EP3983011A1 - Protéines de fusion pour vaccins contre la tuberculose - Google Patents

Protéines de fusion pour vaccins contre la tuberculose

Info

Publication number
EP3983011A1
EP3983011A1 EP20731142.4A EP20731142A EP3983011A1 EP 3983011 A1 EP3983011 A1 EP 3983011A1 EP 20731142 A EP20731142 A EP 20731142A EP 3983011 A1 EP3983011 A1 EP 3983011A1
Authority
EP
European Patent Office
Prior art keywords
seq
fusion protein
bcg
antigens
esat
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20731142.4A
Other languages
German (de)
English (en)
Inventor
Rasmus MORTENSEN
Claus Aagaard
Peter Lawætz ANDERSEN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Statens Serum Institut SSI
Original Assignee
Statens Serum Institut SSI
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Statens Serum Institut SSI filed Critical Statens Serum Institut SSI
Publication of EP3983011A1 publication Critical patent/EP3983011A1/fr
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6018—Lipids, e.g. in lipopeptides
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6068—Other bacterial proteins, e.g. OMP
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag

Definitions

  • the present invention relates to fusion proteins based on antigenic polypeptides from Mycobacterium tuberculosis for preventing, inhibiting or treating infections and/or disease caused by a species of the tuberculosis complex.
  • the present invention relates to fusion proteins comprising antigens that do not prime an immune response against BCG and/or ESAT-6 repeats.
  • the fusion proteins may comprise a combination of early and late antigens.
  • the present invention relates to vaccines, immunogenic compositions and pharmaceutical compositions comprising the fusion proteins.
  • M . tuberculosis Human tuberculosis caused by Mycobacterium tuberculosis ( M . tuberculosis) is a severe global health problem, responsible for millions of deaths annually, according to the WHO. During the 1960s and 1970s, incidences of new
  • tuberculosis cases were on a decline, but the positive tendency has been broken, partly due to the advent of AIDS and the appearance of multidrug resistant strains of M. tuberculosis.
  • BCG Baciiie Calmette- Guerin
  • BCG Baciiie Calmette- Guerin
  • BCG Baciiie Calmette- Guerin
  • An alternative strategy for providing an effective tuberculosis vaccine revolves around subunit vaccines based on fusion proteins of M. tuberculosis antigens.
  • the subunit approach has been considered to hold a number of advantages, such as increased safety and stability as well as the ability to boost prior BCG vaccination. Efforts have been put into combining the fusion proteins of a variety of M.
  • tuberculosis antigens with suitable adjuvants to induce robust cellular immune responses.
  • W02014063704 A2 and WO2015161853 A1 all disclose fusion proteins against tuberculosis infection and disease, wherein a variety of M. tuberculosis antigens have been mixed to provide subunit vaccines capable of inducing strong immune responses.
  • common to the above fusion proteins is that none of the corresponding subunit vaccines have yet resulted in a commercially available vaccine against tuberculosis.
  • tuberculosis vaccine with the potential to result in a clinically acceptable vaccine would be advantageous.
  • fusion proteins capable of inducing a strong high quality immune response and therefore the ability to enhance protection would be advantageous.
  • fusion proteins that are conceptually different from previously developed fusion proteins for preventing, inhibiting or treating tuberculosis infection and/or disease.
  • the fusion proteins described herein are based on antigens that do not prime an immune response against BCG (herein termed "BCG- antigens") and/or "ESAT-6 repeats", wherein more than one copy of the ESAT-6 antigen are present in the fusion protein.
  • BCG- antigens antigens that do not prime an immune response against BCG
  • ESAT-6 repeats wherein more than one copy of the ESAT-6 antigen are present in the fusion protein.
  • the fusion proteins may comprise a combination of early and late antigens.
  • the fusion proteins provided herein yield improved immune responses compared to existing tuberculosis vaccines.
  • an object of the present invention relates to the provision of fusion proteins comprising BCG- antigens and/or ESAT-6 repeats that induce strong cellular immune response, while at the same time improving the quality of the T cells.
  • Another object of the present invention relates to the the provision of fusion proteins comprising a combination of early and late tuberculosis antigens that both accelerate the immune response after Mtb infection and induce T cells with potential of specifically recognizing the bacteria in the late chronic phase of infection.
  • a further object of the present invention is to provide fusion proteins that may be effectively used in a vaccine or immunogenic composition for prevention, inhibition or treatment of tuberculosis infection and/or disease, either as a stand- alone vaccine or in combination with BCG.
  • one aspect of the invention relates to a fusion protein comprising at least five antigens that originate from M. tuberculosis.
  • An embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises both early and late antigens.
  • Another embodiment of the present invention relates to the fusion protein as described herein, wherein the antigens are deleted from, non-secreted or have low-expression in BCG.
  • a further embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises at least two ESAT-6 repeats, such as at least three ESAT-6 repeats, such as at least four ESAT-6 repeats, such as at least five ESAT-6 repeats.
  • a further aspect of the present invention is to provide a nucleic acid sequence comprising a sequence encoding a fusion protein as described herein.
  • An even further aspect of the present invention is to provide a recombinant expression vector comprising a nucleotide sequence as described herein operatively linked to one or more control sequences suitable for directing the production of the fusion protein in a suitable host.
  • Another aspect of the present invention is to provide a recombinant host cell comprising an expression vector as described herein.
  • Figure 1 shows immune recognition of selected antigens that M. bovis BCG Danish do not induce a T cell response against (BCG- antigens). Immune responses after (A) M. bovis BCG Danish immunization, (B) M. tuberculosis Erdman infection, or (C) immunization with the individual antigens.
  • Figure 2 shows schematic overview of selected fusion proteins containing BCG- antigens.
  • Figure 3 shows schematic overview of additional selected fusion proteins described herein.
  • Figure 4 shows immune kinetic recognition of ESAT-6 and MPT70 during an Mtb infection.
  • A Antigen specific interferon-g release or
  • B fold increase in the frequency of antigen specific CD4 T cells was measured in the lung 3, 12 and 20 weeks after Mtb Erdman infection.
  • Figure 5 shows that vaccines containing late stage expressed antigens induce long term protection.
  • H105 includes the antigens MPT64, MPT70, and MPT83.
  • Figure 6 shows that co-administration of BCG and H107/CAF01 increased the specific H107 immune response in two vaccination models.
  • the frequency of H107 specific CD4 T cells was measured in spleens three weeks after H107/CAF01 immunization in (A) a standard preventive model and (B) a BCG re-vaccination model were M. bovis BCG Danish was administered 12 months prior to the H107 vaccine
  • FIG. 7 shows that BCG co-administration affects the differentiation of fusion protein specific CD4 T cells depending on whether the fusion protein contains BCG- antigens (H107) or BCG + antigens (H65).
  • Figure 9 shows that co-vaccination with M. bovis BCG and the H104-H107 fusion proteins formulated in CAFOl induced significant protection against Mtb infection. Lung CFU's at week four post Mtb Erdman infection are shown.
  • FIG 10 shows that addition of free ESAT-6 protein into the vaccine formulation did not increase the vaccine primed immune response nor did it reduce the mycobacteria number after Mtb infection.
  • the number of ESAT-6 specific CD4 T cells was determined in the vaccination groups (A) three weeks after
  • Figure 11 shows that repeating ESAT-6 in a fusion protein resulted in increased immune responses against ESAT-6 and better protection in a preventive vaccination model.
  • A Antigen specific responses were measured after H64 (one ESAT-6 copy) and H76 (five ESAT-6 copies) immunization in spleens three weeks after immunization.
  • B The number of mycobacteria was determined in the lungs six weeks after infection with Mtb.
  • Figure 12 shows that repeating ESAT-6 in a fusion molecule improved recruitment of ESAT-6 specific T cells to the site of infection and improved the protective efficacy of the vaccine in a post-exposure vaccination model.
  • Animals infected with Mtb Erdman were treated with antibiotics. Two weeks prior to treatment termination immunization with H83/CAF01 (one ESAT-6 copy) and H84/CAF01 (four ESAT-6 copies) was started.
  • A Number of ESAT-6 specific CD4 T cells in the lungs two weeks after immunization.
  • B Number of mycobacteria in lungs 22 and 35 weeks after infection (6 and 19 weeks after the last immunization).
  • Figure 13 shows that repeating ESAT-6 in the fusion protein increased immune responses and improved the protective efficacy of the vaccine.
  • Figure 14 shows that the H107 fusion protein formulated in CAFOl adjuvant was a better vaccine than H56 formulated in CAFOl.
  • Figure 15 shows that already three weeks after the first immunization (one week after the second H107) an increased TB10.4 response was observed in the
  • Figure 16 shows a schematic diagram of the composition of the H107 and H107e fusion proteins.
  • Figure 16A shows SDS-PAGE and western blots of the H107 and H107e fusion protein expression from E.coli. H107e has increased protein expression compared to H107.
  • Figure 17 shows SDS-PAGE and western blots of the H107 and H107e fusion protein expression from E.coli. H107e has increased protein expression compared to H107.
  • FIG. 17B shows that H107e induces immune responses to the same individual antigens as H107.
  • Figure 17C shows that, after infection, H107e confers
  • Figure 17D shows that, in BCG-memory mice, H107e (BCG-) vaccination leads to less differentiated CD4 T cells (better quality) compared to H65 (BCG+), measured by functional differentiation score, FDS as well as proportions of IL-17 producing CD4 T cells.
  • the term "antigen" refers to a molecule, such as an immunogenic polypeptide, that is capable of inducing an immune response.
  • the immune response generated by the antigen may be B cell driven (antibody- mediated immune response) and/or T cell driven (cellular immune response).
  • the antigens described herein originate form Mycobacterium tuberculosis ( M . tuberculosis or Mtb). Early and late antigens
  • M. tuberculosis infection runs essentially through 3 phases; (i) the acute phase, (ii) the latent/chronic phase and possibly (iii) the reactivation phase.
  • the gene expression pattern of M. tuberculosis changes during these phases.
  • the term "early antigen” refers to antigens expressed primarily during the acute phase
  • late antigens refers to antigens expressed primarily during the latent/chronic phase.
  • fusion proteins refers to polypeptides comprising a random order of two or more antigens from M. tuberculosis or analogues thereof.
  • the antigens may be fused together with or without an amino acid linker of varying length and sequence. Fusion proteins may be produced by operatively linking two or more heterologous nucleic acid sequences encoding the amino acid sequences of the antigens of interest.
  • all cysteines in the fusion protein may be replaced with any amino acid, but serine is the preferred substitute because of its high structural similarity with cysteine.
  • the fusion proteins or antigens may comprise appropriate purification tags (or affinity tag) to allow purification from the crude biological source (e.g.
  • Purification tags include, but are not limited to, His-tag, chitin binding protein (CBP), maltose binding protein (MBP) and
  • GST glutathione-S-transferase
  • fusion protein may be used interchangeably with the term "subunit vaccine”.
  • polypeptide refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and/or synthetic non-naturally occurring analogs thereof linked via peptide bonds.
  • polypeptide sequences the left- hand end of a polypeptide sequence is the amino-terminus (N-terminus); the right-hand end of a polypeptide sequence is the carboxyl-terminus (C-terminus).
  • the polypeptide may be chemically modified by glycosylation, lipidation, prosthetic groups, or by containing additional amino acids such as e.g. a purification tag (e.g. his-tag) or a signal peptide.
  • Purification tags are used to obtain highly pure protein preparations.
  • the His-tag may comprise a methionine as the first amino acid followed by 6-8 histidines if used N-terminal, and 6-8 histidines followed by a STOP- codon if used C-terminal. When used N-terminal, the methionine start codon in the gene coding for the polypeptide fusion may be deleted to avoid false translational start sites.
  • Each polypeptide is encoded by a specific nucleic acid sequence. It will be under stood that such sequences include analogues and variants thereof, wherein such nucleic acid sequences have been modified by substitution, insertion, addition or deletion of one or more nucleic acid. Substitutions are preferably conservative substitutions in the codon usage, which will not lead to any change in the amino acid sequence, but may be introduced to enhance the expression of the protein. Polypeptides may be produced recombinantly or synthetically, for example, using an automated polypeptide synthesizer.
  • M. tuberculosis refers to the pathogenic bacterial species Mycobacterium tuberculosis of the family Mycobacteriaceae, which can be the causative agent of tuberculosis infection and disease.
  • Mycobacterium tuberculosis may be abbreviated as Mtb herein.
  • infection refers to any one of these 3 phases, i.e. vaccination or immunization against disease caused by a virulent mycobacterium may include the acute phase, latent/chronic phase and reactivation phase.
  • BCG Bacille Calmette-Guerin
  • BCG was developed by attenuation of Mycobacterium bovis at the Institute Pasteur almost 100 years ago and during this process, the virulent strain lost one important gene segment encoding virulence associated antigens, such as ESAT-6. This original mutation is referred to as RD1.
  • the BCG vaccine was in the subsequent 30-40 years distributed to various laboratories and production facilities worldwide. This gave rise to various vaccine substrains often named after the location of the laboratory in which it is produced (BCG Danish, Moscow, Tokyo etc). As many of these strains initially were propagated by continuous cultures, a large number of deletions of the original genome has been observed and with different distribution in different substrains. In total, at least 12 major
  • RDl-11 and the SigK mutation are reported among the BCG vaccine strains that have been analysed. Some of these deletions contain immunologically important antigens with vaccine potential and some deletions are immunologically silent.
  • the original RD1 deletion is an example of a region that contains immune dominant antigens of great importance for vaccines.
  • antigens that are strongly recognized during the natural infection; RD1, RD2 and the antigens whose expression is regulated by SigK.
  • the inclusion of antigens from all three regions has provided both a sufficiently large number of antigens to construct a powerful polyprotein-based vaccine, as well as antigens expressed in different stages of infection.
  • BCG strains may be divided into two main groups; early BCG strains and late BCG strains. Early and late BCG strains are not to be confused with early and late antigens.
  • the early BCG strains lack the RD1 region.
  • the early BCG strains comprises BCG Russia, BCG Japan, BCG Moreau, BCG Sweden and BCG Birkhaug.
  • the late BCG strains lack the RD1 and RD2 regions, and have a mutation in sigK.
  • the late BCG strains comprise BCG Tice, BCG Frappier, BCG Pasteur, BCG Danish, BCG Glaxo, BCGska, BCG China as well as the genetically modified BCG strain, VPM1002.
  • the late and early BCG strains does not possess identical genotypes and phenotypes. Thus, in the late BCG strains some antigens encoded by the RD2 region have been deleted and other antigens are poorly expressed or non- secreted. Consequently, for these missing, poorly expressed or non-secreted antigens, no immune response will be induced upon vaccination with the late BCG strains.
  • BCG + antigen refers to an antigen that is expressed by a given BCG strain and primes an immune response (as measured by IFN-y release (e.g. ELISA) in cultures of stimulated cells) upon vaccination with the given BCG strain.
  • IFN-y release e.g. ELISA
  • fusion proteins comprising BCG + antigens, when administered following the initial vaccination with a given BCG strain, may be used to boost an immune response previously induced by said given BCG strain.
  • BCG + antigens may be defined in relation to BCG strains selected from the group consisting of BCG Danish, BCG Pasteur and BCGska and/or any derivatives of these strains (e.g. VPM1002).
  • the BCG + antigens include, but are not limited to, Rvl886c (Ag85b), Rv3804c (Ag85a), Rv0288 (TB10.4), Rv0287 (EsxG), Rv3478 (PPE60), Rv0475 (HBHA), Rv3890c (EsxC), Rv3891c (EsxD), Rvl284 (CanA), Rv3019c (EsxR), Rv3020c (EsxS), Rv3017c (EsxQ), Rv2031c (HspX), Rv0983 (PepD), Rvll96 (PPE18), Rv2608 (PPE42), Rv3619 (EsxV) and Rv3620 (EsxW), and variants thereof.
  • BCG- antigen refers to an antigen that does not prime an immune response (as measured by IFN-y release) upon vaccination with a given BCG strain because it is either deleted, non-secreted or has a low expression level.
  • fusion proteins comprising BCG- antigens, when
  • BCG- antigens may be defined as functionally negative.
  • BCG antigens may be defined in relation to BCG strains selected from the group consisting of BCG Danish, BCG Pasteur and BCGska and/or any derivatives of these strains (e.g. VPM1002). Therefore, BCG antigens may be defined as functionally negative in relation to these BCG strains.
  • the BCG antigens include, but are not limited to, Rv3875 (ESAT-6), Rv3873 (PPE68), Rv3876 (espl), Rv3615c (espC), Rv3616c (espA), Rvl980c (MPT64), Rv2875 (MPT70), Rv2873 (MPT83), and variants thereof.
  • BCG antigens may be used to prime a complementary immune response, i.e. an immune response against Mtb antigens different from antigens of an initial BCG vaccination.
  • Immunogenic epitope i.e. an immune response against Mtb antigens different from antigens of an initial BCG vaccination.
  • An immunogenic epitope of a polypeptide is a part of the polypeptide, which elicits an immune response in an animal or a human being, and/or in a biological sample determined by any of the biological assays described herein.
  • the immunogenic epitope of a polypeptide may be a T-cell epitope or a B-cell epitope.
  • Immunogenic epitope can be related to one or a few relatively small parts of the polypeptide, they can be scattered throughout the polypeptide sequence or be situated in specific parts of the polypeptide. For a few polypeptides epitopes have even been demonstrated to be scattered throughout the polypeptide covering the full sequence (Ravn, 1999).
  • T-cell epitopes which are recognized during an immune response
  • a "brute force” method Since T-cell epitopes are linear, deletion mutants of the polypeptide will, if constructed systematically, reveal what regions of the polypeptide are essential in immune recognition, e.g. by subjecting these deletion mutants e.g. to the IFN-g assay described herein.
  • Another method utilises overlapping peptides for the detection of MHC class II epitopes, preferably synthetic, having a length of e.g. 20 amino acid residues derived from the polypeptide. These peptides can be tested in biological assays (e.g.
  • the IFN-g assay as described herein will give a positive response (and thereby be immunogenic) as evidence for the presence of a T cell epitope in the peptide.
  • MHC class I epitopes it is possible to predict peptides that will bind (Stryhn, 1996) and hereafter produce these peptides synthetic and test them in relevant biological assays e.g. the IFN-g assay as described herein.
  • the peptides preferably having a length of e.g. 8 to 11 amino acid residues derived from the polypeptide.
  • a polypeptide fragment of the invention has a length of at least 7 amino acid residues, such as at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, and at least 30 amino acid residues.
  • a polypeptide fragment has a length of at most 50 amino acid residues, such as at most 40, 35, 30, 25, and 20 amino acid residues. It is expected that the peptides having a length of between 10 and 30 amino acid residues will prove to be most efficient as MHC class II epitopes and therefore especially preferred lengths of the polypeptide fragment used in the inventive method are 18, such as 15, 14, 13, 12 and even 11 amino acid residues. It is expected that the peptides having a length of between 7 and 12 amino acid residues will prove to be most efficient as MHC class I epitopes and therefore especially preferred lengths of the polypeptide fragment used in the inventive method are 11, such as 10, 9, 8 and even 7 amino acid residues.
  • Immunogenic portions fragment comprising immunogenic epitopes of polypeptides, comprising the immunogenic epitope, may be recognized by a broad part (high frequency) or by a minor part (low frequency) of the genetically heterogenic human population.
  • some immunogenic portions induce high immunological responses (dominant), whereas others induce lower, but still significant, responses (subdominant).
  • High frequencyxlow frequency can be related to the immunogenic portion binding to widely distributed MHC molecules (H LA type) or even by multiple MHC molecules (Sinigaglia, 1988; Kilgus, 1991).
  • Fragments comprising immunogenic epitopes from said polypeptides can be present as overlapping peptides of at least 10 amino acid length thereby spanning several epitopes.
  • variable refers to antigens, polypeptides or fusion proteins as described herein which are “substantially homologous” to and/or retain at least a substantial amount of the immunogenicity of the antigens, polypeptides or fusion protein to which it refers.
  • substantially homologous refers to an amino acid sequence of an antigen, polypeptide or fusion protein, which have at least 80% sequence identity, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to the antigen, polypeptide or fusion protein to which it refers.
  • the term "substantial amount of the immunogenicity” refers to an antigen, polypeptide or fusion protein, which retains at least 80% of the immunogenicity, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%
  • variants of antigens, polypeptides or fusion proteins as described herein may have the same immunogenicity or at least the same immunogenicity as the antigen, polypeptide or fusion protein to which it refers.
  • antigens, polypeptides or fusion proteins with a defined sequence identity to an antigen, polypeptide or fusion protein as described herein may have the same
  • immunogenicity or at least the same immunogenicity as the antigen, polypeptide or fusion protein to which it refers.
  • the term "antigen repeat" refers to antigens in which more than one copy of the antigen is present in a fusion protein.
  • the distinct antigen copies of the antigen repeat may be positioned (i) consecutively, (ii) alternately with antigens different from the antigen repeat, or (iii) separated by antigens different from the antigen repeat in the fusion protein.
  • Antigen repeats may comprise at least two antigen repeats, such as at least three antigen repeats, such as at least four antigen repeats, such as at least five antigen repeats.
  • a fusion protein with e.g. at least three antigen repeats comprises at least three copies of said antigen.
  • fusion proteins comprising antigen repeats of ESAT-6, i.e. ESAT-6 repeats, are preferred.
  • linker molecule refers to peptide sequences that occur between antigens in a fusion protein.
  • Linkers are often composed of flexible residues like glycine and serine so that the adjacent domains of the fusion protein are free to move relative to one another and for independent proper folding during secretion/manufacturing. Longer linkers may be used, if necessary, to ensure that two adjacent domains of the fusion protein do not sterically interfere with one another. It is to be understood that linkers at the genetic level are composed of nucleic acids. Thus, nucleic acids encoding the fusion proteins as described herein may comprise linkers represented by nucleic acid sequences.
  • co-vaccination refers to either the simultaneous administration of two distinct vaccines and/or immunogenic compositions, e.g. simultaneous administration of a subunit vaccine (or fusion protein) as described herein and a BCG vaccine, or vaccination with BCG followed by subunit
  • the terms “vaccine” and “immunogenic composition” refer to a composition comprising at least one antigen which is capable of providing active acquired immunity to tuberculosis infection or disease.
  • the "vaccine” or “immunogenic composition” may preferably comprise a fusion protein as described herein, which is capable of providing active acquired immunity to tuberculosis infection or disease.
  • a vaccine or immunogenic composition as described herein is able to decrease bacterial load in target organs, prolong survival times and/or diminish weight loss of an animal after challenge with a virulent
  • Mycobacterium compared to non-vaccinated animals.
  • the vaccine or immunogenic composition may comprise an immunologically and pharmaceutically acceptable carrier or vehicle.
  • Suitable carriers include, but are not limited to, polymers to which the polypeptide is bound by hydrophobic non- covalent interaction, such as a plastic, e.g. polystyrene, or polymers to which the polypeptide is covalently bound, such as a polysaccharide, or polypeptides, e.g. bovine serum albumin, ovalbumin or keyhole limpet haemocyanin.
  • Suitable vehicles include, but are not limited to, diluents and suspending agents.
  • a vaccine or immunogenic composition preferably comprises one or more adjuvants.
  • Adjuvants include, but are not limited to, polymers to which the polypeptide is bound by hydrophobic non- covalent interaction, such as a plastic, e.g. polystyrene, or polymers to which the polypeptide is covalently bound, such as a polysaccharide, or polypeptide
  • adjuvant refers to a compound or mixture that enhances the immune response to an antigen.
  • An adjuvant can serve as a tissue depot that slowly releases the antigen and as a lymphoid system activator, which non-specifically enhances the immune response.
  • a primary challenge with an antigen alone, in the absence of an adjuvant will fail to elicit a humoral or cellular immune response.
  • Adjuvants include, but are not limited to, neutral adjuvant formulations, anionic adjuvant formulations, cationic adjuvant formulations (e.g. "CAFOl”, “CAF04”, “CAF09” and “CAF10”), cationic liposomes (e.g.
  • DDA dimethyldioctadecylammonium bromide
  • Quil A QS21, poly I :C, aluminium hydroxide, Freund's incomplete adjuvant, IFN-g, IL-2, IL-12, monophosphoryl lipid A (MPL), Trehalose Dimycolate (TDM), Trehalose Dibehenate (TDB), Muramyl Dipeptide (MDP), monomycolyl glycerol (MMG), CpG and "IC31" or combinations hereof.
  • expression vector refers to a DNA molecule used as a vehicle to transfer recombinant genetic material into a host cell.
  • the four major types of expression vectors are plasmids, bacteriophages and other viruses, cosmids, and artifical chromosomes.
  • the expression vector itself is generally a DNA sequence that consists of an insert (a heterologous nucleic acid sequence, transgene) and a larger sequence that serves as the "backbone" of the expression vector.
  • the purpose of an expression vector, which transfers genetic information to the host is typically to isolate, multiply, or express the insert in the target cell.
  • Expression vectors are specifically adapted for the expression of the heterologous sequences in the target cell, and generally have a promoter sequence that drives expression of the heterologous sequences. Operatively linked
  • operatively linked refers to the connection of elements being a part of a functional unit such as a gene or an open reading frame. Accordingly, by operatively linking a promoter to a nucleic acid sequence encoding a polypeptide the two elements becomes part of the functional unit - a gene.
  • the linking of the expression control sequence (promoter) to the nucleic acid sequence enables the transcription of the nucleic acid sequence directed by the promoter.
  • promoter promoter
  • the sequences becomes part of the functional unit - an open reading frame encoding a fusion protein comprising the amino acid sequences encoding by the heterologous nucleic acid sequences.
  • operatively linking two amino acids sequences the sequences become part of the same functional unit - a polypeptide. Operatively linking two heterologous amino acid sequences generates a hybrid (fusion) polypeptide.
  • mammal refers to any animal belonging to the class Mammalia including, but not limited to, rodents, primates, ungulates and carnivores. Ungulates include, but are not limited to cattle, horses, pigs, sheep, goat and camels.
  • the mammal is preferably a human or a domestic animal.
  • sequence identity refers to the sequence identity between genes or proteins at the nucleotide, base or amino acid level, respectively. Specifically, a DNA and a RNA sequence are considered identical if the transcript of the DNA sequence can be transcribed to the identical RNA sequence.
  • sequence identity is a measure of identity between proteins at the amino acid level and a measure of identity between nucleic acids at nucleotide level.
  • the protein sequence identity may be determined by comparing the amino acid sequence in a given position in each sequence when the sequences are aligned.
  • the nucleic acid sequence identity may be determined by comparing the nucleotide sequence in a given position in each sequence when the sequences are aligned.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the two sequences are of different length and gaps are seen as different positions.
  • One may manually align the sequences and count the number of identical amino acids.
  • alignment of two sequences for the determination of percent identity may be accomplished using a mathematical algorithm.
  • Such an algorithm is incorporated into the NBLAST and XBLAST programs of (Altschul et al. 1990).
  • Gapped BLAST may be utilized.
  • PSI-Blast may be used to perform an iterated search, which detects distant relationships between molecules.
  • NBLAST, XBLAST, and Gapped BLAST programs the default parameters of the respective programs may be used. See http://www.ncbi.nlm.nih.gov.
  • sequence identity may be calculated after the sequences have been aligned e.g. by the BLAST program in the EMBL database (www.ncbi.nlm.gov/cgi-bin/BLAST).
  • the default settings with respect to e.g. "scoring matrix” and "gap penalty” may be used for alignment.
  • the BLASTN and PSI BLAST default settings may be advantageous.
  • the percent identity between two sequences may be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted.
  • An embodiment of the present invention thus relates to sequences of the present invention that has some degree of sequence variation.
  • fusion proteins comprising a selection of antigens for production of a tuberculosis subunit vaccine.
  • This type of tuberculosis vaccine is believed to possess a series of advantages over other vaccine strategies, including improved safety and stability of the vaccine.
  • By careful design of the fusion proteins strong cellular immune responses of high quality T cells can be induced.
  • fusion proteins that may be effectively used in a vaccine or immunogenic composition for prevention, inhibition or treatment of tuberculosis infection and/or disease.
  • an aspect of the present invention relates to a fusion protein comprising at least two antigens that originate from M. tuberculosis.
  • the genome of Mycobacterium tuberculosis contains approximately 4000 genes, many of which encode proteins that are not suitable as antigens in a subunit vaccine against tuberculosis infection or disease as they do not induce a immune response of sufficient strength or quality.
  • the antigens of the fusion proteins as described herein are carefully selected for their immunogenicity, and include, but are not limited to, Rv3875 (ESAT-6), Rv3873 (PPE68), Rv3876 (espl),
  • Rv3615c espC
  • Rv3616c espA
  • Rvl980c MPT64
  • Rv2875 MPT70
  • Rv2873 MPT83
  • Rvl886c Ag85b
  • Rv3804c Ag85a
  • Rv0288 T10.4
  • Rv0287 EsxG
  • Rv3478 PPE60
  • Rv0475 HBHA
  • Rv3890c (EsxC Rv3891c (EsxD
  • Rvl284 CanA
  • Rv3019c EsxR
  • Rv3020c EsxS
  • Rv3017c EsxQ
  • Rv2031c HspX
  • Rv0983 PepD
  • Rvll96 PPE18
  • Rv2608 PPE42
  • Rv3619 EsxV
  • Rv3620 EsxW
  • Rv2660c Rv3614 (EspD)
  • an embodiment of the present invention relates to the fusion protein as described herein, wherein the antigens are selected from the group consisting of SEQ ID NO: 1 (ESAT-6), SEQ ID NO: 2 (PPE68), SEQ ID NO: 3 (espl), SEQ ID NO:4 (espC), SEQ ID NO: 5 (espA), SEQ ID NO: 6 (MPT64), SEQ ID NO: 7 (MPT70), SEQ ID NO: 8 (MPT83), SEQ ID NO: 10 (Ag85b), SEQ ID NO: 11 (Ag85a), SEQ ID NO: 12 (TB10.4), SEQ ID NO: 13 (EsxG), SEQ ID NO: 14 (PPE60), SEQ ID NO: 15 (HBHA), SEQ ID NO: 16 (EsxC), SEQ ID NO: 17 (EsxD), SEQ ID NO: 18 (CanA), SEQ ID NO: 19 (EsxR), SEQ ID NO: 20 (EsxS), SEQ ID NO:
  • Another embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises at least three antigens, such as at least four antigens, such as at least five antigens, such as at least six antigens, such as at least seven antigens, such as at least eight antigens.
  • M. tuberculosis The course of a M. tuberculosis infection runs essentially through 3 phases. During the acute phase, the bacteria proliferate in the organs, until the immune response increases. Specifically sensitized CD4 T lymphocytes mediate control of the infection, and the most important mediator molecule seems to be interferon gamma (IFN-gamma). The bacterial loads start to decline and a latent/chronic phase is established in which the bacterial load is kept stable at a low level. In this phase M. tuberculosis goes from active multiplication to dormancy, essentially becoming non-replicating and remaining inside the granuloma. In some cases, the infection goes to the reactivation phase, where the dormant bacteria starts replicating again.
  • IFN-gamma interferon gamma
  • a fusion protein comprising both antigens that are highly expressed in the early and late stage of infection, respectively, may be employed in a potent vaccine that may (i) accelerate the immune response after Mtb infection and (ii) induce T cells with the potential of specifically recognizing the bacteria in the late chronic phase.
  • the benefits of such a fusion protein design extend also to improved epitope coverage and the ability to target both acute and latent/chronic infections.
  • an embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises both early and late antigens.
  • Another embodiment of the present invention relates to the fusion protein as described herein, wherein the early antigens are selected from the group consisting of Rv3875 (ESAT-6), Rv3873 (PPE68), Rv3876 (espl), Rv3615c (espC), Rv3616c (espA), Rvl886c (Ag85b), Rv3804c (Ag85a), Rv0288 (TB10.4), Rv0287 (EsxG), Rv3478 (PPE60), Rv0475 (HBHA), Rv3890c (EsxC), Rv3891c (EsxD), Rvl284 (CanA), Rv3019c (EsxR), Rv3020c (EsxS), Rv3017c (EsxQ), Rv0983 (PepD), Rvll
  • a further embodiment of the present invention relates to the fusion protein as described herein, wherein the late antigens are selected from the group consisting of Rv2875 (MPT70), Rv2873 (MPT83), Rv2031c (HspX) and Rv2660c, and variants thereof.
  • BCG + antigens tuberculosis antigens that are expressed by BCG and have already primed an immune response upon initial BCG vaccination.
  • BCG + antigens tuberculosis antigens that are expressed by BCG and have already primed an immune response upon initial BCG vaccination.
  • BCG + antigens tuberculosis antigens that are expressed by BCG and have already primed an immune response upon initial BCG vaccination.
  • BCG + antigens Such antigens are herein referred to as BCG + antigens and may be administered as part of a subunit vaccine with the aim of boosting the immune response induced by the initial BCG vaccination.
  • mycobacterial infections including BCG vaccination and Mtb infection, induce T cells with poor T cell quality and these have proven to be extremely difficult to reprogram upon boosting with subunit vaccines with disappointing efficacy as the result.
  • tuberculosis antigens which are functionally negative with regards to BCG, i.e. antigens that do not prime an immune response upon vaccination with BCG, may be utilized in a fusion protein to provide a markedly enhanced protection against M. tuberculosis.
  • antigens are herein referred to as BCG- antigens and may be used in a fusion protein as part of a stand-alone vaccine or in combination with BCG to prime a complementary immune response against BCG antigens different from antigens of a primary BCG vaccination.
  • a BCG vaccination is principally a mycobacterium infection, which due to chronic antigen stimulation induces short lived T cells that only to a poor degree are capable of penetrating the lung tissue and combat the infection.
  • This footprint or immunological heritage left behind by the primary immune response of the BCG vaccination is very difficult to change subsequently.
  • the fusion proteins disclosed herein are in variations based on BCG- antigens, which are not constrained by this immunological heritage and can therefore circumvent a barrier for T cell quality that is normally limiting for traditional subunit vaccines based on BCG + antigens.
  • variations of the fusion proteins described herein are based on antigens that do not prime an immune response against BCG, i.e. BCG- antigens.
  • the fusion proteins provided herein yield improved immune responses compared to existing tuberculosis vaccines.
  • an embodiment of the present invention relates to the fusion protein as described herein, wherein at least one antigen does not prime an immune response against BCG.
  • Another embodiment of the present invention relates to the fusion protein as described herein, wherein at least one antigen is functionally negative with regards to BCG.
  • a further embodiment of the present invention relates to the fusion protein as described herein, wherein at least one antigen is a BCG- antigen.
  • an embodiment of the present invention relates to the fusion protein as described herein, wherein BCG is selected from a late BCG strain, preferably BCG Danish, BCG Pasteur or BCG Prague.
  • Another embodiment of the present invention relates to the fusion protein as described herein, wherein at least one antigen does not prime an immune response against a late BCG strain.
  • a further embodiment of the present invention relates to the fusion protein as described herein, wherein at least one antigen does not prime an immune response against a BCG strain selected from the group consisting of BCG Danish, BCG Pasteur and BCG Prague.
  • the late BCG strains may also be characterized by (i) the absence of some antigens encoded by the RD2 region which have been deleted and (ii) poor expression or non-secretion of other antigens. Consequently, for these missing, poorly expressed or non-secreted antigens, no immune response will be induced upon vaccination with the late BCG strains.
  • an embodiment of the present invention relates to the fusion protein as described herein, wherein the antigens are deleted from, non-secreted or have low-expression in BCG.
  • Another embodiment of the present invention relates to the fusion protein as described herein, wherein the antigens are deleted from, non-secreted or have low- expression in a late BCG strain, preferably the late BCG strain is selected from the group consisting of BCG Danish, BCG Pasteur and BCGska.
  • a further embodiment of the present invention relates to the fusion protein as described herein, wherein the antigens are selected from the group consisting of antigens located within RD1, antigens located within RD2 and antigens whose expression is regulated by SigK, and combinations thereof.
  • BCG- antigens that are suitable to be included in a fusion protein for use in a vaccine against tuberculosis infection and/or disease.
  • an embodiment of the present invention relates to the fusion protein as described herein, wherein the antigens are selected from the group consisting of Rv3875 (ESAT-6), Rv3873 (PPE68), Rv3876 (espl), Rv3615c (espC), Rv3616c (espA), Rvl980c (MPT64), Rv2875 (MPT70) and Rv2873 (MPT83), and variants thereof. It is to be understood that variants of antigens, polypeptides or fusion proteins as described herein may have the same immunogenicity, i.e.
  • SEQ ID NO: 1 amino acid sequences selected from the group consisting of SEQ ID NO: 1 (ESAT-6), SEQ ID NO: 2 (PPE68), SEQ ID NO: 3 (espl), SEQ ID NO:4 (espC), SEQ ID NO: 5 (espA), SEQ ID NO: 6 (MPT64), SEQ ID NO: 7
  • an embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises an amino acid sequence selected from :
  • a preferred embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises:
  • SEQ ID NO: 1 ESAT-6
  • SEQ ID NO: 2 PPE68
  • SEQ ID NO: 3 espl
  • SEQ ID NO:4 espC
  • SEQ ID NO: 5 espA
  • Another embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises an amino acid sequence selected from :
  • a further embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises:
  • SEQ ID NO: 1 SEQ ID NO: 6
  • PPE68 SEQ ID NO: 3
  • SEQ ID NO:4 espC
  • SEQ ID NO: 5 espA
  • SEQ ID NO: 6 MPT64
  • SEQ ID NO: 7 MPT70
  • SEQ ID NO: 8 MPT83
  • Yet another embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises an amino acid sequence selected from :
  • SEQ ID NO: 37 H105 or variants or immunogenic epitopes thereof, or b) amino acid sequence having at least 80% sequence identity, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to any one of the amino acid sequences of a).
  • a further embodiment of the present invention relates to the fusion protein as described herein, wherein the amino acid sequences of b) have at least 90% sequence identity, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to any one of the amino acid sequences of a).
  • antigens, polypeptides or fusion proteins with a defined sequence identity to an antigen, polypeptide or fusion protein as described herein may have the same immunogenicity, i.e. they are equally effective in inducing an immune response, or at least the same immunogenicity as the antigen, polypeptide or fusion protein to which it refers.
  • a preferred embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises both early and late antigens that does not prime an immune response against BCG ⁇ i.e. BCG- antigens).
  • Another embodiment of the present invention relates to the fusion protein as described herein, wherein the late antigen that does not prime an immune response against BCG is Rv2875 (MPT70) and/or Rv2873 (MPT83), and variants thereof.
  • a further embodiment of the present invention relates to the fusion protein as described herein, wherein the early antigen that does not prime an immune response against BCG is selected from the group consisting of Rv3875 (ESAT-6), Rv3873 (PPE68), Rv3876 (espl), Rv3615c (espC) and Rv3616c (espA), and variants thereof.
  • the early antigen that does not prime an immune response against BCG is selected from the group consisting of Rv3875 (ESAT-6), Rv3873 (PPE68), Rv3876 (espl), Rv3615c (espC) and Rv3616c (espA), and variants thereof.
  • fusion proteins disclosed herein are restricted to containing exclusively BCG- antigens.
  • an embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises only antigens that does not prime an immune response against BCG ⁇ i.e. BCG- antigens).
  • ESAT-6 6 kDa early secretory antigenic target
  • ESAT-6 protein which is constitutively expressed and secreted in large amount by the bacterium, is recognized in both humans and animals following infection. Thus, ESAT-6 is acknowledged to be an important antigen that may be advantageously included in subunit vaccines against tuberculosis infection and disease. However, since the native ESAT-6 molecule is a small protein of only 95 amino acids, the magnitude of the immune response raised against ESAT-6 in humans and animals is relative low.
  • fusion proteins which comprise several copies of ESAT-6 so that ESAT-6 relatively represents a larger fraction of the fusion protein.
  • ESAT-6 relatively represents a larger fraction of the fusion protein.
  • increasing the relative content of ESAT-6 antigen in the fusion proteins mitigates the challenge with native ESAT-6 having low immunogenicity.
  • each occurrence of the antigen is referred to as an ESAT-6 repeat, e.g. a fusion protein comprising four copies of the ESAT-6 antigen is said to comprise four ESAT-6 repeats.
  • an embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises at least two ESAT-6 repeats, such as at least three ESAT-6 repeats, such as at least four ESAT-6 repeats, such as at least five ESAT-6 repeats.
  • a preferred embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises at least four ESAT-6 repeats.
  • Another embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises four ESAT-6 repeats.
  • each ESAT-6 repeat is represented by SEQ ID NO: l or an amino acid sequence having at least 80 % sequence identity, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to SEQ ID NO: 1.
  • the ESAT-6 repeats may in principal be distributed in any order in the fusion proteins, such as at the C-terminal, N-terminal, consecutively, alternating, or separated.
  • An embodiment of the present invention relates to the fusion protein as described herein, wherein the ESAT-6 repeats are separated by at least one antigen different from ESAT-6.
  • Another embodiment of the present invention relates to the fusion protein as described herein, wherein the ESAT-6 repeats are positioned alternately with antigens different from ESAT-6.
  • Antigens suitable for use in fusion proteins with ESAT-6 repeats are preferably highly immunogenic and is recognized in both humans and animals following tuberculosis infection.
  • antigens suitable for use in a fusion protein together with ESAT-6 repeats include, but are not limited to Rv3873 (PPE68), Rv3876 (espl), Rv3615c (espC), Rv3616c (espA), Rvl980c (MPT64), Rv2875 (MPT70), Rv2873 (MPT83), Rvl886c (Ag85b), Rv3804c (Ag85a), Rv0288 (TB10.4), Rv0287 (EsxG), Rv3478 (PPE60), Rv0475 (HBHA), Rv3890c (EsxC), Rv3891c (EsxD), Rvl284 (CanA), Rv3019c (EsxR), Rv3020c (EsxS), Rv3017c (EsxQ), Rv2031c (HspX), Rv0983 (PepD), Rvll96 (PPE18), Rv2608
  • an embodiment of the present invention relates to the fusion protein as described herein, wherein the antigens are selected from the group consisting of SEQ ID NO: 2 (PPE68), SEQ ID NO: 3 (espl), SEQ ID NO:4 (espC), SEQ ID NO: 5 (espA), SEQ ID NO: 6 (MPT64), SEQ ID NO: 7 (MPT70), SEQ ID NO: 8 (MPT83),
  • SEQ ID NO: 10 (Ag85b), SEQ ID NO: 11 (Ag85a), SEQ ID NO: 12 (TB10.4), SEQ ID NO: 13 (EsxG), SEQ ID NO: 14 (PPE60), SEQ ID NO: 15 (HBHA), SEQ ID NO: 16 (EsxC), SEQ ID NO: 17 (EsxD), SEQ ID NO: 18 (CanA), SEQ ID NO: 19 (EsxR), SEQ ID NO: 20 (EsxS), SEQ ID NO: 21 (EsxQ), SEQ ID NO: 22 (HspX), SEQ ID NO: 23 (PepD), SEQ ID NO: 24 (PPE18), SEQ ID NO: 25 (PPE42), SEQ ID NO: 26 (EsxV), SEQ ID NO: 27 (EsxW), SEQ ID NO: 28 (Rv2660c), SEQ ID NO: 29 (Rv3614), SEQ ID NO: 30 (Rv3865), SEQ ID NO: 31 (Rv
  • the fusion proteins as described herein include also fusion proteins comprising both early and late antigens or BCG- antigens in combination with ESAT-6 repeats. Therefore, a preferred embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises both early and late antigens, and at least two ESAT-6 repeats, such as at least three ESAT-6 repeats, such as at least four ESAT-6 repeats, such as at least five ESAT-6 repeats.
  • Another preferred embodiment of the present invention relates to the fusion protein as described herein, wherein at least one antigen does not prime an immune response against BCG, and wherein the fusion protein comprises at least two ESAT-6 repeats, such as at least three ESAT-6 repeats, such as at least four ESAT-6 repeats, such as at least five ESAT-6 repeats.
  • An embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises:
  • SEQ ID NO: 1 SEQ ID NO: 6
  • PPE68 SEQ ID NO: 3
  • SEQ ID NO:4 espC
  • SEQ ID NO: 5 espA
  • SEQ ID NO: 6 MPT64
  • SEQ ID NO: 7 MPT70
  • SEQ ID NO: 8 MPT83
  • the fusion protein comprises four ESAT-6 repeats positioned alternately with antigens different from ESAT-6.
  • Another particularly preferred embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises both early and late BCG- antigens in combination with ESAT-6 repeats.
  • a variant of such a fusion protein is termed H107 (SEQ ID NO:9).
  • fusion protein as described herein, wherein the fusion protein comprises the amino acid sequence represented by SEQ ID NO:9.
  • fusion protein comprises:
  • SEQ ID NO:9 H107 or variants or immunogenic epitopes thereof, or b) amino acid sequence having at least 80% sequence identity, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to any one of the amino acid sequences of a), and
  • amino acid sequence of b) have the same immunogenicity or at least the same immunogenicity as SEQ ID NO:9.
  • H107e A variant of the H107 fusion protein is termed H107e (SEQ ID NO:91).
  • a preferred embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises the amino acid sequence represented by SEQ ID NO:91.
  • Another embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises:
  • SEQ ID NO:91 H107e or variants or immunogenic epitopes thereof, or b) amino acid sequence having at least 80% sequence identity, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to any one of the amino acid sequences of a), and
  • amino acid sequence of b) have the same immunogenicity or at least the same immunogenicity as SEQ ID NO:91.
  • the antigens of the fusion proteins described herein may be connected through linkers, such as peptide linkers.
  • linkers such as peptide linkers.
  • an embodiment of the present invention relates to the fusion protein as described herein, wherein the antigens of the fusion proteins are connected with a linker molecule.
  • an embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises a purification tag.
  • the purification tag is selected from the group consisting of His-tag, chitin binding protein (CBP), maltose binding protein (MBP) and glutathione-S-transferase (GST).
  • CBP chitin binding protein
  • MBP maltose binding protein
  • GST glutathione-S-transferase
  • a further embodiment of the present invention relates to the fusion protein as described herein, wherein the purification tag is a His-tag.
  • Variations of the fusion proteins may include BCG + antigens. Therefore, an embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises at least one antigen that does prime an immune response against BCG. Another embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises at least one BCG + antigen.
  • Yet another embodiment of the present invention relates to the fusion protein as described herein, wherein the at least one antigen that does prime an immune response against BCG is selected from the group consisting of Rvl886c (Ag85b), Rv3804c (Ag85a), Rv0288 (TB10.4), Rv0287 (EsxG), Rv3478 (PPE60), Rv0475 (HBHA), Rv3890c (EsxC), Rv3891c (EsxD), Rvl284 (CanA), Rv3019c (EsxR), Rv3020c (EsxS), Rv3017c (EsxQ), Rv2031c (HspX), Rv0983 (PepD), Rvll96 (PPE18), Rv2608 (PPE42), Rv3619 (EsxV) and Rv3620 (EsxW), and variants thereof.
  • a further embodiment of the present invention relates to the fusion protein as described herein, where
  • the fusion proteins disclosed herein are immunogenic and may be used effectively used in a vaccine or immunogenic composition for prevention, inhibition or treatment of tuberculosis infection and/or disease.
  • a vaccine or immunogenic composition for prevention, inhibition or treatment of tuberculosis infection and/or disease.
  • immunogenic composition may be used prophylactically or therapeutically.
  • an aspect of the present invention relates to a vaccine or immunogenic composition comprising a fusion protein as described herein.
  • the vaccine or immunogenic composition comprises an immunologically and
  • an embodiment of the present invention relates to the vaccine or immunogenic composition as described herein, wherein the vaccine or immunogenic composition further comprises an immunologically and pharmaceutically acceptable carrier, vehicle or adjuvant.
  • Another embodiment of the present invention relates to the vaccine or immunogenic composition as described herein, wherein the vaccine or
  • immunogenic composition further comprises one or more adjuvants.
  • adjuvants are selected from the group consisting of neutral adjuvant formulations, anionic adjuvant formulations, cationic adjuvant formulations, cationic liposomes (e.g.
  • dimethyldi- octadecylammonium bromide DDA
  • Quil A QS21, poly I :C, aluminium hydroxide
  • Freund's incomplete adjuvant IFN-g, IL-2, IL-12, monophosphoryl lipid A (MPL), Trehalose Dimycolate (TDM), Trehalose Dibehenate (TDB), Muramyl Dipeptide (MDP), monomycolyl glycerol (MMG), CpC and "IC31", or combinations hereof.
  • An embodiment of the present invention relates to the vaccine or immunogenic composition as described herein, wherein the adjuvant is cationic adjuvant formulation 1 (CAFOl).
  • the adjuvant is cationic adjuvant formulation 10 (CAF10) comprising DDA, MMG and CpG.
  • cationic adjuvant formulations which may be used as an adjuvant in a vaccine or immunogenic composition according to the present invention are listed in table 1 below:
  • DDA N,N-d imethyl-N,N-d ioctadecyla mmon ium (Bromide sa lt) .
  • DSPC l ,2-Distea royl-sn-g lycero-3-phosphochol ine.
  • DSPE-PEG 1,2- d istea royl- sn-glycero-3-phosphoethanolamine-N- [carboxy(polyethyleneglycol)-2000] (sodium salt).
  • TDB a,a-trehalose 6,6'-dibehenate.
  • MMG Synthetic mono-mycolyl glycerol.
  • Poly-IC Polyinosinic-polycytidylic acid (sodium salt).
  • CpG 5'-C-phosphate-G-3' oligonucleotide.
  • MPL Monophosphoryl lipid A.
  • fusion proteins disclosed herein may be used either in a stand-alone vaccine or in combination with BCG vaccine.
  • an embodiment of the present invention relates to the vaccine or immunogenic composition as described herein, wherein the vaccine or immunogenic composition further comprises BCG.
  • Another aspect of the present invention relates to a fusion protein as described herein or a vaccine or immunogenic composition as described herein for use in vaccination or immunization of a subject against infections and/or disease caused by a virulent mycobacterium.
  • Virulent mycobacteria may infect a wide variety of animals and can in some settings pose a challenge for e.g. farm animals.
  • an embodiment of the present invention relates to the fusion protein, vaccine or immunogenic
  • a preferred embodiment of the present invention relates to the fusion protein, vaccine or immunogenic composition for use as described herein, wherein the mammal is a human.
  • Tuberculosis may be caused by different virulent mycobacteria. Therefore, an embodiment of the present invention relates to the fusion protein, vaccine or immunogenic composition for use as described herein, wherein the virulent mycobacterium is selected from the group consisting of M. tuberculosis , M. bovis , M. africanum , M. canetti, and M. microti, preferably M. tuberculosis.
  • a preferred embodiment of the present invention relates to the fusion protein, vaccine or immunogenic composition for use as described herein, wherein the virulent mycobacterium is M. tuberculosis.
  • Any of the conventional methods for administration of a vaccine are applicable and include, but are not limited to, oral formulations, suppositories, parenterally, and by injection, such as subcutaneously or intramuscularly.
  • An embodiment of the present invention relates to the fusion protein, vaccine or immunogenic composition for use as described herein, wherein the fusion protein, vaccine or immunogenic composition is administered by a route selected from the group consisting of orally, parenterally, subcutaneously and intramuscularly.
  • the dosage of the vaccine will depend on the route of administration and varies according to the age of the person to be vac cinated and, to a lesser degree, the size of the person to be vaccinated.
  • the fusion protein, vaccine or immunogenic composition may advantageously be administered in combination with BCG vaccine as described herein.
  • an embodiment of the present invention relates to the fusion protein, vaccine or immunogenic composition for use as described herein, wherein BCG is
  • fusion protein, vaccine or immunogenic composition when administered simultaneously with the fusion protein, vaccine or immunogenic composition as disclosed herein, may provide a strong adjuvant effect resulting in a strong immune response of high quality.
  • a preferred embodiment of the present invention relates to the fusion protein, vaccine or immunogenic composition for use as described herein, wherein BCG is administered simultaneously with administration of the fusion protein, vaccine or immunogenic composition.
  • a further embodiment of the present invention relates to the fusion protein, vaccine or immunogenic composition for use as described herein, wherein the subject has previously been vaccinated with BCG.
  • an aspect of the present invention relates to a kit comprising :
  • An embodiment of the present invention relates to the kit as described herein, wherein i) and ii) are for simultaneous, separate or sequential administration.
  • the fusion proteins as disclosed herein may be produced recombinantly by designing expression vector constructs encoding the fusion proteins and
  • an aspect of the present invention relates to a nucleic acid sequence comprising a sequence encoding a fusion protein as described herein.
  • Another aspect of the present invention relates to a recombinant expression vector comprising a nucleotide sequence as described herein operatively linked to one or more control sequences suitable for directing the production of the fusion protein in a suitable host.
  • a further aspect of the present invention relates to a recombinant host cell comprising an expression vector as described herein.
  • Expression vectors suitable for production of the fusion proteins disclosed herein may include nucleic acids selected from the group consisting of SEQ ID NO:46 (ESAT-6), SEQ ID NO:47 (PPE68), SEQ ID NO:48 (espl), SEQ ID NO:49 (espC), SEQ ID NO: 50 (espA), SEQ ID NO: 51 (MPT64), SEQ ID NO: 52 (MPT70), SEQ ID NO: 53 (MPT83), SEQ ID NO: 55 (Ag85b), SEQ ID NO: 56 (Ag85a), SEQ ID NO: 57 (TB10.4), SEQ ID NO: 58 (EsxG), SEQ ID NO: 59 (PPE60), SEQ ID NO: 60 (HBHA), SEQ ID NO: 61 (EsxC), SEQ ID NO: 62 (EsxD), SEQ ID NO: 63 (CanA), SEQ ID NO: 64 (EsxR), SEQ ID NO: 65 (EsxS), SEQ ID NO
  • selected fusion proteins may be encoded by nucleic acids selected from the group consisting of SEQ ID NO: 54 (H107), SEQ ID NO: 79 (H107b), SEQ ID NO: 80 (H107c), SEQ D NO: 92 (H107e), SEQ ID NO: 81 (H106), SEQ ID NO: 82 (H105) and SEQ ID NO: 83 (H104), and variants thereof.
  • Table 2 shows an overview of antigens and fusion proteins along with sequence numbers and expression pattern :
  • a fusion protein comprising at least two antigens that originate from M.
  • fusion protein according to item 1, wherein the fusion protein comprises at least three antigens, such as at least four antigens, such as at least five antigens, such as at least six antigens, such as at least seven antigens, such as at least eight antigens.
  • fusion protein according to any one of items 1 or 2, wherein the fusion protein comprises both early and late antigens.
  • fusion protein according to any one of the preceding items, wherein the antigens are deleted from, non-secreted or have low-expression in BCG.
  • Rv3875 ESAT-6
  • Rv3873 PPE68
  • Rv3876 espl
  • Rv3615c espC
  • Rv3616c espA
  • Rvl980c MPT64
  • Rv2875 MPT70
  • Rv2873 MPT83
  • amino acid sequences selected from the group consisting of SEQ ID NO: 1 (ESAT-6), SEQ ID NO: 2 (PPE68), SEQ ID NO: 3 (espl), SEQ ID NO:4 (espC), SEQ ID NO: 5 (espA), SEQ ID NO: 6 (MPT64), SEQ ID NO: 7 (MPT70) and SEQ ID NO: 8 (MPT83) and variants or immunogenic epitopes thereof, or
  • amino acid sequences having at least 80% sequence identity such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to any one of the amino acid sequences of a).
  • fusion protein according to any one of the preceding items, wherein the fusion protein comprises:
  • SEQ ID NO: 1 (ESAT-6), SEQ ID NO: 2 (PPE68), SEQ ID NO: 3 (espl), SEQ ID NO:4 (espC), and SEQ ID NO: 5 (espA) or variants or immunogenic epitopes thereof, or b) amino acid sequences having at least 80% sequence identity, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to any one of the amino acid sequences of a).
  • fusion protein according to any one of the preceding items, wherein the fusion protein comprises:
  • SEQ ID NO: 1 SEQ ID NO: 6
  • PPE68 SEQ ID NO: 3
  • SEQ ID NO:4 espC
  • SEQ ID NO: 5 espA
  • SEQ ID NO: 6 MPT64
  • SEQ ID NO: 7 MPT70
  • SEQ ID NO: 8 MPT83
  • amino acid sequences having at least 80% sequence identity such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to any one of the amino acid sequences of a).
  • amino acid sequences of b) have at least 90% sequence identity, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to any one of the amino acid sequences of a).
  • fusion protein according to any one of the preceding items, wherein the fusion protein comprises at least two ESAT-6 repeats, such as at least three ESAT- 6 repeats, such as at least four ESAT-6 repeats, such as at least five ESAT-6 repeats.
  • SEQ ID NO: 1 SEQ ID NO: 6
  • PPE68 SEQ ID NO: 3
  • SEQ ID NO:4 espC
  • SEQ ID NO: 5 espA
  • SEQ ID NO: 6 MPT64
  • SEQ ID NO: 7 MPT70
  • SEQ ID NO: 8 MPT83
  • amino acid sequences having at least 80% sequence identity such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to any one of the amino acid sequences of a);
  • the fusion protein comprises four ESAT-6 repeats positioned alternately with antigens different from ESAT-6.
  • fusion protein comprises the amino acid sequence represented by any one selected from the group consisting of:
  • amino acid sequences having at least 80% sequence identity such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to any one of the amino acid sequences of a).
  • fusion protein according to any one of claims, wherein the fusion protein is encoded by nucleic acid sequences selected from the group consisting of:
  • fusion protein according to any one of the preceding items, wherein the fusion protein comprises at least one antigen that does prime an immune response against BCG.
  • the fusion protein according to item 23, wherein the at least one antigen that does prime an immune response against BCG is selected from the group consisting of Rvl886c (Ag85b), Rv3804c (Ag85a), Rv0288 (TB10.4), Rv0287 (EsxG), Rv3478 (PPE60), Rv0475 (HBHA), Rv3890c (EsxC), Rv3891c (EsxD), Rvl284 (CanA), Rv3019c (EsxR), Rv3020c (EsxS), Rv3017c (EsxQ), Rv2031c (HspX), Rv0983 (PepD), Rvll96 (PPE18), Rv2608 (PPE42), Rv3619 (EsxV) and Rv3620 (EsxW), and variants thereof.
  • Rvl886c Ag85b
  • Rv3804c Ag85a
  • Rv0288 TB10.4
  • fusion protein according to any one of items 23 or 24, wherein the at least one antigen that does prime an immune response against BCG is selected from :
  • a vaccine or immunogenic composition comprising a fusion protein according to any one of the preceding items.
  • the adjuvants are selected from the group consisting of neutral adjuvant formulations, anionic adjuvant formulations, cationic adjuvant formulations, cationic liposomes (e.g. dimethyldioctadecylammonium bromide (DDA)), Quil A, QS21, poly I:C, aluminium hydroxide, Freund's incomplete adjuvant, IFN-g, IL-2, IL-12,
  • DDA dimethyldioctadecylammonium bromide
  • MPL monophosphoryl lipid A
  • TDM Trehalose Dimycolate
  • TTB Trehalose Dibehenate
  • MDP Muramyl Dipeptide
  • MMG monomycolyl glycerol
  • immunogenic composition according to any one of items 26-29 for use in vaccination or immunization of a subject against infections and/or disease caused by a virulent mycobacterium.
  • fusion protein, vaccine or immunogenic composition for use according to item 31, wherein the mammal is a human.
  • the fusion protein, vaccine or immunogenic composition for use according to any one of items 30-32, wherein the virulent mycobacterium is selected from the group consisting of M. tuberculosis , M. bovis, M. africanum , M. canetti, and M. microti, preferably M. tuberculosis.
  • the fusion protein, vaccine or immunogenic composition for use according to any one of items 30-33, wherein BCG is administered prior to, simultaneously or subsequent to administration of the fusion protein, vaccine or immunogenic composition.
  • fusion protein, vaccine or immunogenic composition for use according to item 34, wherein BCG is administered simultaneously with administration of the fusion protein, vaccine or immunogenic composition.
  • a kit comprising :
  • a nucleic acid sequence comprising a sequence encoding a fusion protein according to any one of items 1-25.
  • a recombinant expression vector comprising a nucleotide sequence according to item 39 operatively linked to one or more control sequences suitable for directing the production of the fusion protein in a suitable host.
  • a recombinant host cell comprising an expression vector according to item 40.
  • Example 1 BCG vaccination does not prime an immune response against the M. tuberculosis antigens of the H104-H107 fusion proteins
  • mice Six to ten-week old female CB6F1 mice were immunized with M. bovis BCG Danish or infected with 25-50 CFU virulent Mtb Erdman by the aerosol route or
  • concentration of cytokine IFN-g was measured by sandwich ELISA in the cell supernatants.
  • BCG non-BCG
  • M. bovis BCG Danish immunization did not induce a cellular immune response against any of the eight vaccine antigens candidates.
  • all antigen candidates gave rise to an immune response after Mtb infection or single protein immunization.
  • the antigens of H104-H107 do not share antigens with M. bovis BCG Danish and are specific for Mtb.
  • BCG- antigens antigens that BCG immunization raises a T cell response against are herein referred to as BCG + antigens, e.g. TB10.4.
  • Mycobacterium tuberculosis strain H37Rv The synthetic DNA sequences were codon optimized for expression in Escherichia coli and cloned into the pJ411 expression vector under the control of the inducible promoter T7 promoter and transformed into Escherichia coli.
  • the pJ411 vector encodes an N-terminal His-tag in frame with the coding sequence for the inserted recombinant protein. For each protein, the expression of the His-tagged recombinant protein was induced when the in vitro culture reached a density of ⁇ 0.5 OD6oo. The following day the bacteria were harvested, lysed, and the recombinant protein was purified using a three-step purification process. These included removing soluble E.
  • coli protein from the precipitated recombinant proteins (inclusion bodies), immobilized metal affinity chromatography and either anion or cation exchange, depending on the protein charge. After purification, the identity of the purified protein was confirmed by mass-spectroscopy and the purity by scanning Coomassie stained SDS-gels. Finally, the protein concentration was determined. Results
  • fusion proteins with BCG- antigens were designed.
  • a selection of recombinant fusion proteins are schematically illustrated in ( Figure 2 and 3).
  • the yield of purified fusion protein from 6L starting culture varied from 0.6 mg to 20 mg.
  • the concentration of the purified protein varied from 0.2 mg/ml to 0.7 mg/ml.
  • the purity was 95% or better.
  • MTP70 is an antigen wherein the expression is uprequlated during late stage Mtb infection, i.e. a "late antigen"
  • mice Female CB6F1 mice were aerosolly infected with 25-50 CFU virulent Mtb Erdman. Three, twelve and twenty weeks later mice were sacrificed for immunological analysis. Lungs were mildly homogenized using T-tubes GentleMACS (Miltenyi Biotec) and digested using collagenase IV (Sigma-Aldrich) for 30-60 minutes at 37°C. Single cell suspensions were obtained by passing the tissue through 100 pm cell strainers. Cells were washed twice in RPMI before antigen stimulation for intracellular cytokine staining (ICS) and ELISA.
  • ICS cytokine staining
  • ICS A total of 1-2 x 10 6 lung cells were stimulated in vitro for 1 hour in RPMI + 10% FCS containing 1 pg/ml anti-CD28 and anti-CD49d with or without 5 pg of ESAT-6 or MPT70 pepmix followed by 5 hours in the presence of 10 pg/ml brefeldin A (Sigma-Aldrich) at 37°C in an automated heater that cooled the cells to 4°C after incubation. The next day, cells were washed in FACS buffer (lx PBS containing 1% FCS) and stained at 4°C for surface markers using anti-CD3, anti- CD4 and anti-CD44.
  • FACS buffer lx PBS containing 1% FCS
  • ELISA Lung cells were stimulated with ESAT-6 or MPT70 pepmix at 37°C and 5% CO 2 . After three days, secreted IFN-y was measured by sandwich ELISA of culture supernatants.
  • ESAT-6 is an "early antigen”
  • MPT70 is a “late antigen”, for which expression is upregulated during late stage infection.
  • mice were immunized three times with 2 mg of fusion protein
  • CAFOl cationic adjuvant formulation 1
  • Mycobacterium tuberculosis strain Erdman Four or eighteen weeks later the number of mycobacteria was determined in individual lungs by plating of serial dilution of lung homogenate.
  • BCG+H107 BCG+H107
  • BCG+H107 BCG+H107
  • the animals were vaccinated with H107 again after two weeks (right side) and four weeks (left side).
  • Two weeks after the last immunization animals were euthanized and single cell suspensions were obtained by passing spleens through 100 pm cell strainers.
  • Cells were washed twice in RPMI before antigen stimulation for ICS as described in example 3, except that this time, anti-KLRGl was included in the surface stain.
  • bovis BCG Danish immunized and rested for twelve months. At this time point the BCG immunized animals were divided into four groups (n 4 per group) and re-immunized once with M. bovis BCG or three times with H107/CAF01 or saline. Three weeks after the third immunization, animals were euthanized and single cell suspensions obtained from spleens and stimulated for ICS (as described in example 3) using H107 as the antigen.
  • mice were co-immunized (as described above) with BCG and either H104-H107 (BCG- antigens), H74 (BCG- antigens, SEQ ID NO:42) or H65 (BCG + antigens, SEQ ID NO:43).
  • the control groups were immunised with H107/CAF01 or BCG or injected with saline.
  • the bacteria load was measured in individual lungs 4 and 18 weeks after Mtb strain Erdman infection.
  • Vaccination with live BCG induces CD4 T cells with a highly differentiated phenotype, which are known to have a poor protective capacity. In contrast, subunit vaccines elicit less differentiated CD4 T cells. Highly differentiated CD4 T cells (effector memory and effector cells) express IFN-g but gradually loose co expression of TNF-a and IL-2 in contrast to less differentiated T cells (e.g. central memory cells) ( Figure 7A).
  • a functional differentiation score FDS may be defined as IFN-gH- cells divided by IFN-g- cells.
  • H107 BCG- antigens
  • H65 BCG + antigens
  • mice were immunized three times with either 5 pg of H56 (SEQ ID NO:45) in CAFOl or 5 pg H56+5 pg ESAT-6 in CAF01.
  • splenocytes were isolated from two animals per group and 2 x 10 5 cells/well were stimulated in vitro with ESAT-6 protein for six hours at 37°C.
  • the number of CD4 T cells producing either cytokine IFN-g, TNF-a or IL-2 in response to the stimulation was determined by ICS.
  • mice were immunized with H64/CAF01 or H76/CAF01 containing one (H64, SEQ ID NO:44) or five (H76, SEQ ID NO:41) copies of the ESAT-6 molecule, respectively.
  • H64/CAF01 or H76/CAF01 containing one (H64, SEQ ID NO:44) or five (H76, SEQ ID NO:41) copies of the ESAT-6 molecule, respectively.
  • splenocytes were stimulated with the single antigens present in both the H64 and H76 fusion protein and the frequency of IFN-g, TNF-a or IL-2 producing CD4 T cells was determined by ICS.
  • Stimulation with the Rv0287 protein was included as a negative control.
  • the number of bacteria was
  • free ESAT-6 is ESAT-6 protein that is not part of a fusion protein
  • ESAT-6 containing fusion protein Figure 10
  • incorporating more copies of ESAT-6 into the fusion protein increased the number of ESAT-6 specific CD4 T cells primed by the vaccine and improved protection in different animal models ( Figure 11 and 12).
  • mice Female 129/Sv and CB6F1 mice, respectively, were immunized three times with 1 pg of either H74 (SEQ ID NO:42), H105 (SEQ ID NO: 37) or H107 (SEQ ID NO:9) in CAF01.
  • H74 SEQ ID NO:42
  • H105 SEQ ID NO: 37
  • H107 SEQ ID NO:9
  • splenocytes were isolated from four animals per group and 1-2 x 10 6 cells/well were stimulated in vitro with ESAT-6 peptides for six hours at 37°C before measuring the number of cytokine producing CD4 T cells by ICS.
  • the level of IFN-g secretion was measured in 3-day culture supernatants by ELISA as previously described.
  • splenocytes were isolated from four animals per group and 1-2 x 10 6 cells/well were stimulated in vitro with ESAT-6 peptides for six hours at 37°C before measuring the number of cytokine producing CD4 T cells by ICS.
  • the level of IFN-g secretion was measure in 3-day culture supernatants by ELISA as described herein.
  • all animals were aerosol infected with Mtb strain Erdman and lung bacterial burden was measured by plating organ homogenates after 4 weeks infection.
  • Example 8 H107 is a better stand-alone vaccine than state of the art subunit vaccine H56
  • mice were immunized three times with 2 pg of fusion protein
  • CAFOl cationic adjuvant formulation 1
  • Mycobacterium tuberculosis strain Erdman Four to twelve weeks later the number of mycobacteria was determined in individual lungs by plating of serial dilution of lung homogenate. Results
  • H107 SEQ ID NO:9
  • H56 current state-of-the-art subunit vaccine
  • the H107/CAF01 vaccine provides superior protection against an aerosol challenge with virulent Mtb when compared to a state-of-the-art subunit TB vaccine (H56/CAF01) ( Figure 14).
  • Example 9 BCG+H107 co-administration increases BCG-specific immune responses
  • Example 10 H107e has increased protein expression in E. coli compared to H107
  • the yield of recombinant protein expression is, amongst other things, dependent on the amino acid sequence of the protein and large scale vaccine-manufacturing would require the most optimal expression process.
  • a high- expressing version of H107, H107e was developed. Material and methods
  • H107e SEQ ID NO:91
  • DNA sequences corresponding to H107 (SEQ ID NO: 54) and H107e (SEQ ID NO:92) were made by chemical synthesis inserted into the pJ 411 expression vector (ATUM, Menlo Park, CA, US) and transformed into E. coli BL21 (DE3) strain
  • H107e is as immunogenic as H107 and also acts svnerqisticallv with BCG
  • the bacterial load was measured in individual lungs 8 weeks after Mtb strain Erdman infection.
  • H107e and H107 have the same immunogenicity measured by both cytokine expressing CD4 T cells ( Figure 17A, left) and IFN-g release by ELISA ( Figure 17A, right). It was also confirmed that H107e induces immune responses to the same individual antigens as H107 ( Figure 17B). It was noticeable that the deletion in the Rv3876-part in H107e led to a minor decrease in the Rv3876-specific immune response, but an increase in the immune responses against MPT70 and MPT83 ( Figure 17B). After infection, H107e conferred protection similar to, or better than, BCG and BCG+H107e co- vaccination led to a significant increase in protection compared to both BCG and H107e alone ( Figure 17C), as was previously observed with H107 in example 9.
  • H107e has similar immunogenicity to H107 and induces significant protection after Mtb challenge. Like H107, H107e acts synergistically with BCG and co-vaccination with BCG+H107e protective levels that are significantly higher than H107e and BCG alone. In BCG primed animals, H107e (BCG-) vaccination induce less differentiated T cells (like H107) and increase the proportion of Thl7 cells, measured by IL-17 expressing CD4 T cells compared to H65 (BCG+) .

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Communicable Diseases (AREA)
  • Pulmonology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Oncology (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des protéines de fusion basées sur des polypeptides antigéniques provenant de Mycobacterium tuberculosis (Mtb) destinées à prévenir, inhiber ou traiter des infections et/ou des maladies provoquées par une espèce du complexe tuberculosis. En particulier, la présente invention concerne des protéines de fusion comprenant des antigènes qui n'entraînent pas une réponse immunitaire contre les répétitions BCG et/ou ESAT-6. Les protéines de fusion peuvent comprendre une combinaison d'antigènes précoces et tardifs. En outre, la présente invention concerne des vaccins, des compositions immunogènes et des compositions pharmaceutiques comprenant les protéines de fusion.
EP20731142.4A 2019-06-14 2020-06-12 Protéines de fusion pour vaccins contre la tuberculose Pending EP3983011A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP19180280 2019-06-14
PCT/EP2020/066343 WO2020249756A1 (fr) 2019-06-14 2020-06-12 Protéines de fusion pour vaccins contre la tuberculose

Publications (1)

Publication Number Publication Date
EP3983011A1 true EP3983011A1 (fr) 2022-04-20

Family

ID=66999556

Family Applications (1)

Application Number Title Priority Date Filing Date
EP20731142.4A Pending EP3983011A1 (fr) 2019-06-14 2020-06-12 Protéines de fusion pour vaccins contre la tuberculose

Country Status (5)

Country Link
EP (1) EP3983011A1 (fr)
KR (1) KR20220032054A (fr)
CN (1) CN114222762A (fr)
BR (1) BR112021025172A2 (fr)
WO (1) WO2020249756A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112852848B (zh) * 2021-03-17 2022-04-29 中国农业大学 一种含密码子优化的结核分枝杆菌融合蛋白ah疫苗
CN116769056A (zh) * 2023-07-07 2023-09-19 武汉大学 融合蛋白及纯化制备方法和应用

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101248084B (zh) * 2005-06-23 2013-11-06 国家血清研究中心 包含潜伏感染阶段期间所表达的抗原的结核疫苗
ES2647070T3 (es) * 2005-06-23 2017-12-19 Statens Serum Institut Vacunas para tuberculosis que comprenden antígenos expresados durante la fase de infección latente
US20100015171A1 (en) 2008-07-15 2010-01-21 Statens Serum Institute Vaccines comprising tb 10.4
US20110206712A1 (en) * 2008-07-25 2011-08-25 James Brown Tuberculosis rv2386c protein, compositions and uses thereof
EA029492B1 (ru) * 2012-07-10 2018-04-30 Трансген Са Вакцина на основе микобактериальных антигенов
IN2015DN03962A (fr) 2012-10-23 2015-10-02 Statens Seruminstitut
WO2015161853A1 (fr) 2014-04-24 2015-10-29 Statens Serum Institut Nouveaux vaccins contre m. tuberculosis

Also Published As

Publication number Publication date
CN114222762A (zh) 2022-03-22
BR112021025172A2 (pt) 2022-01-25
WO2020249756A1 (fr) 2020-12-17
KR20220032054A (ko) 2022-03-15

Similar Documents

Publication Publication Date Title
AU2006261445B2 (en) Tuberculosis vaccines comprising antigens expressed during the latent infection phase
EP0652962B1 (fr) Expression de proteines recombinantes fusionnees dans des bacteries attenuees
JP5010079B2 (ja) マイコバクテリウム・ツベルクローシスｅｓａｔ−６遺伝子ファミリーベースの結核ワクチン及び診断法
Olive et al. Potential of lipid core peptide technology as a novel self-adjuvanting vaccine delivery system for multiple different synthetic peptide immunogens
ES2291810T3 (es) Fragmentos de acido nucleico y fragmentos de polpeptidos derivados de m. tuberculosis.
JP2015536309A (ja) 結核菌（ｍ．ｔｕｂｅｒｃｕｌｏｓｉｓ）ワクチン
CN107847575B (zh) 用于猪生殖与呼吸综合症及猪圆环病毒相关疾病的疫苗组合物
JP2011502165A (ja) 鞭毛細菌に対する免疫応答を強化する組成物および方法
JP2017513860A (ja) 新規結核菌（ｍ．ｔｕｂｅｒｃｕｌｏｓｉｓ）ワクチン
EP3983011A1 (fr) Protéines de fusion pour vaccins contre la tuberculose
WO2005061534A2 (fr) Vaccins antituberculeux ameliores
JP2017520556A (ja) 抗原ペプチドを投与するための組成物、方法、及び療法
Fan et al. Construction and immunogenicity of a T cell epitope-based subunit vaccine candidate against Mycobacterium tuberculosis
CN101248084A (zh) 包含潜伏感染阶段期间所表达的抗原的结核疫苗
AU2012202486B2 (en) Tuberculosis vaccine and diagnostics based on the Mycobacterium tuberculosis esat-6 gene family
US20220118079A1 (en) Immunogenic Antigens
US20230226166A1 (en) Immunogenic Antigens
Hernández et al. Immunization of mice with a Mycobacterium tuberculosis genomic expression library results in lower bacterial load in lungs after challenge with BCG
JP5075969B2 (ja) マイコバクテリウム・ツベルクローシスｅｓａｔ−６遺伝子ファミリーベースの結核ワクチン及び診断法
Kristof Development and characterisation of a novel vaccine for Bordetella pertussis (whooping cough)
AU2011203012A1 (en) Tuberculosis vaccines comprising antigens expressed during the latent infection phase
AAGAARD et al. Sommaire du brevet 2836319
AAGAARD et al. Sommaire du brevet 2612900

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: UNKNOWN

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20220113

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20231207