23 kDa form which is found in the culture media (Harboe et al., 1998).
  • the structure of MPB70 has recently been solved and superimposition of MPB83 on the MPB70 structure confirmed the overall homology of the antigens (Carr et al., 2003).
  • MBP70 and MPB83 have been developed as candidates for novel TB vaccine development (Chambers et al., 2000; Morris et al., 2000; Al Attiyah ef al., 2003; Tollefsen ef al., 2003; Xue ef al., 2004).
  • extracellular antigens have consistently been implicated in the induction of a protective immune response against M.
  • tuberculosis H37Rv genome tuberculosis H37Rv genome.
  • these regulatory elements mediate responses to changing external conditions (Manganelli et al., 2001 ; 2004b; Ando et al., 2003; Hu et al., 2004), with a common feature being their control over relatively small regulons (Bashyam and Hasnain, 2004).
  • the regulon of sigC has been estimated to contain 13, 14 and 18 genes, in exponential, early and late stationary phase growth, respectively (Sun et al., 2004), consistent with present observations of two regions, comprising 13 genes, being consistently upregulated in wild-type s/gK-complemented strains.
  • tuberculosis H37Rv and BCG Pasteur and BCG Pasteur is now shown to be functionally deficient in this regulon, therefore the impact of mutations in these genes may have been minimized.
  • An epidemiologic study of M. tuberculosis isolates in San Francisco used genomic hybridization studies to determine deletions in strains that had successfully caused TB, thereby generating a list of genes that are apparently nonessential for disease (Tsolaki et al., 2004). Of 224 genes disrupted in at least one clinical isolate, none of the genes implicated in the sigK nor the mpb83/70 regions is featured, suggesting that loss of these genes may be detrimental to disease causation.
  • s/ ' gK-regulated genes are supported by transcriptome analysis of M. tuberculosis during intracellular conditions, where mpblO and mpb83 figure among the most highly induced genes, across time points and in both activated and non-activated macrophages (Schnappinger et al., 2003). Additionally, in a microarray-based study of M. tuberculosis expression during murine infection, sigK and mpt53 were among those genes noted as significantly dysregulated in vivo (Talaat et al., 2004). Together, these results point to a potential role of the sigK regulon in the pathogenesis of TB that merits further attention.
  • MPB70 and MPB83 are also differentially produced by M. tuberculosis and M. bovis. Although these organisms have identical, wild-type AUG sigK start codons, in vitro expression is low (although inducible) in M. tuberculosis and constitutively high in M. bovis (Wiker et al., 1996). The constitutive in vitro production of MBP70 and MPB83 observed in M. bovis may therefore stem from unregulated activity of SigK.
  • Activity of an ECF sigma can be mediated by a second protein, the anti-sigma factor, that functions post- translationally as a negative regulator to prevent constitutive expression of the target regulon (Helmann, 2002; Manganelli et al., 2004a).
  • the sigma/anti-sigma pair are usually adjacent and co-transcribed genes; for instance, in M. tuberculosis, RshA (Rv3221A) is the anti-sigma factor for SigH (Rv3223c) while UsfX ⁇ Rv3287c) is the anti-sigma factor for SigF (Rv3286c) (Beaucher et al., 2002; Song et al., 2003).
  • RvO444c may encode the anti-sigma factor for sigK (RvO445c), and by extension, mutations in RvO444c might result in unregulated expression of sigK.
  • RvO445c anti-sigma factor for sigK
  • mutations in RvO444c might result in unregulated expression of sigK.
  • Ongoing investigations are pursuing this possibility, based on the presence of two non-synomyous SNPs in RvO444c restricted to M. tuberculosis complex species presenting constitutively high MPB70 production (data not shown).
  • Example 1 IMMUNIZATION EXPERIMENTS WITH GUINEA PIGS
  • Hartley guinea pigs were vaccinated with 10 3 CFU of recombinant BCG. Guinea pigs were rested for 10 weeks and then infected with a low dose aerosol of M. tuberculosis H37Rv. Viable count was performed at day 30 post challenge on 5 guinea pigs per group ( Figure 5).
  • mice The protocol used for mice was similar to that used for the guinea pigs described in Example 1. Vaccination was with 10 6 bacteria injected sub- cutaneously. The mice were challenged with 10 4 of Mycobacterium tuberculosis 10 weeks later. Results appeared 4 and 8 weeks after challenge (Figure 6).
  • the vaccine was once again 10 6 bacteria injected sub-cutaneously. Spleens were harvested 28 days later, splenocytes were isolated and plated in tissue culture plates, then stimulated with either nothing (control), PHA,
  • BCG Pasteur T A Table 3 Genes whose expression was changed upon complementation of BCG Pasteur with wild-type sigK.
  • BCG Pasteur :pSIGK-BIRK vs.
  • BCG Pasteur :: pSIGK-Rv vs.
  • MPT83 a seroreactive antigen of Mycobacterium tuberculosis with homology to MPT70. Scand J Immunol 43: 490- 499.
  • RNA encoding the MPT83 antigen induces protective immune responses against Mycobacterium tuberculosis infection. Infect lmmun 72: 6324-6329.

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Abstract

L'invention concerne un vaccin contre la tuberculose (TB) amélioré et sa méthode de fabrication. L'invention concerne également une méthode pour déterminer la puissance de souches de TB. Des souches de bacilles de Calmette-Guerin (BCG), Mycobacterium bovis, sont génétiquement et phénotypiquement hétérogènes. L'expression des protéines antigènes MPB70 et MPB83 est connue pour varier considérablement dans les souches BCG; toutefois, la raison pour cette différence phénotypique est restée inconnue. Comme l'histoire de la dissémination des souches BCG est connue, il est possible de déterminer précisément la chronologie des changements génétiques spécifiques des souches BCG (Behr et Small, 1999). Un certain nombre de ces mutations affectent des gènes de régulation putatifs (Behr et al., 1999; Brosch et al ., 2000; Spreadbury et al ., 2005), de sorte qu'une hypothèse a été lancée selon laquelle il était probable qu'un gène de régulation soit responsable pour la production variable de MPB70 et de MPB83. La production de MPB70 et de MPB83 dans un ensemble de souches BCG a par conséquent été déterminée de sorte à attribuer la chronologie de ce changement phénotypique et à guider des études vers l'identification de la mutation possible. Il est intéressant de constater que les données impliquent une mutation du codon de départ dans le facteur K de M. tuberculosis sigma (RvO445c ou sigK) et dévoilent une liaison hautement spécifique entre sigK et l'expression de MPB70 et de MPB83.
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WO2012140068A1 (fr) * 2011-04-11 2012-10-18 Alarum Development Ltd. Nouveaux vaccins destinés à la prévention et au traitement de la tuberculose
EP2754452A1 (fr) 2013-01-09 2014-07-16 LIONEX Diagnostics and Therapeutics GmbH SigC pour la modulation de réponses immunitaires
CN110506108A (zh) * 2017-04-07 2019-11-26 成都永安制药有限公司 过表达phoP-phoR的重组BCG

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WO2012140068A1 (fr) * 2011-04-11 2012-10-18 Alarum Development Ltd. Nouveaux vaccins destinés à la prévention et au traitement de la tuberculose
US10478482B2 (en) 2011-04-11 2019-11-19 Alarum Development Ltd Vaccines for prevention and treatment of tuberculosis
EP2754452A1 (fr) 2013-01-09 2014-07-16 LIONEX Diagnostics and Therapeutics GmbH SigC pour la modulation de réponses immunitaires
CN110506108A (zh) * 2017-04-07 2019-11-26 成都永安制药有限公司 过表达phoP-phoR的重组BCG
CN110506108B (zh) * 2017-04-07 2023-10-24 成都安永鼎业生物技术有限公司 过表达phoP-phoR的重组BCG

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