EP3983011A1

EP3983011A1 - Fusion proteins for tuberculosis vaccines

Info

Publication number: EP3983011A1
Application number: EP20731142.4A
Authority: EP
Inventors: Rasmus MORTENSEN; Claus Aagaard; Peter Lawætz ANDERSEN
Original assignee: Statens Serum Institut SSI
Current assignee: Statens Serum Institut SSI
Priority date: 2019-06-14
Filing date: 2020-06-12
Publication date: 2022-04-20
Also published as: CN114222762A; BR112021025172A2; KR20220032054A; PH12021553146A1; WO2020249756A1

Abstract

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. In particular, 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. Further, the present invention relates to vaccines, immunogenic compositions and pharmaceutical compositions comprising the fusion proteins.

Description

Fusion proteins for tuberculosis vaccines

Technical field of the invention

Background of the invention

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.

The only vaccine presently available for clinical use is BCG ( Baciiie Calmette- Guerin ), a vaccine whose efficacy remains a matter of controversy. BCG is a live, attenuated vaccine that is widely administered to infants in most areas endemic for tuberculosis. BCG generally induces a high level of acquired resistance in animal models of tuberculosis, and in children it is protective against disseminated forms of tuberculosis such as meningitis and miliary tuberculosis. When given to young children, it is protective against tuberculosis for years but then the efficacy vanes. Comparison of various controlled trials has revealed that the protective efficacy of BCG in adults varies dramatically with an efficacy range from

ineffective to 80% protection. This makes the development of a new and improved vaccine against M. tuberculosis an urgent matter, which has been given a very high priority by the WHO. Currently, there are several new tuberculosis vaccines in clinical trials. Since there is a general consensus that a strong cellular immune response is required for protection against tuberculosis infection and disease, the majority of current clinical tuberculosis vaccine candidates aims at inducing classical TH1 cytokines such as IFN-y or TNF-a from either CD4+ or CD8+ T cells and do not address issues about improving T cell quality, which plays a significant role in protective immunity. Current tuberculosis vaccine candidates include various vaccine platforms, such as inactivated whole cells or whole cell extracts, viral vector- based vaccines, live recombinant BCG or attenuated BCG vaccines, and DNA vaccines. However, none of the above vaccine platforms has this far yielded convincing clinical results.

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.

In W02010006607 A2 it was demonstrated that a simple fusion consisting of only two M. tuberculosis antigens could function as a tuberculosis vaccine when administered together with the adjuvant IC31. WO2006136162 A2,

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. However, common to the above fusion proteins is that none of the corresponding subunit vaccines have yet resulted in a commercially available vaccine against tuberculosis.

Hence, an improved tuberculosis vaccine with the potential to result in a clinically acceptable vaccine would be advantageous. In particular, fusion proteins capable of inducing a strong high quality immune response and therefore the ability to enhance protection would be advantageous. Summary of the invention

Herein is provided 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. 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.

Thus, 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.

Thus, 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.

Another aspect of the present invention relates to a vaccine or immunogenic composition comprising a fusion protein as described herein. Yet another aspect of the present invention is to provide 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. Still another aspect of the present invention is to provide a kit comprising :

i) A fusion protein as described herein or a vaccine or immunogenic

composition as described herein,

ii) BCG, and

iii) optionally, instructions for use.

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.

Brief description of the figures

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. Lung CFU's in Mtb infected animals immunized with H104 or H105 or injected with saline (A) 4 weeks and (B) 18 weeks after aerosol Mtb infection. 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

Figure 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). (A) Overview showing T cell

differentiation based on cytokine expression by the single cell, adapted from Seder et al (2008). (B) Cytokine expression profile of CD4 cells located in the spleen based on single cell expression of the cytokines IFN-g, TNF-a, and/or IL-2. (C) Frequency of fusion protein specific CD4 T cells that expressed KLRG1. Figure 8 shows that protection against Mtb infection was increased when the fusion protein containing BCG- antigens (H107) was co-administered with BCG. Lung CFU's four (A) and eighteen weeks (B) after Mtb Erdman infection.

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.

Figure 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

immunization and (B) three weeks after Mtb infection. (C) After six weeks infection the number of mycobacteria was determined in lungs.

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. Specific ESAT-6 responses in spleens isolated from (A) vaccinated 129sc mice and (B) CB6F1 mice. (C) ESAT-6 specific immune responses in spleens from BCG and subunit co immunized mice and the protective efficacy in the lungs after four weeks infection.

Figure 14 shows that the H107 fusion protein formulated in CAFOl adjuvant was a better vaccine than H56 formulated in CAFOl. The protective efficacy

(DI ogioCFUsa line- 1 ogioCFU immunized) was determined in lungs 4 to 12 weeks after Mtb infection. Results are pooled data from five independent experiments.

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

BCG+H107 group compared to BCG alone meaning that H107+BCG co

administration increases BCG-specific immune responses and therefore that H107 is acting as an adjuvant for BCG. 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

Immunogenicity measured by both cytokine expressing CD4 T cells (Figure 17A, left) and IFNy release by ELISA (Figure 17A, right) after co-vaccination with BCG. H107 and H107e induce similar magnitudes of immune responses.

Figure 17B shows that H107e induces immune responses to the same individual antigens as H107. Figure 17C shows that, after infection, H107e confers

protection similar to or better than BCG and BCG+H107e co-vaccination leads to a significant increase in protection compared to both BCG and H107e alone.

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 present invention will now be described in more detail in the following. Detailed description of the invention

Definitions

Prior to discussing the present invention in further details, the following terms and conventions will first be defined :

Antigen

In the present context, 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

The course of a 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. Thus, in the present context, the term "early antigen" refers to antigens expressed primarily during the acute phase, whereas "late antigens" refers to antigens expressed primarily during the latent/chronic phase.

Fusion protein

In the present context, the term "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. To avoid protein aggregation in the down-stream production 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.

recombinant expression system). Purification tags include, but are not limited to, His-tag, chitin binding protein (CBP), maltose binding protein (MBP) and

glutathione-S-transferase (GST).

Herein, the term "fusion protein" may be used interchangeably with the term "subunit vaccine".

Polypeptide

In the present context, the term "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.

Conventional notation is used herein to portray 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

In the present context, the term "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.

As described herein a M. tuberculosis infection runs through 3 phases. In the present context, the term "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.

Bacille Calmette- Guerin (BCG)

In the present context, the term "Bacille Calmette-Guerin (BCG)" refers to a strain of live, attenuated tuberculosis bacteria derived from Mycobacterium bovis. BCG is currently the only commercially available vaccine against tuberculosis infection and disease.

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, Prague, 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

alterations/deletions (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. Herein are disclosed 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. In the present context, 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, BCG Prague, 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

In the present context, the term "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. Thus, 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. Herein BCG⁺ antigens may be defined in relation to BCG strains selected from the group consisting of BCG Danish, BCG Pasteur and BCG Prague and/or any derivatives of these strains (e.g. VPM1002). Thus, 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

In the present context, the term "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. Thus, fusion proteins comprising BCG- antigens, when

administered following the initial vaccination with a given BCG strain, will not boost an immune response previously induced by said given BCG strain. Thus, BCG- antigens may be defined as functionally negative. Herein BCG antigens may be defined in relation to BCG strains selected from the group consisting of BCG Danish, BCG Pasteur and BCG Prague 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. Thus, 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

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).

In order to identify relevant T-cell epitopes which are recognized during an immune response, it is possible to use 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) and some of these will give a positive response (and thereby be immunogenic) as evidence for the presence of a T cell epitope in the peptide. For the detection of 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.

Although the minimum length of a T-cell epitope has been shown to be at least 6 amino acids, it is normal that such epitopes are constituted of longer stretches of amino acids. Hence, it is preferred that 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. Hence, in important

embodiments of invention, it is preferred that 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 (fragments 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. In addition, 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.

Variants

In the present context, the term "variant" 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.

In the present context, the term "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.

In the present context, 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%

immunogenicity compared to the antigen, polypeptide or fusion protein to which it refers.

Thus, 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. Similarly, 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.

Antigen repeats

In the present context, 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. Thus, a fusion protein with e.g. at least three antigen repeats comprises at least three copies of said antigen.

Herein, fusion proteins comprising antigen repeats of ESAT-6, i.e. ESAT-6 repeats, are preferred.

Linker molecule

In the present context, the term "linker molecule" or "linker" 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

In the present context, the term "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

vaccination within the period of time in which BCG can be expected to persist in the host.

The terms "co-vaccination", "co-immunization" and co-administration" are used interchangeably herein.

Vaccine and immunogenic composition

In the present context, 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. Thus, 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. Moreover, a vaccine or immunogenic composition preferably comprises one or more adjuvants. Adjuvants

In the present context, the term "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. Often, 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. dimethyldioctadecylammonium 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), CpG and "IC31" or combinations hereof.

Expression vector

In the present context, the term "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

In the present context, the term "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. By operatively linking two heterologous nucleic acid sequences encoding a polypeptide 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. By 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

In the present context, the term "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.

In the present context, the mammal is preferably a human or a domestic animal.

Sequence identity

In the present context, the term "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.

Thus, in the present context "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. Similarly, 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.

To determine the percent identity of two amino acid sequences or of two nucleic acids, 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 percent identity between the two sequences is a function of the number of identical positions shared by the sequences (/.e., % identity = # of identical positions/total # of positions (e.g., overlapping positions) x 100). In one embodiment, the two sequences are the same length.

In another embodiment, 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. Alternatively, 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). BLAST nucleotide searches may be performed with the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches may be performed with the XBLAST program, score = 50, wordlength =

3 to obtain amino acid sequences homologous to a protein molecule of the invention.

To obtain gapped alignments for comparison purposes, Gapped BLAST may be utilized. Alternatively, PSI-Blast may be used to perform an iterated search, which detects distant relationships between molecules. When utilizing the NBLAST, XBLAST, and Gapped BLAST programs, the default parameters of the respective programs may be used. See http://www.ncbi.nlm.nih.gov. Alternatively, 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).

Generally, the default settings with respect to e.g. "scoring matrix" and "gap penalty" may be used for alignment. In the context of the present invention, 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 for tuberculosis vaccines

About one-quarter of the world's population is estimated to be infected with tuberculosis, with the disease causing more than a million deaths annually. This makes tuberculosis the deadliest infectious disease globally. Despite this fact, no efficient vaccine has been developed to replace and enhance the controversial and unreliable BCG vaccine developed a hundred years ago. Strategies based on different platforms, such as inactivated whole cells or whole cell extracts, viral vector-based vaccines, live recombinant BCG, attenuated BCG vaccines, or DNA vaccines, have largely failed to provide encouraging results with the promise of an enhanced tuberculosis vaccine.

An alternative approach is the design of 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. Thus, herein are provided fusion proteins that may be effectively used in a vaccine or immunogenic composition for prevention, inhibition or treatment of tuberculosis infection and/or disease.

Thus, 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. Thus, 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 (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), Rv3620 (EsxW), Rv2660c, Rv3614 (EspD), Rv3865 (EspF), Rv3849 (EspR), Rv3872 (PE35) and Rv3881 (EspB).

Thus, 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: 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 (Rv3849), SEQ ID NO: 32 (Rv3872) and SEQ ID NO: 33 (Rv3881) and variants thereof.

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.

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.

The transition of M. tuberculosis from primary infection to latency is accompanied by changes in gene expression. It is also likely that changes in the antigen- specificity of the immune response occur, as the bacteria modulates gene expression during its transition from active replication to dormancy. The full nature of the immune response that controls latent/chronic infection and the factors that lead to reactivation are not completely elucidated. However, there is some evidence for a shift in the dominant cell types responsible. While CD4 T cells are essential and sufficient for control of infection during the acute phase, studies suggest that CD8 T cell responses are more important in the latent/chronic phase.

Currently there are several new TB vaccines in clinical trials. However, they are primarily classical preventive vaccines based on a limited number of antigens expressed in the early stage of infection. However, as a direct consequence of the expression dynamic described above, the epitope pattern that is presented to T cells changes radically over time. Thus, without being bound by theory, it is herein contemplated that 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.

Therefore, 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), Rvll96 (PPE18), Rv2608 (PPE42), Rv3619 (EsxV), Rv3620 (EsxW), Rv3614 (EspD), Rv3865 (EspF), Rv3849 (EspR), Rv3872 (PE35) and Rv3881 (EspB), and variants thereof.

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.

The majority of the population in countries plagued by tuberculosis disease are vaccinated with BCG - a strain of live, attenuated tuberculosis bacteria derived from Mycobacterium bovis. A prevalent strategy for enhancing protection against tuberculosis, is to boost the immune response raised as a reaction to the initial BCG vaccination. This may be sought accomplished by administrating tuberculosis antigens that are expressed by BCG and have already primed an immune response upon initial BCG vaccination. 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. However, 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.

Herein are disclosed fusion protein variations that are conceptually different from the BCG⁺ boost vaccines described above. Surprisingly, it has been found that 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. Such 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.

Without being bound by theory, it is contemplated that at least part of the increased protection is due to the quality of the immune response induced by the fusion proteins disclosed herein. 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.

Thus, 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.

Therefore, 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.

It is to be noted that a variety of different BCG strains exists, with genotype and phenotype varying between different strains. Thus, for the purpose of determining whether an antigen is a BCG⁺ or BCG- antigen, herein reference is made to the late BCG strains, preferably BCG Danish, BCG Pasteur, BCG Prague or VPM1002. Therefore, 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. Thus, 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 BCG Prague.

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.

While the genome of Mycobacterium tuberculosis contains approximately 4000 genes, there is large overlap of approximately 98% with the genome of BCG. Consequently, the number of potential BCG- antigens is limited to about 100 antigens which BCG does not prime a T cell response against. Herein are identified BCG- antigens that are suitable to be included in a fusion protein for use in a vaccine against tuberculosis infection and/or disease. Thus, 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. 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. Yet another embodiment of the present invention relates to the fusion protein as described herein, wherein the antigens are selected from :

a) 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

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).

Some fragments of antigens have been found to be advantageous. Thus, 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) SEQ ID NO: 34 (H107b), SEQ ID NO: 35 (H107c) or SEQ ID NO:91

(H107e) or variants or immunogenic epitopes thereof, or

A preferred embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises:

a) 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

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) SEQ ID NO: 36 (H106) or SEQ ID NO: 38 (H104) or variants or

immunogenic epitopes thereof, or

A further embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises:

a) 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) or variants or immunogenic epitopes thereof, or

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 :

a) 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). It is to be understood that 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.

As described herein, it is contemplated that vaccination with both early and late antigens results in an improved immune response. Herein are identified both early and late antigens out of the limited number of BCG- antigens. 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 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.

Some variations of the fusion proteins disclosed herein are restricted to containing exclusively BCG- antigens. Thus, 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).

The fusion proteins as described herein may also be improved by altering the content of the 6 kDa early secretory antigenic target (ESAT-6). ESAT-6 is an important secretory protein and potent T cell antigen of Mycobacterium

tuberculosis. The 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.

Herein are disclosed variations of fusion proteins which comprise several copies of ESAT-6 so that ESAT-6 relatively represents a larger fraction of the fusion protein. Without being bound by theory, it is contemplated herein that increasing the relative content of ESAT-6 antigen in the fusion proteins mitigates the challenge with native ESAT-6 having low immunogenicity. For fusion proteins comprising more than one copy of ESAT-6, 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. Thus, 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. A further embodiment of the present invention relates to the fusion protein as described herein, wherein 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. Thus, 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 (PPE42), Rv3619 (EsxV), Rv3620 (EsxW), Rv2660c, Rv3614 (EspD), Rv3865 (EspF), Rv3849 (EspR), Rv3872 (PE35) and Rv3881 (EspB).

Thus, 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 (Rv3849), SEQ ID NO: 32 (Rv3872) and SEQ ID NO: 33 (Rv3881) and variants thereof, 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.

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:

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

wherein 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). Thus, 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:9. Another embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises:

a) 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

wherein the amino acid sequence of b) have the same immunogenicity or at least the same immunogenicity as SEQ ID NO:9.

A variant of the H107 fusion protein is termed H107e (SEQ ID NO:91). Thus, 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:

a) 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

wherein the 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. Thus, 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.

For enabling production of the fusion proteins or antigens, they may comprise appropriate purification tags to allow purification from e.g. a recombinant expression system. Thus, an embodiment of the present invention relates to the fusion protein as described herein, wherein the fusion protein comprises a purification tag. Another embodiment of the present invention relates to the fusion protein as described herein, wherein 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). 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, wherein the at least one antigen that does prime an immune response against BCG is selected from :

a) amino acid selected from the group consisting of 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) and SEQ ID NO: 27 (EsxW) and variants or immunogenic epitopes thereof, or

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. Such a vaccine or

immunogenic composition may be used prophylactically or therapeutically.

Thus, an aspect of the present invention relates to a vaccine or immunogenic composition comprising a fusion protein as described herein.

In order to ensure optimum performance of the vaccine or immunogenic composition, it is preferred that it comprises an immunologically and

pharmaceutically acceptable carrier, vehicle or adjuvant. Thus, 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. A further embodiment of the present invention relates to the vaccine or immunogenic composition as described herein, wherein the 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). In another preferred embodiment, the adjuvant is cationic adjuvant formulation 10 (CAF10) comprising DDA, MMG and CpG.

Further 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:

Ta ble 1

CAF adj uva nt nomenclature.

Delivery system Immunostimulators

Type Surfactant(s) Oil CLR-ligand TLR-ligand Other CAFOl Liposome DDA TDB

CAF04 Liposome DDA MMG

CAF05 Liposome DDA TDB Poly-IC - CAF06 Liposome DDA TDB MPL

CAF09 Liposome DDA MMG Poly-IC - CAF10 Liposome DDA MMG CpG

CAF11 Liposome DDA MMG Flagellin - CAF19 Emulsion DDA Squalane MMG

CAF24 Emulsion DDA Squalane MMG pIC

Abbreviations : 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.

The fusion proteins disclosed herein may be used either in a stand-alone vaccine or in combination with BCG vaccine. Thus, 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. Thus, an embodiment of the present invention relates to the fusion protein, vaccine or immunogenic

composition for use as described herein, wherein the subject is a mammal.

However, the main aim with the provision of the fusion proteins disclosed herein is to improve current possibilities to combat the global challenge with tuberculosis infection and/or disease in humans. Thus, 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.

The manner of application may be varied widely. 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. Thus, an embodiment of the present invention relates to the fusion protein, vaccine or immunogenic composition for use as described herein, wherein BCG is

administered prior to, simultaneously or subsequent to administration of the fusion protein, vaccine or immunogenic composition. It has surprisingly been found that BCG, 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. Thus, 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.

As the fusion protein, vaccine or immunogenic composition disclosed herein may work synergistically with BCG vaccine, these may conveniently be supplied together for easy application. Thus, an aspect of the present invention relates to a kit comprising :

i) A fusion protein as described herein or a vaccine or immunogenic

composition as described herein,

ii) BCG, and

iii) optionally, instructions for use.

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

introduction of the expression vector in a suitable recombinant expression system. Thus, 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: 66 (EsxQ), SEQ ID NO: 67 (HspX), SEQ ID NO: 68 (PepD), SEQ ID NO: 69 (PPE18), SEQ ID NO: 70 (PPE42), SEQ ID NO: 71 (EsxV), SEQ ID NO: 72 (EsxW), SEQ ID NO:73 (Rv2660c), SEQ ID NO: 74 (Rv3614), SEQ ID NO: 75 (Rv3865), SEQ ID NO: 76 (Rv3849), SEQ ID NO: 77 (Rv3872) and SEQ ID NO: 78 (Rv3881), and variants thereof.

Moreover, 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 :

All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention. Embodiments and features of the present invention are also outlined in the following items.

Items

1. A fusion protein comprising at least two antigens that originate from M.

tuberculosis. 2. The 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.

3. The fusion protein according to any one of items 1 or 2, wherein the fusion protein comprises both early and late antigens.

4. The fusion protein according to any one of the preceding items, wherein at least one antigen does not prime an immune response against BCG.

5. The fusion protein according to any one of the preceding items, wherein the antigens are deleted from, non-secreted or have low-expression in BCG.

6. The fusion protein according to any one of the preceding items, 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.

7. The fusion protein according to any one of the preceding items, wherein the antigens are selected from :

a) 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

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).

8. The fusion protein according to any one of the preceding items, wherein the fusion protein comprises:

a) 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).

9. The fusion protein according to any one of the preceding items, wherein the fusion protein comprises:

10. The fusion protein according to according to any one of items 7-9, 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).

11. The 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.

12. The fusion protein according to any one of the preceding items, wherein the fusion protein comprises at least four ESAT-6 repeats. 13. The fusion protein according to any one of items 11 or 12, wherein the ESAT- 6 repeats are separated by at least one antigen different from ESAT-6.

14. The fusion protein according to any one of items 11-13, wherein the ESAT-6 repeats are positioned alternately with antigens different from ESAT-6. 15. The fusion protein according to any one of the preceding items, wherein the fusion protein comprises:

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); and

19. The fusion protein according to any one of the preceding claims, wherein the fusion protein comprises the amino acid sequence represented by any one selected from the group consisting of:

a) SEQ ID NO:9, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID

NO: 37, SEQ ID NO: 38 or SEQ ID NO:91 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). 20. The fusion protein according to any one of the preceding items, wherein the fusion protein comprises the amino acid sequence represented by SEQ ID NO:9 or SEQ ID NO:91.

21. The fusion protein according to any one of claims, wherein the fusion protein is encoded by nucleic acid sequences selected from the group consisting of:

a) SEQ ID NO: 54 (H107), SEQ ID NO: 79 (H107b), SEQ ID NO: 80

(H107c), SEQ ID NO:92 (H107e) SEQ ID NO:81 (H106), SEQ ID NO: 82 (H105) and SEQ ID NO:83 (H104), or variants or fragments thereof; or b) nucleic 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).

22. The fusion protein according to any one of the preceding items, wherein the antigens of the fusion proteins are connected with a linker molecule.

23. The 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.

24. 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.

25. The 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) amino acid selected from the group consisting of 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) and SEQ ID NO: 27 (EsxW)and 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). 26. A vaccine or immunogenic composition comprising a fusion protein according to any one of the preceding items.

27. The vaccine or immunogenic composition according to item 26, wherein the vaccine or immunogenic composition further comprises one or more adjuvants.

28. The vaccine or immunogenic composition according to item 27, wherein 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,

monophosphoryl lipid A (MPL), Trehalose Dimycolate (TDM), Trehalose Dibehenate (TDB), Muramyl Dipeptide (MDP), monomycolyl glycerol (MMG), CpG and "IC31", or combinations hereof.

29. The vaccine or immunogenic composition according to any one of items 26- 28, wherein the vaccine or immunogenic composition further comprises BCG.

30. A fusion protein according to any one of items 1-25 or a vaccine or

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.

31. The fusion protein, vaccine or immunogenic composition for use according to item 30, wherein the subject is a mammal.

32. The fusion protein, vaccine or immunogenic composition for use according to item 31, wherein the mammal is a human.

33. 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. 34. 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.

35. The 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.

36. The fusion protein, vaccine or immunogenic composition for use according to any one of items 30-35, wherein the subject has previously been vaccinated with BCG.

37. A kit comprising :

i) A fusion protein according to any one of items 1-25 or a vaccine or

immunogenic composition according to any one of items 26-29, ii) BCG, and

iii) optionally, instructions for use.

38. The kit according to item 37, wherein i) and ii) are for simultaneous, separate or sequential administration.

39. A nucleic acid sequence comprising a sequence encoding a fusion protein according to any one of items 1-25.

40. 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.

41. A recombinant host cell comprising an expression vector according to item 40.

The invention will now be described in further details in the following non-limiting examples. Examples

Example 1 : BCG vaccination does not prime an immune response against the M. tuberculosis antigens of the H104-H107 fusion proteins

Material and methods

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

immunized three times with 2 mV of single proteins formulated in cationic adjuvant formulation 1 (CAFOl) in a total volume of 200 mI_. Each protein vaccine was spaced with two weeks. Ten weeks after BCG immunization or eighteen weeks after Mtb infection or two weeks after the last single protein immunization, single cell suspensions of splenocytes were obtained from individual animals by passing the spleens through 100 pm cell strainers. Cells were washed twice in RPMI followed by determination of the number of cells obtained from each spleen and adjustment to 2 x 10⁶ cells/ml. To stimulate the cells, 100 pL (2 x 10⁵ cells) were aliquoted into individual wells in 96-well plates and purified recombinant single Mtb antigens (2 pg/ml) were added. The total volume per well was adjusted to 200 mI_ using RPMI medium and the cells were incubated with the individual antigens for 3-5 days at 37°C in a CO2 incubator. At this time point, the

concentration of cytokine IFN-g was measured by sandwich ELISA in the cell supernatants.

Results

A subset of potential antigen candidates were selected for designing subunit vaccines with non-BCG (BCG ) antigens and it was investigated whether they gave rise to an immune response after BCG vaccination. Splenocytes isolated from mice were stimulated with the indicated single antigens (2 pg/ml) purified as

recombinant proteins (see also example 2). After stimulation, cell release of cytokine IFN-y into the medium was measured. The cytokine IFN-y concentration measured in media from cells incubated without antigens ("none") established the baseline for non-stimulated cells. In the BCG immunized animals, none of the antigen candidates gave rise to immune responses (Figure 1A). In contrast, the TB10.4 antigen, which is expressed by BCG, gave rise to a substantial immune response (Figure 1A). In Mtb infected animals, all of the antigens gave rise to an immune response, validating that these antigens are immunogenic and expressed during Mtb infection (Figure IB). Similarly, in animals vaccinated with the individual proteins, all the antigens gave rise to an immune response,

demonstrating that these antigens are immunogenic in the CB6F1 mouse strain

(Figure 1C).

Conclusion

M. bovis BCG Danish immunization did not induce a cellular immune response against any of the eight vaccine antigens candidates. In contrast, all antigen candidates gave rise to an immune response after Mtb infection or single protein immunization. Thus, the antigens of H104-H107 do not share antigens with M. bovis BCG Danish and are specific for Mtb. Herein such antigens are referred to as BCG- antigens. In contrast, antigens that BCG immunization raises a T cell response against are herein referred to as BCG⁺ antigens, e.g. TB10.4.

Example 2: Construction of fusion proteins

Material and methods

Genes encoding single and fusion proteins were made synthetically based on the protein sequences translated from the published genome sequence of

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

Based on the results of example 1, 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.

Conclusion

All single and fusion proteins were produced in sufficient quantities and qualities to be antigens suitable for a vaccine and to perform all the needed immunizations and cell stimulations described in the examples below.

Example 3: MTP70 is an antigen wherein the expression is uprequlated during late stage Mtb infection, i.e. a "late antigen"

Material and methods

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: A total of 1-2 x 10⁶ 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. After 15-30 min of surface staining, cells were washed in FACS buffer, permeabilized using the Cytofix/Cytoperm kit (BD) according to the manufacturer's instructions, and stained intracellularly with anti-IFN-g, anti-TNF- ot, anti-IL-2, anti-IL-17A. Cells were subsequently washed, resuspended in FACS buffer and analysed with a LSR FACS Fortessa (BD). All flow cytometry data was analysed with FlowJo software v. lO (Tree Star, Ashland, OR, USA).

ELISA: Lung cells were stimulated with ESAT-6 or MPT70 pepmix at 37°C and 5% CO². After three days, secreted IFN-y was measured by sandwich ELISA of culture supernatants.

Results

Three weeks post Mtb infection there was a substantial ESAT-6 response measured by both IFN-g ELISA (Figure 4A) and cytokine secreting CD4 T cells (Figure 4B). However, at this time point, there was no immune response against MPT70. At 12 weeks post infection, there was a small, but detectable immune response against MPT70 that was increased at week 20. This supports ESAT-6 as an "early antigen" and MPT70 as a "late antigen", which is characterized by immune recognition in the late/chronic stages of infection.

Conclusion

The kinetics of the immune responses against ESAT-6 and MPT70 support that, within BCG- antigens, ESAT-6 is an "early antigen", while MPT70 is a "late antigen", for which expression is upregulated during late stage infection.

Example 4: Late-staoe antigens improve long term protection

Material and methods

Groups of mice were immunized three times with 2 mg of fusion protein

formulated in cationic adjuvant formulation 1 (CAFOl) in a total volume of 200 yL. Each vaccination round was separated by 2 weeks. Six weeks after the third immunization, all animals were aerosol challenged (20-50 CFU) with

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.

Results

To test the importance of "late antigens" for long-term protection, the bacterial load in infected animals that were immunized with a fusion protein containing both early and late stage antigens (H105) (SEQ ID NO: 37) were compared with infected animals immunized with a fusion protein containing only early expressed antigens (H104) (SEQ ID NO: 38). In the early stage of infection (four weeks infection), all four fusion proteins induced significant protection with no difference in their protection level (Figure 5A). In the chronic stage of infection (18 weeks) the addition of the late stage antigens made a difference as the H105 provided better protection than H104 lacking the late stage antigens (p=0.044) (Figure 5B).

Conclusion

Including late stage antigens in the vaccine improves long-term

protection/containment (Figure 5).

Example 5: BCG antigens are highly efficacious and, in contrast to BCG⁺ antigens, works "svnerqisticallv" with BCG

Material and methods

Groups of CB6F1 mice (n=4) were co-immunized with H107 and BCG

(BCG+H107) by first administering BCG s.c. at the left side at the base of the tail followed by 1 pg H107 (SEQ ID NO: 9) in CAFOl administered at the same injection site the day after. 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. In a second experiment all animals, except a small saline control group (n=4), were M. 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.

In separate experiments, groups of 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.

Results

In saline and BCG immunized animals, there was no immune response against H107 as expected since H107 does not share antigens with BCG. Immunization with H107 led to formation of a robust CD4 T cell response and, importantly, this was significantly enhanced by co-administration of BCG (BCG+H107, Figure 6A). This demonstrates that BCG co-administration can potentiate immune responses of H107 (BCG- antigens), even though antigens are not shared between the two vaccines.

The majority of the world's population are immunized by BCG as newborns and BCG revaccination is gaining increasing interest due to promising results of recent clinical trials. To investigate if the co-adjuvant effect of BCG was seen also in already BCG vaccinated animals, a BCG re-vaccination model was applied in which the two BCG immunization were spaced with one year (Figure 6B). There was no responses against H107 in the saline, BCG, or the BCG re-vaccinated groups as expected. In contrast, vaccination with H107 in primary BCG vaccinated animals resulted in a solid CD4 T cell response that was significantly increased when the H107 was co-administered with a secondary BCG vaccination. This demonstrates that BCG also works as a co-adjuvant in already BCG vaccinated animals.

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). To characterize T cell differentiation, a functional differentiation score (FDS) may be defined as IFN-gH- cells divided by IFN-g- cells. BCG vaccination led predominantly to IFN-g producing effector- and effector memory CD4 T cells (and very few IFN-g cells), yielding a differentiation index of 13.3 (Figure 7B, top). Co-immunization of BCG and a BCG⁺ vaccine (H65) led to some improvements in the cytokine expression profile with more IFN-g cells, yielding a FDS of 1.9 (Figure 7B, middle). However, co-vaccination of BCG with H107 (BCG- antigens) led to an even greater improvement of the cytokine expression profile with predominantly IFN-g central memory CD4 T cells, yielding a FDS of 0.5. This illustrates that an improved T cell quality can be obtained when using BCG- antigens compared to BCG⁺ antigens when the vaccines are

administered together with BCG. Highly differentiated T cells are also

characterized by expression of KLRG1 and it was confirmed that BCG+H107 resulted in the lowest frequency of KLRG1 expression CD4 T cells compared to both BCG and BCG+H65 (Figure 7C).

To compare the protective efficacy of BCG co-immunization with either BCG- antigens (H107) or BCG⁺ antigens (H65), immunized mice were aerosol infected with Mtb Erdman. H74 was included as a reference to a previously described fusion protein (WO2015161853 Al). After four weeks infection, co-immunization with both H107 and H74 added to the protective efficacy of the M. bovis BCG vaccine whereas H65 did not. This increase was significant for H107, not only compared to H107 or BCG alone (p<0.0001) but also to BCG co-immunization with H74 (p=0.0489) and H65 (p<0.0001) (Figure 8A). Notably, H107

administered as a stand-alone vaccine was more efficacious than BCG and

BCG+H65 and on par with BCG+H74. At the late time point (Figure 8B), the protective efficacy of BCG alone and BCG+H65 co-vaccination was lost, but H107/CAF01 still protected significantly against Mtb (p<0.0001) and co

vaccination with BCG and H107 fusion protein remained the best protected group. The other BCG- fusions, H104-H106, induced similar levels of protection when these vaccines were co-administered with BCG at week 4 (Figure 9).

Conclusion

Co-administration of M. bovis BCG with subunit vaccines consisting of BCG- antigens, resulted in enhanced immune responses against the subunit vaccine both in terms of magnitude (Figure 6) and quality (Figure 7) and improved protection after aerosol Mtb infection (Figure 8 and Figure 9). H107 as a stand alone vaccine was more efficacious than BCG and BCG+H65 and on par with BCG+H74, demonstrating that the BCG- antigen combinations disclosed herein are potent when co-administered together with BCG as well as stand-alone vaccines. Example 6: ESAT-6 repeats in fusion proteins increase immunoqenicitv

Material and methods

In a first experiment, groups of female CB6F1 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. Three weeks after third immunization, splenocytes were isolated from two animals per group and 2 x 10⁵ 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. Six weeks after immunization the mice were infected with Mtb strain Erdman and three weeks later lung cells were isolated from four animals per group and treated as the splenocytes above to determine the number of cytokine-producing CD4 T cells. After six weeks of infection, the number of bacteria was determined by plating lung homogenates from individual mice on 7H11 agar (n = 8 per group) (Figure 10).

In a second experiment (Figure 11), groups of 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. Three weeks after third immunization, 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

determined in the lungs six weeks after Mtb strain Erdman infection.

In a third experiment (Figure 12), the influence of ESAT-6 repeats in another molcular backbone was investigated using an Mtb relapse model. Six weeks after Mtb Erdman aerosol infection, mice were given isoniazid (0.1 g/L) and rifabutin (0.1 g/L) in the drinking water ad libitum for six weeks. Immunizations with 0.5 pg of H83 (one ESAT-6 copy, SEQ ID NO:40) and H84 (four ESAT-6 copies, SEQ ID NO: 39) or saline (control) were commenced at week 10 after infection and given three times with three weeks interval. Lungs were taken for immune analysis by ICS two weeks after the last immunization (week 18 of the infection) and lung bacterial load was evaluated by plating homogenates on 7H11 agar at week 22 and 35 post infection. Results

ESAT-6 is a low immunogenic antigen after protein vaccination and it is therefore desirable to increase the ESAT-6 specific immunogenicity. It was first investigated whether adding more free ESAT-6 in combination with an ESAT-6 containing fusion protein (H56) would increase the immune response. Three weeks after vaccination, the ESAT-6 specific immune response was higher in mice that received H56 alone compared to mice that received H56+ESAT-6 (Figure 10A). This difference was even more pronounced in the lungs three weeks after aerosol infection. At this time point, the difference in the number of cytokine-producing CD4 T cells was significant (p=0.0060) (Figure 10B). Both vaccination regimes gave similar significant protection in the lung six weeks after infection (p=0.0016 and p=0.0121 relative to non-vaccinated animals). This demonstrates that adding more ESAT-6 protein to the vaccine does not increase immunogenicity or protection.

As a solution to ESAT6 being low-immunogenic, fusion proteins in which the ESAT-6 sequence is repeated throughout the molecular construct were designed. Comparing the immune response induced by H64, containing one copy of the ESAT-6 molecule, to the H76 fusion protein, containing five copies of the ESAT-6 molecule, showed that the number of vaccine-primed ESAT-6 specific CD4 T cells was three to five-fold higher in H76 immunized animals (Figure 11A). The increased immune response specific for ESAT-6 also resulted in improved protection when we compared the bacteria load in the H64 and H76 immunized animals six weeks after aerosol Mtb Erdman infection (Figure 11B).

Similarly, with a different molecular construct, increasing the number of ESAT-6 copies from one (H83) to four (H84) increased the immunogenicity and protection in an Mtb relapse model, using partial antibiotic therapy. Two weeks after the last immunization, the ESAT-6 specific CD4 T cell response was significantly increased in the H84 group compared to the H83 group (Figure 12A). After the end of partial antibiotic treatment, the bacterial load in control and H83 vaccinated animals increased from week 22 to week 35 (relapse) (Figure 12B). In contrast, the bacterial load decreased from week 22 to week 35 in the H84 vaccinated animals (Figure 12B), demonstrating that the ESAT-6 repeat pattern both increased ESAT-6 specific immune responses as well as protective capacity in the Mtb relapse model.

Conclusion

It is not possible to increase the vaccine-primed ESAT-6 response by adding free ESAT-6 protein ("free ESAT-6" is ESAT-6 protein that is not part of a fusion protein) in the vaccine formulation together with an ESAT-6 containing fusion protein (Figure 10). Instead, 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).

Example 7: Fusion proteins work both alone and with BCG

Material and methods

In a first (Figure 13A) and second experiment (Figure 13B), 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. Two weeks after third immunization, splenocytes were isolated from four animals per group and 1-2 x 10⁶ 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.

In a third experiment (Figure 13C), female CB6F1 mice were co-immunized with BCG and 1 pg of either H105 or H107 in CAFOl (BCG+H105 or BCG+H107) by administering BCG s.c. at the left side at the base of the tail followed by H105 or H107 in CAFOl at the same injection site the following day. The animals were subsequently immunized with the subunit vaccine again after two (right side) and four weeks (left side). Two weeks after third immunization, splenocytes were isolated from four animals per group and 1-2 x 10⁶ 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. Six weeks after the last immunization, all animals were aerosol infected with Mtb strain Erdman and lung bacterial burden was measured by plating organ homogenates after 4 weeks infection.

Results

In 129/Sv mice, increasing the number of ESAT-6 copies from one (H74 and H105) to four (H107) led to enhanced ESAT-6 responses (Figure 13A). This was also true in CB6F1 mice, measured by both ICS and ELISA (Figure 13B). Finally, it was tested, whether this was also true when the vaccines were co-administered with BCG. In this regimen, again, H107 significantly increased the ESAT-6 specific immune response compared to H105, which also led to increased protection against Mtb aerosol infection (Figure 13C).

Conclusion

In three independent experiments (with two different mouse strains), it was demonstrated that increasing the number of ESAT-6 copies from one (H74 and H105) to four (H107) increased the ESAT-6 specific immune response. This was also true when the vaccines were co-administered with BCG. This also decreased bacterial load, showing that repeating ESAT-6 in the vaccine backbone is a robust method to improve both immunogenicity and vaccine mediated protection. Also, the data support that the fusions are efficacious both as stand-alone vaccines and together with BCG.

Example 8: H107 is a better stand-alone vaccine than state of the art subunit vaccine H56

Material and methods

Groups of mice were immunized three times with 2 pg of fusion protein

formulated in cationic adjuvant formulation 1 (CAFOl) in a total volume of 200 pL. Each vaccination round was separated by 2 weeks. Six weeks after the third immunization, all animals were aerosol challenged (20-50 CFU) with

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

To compare the protective efficacy of H107 (SEQ ID NO:9) against the current state-of-the-art subunit vaccine (H56; SEQ ID NO:45), groups of mice were immunized with the fusion proteins in CAFOl adjuvant in two (H107) and five (H56) independent experiments. The animals were challenged with Mtb strain Erdman and the number of bacteria was enumerated in the lung of individual animals (Figure 14). Depending on the experiment, the protective efficacy was determined four to twelve weeks after Mtb strain Erdman challenge. On average the H107 vaccine gave 2.0 loglO protection corresponding to a reduction in bacterial load of 100-fold. In comparison, the H56 vaccine reduced on average the bacterial load ~7-fold (0.82 loglO).

Conclusion

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

Material and methods

Groups naive CB6F1 mice (n=4 per time point) were co-immunized with BCG and three times 1 pg of H107 (SEQ ID NO:9) as described in previous examples.

Animals were euthanized at different time points after immunization 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 previous examples. TB10.4 was used for stimulation to asses BCG-specific immune responses as this antigen is included in BCG, but not in H107.

Results

Already three weeks after the first immunization (one week efter the second H107) an increased TB10.4 response was observed in the BCG+H107 group compared to BCG alone (Figure 15). This difference was maintained up until week 9, where the experiment was terminated. This demonstrates that co administration of H107 with BCG increases the BCG-speciffic immune responses even though they do not share antigens. Conclusion :

Previous examples have shown that BCG+H107 co-administration leads to increased H107 responses (BCG is acting as an adjuvant for H107). This examples shows that H107+BCG co-administration also increases BCG-specific immune responses, meaning that H107 is acting as an adjuvant for BCG (Figure 15). In this way, there is a true synergy between the two vaccines.

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. To address this, a high- expressing version of H107, H107e was developed. Material and methods

To optimize expression, a proline rich sequence in the Rv3876-part of H107 (SEQ ID NO:9), AA298-AA517, was deleted to make 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

(Agilent, DK) that was grown in Luria-Bertani (LB) medium. Recombinant protein expression levels were evaluated by SDS-PAGE or western-blot (using a primary antibody recognizing the MPT70-part of H107/H107e) at 0, 1 and 3 hours after induction of the culture with 1 mM Isopropyl b-D-l-thiogalactopyranoside (IPTG). The same amount of cells (same OD600) were loaded in the lanes on the gels. After expression, recombinant antigen was purified from inclusion bodies and subjected to metal ion affinity chromatography with a 5ml HisTrap HP column (GE Healthcare) followed by anion exchange using a HiTrap Q HP column (GE

Healthcare). Final protein yield was estimated by the bicinchoninic acid assay.

Results

From culture induction experiments, it was clear that H107e had a significantly increased expression in E. coli. After both 1 and 3 hours after induction, the bands corresponding to H107e was much stronger compared to the ones for H107 on both the SDS-PAGE gel and in the western blot (Figure 16A). For comparison, the yield of purified protein from a 6L fermentation went from 2.4-7.2 mg for H107 to 15.2 mg for H107e.

Conclusion :

Altering the sequence in H107e led to significant increases in protein expression and yield compared to H107.

Example 11 : H107e is as immunogenic as H107 and also acts svnerqisticallv with BCG

Materials and methods

Groups naive CB6F1 mice (n=8) were co-immunized with BCG and 1 pg of either H107 (SEQ ID NO:9) or H107e (SEQ ID NO:91) as described in previous examples. 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 and IFN-g ELISA as described in previous examples.

In a second experiment, groups of naive CB6F1 mice (n = 6) were co-immunized with BCG and H107e as above and the control groups were immunized with H107e/CAF01 or BCG or injected with saline. The bacterial load was measured in individual lungs 8 weeks after Mtb strain Erdman infection.

In a third experiment, groups of BCG-memory CB6F1 mice (n = 6) were immunized 3 times s.c. with 2 pg H107e or H65 at the base of the tail. Two weeks after the last immunization, animals were euthanized and single cell suspensions were obtained by passing inguinal lymph nodes through 100 pm cell strainers. Cells were washed twice in RPMI before antigen stimulation for ICS.

Results

After co-vaccination with BCG, it was confirmed that 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.

In BCG-memory mice, H107e (BCG-) vaccination led to less differentiated CD4 T cells (better quality) compared to H65 (BCG+), measured by functional

differentiation score, FDS (Figure 17D). Additionally, as Thl7 cells have gained increased interest during the past decade, and Thl7 cells are suggested to have protective properties during Mtb infection, IL-17 expressing CD4 T cells were measured by flow cytometry. H107e induced a much higher proportion of Thl7 cells compared to H65, demonstrating that BCG- vaccines yields an overall broader immune response than traditional BCG+ vaccines in BCG-primed individuals (Figure 17D).

Conclusion

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+) .

References

· W02010006607 A2

• WO2006136162 A2

• W02014063704 A2

• WO2015161853 A1

• Seder ef al (2008), Nat. Rev. Immunol. 8, 247-258

Claims

1. A fusion protein comprising at least five antigens antigens that originate from M. tuberculosis.

2. The fusion protein according to claim 1, wherein the fusion protein comprises at least six antigens, such as at least seven antigens, such as at least eight antigens.

3. The fusion protein according to any one of claims 1 or 2, wherein the fusion protein comprises both early and late antigens.

4. The fusion protein according to any one of the preceding claims, wherein at least one antigen does not prime an immune response against BCG.

5. The fusion protein according to any one of the preceding claims, wherein the antigens are deleted from, non-secreted or have low-expression in BCG.

6. The fusion protein according to any one of the preceding claims, wherein the antigens are selected from :

7. The fusion protein according to any one of the preceding claims, wherein the fusion protein comprises:

8. The fusion protein according to any one of the preceding claims, wherein the fusion protein comprises:

a) 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) or variants or immunogenic epitopes thereof , or

9. The fusion protein according to according to any one of claims 5-7, 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).

10. The fusion protein according to any one of the preceding claims, 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.

11. The fusion protein according to any one of the preceding claims, wherein the fusion protein comprises at least four ESAT-6 repeats.

12. The fusion protein according to any one of claims 10 or 11, wherein the ESAT- 6 repeats are separated by at least one antigen different from ESAT-6.

13. The fusion protein according to any one of claims 10-12, wherein the ESAT-6 repeats are positioned alternately with antigens different from ESAT-6.

14. The fusion protein according to any one of the preceding claims, wherein the fusion protein comprises:

a) 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) 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), and

15. The fusion protein according to any one of the preceding claims, wherein the fusion protein comprises the amino acid sequence represented by any one selected from the group consisting of:

a) SEQ ID NO:9, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID

16. The fusion protein according to any one of claims 1-15, wherein the fusion protein comprises the amino acid sequence represented by SEQ ID NO: 9 or SEQ ID NO:91.

17. The fusion protein according to any one of claims, wherein the fusion protein is encoded by nucleic acid sequences selected from the group consisting of:

a) SEQ ID NO: 54 (H107), SEQ ID NO: 79 (H107b), SEQ ID NO: 80

(H107c), SEQ ID NO:92 (H107e) SEQ ID NO: 81 (H106), SEQ ID NO: 82 (H105) and SEQ ID NO:83 (H104), or variants or fragments thereof; or b) nucleic 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).

18. The fusion protein according to any one of the preceding claims, wherein the antigens of the fusion proteins are connected with a linker molecule.

19. The fusion protein according to any one of the preceding claims, wherein the fusion protein comprises at least one antigen that does prime an immune response against BCG.

20. The fusion protein according to any one of claim 19, wherein the at least one antigen that does prime an immune response against BCG is selected from :

21. A vaccine or immunogenic composition comprising a fusion protein according to any one of the preceding claims.

22. The vaccine or immunogenic composition according to claim 21, wherein the vaccine or immunogenic composition further comprises one or more adjuvants.

23. The vaccine or immunogenic composition according to claim 22, wherein 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, monophosphoryl lipid A (MPL), Trehalose Dimycolate (TDM), Trehalose Dibehenate (TDB), Muramyl Dipeptide (MDP), monomycolyl glycerol (MMG), CpG and "IC31", or combinations hereof.

24. The vaccine or immunogenic composition according to any one of claims 21- 23, wherein the vaccine or immunogenic composition further comprises BCG.

25. A fusion protein according to any one of claims 1-20 or a vaccine or immunogenic composition according to any one of claims 21-24 for use in vaccination or immunization of a subject against infections and/or disease caused by a virulent mycobacterium.

26. The fusion protein, vaccine or immunogenic composition for use according to claim 25, wherein the subject is a mammal.

27. The fusion protein, vaccine or immunogenic composition for use according to claim 26, wherein the mammal is a human.

28. The fusion protein, vaccine or immunogenic composition for use according to any one of claims 25-27, 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.

29. The fusion protein, vaccine or immunogenic composition for use according to any one of claims 25-28, wherein BCG is administered prior to, simultaneously or subsequent to administration of the fusion protein, vaccine or immunogenic composition.

30. The fusion protein, vaccine or immunogenic composition for use according to claim 29, wherein BCG is administered simultaneously with administration of the fusion protein, vaccine or immunogenic composition.

31. The fusion protein, vaccine or immunogenic composition for use according to any one of claims 25-30, wherein the subject has previously been vaccinated with BCG.

32. A kit comprising :

i) A fusion protein according to any one of claims 1-20 or a vaccine or

immunogenic composition according to any one of claims 21-24, ii) BCG, and

iii) optionally, instructions for use.

33. The kit according to claim 32, wherein i) and ii) are for simultaneous, separate or sequential administration.

34. A nucleic acid sequence comprising a sequence encoding a fusion protein according to any one of claims 1-20.

35. A recombinant expression vector comprising a nucleotide sequence according to claim 34 operatively linked to one or more control sequences suitable for directing the production of the fusion protein in a suitable host.

36. A recombinant host cell comprising an expression vector according to claim 35.