WO2008043180A1 - Immunostimulatory composition comprising recombinant bcg mycobacterium expressing mammalian cysteine protease - Google Patents

Immunostimulatory composition comprising recombinant bcg mycobacterium expressing mammalian cysteine protease Download PDF

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WO2008043180A1
WO2008043180A1 PCT/CA2007/001818 CA2007001818W WO2008043180A1 WO 2008043180 A1 WO2008043180 A1 WO 2008043180A1 CA 2007001818 W CA2007001818 W CA 2007001818W WO 2008043180 A1 WO2008043180 A1 WO 2008043180A1
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bcg
cathepsin
cells
nucleic acid
acid molecule
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PCT/CA2007/001818
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French (fr)
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Zakaria Hmama
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Zakaria Hmama
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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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • C12Y304/22027—Cathepsin S (3.4.22.27)
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to immunogenic compositions that derive from recombinant strains of Mycobacteria. More specifically, the present invention relates to immunogenic compositions of Mycobacterium bovis Bacille Calmette-Guerin (BCG) that express and secrete a mammalian cysteine protease.
  • BCG Bacille Calmette-Guerin
  • Tuberculosis is primarily caused by infection with Mycobacterium tuberculosis and remains as a leading cause of mortality from bacterial infection; a third of the world's population is currently infected and 2-3 million deaths occur each year.
  • M. tuberculosis infections are increasing, largely due to two lethal developments: the association of TB with HIV-infected individuals and the emergence of multidrug-resistant strains of M. tuberculosis (MDR-TB).
  • the current standard chemotherapy for TB involves a 6-month treatment program and a cocktail of drugs: an initial 2-month treatment with 4 drugs (isoniazid (INH), rifampin (RIF), pyrazinamide and ethambutol) followed by an additional 4- month treatment with INH and RIF.
  • the inadequacies of this chemotherapy include its toxicity, poor patient compliance with the lengthy treatment, and its ineffectiveness against MDR-strains.
  • Chemotherapy against MDR-TB involves more toxic drugs, may last up to 2 years, and is expensive, with the additional complication of even poorer patient compliance.
  • BCG Bacillus Calmette-Guerin
  • M. bovis mycobacteria
  • BCG Bacillus Calmette-Guerin
  • the vaccine originated from a virulent isolate of M. bovis, which causes TB in cows, and was obtained after undefined number of subcultures over a period of ⁇ 10 years leading to attenuation of the bacterium.
  • BCG was first administered to humans in 1921.
  • BCG is still widely used to provide protection against childhood forms of TB meningitis and military TB, but has shown inconsistent efficacy against pulmonary TB in adults (ter Dam 1984 Adv.Tuberc.Res 21 :79-106; Fine 1995. Lancet 346: 1339-1345).
  • the reasons behind the lack of protection are not completely understood but may relate to
  • DM VAN/253729-16266/67491962 prior exposure to environmental mycobacteria, a waning immune response after vaccination or antigenic differences that exist between BCG and M. tuberculosis.
  • Mycobacteria are adept intracellular pathogens that employ a number of different means to evade the host immune response.
  • An example of these virulence strategies is the ability of the bacteria to modulate the presentation of mycobacterial - derived antigens to the T lymphocyte population of the host.
  • DM VAN/253729-16266/67491962 Compositions that improve the level of antigen presentation and increase the immunogenicity of vaccines directed against mycobacteria, particularly M. tuberculosis are needed.
  • aspects of the present invention provide vectors comprising nucleotides that encode a protease enzyme.
  • the present invention also provides recombinant bacteria that comprise these vectors and secrete a protease enzyme.
  • compositions that stimulate an immune response by increasing the level of antigen presentation following administration to a mammalian host.
  • compositions described herein relate to strains of mycobacteria that stimulate antigen presentation by the major histocompatibility complex class II (MHC-II) molecules found on the surface of the antigen presenting cells (APC) of the host.
  • MHC-II major histocompatibility complex class II
  • a recombinant strain of mycobacteria capable of expressing a mammalian cysteine protease.
  • the recombinant strain of mycobacteria may be further engineered to attenuate the virulence of the bacteria.
  • the recombinant mycobacteria may be M. bovis, M. tuberculosis or M. bovis, strain BCG.
  • the mammalian cysteine protease may further be secreted by the mycobacteria.
  • the mammalian cysteine protease may be cathepsin S (SEQ ID: 9).
  • expression of the mammalian cysteine protease is under the control of a constitutive or an inducible bacterial promoter.
  • the cathepsin S coding region consists only of those nucleotides that code for the mature enzyme (SEQ ID NO: 1).
  • the cathepsin S coding region is in-frame with a nucleotide region that encodes a signal peptide (SEQ ID NO: 2).
  • the recombinant strain of mycobacteria encodes a cathepsin S coding region and at least one other nucleotide sequence that encodes an immunogenic polypeptide that is derived from mycobacteria.
  • the immunogenic polypeptide may originate from M. bovis, M. tuberculosis or M. bovis, strain BCG.
  • the recombinant strain of mycobacteria encodes a cathepsin S coding region and at least one other nucleotide sequence that encodes a mammalian cytokine.
  • Fig. 1 shows the human (SEQ ID NO: 9) and murine (SEQ ID NO: 11) amino acid sequences for the cathepsin S precursor protein. The site of cleavage for the release of the mature active enzyme from the full-length precursor protein is also indicated;
  • Fig. 2 depicts the pSMT-CatS vector.
  • the mycobacterial promoter from the hsp60 gene (Mulder M.A. 1997 Tuber Lung Dis 78:211-23), is used to drive transcription of the transgene.
  • the transgene comprises a ribosomal binding site and signal peptide (SEQ ID NO: 3) from the fbpB gene (SS-aAg85) and a sequence encoding the mature human cathepsin S protein (Active huCatS) (SEQ ID NO: 6);
  • Fig. 3 illustrates growth curves for wild type BCG (non-transformed; empty circle), BCG transformed with an empty pSMT-3 vector (pSMT3; solid square) and a recombinant BCG strain expressing the truncated human cathepsin S polypeptide (BCG-hcs); solid circle);
  • Fig. 4 depicts cathepsin S enzyme activity in culture supernatant and bacterial cell pellets from the parental wild type (BCG), BCG transformed with the pSMT3 vector (pSMT3) or the rBCG-CatS strain (pSMT3-hcs);
  • Fig. 5 depicts cathepsin S enzyme activity from THP-I macrophage and bone- marrow derived macrophage (BMM0) lysates that were left uninfected or infected with either BCG or the recombinant BCG strain.
  • THP-I cells were infected with the rBCG-CatS strain expressing the human cathepsin S (rBCG-hcs) while BMM0 were infected with the recombinant BCG strain expressing the murine cathepsin S (rBCG- mcs).
  • Lane 1 control non-treated cells
  • Lane 2 stimulation with IFN- ⁇ .
  • Lane 3 infection with BCG (OfN, then stimulation with IFN- ⁇ .)
  • Lane 4 infection with rBCG-hcs (THP- 1 ) or rBCG-mcs (BMM0);
  • Fig. 6 shows a western blot depicting the expression of cathepsin S from macrophages cell lysates.
  • THP-I cells were left uninfected or infected with either wild type BCG or rBCG-CatS.
  • Lane 1 control non-treated cells
  • Lane 2 stimulation with IFN- ⁇ . (200 U/ml, 24 h)
  • Lane 3 infection with BCG (O/N, then stimulation with IFN- ⁇ )
  • Lane 4 infection with rBCG-catS and no stimulation with IFN- ⁇ ;
  • Fig. 7 depicts IL-2 production from the DBl hybridoma T cells in response to co-incubation with THP-I macrophages that were either i) untreated ii) treated with IFN- ⁇ only, iii) treated with IFN- ⁇ and infected with BCG (WT BCG) or iv) treated with IFN- ⁇ and infected with rBCG-hcs;
  • Fig. 8 shows the maturation of THP-I -derived DC following exposure to apoptotic vesicles.
  • THP-I -derived DC were either left untreated (control) or exposed to cell-free culture medium from macrophages infected with either rBCG-Cats (rBCGhcs) or wild type BCG (BCG).
  • the cells were stained with FITC conjugated antibodies and analyzed by FACS.
  • the mean fluorescence intensity (MFI) indices were deducted from fluorescence histograms.
  • the L243 mAb recognizes the mature THP-I -derived DC following exposure to apoptotic vesicles.
  • the LN2 mAB recognizes the invariant chain, the HBl 5.
  • e mAb recognizes the DC maturation marker B7.1 (CD83) and the IT2.2 mAb recognizes the B7.2 (CD86) marker of DC maturation; and
  • Fig. 9 illustrates FACS analysis to determine the level of apoptosis within RAW 264.7 macrophage monolayers infected the wild type (BCG) or the recombinant strain of BCG expressing the murine cathepsin S (rBCG-mcs).
  • the negative control (control) illustrates the level of apoptosis in uninfected RAW 264.7 macrophages while macrophages treated with IFN- ⁇ illustrates the positive control for apoptosis.
  • Various embodiments of the present invention are based on the surprising discovery that BCG interferes with antigen processing and antigen presentation by the antigen presenting cells (APC) of the host immune system.
  • APC antigen presenting cells
  • Expression of cathepsin S by recombinant BCG may enhance antigen presentation to host lymphocytes and may elicit a protective T cell response following administration to a subject such a recombinant BCG strain.
  • vector refers to a polynucleotide composition used for introducing exogenous or endogenous polynucleotide sequences into a host cell.
  • a host cell may be eukaryotic or prokaryotic.
  • a vector comprises a nucleotide sequence which may encode one or more polypeptide molecules. Plasmids, cosmids, viruses and bacteriophages, in a natural state or which have undergone recombinant engineering, are non-limiting examples of commonly used vectors to provide recombinant vectors comprising at least one desired isolated polynucleotide molecule.
  • vacun refers to a composition that, upon administration to a subject, stimulates an adaptive immune response, but is incapable of causing a severe infection.
  • a vaccine may comprise, for example, a live attenuated or killed pathogen, or a preparation of a pathogen-specific structure, or other similar
  • DM VAN/253729-16266/6749196 2 agent an adjuvant, excipient or other additives.
  • an agent for stimulation of the immune response may be encapsulated in a liposome or other microparticle.
  • the pathogen may be a virus, eukaryotic parasite, or a bacterium.
  • Adjuvants, excipients, and other additives for inclusion in a composition for use in a vaccine and methods of preparing such compositions will be known to those of skill in the art.
  • a vaccine may be administered in accordance with two general strategies - before exposure of the subject to the pathogen (pre-exposure or prophylactic vaccination) or after exposure of the subject to the pathogen (post-exposure or therapeutic vaccination).
  • a therapeutic vaccine may be administered alone or in combination with a drug therapy regimen.
  • Prophylactic or therapeutic vaccines may be administered as a single-dose schedule or as a multiple-dose schedule.
  • a course of vaccination may include at least two doses followed by other doses at subsequent time intervals required to maintain or reinforce the immune response.
  • each does may be administered about 2-4 months apart.
  • the vaccinated subject may receive additional doses months or years following the dose schedule, or upon exposure to the pathogen.
  • the necessary dosage, timing of doses and quantity of doses may be dependent on the subject (for example, a child or an adult) and will be familiar to a skilled individual, frequently a medical practitioner.
  • Methods of administration of a vaccine include, for example, intramuscular, subcutaneous or intraperitoneal injection, oral, inhalation, suppository, transdermal routes and the like.
  • MHC protein is encoded by the major histocompatibility complex of a subject, with a key role in antigen presentation for the immune system. MHC proteins may be found on several cell types, including APCs such as macrophages or dendritic cells, or other cells found in a mammal. Epitopes associated with MHC Class I may range from about 8-11 amino acids in length, while epitopes associated MHC Class II may be longer, ranging from about 9-25 amino acids in length. MHC Class I molecules present peptides generated in the cytosol to CD8 T cells, and the MHC Class II molecules present peptides degraded in cellular vesicles to CD4 T cells. The cellular processes involved in MHC epitope processing and presentation and the
  • DM VAN/253729-16266/67491962 interaction with other cells is well-described in the art, in various texts and references. See, for example, Roitt's Essential Immunology. IM Roitt, PJ Delves. Oxford, Blackwell Science Publishers, 2001.
  • an “antigen” is a substance which may induce a specific immune response.
  • Immunogen is a type of antigen that provokes an immune response when introduced into a subject. Immunogens may have varying ability to stimulate an immune reaction, depending on the commonalities or differences with epitopes found in the subject, size of the immunogen, chemical composition, and the like.
  • An "adaptive immune response” is an immune response involving a lymphocyte.
  • B-cells and T-cells are examples of the two major types of lymphocytes.
  • the cellular processes involved in stimulation of B-cells and T-cells are well described in the art, in various texts and references. See, for example, Roitt's Essential Immunology. IM Roitt, PJ Delves. Oxford, Blackwell Science Publishers 2001.
  • 'peptide', 'polypeptide' and protein' may be used interchangeably, and refer to a macromolecule comprised of at least two amino acid residues covalently linked by peptide bonds or modified peptide bonds, for example peptide isosteres (modified peptide bonds) that may provide additional desired properties to the peptide, such as increased half-life.
  • a peptide may comprise at least two amino acids.
  • the amino acids comprising a peptide or protein described herein may also be modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art.
  • Modifications can occur anywhere in a peptide, including, for example, the peptide backbone, the amino acid side-chains and the amino or carboxyl termini, and the like. It will be understood by one of skill in the art that the same type of modification may be present in the same or varying degrees at several sites in a given peptide.
  • M. tuberculosis exerts several pro-pathogen survival influences on the host cell that it infects, primarily macrophages and other antigen-presenting cells. These include the inhibition of phagosomal maturation which impedes the accumulation
  • the process of phagosomal maturation is also essential for loading microbial-derived peptides onto mature MHC-II molecules that are subsequently exported to the APC surface.
  • the presentation of pathogen-derived peptide antigens in complex with MHC-II molecules is required for the stimulation of host CD4+ T cells. Since pathogenic mycobacteria subvert this maturation process, there is a decreased ability for the adaptive immune system to respond to infection or vaccination (reviewed in Hestvik et al 2005. FEMS Microbiology Reviews 29: 1041-1050; Skeiky and Sadoff 2006. Nat. Rev. Microbiol 4:469-476).
  • M. tuberculosis also interferes with host apoptotic signaling pathways as a means to maintain its intracellular niche.
  • Previous studies have shown that avirulent strains of M. tuberculosis induce higher levels of apoptosis than virulent M tuberculosis (Keane J. et al. 2000 J Immunol. 15;164(4):2016-20).
  • Apoptotic vesicles from infected cells can be internalized by dendritic cells.
  • the DCs process and display the microbial antigens via MHC class I and CDl which allows for CD8 T cell stimulation (Winau F et al. 2004 Cell Microbiol. 6(7):599-607).
  • the inhibition of macrophage apoptosis by virulent M. tuberculosis may impede antigen presentation by bystander APCs.
  • Cathepsin S is a protease that is mainly expressed within the lysosomal compartments of antigen presenting cells (APC) and its expression is inducible in the presence of interferon- ⁇ (IFN- ⁇ ). Cathepsin S participates in the proteolytic degradation of the invariant chain (Ii) which is associated with immature MHC-II molecules. Thus, cathepsin S activity is required for the maturation of MHC-II molecules and the subsequent peptide loading that precedes antigen presentation at the APC surface (Hsieh et al 2002. J. Immunol 168:2618-2625; Bania et al. 2003. Proc. Natl. Acad. Sci. USA 100:6664-6669). Sendide et al. (Sendide et al 2005. J. Immunol 175: 5324-5332) illustrated that cathepsin S enzyme activity and cathepsin S gene expression are reduced in BCG-infected cells.
  • compositions with enhanced antigen presentation Cathepsin S is a cysteine protease that plays a key role in the normal processing and maturation of MHC class II molecules in APCs. Pathogenic
  • the present invention comprises a recombinant BCG strain that expresses and secretes the active cathepsin S enzyme during infection of APCs.
  • the cathepsin S coding region was restricted to those sequences that encode the mature enzyme rather than the full-length preprotein.
  • the cathepsin S coding region was fused to a bacterial signal peptide that is recognized by mycobacteria to generate the cathepsin S transgene.
  • the presence of the signal peptide ensures the active secretion of the mammalian protease from the bacteria.
  • Transcription of the transgene was regulated by a mycobacterial promoter that derived from the hsp60 gene from BCG.
  • the hsp60 promoter sequence is constitutively active, thus allowing for the continuous expression of the transgene.
  • the recombinant expression of the transgene by BCG resulted in several desirable characteristics. Macrophage infection with the recombinant bacteria led to enhanced maturation of MHC-II molecules due to the increased degradation of the invariant chain. Furthermore, the expression of cathepsin S by the recombinant BCG strain increased the presentation of mycobacterial-derived antigens by APCs. The increased presentation of bacterial antigens in complex with mature MHC-II molecules may lead to stimulation of T lymphocytes that are specific for bacterial epitopes. This stimulation of T lymphocytes and the subsequent cytokine secretion may influence the immune response to vaccination, and may further allow development of protective immunity.
  • cathepsin S by the recombinant BCG induced the apoptotic response in infected macrophages.
  • Apoptosis of mycobacteria-infected macrophages may result in a decline in the number of viable bacteria and promote the cross- presentation of mycobacterial antigens by bystander APCs.
  • Apoptotic vesicles from infected macrophages can be engulfed by other APCs leading to the processing and presentation of microbial antigens to T lymphocytes.
  • mycobacterial antigens may be presented in context with MHC-I molecules leading to the stimulation of CD8 T cells.
  • expression of the mature cathepsin S during APC infection by mycobacteria may overcome the microbial-induced block in antigen presentation that occurs with the current BCG vaccine.
  • a cytokine is a protein that affects the behaviour of other cells by acting on specific, cell-surface cytokine receptors. Cytokines made by lymphocytes are often called interleukins. Examples of proinflammatory cytokines are known in the art and include IL-2, IL-18, IFN- ⁇ , GM-CSF, IL- 12 and the like.
  • the BCG mycobacterial vaccine strain comprises a vector for expression of both cathepsin S and at least one mammalian proinflammatory cytokine. Such expression may improve the antigenicity and/or immunogenicity of recombinant BCG by enhancing the presentation of microbial antigens.
  • the BCG mycobacterial vaccine strain comprises a vector for expression of both cathepsin S and at least one other immunodominant mycobacterial antigen. Such expression may improve the may improve the antigenicity and/or immunogenicity of recombinant BCG by enhancing the presentation of microbial antigens.
  • the transcription of the cathepsin S transgene may be under the control of a constitutive bacterial promoter.
  • the strength of a promoter is dictated by the interaction between the RNA polymerase and the promoter sequence.
  • constitutive mycobacterial promoters include but are not limited to the mycobacterial hsp60 and hsp70 promoters.
  • the transcription of the cathepsin S transgene may be under the control of an inducible promoter.
  • DM VAN/253729-16266/6749196.2 inducible promoter typically requires the participation of alternate sigma factors to induce transcription.
  • the expression of the alternate sigma factors may be limited to specific environmental conditions. Examples of such environmental conditions include but are not limited to iron depletion, reduced oxygen tension or the presence of other environmental stresses known in the art.
  • Examples of inducible mycobacterial promoters include but are not limited to the mycobacterial icl promoter and the mycobacterial acetamidase promoter.
  • RPMI 1640 Hank's buffered salt solution (HBSS), phorbol-12-myristate-13- acetate (PMA), protease inhibitor mixture, PMSF, and trypsin- EDTA were obtained from Sigma- Aldrich.
  • Monoclonal anti-Cathepsin S was from Calbiochem
  • anti-Ii mAb (clone LN2) was from BD Pharmingen.
  • Anti-human HLA-DR mAb (clone L243) was from BD Pharmingen.
  • Anti-B7.1 mAb (clone HBl 5.e) was from BD Pharmingen.
  • Anti-B7.2 mAb (clone IT.2) was from BD Pharmingen.
  • Anti-FITC- conjugated F(ab')2 goat anti-mouse IgG and HRP-conjugated goat anti-mouse IgG were from Sigma-Aldrich.
  • Human recombinant IFN- ⁇ was from Genentech (South San Francisco, CA).
  • Cathepsin S inhibitor Z-Phe-Leu-COCHO and the recombinant active form of human Cathepsin S were from Calbiochem.
  • Cathepsin S substrate Z- Val-Val-Arg-AMC was from Bachem Bioscience.
  • Human IL-2 ELISA kit was from eBioscience.
  • M. bovis BCG "Pasteur strain 1173P2" was grown in Middlebrook 7H9 broth (Difco) supplemented with 10% (v/v) OADC (oleic acid, albumin and dextrose solution; Difco) and 0.05% (v/v) Tween 80 (Sigma-Aldrich) (7H9-OADC) at 37°C on a rotating platform (50 rpm).
  • Mycobacteria were also cultured on 7H10 agar supplemented with OADC (BD Biosciences). To monitor bacterial growth in vitro, the turbidity of mycobacterial broth cultures was determined by measuring the optical density at 600 nm (OD600) over time. For infection experiments, bacteria were cultured in 7H9-OADC until an OD600 of 0.5. Bacteria were harvested by centrifugation and pellets were suspended in complete media plus 10% glycerol.
  • DM VAN/253729-16266/6749196 2 Mycobacterial cultures were stored in aliquot ( ⁇ 5 x 10 8 /vial) at -70 0 C. Before infection, bacteria were grown 48 h in 7H9-OADC and opsonized as follows: 10 9 mycobacteria were suspended in 1 ml of RPMI 1640 containing 50% human serum (AB+ and PPD-) and rocked for 30 min at 37 0 C. Bacteria were then pelleted and suspended in 1 ml of RPMI 1640 and clumps were disrupted by multiple passages through a 25-gauge needle.
  • cDNA was synthesized using Superscript First-Strand Synthesis system (Invitrogen) with the following primers; CatS-Forward (5'- ATCTAAGCTTTTGCCTGATTCTGTC-3 ' ; SEQ ID NO:4) which contains a HwJIII adaptor (underlined) and CatS-Reverse (5'- TTGGGATCGATCTAGATTTCTGGGTA-S ' ; SEQ ID NO : 5) which contains a CM adaptor (underlined).
  • the amplicon was digested with Hindlll and Clal (SEQ ID NO: 6) and cloned into the E. coli-Mycobacterium shuttle vector pSMT-3 (Herrmann JL et al. 1996 EMBO J. 15(14):3547-54) that had been previously digested with Hindlll and Clal to generate the pSMT-3-CatS vector.
  • the signal peptide and ribosomal binding site of the fbpB gene (SEQ ID NO:
  • the PCR product was cloned into pSMT-3-CatS vector that had been previously digested with BamHl and Pstl to generate pSMT- fbpB signal peptide-CatS (pSMT-CatS).
  • the pSMT-CatS vector comprising the human cathepsin S coding region was electroporated into BCG and transformants were selected onto 7H10 agar medium supplemented with 100 ug/ml of hygromycin B (Invitrogen). The presence of the
  • DM VAN/253729-16266/67491962 transgene comprising the signal peptide from the fbpB and the human cathepsin S coding regions was confirmed by PCR and restriction endonuclease digestion of the recombinant BCG strain, rBCG-hcs.
  • the amplicon was digested with Pstl and Clal and cloned into the E. coli-Mycobacterium shuttle vector pSMT-3 (Herrmann JL et al. 1996 EMBO J. 15(14):3547-54) that had been previously digested with Pstl and Clal to generate the pSMT-mCatS vector.
  • the signal peptide and ribosomal binding site of the fbpB gene (SEQ ID NO:
  • the PCR product was digested using Pstl and BamHl and cloned into pSMT-3 that had been previously digested with the same enzymes, to generate pSMT-mcs.
  • the pSMT-mcs vector comprising the murine cathepsin S coding region fused to the signal peptide was electroporated into BCG and transformants were selected onto 7H10 agar medium supplemented with 100 ug/ml of hygromycin B (Invitrogen). The presence of the transgene comprising the signal peptide from the fbpB and the murine cathepsin S coding regions was confirmed by PCR and restriction endonuclease digestion of the recombinant BCG strain, rBCG-mcs.
  • the monocytic cell line THP-I (ATCC TIB-202) was cultured in RPMI 1640 supplemented with 5% Fetal Calf Serum (FCS) (Invitrogen Life Technologies), L- glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 ⁇ g/ml).
  • FCS Fetal Calf Serum
  • L- glutamine 2 mM
  • penicillin 100 U/ml
  • streptomycin 100 ⁇ g/ml
  • THP-I cells were treated with PMA (10 ng/ml) for 24h followed by treatment with IL-4 (1000 units/ml) for 96 h.
  • Bone marrow cells from the femur and tibia of BALB/C mice were culture in 10-cm diameter cell culture dishes for 4-5 days at 37°C and 5% CO2 in RPMI 1640 medium supplemented with 10% heat-inactivated FCS (HyClone, Logan, Utah), 2 mM L-glutamine, 10 mM HEPES, 50 ⁇ M 2-ME, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin (all from Stemcell Technologies, Vancouver, BC) and containing 10% of L929 conditioned media (LCM) or 10 ng/ml M-CSF (R&D Systems, Minneapolis, MN) in order to differentiate bone marrow cells into macrophages.
  • FCS heat-inactivated FCS
  • 2-ME 100 U/ml penicillin
  • streptomycin all from Stemcell Technologies, Vancouver, BC
  • L929 conditioned media L929 conditioned media
  • 10 ng/ml M-CSF R&D Systems,
  • L929 cells (ATCC #CCL-1) were cultured in RPMI media plus 2 mM L- glutamine, 10 mM HEPES, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin and 10% FCS. LCM was collected from cells grown for 1 week and kept in 5 ml aliquots at -70 0 C).
  • RAW 264.7 cells (ATCC TIB-71 ) were cultured in 10-cm diameter cell culture dishes as recommended by the cell line supplier ATCC (Manassas, VA).
  • the medium was Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 10 mM HEPES, 50 ⁇ M 2-ME, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin and containing 1 % non-essential amino acids (complete media).
  • DMEM Dulbecco's modified Eagle's medium
  • Cathepsin S activity was measured using the fluorogenic substrate Z- VaI- VaI- Arg-NHMec as described Sendide et al. (Sendide et al 2005. J. Immunol 175: 5324- 5332).
  • Adherent THP-I cells were scraped into extraction buffer (0.01% Triton X-100 in 0.1 M potassium phosphate buffer containing 1 mM EDTA, pH 7.5) and frozen/thawed three times. Samples were sonicated for 5 s in a Sonic Dismembrator 60 (Fisher Scientific) in ice. Cell debris and membranes were removed by centrifugation at 12,000 x g for 30 min at 4 0 C.
  • Cathepsin S activities were calculated by reference to a standard curve using a range (20 to 20,000 U/ml) of active recombinant human cathepsin S. Reaction mixtures with bovine serum albumin instead of cell lysate were used as controls. Results were expressed after subtracting the fluorescence value corresponding to this control.
  • Control and infected cells were washed with HBSS and whole cell lysates were prepared in Nonidet P-40 lysis buffer (50 mM sodium acetate, 5 mM MgC12, 0.5% Nonidet P-40, pH 7.4) in the presence of protease inhibitors mixture and PMSF. Proteins (50 ⁇ g/lane) were separated by 12% SDS-PAGE then transferred to nitrocellulose membrane and probed with anti-cathepsin S mAb. To assess the amount of individual proteins in each sample, after detection of bound anti-cathepsin S mAb, membranes were stripped and reprobed with anti-actin Abs and developed by ECL as described (Hmama 1999, supra).
  • macrophage or dendritic cells were scraped with a rubber policeman and cells were collected in HBSS containing 0.1% NaN3 and 1% FCS (staining buffer). Cells were then labelled with anti-class II mAbs, anti-Ii mAbs, anti-B7.1 mAbs, anti-B7.2 mAbs or irrelevant isotype-matched IgG for 20 min. Cells were then washed twice with staining buffer and labelled with FITC -conjugated F(ab')2 goat anti-mouse IgG for 20 min.
  • RAW 264.7 cells were grown at ⁇ 5 x 10 6 cells in 10 ml complete medium in 10-cm culture plates and infected with mycobacteria at an MOI of 20:1 for 24h. Cells were washed 3 times with warm HANKS and flushed gently with cold HANKS to detach cells from plates without damaging cell membrane. Cells were stained 30 min on ice with propidium iodide (PI, Sigma P-4170) at 1 : 100 and FITC-AnnexinV (Calbiochem, PF032) at 1 :50 in HBSS 5% FCS. Cells were washed twice with HBSS 5% FCS and immediately analyzed for expression of dual, red (PI) and green (AnnexinV), fluorescence in a BD FACScalibur cytofluorometer.
  • PI propidium iodide
  • FITC-AnnexinV Calbiochem, PF032
  • DBl T hybridoma cells (Gehring et al. 2003 Infect Immun.; 71(8): 4487- 4497) were cultured in DMEM supplemented with 5% Fetal Calf Serum (FCS) (Invitrogen Life Technologies), L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 ⁇ g/ml).
  • FCS Fetal Calf Serum
  • L-glutamine 2 mM
  • penicillin 100 U/ml
  • streptomycin 100 ⁇ g/ml
  • DM VAN/253729-16266/6749196 2 allowed to adhere and differentiate in the presence of PMA (20 ng/ml) at 37°C in a humidified atmosphere of 5% CO2 for 24 h.
  • the differentiated THP-I cells were left untreated or infected with either BCG of rBCG-CatS overnight. The cells were then washed and incubated with 200 U of recombinant human IFN- ⁇ for 24 h. The cells were then fixed in 1% paraformaldehyde, followed by incubation with DBl T hybridoma cells (10 6 cells/well). Supernatants were harvested after 2Oh and the amount of IL-2 produced by T hybridoma cells was measured by ELISA specific for IL-2.
  • Hybridization was performed using Operon's Human Genome OpArray version 4 slides.
  • the slides were scanned using the GenePix ® Autoloader 200AL (GenePix is a registered trademark of Axon Instruments Inc., Union City, CA, USA) and data analysis was done with Imagene ® software (Imagene is a registered trademark of BioDiscovery Inc., Los Angeles CA, USA) and GenSpring version GX (Agilent Technologies) software.
  • the cathepsin S protein is synthesized as a 331 amino acid preprotein (SEQ ID NO: 9) and the mature enzyme is generated following cleavage between amino acids 114 and 115, (SEQ ID NO: 1) as depicted in Fig. 1.
  • a vector was constructed that encoded a mycobacterial promoter to control the expression of the human cathepsin S coding region that was fused to a mycobacterial signal peptide.
  • DM VAN/253729-16266/6749196.2 the human cathepsin S nucleotide coding region that encoded the amino acids 115- 331 was cloned in-frame with the coding region of the Ag85B signal peptide.
  • the resulting transgene was inserted into the pSMT3 shuttle vector which contained a mycobacterial Hsp60 promoter region to drive the transcription of the transgene.
  • the recombinant vector comprising the promoter and the transgene referred as pSMT- CatS (Fig. 2), was electroporated into BCG to generate the recombinant strain known as rBCG-CatS.
  • the in vitro growth curves of the parental wild type and rBCG-CatS were compared by measurement of the optical density of broth cultures. Analysis of the growth curves (Fig. 3) indicates that the wild type and recombinant strain have nearly identical growth characteristics in broth.
  • the enzyme activity of cathepsin S was measured in the culture supernatant and bacterial pellet for the rBCG-CatS and the wild type BCG.
  • the culture supernatant of the recombinant strain contained high levels of cathepsin S enzyme activity as compared to wild type BCG and minimal activity was detected in the bacterial pellets, indicating that the rBCG-CatS strain secretes the active cathepsin S enzyme during culture in vitro (Fig. 4).
  • the level of cathepsin S expression by the recombinant strain was also determined following macrophage infection.
  • Levels of cathepsin S enzyme activity and protein expressed were compared in macrophage cell lysates from THP-I monolayers that were either i) uninfected, ii) infected with BCG or iii) infected with rBCG-CatS.
  • cell lysates were collected from murine macrophage monolayers were either i) uninfected, ii) infected with BCG or iii) infected with rBCG-mcs.
  • Cathepsin S enzyme activity was significantly increased in the cell lysates from macrophages infected with the recombinant BCG (Fig. 5) indicating that the rBCG-CatS and rBCG-mcs strains actively secrete cathepsin S following macrophage infection.
  • the level of cathepsin S expression in the THP-I cell lysates was assessed by Western blot and showed that macrophages infected with rBCG-CatS have increased levels of cathepsin S protein as compared to BCG-infected macrophages (Fig. 6).
  • Macrophages infected with rBCG-CatS exhibit increased presentation of mycobacterial protein antigens via MHC-II
  • the stimulation of T lymphocytes was used to determine if the secretion of cathepsin S by rBCG-CatS would affect antigen processing and presentation by the APC.
  • the DBl T cell hybridoma specifically recognizes an epitope from the mycobacterial Ag85B protein in the context of MHC class II molecules on the APC surface.
  • the recognition of the peptide:MHC complex by the T cell receptor causes the release of IL-2 from the T lymphocyte which is used as a marker for T cell stimulation.
  • Macrophages infected with the rBCG-CatS strain showed an increased ability to induce IL-2 release from the T cells, whereas the BCG-infected macrophages were only modest inducers of IL-2 (Fig. 7). These results demonstrate that the recombinant expression of cathepsin S by BCG facilitated the presentation of mycobacterial antigens in complex with MHC-II to T lymphocytes.
  • Macrophages infected with rBCG-CatS show increased transcription of MHC class II genes
  • Microarray analysis was used to compare the transcriptional profiles of macrophages infected with either wild type BCG or the rBCG-CatS strain. Infection with the recombinant strain of BCG led to increased expression of genes that are known to be involved in phagosome maturation and apoptosis. In addition, macrophage infection with rBCG-CatS also resulted in the increased expression of MHC-II-related genes. The values represent fold changes in gene expression in macrophages infected wild type BCG relative to uninfected cells (BCG/control) and fold changes in gene expression of macrophages infected with rBCG-catS relative to macrophages infected with wild type BCG (rBCG- hcs/BCG).
  • a list of exemplary genes that were upregulated and are known to be involved in phagosome maturation, antigen presentation and apoptosis is provided in Table 1.
  • Table 1 A cluster of genes related to phagosome maturation, apoptosis and Ag presentation were significantly up-regulated in M0 infected with rBCG-hcs opposed to wild-type BCG. The later was rather inducing expression of genes related to apoptosis inhibition.
  • THP-I -derived immature dendritic cells were infected with either BCG or rBCG-CatS to determine whether the bacterial expression of cathepsin S would lead to an increase in the surface expression of MHC-II molecules.
  • the rBCG-CatS-infected dendritic cells had increased levels of MHC-II molecules and increased levels of other dendritic cell differentiation markers (B7.1 and B7.2) as observed by FACS analysis.
  • Macrophages infected with rBCG-CatS undergo apoptosis and the apoptotic blebs enable cross priming by dendritic cells
  • Macrophages were either i) left untreated; ii) stimulated with EFN- ⁇ for 24 h or; iii) infected overnight with either wild type BCG or rBCG-mcs.
  • the infected cells were stained with annexin V-FITC and PI and analyzed by FACS.
  • Fig. 9 murine macrophages infected with rBCG-mcs showed increased annexin V-staining, similar to levels achieved with IFN- ⁇ treatment.
  • THP-I -derived dendritic cells were either i) left untreated or ii) exposed to the cell-free overlying medium from macrophages that had been infected with either BCG or rBCG-CatS. As shown in Fig. 10, the exposure of immature dendritic cells to the apoptotic blebs derived from rBCG-CatS-infected macrophages was sufficient to induce the expression of MHC-II molecules and markers of dendritic cell maturation.

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Abstract

A nucleic acid molecule comprising a nucleotide sequence configured for controllable expression of a mammalian cysteine protease suitable for enhancing an immune response directed against Mycobacterium A recombinant Mycobacterium Bacille Calmette-Guerm (BCG) provided with the nucleic acid molecule and pharmaceutical compositions comprising the recombinant BCG are suitable for use in immunotherapy and in vaccinations against tuberculosis

Description

BCG-BASED IMMUNOSTIMULATORY COMPOSITION
TECHNICAL FIELD
The invention relates to immunogenic compositions that derive from recombinant strains of Mycobacteria. More specifically, the present invention relates to immunogenic compositions of Mycobacterium bovis Bacille Calmette-Guerin (BCG) that express and secrete a mammalian cysteine protease.
BACKGROUND ART
Tuberculosis (TB) is primarily caused by infection with Mycobacterium tuberculosis and remains as a leading cause of mortality from bacterial infection; a third of the world's population is currently infected and 2-3 million deaths occur each year. After years in decline, M. tuberculosis infections are increasing, largely due to two lethal developments: the association of TB with HIV-infected individuals and the emergence of multidrug-resistant strains of M. tuberculosis (MDR-TB).
The current standard chemotherapy for TB involves a 6-month treatment program and a cocktail of drugs: an initial 2-month treatment with 4 drugs (isoniazid (INH), rifampin (RIF), pyrazinamide and ethambutol) followed by an additional 4- month treatment with INH and RIF. The inadequacies of this chemotherapy include its toxicity, poor patient compliance with the lengthy treatment, and its ineffectiveness against MDR-strains. Chemotherapy against MDR-TB involves more toxic drugs, may last up to 2 years, and is expensive, with the additional complication of even poorer patient compliance.
The only vaccine against TB that is currently available is the live attenuated strain of mycobacteria known as BCG (Bacille Calmette-Guerin). The vaccine originated from a virulent isolate of M. bovis, which causes TB in cows, and was obtained after undefined number of subcultures over a period of ~ 10 years leading to attenuation of the bacterium. BCG was first administered to humans in 1921. BCG is still widely used to provide protection against childhood forms of TB meningitis and military TB, but has shown inconsistent efficacy against pulmonary TB in adults (ter Dam 1984 Adv.Tuberc.Res 21 :79-106; Fine 1995. Lancet 346: 1339-1345). The reasons behind the lack of protection are not completely understood but may relate to
DM VAN/253729-16266/67491962 prior exposure to environmental mycobacteria, a waning immune response after vaccination or antigenic differences that exist between BCG and M. tuberculosis.
Recent approaches have attempted to improve the immunogenicity of the BCG vaccine. Recombinant BCG strains that overexpress bacterial antigens are described in U.S. Patent 6,924,118 and U.S. Patent 6,471,967 which provides recombinant attenuated mycobacteria that express extracellular proteins derived from pathogenic mycobacteria. Examples of immunodominant antigens may include Ag85B, Ag85A, CFP-IO, and ESAT6 and may include proteins that are secreted by the bacteria or are localized to the bacterial cell surface. An alternate approach is outlined in U.S. Patent 5,591,632 which provides recombinant BCG strains that secrete mammalian cytokines to further stimulate an immune response. Other examples of recombinant strains of mycobacteria that express cytokines can be found in Murray PJ et al. 1996 Proc Natl Acad Sci U S A. Jan 23;93(2):934-9; Biet F et al.2002 Infect Immun. Dec;70(12):6549-57; Young SL et al. 2002 Int Immunol. Jul;14(7):793-800. Other approaches, as outlined in PCT application WO 03/089462, attempt to increase the survival and persistence of BCG within the host in order to induce a long term protective immunity.
Complications from the BCG vaccination have been reported in immunocompromised individuals such as HIV-infected children and adults. The inability of this patient population to mount an effective cell-mediated immune response can lead to the development of disseminated BCG disease. The development of severely attenuated strains of BCG or M. tuberculosis has been suggested as a means of providing a safe and effective vaccine for immunocompromised populations (Sambandamurthy VK et al. 2006 Vaccine. Sep 11;24 (37-39):6309-20; Martin C. et al. 2006 Vaccine. Apr 24;24 (17):3408-19).
Mycobacteria are adept intracellular pathogens that employ a number of different means to evade the host immune response. An example of these virulence strategies is the ability of the bacteria to modulate the presentation of mycobacterial - derived antigens to the T lymphocyte population of the host.
DM VAN/253729-16266/67491962 Compositions that improve the level of antigen presentation and increase the immunogenicity of vaccines directed against mycobacteria, particularly M. tuberculosis are needed.
DISCLOSURE OF THE INVENTION Aspects of the present invention provide vectors comprising nucleotides that encode a protease enzyme. The present invention also provides recombinant bacteria that comprise these vectors and secrete a protease enzyme.
In accordance with one aspect of the invention, compositions are provided that stimulate an immune response by increasing the level of antigen presentation following administration to a mammalian host.
The compositions described herein relate to strains of mycobacteria that stimulate antigen presentation by the major histocompatibility complex class II (MHC-II) molecules found on the surface of the antigen presenting cells (APC) of the host.
In accordance with another aspect of the invention, there is provided a recombinant strain of mycobacteria capable of expressing a mammalian cysteine protease.
In accordance with another aspect of the invention, the recombinant strain of mycobacteria may be further engineered to attenuate the virulence of the bacteria.
The recombinant mycobacteria may be M. bovis, M. tuberculosis or M. bovis, strain BCG.
The mammalian cysteine protease may further be secreted by the mycobacteria. The mammalian cysteine protease may be cathepsin S (SEQ ID: 9).
In accordance with another aspect of the invention, expression of the mammalian cysteine protease is under the control of a constitutive or an inducible bacterial promoter.
DM VAN/253729-16266/6749196 2 In accordance with another aspect of the invention, the cathepsin S coding region consists only of those nucleotides that code for the mature enzyme (SEQ ID NO: 1).
In accordance with another aspect of the invention, the cathepsin S coding region is in-frame with a nucleotide region that encodes a signal peptide (SEQ ID NO: 2).
In accordance with another aspect of the invention, the recombinant strain of mycobacteria encodes a cathepsin S coding region and at least one other nucleotide sequence that encodes an immunogenic polypeptide that is derived from mycobacteria.
The immunogenic polypeptide may originate from M. bovis, M. tuberculosis or M. bovis, strain BCG.
In accordance with another aspect of the invention, the recombinant strain of mycobacteria encodes a cathepsin S coding region and at least one other nucleotide sequence that encodes a mammalian cytokine.
Other aspects and features of the present invention will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the human (SEQ ID NO: 9) and murine (SEQ ID NO: 11) amino acid sequences for the cathepsin S precursor protein. The site of cleavage for the release of the mature active enzyme from the full-length precursor protein is also indicated;
Fig. 2 depicts the pSMT-CatS vector. The mycobacterial promoter from the hsp60 gene (Mulder M.A. 1997 Tuber Lung Dis 78:211-23), is used to drive transcription of the transgene. The transgene comprises a ribosomal binding site and signal peptide (SEQ ID NO: 3) from the fbpB gene (SS-aAg85) and a sequence encoding the mature human cathepsin S protein (Active huCatS) (SEQ ID NO: 6);
DM VAN/253729-16266/67491962 Fig. 3 illustrates growth curves for wild type BCG (non-transformed; empty circle), BCG transformed with an empty pSMT-3 vector (pSMT3; solid square) and a recombinant BCG strain expressing the truncated human cathepsin S polypeptide (BCG-hcs); solid circle);
Fig. 4 depicts cathepsin S enzyme activity in culture supernatant and bacterial cell pellets from the parental wild type (BCG), BCG transformed with the pSMT3 vector (pSMT3) or the rBCG-CatS strain (pSMT3-hcs);
Fig. 5 depicts cathepsin S enzyme activity from THP-I macrophage and bone- marrow derived macrophage (BMM0) lysates that were left uninfected or infected with either BCG or the recombinant BCG strain. THP-I cells were infected with the rBCG-CatS strain expressing the human cathepsin S (rBCG-hcs) while BMM0 were infected with the recombinant BCG strain expressing the murine cathepsin S (rBCG- mcs). Lane 1 : control non-treated cells, Lane 2: stimulation with IFN-γ. (200 U/ml, 24 h), Lane 3: infection with BCG (OfN, then stimulation with IFN-γ.), Lane 4: infection with rBCG-hcs (THP- 1 ) or rBCG-mcs (BMM0);
Fig. 6 shows a western blot depicting the expression of cathepsin S from macrophages cell lysates. THP-I cells were left uninfected or infected with either wild type BCG or rBCG-CatS. Lane 1 : control non-treated cells, Lane 2: stimulation with IFN-γ. (200 U/ml, 24 h), Lane 3: infection with BCG (O/N, then stimulation with IFN-γ), Lane 4: infection with rBCG-catS and no stimulation with IFN-γ;
Fig. 7 depicts IL-2 production from the DBl hybridoma T cells in response to co-incubation with THP-I macrophages that were either i) untreated ii) treated with IFN-γ only, iii) treated with IFN-γ and infected with BCG (WT BCG) or iv) treated with IFN-γ and infected with rBCG-hcs;
Fig. 8 shows the maturation of THP-I -derived DC following exposure to apoptotic vesicles. THP-I -derived DC were either left untreated (control) or exposed to cell-free culture medium from macrophages infected with either rBCG-Cats (rBCGhcs) or wild type BCG (BCG). The cells were stained with FITC conjugated antibodies and analyzed by FACS. The mean fluorescence intensity (MFI) indices were deducted from fluorescence histograms. The L243 mAb recognizes the mature
DM VAN/253729-16266/6749196 2 MHC class II, the LN2 mAB recognizes the invariant chain, the HBl 5. e mAb recognizes the DC maturation marker B7.1 (CD83) and the IT2.2 mAb recognizes the B7.2 (CD86) marker of DC maturation; and
Fig. 9 illustrates FACS analysis to determine the level of apoptosis within RAW 264.7 macrophage monolayers infected the wild type (BCG) or the recombinant strain of BCG expressing the murine cathepsin S (rBCG-mcs). The negative control (control) illustrates the level of apoptosis in uninfected RAW 264.7 macrophages while macrophages treated with IFN-γ illustrates the positive control for apoptosis.
BEST MODES FOR CARRYING OUT THE INVENTION Various embodiments of the present invention are based on the surprising discovery that BCG interferes with antigen processing and antigen presentation by the antigen presenting cells (APC) of the host immune system. Expression of cathepsin S by recombinant BCG may enhance antigen presentation to host lymphocytes and may elicit a protective T cell response following administration to a subject such a recombinant BCG strain.
Any terms not directly defined herein shall be understood to have the meanings commonly associated with them as understood within the art of the invention. As employed throughout the specification, the following terms, unless otherwise indicated, shall be understood to have the following meanings.
As used herein, the term "vector" refers to a polynucleotide composition used for introducing exogenous or endogenous polynucleotide sequences into a host cell. A host cell may be eukaryotic or prokaryotic. A vector comprises a nucleotide sequence which may encode one or more polypeptide molecules. Plasmids, cosmids, viruses and bacteriophages, in a natural state or which have undergone recombinant engineering, are non-limiting examples of commonly used vectors to provide recombinant vectors comprising at least one desired isolated polynucleotide molecule.
As used herein, the term "vaccine" refers to a composition that, upon administration to a subject, stimulates an adaptive immune response, but is incapable of causing a severe infection. A vaccine may comprise, for example, a live attenuated or killed pathogen, or a preparation of a pathogen-specific structure, or other similar
DM VAN/253729-16266/6749196 2 agent, and an adjuvant, excipient or other additives. Alternatively, an agent for stimulation of the immune response may be encapsulated in a liposome or other microparticle. The pathogen may be a virus, eukaryotic parasite, or a bacterium. Adjuvants, excipients, and other additives for inclusion in a composition for use in a vaccine and methods of preparing such compositions will be known to those of skill in the art.
A vaccine may be administered in accordance with two general strategies - before exposure of the subject to the pathogen (pre-exposure or prophylactic vaccination) or after exposure of the subject to the pathogen (post-exposure or therapeutic vaccination). A therapeutic vaccine may be administered alone or in combination with a drug therapy regimen. Prophylactic or therapeutic vaccines may be administered as a single-dose schedule or as a multiple-dose schedule. In a multiple-dose schedule, a course of vaccination may include at least two doses followed by other doses at subsequent time intervals required to maintain or reinforce the immune response. For example, in a 3-dose schedule, each does may be administered about 2-4 months apart. The vaccinated subject may receive additional doses months or years following the dose schedule, or upon exposure to the pathogen. The necessary dosage, timing of doses and quantity of doses may be dependent on the subject (for example, a child or an adult) and will be familiar to a skilled individual, frequently a medical practitioner.
Methods of administration of a vaccine include, for example, intramuscular, subcutaneous or intraperitoneal injection, oral, inhalation, suppository, transdermal routes and the like.
An "MHC" protein is encoded by the major histocompatibility complex of a subject, with a key role in antigen presentation for the immune system. MHC proteins may be found on several cell types, including APCs such as macrophages or dendritic cells, or other cells found in a mammal. Epitopes associated with MHC Class I may range from about 8-11 amino acids in length, while epitopes associated MHC Class II may be longer, ranging from about 9-25 amino acids in length. MHC Class I molecules present peptides generated in the cytosol to CD8 T cells, and the MHC Class II molecules present peptides degraded in cellular vesicles to CD4 T cells. The cellular processes involved in MHC epitope processing and presentation and the
DM VAN/253729-16266/67491962 interaction with other cells is well-described in the art, in various texts and references. See, for example, Roitt's Essential Immunology. IM Roitt, PJ Delves. Oxford, Blackwell Science Publishers, 2001.
An "antigen" is a substance which may induce a specific immune response.
An "immunogen" is a type of antigen that provokes an immune response when introduced into a subject. Immunogens may have varying ability to stimulate an immune reaction, depending on the commonalities or differences with epitopes found in the subject, size of the immunogen, chemical composition, and the like.
An "adaptive immune response" is an immune response involving a lymphocyte. B-cells and T-cells are examples of the two major types of lymphocytes. The cellular processes involved in stimulation of B-cells and T-cells are well described in the art, in various texts and references. See, for example, Roitt's Essential Immunology. IM Roitt, PJ Delves. Oxford, Blackwell Science Publishers 2001.
The terms 'peptide', 'polypeptide' and protein' may be used interchangeably, and refer to a macromolecule comprised of at least two amino acid residues covalently linked by peptide bonds or modified peptide bonds, for example peptide isosteres (modified peptide bonds) that may provide additional desired properties to the peptide, such as increased half-life. A peptide may comprise at least two amino acids. The amino acids comprising a peptide or protein described herein may also be modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in a peptide, including, for example, the peptide backbone, the amino acid side-chains and the amino or carboxyl termini, and the like. It will be understood by one of skill in the art that the same type of modification may be present in the same or varying degrees at several sites in a given peptide.
Mycobacterial infection and host cell response
M. tuberculosis exerts several pro-pathogen survival influences on the host cell that it infects, primarily macrophages and other antigen-presenting cells. These include the inhibition of phagosomal maturation which impedes the accumulation
DM VAN/253729-16266/6749196.2 lysosomal markers and blocks the acidification of the mycobacterial phagosome. The process of phagosomal maturation is also essential for loading microbial-derived peptides onto mature MHC-II molecules that are subsequently exported to the APC surface. The presentation of pathogen-derived peptide antigens in complex with MHC-II molecules is required for the stimulation of host CD4+ T cells. Since pathogenic mycobacteria subvert this maturation process, there is a decreased ability for the adaptive immune system to respond to infection or vaccination (reviewed in Hestvik et al 2005. FEMS Microbiology Reviews 29: 1041-1050; Skeiky and Sadoff 2006. Nat. Rev. Microbiol 4:469-476).
M. tuberculosis also interferes with host apoptotic signaling pathways as a means to maintain its intracellular niche. Previous studies have shown that avirulent strains of M. tuberculosis induce higher levels of apoptosis than virulent M tuberculosis (Keane J. et al. 2000 J Immunol. 15;164(4):2016-20). Apoptotic vesicles from infected cells can be internalized by dendritic cells. Subsequently, the DCs process and display the microbial antigens via MHC class I and CDl which allows for CD8 T cell stimulation (Winau F et al. 2004 Cell Microbiol. 6(7):599-607). The inhibition of macrophage apoptosis by virulent M. tuberculosis may impede antigen presentation by bystander APCs.
Cathepsin S is a protease that is mainly expressed within the lysosomal compartments of antigen presenting cells (APC) and its expression is inducible in the presence of interferon-γ (IFN-γ). Cathepsin S participates in the proteolytic degradation of the invariant chain (Ii) which is associated with immature MHC-II molecules. Thus, cathepsin S activity is required for the maturation of MHC-II molecules and the subsequent peptide loading that precedes antigen presentation at the APC surface (Hsieh et al 2002. J. Immunol 168:2618-2625; Bania et al. 2003. Proc. Natl. Acad. Sci. USA 100:6664-6669). Sendide et al. (Sendide et al 2005. J. Immunol 175: 5324-5332) illustrated that cathepsin S enzyme activity and cathepsin S gene expression are reduced in BCG-infected cells.
Compositions with enhanced antigen presentation Cathepsin S is a cysteine protease that plays a key role in the normal processing and maturation of MHC class II molecules in APCs. Pathogenic
DM VAN/253729-16266/6749196.2 mycobacteria, including M. tuberculosis and BCG, are able to interfere with the antigen processing and presentation mechanisms of host cells partly by inhibition of cathepsin S activity. Given that efficient stimulation of a specific immune response by APC is a prerequisite for successful immunization, the present invention comprises a recombinant BCG strain that expresses and secretes the active cathepsin S enzyme during infection of APCs. To enable the expression of cathepsin S by the bacterial host, the cathepsin S coding region was restricted to those sequences that encode the mature enzyme rather than the full-length preprotein.
In addition, the cathepsin S coding region was fused to a bacterial signal peptide that is recognized by mycobacteria to generate the cathepsin S transgene. The presence of the signal peptide ensures the active secretion of the mammalian protease from the bacteria. Transcription of the transgene was regulated by a mycobacterial promoter that derived from the hsp60 gene from BCG. The hsp60 promoter sequence is constitutively active, thus allowing for the continuous expression of the transgene.
The recombinant expression of the transgene by BCG resulted in several desirable characteristics. Macrophage infection with the recombinant bacteria led to enhanced maturation of MHC-II molecules due to the increased degradation of the invariant chain. Furthermore, the expression of cathepsin S by the recombinant BCG strain increased the presentation of mycobacterial-derived antigens by APCs. The increased presentation of bacterial antigens in complex with mature MHC-II molecules may lead to stimulation of T lymphocytes that are specific for bacterial epitopes. This stimulation of T lymphocytes and the subsequent cytokine secretion may influence the immune response to vaccination, and may further allow development of protective immunity.
The expression of cathepsin S by the recombinant BCG induced the apoptotic response in infected macrophages. Apoptosis of mycobacteria-infected macrophages may result in a decline in the number of viable bacteria and promote the cross- presentation of mycobacterial antigens by bystander APCs. Apoptotic vesicles from infected macrophages can be engulfed by other APCs leading to the processing and presentation of microbial antigens to T lymphocytes. In this context, mycobacterial antigens may be presented in context with MHC-I molecules leading to the stimulation of CD8 T cells.
DM VAN/253729-16266/6749196 2 In another embodiment, expression of the mature cathepsin S during APC infection by mycobacteria may overcome the microbial-induced block in antigen presentation that occurs with the current BCG vaccine.
Alternate embodiments of the invention Protective immunity against M. tuberculosis necessitates the development of a strong ThI response (Stenger and Modlin 1999 Curr Opin Microbiol. Feb;2(l):89-9; Boom WH et al. 2003 Tuberculosis (Edinb).;83(l-3):98-106). In particular, the increased expression of proinflammatory cytokines is required to direct T cell activation toward a T-helper 1 (ThI) phenotype.
A cytokine is a protein that affects the behaviour of other cells by acting on specific, cell-surface cytokine receptors. Cytokines made by lymphocytes are often called interleukins. Examples of proinflammatory cytokines are known in the art and include IL-2, IL-18, IFN-γ, GM-CSF, IL- 12 and the like.
In an alternate embodiment of the invention, the BCG mycobacterial vaccine strain comprises a vector for expression of both cathepsin S and at least one mammalian proinflammatory cytokine. Such expression may improve the antigenicity and/or immunogenicity of recombinant BCG by enhancing the presentation of microbial antigens.
In an alternate embodiment of the present invention, the BCG mycobacterial vaccine strain comprises a vector for expression of both cathepsin S and at least one other immunodominant mycobacterial antigen. Such expression may improve the may improve the antigenicity and/or immunogenicity of recombinant BCG by enhancing the presentation of microbial antigens.
In another embodiment of the present invention, the transcription of the cathepsin S transgene may be under the control of a constitutive bacterial promoter. The strength of a promoter is dictated by the interaction between the RNA polymerase and the promoter sequence. Examples of constitutive mycobacterial promoters include but are not limited to the mycobacterial hsp60 and hsp70 promoters.
In another embodiment of the present invention, the transcription of the cathepsin S transgene may be under the control of an inducible promoter. An
DM VAN/253729-16266/6749196.2 inducible promoter typically requires the participation of alternate sigma factors to induce transcription. The expression of the alternate sigma factors may be limited to specific environmental conditions. Examples of such environmental conditions include but are not limited to iron depletion, reduced oxygen tension or the presence of other environmental stresses known in the art. Examples of inducible mycobacterial promoters include but are not limited to the mycobacterial icl promoter and the mycobacterial acetamidase promoter.
Methods and Materials
Reagents and chemicals RPMI 1640, Hank's buffered salt solution (HBSS), phorbol-12-myristate-13- acetate (PMA), protease inhibitor mixture, PMSF, and trypsin- EDTA were obtained from Sigma- Aldrich. Monoclonal anti-Cathepsin S was from Calbiochem, anti-Ii mAb (clone LN2) was from BD Pharmingen. Anti-human HLA-DR mAb (clone L243) was from BD Pharmingen. Anti-B7.1 mAb (clone HBl 5.e) was from BD Pharmingen. Anti-B7.2 mAb (clone IT.2) was from BD Pharmingen. Anti-FITC- conjugated F(ab')2 goat anti-mouse IgG and HRP-conjugated goat anti-mouse IgG were from Sigma-Aldrich. Human recombinant IFN-γ was from Genentech (South San Francisco, CA). Cathepsin S inhibitor Z-Phe-Leu-COCHO and the recombinant active form of human Cathepsin S were from Calbiochem. Cathepsin S substrate Z- Val-Val-Arg-AMC was from Bachem Bioscience. Human IL-2 ELISA kit was from eBioscience.
Mycobacterial strains
M. bovis BCG "Pasteur strain 1173P2" was grown in Middlebrook 7H9 broth (Difco) supplemented with 10% (v/v) OADC (oleic acid, albumin and dextrose solution; Difco) and 0.05% (v/v) Tween 80 (Sigma-Aldrich) (7H9-OADC) at 37°C on a rotating platform (50 rpm). Mycobacteria were also cultured on 7H10 agar supplemented with OADC (BD Biosciences). To monitor bacterial growth in vitro, the turbidity of mycobacterial broth cultures was determined by measuring the optical density at 600 nm (OD600) over time. For infection experiments, bacteria were cultured in 7H9-OADC until an OD600 of 0.5. Bacteria were harvested by centrifugation and pellets were suspended in complete media plus 10% glycerol.
DM VAN/253729-16266/6749196 2 Mycobacterial cultures were stored in aliquot (~5 x 108/vial) at -700C. Before infection, bacteria were grown 48 h in 7H9-OADC and opsonized as follows: 109 mycobacteria were suspended in 1 ml of RPMI 1640 containing 50% human serum (AB+ and PPD-) and rocked for 30 min at 370C. Bacteria were then pelleted and suspended in 1 ml of RPMI 1640 and clumps were disrupted by multiple passages through a 25-gauge needle.
Construction of rBCG-hcs
For the amplification of the human cathepsin S coding region, total RNA was isolated from IFN-γ-stimulated THP-I cells using the RNeasy® Minikit (RNeasy is a registered trademark of Qiagen GmbH Corp., Hilden, Fed. Rep. Germany) according to the manufacturer's protocol. cDNA was synthesized using Superscript First-Strand Synthesis system (Invitrogen) with the following primers; CatS-Forward (5'- ATCTAAGCTTTTGCCTGATTCTGTC-3 ' ; SEQ ID NO:4) which contains a HwJIII adaptor (underlined) and CatS-Reverse (5'- TTGGGATCGATCTAGATTTCTGGGTA-S ' ; SEQ ID NO : 5) which contains a CM adaptor (underlined). The amplicon was digested with Hindlll and Clal (SEQ ID NO: 6) and cloned into the E. coli-Mycobacterium shuttle vector pSMT-3 (Herrmann JL et al. 1996 EMBO J. 15(14):3547-54) that had been previously digested with Hindlll and Clal to generate the pSMT-3-CatS vector.
The signal peptide and ribosomal binding site of the fbpB gene (SEQ ID NO:
3) from mycobacteria was amplified by PCR from the genomic DNA of BCG using the following primers; Alpha-forward (5'-
GACCGGATCCCGACGACATACAGGACAAAGGGG-S1; SEQ ID NO:7) which contains a BamHl adaptor (underlined) and Alpha-Reverse (5,- CCGGCTGCAGCGCGCCCGCGGTTGCCGCTCCG-B': SEQ ID NO:8) which contains a Pstl adaptor (underlined). The PCR product was cloned into pSMT-3-CatS vector that had been previously digested with BamHl and Pstl to generate pSMT- fbpB signal peptide-CatS (pSMT-CatS).
The pSMT-CatS vector comprising the human cathepsin S coding region was electroporated into BCG and transformants were selected onto 7H10 agar medium supplemented with 100 ug/ml of hygromycin B (Invitrogen). The presence of the
DM VAN/253729-16266/67491962 transgene comprising the signal peptide from the fbpB and the human cathepsin S coding regions was confirmed by PCR and restriction endonuclease digestion of the recombinant BCG strain, rBCG-hcs.
Construction of rBCG-mcs For the amplification of the murine cathepsin S coding region, total RNA was isolated from IFN-γ-stimulated RAW 264.7 cells using the RNeasy® Minikit (Qiagen) according to the manufacturer's protocol. cDNA was synthesized using Superscript First-Strand Synthesis system (Invitrogen) with the following primers: Forward (5' CTAATCTGCAGTTGCCTGACACTGT 3' ; SEQ ID NO: 14) which contains a Pstl adaptor (underlined) and Reverse (5 ' TTGGGATCGATCTAGATTTCTGGGT 3 ' ; SEQ ID NO: 15) which contains a Clal adaptor (underlined). The amplicon was digested with Pstl and Clal and cloned into the E. coli-Mycobacterium shuttle vector pSMT-3 (Herrmann JL et al. 1996 EMBO J. 15(14):3547-54) that had been previously digested with Pstl and Clal to generate the pSMT-mCatS vector.
The signal peptide and ribosomal binding site of the fbpB gene (SEQ ID NO:
3) from mycobacteria was amplified by PCR from the genomic DNA of BCG using the following primers; Alpha-forward (5'-
GACCGGATCCCGACGACATACAGGACAAAGGGG -31; SEQ ID NO:7) which contains a BamHl restriction site and Alpha-Reverse (5'- CCGGCTGCAGCGCGCCCGCGGTTGCCGCTCCG -3'; SEQ ID NO:8) which contains a .Ps/lrestriction site. The PCR product was digested using Pstl and BamHl and cloned into pSMT-3 that had been previously digested with the same enzymes, to generate pSMT-mcs.
The pSMT-mcs vector comprising the murine cathepsin S coding region fused to the signal peptide was electroporated into BCG and transformants were selected onto 7H10 agar medium supplemented with 100 ug/ml of hygromycin B (Invitrogen). The presence of the transgene comprising the signal peptide from the fbpB and the murine cathepsin S coding regions was confirmed by PCR and restriction endonuclease digestion of the recombinant BCG strain, rBCG-mcs.
Differentiation and infection of macrophages and dendritic cells
DM VAN/253729-16266/6749196.2 The monocytic cell line THP-I (ATCC TIB-202) was cultured in RPMI 1640 supplemented with 5% Fetal Calf Serum (FCS) (Invitrogen Life Technologies), L- glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 μg/ml). For differentiation into a macrophage-like phenotype, cells were seeded at a density of 105/cm2 and allowed to adhere and differentiate in the presence of PMA (20 ng/ml) at 37°C in a humidified atmosphere of 5% CO2 for 24 h. For differentiation into a dendritic cell-like phenotype, THP-I cells were treated with PMA (10 ng/ml) for 24h followed by treatment with IL-4 (1000 units/ml) for 96 h.
Bone marrow cells from the femur and tibia of BALB/C mice were culture in 10-cm diameter cell culture dishes for 4-5 days at 37°C and 5% CO2 in RPMI 1640 medium supplemented with 10% heat-inactivated FCS (HyClone, Logan, Utah), 2 mM L-glutamine, 10 mM HEPES, 50 μM 2-ME, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Stemcell Technologies, Vancouver, BC) and containing 10% of L929 conditioned media (LCM) or 10 ng/ml M-CSF (R&D Systems, Minneapolis, MN) in order to differentiate bone marrow cells into macrophages.
L929 cells (ATCC #CCL-1) were cultured in RPMI media plus 2 mM L- glutamine, 10 mM HEPES, 100 U/ml penicillin, and 100 μg/ml streptomycin and 10% FCS. LCM was collected from cells grown for 1 week and kept in 5 ml aliquots at -700C).
RAW 264.7 cells (ATCC TIB-71 ) were cultured in 10-cm diameter cell culture dishes as recommended by the cell line supplier ATCC (Manassas, VA). The medium was Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 10 mM HEPES, 50 μM 2-ME, 100 U/ml penicillin, and 100 μg/ml streptomycin and containing 1 % non-essential amino acids (complete media). Cells were cultured at 37°C and 5% CO2. Cultures were split after 70-80% confluence.
For infection experiments, cells were washed three times with HBSS and adherent monolayers were replenished with culture medium without antibiotics and infected with opsonized mycobacteria (bacteria to cell ratio of 25: 1). After a period of 3 h, partially attached noningested bacteria were removed by 5 min treatment with
DM VAN/253729-16266/6749196.2 trypsin-EDTA and extensive washing with HBSS. This procedure resulted in an infection rate of 80-90% with an approximate range from 5 to 10 bacteria per cell.
Measurement of Cathepsin S activity
Cathepsin S activity was measured using the fluorogenic substrate Z- VaI- VaI- Arg-NHMec as described Sendide et al. (Sendide et al 2005. J. Immunol 175: 5324- 5332). Adherent THP-I cells were scraped into extraction buffer (0.01% Triton X-100 in 0.1 M potassium phosphate buffer containing 1 mM EDTA, pH 7.5) and frozen/thawed three times. Samples were sonicated for 5 s in a Sonic Dismembrator 60 (Fisher Scientific) in ice. Cell debris and membranes were removed by centrifugation at 12,000 x g for 30 min at 40C. Fifty microliters of the soluble fraction (-100 μg of protein) were added to 50 μl of the reaction buffer (0.1M potassium phosphate buffer, 5 mM EDTA, pH 7.5, and 5 mM DTT), and samples were incubated for 45 min at 400C to inactivate cathepsin L. Thereafter, 50 μl of 12.5 μM Z-Val-Val-Arg-AMC (Cat S/L substrate) were added to the mixtures. After additional incubation at 4O0C for 10 min in the dark, fluorescence was measured at .ex/em 360/460 nm in a VersaFluor instrument (Bio-Rad). Cathepsin S activities were calculated by reference to a standard curve using a range (20 to 20,000 U/ml) of active recombinant human cathepsin S. Reaction mixtures with bovine serum albumin instead of cell lysate were used as controls. Results were expressed after subtracting the fluorescence value corresponding to this control.
Western blotting of Cathepsin S
Control and infected cells were washed with HBSS and whole cell lysates were prepared in Nonidet P-40 lysis buffer (50 mM sodium acetate, 5 mM MgC12, 0.5% Nonidet P-40, pH 7.4) in the presence of protease inhibitors mixture and PMSF. Proteins (50 μg/lane) were separated by 12% SDS-PAGE then transferred to nitrocellulose membrane and probed with anti-cathepsin S mAb. To assess the amount of individual proteins in each sample, after detection of bound anti-cathepsin S mAb, membranes were stripped and reprobed with anti-actin Abs and developed by ECL as described (Hmama 1999, supra).
DMJVAN/253729-16266/67491962 Cell surface staining and flow cytometry
To measure cell surface expression of MHC class II or markers of DC differentiation, macrophage or dendritic cells were scraped with a rubber policeman and cells were collected in HBSS containing 0.1% NaN3 and 1% FCS (staining buffer). Cells were then labelled with anti-class II mAbs, anti-Ii mAbs, anti-B7.1 mAbs, anti-B7.2 mAbs or irrelevant isotype-matched IgG for 20 min. Cells were then washed twice with staining buffer and labelled with FITC -conjugated F(ab')2 goat anti-mouse IgG for 20 min. To control for cell viability, cells were incubated with propidium iodide (0.5 μg/ml staining buffer) for 10 min, and cells were then washed twice and fixed in 2% paraformaldehyde in staining buffer. Cell fluorescence was analyzed using a FACS Calibur flow cytometer (BD Biosciences). Viable cells were identified by exclusion of propidium iodide. Relative fluorescence intensities of 10,000 cells were recorded as single-parameter histograms (log scale, 1024 channels, and 4 decades) and the mean fluorescence intensity (MFI) was calculated for each histogram. Results are expressed as MFI index, which corresponds to the ratio calculated using: [(MFI of cells + specific Ab)/ (MFI of cells + irrelevant isotype- matched IgG)].
Apoptosis studies by FACS
RAW 264.7 cells were grown at ~ 5 x 106 cells in 10 ml complete medium in 10-cm culture plates and infected with mycobacteria at an MOI of 20:1 for 24h. Cells were washed 3 times with warm HANKS and flushed gently with cold HANKS to detach cells from plates without damaging cell membrane. Cells were stained 30 min on ice with propidium iodide (PI, Sigma P-4170) at 1 : 100 and FITC-AnnexinV (Calbiochem, PF032) at 1 :50 in HBSS 5% FCS. Cells were washed twice with HBSS 5% FCS and immediately analyzed for expression of dual, red (PI) and green (AnnexinV), fluorescence in a BD FACScalibur cytofluorometer.
Antigen Presentation Assays
DBl T hybridoma cells (Gehring et al. 2003 Infect Immun.; 71(8): 4487- 4497) were cultured in DMEM supplemented with 5% Fetal Calf Serum (FCS) (Invitrogen Life Technologies), L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 μg/ml). THP-I cells were seeded at a density of 105/cm2 and
DM VAN/253729-16266/6749196 2 allowed to adhere and differentiate in the presence of PMA (20 ng/ml) at 37°C in a humidified atmosphere of 5% CO2 for 24 h. The differentiated THP-I cells were left untreated or infected with either BCG of rBCG-CatS overnight. The cells were then washed and incubated with 200 U of recombinant human IFN-γ for 24 h. The cells were then fixed in 1% paraformaldehyde, followed by incubation with DBl T hybridoma cells (106 cells/well). Supernatants were harvested after 2Oh and the amount of IL-2 produced by T hybridoma cells was measured by ELISA specific for IL-2.
Microarray Analysis RNA was isolated using the Qiagen RNeasy mini kit and the RNA quality was assessed using a BioAnalyzer (Agilent technologies). RNA samples were first reverse transcribed into cDNA using the Superscript II Reverse transcriptase (Invitrogen) according to the manufacturer's recommendations. Each sample was hybridized with the cDNA derived from a universal RNA (Stratagene). cDNA labeling was carried out using Genisphere's Array 350 Expression Array Detection Kit (Genisphere Inc.
Hatfield, PA) following the manufacturer's instructions. Hybridization was performed using Operon's Human Genome OpArray version 4 slides. The slides were scanned using the GenePix® Autoloader 200AL (GenePix is a registered trademark of Axon Instruments Inc., Union City, CA, USA) and data analysis was done with Imagene® software (Imagene is a registered trademark of BioDiscovery Inc., Los Angeles CA, USA) and GenSpring version GX (Agilent Technologies) software.
Statistical analysis
All data are expressed as the mean +/- SD. Statistical analysis was performed using Student's t test. Values of p < 0.05 were considered to be significant.
EXAMPLE 1
Construction of a recombinant BCG strain that secretes active CatS
The cathepsin S protein is synthesized as a 331 amino acid preprotein (SEQ ID NO: 9) and the mature enzyme is generated following cleavage between amino acids 114 and 115, (SEQ ID NO: 1) as depicted in Fig. 1. A vector was constructed that encoded a mycobacterial promoter to control the expression of the human cathepsin S coding region that was fused to a mycobacterial signal peptide. Briefly,
DM VAN/253729-16266/6749196.2 the human cathepsin S nucleotide coding region that encoded the amino acids 115- 331 was cloned in-frame with the coding region of the Ag85B signal peptide. The resulting transgene was inserted into the pSMT3 shuttle vector which contained a mycobacterial Hsp60 promoter region to drive the transcription of the transgene. The recombinant vector comprising the promoter and the transgene, referred as pSMT- CatS (Fig. 2), was electroporated into BCG to generate the recombinant strain known as rBCG-CatS.
The in vitro growth curves of the parental wild type and rBCG-CatS were compared by measurement of the optical density of broth cultures. Analysis of the growth curves (Fig. 3) indicates that the wild type and recombinant strain have nearly identical growth characteristics in broth. To assess whether the rBCG-CatS strain was producing the active cathepsin S protein, the enzyme activity of cathepsin S was measured in the culture supernatant and bacterial pellet for the rBCG-CatS and the wild type BCG. The culture supernatant of the recombinant strain contained high levels of cathepsin S enzyme activity as compared to wild type BCG and minimal activity was detected in the bacterial pellets, indicating that the rBCG-CatS strain secretes the active cathepsin S enzyme during culture in vitro (Fig. 4).
The level of cathepsin S expression by the recombinant strain was also determined following macrophage infection. Levels of cathepsin S enzyme activity and protein expressed were compared in macrophage cell lysates from THP-I monolayers that were either i) uninfected, ii) infected with BCG or iii) infected with rBCG-CatS. Similarly, cell lysates were collected from murine macrophage monolayers were either i) uninfected, ii) infected with BCG or iii) infected with rBCG-mcs. Cathepsin S enzyme activity was significantly increased in the cell lysates from macrophages infected with the recombinant BCG (Fig. 5) indicating that the rBCG-CatS and rBCG-mcs strains actively secrete cathepsin S following macrophage infection. The level of cathepsin S expression in the THP-I cell lysates was assessed by Western blot and showed that macrophages infected with rBCG-CatS have increased levels of cathepsin S protein as compared to BCG-infected macrophages (Fig. 6).
DM VAN/253729-16266/6749196.2 EXAMPLE 2
Macrophages infected with rBCG-CatS exhibit increased presentation of mycobacterial protein antigens via MHC-II
The stimulation of T lymphocytes was used to determine if the secretion of cathepsin S by rBCG-CatS would affect antigen processing and presentation by the APC. The DBl T cell hybridoma specifically recognizes an epitope from the mycobacterial Ag85B protein in the context of MHC class II molecules on the APC surface. The recognition of the peptide:MHC complex by the T cell receptor causes the release of IL-2 from the T lymphocyte which is used as a marker for T cell stimulation.
Macrophages infected with the rBCG-CatS strain showed an increased ability to induce IL-2 release from the T cells, whereas the BCG-infected macrophages were only modest inducers of IL-2 (Fig. 7). These results demonstrate that the recombinant expression of cathepsin S by BCG facilitated the presentation of mycobacterial antigens in complex with MHC-II to T lymphocytes.
EXAMPLE 3
Macrophages infected with rBCG-CatS show increased transcription of MHC class II genes
Microarray analysis was used to compare the transcriptional profiles of macrophages infected with either wild type BCG or the rBCG-CatS strain. Infection with the recombinant strain of BCG led to increased expression of genes that are known to be involved in phagosome maturation and apoptosis. In addition, macrophage infection with rBCG-CatS also resulted in the increased expression of MHC-II-related genes. The values represent fold changes in gene expression in macrophages infected wild type BCG relative to uninfected cells (BCG/control) and fold changes in gene expression of macrophages infected with rBCG-catS relative to macrophages infected with wild type BCG (rBCG- hcs/BCG). A list of exemplary genes that were upregulated and are known to be involved in phagosome maturation, antigen presentation and apoptosis is provided in Table 1. A cluster of genes related to phagosome maturation, apoptosis and Ag presentation were significantly up-regulated in M0 infected with rBCG-hcs opposed to wild-type BCG. The later was rather inducing expression of genes related to apoptosis inhibition.
DM VAN/253729-16266/6749196 2 EXAMPLE 4
Infection of immature dendritic cells with rBCG-CatS leads to increased expression of mature MHC-II
THP-I -derived immature dendritic cells were infected with either BCG or rBCG-CatS to determine whether the bacterial expression of cathepsin S would lead to an increase in the surface expression of MHC-II molecules. As depicted in Fig. 8, the rBCG-CatS-infected dendritic cells had increased levels of MHC-II molecules and increased levels of other dendritic cell differentiation markers (B7.1 and B7.2) as observed by FACS analysis. These results strongly suggest that restoration of cathepsin activity in APC by infection with rBCG-CatS would contribute to the induction of an efficient protective T cell response.
DM VAN/253729-16266/6749196.2 Table I: Macrophage transcriptome in response rBCG-hcs*.
Figure imgf000024_0001
* The values (averages of 2 independent expeπments) represent fold changes in gene expression in M0 infected with wild-type BCG relative to control non-infected cells (BCG/control) and fold changes in gene expression of M0 infected with rBCG-hcs relative to cells infected with wild-type BCG (rBCG-hcs/BCG) The bold represents increases and underlined values represent decreases in gene expressions Values < 2 represent no significant change
DM VAN/253729-16266/6749196 2 EXAMPLE 5
Macrophages infected with rBCG-CatS undergo apoptosis and the apoptotic blebs enable cross priming by dendritic cells
Macrophages were either i) left untreated; ii) stimulated with EFN-γ for 24 h or; iii) infected overnight with either wild type BCG or rBCG-mcs. The infected cells were stained with annexin V-FITC and PI and analyzed by FACS. As shown in Fig. 9. murine macrophages infected with rBCG-mcs showed increased annexin V-staining, similar to levels achieved with IFN-γ treatment. These results indicate that unlike BCG, the recombinant BCG strain expressing cathepsin S readily induces apoptosis of the infected macrophage. The induction of apoptosis in the macrophages infected with the rBCG-mcS strain led to the release of apoptotic blebs into the overlying cell culture medium.
Immature THP-I -derived dendritic cells were either i) left untreated or ii) exposed to the cell-free overlying medium from macrophages that had been infected with either BCG or rBCG-CatS. As shown in Fig. 10, the exposure of immature dendritic cells to the apoptotic blebs derived from rBCG-CatS-infected macrophages was sufficient to induce the expression of MHC-II molecules and markers of dendritic cell maturation. In addition, the exposure of dendritic cells to the rBCG-CatS-derived apoptotic blebs from infected macrophages resulted in the decreased exposure of the Ii chain, which is further evidence of the induced maturation of MHC-II molecules.
While specific exemplary embodiments of the invention have been described and illustrated, such embodiments should be considered illustrative of the invention only and not as limiting the invention.
DM VAN/253729-16266/6749196.2

Claims

1. A nucleic acid molecule comprising a nucleotide sequence configured for controllable expression of a cysteine protease.
2. A nucleic acid molecule according to claim 1, wherein the cysteine protease is a mammalian cysteine protease.
3. A nucleic acid molecule according to claim 1, wherein the cysteine protease is one of a human cathespin S protein or a murine cathespin S protein.
4. A nucleic acid molecule according to claim 1 , wherein the nucleotide sequence encodes at least the human sequence
LPDSVDWREKGCVTEVKYQGSCGACWAFSAVGALEAQLKLKTGKLVSLSAQNLVD CSTEKYGNKGCNGGFMTTAFQYIIDNKGIDSDASYPYKAMDLKCQYDSKYRAATCS KYTELPYGREDVLKEAANKGPVSVGVDARHPSFFLYRSGVYYEPSCTQNVNHGVLV VGYGDLNGKEYWLVKNSWGHNFGEEGYIRMARNKGNHCGIASFPSYPEI.
5. A nucleic acid molecule according to claim 1, wherein the nucleotide sequence encodes at least the murine sequence
LPDTVDWREKGCVTEKYQGSCGACWAFSAVGALEGQLKLKTGKLISLSAQNLVDCS NEEKYGNKGCGGGYMTEAFQYIIDNGGIEADAS YPYKAMDEKCHYNSKNRAATCS RYIQLPFGDEDALKEAVATKGPVSVGIDASHSFFFYKSGVYDDPSCTGNVNHGVLW GYGTLDGKDYWLVKNSWGLNFGDQGYIRMARNNKNHCGIASYCSYPEI.
6. A nucleic acid molecule according to claim 4 or claim 5, wherein the nucleotide sequence additionally encodes a signal peptide.
7. A nucleic acid molecule according to claim 6, wherein the nucleotide sequence encodes the signal peptide
MTDVSRKTRAWGRRLMIGTAAA WLPGLVGLAGGAATAGA.
8. A recombinant microorganism comprising the nucleic acid molecule of claim 1.
DM VAN/253729-16266/6749196 2
9. A recombinant microorganism according to claim 8, wherein said microorganism is a strain selected from the group consisting of Mycobacterium bovis, Mycobacterium bovis strain BCG, and Mycobacterium tuberculosis.
10. A recombinant microorganism according to claim 8, wherein said microorganism is a strain is a Mycobacterium bovis strain BCG.
11. A pharmaceutical composition comprising a nucleic acid molecule according to claim 1 , and a pharmceutically acceptable carrier.
12. A pharmaceutical composition according to claim 11, additionally comprising an adjuvant.
13. A pharmaceutical composition according to claim 11, additionally comprising a pharmaceutically acceptable excipient.
14. A pharmaceutical composition according to claim 11, additionally comprising a pharmaceutically acceptable diluent.
15. A pharmaceutical composition comprising a recombinant microorganism according to claim 7, and a pharmceutically acceptable carrier.
16. A pharmaceutical composition according to claim 15, additionally comprising an adjuvant.
17. A pharmaceutical composition according to claim 15, additionally comprising a pharmaceutically acceptable excipient.
18. A pharmaceutical composition according to claim 15, additionally comprising a pharmaceutically acceptable diluent.
19. Use of a pharmaceutical composition according to claim 11, in vaccination and/or immunotherapy.
20. The use of claim 18 for vaccination against tuberculosis.
21. Use of a pharmaceutical composition according to claim 15, in vaccination and/or immunotherapy.
DM VAN/253729-16266/6749196.2
22. The use of claim 21 for vaccination against tuberculosis.
DM VAN/253729-16266/6749196 2
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BROEMME D. ET AL.: "Functional expression of human cathepsin S in Saccharomyces cerevisiae. Purification and characterization of the recombinant enzyme", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 268, 1993, pages 4832 - 4838, XP002249184 *
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