The present invention relates to immunogenic compositions comprising viral vectors capable of expressing novel combinations of antigens. The present invention also provides fusion proteins comprising novel combinations of antigens, and their use in the treatment and prevention of diseases such as tuberculosis.

All publications, patents and patent applications cited herein are incorporated by reference.

Background to the invention

Tuberculosis (TB) is caused by the respiratory pathogen Mycobacterium

tuberculosis and kills 2 million people each year, predominantly in the developing world. The only licensed vaccine against M. tuberculosis, Bacille Calmette-Guerin (BCG) ¹ , is an attenuated strain of Mycobacterium bovis, which is typically administered intradermally as a single dose to newborn infants. However, protective efficacy against adult pulmonary disease varies from 0-80% ² and wanes with time ³ . Bovine tuberculosis (όΊ ) also represents a significant cause of mortality and morbidity amongst cattle. It is controlled in the UK through the test and slaughter policy. However, since the 1980s the incidence of bTB has been increasing steadily. This has had economic consequences and raises potential human and animal welfare issues. Vaccination in areas of high incidence has been proposed as a sustainable control measure, particularly in countries where there exists a wildlife reservoir of tuberculosis infection.

Administration of a viral vector expressing mycobacterial antigens, and BCG vaccination followed by immunisation with viral vectors expressing the

mycobacterial Ag85A antigen, have been shown to confer protection against challenge with Mycobacteria bovis and Mycobacterium tuberculosis \ Radosevic et at have demonstrated that vaccination with a recombinant adenovirus vector serotype 35 (rAd35) expressing a fusion protein composed of Ag85A, Ag85B and TB10.4 confers protection against Mycobacterium tuberculosis when administered to mice through an intranasal or intramuscular route. Magalhaes et at have demonstrated the immunogenicity of a similar vaccine when used as a booster in

BCG-primed rhesus macaques. Jeyanathan et af have demonstrated that the use of a bivalent Adenovirus-vectored vaccine expressing Ag85a and TBI 0.4 antigens as a fusion protein can induce robust T-cell responses towards the respective antigens within the airway lumen and spleen.

However, the antigen composition, strength and durability of the immune responses induced by these vaccines has yet to be optimized and, in the face of the global TB epidemic, there remains a need to develop increasingly effective vaccines. The present invention provides immunogenic compositions comprising a viral vector expressing a unique combination of at least four different antigens which induce a surprisingly strong and durable immune response.

Description According to a first aspect of the present invention there is provided an immunogenic composition comprising a non-replicating or replication impaired viral vector capable of expressing the translation product of mycobacterial Ag85A, TB9.8, TB10.4 and Acr2 genes. The Ag85A, TB9.8, TB10.4 and Acr2 genes are collectively referred to herein as "the antigen genes" and the translation products of these genes as "the antigens".

The 85A antigen (Ag85A) (Accession Nos. CAA17868 and BX842584) is a member of the Ag85 complex, a family of proteins secreted by M. tuberculosis, BCG, M.bovis and many other species of mycobacteria. Ag85A is encoded by the βρΑ gene. The Ag85A from M. tuberculosis is listed in SEQ ID NO. 1 and SEQ ID NO.2. It is highly conserved amongst all mycobacterial species and is

immunodominant in animal and human studies. Preferably, the Ag85A antigen comprises a C terminal truncation of the wild type sequence. More preferably, the Ag85A antigen comprises a 15 amino acid C terminal truncation. In one

embodiment, the Ag85A antigen comprises the sequence of SEQ ID NO.3.

Preferably, the Ag85A antigen is encoded by the nucleotide sequence of SEQ ID NO. 4.

TB9.8, also known as Rv0287, is a member of the ESAT-6 family and is relatively conserved in a number of mycobacteria including M, tuberculosis, BCG and M. bovis. In one embodiment, the TB9.8 antigen comprises the sequence of SEQ ID NO.5. Preferably, the TB9.8 antigen is encoded by the nucleotide sequence of SEQ ID NO. 6. Acr2, also known as Rv0251c, is an alpha-crystallin homologue HSP which is conserved amongst M. tuberculosis, BCG, M. bovis and many other species of mycobacteria. In one embodiment, the Acr 2 antigen comprises the sequence of SEQ ID NO.7. Preferably, the Acr2 antigen is encoded by the nucleotide sequence of SEQ ID NO. 8.

TB10.4, also known as Rv0288, is a member of the ESAT-6 family and relatively conserved in many species of mycobacteria including M. tuberculosis, BCG and M. bovis. In one embodiment, the TB10.4 antigen comprises the sequence of SEQ ID NO. . Preferably, the TB10.4 antigen is encoded by the nucleotide sequence of SEQ ID NO. 10.

The term "antigen" as used herein also includes fragments and variants such as derivatives, analogues, homologues and functional equivalents of the parent antigen. As used herein, "parent antigen" encompasses the wildtype antigen or antigen sequences exemplified herein.These fragments and variants retain essentially the same biological activity or function as the parent antigen. Preferably, they retain or improve upon the same antigenicity and/or immunogenicity as the parent antigen. Generally, "antigenic" is taken to mean that the protein or polypeptide is capable of being used to raise antibodies or indeed is capable of inducing an antibody response in a subject. "Immunogenic" is taken to mean that the protein or polypeptide is capable of eliciting a potent and preferably a protective immune response in a subject. Thus, in the latter case, the protein or polypeptide may be capable of generating an antibody response and a non-antibody based immune response.

Preferably, fragments of the antigens comprise at least n consecutive amino acids from the sequence of the parent antigen, wherein n is preferably 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 57, 58, 59, 60, 70, 80, 90, 100 or more. The fragments preferably include one or more epitopic regions from the parent antigen, or are sufficiently similar to such regions to retain their antigemc/irnmunogenic properties.

The antigens of the present invention include variants such as derivatives, analogues, homologues or functional equivalents of the parent antigen. Particularly preferred are derivatives, analogues, homologues or functional equivalents having an amino acid sequence similar to that of the parent antigen, in which one or more amino acid residues are substituted, deleted or added in any combination.

Especially preferred are silent substitutions, additions and deletions, which do not alter the properties and activities of the protein of the present invention. Various amino acids have similar properties, and one or more such amino acids of a substance can often be substituted by one or more other such amino acids without eliniinating a desired activity of that substance. Thus the amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains). Of these possible substitutions it is preferred that glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic). Other amino acids which can often be substituted for one another include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulphur containing side chains). Variants include naturally occurring and artificial variants. Artificial variants may be generated using mutagenesis techniques, including those applied to nucleic acid molecules, cells or organisms. Preferably, these variants retain an antigenic determinant or epitope in common with the parent antigen.

Preferably, the derivatives, analogues, homologues, and functional equivalents have an amino acid sequence at least 50%, 60%, 70%, 80%, 90% , 95% or 99 % identical to amino acid sequence of the parent antigen. One can use a program such as the CLUSTAL program to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score.

One or more of the antigens or antigen genes may be truncated at the C-terminus and/or the N-terminus. This may facilitate cloning and construction of the vectored vaccine and/or enhance the immunogenicity or antigenicity of the antigen. Methods for truncation will be known to those of skill in the art. For example, various well- known techniques of genetic engineering can be used to selectively delete the encoding nucleic acid sequence at either end of the antigen gene, and then insert the desired coding sequence into the viral vector. For example, truncations of the candidate protein are created using 3 ' and/or 5 ' exonuclease strategies selectively to erode the 3' and/or 5' ends of the encoding nucleic acid, respectively. Preferably, the wild type gene sequence is truncated such that the expressed antigen is truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids relative to the parent antigen. Most preferably, the antigen gene is truncated by 15 amino acids at the C- terminus relative to the wild type antigen. Preferably, the Ag8 A antigen is C-terminally truncated.

One or more of the antigen genes may also comprise a leader sequence. The leader sequence may affect processing of the primary transcript to mRNA, translation efficiency, mRNA stability, and may enhance expression and/or immunogenicity of the antigen. Preferably, the leader sequence is tissue plasminogen activator (tPA)\ Preferably, the tPA leader sequence comprises the sequence of SEQ ID NO. 11, or a sequence 80% , 90% , 95 % or 99% identical thereto. Preferably, the tPA leader sequence comprises the nucleotide sequence of SEQ ID NO. 12 or a sequence 80%, 90%, 95 % or 99% identical thereto. Preferably, the tPA leader sequence is positioned N-terminal to the Ag85A antigen.

The tPA leader sequence may be linked to the sequence of the Ag85A antigen via a peptide linker. Peptide linkers are generally from 2 to about 50 amino acids in length, and can have any sequence, provided that it does not form a secondary structure that would interfere with domain folding of the fusion protein. Preferably, the linker comprises the sequence of SEQ ID NO. 13. Preferably, the linker sequence is encoded by the nucleotide sequence of SEQ ID NO. 14.

One or more of the antigen genes may comprise a marker such as the Green Fluorescent Protein (GFP) marker to facilitate detection of the expressed product of the inserted gene sequence.

One or more of the antigen genes may comprise a nucleic acid sequence encoding a tag polypeptide that is covalently linked to the antigen upon translation. Preferably the tag polypeptide is selected from the group consisting of a PK tag, a FLAG tag, a MYC tag, a polyhistidine tag or any tag that can be detected by a monoclonal antibody. The nucleic acid sequence encoding the tag polypeptide may be positioned such that, following translation, the tag is located at the C-terminus or the N- terminus of the expressed antigen or may be internal to the expressed antigen. Preferably, the tag is located at the C-terminus of the expressed antigen. In a preferred embodiment, one or more of the antigen genes encode a PK tag with the sequence of SEQ ID NO. 15.

A tag of this type may facilitate detection of antigen expression and clones expressing the antigen, and/or enhance the immunogenicity or antigenicity of the antigen.

If a tag polypeptide is used, nucleotides encoding a linker sequence are preferably inserted between the nucleic acid encoding the tag polypeptide and the nucleic acid encoding the expressed antigen. Preferably, the linker sequence comprises the sequence of SEQ ID NO. 16.

The antigen genes encoding the Ag85A, TB9.8, TB10.4 and Acr2 antigens may comprise separate genes at separate locations within the viral vector. In such an embodiment, the translation product of each gene is a separate polypeptide corresponding to an individual antigen.

Alternatively, the antigen genes encoding Ag85A, TB9.8, TB10.4 and Acr2 may comprise a fusion gene, the translation product of which is a fusion protein comprising Ag85A, TB9.8, TB10.4 and Acr2. The vector used in the present invention is preferably a non-replicating or replication-impaired viral vector. The term "non-replicating" or "replication- impaired" as used herein means not capable of replication to any significant extent in the majority of normal human cells. Viruses which are non-replicating or replication-impaired may have become so naturally (i.e. they may be isolated as such from nature) or artificially (e.g. by breeding in vitro or by genetic manipulation, for example deletion of a gene which is critical for replication). There will generally be one or a few cell types in which the viruses can be grown, such as CEF cells for modified virus Ankara (MVA). Replication of a virus is generally measured in two ways: 1) DNA synthesis and 2) viral titre. More precisely, the term "non-replicating or replication-impaired" implies that the viruses satisfy either or both of the following criteria:

1) exhibit a 1 log (10 fold) reduction in DNA synthesis compared to the Copenhagen strain of vaccinia virus in MRC-5 cells (a human cell line);

2) exhibit a 2 log reduction in viral titre in HELA cells (a human cell line) compared to the Copenhagen strain of vaccinia virus.

It is preferred that the viral vector is incapable of causing a serious infection in the human patient. However, the viral vector is preferably capable of stimulating a T cell response.

As used herein, viral vectors "capable of expressing" the translation product of the antigen genes encompasses those viral vectors which are expressing the translation product, and those viral vectors which are not in the process of expressing the translation product, but which comprise the necessary nucleic acid sequences encoding the antigens.

Examples of viral vectors that are useful in this context are poxvirus vectors, adenovirus vectors, alphavirus vectors, herpes viral vectors, flavivirus vectors, arenavirus vectors, lentivirus vectors, retroviral vectors and influenza virus vectors.

Preferably, the viral vector comprises a poxvirus vector or an adenovirus vector.

Poxvirus vectors include vaccinia virus vectors and avipox vectors. Avipox vectors include canarypox or fowlpox vectors. Particularly suitable as an avipox vector is a strain of canarypox known as ALVAC {commercially available as Kanapox), strains derived from ALVAC, and a fowipox strain known as FP9. Particularly preferred poxvirus vectors include MVA, strains derived from MVA, and NYVAC.

Particularly preferred advenovirus vectors include both human and non-human adenovirus vectors such as Adenovirus 5 (Ad5) and AdCh63. Adenoviral vectors are particularly suitable for use in the present invention. It has been found that the use of an adenovirus vector induces a very strong CD 8 memory T cell response in addition to a very strong CD4 memory T cell response. The induction of both a CD 8 and a CD4 memory T cell response by the same vaccine is likely to be of benefit in both the prophylaxis and treatment of mycobacterial disease.

The immunogenic composition of the present invention may comprise more than one type of viral vector. Preferably, the immunogenic composition comprises 1, 2, 3, 4 or more different viral vectors. In one embodiment, the immunogenic composition of the present invention comprises both a pox virus vector and an adenoviral vector. Preferably, the immunogenic composition comprises MVA, or a strain derived from MVA, and Ad5.

The immunogenic composition according to the first aspect may comprise one or more additional active ingredients.

The immunogenic composition may comprise one or more additional antigens or epitopes derived from disease-causing agents. Preferably, the one or more additional antigens is/are derived from M. tuberculosis, Plasmodium sp, influenza virus, HIV, Hepatitis C virus, Cytomegalovirus, Human papilloma virus, malaria, leishmania parasites or, preferably, any mycobacteria spp..

The immunogenic composition may comprise one or more antimicrobial compounds. Examples of antimicrobials suitable for use in the compositions of the invention are antiruberculous chemotherapeutics such as rifampicin, isoniazid, ethambutol, pyrizinamide, etc. Suitable additional excipients, diluents, adjuvants or the like may be included.

Suitable carriers and/or diluents are well known in the art and include

pharmaceutical grade starch, mannitol, lactose, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, (or other sugar), magnesium carbonate, gelatin, oil, alcohol, detergents, emulsifiers or water (preferably sterile). The composition may be a mixed preparation of a composition or may be a combined preparation for simultaneous, separate or sequential use (including administration).

The composition according to the invention for use in the aforementioned indications may be administered by any convenient method, for example by oral (including by inhalation), parenteral, mucosal (e.g. buccal, sublingual, nasal, aerosol), rectal or transdermal administration and the compositions adapted accordingly. Preferably, the immunogenic compositions are administered by injection or by aerosol or mucosal methods.

For oral administration, the composition can be formulated as liquids or solids, for example solutions, syrups, suspensions or emulsions, tablets, capsules and lozenges.

A liquid formulation will generally consist of a suspension or solution of the compound or physiologically acceptable salt in a suitable aqueous or non-aqueous liquid carrier(s) for example water, ethanol, glycerine, polyethylene glycol or oil. The formulation may also contain a suspending agent, preservative, flavouring or colouring agent.

A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations. Examples of such carriers include magnesium stearate, starch, lactose, sucrose and microcrystalline cellulose.

A composition in the form of a capsule can be prepared using routine encapsulation procedures. For example, powders, granules or pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatine capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatine capsule.

Compositions for oral administration may be designed to protect the active ingredient against degradation as it passes through the alimentary tract, for example by an outer coating of the formulation on a tablet or capsule. Typical parenteral compositions consist of a solution or suspension of the compound or physiologically acceptable salt in a sterile aqueous or non-aqueous carrier or parenteraily acceptable oil, for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil. Alternatively, the solution can be lyophilised and then reconstituted with a suitable solvent just prior to administration.

Compositions for nasal or oral administration may conveniently be formulated as aerosols, drops, gels and powders. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomising device. Alternatively the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve, which is intended for disposal once the contents of the container have been exhausted. Where the dosage form comprises an aerosol dispenser, it will contain a pharmaceutically acceptable propellant. The aerosol dosage forms can also take the form of a pump-atomiser. Compositions suitable for transdermal adrriinistration include ointments, gels, patches and injections including powder injections. Conveniently the composition is in unit dose form such as a tablet, capsule or ampoule.

The pharmaceutical composition is preferably sterile. It is preferably pyrogen-free. It is preferably buffered e.g. at between pH 6 and pH 8, generally around pH 7. Preferably, the composition is substantially isotonic with humans.

The immunogenic compositions according to the present invention may be prophylactic (i.e. to prevent infection), post-exposure (i.e. to treat after infection but before disease) or therapeutic (i.e. to treat disease). Preferably, the vaccines are prophylactic or post-exposure. Where the immunogenic composition is for prophylactic use, the subject is preferably an infant, young child, older child or teenager. Where the immunogenic composition is for therapeutic use, the subject is preferably an adult. The immunogenic compositions of the invention deliver an immunologically effective amount of the antigens to the subject. By 'immunologically effective amount' , it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the capacity of the individual's immune system, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. Preferably, the immunogenic composition according to the first aspect of the present invention is a vectored vaccine. According to a second aspect of the present invention, there is provided a fusion protein comprising the Ag85A, TB9.8, Acr2 and TB10.4 antigens. The term "fusion protein" as used herein is taken to mean one or more proteins joined together by chemical means and/or by peptide bonds through protein synthesis. Translation of the poly-antigen fusion gene encoding the fusion protein of the present invention results in the production of a single polypeptide with functional properties derived from all four of the original antigens.

The antigens may be arranged in any order within the fusion protein. Preferably, the Ag85A antigen is positioned N-terminal to the TB9.8, Acr2 and TB10.4 antigens. Preferably, the antigens are arranged, from the N terminus, in the order: Ag85A, TB9.8, Acr2 and TB10.4.

The fusion protein may comprise one or more additional antigens, including further mycobacterial antigens.

Peptide linkers may be employed to separate two or more of the antigens in the fusion protein of the present invention. Peptide linkers are classically used in fusion proteins in order to ensure their correct folding into secondary and tertiary structures. They are generally from 2 to about 50 amino acids in length, and can have any sequence, provided that it does not form a secondary structure that would interfere with domain folding of the fusion protein. Preferably, a peptide linker separates the Ag85A antigen from the adjacent antigens. More preferably, a peptide linker separates the Ag85A antigen from the TB9.8 antigen. In one embodiment, the peptide linker comprises the sequence of SEQ ID NO. 13.

The fusion protein may also comprise a leader sequence N-terminal to the Ag85 A antigen. Preferably, the leader sequence is a tPA leader sequence as described above. In a particularly preferred embodiment, the fusion protein comprises a tPA leader sequence, a C-terminally truncated Ag85a antigen, TB9.8 antigen, TB10.4 antigen and Acr2 antigen. Preferably, the fusion protein comprises the sequence of SEQ ID NO.17.

According to a third aspect of the present invention there is provided a recombinant polynucleotide comprising a sequence encoding the fusion protein of the second aspect of the present invention. The recombinant polynucleotide may comprise:

(i) a DNA sequence which encodes the fusion protein of the second aspect of the present invention or its RNA equivalent;

(ii) a sequence which is complementary to any of the sequences of (i);

(ii) a sequence which has substantial identity with those of (i) or (ii)

The recombinant polynucleotide, or nucleic acid construct, may comprise any length of nucleic acid which may be DNA, cDNA or RNA such as mRNA obtained by cloning or produced by chemical synthesis. The DNA may be single or double stranded. Single stranded DNA may be the coding sense strand, or it may be the non-coding or anti-sense strand. For therapeutic use, the nucleic acid construct is preferably in a form capable of being expressed in the subject to be treated.

The recombinant polynucleotide preferably comprises a first nucleic acid sequence encoding Ag85A, a second nucleic acid sequence encoding TBS ⁾ .8, a third nucleic acid sequence encoding Acr2 and a fourth nucleic acid sequence encoding TB104. The recombinant polynucleotide may additionally comprise nucleic acid sequences encoding further antigens, leader sequences, linker sequences and/or tags.

The recombinant polynucleotide preferably includes a promoter or other regulatory sequence which controls expression of the nucleic acid. Promoters and other regulatory sequences which control expression of a nucleic acid have been identified and are known in the art. When the viral vector comprises an adenovirus, the promoter is preferably selected from the group comprising human CMV promoters, murine CMV promoters, ubiquitin, actin and other mammalian promoters. Most preferred are human CMV promoters. When the viral vector comprises a poxvirus, a poxvirus promoter such as p7.5 or SSP is preferred.

Preferably, the recombinant polynucleotide of the present invention comprises the sequence of SEQ ID NO. 18. One of skill in the art will appreciate that the present invention can include variants of those particular nucleic acid molecules which are exemplified herein. These may occur in nature, for example because of strain variation. For example, additions, substitutions and/or deletions are included. One of skill in the art will also appreciate that variation from the particular nucleic acid molecules exemplified herein will be possible in view of the degeneracy of the genetic code.

Preferably, the variants have substantial identity to the nucleic acid molecules exemplified herein. Sequences which have substantial identity preferably have at least 50%, 60% , 70%, 80% , 90% , 95 % or 99% identity with said sequences.

When comparing nucleic acid sequences for the purposes of determining the degree of homology or identity one can use programs such as BESTFIT and GAP (both from the Wisconsin Genetics Computer Group (GCG) software package) BESTFIT, for example, compares two sequences and produces an optimal alignment of the most similar segments. GAP enables sequences to be aligned along their whole length and finds the optimal alignment by inserting spaces in either sequence as appropriate. Suitably, in the context of the present invention compare when discussing identity of nucleic acid sequences, the comparison is made by alignment of the sequences along their whole length. The recombinant polynucleotide of the present invention may comprise a polyvalent insert. Such a polyvalent insert may be inserted by one of skill in the art into any appropriate viral vector. According to a fourth aspect of the present invention there is provided a viral vector comprising the recombinant polynucleotide or polyvalent insert of the fourth aspect of the present invention. The recombinant polynucleotide or polyvalent insert is preferably expressed from the viral vector at a suitable insertion site, using a suitable promoter. Suitable insertion sites are well known to one of skill in the art. Preferred insertion sites include the El region in adenoviral vectors and the Tk locus in poxvirus vectors. Suitable promoters are well known in the art and preferred promoters are discussed above.

The fourth aspect of the present invention also provides a viral vector comprising two, three or four separate polynucleotides encoding the Ag85A, TB9.8, Acr2 and TBI 0.4 antigens, which may be expressed from the viral vector from different insertion sites.

According to a fifth aspect of the present invention there is provided a method of treating or preventing a disease in a subject comprising administering to the subject an immunogenic composition according to the first aspect of the present invention, the fusion protein according to the second aspect of the present invention, the

recombinant polynucleotide according to the third aspect of the present invention and/or the viral vector according to the fourth aspect of the present invention.

The treatment or prevention of mycobacterial diseases such as tuberculosis is associated with the maintenance of a strong cell-mediated response to infection involving both CD4+ and CD8+T cells and the ability to respond with Thl-type cytokines, particularly IFN-γ ⁹ . It is desirable to induce a memory immune response. Memory immune responses are classically attributed to the reactivation of long-lived, antigen-specific T lymphocytes that arise directly from differentiated effector T cells and persist in a uniformly quiescent state. Memory T cells have been shown to be heterogeneous and to comprise at least two subsets, endowed with different migratory capacity and effector function; effector memory T cells (TEM) and central memory T cells (CTM). TEM resemble the effector cells generated in the primary response in that they lack the lymph node-homing receptors L-selectin and CCR7 and express receptors for migration into inflamed tissues. Upon re-encounter with antigen, these TEM can rapidly produce IFN-γ or IL-4 or release pre- stored perform. TCM express L-selectin and CCR7 and lack immediate effector function. These cells have a low activation threshold and, upon restimulation in secondary lymphoid organs, proliferate and differentiate to effectors.

Preferably, the method according to the fifth aspect of present invention comprises a method of generating a T cell immune response in a subject. Preferably, the T cell immune response is a protective T cell immune response. Preferably, the T cell immune response is long lasting and persists for at least 1, 2, 5, 10, 15, 20 , 25 or more years.

As demonstrated in the example below, the immunogenic composition of the present invention induces a surprisingly strong response to recombinant mycobacterial antigens and purified protein derivative (PPD-B). This response is significantly stronger than that induced by immunogenic compositions comprising viral vectors expressing only Ag85A. An unexpected synergistic interaction between the four antigens has been demonstrated, which may be responsible for the strength of the immune response induced.

The immunogenic composition according to the first aspect of the present invention, the fusion protein according to the second aspect of the present invention, the recombinant polynucleotide according to the third aspect of the present invention and/or the viral vector according to the fourth aspect of the present invention may be administered either as a single immunisation or multiple immunisations.

Preferably, the immunogenic composition according to the first aspect of the present invention, the fusion protein according to the second aspect of the present invention, the recombinant polynucleotide according to the third aspect of the present invention and/or the viral vector according to the fourth aspect of the present invention are administered as part of a single, double or triple vaccination strategy. They may also be administered as part of a homologous or heterologous prime-boost immunisation regime.

The methods according to the fifth aspect of the present invention have been found to be particularly effective in subjects that have been previously exposed to a mycobacterial antigen. The fifth aspect of the present invention therefore includes a method of treating or preventing a disease in a subject comprising administering to the subject at least one mycobacterial antigen in combination with an immunogenic composition according to the first aspect of the present invention, a fusion protein according to the second aspect of the present invention, a recombinant

polynucleotide according to the third aspect of the present invention or a viral vector according to the fourth aspect of the present invention. Thus, the fifth aspect of the present invention provides a method of treating or preventing a disease in a subject comprising the steps of: (a) administering at least one mycobacterial antigen; and

(b) administering animmunogenic composition according to the first aspect of the present invention, a fusion protein according to the second aspect of the present invention, a recombinant polynucleotide according to the third aspect of the present invention or a viral vector according to the fourth aspect of the present invention.

The mycobacterial antigen and the immunogenic composition, fusion protein, recombinant polynucleotide or viral vector according to the present invention may be administered simultaneously, sequentially or separately. For example, the immunogenic composition according to the first aspect of the present invention, fusion protein according to the second aspect of the present invention, recombinant polynucleotide according to the third aspect of the present invention or viral vector according to the fourth aspect of the present invention may be administered to prime the patient before administration of the mycobacterial antigen(s) or after the administration of the mycobacterial antigen(s) to boost the patient's immune response to that antigen.

The mycobacterial antigen may be from M. tuberculosis and/or from one or more other bacteria such as M. avium-intracellulare, M. kansasii, M. marinum and M. ulcerans.

Alternatively or additionally, the subject may have been pre-exposed to one or more mycobacteria themselves. The subject may be latently infected with one or more mycobacteria. In particular, the subject may have been pre-exposed to and/or be latently infected with M. tuberculosis, M. avium-intracellulare, M. kansasii, M. marinum and/or M. ulcerans,

Pre-exposure may comprise previous inoculation with the BCG (Bacille Calmette- Guerin) vaccine. 80% of infants throughout the world receive BCG each year. However, the efficacy of the vaccine wanes as the individual reaches 10 to 15 years of age. Although repeated vaccination with BCG does not appear to further enhance protection against TB, incorporating BCG into a heterologous prime-boost regime may retain the protective effects of BCG. In a preferred embodiment, immunogenic composition according to the first aspect of the present invention, the fusion protein according to the second aspect of the present invention, the recombinant polynucleotide according to the third aspect of the present invention or the viral vector according to the fourth aspect of the present inventionare administered to BCG-primed subjects. Thus, the fifth aspect of the present invention provides a method of treating or preventing a disease in a subject comprising the steps of:

(a) vaccinating the subject with a BCG vaccine; (b) administering an immunogenic composition according to the first aspect of the present invention, a fusion protein according to the second aspect of the present invention, a recombinant polynucleotide according to the third aspect of the present invention or a viral vector according to the fourth aspect of the present invention.

The BCG vaccine and the immunogenic composition, fusion protein, recombinant polynucleotide or viral vector according to the present invention may be

administered simultaneously, sequentially or separately

Administration of the immunogenic composition, fusion protein, recombinant polynucleotide or viral vector according to the present invention may occur up to or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more weeks or 0.25 , 0.5 , 0.75, 1, 5, 10, 15, 20, 25, 30, 35 or 40 or more years after vaccination with the BCG vaccine. Preferably, the immunogenic composition, fusion protein, recombinant

polynucleotide or viral vector according to the present invention is administered at least 4 weeks after the BCG vaccination. Preferably, the immunogenic composition, fusion protein, recombinant polynucleotide or viral vector according to the present invention is administered up to 20 years after the BCG vaccination.

The immunogenic composition may comprise a single viral vector, or a mixture of viral vectors, as described above.

The method may include second or subsequent administrations of the immunogenic composition, fusion protein, recombinant polynucleotide or viral vector according to the present invention. The second administration can be administered over a short time period or over a long time period. The doses may be administered over a period of hours, days, weeks, months or years, for example up to or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more weeks or 0.25, 0.5, 0.75, 1, 5, 10, 15, 20, 25, 30, 35 or 40 or more years after the first adrrunistration. Preferably, the second administration occurs at least 2 months after the first administration. Preferably, the second administration occurs up to 10 years after the first administration. These time intervals preferably apply mutatis mutandis to the period between any subsequent doses. The first and subsequent administrations of the immunogenic composition according to the present invention may comprise administration of the same immunogenic composition, or of different immunogenic compositions. Where different immunogenic compositions are used, these may include immunogenic compositions comprising different viral vectors. For example, the first administration may comprise an immunogenic composition according to the present invention comprising a first viral vector, whilst the second administration may comprise an immunogenic composition according to the present invention comprising a second viral vector. Preferably, the first viral vector is a poxvirus vector and the second viral vector is an adenoviral vector, or vice versa. More preferably, the first viral vector is MVA and the second viral vector is ADS, or vice versa. Alternatively, the first and subsequent adrninistrations may comprise administrations of

immunogenic compositions comprising a mix of two or more different viral vectors.

The subject to be treated is preferably mammalian. Preferred mammals include cows, sheep, goats, pigs, wild boar, buffalo, bison, horses, camelids, deer, elephants, badgers, possums, cats, lions, monkeys and humans.

The Ag85A, TB9.8, Acr2 and TB10.4 antigens are highly conserved amongst mycobacterial species. The immunogenic composition of the present invention can therefore be used to treat or prevent a variety of diseases. Preferably, the disease is a mycobacterial disease. Preferably, the disease in question is selected from the group comprising tuberculosis, leprosy, Mycobacterium avium infection, non- tuberculosis mycobacterial infection, Buruli ulcer, Mycobacterium bovis infection or disease, Mycobacterium paratuberculosis infection, Mycobacterium ulcerans infection, Mycobacterium Kansasii infection, Mycobacterium marinum infection or related disease. Where the viral vector comprises MVA, the disease in question may also be selected from smallpox and monkeypox. Numerous other diseases and conditions are believed to be linked to mycobacterial infection, or are known to be treated or prevented using other mycobacterial vaccines. The immunogenic composition of the present invention is therefore also provided for the treatment or prevention of other, non-mycobacterial diseases including inflammatory bowel disease, Crohns disease, autoimmune disease, cancer and bladder cancer.

The fifth aspect of the present invention encompasses the immunogenic composition of the first aspect of the present invention, the fusion protein according to the second aspect of the present invention, the recombinant polynucleotide according to the third aspect of the present invention or the viral vector according to the fourth aspect of the present invention for use in the treatment or prevention of a disease in a subject. The fifth aspect of the present invention also includes the use of the immunogenic composition of the first aspect of the present invention, the fusion protein according to the second aspect of the present invention, the recombinant polynucleotide according to the third aspect of the present invention or the viral vector according to the fourth aspect of the present invention in the manufacture of a medicament for the treatment or prevention of a disease in a subject.

According to a sixth aspect of the present invention there is provided a kit comprising the immunogenic composition according to the first aspect of the present invention, the fusion protein according to the second aspect of the present invention, the recombinant polynucleotide according to the third aspect of the present invention and/or the viral vector according to the fourth aspect of the present invention. The kit may additionally comprise a further mycobacterial antigen, or a BCG vaccine as described above, for simultaneous, sequential or separate administration with the immunogenic composition, fusion protein, recombinant polynucleotide or viral vector of the present invention. The specific details recounted above and described in the example apply mutatis mutandis to all aspects and embodiments of the present invention.

Figures

Figure 1 shows IFN-γ secretion in whole blood cultures 24 hr after stimulation with: (A) PPD-B, (B) Ag85, (C) TB9.8 or (D) TB10.4. The Figure shows the median with the interquartile range production of IFN-γ secreted into superaatants in whole blood assays for each group against the listed antigens. 'Poly' indicates expression of the Ag85A, TB9.8, Acr2 and TB10.4 antigens. 'A' indicates an Adenoviral vectored vaccine. 'M' indicates an MVA vectored vaccine. 'Mix' indicates a mix of

Adenoviral and MVA vectored vaccines.

Figure 2 shows ex- vivo IFN-γ ELISPOT after overnight stimulation of PBMC cultures with (A) PPD-B, (B) Ag85, (C) TB9.8 or (D) TB10.4. The Figure shows the median with the interquartile range production of IFN-γ trapped by membrane bound antibodies in ELISPOT for each group against the listed antigens. 'Poly' indicates expression of the Ag85A, TB9.8, Acr2 and TB10.4 antigens. 'A' indicates an

Adenoviral vectored vaccine 'M' indicates an MVA vectored vaccine. 'Mix' indicates a mix of Adenoviral and MVA vectored vaccines.

The invention will now be described by way of the following non-limiting example.

Example

Materials and Methods

In summary, the immune response generated by the immunogenic compositions of the present invention were tested by vaccinating cattle with BCG and, 8 weeks later, administering recombinant Adveno virus 5 (Ad5), Modified Vaccinia Ankara virus (MVA) or a mixture thereof. Each viral vector expressed the mycobacterial mycolyl transferase Ag85A alone or as part of a fusion protein comprising TB9.8, TBI 0.4 and Acr2. The fusion protein of the present invention used in this example had a sequence corresponding to SEQ ID NO.17. Four weeks after the first viral administration, those cattle that were treated with Ad5 were administered with MVA, whilst those first treated with MVA were administered Ad5. Those first treated with a mixture of Ad5 and MVA were administered a further mixture of Ad5 and MVA.

Specifically, 40 Friesian calves of 5-7 months of age were divided into 8 groups of 5 each:

1. Non- vaccinated controls;

2. BCG (SSI, Copenhagen, DK);

3. BCG + Ad85-MVA85& + Ad85-MVA85&;

4. BCG +Ad85 + MVA85;

5. BCG + MVA85 +Ad85;

6. BCG + Ad(Poly)-MVA(Poly)* + Ad(Poly)-MVA(Poly)*;

7. BCG +Ad(Poly) +MVA(Poly);

8. BCG + MVA(Poly) +Ad(Poly).

(& Indicates expression of mycobaterial Ag85A)

(* Poly indicates expression of Ag85A and antigens TB9.8, TB10.4 and Acr2)

BCG was applied subcutaneously at c 2xl0 ^fi cfu per dose. MVA constructs were used at 5xl0 ^a pfu per dose. Adenovirus constructs were used at 4xl0 ⁹ infectious particles. Viral constructs were applied intramuscularly. Time line of animal vaccination protocol

0 w feeks 8 w Ϊeeks 12 w Feek ^" s

BCG 1 ^st viral administration

Assays

Immune responses were evaluated by assessing antigen-specific 3-H-TdR

incorporation, secretion of IFN-γ by ELIS A and ELISPOT and determination of the cells producing IFN-γ by intracellular staining. The IFN-γ ELISPOT assay is a particularly useful assay, as the secretion of IFN-γ from antigen specific T cells is the best available correlate of protection against M.tuberculosis. Furthermore, the ELISPOT assay is a very reproducible and sensitive method of quantifying the number of IFN-γ secreting antigen specific T cells.

Whole-blood IF -γ assay (Bovigam assay):

Blood samples were collected from vaccinated or M. Z>ovz ^' i-infected cattle into heparinized Vacutainers (Becton Dickinson, Oxford, United Kingdom). The Bovigam assay was performed according to the manufacturer's instructions (Prionics, Schlieren, Switzerland). Briefly, whole-blood cultures (0.2 ml) were performed in 96-well tissue culture plates. Cultures contained 0.10 ml heparinized blood and 0.1 ml RPMI 1640 (no antigen), tuberculin at 10 μg/ml, recombinant Ag85 A at 10 μg ml ( (Lionex Ltd. (Braunschweig, Germany)), TB9.8 at 10 μg/ml _J TBI 0.4 at 5 ^ηιΐ or Acr2 at 10 μg/ml (Proteix, Biotechnologies, Jesenice, Czech Republic) . After 24 h of culture at 37°C, plasma supematants (100 μΐ/well) were transferred to a 96-well plate by using a sterile pipette and stored at -20°C until tested by IFN-γ enzyme-linked

immunosorbent assay (ELISA). Bovigam IFN-γ ELISA was performed according to the supplier's instructions. Color change in the ELISA reaction was measured as optical density at 450 nm (OD ₄₅ o) ¹⁰ . IFN-γ ELISPOT assay:

Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by Histopaque-1077 (Sigma- Aldrich) gradient centrifugation and suspended in tissue culture medium (RPMI 1640 [Sigma- Aldrich] supplemented with 10% fetal calf serum [Sigma-Aldrich], nonessential amino acids [Sigma- Aldrich] , 5 x 10 ^-5 M 2- mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate). Prior to the addition of cells, ELISPOT plates (Immobilon-P polyvinylidene difluoride membranes; Millipore, Molsheim, France) were prepared by coating them overnight at 4°C with a bovine IFN-γ specific monoclonal antibody (Mabtech, Stockholm, Sweden). Unbound antibody was then removed by washing, and the wells were blocked with 10% fetal calf serum in RPMI 1640 medium. Cells were added in 200 μΐ of medium at a concentration of 10 ⁶ cells/ml and stimulated with antigen as described for whole blood IFN-γ assay. After 24 h-of-incubation in the presence of cells, spots were developed with a biotinylated bovine IFN-y-specific monoclonal antibody

(Mabtech) followed by alkaline phosphatase-conjugatedstreptavidin (Mabtech). The spots were visualized with 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium substrate (Sigma-Aldrich). Spots were counted in an AID ELSIPOT reader ⁴ .

Statistical analysis was carried out using GraphPad Prism 5 using Kruskall-Wallis ANOVA with Dunns multiple comparison test carried our using Prism V5.0

(GraphPad, San Diego, CA, USA). Results

The results demonstrate an unexpected synergistic interaction between the four antigens in the fusion protein. Table 1 : IFN-γ secretion in whole blood cultures 24 hr after stimulation with: (A) PPD-B, (B) Ag85, (C) TB9.8 or (D) TBI 0.4. The data are the means of the responses from triplicate samples from each individual animal at each time point (0, 4, 9 and 13 weeks). In the title row of each sub-table, 'NC indicates non- vaccinated control, 'Poly' indicates expression of the Ag85A, TB9.8, Acr2 and TB10.4 antigens, 'A' indicates an Adenoviral vectored vaccine, 'M' indicates an MVA vectored vaccine and 'Mix' indicates a mix of Adenoviral and MVA vectored vaccines.

(A) PPD-B

BCG-Mix-8 15

C:Y1 C:Y2 C:Y3 C:Y4 C:Y5

0 1.719687 0.161359 0 0.159091 0

4 1.881787 0.33633 2.527784 8.451667 9.234028

9 2.693577 6.859628 0.511197 95.05209 9.963888

13 13.73415 9.84493 3.581287 48.60583 6.905903

BCG-A-M-1 55

D:Y1 D:Y2 D:Y3 D:Y4 D:Y5

0 0.266285 0.083315 0 0.014394 0.352273

4 0.226098 0.048939 16.4375 54.76167 34.4618

9 3.017103 7.221291 7.683333 21.47986 21.06389

13 65.40578 2.751401 41.08438 71.15916 34.10312 BCG-A-M-J 55

D:Y1 D:Y2 D:Y3 D:Y4 D:Y5

0.266285 0.083315 0 0.014394 0.352273 0.226098 0.048939 16.4375 54.76167 34.4618 3.017103 7.221291 7.683333 21.47986 21.06389 65.40578 2.751401 41.08438 71.15916 34.10312

(B) Ag85-A

BCG-A-M-85

D:Y1 D:Y2 D:Y3 D:Y4 D:Y5

0 0.235802 0.022167 0 0 0 4 0 0.012961 0 0 0.411806 9 0.678655 5.772434 7.402381 1.949306 24.61389 13 48.10206 3.873227 42.81771 80.6625 46.39201

BCG-M-A-85

E:Y1 E:Y2 E:Y3 E:Y4 E:Y5

0 1.079333 0 0 0.131061 0 4 0.21665 0.140731 0 0.026958 0 9 0.062739 0.076729 8.673721 13.25521 18.51771 13 0.065165 0.678566 18.03342 1.570139 14.30833

(C) TB9.8

BCG-Mix-85

C:Y1 C:Y2 C:Y3 C:Y4 C:YS

0 1.507466 0.226078 0 0.096212 0 4 0.701658 0.120142 0.193168 0.485 0 9 0.266759 7.562166 0 101.2743 3.952778 13 2.478655 4.720289 0.813023 54.7925 1.575347

BCG-A-M-85

D:Y1 D:Y2 D:Y3 D:Y4 D:Y5

0 0.235802 0.022167 0 0 0 4 0 0.012961 0 0 0.411806 9 0.678655 5.772434 7.402381 1.949306 24.61389 13 48.10206 3.873227 42.81771 80.6625 46.39201 BCG-M-A-85

E:Y1 E:Y2 E:Y3 E:Y4 E:Y5

0 1.079333 0 0 0.131061 0 4 0.21665 0.140731 0 0.026958 0 9 0.062739 0.076729 8.673721 13.25521 18.51771 13 0.065165 0.678566 18.03342 1.570139 14.30833

BCG

B:Y1 B:Y2 B:Y3 B:Y4 B:Y5

0 0.042739 0 0.232576 0.371212 0

4 0.450818 0.053136 0.069292 0.393056 3.879167

9 4.840702 0.440248 1.619097 0.342708 7.251041

13 9.138057 0.686168 19.46458 3.866667 6.077778 BCC-Mix-85

C:Y1 C:Y2 C:Y3 C:Y4 C:Y5

0 2.026024 0.126226 0 0 2.781061

4 1.354756 0.062491 0.245912 0.058333 1.395139

9 5.247907 0.194755 0.008708 1.654861 0.622222

13 41.88423 0.364618 1.244693 0.161667 1.039236

Table 2: Ex-vivo IFN-γ ELISPOT after overnight stimulation of PBMC cultures with (A) PPD-B, (B) Ag85, (C) TB9.8 or (D) TB10.4. The data are the means of the responses from triplicate samples from each individual animal at each time point (0, 4, 9 and 13 weeks). In the title row of each sub-table, 'NC indicates non- vaccinated control, 'Poly' indicates expression of the Ag85A, TB9.8, Acr2 and TB10.4 antigens, 'A' indicates an Adenoviral vectored vaccine, 'M' indicates an MVA vectored vaccine and 'Mix' indicates a mix of Adenoviral and MVA vectored vaccines.

(A) PPD-B

BCG-M A-85

E:Y1 E:Y2 E:Y3 E:Y4 E:Y5

0 0 50 150 1558 175

4 1115 147 203 1657 675

9 993 733 1768 4537 1783

13 205 783 1020 3697 2260

(B) Ag85A

BCG-Mix-85

C:Y1 C:Y2 C:Y3 C:Y4 C:Y5

0 197 52 0 168 160

4 0 542 0 703 285

9 0 1093 105 4053 810

13 0 1178 17 3273 453 BCG-A-M-85

D:Y1 D:Y2 D:Y3 D:Y4 D:YS

0 18 0 197 48 773

4 128 0 297 62 255

9 375 0 1413 3267 1063

13 1312 207 2200 3623 1147

(C) TB9.8

NC

A:Y1 A:Y2 A:Y3 A:Y4 A:Y5

0 0 142 138 41.66667 48.33333

4 242 72 0 0 418.3333

9 0 0 7 1203.333 33.33333

13 0 0 15 330 446.6667 BCG

B:Y1 B:Y2 B:Y3 B:Y4 B:Y5

0 53 151.6667 186.6667 613.3333

352 140 1360 1096.667 788.3333

0 372 0 576.6667 1420

0 0 0 180 1236.667

(D) TBI 0.4

I tCG-M-A-85

E:Y1 E:Y2 E:Y3 E:Y4 E:Y5

0 27 95 577 1055 115

4 412 43 453 2920 215

9 0 0 0 3490 197

13 43 0 350 3447 310

Figure 1 shows IFN-γ secretion in whole blood cultures 24 hr after stimulation with: (A) PPD-B, (B) Ag85 ₅ (C) TB9.8 or (D) TBI 0.4. This graph was generated using the data in Table 1. Groups vaccinated with vectors encoding the Poly antigens show stronger responses to recombinant mycobacterial antigens as well as to PPD-B.

Figure 2 shows ex- vivo IFN-γ ELISPOT after overnight stimulation of PBMC cultures with (A) PPD-B, (B) Ag85, (C) TB9.8 or (D) TB10.4. This graph was generated using the data in Table 2. As with whole blood cultures, groups vaccinated with vectors encoding the Poly antigens show stronger responses to recombinant mycobacterial antigens as well as to PPD-B.

Figures 1 and 2 both illustrate measurements taken at the key timepoints, i.e. before BCG vaccination, and at the peak of the immune response induced by the BCG vaccination and after each booster vaccination. In the whole blood IFN-γ assay, statistically significant differences in the responses to PPD-B, TB9.8 and TB10.4 were found at weeks 9 and 13. In the PBMC

ELI SPOT assay, statistically significant differences in the responses to TB 9.8 and TB10.4 were found at weeks 9, 12, 13 and 14.

Immunisation of cattle of 5-7 months of age with the immunogenic compositions of the present invention would therefore appear to boost the immune responses to recombinant mycobacterial antigens. In particular, the claimed recombinant viral vectors enhance the immune response to the encoded mycobacterial antigens elicited by the BCG vaccination. Although not statistically significant, secreted IFN-γ responses in whole blood appear to be stronger following boosting with MVA compare to Ad5 constructs. No differences in the order of vaccination were apparent in the ELISPOT assays. Poly encoding vectors induced stronger responses to PPD-B than Ag85A encoding vectors.

Besides the inherent importance of bovine TB, the results from these experiments also inform on the development of vaccines against TB in humans.

Sequences Sequences SEP ID NO. 1

(Amino acid sequence of wildtype M. tuberculosis Ag85A antigen)

MQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLP VEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFE WYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWL QANRHVKPTGSAWGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMG PTLIGLAMGDAGGYKASDMWGPKEDP AWQRNDPLLNVGKLIA NTRVWVY CGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTS WEYW GAQLNAMKPDLQRALGATPNTGPAPQGA

SEP ID NO. 2

(Exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NP. 1)

ATGCAGCTTGTTGACAGGGTTCGTGGCGCCGTCACGGGTATGTCGCGTCG ACTCGTGGTCGGGGCCGTCGGCGCGGCCCTAGTGTCGGGTCTGGTCGGCG CCGTCGGTGGCACGGCGACCGCGGGGGCATTTTCCCGGCCGGGCTTGCCG GTGGAGTACCTGCAGGTGCCGTCGCCGTCGATGGGCCGTGACATCAAGGT CCAATTCCAAAGTGGTGGTGCCAACTCGCCCGCCCTGTACCTGCTCGACG GCCTGCGCGCGCAGGACGACTTCAGCGGCTGGGACATCAACACCCCGGCG TTCGAGTGGTACGACCAGTCGGGCCTGTCGGTGGTCATGCCGGTGGGTGG CCAGTCAAGCTTCTACTCCGACTGGTACCAGCCCGCCTGCGGCAAGGCC GGTTGCCAGACTTACAAGTGGGAGACCTTCCTGACCAGCGAGCTGCCGGG GTGGCTGCAGGCCAACAGGCACGTCAAGCCCACCGGAAGCGCCGTCGTCG GTCTTTCGATGGCTGCTTCTTCGGCGCTGACGCTGGCGATCTATCACCCCC AGCAGTTCGTCTACGCGGGAGCGATGTCGGGCCTGTTGGACCCCTCCCAG GCGATGGGTCCCACCCTGATCGGCCTGGCGATGGGTGACGCTGGCGGCTA CAAGGCCTCCGACATGTGGGGCCCGAAGGAGGACCCGGCGTGGCAGCGC AACGACCCGCTGTTGAACGTCGGGAAGCTGATCGCCAACAACACCCGCGT CTGGGTGTACTGCGGCAACGGCAAGCCGTCGGATCTGGGTGGCAACAAC CTGCCGGCCAAGTTCCTCGAGGGCTTCGTGCGGACCAGCAACATCAAGTT CCAAGACGCCTACAACGCCGGTGGCGGCCACAACGGCGTGTTCGACTTCC CGGACAGCGGTACGCACAGCTGGGAGTACTGGGGCGCGCAGCTCAACGC TATGAAGCCCGACCTGCAACGGGCACTGGGTGCCACGCCCAACACCGGGC CCGCGCCCCAGGGCGCCTAG

SEP ID NO. 3

(Exemplary amino acid sequence of C-terminally truncated Ag85A antigen)

MQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLP VEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFE WYDQSGLSWMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWL QANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAM GPTLIGLAMGD AGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVW VYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDS GTHSWEYWGAQLNAMKPDLQR

SEP ID NO. 4

(Exemplary nucleotide sequence encoding the amino acids sequence of SEQ ID NO. 3)

ATGCAGCTTGTTGACAGGGTTCGTGGCGCCGTCACGGGTATGTCGCGTCG ACTCGTGGTCGGGGCCGTCGGCGCGGCCCTAGTGTCGGGTCTGGTCGGC GCCGTCGGTGGCACGGCGACCGCGGGGGCATTTTCCCGGCCGGGCTTGC CGGTGGAGTACCTGCAGGTGCCGTCGCCGTCGATGGGCCGTGACATCAA GGTCCAATTCCAAAGTGGTGGTGCCAACTCGCCCGCCCTGTACCTGCTCG ACGGCCTGCGCGCGCAGGACGACTTCAGCGGCTGGGACATCAACACCCC GGCGTTCGAGTGGTACGACCAGTCGGGCCTGTCGGTGGTCATGCCGGTG GGTGGCCAGTCAAGCTTCTACTCCGACTGGTACCAGCCCGCCTGCGGCAA GGCCGGTTGCCAGACTTACAAGTGGGAGACCTTCCTGACCAGCGAGCTG CCGGGGTGGCTGCAGGCCAACAGGCACGTCAAGCCCACCGGAAGCGCCG TCGTCGGTCTTTCGATGGCTGCTTCTTCGGCGCTGACGCTGGCGATCTAT CACCCCCAGCAGTTCGTCTACGCGGGAGCGATGTCGGGCCTGTTGGACCC CTCCCAGGCGATGGGTCCCACCCTGATCGGCCTGGCGATGGGTGACGCT GGCGGCTACAAGGCCTCCGACATGTGGGGCCCGAAGGAGGACCCGGCGT GGCAGCGCAACGACCCGCTGTTGAACGTCGGGAAGCTGATCGCCAACAA CACCCGCGTCTGGGTGTACTGCGGCAACGGCAAGCCGTCGGATCTGGGT GGCAACAACCTGCCGGCCAAGTTCCTCGAGGGCTTCGTGCGGACCAGCA ACATCAAGTTCCAAGACGCCTACAACGCCGGTGGCGGCCACAACGGCGT GTTCGACTTCCCGGAC AGCGGTACGC AC AGCTGGGAGTACTGGGGCGCG CAGCTCAACGCTATGAAGCCCGACCTGCAACGT

SEP ID NO. 5

(Exemplary amino acid sequence of TB9.8 antigen)

MSLLDAHIPQLVASQSAFAAKAGLMRHTIGQAEQAAMSAQAFHQGESSAAF QAAHARFVAAAAKVNTLLDVAQANLGEAAGTYVAADAAAASTYTGF

SEQ ID NO. 6

(Exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 5)

ATGAGCCTGCTGGACGCCCACATCCCCCAGCTGGTCGCCAGCCAGAGCG CCTTTGCCGCCAAGGCTGGACTGATGAGACACACCATTGGCCAGGCCGA GCAGGCCGCCATGAGCGCCCAGGCCTTCCACCAGGGCGAGAGCAGCGCC GCCTTCCAGGCCGCCCACGCCAGATTTGTGGCCGCTGCCGCAAAAGTGA ACACCCTGCTGGATGTGGCCCAGGCCAACCTGGGCGAAGCCGCCGGAAC CTACGTGGCCGCCGATGCCGCAGCTGCCTCCACCTACACCGGCTTC

SEQ ID NO. 7

(Exemplary amino acid sequence of Acr2 antigen) MNNLALWSRPVWDVEPWDRWLRDFFGPAATTDWYRPVAGDFTPAAEIVK DGDDAVVRLELPGIDVDKDVNVELDPGQPVSRLVIRGEHRDEHTQDAGDKD GRTLREIRYGSFRRSFRLPAHVTSEAIAASYDAGVLTVRVAGAYKAPAETQA QRIAITK

SEP ID NO. 8

(Exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 7)

ATGAACAACCTGGCCCTGTGGAGCAGGCCCGTGTGGGACGTGGAGCCCT GGGACCGGTGGCTGCGGGACTTCTTCGGCCCTGCCGCCACCACCGACTG GTACAGGCCCGTGGCCGGCGACTTTACCCCTGCCGCCGAGATCGTGAAG GATGGCGACGACGCTGTGGTGCGGCTGGAACTGCCCGGCATCGACGTGG AC AAGGACGTGAACGTGGAGCTGGACCCCGGCC AGCCCGTGAGCCGGCT GGTGATCCGGGGCGAGCACCGGGACGAGCACACCCAGGATGCTGGCGAC AAGGACGGCCGGACCCTGCGGGAGATCAGATACGGCAGCTTTAGACGGA GCTTCCGGCTGCCCGCCCACGTGACCAGCGAGGCCATCGCCGCCAGCTA CGACGCCGGAGTGCTGACCGTGAGGGTGGCCGGAGCCTACAAGGCCCCT GCCGAGACCCAGGCCCAGCGGATCGCCATCACCAAG

SEP ID NO. 9

(Exemplary amino acid sequence of TB10.4 antigen) MSQIMYNYPAMLGHAGDMAGYAGTLQSLGAEIAVEQAALQSAWQGDTGIT YQAWQAQWNQAMEDLVRAYHAMSSTHEANTMAMMARDTAEAAKWGG

SEP ID NO.10

(Exemplary nucleotide sequence encoding the amino acids sequence of SEQ ID NO. 9) ATGAGCCAGATCATGTACAACTACCCCGCCATGCTGGGCCACGCTGGCG ATATGGCAGGCTACGCCGGCACCCTGCAGAGCCTGGGCGCCGAGATTGC CGTGGAACAGGCCGCTCTGCAGTCCGCCTGGCAGGGAGACACCGGCATC ACCTATCAGGCTTGGCAGGCCCAGTGGAACCAGGCCATGGAGGACCTCG TGCGGGCCTACCACGCCATGAGCAGCACCCACGAGGCCAACACCATGGC CATGATGGCCCGGGACACCGCCGAGGCCGCCAAGTGGGGCGGCTGA

SEP ID NO. 11

(Exemplary amino acid sequence of tPA leader sequence)

MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRR SEP ID NO. 12

(Exemplary nucleotide sequence encoding the arnino acid sequence of SEQ ID NP. 11)

ATGGACGCCATGAAGCGGGGCCTGTGCTGCGTGCTGCTGCTGTGTGGCGC CGTGTTCGTGAGCCCCAGCCAGGAAATCCACGCCCGGTTCAGGCGG SEP ID NO. 13

(Examplary amino acid sequence of linker sequence)

GS SEP ID NP. 14

(Examplary nucleotide sequence encoding the amino acid sequence of SEQ ID NP. 13)

GGATCT SEP ID NO. 15

(Exemplary amino acid sequence of PK tag) PNPLGLD

SEP ID NO. 16

(Exemplary amino acid sequence of linker sequence) GSI

SEP ID NO. 17

(Amino acid sequence of exemplary fusion protein comprising tPA leader sequence, C-terminally truncated Ag85A antigen and TB9.8, TB10.4 and Acr2 antigens) MDAMKRGLCCVLLLCGAVFVSPSQEIHARFRRGSMQLVDRVRGAVTGMSR RLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLPVEYLQVPSPSMGRDIKV QFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGG QSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAWG LSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYK ASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNL PAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAM KPDLQRGSMSLLDAHIPQLVASQSAFAAKAGLMRHTIGQAEQAAMSAQAFH QGESSAAFQAAHARFVAAAAKVNTLLDVAQANLGEAAGTYVAADAAAAST YTGFMNNLALWSRPVWDVEPWDRWLRDFFGPAATTDWYRPVAGDFTPAA EIVKDGDDAWRLELPGIDVDKDVNVELDPGQPVSRLVIRGEHRDEHTQDA GDKDGRTLREIRYGSFRRSFRLPAHVTSEAIAASYDAGVLTVRVAGAYKAPA ETQAQRIAITKMSQIMYNYPAMLGHAGDMAGYAGTLQSLGAEIAVEQAALQ SAWQGDTGITYQAWQAQWNQAMEDLVRAYHAMSSTHEANTMAMMARDT AEAAKWGG SEQ ID NO. 18

(Exemplary nucleotide sequence encoding the amino acids sequence of SEQ ID NO. 17) ATGGACGCCATGAAGCGGGGCCTGTGCTGCGTGCTGCTGCTGTGTGGCGC CGTGTTCGTGAGCCCCAGCCAGGAAATCCACGCCCGGTTCAGGCGGGGA TCTATGCAGCTTGTTGACAGGGTTCGTGGCGCCGTCACGGGTATGTCGCG TCGACTCGTGGTCGGGGCCGTCGGCGCGGCCCTAGTGTCGGGTCTGGTCG GCGCCGTCGGTGGCACGGCGACCGCGGGGGCATTTTCCCGGCCGGGCTT GCCGGTGGAGTACCTGC AGGTGCCGTCGCCGTCGATGGGCCGTGACATC AAGGTCCAATTCCAAAGTGGTGGTGCCAACTCGCCCGCCCTGTACCTGCT CGACGGCCTGCGCGCGCAGGACGACTTCAGCGGCTGGGACATCAACACC CCGGCGTTCGAGTGGTACGACCAGTCGGGCCTGTCGGTGGTCATGCCGGT GGGTGGCCAGTCAAGCTTCTACTCCGACTGGTACCAGCCCGCCTGCGGCA AGGCCGGTTGCCAGACTTACAAGTGGGAGACCTTCCTGACCAGCGAGCT GCCGGGGTGGCTGCAGGCCAACAGGCACGTCAAGCCCACCGGAAGCGCC GTCGTCGGTCTTTCGATGGCTGCTTCTTCGGCGCTGACGCTGGCGATCTA TCACCCCCAGCAGTTCGTCTACGCGGGAGCGATGTCGGGCCTGTTGGACC CCTCCCAGGCGATGGGTCCCACCCTGATCGGCCTGGCGATGGGTGACGCT GGCGGCTACAAGGCCTCCGACATGTGGGGCCCGAAGGAGGACCCGGCGT GGCAGCGCAACGACCCGCTGTTGAACGTCGGGAAGCTGATCGCCAACAA CACCCGCGTCTGGGTGTACTGCGGCAACGGCAAGCCGTCGGATCTGGGT GGCAACAACCTGCCGGCCAAGTTCCTCGAGGGCTTCGTGCGGACCAGCA ACATCAAGTTCCAAGACGCCTACAACGCCGGTGGCGGCCACAACGGCGT GTTCGACTTCCCGGACAGCGGTACGCACAGCTGGGAGTACTGGGGCGCG CAGCTCAACGCTATGAAGCCCGACCTGCAACGTGGATCCATGAGCCTGC TGGAC GC CC AC ATCCCCC AGCTGGTCGCC AGCC AGAGC GCCTTTGCCGCC AAGGCTGGACTGATGAGACACACCATTGGCCAGGCCGAGCAGGCCGCCA TGAGCGCCCAGGCCTTCCACCAGGGCGAGAGCAGCGCCGCCTTCCAGGC CGCCCACGCCAGATTTGTGGCCGCTGCCGCAAAAGTGAACACCCTGCTG GATGTGGCCCAGGCCAACCTGGGCGAAGCCGCCGGAACCTACGTGGCCG CCGATGCCGCAGCTGCCTCCACCTACACCGGCTTCATGAACAACCTGGCC

CTGTGGAGCAGGCCCGTGTGGGACGTGGAGCCCTGGGACCGGTGGCTGC

GGGACTTCTTCGGCCCTGCCGCCACCACCGACTGGTACAGGCCCGTGGCC

GGCGACTTTACCCCTGCCGCCGAGATCGTGAAGGATGGCGACGACGCTG

TGGTGCGGCTGGAACTGCCCGGCATCGACGTGGACAAGGACGTGAACGT

GGAGCTGGACCCCGGCCAGCCCGTGAGCCGGCTGGTGATCCGGGGCGAG

CACCGGGACGAGCACACCCAGGATGCTGGCGACAAGGACGGCCGGACCC

TGCGGGAGATCAGATACGGCAGCTTTAGACGGAGCTTCCGGCTGCCCGC

CCACGTGACCAGCGAGGCCATCGCCGCCAGCTACGACGCCGGAGTGCTG

ACCGTGAGGGTGGCCGGAGCCTACAAGGCCCCTGCCGAGACCCAGGCCC

AGCGGATCGCCATCACCAAGATGAGCCAGATCATGTACAACTACCCCGC

CATGCTGGGCCACGCTGGCGATATGGCAGGCTACGCCGGCACCCTGCAG

AGCCTGGGCGCCGAGATTGCCGTGGAACAGGCCGCTCTGCAGTCCGCCT

GGCAGGGAGACACCGGCATCACCTATCAGGCTTGGCAGGCCCAGTGGAA

CCAGGCCATGGAGGACCTCGTGCGGGCCTACCACGCCATGAGCAGCACC

CACGAGGCCAACACCATGGCCATGATGGCCCGGGACACCGCCGAGGCCG

CCAAGTGGGGCGGCTGA

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