A pulmonary vaccine to prevent tuberculosis

Abstract

A vaccine composition comprising an immunogen comprising one or more SAP components or at least one nucleic acid encoding one or more SAP components; and an expression vector that encodes a chimeric CD40L polypeptide or functional equivalent thereof.

Description

A pulmonary vaccine to prevent tuberculosisCross-reference to earlier application

[0001] The application claims priority from US provisional patent application no. 63 / 568879, the entire contents of which is incorporated herein by reference.Field of the disclosure

[0002] The present disclosure relates to vaccine compositions for the treatment and / or prevention of a mycobacterial infection, such as Mycobacterium tuberculosis infection, and methods of use thereof.Background of the disclosure

[0003] Tuberculosis (TB) is a significant global health problem with approximately 10 million new cases and 1.3 million deaths annually. Mycobacterium tuberculosis, the causative agent of TB, is a highly virulent pulmonary pathogen, that is spread by human- to-human aerosol transmission. In addition, two billion people are estimated to have a latent TB infection (LTBI) that is asymptomatic. The potential reactivation of LTBI into destructive pulmonary disease is a challenge to efforts to eradicate TB.

[0004] Immunosuppressive conditions, most notably coinfection with human immunodeficiency virus (HIV) and type 2 diabetes (T2D), are strongly associated with LTBI reactivation. A highly efficacious vaccine is critical to prevent initial TB infection, and to reduce LTBI burden.

[0005] Bacille Calmette-Guerin (BCG) the only licensed TB vaccine, which prevents TB in children, has low efficacy against pulmonary TB in adults, and is contraindicated in people with impaired immunity. In efforts to overcome the variability and waning of BCG- induced immunity, those working in the field have investigated multiple different approaches for the development of new vaccine under development, including the use of viral vectored vaccines, adjuvant protein subunit vaccines, and whole-cell vaccines with heat-inactivated, fragmented or genetically modified mycobacteria. Despite these global efforts, most new anti-TB vaccine candidates have not shown superiority over BCG.

[0006] Most new vaccine approaches aim to generate strong inflammatory responses in order to ‘kick-start’ the immune system to generate protective adaptive immune responses to M. tuberculosis infection. This strategy is facilitated by use of adjuvant molecules designed to cause local inflammation and release inflammatory cytokines. Unfortunately, such systemic inflammatory responses are also associated with high levels of reactogenicity and toxicity. This is a particular problem for protection against pulmonary pathogens such as TB, where ideally the vaccine response should be induced locally to focus protection on the respiratory tract.

[0007] In addition, effective delivery of TB vaccine candidates to the primary site of TB infection, including LTBI, remains a challenge for vaccines that rely upon intramuscular administration, and likely contributes to the poor protective efficacy of existing TB vaccine candidates.

[0008] There remains, therefore, a need for new vaccines for the prevention of TB, for reducing the severity of TB disease, for reducing the likelihood and occurrence of LTBI, and / or reducing the re-activation of LTBI.

[0009] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and / or combined with other pieces of prior art by a skilled person in the art.Summary of the disclosure

[0010] The disclosure provides a vaccine composition comprising:(i) an immunogen comprising one or more sulfate-assimilation pathway (SAP) components, or at least one nucleic acid sequence encoding one or more SAP components; and(ii) a chimeric CD40L polypeptide or functional equivalent thereof, or at least one nucleic acid sequence encoding a chimeric CD40L polypeptide or functional equivalent thereof.

[0011] In any embodiment, the immunogen comprises or consists of the amino acid sequence set forth in SEQ ID NO:2, or variants thereof that are at least 80% identicalthereto. Preferably, the immunogen comprises an amino acid sequence that is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 2.

[0012] In any embodiment, the immunogen comprises or consists of fusion polypeptide comprising CysD. In some embodiments, the immunogen may comprise or consist of a fusion polypeptide comprising CysD and an additional mycobacterial antigen, preferably Ag85B antigen or a functional variant thereof. In preferred embodiments, the immunogen comprises or consists of the fusion polypeptide CysVac2, or a functional equivalent thereof.

[0013] In any embodiment, the immunogen comprises a bacterial Ag85B antigen comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4, or variants thereof that are at least 80% identical thereto. Preferably, the Ag85B amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 4.

[0014] In any embodiment, the immunogen comprises or consists of a fusion polypeptide comprising of consisting of the amino acid sequence set forth in SEQ ID NO: 5, or variants thereof that are at least 80% identical thereto. Preferably, the fusion protein amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 5.

[0015] In any embodiment, the immunogen comprises or consists of a synthetic or recombinant polypeptide or functional equivalent thereof, optionally purified protein.

[0016] In some embodiments, the immunogen comprises at least one nucleic acid sequence encoding CysD. Optionally, the nucleic acid sequence encodes a fusion protein including CysD. In most preferred embodiments, the immunogen comprises or consists of a heterologous nucleic acid sequence encoding the fusion polypeptide CysVac2, or an immunogenic fragment or homolog thereof.

[0017] In any embodiment, the immunogen comprises or consists of the nucleic acid sequence set forth in any one or more or SEQ ID NO: 1 or SEQ ID NO: 6 or variants thereof that are at least 80% identical thereto; or the immunogen comprises or consists of the nucleic acid sequence that encodes the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 5, or variants thereof that are at least 80% identical thereto.

[0018] In any embodiment, the immunogen comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 1 , or variants thereof that are at least 80% identical thereto. Preferably, the heterologous nucleic acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 1 .

[0019] In any embodiment, the immunogen comprises or consists of the nucleic acid sequence that encodes the amino acid sequence set forth in SEQ ID NO: 2, or variants thereof that are at least 80% identical thereto. Preferably, the amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 2.

[0020] In any embodiment, the immunogen further comprises one or more additional Mycobacterium bacterial antigen (also referred to as a mycobacterial antigen) orfunctional variant thereof, or at least one nucleic acid sequence encoding one or more additional Mycobacterium bacterial antigen, wherein the additional Mycobacterium bacterial antigen is not a sulfate-assimilation pathway (SAP) component. In some embodiments, the one or more additional mycobacterial antigen is selected from: Ag85B, PE13, PPE15, MPT83, EsxA, EsxB, EsxH, EspC, EsxR, and PPE18; or a functional variant or derivative thereof; preferably selected from Ag85B, PE13, PPE15, and MPT83 or a functional variant or derivative thereof.

[0021] In some embodiments, the one or more additional mycobacterial antigen is provided as a fusion polypeptide with one or more sulfate-assimilation pathway (SAP) components, or one or more nucleic acids encoding the same. For example, Ag58B may be provided as a fusion protein with CysD (CysVac2). A fusion polypeptide may comprise a linker between the one or more SAP components and the one or more additional mycobacterial antigen. Suitable linker sequences are known to those in the art. The one or more additional mycobacterial antigen may be fused to the one or more SAP components at the N-terminus or the C-terminus.

[0022] In some embodiments, the one or more additional Mycobacterium bacterial antigen is at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or all ten of: Ag85B, PE13, PPE15, MPT83, EsxA, EsxB, EsxH, EspC, EsxR, and PPE18.

[0023] In some embodiments, the one or more additional Mycobacterium bacterial antigen is selected from: Ag85B, PE13, PPE15, and MPT83, or any combination thereof. In some embodiments, the one or more additional Mycobacterium bacterial antigen is at least one, at least two, at least three, or all four of: Ag85B, PE13, PPE15, and MPT83.

[0024] Preferably, the one or more additional Mycobacterium bacterial antigen includes Ag85B or a nucleic acid sequence encoding the same. More preferably, Ag85B is provided as the fusion protein CysVac2 or functional variant thereof, or a nucleic acid encoding the same.

[0025] In preferred embodiments, the immunogen comprises or consists of CysVac2 (a fusion protein of CysD and Ag85B), PE13, PPE15, and MPT83; or one or more nucleic acid sequences encoding the same.

[0026] In any embodiment, the vaccine composition may further comprise a Ag85B antigen or a functional variant thereof. The Ag85B antigen of functional variant thereof may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 4, or variants thereof that are at least 80% identical thereto. Preferably, the amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 4.

[0027] In any embodiment, the vaccine composition may further comprise at least one nucleic acid encoding an Ag85B antigen or a functional variant thereof. In any embodiment, the immunogen further comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 3, or variants thereof that are at least 80% identical thereto. Preferably, the nucleic acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 3.

[0028] In any embodiment, the vaccine composition further comprises a nucleic acid that comprises or consists of the nucleic acid sequence that encodes the Ag58B amino acid sequence set forth in SEQ ID NO: 4, or variants thereof that are at least 80% identical thereto. Preferably, the amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 4.

[0029] In any embodiment, the vaccine composition may further comprise PE13 antigen or a functional variant thereof. The PE13 antigen of functional variant thereof may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 11 , or variantsthereof that are at least 80% identical thereto. Preferably, the amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 1 1.

[0030] In any embodiment, the vaccine composition may further comprise at least one nucleic acid encoding PE13 antigen or a functional variant thereof. In any embodiment, the immunogen further comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 10, or variants thereof that are at least 80% identical thereto. Preferably, the nucleic acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 10.

[0031] In any embodiment, the vaccine composition further comprises a nucleic acid that comprises or consists of the nucleic acid sequence that encodes the PE13 amino acid sequence set forth in SEQ ID NO: 1 1 , or variants thereof that are at least 80% identical thereto. Preferably, the amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 1 1 .

[0032] In any embodiment, the vaccine composition may further comprise a PPE15 antigen or a functional variant thereof. The PPE15 antigen of functional variant thereof may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 13, or variants thereof that are at least 80% identical thereto. Preferably, the amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, atleast about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 13.

[0033] In any embodiment, the vaccine composition may further comprise at least one nucleic acid encoding PPE15 antigen or a functional variant thereof. In any embodiment, the immunogen further comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 12, or variants thereof that are at least 80% identical thereto. Preferably, the nucleic acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 12.

[0034] In any embodiment, the vaccine composition further comprises a nucleic acid that comprises or consists of the nucleic acid sequence that encodes the PPE15 amino acid sequence set forth in SEQ ID NO: 13, or variants thereof that are at least 80% identical thereto. Preferably, the amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 13.

[0035] In any embodiment, the vaccine composition may further comprise a MPT83 antigen or a functional variant thereof. The MPT83 antigen of functional variant thereof may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 15, or variants thereof that are at least 80% identical thereto. Preferably, the amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 15.

[0036] In any embodiment, the vaccine composition may further comprise at least one nucleic acid encoding MPT83 antigen or a functional variant thereof. In any embodiment, the immunogen further comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 14, or variants thereof that are at least 80% identical thereto. Preferably, the nucleic acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 14.

[0037] In any embodiment, the vaccine composition further comprises a nucleic acid that comprises or consists of the nucleic acid sequence that encodes the MPT83 amino acid sequence set forth in SEQ ID NO: 15, or variants thereof that are at least 80% identical thereto. Preferably, the amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 15.

[0038] In any embodiment, the vaccine composition may further comprise an EsxA antigen or a functional variant thereof. The EsxA antigen of functional variant thereof may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 17, or variants thereof that are at least 80% identical thereto. Preferably, the amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 17.

[0039] In any embodiment, the vaccine composition may further comprise at least one nucleic acid encoding EsxA antigen or a functional variant thereof. In any embodiment, the immunogen further comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 16, or variants thereof that are at least 80% identical thereto. Preferably, the nucleic acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 16.

[0040] In any embodiment, the vaccine composition further comprises a nucleic acid that comprises or consists of the nucleic acid sequence that encodes the EsxA amino acid sequence set forth in SEQ ID NO: 17, or variants thereof that are at least 80% identical thereto. Preferably, the amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 17.

[0041] In any embodiment, the vaccine composition may further comprise an EsxB antigen or a functional variant thereof. The EsxB antigen of functional variant thereof may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 19, or variants thereof that are at least 80% identical thereto. Preferably, the amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 19.

[0042] In any embodiment, the vaccine composition may further comprise at least one nucleic acid encoding EsxB antigen or a functional variant thereof. In any embodiment, the immunogen further comprises or consists of the nucleic acid sequence set forth inSEQ ID NO: 17, or variants thereof that are at least 80% identical thereto. Preferably, the nucleic acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 18.

[0043] In any embodiment, the vaccine composition further comprises a nucleic acid that comprises or consists of the nucleic acid sequence that encodes the EsxB amino acid sequence set forth in SEQ ID NO: 19, or variants thereof that are at least 80% identical thereto. Preferably, the amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 19.

[0044] In any embodiment, the vaccine composition may further comprise an EsxH antigen or a functional variant thereof. The EsxH antigen of functional variant thereof may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 21 , or variants thereof that are at least 80% identical thereto. Preferably, the amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 21.

[0045] In any embodiment, the vaccine composition further comprises a nucleic acid that comprises or consists of the nucleic acid sequence that encodes the EsxH amino acid sequence set forth in SEQ ID NO: 20, or variants thereof that are at least 80% identical thereto. Preferably, the amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, atleast about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 20.

[0046] In any embodiment, the vaccine composition may further comprise at least one nucleic acid encoding EsxH antigen or a functional variant thereof. In any embodiment, the immunogen further comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 21 , or variants thereof that are at least 80% identical thereto. Preferably, the nucleic acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 21 .

[0047] In any embodiment, the vaccine composition may further comprise an EspC antigen or a functional variant thereof. The EspC antigen of functional variant thereof may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 23, or variants thereof that are at least 80% identical thereto. Preferably, the amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 23.

[0048] In any embodiment, the vaccine composition further comprises a nucleic acid that comprises or consists of the nucleic acid sequence that encodes the EspC amino acid sequence set forth in SEQ ID NO: 22, or variants thereof that are at least 80% identical thereto. Preferably, the amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 22.

[0049] In any embodiment, the vaccine composition may further comprise at least one nucleic acid encoding EspC antigen or a functional variant thereof. In any embodiment, the immunogen further comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 23, or variants thereof that are at least 80% identical thereto. Preferably, the nucleic acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 23.

[0050] In any embodiment, the vaccine composition may further comprise an EsxR antigen or a functional variant thereof. The EsxR antigen of functional variant thereof may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 25, or variants thereof that are at least 80% identical thereto. Preferably, the amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 25.

[0051] In any embodiment, the vaccine composition further comprises a nucleic acid that comprises or consists of the nucleic acid sequence that encodes the EsxR amino acid sequence set forth in SEQ ID NO: 24, or variants thereof that are at least 80% identical thereto. Preferably, the amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 24.

[0052] In any embodiment, the vaccine composition may further comprise at least one nucleic acid encoding EsxR antigen or a functional variant thereof. In any embodiment, the immunogen further comprises or consists of the nucleic acid sequence set forth inSEQ ID NO: 25, or variants thereof that are at least 80% identical thereto. Preferably, the nucleic acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 25.

[0053] In any embodiment, the vaccine composition may further comprise an PPE18 antigen or a functional variant thereof. The PPE18 antigen of functional variant thereof may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 27, or variants thereof that are at least 80% identical thereto. Preferably, the amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 27.

[0054] In any embodiment, the vaccine composition may further comprise at least one nucleic acid encoding PPE18 antigen or a functional variant thereof. In any embodiment, the immunogen further comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 26, or variants thereof that are at least 80% identical thereto. Preferably, the nucleic acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 26.

[0055] In any embodiment, the vaccine composition further comprises a nucleic acid that comprises or consists of the nucleic acid sequence that encodes the PPE18 amino acid sequence set forth in SEQ ID NO: 27, or variants thereof that are at least 80% identical thereto. Preferably, the amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 27.

[0056] In any embodiment, the immunogen comprises or consists of the heterologous nucleic acid sequence set forth in SEQ ID NO: 6, or variants thereof that are at least 80% identical thereto. Preferably, the heterologous nucleic acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 6.

[0057] In any embodiment, the immunogen comprises or consists of a fusion protein encoded by the heterologous nucleic acid comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 5, or variants thereof that are at least 80% identical thereto. Preferably, the fusion protein amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence set forth in SEQ ID NO: 5.

[0058] In any embodiment, the chimeric CD40L polypeptide or functional equivalent thereof is a polypeptide that comprises or consists of an ISF35 amino acid sequence at set forth in SEQ ID NO:8, or variants thereof that are at least 80% identical thereto. Preferably, the ISF35 amino acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to CD40L ISF35 amino acid sequence set forth in SEQ ID NO:7.

[0059] In any embodiment, the chimeric CD40L polypeptide or functional equivalent thereof is encoded by a nucleotide sequence comprising or consisting of a nucleotide sequence as set forth in SEQ ID NO:7, or variants thereof that are at least 80% identical thereto. Preferably, the nucleotide sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the nucleotide sequence set forth in SEQ ID NO: 8.

[0060] The skilled person will appreciate that the vaccine composition may comprise an expression vector comprising a nucleotide sequence that encodes the chimeric CD40L polypeptide or functional equivalent thereof. Alternatively, the vaccine composition may comprise a synthetic or recombinant chimeric CD40L polypeptide or functional equivalent thereof, optionally a purified synthetic or recombinant polypeptide or functional equivalent thereof.

[0061] In preferred embodiments, the vaccine composition comprises an expression vector comprising a nucleotide sequence that encodes a chimeric CD40L polypeptide or functional equivalent thereof. The skilled person will be familiar with suitable expression vectors available in the art.

[0062] In any embodiment of any aspect of the disclosure, the expression vector is a non-viral or a viral vector. Examples of non-viral vectors include but are not limited to plasmids, cosmids, bacmids, mRNA vectors, linear DNA (eg doggybone DNA (dbDNA)). Examples of viral vectors include but are not limited to vectors derived from adenoviruses (eg Ad5), adeno-associated viruses (AAVs) (including recombinant AAVs (rAAVs) and self-complementary AAVs (scAAVs)), retroviruses, replication-deficient retroviruses, vaccinia viruses, baculoviruses. In preferred embodiments, the viral expression vector is a replication-deficient viral vector. In preferred embodiments, the viral vector is an adenoviral vector, more preferably Ad5.

[0063] In preferred embodiments, the expression vector comprising a nucleotide sequence that encodes the chimeric CD40L polypeptide or functional equivalent thereof is an adenoviral expression vector that encodes a chimeric CD40 ligand (CD40L) proteinISF35. In most preferred embodiments, the expression vector that encodes the chimeric CD40L polypeptide or functional equivalent thereof is a vector that comprises or consists of the adjuvant vector MemVax (as described previously, eg W02022 / 056302 incorporated herein by reference) (the nucleotide sequence set forth in SEQ ID NO:9).

[0064] In any embodiment, the expression vector that encodes the chimeric CD40L polypeptide or functional equivalent thereof comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 9, or variants thereof that are at least 80% identical thereto. Preferably, the nucleic acid sequence is at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 9.

[0065] In any embodiment, (i) the chimeric CD40L polypeptide or functional equivalent thereof, or nucleic acid sequence encoding the chimeric CD40L polypeptide or functional equivalent thereof, and (ii) the immunogen of the vaccine composition are provided separately in the vaccine composition. In some embodiments, the vaccine composition comprises an expression vector encoding a chimeric CD40L polypeptide or functional equivalent thereof that is mixed, formulated or otherwise combined with the immunogen component. Preferably, the vaccine composition mixture comprises an expression vector encoding the chimeric CD40L polypeptide or functional equivalent thereof, most preferably MemVax, and the immunogen, most preferably CysVac2.

[0066] In preferred embodiments, the vaccine composition comprises a single expression vector that encodes both (i) the immunogen and (ii) the chimeric CD40L polypeptide or functional equivalent thereof. In preferred embodiments, the MemVax vector is modified to include one or more nucleic acid sequences encoding the immunogen, using molecular cloning techniques known to those in the art. Optionally, the vaccine composition comprises a single expression vector wherein the expression of the chimeric CD40L polypeptide or functional equivalent thereof and expression of the immunogen are controlled by one or more separate or shared regulatory elements (eg promoter (such as a cell-specific, tissue-specific, or temporal-specific promoter),enhancer, internal ribosome entry site, transcription terminator). The skilled person will be familiar with suitable regulatory elements available in the art.

[0067] In any embodiment of any aspect, an expression vector or nucleic acid sequence may further comprise a suitable marker to detect expression in a cell and / or tissue of interest. Exemplary markers include but are not limited to fluorescent labels, antibiotic resistance genes, restriction enzyme sites. The skilled person will be familiar with suitable markers available in the art.

[0068] In any embodiment of any aspect, the amino acid sequence may be codon- optimised for expression in a mammalian cell, preferably a human cell.

[0069] In any embodiment, the vaccine composition may further comprise one or more additional adjuvants for potentiating an immune response to M. tuberculosis. Additional adjuvants include but are not limited to aluminium-based adjuvants (such as aluminium hydroxide, aluminium phosphate), calcium phosphate hydroxide, beryllium, monophsphoryl lipid A, cytokines (eg IL-2), oils (such as paraffin oil), polysaccharidederived adjuvants (eg Advax), Freund's complete adjuvant, and Freund's incomplete adjuvant, adjuvants using CpG oligonucleotides (eg AdvaxcPG), and combinations thereof. The skilled person will be familiar with additional adjuvants commonly used in the art.

[0070] In any embodiment in any aspect, the vaccine composition may further comprise a delivery system to facilitate delivery of the composition to a cell or tissue of interest, preferably a human cell or human tissue of interest. Exemplary delivery systems suitable for vaccine compositions include but are not limited to lipid nanoparticles (LNPs), nanoparticles, liposomes, exosomes, extracellular vesicles, microvesicles.

[0071] In any embodiment, the vaccine composition may be formulated for targeted delivery to cell or tissue of interest. For example, the vaccine composition may be delivered using a delivery system that comprises one or more targeting moieties (eg cell penetrating peptide, cell-specific or tissue-specific ligand, cell-specific or tissues-specific receptor). For example, the vaccine composition may further comprise a targeting moiety (eg cell penetrating peptide (CPP), cell-specific or tissue-specific ligand, cell-specific or tissues-specific receptor).

[0072] In any embodiment of any aspect of the disclosure, the vaccine composition may comprise pharmaceutically acceptable excipients, diluents, stabilizers, and / or carriers. "Pharmaceutically acceptable carrier" refers to a carrier or excipient that can be included in the compositions described herein and that causes no significant adverse toxicological effect on the subject. The skilled person will be familiar with suitable excipients, diluents and carriers known to the art. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCI, normal saline solutions, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors and colors, liposomes, dispersion media, microcapsules, cationic lipid carriers, isotonic and absorption delaying agents, and the like. The carrier may also be substances for providing the formulation with stability, sterility and isotonicity (e.g. antimicrobial preservatives, antioxidants, chelating agents and buffers), for preventing the action of microorganisms (e.g. antimicrobial and antifungal agents, such as parabens, chlorobutanol, sorbic acid and the like) or for providing the formulation with an edible flavor etc.

[0073] In some embodiments, the vaccine composition comprises a buffer. Preferably, the buffer comprises phosphate-buffered saline (PBS). In some embodiments, the vaccine composition is formulated for a particular mode of administration. For example, the vaccine composition is formulated for intramuscular administration, inhalation, or mucosal administration (such as intranasal administration). Preferably, the vaccine composition is formulated for intranasal administration.

[0074] The present disclosure also provides a method for inducing an immune response to a mycobacterial SAP component in a subject in need thereof, the method comprising administering an effective amount of the vaccine composition described herein to the subject, thereby inducing an immune response to a SAP protein from a Mycobacterium species in the subject. Preferably, the SAP component comprises a CysD protein from M. tuberculosis, optionally a fusion protein of the SAP component CysD and one or more additional mycobacterial antigens. More preferably, the SAP component comprises CysVac2 (a fusion protein of CysD and Ag85B).

[0075] The present disclosure also provides a method for inducing an immune response to M. tuberculosis in a subject in need thereof, the method comprising administering aneffective amount of the vaccine composition described herein to the subject, thereby inducing an immune response to M. tuberculosis in the subject.

[0076] In still further embodiments, the disclosure provides a method for inducing an immune response to an mycobacterial SAP component in a subject, the method comprising administering to a subject in need thereof, an amount of (i) an immunogen to which an immune response is desired and an amount of (ii) a chimeric CD40L polypeptide or functional equivalent thereof, or at least one nucleic acid sequence encoding a chimeric CD40L polypeptide or functional equivalent thereof, preferably an expression vector that encodes a chimeric CD40L polypeptide or functional equivalent thereof; sufficient to potentiate an immune response to the immunogen, thereby stimulating an immune response to the immunogen in the subject; wherein the immunogen comprises one or more SAP components, or at least one nucleic acid sequence encoding one or more SAP components.

[0077] The (i) immunogen, preferably comprising CysVac2, and the (ii) a chimeric CD40L polypeptide or functional equivalent thereof, or at least one nucleic acid sequence encoding a chimeric CD40L polypeptide or functional equivalent thereof, preferably an expression vector that encodes a chimeric CD40L polypeptide or functional equivalent thereof; may be administered concomitantly or sequentially (in either order).

[0078] The skilled person will appreciate that for sequential administration, (i) and (ii) would be administered temporally such that the adjuvant component (ii) will be able to enhance the immune response induced by the immunogen (i) (such as within 15, 10, 5 or 2 minutes of each other). The vaccine composition could be administered with (i) and (ii) delivered as separate formulations, for example, two separate nasal sprays or two separate injections. Preferably, where there are separate formulations, they are for the same route of administration. Preferably, where there are separate formulations, they are administered in the same location, for example, two injections in the same region (such as within a 10 or 5 cm2area).

[0079] In preferred embodiments, the (i) immunogen and the (ii) chimeric CD40L polypeptide or functional equivalent thereof, or at least one nucleic acid sequence encoding a chimeric CD40L polypeptide or functional equivalent thereof, are administered concomitantly, optionally as a composition comprising (i) and (ii) (such as a mixture orformulation). Concomitant use also covers administration of two separate formulations at the same time. In preferred embodiments, concomitant administration is of a single vector encoding both (i) and (ii).

[0080] The immunogen may comprise at least one nucleic acid sequence encoding a Ag85B antigen or a functional variant thereof; or comprise the Ag85B antigen or a functional variant thereof. Optionally, the immunogen comprises a nucleic acid sequence that encodes Ag85B antigen or a functional variant thereof, preferably the nucleic acid sequence encodes a fusion polypeptide of the SAP component and Ag85B antigen or functional variant thereof. Most preferably, the immunogen is an amino acid sequence comprising or consisting of the amino acid sequence of the fusion polypeptide CysVac 2, or a nucleic acid sequence comprising or consisting of a nucleic acid sequence encoding the fusion polypeptide CysVac2.

[0081] The vaccine composition of the disclosure is optionally administered via mucosal administration, oral administration, subcutaneous administration, inhalation, or parentally such as intramuscularly. Preferably, the vaccine composition of the invention is administered mucosally (i.e. via the mucosa). More preferably, the vaccine composition is administered intranasally (i.e. via the nasal mucosa). Alternatively, the vaccine composition is administered parentally, such as intramuscularly. The administration via inhalation is optionally aerosol inhalation. Other modes of administration include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.

[0082] Further, the present disclosure provides for the i) an immunogen comprising one or more sulfate-assimilation pathway (SAP) components, or at least one nucleic acid sequence encoding one or more SAP components; and (ii) a chimeric CD40L polypeptide or functional equivalent thereof, or at least one nucleic acid sequence encoding a chimeric CD40L polypeptide or functional equivalent thereof, preferably an expression vector that encodes a chimeric CD40L polypeptide or functional equivalent thereof; in the manufacture of a vaccine composition for inducing an immune response to a mycobacterial SAP component in a subject.

[0083] The present disclosure also provides for the use of an immunogen comprising one or more sulfate-assimilation pathway (SAP) components, or at least one nucleic acidsequence encoding one or more SAP components in the manufacture of a vaccine composition for inducing an immune response to a mycobacterial SAP component in a subject; wherein the vaccine composition further comprises a chimeric CD40L polypeptide or functional equivalent thereof, or at least one nucleic acid sequence encoding a chimeric CD40L polypeptide or functional equivalent thereof; or wherein the vaccine composition is to be administered in combination with a chimeric CD40L polypeptide or functional equivalent thereof, or at least one nucleic acid sequence encoding a chimeric CD40L polypeptide or functional equivalent thereof.

[0084] The present disclosure also provides for the use a chimeric CD40L polypeptide or functional equivalent thereof, or at least one nucleic acid sequence encoding a chimeric CD40L polypeptide or functional equivalent thereof, preferably an expression vector that encodes a chimeric CD40L polypeptide or functional equivalent thereof; in the manufacture of a vaccine composition for inducing an immune response to a mycobacterial SAP component in a subject; wherein the vaccine composition further comprises an immunogen comprising one or more sulfate-assimilation pathway (SAP) components, or at least one nucleic acid sequence encoding one or more SAP components; or wherein the vaccine composition is to be administered in combination with an immunogen comprising one or more sulfate-assimilation pathway (SAP) components, or at least one nucleic acid sequence encoding one or more SAP components.

[0085] The present disclosure also provides for the use of (i) an immunogen comprising one or more sulfate-assimilation pathway (SAP) components, or at least one nucleic acid sequence encoding one or more SAP components; and (ii) a chimeric CD40L polypeptide or functional equivalent thereof, or at least one nucleic acid sequence encoding a chimeric CD40L polypeptide or functional equivalent thereof, preferably an expression vector that encodes a chimeric CD40L polypeptide or functional equivalent thereof; in the manufacture of a vaccine composition for inducing an immune response to M. tuberculosis in a subject.

[0086] The present disclosure also provides for the use of an immunogen comprising one or more sulfate-assimilation pathway (SAP) components, or at least one nucleic acid sequence encoding one or more SAP components in the manufacture of a vaccine composition for inducing an immune response to M. tuberculosis in a subject, whereinthe vaccine composition further comprises a chimeric CD40L polypeptide or functional equivalent thereof, or at least one nucleic acid sequence encoding a chimeric CD40L polypeptide or functional equivalent thereof; or wherein the vaccine composition is to be administered in combination with a chimeric CD40L polypeptide or functional equivalent thereof, or at least one nucleic acid sequence encoding a chimeric CD40L polypeptide or functional equivalent thereof.

[0087] The present disclosure also provides for the use a chimeric CD40L polypeptide or functional equivalent thereof, or at least one nucleic acid sequence encoding a chimeric CD40L polypeptide or functional equivalent thereof, preferably an expression vector that encodes a chimeric CD40L polypeptide or functional equivalent thereof; in the manufacture of a vaccine composition for inducing an immune response to M. tuberculosis in a subject, wherein the vaccine composition further comprises an immunogen comprising one or more sulfate-assimilation pathway (SAP) components, or at least one nucleic acid sequence encoding one or more SAP components; or wherein the vaccine composition is to be administered in combination with an immunogen comprising one or more sulfate-assimilation pathway (SAP) components, or at least one nucleic acid sequence encoding one or more SAP components.

[0088] The disclosure provides for a vaccine composition described herein, for use in inducing an immune response to a mycobacterial SAP component in a subject. Further, the disclosure provides for a vaccine composition described herein, for use in inducing an immune response to M. tuberculosis in a subject.

[0089] The term "effective amount" or "sufficient amount" refers to the amount of a modified cancer cell or other composition that is sufficient to effect beneficial or desired results. The therapeutically effective amount may vary depending upon one or more of:, the weight and age of the subject, the mode of administration and the like, which can readily be determined by one of ordinary skill in the art. The specific amount may vary depending on one or more of: the particular agent chosen, the dosing regimen to be followed, timing of administration, and the physical delivery system in which it is carried.

[0090] In some embodiments, the dosage of immunogen is between at least about 3 microgram and about 30 micrograms. In some embodiments, the dosage of viralexpression vector encoding a chimeric CD40L polypeptide is between at least about 1 x 1010and 1 x 1011particles, such as 3.3 x 1010.

[0091] The term "administered" means administration of a therapeutically effective dose of a vaccine composition as described herein to the subject.

[0092] “Stimulating an immune response to an immunogen” will be understood by the skilled person to mean that an immune response to the immunogen is generated upon administration of the vaccine composition to a subject. “Inducing an immune response to M. tuberculosis" will be understood by the skilled person to mean that an immune response to M. tuberculosis is generated upon administration of the vaccine composition to a subject. In any of these embodiments, the immune response may be a protective immune response. The immune response may comprise an alteration (such as an increase in number and / or activity) in protective macrophages and / or T cells. The immune response may comprise an elevation in the production of protective cytokines, including but not limited to IL-2, IL-17, IFN-y, TNF. Preferably, the protective immune response is at the site of mycobacterial infection, most preferably the pulmonary infection site.

[0093] In certain embodiments, the infection is an infection with a bacteria selected from: M. tuberculosis, M. leprae, Mycobacterium avium-intracellulare (also known as Mycobacterium Avium Complex, or MAC M. kansasii, M. scrofulaceum, M. fortuitum, M. marinum, M. abscessus, M. ulcerans, or M. chelonae. In certain embodiments, the mycobacterial infection may be an infection that is not associated with tuberculosis disease. Preferably, the mycobacterial infection is an infection with M. tuberculosis (Mtb).

[0094] In any embodiment, the subject may have already been exposed to M. tuberculosis, or may be suspected of having been exposed to M. tuberculosis. In certain embodiments, the subject is an individual with impaired immunity that is not eligible for the BCG vaccine. In certain embodiments, the subject is an individual that has a pre-existing immunosuppressive condition such as human immunodeficiency virus (HIV) or type 2 diabetes (T2D).

[0095] The disclosure also provides for an expression vector or combination of expression vectors encoding a vaccine composition as described herein.

[0096] The disclosure also provides a kit comprising a vaccine composition described herein. The kit may optionally comprise written instructions for the use of the kit in a method or use as described herein. In one embodiment, the disclosure provides a kit described herein when used in a method described herein.

[0097] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

[0098] Further aspects of the present disclosure and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.Brief description of the drawings

[0099] Figure 1 : Pulmonary vaccination with MemTBVax demonstrates improved protection against M. tuberculosis infectionC57BL / 6 mice (n = 5-6 / group) were vaccinated three times, 2 weeks apart by either the intramuscular (IMSC) or intranasal (INSL) route with MemVax alone, MemVax combined with the CysVac2 antigen (MemTBVax) or the BCG vaccine (standard of care). Control unvaccinated mice were also included. Mice were challenged four weeks after the last vaccination with M. tuberculosis H37Rv by aerosol (-100 CFU) and the bacterial burden in the lung (A) or spleen (B) was enumerated 4 weeks later. M. tuberculosis bacterial load is presented as Iog10 of the mean CFU ± SEM. The significance of differences between the groups was determined by ANOVA (*p < 0.05; **p < 0.01 ; ****p < 0.001 ).

[0100] Figure 2: Protection afforded by pulmonary vaccination with MemTBVax is associated with enhanced activation of antigen-specific CD4+ T cells. C57BL / 6 mice (n = 5-6 / group) were vaccinated three times, 2 weeks apart by either the intramuscular (IMSC) or intranasal (INSL) route with MemVax alone, MemVax combined with the CysVac2 antigen (MemTBVax) or the BCG vaccine (standard of care). Control unvaccinated mice were also included. Mice were challenged with M. tuberculosis H37Rv by aerosol (-100 CFU) four weeks after the last vaccination, and 4 weeks later lung cells were restimulated ex vivo with the CysVac2 antigen, and the production of cytokines (IFN- y, IL-2, IL-17, TNF), by (A) CD4+ T cells or (B) CD8+ T cells was determined by flow cytometry. Data are represented as the percentage of cytokine-producing T cells ± SEM.The significance of differences between the unvaccinated and other groups was determined by ANOVA (*p < 0.05; **p < 0.01 ; ***p < 0.005; ****p < 0.001 ).

[0101] Figure 3: Enhanced number of lung alveolar macrophages and CD103+ T cells correlates with protection afforded by MemTBVax. C57BL / 6 mice (n = 5- 6 / group) were vaccinated three times, 2 weeks apart by either the intramuscular (IMSC) or intranasal (INSL) route with MemVax alone, MemVax combined with the CysVac2 antigen (MemTBVax) or the BCG vaccine (standard of care). Control unvaccinated mice were also included. Mice were challenged with M. tuberculosis H37Rv by aerosol (~100 CFU) four weeks after the last vaccination, and the composition of lung cells types were determined 4 weeks later. Shown are the proportion of (A) alveolar macrophages, (B) interstitial macrophages, and (C) CD103+ T cells, in the lung. The significance of differences between the unvaccinated and other groups was determined by ANOVA (***p < 0.005; ****p < 0.001 ).

[0102] Figure 4: Increased release of cytokines by CD4+ T cells after vaccination of mice with MemTBVax compared to CysVac2 delivered with the AdvaxcPGadjuvant. C57BL / 6 mice (n = 5-6) were vaccinated three times, 2 weeks apart by the intramuscular route with CysVac2 antigen combined with either MemVax adjuvant (MemTBVax) or the AdvaxcPGadjuvant. Other groups were vaccinated with the BCG vaccine (standard of care) or left unvaccinated. Four weeks after the last vaccination, intracellular cytokine staining was performed on PBMCs after re-stimulation with CysVac2 and Brefeldin A, and the frequency of IFN-y, IL-2, IL-17 or TNF-positive CD4+ T cells was assessed by flow cytometry. The significance of differences between the groups was determined by ANOVA (*p<0.05; ***p < 0.005; ****p < 0.001 ).

[0103] Figure 5: Immunogenicity afforded by the TB-Multi vector. C57BL / 6 mice (n=4-5) were vaccinated twice intranasally, 2 weeks apart with the modified MemVax vector (Ad5 expressing CD40L) expressing 5 Mycobacterium tuberculosis antigens (the CysVac2 fusion protein of CysD + Ag85B, together with PE13, PPE15 and MPT83). The combined vaccine was termed TB-Multi. Four weeks after the last vaccination spleen cells were stimulated ex v / vo with the CysVac2 (A), PE13 (B), PPE15 (C) or MPT83 antigens (D) and the production of IL-2 by CD4+T cells determined by flow cytometry. Data are represented as the percentage of cytokine-producing T cells ± SEM. The significance ofdifferences between the unvaccinated and other groups was determined by ANOVA (*p < 0.05).Sequence information

[0104] Table 1 : Sequence informationDetailed description of the embodiments

[0105] Reference will now be made in detail to certain embodiments of the disclsoure. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the disclosure is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

[0106] The vaccine composition comprises an immunogen comprising one or more SAP components; or at least one nucleic acid encoding one or more SAP components. Preferably, one or more SAP components is CysD, or an immunogenic or antigenic fragment or homolog thereof. In one embodiment, a given SAP component (such as CysD, CysNC, CysH, SirA, CysE, CysK1 proteins and their encoding genes) may have a conserved function in terms of activity in the sulphate assimilation pathway and yet have a diverged sequence. These proteins or nucleic acids are referred to as homologs.

[0107] In certain embodiments a given SAP component is one having at least 75%, preferably 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98% or 99% identity to a given SAP component. For example a CysD immunogen may be one having at least 75%, preferably 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98% or 99% identity to a CysDprotein shown in SEQ ID NO: 2. The nucleic acid sequence encoding the CysD immunogen may be one having at least 75%, preferably 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98% or 99% identity to a cysD gene shown in SEQ ID NO: 1 .

[0108] It will be understood that the immunogen may further include other recombinant or synthetic Mycobacterium antigens. Particular examples of suitable antigens are described in WO2013 / 091004 and W02009 / 070700 incorporated by reference in their entirety.

[0109] CysVac2 is a composite immunogenic polypeptide previously identified by the inventors, and comprises bacterial antigens expressed during different stages of the M. tuberculosis life cycle. CysVac2 is a fusion of CysD and the immunogenic Ag85B bacterial antigen of M. tuberculosis (see Counoupas et al. (2016). Npj Vaccines 1 , 16012, https: / / doi.org / 10.1038 / npjvaccines.2016.12; incorporated by reference in its entirety).

[0110] The present inventors have developed a new TB vaccine composition by combining the expression vector MemVax (an adenovirus type 5 vector expressing chimeric human CD40L, initially developed for cancer therapy) with the bacterial immunogen CysVac2. MemVax is a replication deficient adenovirus encoding a chimeric CD40 ligand transgene (synonyms include ISF35 or Ad-CD40 ligand). The resultant vaccine composition is referenced herein as MemTBVax. The inventors have additionally developed a single-vector TB vaccine composition comprising a modified MemVax vector, modified to encode an immunogen comprising multiple mycobacterial antigens, including CysVac2. The resultant vaccine composition is referenced herein as TB-Multi.

[0111] The present inventors have determined that MemVax is a preferable adjuvant for vaccine compositions comprising a mycobacterial immunogen comprising a SAP component, such as CysVac2. The inventors found that using MemVax, encoding a chimeric CD40L polypeptide, results in a very strong immune responses, particularly T- cell responses. A strong T-cell response is important for the vaccinating against tuberculosis. However, a high T-cell response does not always translate to better protection from the vaccine. This preferred combination had highly unexpected and impressive capacity to both strengthen T cell responses and reduce bacterial load in thelung. Specifically, there was a 100-fold decrease in bacterial load where other combinations resulted in only 5-10-fold reduction.

[0112] The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001 ); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, N.J.; Handbook of Biochemistry: Section A Proteins, Vol I, CRC Press (1976); Handbook of Biochemistry: Section A Proteins, Vol II, CRC Press (1976).

[0113] Polypeptides

[0114] As used herein "polypeptide" refers to any sequence of two or more amino acids, regardless of length, post-translation modification, or function. Polypeptides can include natural amino acids and non-natural amino acids. Polypeptides can also be modified in any of a variety of standard chemical ways (e.g., an amino acid can be modified with a protecting group; the carboxy-terminal amino acid can be made into a terminal amide group; the amino-terminal residue can be modified with groups to, e.g., enhance lipophilicity; or the polypeptide can be chemically glycosylated or otherwise modified to increase stability or in vivo half-life). Polypeptide modifications can include the attachment of another structure such as a cyclic compound or other molecule to the polypeptide and can also include polypeptides that contain one or more amino acids in an altered configuration (i.e., R or S; or, L or D).

[0115] Exemplary cells used for expressing a polypeptide are CHO cells, myeloma cells or HEK cells. Host cells used to produce a polypeptide may be cultured in a variety of media, depending on the cell type used. Commercially available media such as Ham's FI0 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing mammalian cells. Media for culturing other cell types discussed herein are known in the art.

[0116] In the case of a recombinant immunogen polypeptide, a nucleic acid encoding same can be cloned into expression constructs or vectors, which are then transfected into host cells, such as E. coli cells, yeast cells, insect cells, or mammalian cells, such as simian COS cells, Chinese Hamster Ovary (CHO) cells, human embryonic kidney (HEK) cells, or myeloma cells that do not otherwise produce the immunogen polypeptide. Molecular cloning techniques to achieve these ends are known in the art and described, for example in Ausubel et aL, (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-lnterscience (1988, including all updates until present) or Sambrook et aL, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A wide variety of cloning and in vitro amplification methods are suitable for the construction of recombinant nucleic acids.

[0117] Methods for isolating a polypeptide or fusion polypeptide are known in the art and / or described herein.

[0118] The skilled artisan will be aware that a polypeptide can be modified to include a tag to facilitate purification or detection, e.g., a poly-histidine tag, e.g., a hexa-histidine tag, or a influenza virus hemagglutinin (HA) tag, or a Simian Virus 5 (V5) tag, or a FLAG tag, or a glutathione S-transferase (GST) tag. The resulting polypeptide is then purified using methods known in the art, such as, affinity purification.

[0119] A recombinant polypeptide prepared from the cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity chromatography or protein G chromatography), or any combination of the foregoing. These methods are known in the art and described, for example in WO99 / 57134 or Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988).

[0120] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1 - 3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (2001 ); and Current Protocols in Molecular Biology, ed. Ausubel et aL, Greene Publishing and Wiley-lnterscience, New York, (1992) (with periodic updates).

[0121] In general, preparation of the fusion proteins can be accomplished by procedures disclosed herein and by recognized recombinant DNA techniques involving, e.g., polymerase chain amplification reactions (PCR), preparation of plasmid DNA, cleavage of DNA with restriction enzymes, preparation of oligonucleotides, ligation of DNA, isolation of mRNA, introduction of the DNA into a suitable cell, transformation or transfection of a host, culturing of the host. Additionally, the fusion proteins can be isolated and purified using chaotropic agents and well known electrophoretic, centrifugation and chromatographic methods.

[0122] In most instances, it will be preferred that each of the fusion protein components encoded by the DNA vector be provided in a "cassette" format. By the term "cassette" is meant that each component can be readily substituted for another component by standard recombinant methods.

[0123] The fusion proteins described herein are preferably produced by standard recombinant DNA techniques. The resultant hybrid DNA molecule can be expressed in a suitable host cell to produce the fusion protein. The DNA molecules are ligated to each other in a 5' to 3' orientation such that, after ligation, the translational frame of the encoded polypeptides is not altered (i.e., the DNA molecules are ligated to each other in-frame). The resulting DNA molecules encode an in-frame fusion protein.

[0124] In most instances, it will be preferred that each of the fusion protein components encoded by the DNA vector be provided in a "cassette" format. By the term "cassette" is meant that each component can be readily substituted for another component by standard recombinant methods.

[0125] The fusion proteins described herein are preferably produced by standard recombinant DNA techniques. The resultant hybrid DNA molecule can be expressed in a suitable host cell to produce the fusion protein. The DNA molecules are ligated to each other in a 5' to 3' orientation such that, after ligation, the translational frame of the encoded polypeptides is not altered (i.e., the DNA molecules are ligated to each other in-frame). The resulting DNA molecules encode an in-frame fusion protein.

[0126] It is preferred that the polypeptides and fusion polypeptides of the present disclosure be substantially pure. That is, the polypeptides have been isolated from cell substituents that naturally accompany it so that the polypeptides are present preferablyin at least 80% or 90% to 95% homogeneity (w / w). polypeptides having at least 98 to 99% homogeneity (w / w) are most preferred for many pharmaceutical, clinical and research applications. Once substantially purified the fusion protein should be substantially free of contaminants for therapeutic applications. Once purified partially or to substantial purity, the soluble fusion proteins can be used therapeutically, or in performing in vitro or in vivo assays as disclosed herein. Substantial purity can be determined by a variety of standard techniques such as chromatography and gel electrophoresis.

[0127] An orthologue as used herein is the equivalent of the protein or peptide used in the fusion protein whose sequence is derived from a non-human animal, preferably a mammal, such as a mouse, rat or pig.

[0128] Functional homologues or variants may be derived by insertion, deletion or substitution of amino acids in, or chemical modification of, the native carboxyl-terminal sequence. Amino acid insertion variants include amino and / or carboxylic terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Insertion amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletion variants are characterised by the removal of one or more amino acids from the sequence.

[0129] A functional variant will be understood by the skilled person to require immunogenic properties suitable for use as a vaccine composition described herein. The skilled person will be familiar with methods, such as those described herein in the Examples, to determine whether or not a variant polypeptide or polynucleotide has suitable immunogenic properties.

[0130] Substitution amino acid variants are those in which at least one amino acid residue in the sequence has been replaced by another of the twenty, primary protein amino acids, or by a non-protein amino acid. In one embodiment substitutions are with conservative amino acids.

[0131] A “conservative amino acid substitution” is one in which the naturally or non- naturally occurring amino acid residue is replaced with a naturally or non-naturally occurring amino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., Lys, Arg, His), acidic side chains (e.g., Asp, Glu), uncharged polar side chains (e.g., Gly, Asn, Gin, Ser, Thr, Tyr, Cys), nonpolar side chains (e.g., Ala, Vai, Leu, He, Pro, Phe, Met, Trp), beta-branched side chains (e.g., Thr, Vai, He) and aromatic side chains (e.g., Phe, Trp, His). Thus, a predicted nonessential amino acid residue, for example, may be replaced with another amino acid residue from the same side chain family. Other examples of acceptable substitutions are substitutions based on isosteric considerations (e.g. norleucine for methionine) or other properties (e.g. 2-thienylalanine for phenylalanine). A full amino acid subclassification is set out in Table 1 and exemplary substitutions are set out in Table 2.Table 1. Amino acid sub-classificationTable 2. Exemplary and Preferred Amino Acid Substitutions

[0132] The polypeptides and peptides of the present disclosure comprise amino acids. Reference to “amino acid” includes naturally occurring amino acids or non- naturally occurring amino acids.

[0133] Peptide compounds are generally and conventionally modifiable by addition of moieties, flanking peptide residues, and substitutions within understood parameters. Peptides can furthermore comprise routine modified backbones, side chains, peptide bond replacements, and terminal modifications using standard peptide chemistries.

[0134] The amino acids incorporated into the amino acid sequence described herein may be L-amino acids, D-amino acids, L- [3 -homo amino acids, D- [3 -homo amino acids or N-methylated amino acids, sugar amino acids, and / or mixtures thereof. Non-natural amino acids may not be recognised by proteases and may therefore alter the half-life.

[0135] Non-naturally occurring amino acids include chemical analogues of a corresponding naturally occurring amino acid. Examples of unnatural amino acids and derivatives include, but are not limited to, 4-amino butyric acid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, t-butylglycine, nor leucine, norvaline, phenylglycine, ornithine, sarcosine, 2-thienyl alanine and / or D-isomers of amino acids.

[0136] In one embodiment, peptides are modified to enhance their pharmacodynamics properties using art recognised modifications. Peptides may be substituted, such as alanine substituted, or substituted with cross linkable moieties and / or linked. Suitable residues may comprise additional alpha-carbon substitutions selected from hetero- lower alkyl, hetero- methyl, ethyl, propyl and butyl. Peptide bond replacements such as trifluoroethylamines are used to produce more stable and active peptidomimetics.

[0137] Nucleic acids

[0138] The terms “nucleic acid” and “polynucleotide,” used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. They include single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers comprising purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0139] A nucleic acid may be isolated.

[0140] A nucleic acid may be recombinant or synthetic.

[0141] Nucleic acids are said to have “5’ ends” and “3’ ends” because mononucleotides are reacted to make oligonucleotides in a manner such that the 5’ phosphate of one mononucleotide pentose ring is attached to the 3’ oxygen of its neighbor in one direction via a phosphodiester linkage. An end of an oligonucleotide is referred to as the “5’ end” if its 5’ phosphate is not linked to the 3’ oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is referred to as the “3’ end” if its 3’ oxygen is not linked to a 5’ phosphate of another mononucleotide pentose ring. A nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5’ and 3’ ends. In either a linear or circular DNA molecule, discrete elements are referred to as being “upstream” or 5’ of the “downstream” or 3’ elements.

[0142] “Codon optimization” may be used and generally includes a process of modifying a nucleic acid sequence for enhanced expression in particular host cells by replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the genes of the host cell while maintaining the native aminoacid sequence. For example, a nucleic acid encoding a protein can be modified to substitute codons having a higher frequency of usage in a given prokaryotic or eukaryotic cell, including a bacterial cell, a yeast cell, a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, a hamster cell, or any other host cell, as compared to the naturally occurring nucleic acid sequence. Codon usage tables are readily available, for example, at the “Codon Usage Database.” These tables can be adapted in a number of ways. See Nakamura etal. (2000) Nucleic Acids Research 28:292, herein incorporated by reference in its entirety for all purposes. Computer algorithms for codon optimization of a particular sequence for expression in a particular host are also available (see, e.g., Gene Forge).

[0143] A nucleic acid molecule as described herein may in any form such as DNA or RNA, including in vitro transcribed RNA or synthetic RNA. Nucleic acids include genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules and modified forms thereof. A nucleic acid molecule may be single stranded or double stranded and linear or closed covalently to form a circle. The RNA may be modified by stabilizing sequences, capping, and polyadenylation. RNA or DNA and may be delivered as plasmids to express the fusion protein or polypeptide. RNA-based approaches are routinely available.

[0144] The term “RNA” relates to a molecule which comprises ribonucleotide residues and preferably being entirely or substantially composed of ribonucleotide residues.“Ribonucleotide” relates to a nucleotide with a hydroxyl group at the 2’-position of a |3-D- ribofuranosyl group. The term includes double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of a RNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in RNA molecules can also comprise non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally-occurring RNA.

[0145] An optimised mRNA based composition could comprise a 5’ and 3’ non translated region (5’-UTR, 3’-UTR) that optimises translation efficiency and intracellular stability as known in the art. In one embodiment, removal of uncapped 5 ‘-triphosphates can be achieved by treating RNA with a phosphatase. RNA may have modified ribonucleotides in order to increase its stability and / or decrease cytotoxicity. For example, in one embodiment, in the RNA, 5-methylcytidine is substituted partially or completely, for cytidine. In one embodiment, the term “modification” relates to providing an RNA with a 5’-cap or 5’-cap analog. The term “5’-cap” refers to a cap structure found on the 5’-end of an mRNA molecule and generally consists of a guanosine nucleotide connected to the mRNA via an unusual 5’ to 5’ triphosphate linkage. In one embodiment, this guanosine is methylated at the 7-position. The term “conventional 5’- cap” refers to a naturally occurring RNA 5’-cap, preferably to the 7-methylguanosine cap. The term “5’-cap” includes a 5’-cap analog that resembles the RNA cap structure and is modified to possess the ability to stabilize RNA and / or enhance translation of RNA. Providing an RNA with a 5’-cap or 5’-cap analog may be achieved by in vitro transcription of a DNA template in the presence of said 5’-cap or 5’-cap analog, wherein said 5’-cap is co- transcriptionally incorporated into the generated RNA strand, or the RNA may be generated, for example, by in vitro transcription, and the 5’-cap may be attached to the RNA post-transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus.

[0146] A further modification of RNA may be an extension or truncation of the naturally occurring poly(A) tail or an alteration of the 5’- or 3 ‘-untranslated regions (UTR) such as introduction of a UTR which is not related to the coding region of said RNA, for example, the exchange of the existing 3’-UTR with or the insertion of one or more, preferably two copies of a 3’-UTR derived from a globin gene, such as alpha2- globin, alphal-globin, beta-globin. RNA having an unmasked poly-A sequence is translated more efficiently than RNA having a masked poly-A sequence. In order to increase stability and / or expression of the RNA it may be modified so as to be present in conjunction with a poly-A sequence, preferably having a length of 10 to 500, more preferably 30 to 300, even more preferably 65 to 200 and especially 100 to 150 adenosine residues. In order to increase expression of the RNA it may be modified within the coding region so as to increase the GC-content to increase mRNA stability and to perform a codon optimization and, thus, enhance translation in cells. ModifiedmRNA may be synthesised enzymatically and packaged into nanoparticles such as lipid nanoparticles and administered, for example intramuscularly.

[0147] As used herein, the term “promoter” is to be taken in its broadest context and includes the transcriptional regulatory sequences of a genomic gene, including the TATA box or initiator element, which is required for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers and silencers) that alter expression of a nucleic acid, e.g., in response to a developmental and / or external stimulus, or in a tissue specific manner. In the present context, the term “promoter” is also used to describe a recombinant, synthetic or fusion nucleic acid sequence, that activates or enhances the expression of a nucleic acid to which it is operably linked. Exemplary promoters can contain additional copies of one or more specific regulatory elements (e.g. enhancers, initiation sequences) to further enhance expression and / or alter the spatial expression and / or temporal expression of said nucleic acid sequence.

[0148] As used herein, the term “operably linked to" means positioning a promoter relative to a nucleic acid such that expression of the nucleic acid is controlled by the promoter.

[0149] Many vectors for expression in cells are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, a sequence encoding a polypeptide (e.g., derived from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence. The skilled artisan will be aware of suitable sequences for expression of a polypeptide. Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, a factor leader, or acid phosphatase leader) or mammalian secretion signals (e.g., herpes simplex gD signal).

[0150] Exemplary promoters active in mammalian cells include cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1 -a promoter (EF1 ), small nuclear RNA promoters (U1 a and U1 b), a-myosin heavy chain promoter, Simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), Adenovirus major late promoter, [3-actin promoter; hybrid regulatory element comprising a CMV enhancer / [3-actin promoter or an immunoglobulin promoter or active fragment thereof. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651 ); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).

[0151] Typical promoters suitable for expression in yeast cells such as for example a yeast cell selected from the group comprising Pichia pastoris, Saccharomyces cerevisiae and S. pombe, include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1 promoter, the PHO5 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.

[0152] Means for introducing the isolated nucleic acid or expression construct comprising same into a cell for expression are known to those skilled in the art. The technique used for a given cell depends on the known successful techniques. Means for introducing recombinant DNA into cells include microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine (Gibco, MD, USA) and / or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation and microparticle bombardment such as by using DNA-coated tungsten or gold particles (Agracetus Inc., Wl, USA) amongst others.

[0153] Compositions

[0154] A vaccine composition as described herein can be administered orally, parenterally, by inhalation spray or powder, adsorption, absorption, topically, rectally, nasally, bucally, vaginally, intraventricularly, via an implanted reservoir in dosage formulations containing conventional non-toxic pharmaceutically-acceptable carriers, or by any other convenient dosage form. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion techniques.

[0155] Methods for preparing a vaccine composition described herein into a suitable form for administration to a subject are known in the art and include, for example, methods as described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990) and U.S. Pharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa., 1984).

[0156] The vaccine compositions of the disclosure are particularly useful for intranasal administration.

[0157] The vaccine compositions for administration will commonly comprise a solution of the polypeptide and / or nucleic acid components dissolved in a pharmaceutically acceptable carrier, for example an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as mixed oils and ethyl oleate may also be used. Liposomes may also be used as carriers. The vehicles may contain minor amounts of additives that enhance isotonicity and chemical stability, e.g., buffers and preservatives.

[0158] The vaccine composition may be aerosolized. The vaccine composition may be formulated as an intranasal spray, and may in this context comprise an additional component or components that serve as aerosolizing agents.

[0159] Administration

[0160] A vaccine composition of the disclosure will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically / prophylactically effective. Formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but other pharmaceutically acceptable forms are also contemplated, e.g., tablets, pills, capsules or other solids for oral administration, suppositories, pessaries, nasal solutions, powders, or sprays, aerosols, inhalants, liposomal forms and the like. Pharmaceutical "slow release" capsules or compositions may also be used.

[0161] It is within the ability of a skilled physician to determine a suitable dosage, e.g., by commencing with a sub-optimal dosage and incrementally modifying the dosage to determine an optimal or useful dosage. Alternatively, to determine an appropriate dosage for treatment / prophylaxis, data from the cell culture assays or animal studies are used, wherein a suitable dose is within a range of circulating concentrations thatinclude the ED50 of the active compound with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. A therapeutically / prophylactically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration or amount of the compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.

[0162] The term “therapeutically effective amount” is the quantity which, when administered to a subject in need of treatment, improves the prognosis and / or state of the subject and / or that reduces or inhibits one or more symptoms of a clinical condition described herein to a level that is below that observed and accepted as clinically diagnostic or clinically characteristic of that condition. The amount to be administered to a subject will depend on the particular characteristics of the condition to be treated, the type and stage of condition being treated, the mode of administration, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, and body weight. A person skilled in the art will be able to determine appropriate dosages depending on these and other factors. Accordingly, this term is not to be construed to limit the present invention to a specific quantity, e.g., weight or amount of protein(s), rather the present invention encompasses any amount of the antigen binding protein(s) sufficient to achieve the stated result in a subject.

[0163] As used herein, the term “prophylactically effective amount” shall be taken to mean a sufficient quantity of a protein to prevent or inhibit or delay the onset of one or more detectable symptoms of a clinical condition. The skilled artisan will be aware that such an amount will vary depending on, for example, the specific antigen binding protein(s), fusion protein(s) or conjugate(s) administered and / or the particular subject and / or the type or severity or level of condition and / or predisposition (genetic or otherwise) to the condition. Accordingly, this term is not to be construed to limit the present invention to a specific quantity, e.g., weight or amount of antigen binding protein(s), rather the present invention encompasses any amount of the antigen binding protein(s) sufficient to achieve the stated result in a subject.

[0164] Prior to administration to humans as a vaccine, the vaccine compositions described under herein are tested according to methods that are well-known to those of skill in the art. For example, tests for toxicity, virulence, safety, etc. are carried out in suitable animal models, e.g. in mice, rabbits, guinea pigs, etc., some of which are immunocompromised. The ability of the vaccine compositions to elicit an immune response is likewise typically tested in suitable animal models, e.g. mice, guinea pigs, etc. such as the testing described herein. In addition, protection studies involving vaccination, boosting, and subsequent challenge with live Mycobacterium (e.g. Mtb) may be carried out using suitable animal models such as mice, guinea pigs, and non-human primates. Finally, those of skill in the art are familiar with the arrangements for carrying out clinical trials in consenting humans, in order to test the efficacy of the vaccine compositions. For details, see, for example, United States patent application 20060121054 (Sun et al.) published June 8, 2006, and references cited therein.

[0165] Kits

[0166] In the case of a kit for therapeutic / prophylactic use, the kit can additionally comprise a pharmaceutically acceptable carrier.

[0167] Optionally a kit is packaged with instructions for use in a method described herein according to any example.

[0168] The disclosure also provides a kit as described herein when used in a method described herein.

[0169] Methods for inducing an immune response

[0170] Generally, the methods comprise administering an effective dose of a vaccine composition of this disclosure to a subject and inducing an immune response that protects against Mycobacterium infection. Protective immunity refers to a body's ability to mount a specific immune response that protects the subject from developing a particular disease or condition that involves the agent against which there is an immune response. An immunologically effective amount is capable of conferring protective immunity to the subject.

[0171] In preferred examples, the methods and uses are for inducing an immune response to a mycobacterial SAP component, from a Mycobacterium species, preferablyM. tuberculosis, in a subject. Accordingly, the disclosure provides methods for: (i) prophylaxis; (ii) treatment; and (iii) boosting immunity to an infection with a Mycobacterium species, preferably to M. tuberculosis. It is in these contexts that the methods of the disclosure minimise the likelihood of development of an infection, either by preventing the infection from developing to a relevant disease or pathology, or by preventing further development of a disease or pathology once an infection has been established.

[0172] The immune response can protect against or treat a subject having, suspected of having, or at risk of developing an infection or related disease, particularly those related to tuberculosis. Further, the compositions of the disclosure may be useful for treating or preventing an infection that is not associated with tuberculosis disease. In certain embodiments, the infection is an infection with M. leprae, Mycobacterium avium- intracellulare (also known as Mycobacterium Avium Complex, or MAC), M. kansasii, M. scrofulaceum, M. fortuitum, M. marinum, M. abscessus or M. chelonae.

[0173] The disclosure provides methods for preventing a subject suspected of or at risk of having a Myobacterium infection. As used herein, the terms “preventing”, “prevent” or “prevention” include administering a vaccine composition of the disclosure to thereby stop or hinder the development of at least one symptom of an infectious disease or condition. Prevention includes inducing a protective immune response that means an infection is less likely to develop.

[0174] It will be understood that the immune response induced may be specifically against the one or more SAP components; or nucleic acid encoding the same. The induced immune response may comprise an alteration (such as an increase in number and / or activity) in protective macrophages and / or T cells. The immune response may comprise an elevation in the production of protective cytokines, including but not limited to IL-2, IL-17, IFN-y, TNF. Preferably, the protective immune response is at the site of mycobacterial infection, most preferably the pulmonary infection site.

[0175] It will be appreciated that the immune response induced may also be useful for inducing non-specific immune responses in a subject. For example, the vaccine compositions of the disclosure may be utilised to reduce the likelihood of a subject developing an infection in response to an infectious agent that has a similar SAPcomponent, or an infectious agent that does not have a similar SAP component but is suppressed by similar immune response.

[0176] A protective immune response may be measured by: detecting increased anti- Myobacterium antibody levels (e.g. in plasma, serum), detecting reduced Myobacterium antigen levels, detecting reduced Myobacterium bacterial load; the production of protective cytokines; detecting cellular proliferation or influx of protective immune cells, such as macrophages and T cells; or a combination thereof.

[0177] Production of protective antibodies or cytokines can be detected using assays known to those in the art; such as ELISA (enzyme-linked immunosorbent assay), PCR (polymerase chain reaction; including quantitative and real-time PCR), western blot, immunohistochemistry, immunoprecipitation, or ELISPOT (enzyme-linked immunospot). Cellular responses can be detected using assays known to those in the art to assess the number and / or proliferation of cell types; such as flow cytometry.

[0178] A routine skin test by injection of tuberculin (or derivative thereof) may also be used to detect the presence or absence of an immune response to Mtb. This test is known to those in the art as the Mantous test or tuberculin skin test (TST). For example, a subject who has not previously had an Mtb infection and displays a negative skin test, who is then administered a vaccine composition of the disclosure, may display a positive skin test indicative of developing anti -M tb immunity.

[0179] The disclosure provides methods for treating a subject having, suspected of having, or at risk of having a Myobacterium infection or related disease.

[0180] “Treating”, “treat”, or “treatment” include administering a vaccine composition of the disclosure to ameliorate one or more signs or symptoms of a Mycobacterium infection, or associated disease. Indicators of successful treatment may include: reduced infectious bacterial load (for example, detected by reduced bacterial number in sputum smear or culture tests; or by molecular testing eg PCR), chest X-ray showing less or no irregular “patches” in the lungs; displaying improved breathing or other symptoms of reduced severity of disease.

[0181] In some embodiments, the Mycobacterium infection is cured, and the subject becomes free of the infectious Mycobacterium.

[0182] As used herein, the term “subject” shall be taken to mean any animal including humans, for example a mammal. Exemplary mammals include a rodent, a mouse, a rat, a rabbit, a dog, a cat, a sheep, a horse, a goat, a llama, cattle, a primate, a pig, and any other mammal. Exemplary subjects include but are not limited to humans and nonhuman primates. For example, the subject is a human.

[0183] In one embodiment, the individual may not have a detectable Mycobacterium infection and / or may not have been previously immunised against Mycobacterium. Such an individual can generally be identified by the Mantoux test which is widely used in the art.

[0184] In another embodiment, the individual may be asymptomatic or have sub-clinical symptoms of infection. An asymptomatic subject more typically, has one or more symptoms (e.g., fever, cough, weight loss). Bacilli may be present and culturable, i.e., can be grown in culture from the above body fluids and individuals may have radiographically evident pulmonary lesions which may include infiltration but without cavitation.

[0185] In another embodiment the individual may have obvious symptoms of infection such as cavitary lesions in the lungs. Bacilli may be culturable from smears of sputum and / or the other body fluids noted above, but also present in sufficient numbers to be detectable as acid-fast bacilli in smears of these fluids.

[0186] In some embodiments, the subject has an active tuberculosis infection.

[0187] In some embodiments, a method or use of the disclosure may further comprise a step or steps to evaluate whether and to what extent an immune response is induced by the vaccine compositions of the disclosure. There are many types of immunoassays that can be implemented. Immunoassays encompassed by the disclosure include, but are not limited to, those described in U.S. Patent 4,367,110 (double monoclonal antibody sandwich assay) and 4,452,901 (western blot). Other assays include immunoprecipitation of labelled ligands and immunocytochemistry, both in vitro and in vivo.

[0188] In some embodiments, a method of the disclosure may further comprise administering an additional therapeutic agent for the prevention or treatment of aMycobacterium infection or related condition. For example, one or more additional therapeutic agents may be selected from: an antibiotic (eg rifampin (RIF), rifabutin, rifapentine, ethambutol, pyrazinamide (PZA), fluoroquinolones (such as moxiflocacin); isoniazid) a therapeutic agent to treat a coexisting medical condition (eg HIV, diabetes; such as an anti-viral or insulin respectively); an anti-inflammatory (eg non-steroidal antiinflammatory drugs, NSAIDs; corticosteroids).

[0189] In other embodiments, the disclosure provides for use of a vaccine composition in the manufacture of a medicament for inducing an immune response to a mycobacterial SAP component in a subject, preferably an immune response to M. tuberculosis. The medicament may be for administration with or further comprise one or more additional therapeutic agents for the prevention or treatment of a Mycobacterium infection or related condition.

[0190] Definitions

[0191] Sulphate assimilation pathway or SAP generally refers to the pathway by which Mycobacteria reduce sulphur, thereby obtaining substrate for the biosynthesis of cysteine and downstream products including mycothiol. In more detail, the pathway involves the formation of adenosine 5’-phosphosulfate (APS) from sulphate, from which APS reductase (encoded by cysH) may produce sulphite, and from which sulfite reductase (encoded by sirA) may produce sulphide, and from which, and with O-Acetyl-L-serine, O- Acetyl -L-Serine Sulfhydalase (encoded by cysK1) may produce cysteine. Key enzymes of SAP include ATP sulfurylase (adenylyl-transferase) (encoded by cysD), GTPase (encoded by cysN), and APS kinase. These enzymes enable the formation of PAS from sulphate, and the formation of 3’ phosphoadenosine 5’-phosphosulfate (PAPS) from APS.

[0192] A SAP component generally refers to a protein or enzyme involved in the reduction of sulphur in Mycobacterium according to the SAP, examples of which include those encoded by cysD, cysNC, cysH, sirA, cysE, cysK1 genes.

[0193] cysD gene generally refers to a nucleic acid encoding a ATP sulfurylase. The nucleic acid may have a nucleotide sequence substantially as shown in SEQ ID NO: 1 herein or otherwise having defined homology and / or identity as defined herein.

[0194] CysD protein generally refers to an ATP sulfurylase. The protein may have an amino acid sequence substantially as shown in SEQ ID NO: 2 herein or otherwise having defined homology and / or identity as defined herein.

[0195] ag85B gene generally refers to a nucleic acid comprising or consisting of the nucleotide sequence set forth in SEQ ID NO: 3 herein or otherwise having defined homology and / or identity as defined herein.

[0196] Ag85B protein generally refers to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4 herein or otherwise having defined homology and / or identity as defined herein. Ag85B protein is expressed by pathogenic mycobacteria and is a component of multiple existing TB vaccines under evaluation in humans.

[0197] CysVac2 antigen generally refers to a polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 5 herein or otherwise having defined homology and / or identity as defined herein.

[0198] CysVac2 encoding nucleic acid sequence generally refers to a nucleic acid sequence comprising or consisting of the sequence set forth in SEQ ID NO: 6 herein or otherwise having defined homology and / or identity as defined herein.

[0199] BCG vaccine refers to the Bacille Calmette Guerin recombinant vaccine comprising attenuated M. bovis.

[0200] By “derivative” is meant an agent or active that has been derived from the polypeptide or fusion protein by modification of the amino acid sequence, or, for example by conjugation or complexing or expression (eg, as a fusion protein) with other chemical moieties or by post-translational modification techniques as would be understood in the art. The term “derivative” also includes within its scope alterations that have been made to a parent sequence including additions, or deletions that provide for functionally equivalent or functionally enhanced molecules.

[0201] By “isolated” is meant material that is substantially or essentially free from components that normally accompany it in its native state.

[0202] The term “subject,” includes patient, and refers to any subject of medical or veterinary interest. Subjects may be a vertebrate subject, such as mammalian subject (e.g, bovines, pigs, dogs, cats, equine, lama, camelids, etc.), non-mammals, reptiles birds, fish. The subject includes a human, for whom prophylaxis or therapy is desired. The subject may be in need of prophylaxis or treatment for a wound care, sarcopenia or other pathology, disease, disorder or condition associated with tissue degeneration or injury, including as described elsewhere herein.

[0203] The term “polynucleotide” or “nucleic acid” as used herein designates mRNA, RNA, cRNA, cDNA or DNA.

[0204] The term sequence “identity” as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present disclosure, “sequence identity” may be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for Windows; available from Hitachi Software Engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Amino acid sequence identity may also be determined using the EMBOSS Pairwise Alignment Algorithms tool available from The European Bioinformatics Institute (EMBL-EBI), which is part of the European Molecular Biology Laboratory. This tool is accessible at the website located at www.ebi.ac.uk / Tools / emboss / align / . This tool utilizes the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970). Default settings are utilized which include Gap Open: 10.0 and Gap Extend 0.5. The default matrix “Blosum62” is utilized for amino acid sequences and the default matrix.

[0205] The term sequence “similarity” refers to the percentage number of amino acids that are identical or constitute conservative amino acid substitutions as defined in Table 2 above. Similarity may be determined using sequence comparison programs such as GAP (Deveraux et al, 1984 Nucleic Acids Research 12: 387-395). In this way, sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

[0206] "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in some instances ±5%, in some instances ±1%, and in some instances ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0207] Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0208] All of the patents and publications referred to herein are incorporated by reference in their entirety.

[0209] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that such prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and / or combined with other pieces of prior art by a skilled person in the art.

[0210] It will be understood that the terms “comprise” and “include” and any of their derivatives (e.g. comprises, comprising, includes, including) as used in thisspecification, and the claims that follow, is to be taken to be inclusive of features to which the term refers, and is not meant to exclude the presence of any additional features unless otherwise stated or implied.

[0211] In some cases, a single embodiment may, for succinctness and / or to assist in understanding the scope of the disclosure, combine multiple features. It is to be understood that in such a case, these multiple features may be provided separately (in separate embodiments), or in any other suitable combination. Alternatively, where separate features are described in separate embodiments, these separate features may be combined into a single embodiment unless otherwise stated or implied. This also applies to the claims which can be recombined in any combination. That is a claim may be amended to include a feature defined in any other claim. Further a phrase referring to “at least one of” a list of items refers to any combination of those items, including single members. As an example, “at least one of: a, b, or c” is intended to cover: a, b, c, a-b, a-c, b-c, and a-b-c.

[0212] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0213] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0214] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.Examples

[0215] Example 1 : Materials and methods

[0216] Bacterial strains and growth conditions: M. tuberculosis H37Rv and M. bovis BCG Pasteur were grown at 37 °C in Middlebrook 7H9 medium (BD) supplemented with 0.5% glycerol, 0.02% Tyloxapol, and 10% albumin-dextrose-catalase (ADC) or on solid Middlebrook 7H11 medium (BD) supplemented with oleic acid-ADC.

[0217] Antigens. CysVac2 were produced in recombinant form from Eschericia coll by Sydney Analytical (University of Sydney). Antigen purity was >90% as assessed by SDS PAGE analysis.

[0218] Vaccination and infection of mice: Female C57BL / 6 (6-8 weeks of age) were maintained in specific pathogen-free condition and experiments were performed with the approval of the Sydney Local Health District Animal Welfare Committee in accordance with relevant guidelines and regulations. Animals were randomly assigned to experimental groups. For protection experiments, mice were vaccinated subcutaneously at the base of the tail either once with 5 x 105CFU of BCG Pasteur (200 pl in PBS), intramuscularly 3 times at 2 weeks interval with MemVax alone, MemVax combined with the CysVac2 antigen (MemTBVax), or intranasally 2 times at 2 weeks interval with the same vaccines. For M. tuberculosis challenge experiments, four weeks after the final vaccination mice were infected with M. tuberculosis H37Rv via the aerosol route using a Middlebrook airborne infection apparatus (Glas-Col) with an infective dose of approximately 100 viable bacilli. Four weeks later the lung and spleen were harvested, homogenized and plated after serial dilution on supplemented Middlebrook 7H1 1 agar plates. Colonies forming units (CFU) were determined approximately 3 weeks later and expressed as Log CFU.

[0219] Assays of cytokine production: PBMCs were isolated by gradient centrifugation of approximately 200 pl of blood per mouse on Histopaque1083 (Sigma) according to manufacturer’s instructions. Lung cells were prepared from vaccinated or infected mice by passage through a cell strainer (BD). Cells were resuspended in buffered ammonium sulfate (ACK buffer; 0.1 mM EDTA (Sigma), 10 mM KHCO3 (Sigma), 150 mM NH4CI (Sigma) to lyse erythrocytes and then washed and resuspended in RPMI 1640 (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (Scientifix, Cheltenham, Australia), 50 pM 2-mercaptoethanol (Sigma), and 100 U ml-1Penicillin / Streptomycin (Sigma). For intracellular cytokine staining, cells were stimulated for 3-4 hours in the presence of the CysVac2 fusion protein (10 pg ml-1) and then for up to 12 hours with brefeldin A (10 pg ml-1). Two million cells were incubated with 1 .25 pg ml-1anti-CD32 / CD16 (eBioscience, San Diego, CA) in FACS wash buffer (PBS / 2% FCS / 0.1 %) for 30 min to block Fc receptors, then washed and incubated for 30 min with either anti-CD4-Alexafluor 700, anti-CD8a-allophycocyanin (APC)-Cy7 oranti-CD44- fluorescein isothiocyanate (FITC). Fixable Blue Dead Cell Stain (Life Technologies) was added to allow dead cell discrimination. Cells were then fixed and permeabilized using the BD Cytofix / Cytoperm™ kit according to the manufacturer’s protocol. Intracellular staining was performed using the following antibodies: anti-IFN-y- phycoerythrin (PE)-Cy7, anti-TNF-PerCP (Biolegend, San Diego, CA), anti-IL-2-PE (clone JES6-5H4) (BD) or anti-IL-17A-BV421 (Biolegend). All samples were acquired on a BD LSR-Fortessa flow cytometer (BD), and analyzed using FlowJo™ analysis software (Treestar, Macintosh Version 9.8, Ashland, OR).

[0220] Assay for identification of macrophages in the lung. Whole lungs from euthanised mice were perfused post-mortem with cold PBS-heparin. For isolation of lung leukocytes, lung tissue in complete RPMI media, consisted of L-glutamine and 25 mM Hepes (Invitrogen, Waltham, CA, USA), FCS (10% v / v), 2-mercaptoethanol (50pM; Sigma-Aldrich) and PenStrep (100U / mL; Invitrogen) was digested with collagenase IV (50U / mL; Sigma-Aldrich) and DNAse I (13pg / mL; Sigma-Aldrich) at 37°C for 45 minutes prior to homogenization (using a GentleMACS Dissociator, Miltenyi Biotec, Germany) and multiple filtration steps through 70pm cell strainers. Erythrocytes were removed using ACK lysis buffer and single cell suspension were prepared and counted by Trypan Blue (0.04%) exclusion followed by dilution in desired concentration. Two million cells were incubated with 1.25 pg ml-1anti-CD32 / CD16 (eBioscience, San Diego, CA) in FACS wash buffer (PBS / 2% FCS / 0.1 %) for 30 min to block Fc receptors, then washed and incubated for 30 min with anti-MHCII-BV421 , anti-CD64-BV510, anti-F4 / 80-BV71 1 , anti- CD103-BV786, anti-Ly6G-BUV396, anti-CD24-BUV793, anti-Ly6C-PerCP5.5, anti- SiglecF-PE, anti-CD3-PECF594, anti-CD64-PECy7, anti-CD1 1 c-AF647, anti-B220- AF700, anti-CD1 1 b-APCy7. All samples were acquired on a BD LSR-Fortessa flow cytometer (BD), and analyzed using FlowJo™ analysis software (Treestar, Macintosh Version 9.8, Ashland, OR).

[0221] Statistical Analysis: The significance of differences between experimental groups was evaluated by one- or two-way analysis of variance (ANOVA), with pairwise comparison of multi-grouped data sets achieved using Tukey or Dunnet post hoc test.Example 2: Decreased bacterial burden in the lungs and spleen after intramuscular or intranasal vaccination with MemVax

[0222] The inventors vaccinated C57BL / 6 mice (n = 5-6 / group) three times, 2 weeks apart by either the intramuscular (IMSC) or intranasal (INSL) route with MemVax alone, MemTBVax or the BCG vaccine (standard of care). Control unvaccinated mice were also followed. Mice were then infected with M. tuberculosis and the bacterial burden in the lung or spleen determined (Figures 1A and 1 B). The lung is the primary site of M. tuberculosis infection in mice and humans. The spleen is a site of bacterial dissemination from the lung.

[0223] Both MemTBVax IMSC-vaccinated and MemTBVax INSL-vaccinated mice displayed reduced bacterial burden in the lung and spleen, when compared to unvaccinated mice and to mice administered with the MemVax viral vector only (Figures 1A and 1 B). This indicates that MemTBVax provides significant immune protection against M. tuberculosis regardless of the administration route.

[0224] An approximate 100-fold decrease in lung bacterial burden was observed in MemTBVax INSL-vaccinated mice compared to unvaccinated mice (Figure 1A), representing a surprisingly high level of protection gained from vaccination. Further, comparing MemTBVax to BCG (as the standard of care), MemTBVax delivered by the INSL route significantly reduced lung bacterial burden (Figure 1 A).

[0225] MemTBVax delivered by the IMSC route could still induce protection against M. tuberculosis infection compared to unvaccinated mice. However, comparing IMSC delivery to INSL delivery, the protection obtained from vaccination was significantly greater when the vaccine was delivered INSL (Figure 1A).

[0226] Thus, pulmonary delivery of MemTBVax via the intranasal route induces unparalleled protection against TB in this model.Example 3: Increased production of cytokines by antigen-specific CD4+ T cells following intramuscular or intranasal vaccination with MemTBVax

[0227] The T cell immune response associated with MemTBVax protection was examined. Mice were vaccinated as above, and lung cells re-stimulated ex v / vo with the CysVac2 antigen. The production of cytokines (IFN-y, IL-2, IL-17, TNF (tumour necrosis factor)) by pulmonary antigen-specific CD4+ T cells (Figure 2A) or CD8+ T cells (Figure 2B) was subsequently determined by flow cytometry.

[0228] Both MemTBVax IMSC-vaccinated and MemTBVax INSL-vaccinated mice displayed significantly increased production of IL-2 and TNF cytokines by CD4+ cells, compared to the unvaccinated, BCG-administered, and vector-only controls (Figure 2A). The greatest elevation of IL-17 and TNF in CD4+ T cells was observed in the MemTBVax INSL group, which was significantly higher than compared to the unvaccinated, BCG- administered, and vector-only controls. The greatest elevation of IFN-y in CD4+ T cells was observed in the MemTBVax IMSC group, but this was not significantly higher than control groups.

[0229] Overall, the greatest release of cytokines by CD4+ T cells was observed after MemTBVax vaccination via the INSL route, particularly for the TNF and IL-17 cytokines (Figure 2A). Notably, IL-17 was only elevated in CD4 T cells from mice vaccinated with MemTBVax via the INSL route, where 5-10% of all CD4+ T cells were observed to secrete IL-17 in the lungs after vaccination. This proportion of IL-17-secreting CD4 T cells in the lungs after vaccination was unexpectedly high, and indicated that induction of CD4+ T cells secreting IL-17 could be used as a correlate or proxy for determining immune protection provided by the MemTBVax vaccine when it is delivered via the INSL route.

[0230] To further explore the effects of MemTBVax on CD4+ T cell cytokine release, mice were vaccinated as above, and intracellular cytokine staining was performed on PBMCs after re-stimulation with CysVac2 and Brefeldin A. The production of IFN-y, IL- 2, IL-17 or TNF-positive CD4+ T cells was again assessed by flow cytometry. MemTBVax delivered by the IMSC route induced significantly increased the production of cytokines IFN-y, IL-2, IL-17, and TNF by antigen-specific CD4+ T cells compared to unvaccinated and BCG-administered groups (Figure 4). Comparing the MemVax adjuvant with the AdvaxCpGadjuvant, IMSC-delivered MemTBVax significantly increased the release of IFN-y, IL-2, IL-17, and TNF by antigen-specific CD4+ T cells, compared to CysVac2 delivered with the AdvaxCpGadjuvant (Figure 4).Example 4: Intranasal vaccination with MemTBVax induces specific cellular immune protection in the lungs

[0231] The inventors examined the composition of immune cell subsets in the lungs of MemTBVax vaccinated mice that had been infected with M. tuberculosis, where again vaccination was performed via either the IMSC or INSL administration route.

[0232] Mice vaccinated with MemTBVax via the intranasal route displayed a unique immune cell signature that has not been previously described for TB vaccines. Alveolar macrophages, the first line of defence in the lung, were only significantly elevated in mice vaccinated with MemTBVax INSL (Figure 3A). Whilst BCG-administered mice displayed some elevation of alveolar macrophages, it was not significant compared to unvaccinated controls.

[0233] Conversely, interstitial macrophages were only significantly decreased in the MemTBVax INSL-vaccinated group, possibly indicating a reduced inflammatory environment (Figure 3B). Finally, CD103-expressing T cells were only significantly elevated in the MemTBVax INSL-vaccinated group. CD103 is a marker for T cells that are important for the clearance of pathogens and tumours.

[0234] Overall, the inventors speculate that the intranasal administration of a vaccine composition (MemTBVax) comprising a bacterial immunogen CysVac2 and an adjuvant MemVax, induces a particularly beneficial, protective immune cell composition in lungs infected by M. tuberculosis.Example 4: Intranasal vaccination with MemTBVax induces specific cellular immune protection in the lungs

[0235] Using standard molecular cloning techniques, the inventors modified the MemVax vector to introduce coding sequences to express CysVac2 (fusion protein of CysD + Ag85B) in addition with 3 other mycobacterial antigens (PE13, PPE15 and MPT83). The single-vector vaccine with combined mycobacterial antigens was termed TB-Multi.

[0236] C57BL / 6 mice (n=4-5) were vaccinated twice intranasally (INSL), 2 weeks apart with TB-Multi. Four weeks after the last vaccination, spleen cells were stimulated ex vivo with the CysVac2 (Figure 5A), PE13 (Figure 5B), PPE15 (Figure 5C) or MPT83 antigen (Figure 5D) and the production of IL-2 by CD4+ T cells determined by flow cytometry.

[0237] The experimental group vaccinated with TB-Multi displayed a significant increase in the production of cytokine-producing CD4+ T cells compared to unvaccinated group provided PBS only, indicating that TB-Multi provides advantageous protective cellular immunity against mycobacterial infection in the lungs.

Claims

CLAIMS1 . A vaccine composition comprising:(i) an immunogen comprising one or more SAP components or functional variants thereof, or at least one nucleic acid sequence encoding one or more SAP components or functional variants thereof; and(ii) a chimeric CD40L polypeptide or functional equivalent thereof, or at least one nucleic acid sequence encoding a chimeric CD40L polypeptide or a functional variant thereof.

2. The vaccine composition of claim 1 , wherein the one or more SAP components, or at least one nucleic acid sequence encoding one or more SAP components comprises a nucleic acid encoding CysD from M. tuberculosis, preferably a nucleic acid encoding a fusion protein including CysD.

3. The vaccine composition of claim 1 , wherein the immunogen comprises or consists of an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:2; or comprises or consists of an amino acid sequence encoded by a nucleic acid sequence at least 80% identical to the nucleic acid sequence of SEQ ID NO:1 .

4. The vaccine composition of claim 1 , wherein the immunogen comprises or consists of a nucleic acid sequence at least 80% identical to the nucleic acid sequence of SEQ ID NO:1.

5. The vaccine composition of any one of claims 1 to 4, wherein the immunogen further comprises one or more additional mycobacterial antigen or functional variant thereof, or one or more nucleic acid sequences encoding one or more additional mycobacterial antigen.

6. The vaccine composition of claim 5, wherein the one or more additional mycobacterial antigen comprises or consists of Ag85B, PE13, PPE15, MPT83, EsxA, EsxB, EsxH, EspC, EsxR, and PPE18, or a functional variant or derivative thereof, or one or more nucleic acid sequences encoding the same; optionally selected from Ag85B, PE13, PPE15, and MPT83 or a functional variant or derivative thereof.

7. The vaccine composition of claim 6, wherein the one or more additional mycobacterial antigen comprises or consists of an amino acid sequence set forth in any one of SEQ ID NO:4, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, or an amino acid sequence at least 80% identical to the amino acid sequence of any one of SEQ ID NO: 4, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27; optionally one or more of SEQ ID NOs: 4, 1 1 , 13, or 15; preferably at least SEQ ID NO:4.

8. The vaccine composition of claim 6, wherein the one or more additional mycobacterial antigen comprises or consists of a nucleic acid sequence set forth in any one of SEQ ID NO: 3, 10, 12, 14, 16, 18, 20, 22, 24, or 26, or a nucleic acid sequence at least 80% identical to the nucleic acid sequence of any one of SEQ ID NOs: 3, 10, 12, 14, 16, 18, 20, 22, 24 or 26 optionally one or more of SEQ ID NOs: 3, 10, 12, or 14; preferably at least SEQ ID NO: 3.

9. The vaccine composition of claim 6, wherein the one or more additional mycobacterial antigen comprises or consists of a Ag85B antigen comprising or consisting of an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 4; comprises or consists of a Ag85B antigen comprising or consisting of an amino acid sequence encoded by a nucleic acid sequence at least 80% identical to the nucleic acid sequence of SEQ ID NO:3.

10. The vaccine composition of claim 6, wherein the one or more additional mycobacterial antigen comprises or consists of at least one nucleic acid encoding a Ag85B antigen or a functional variant thereof, wherein the nucleic acid comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 3, or variants thereof that are at least 80% identical thereto.1 1. The vaccine composition of claim 6, wherein the one or more additional mycobacterial antigen comprises or consists of a PE13 antigen comprising or consisting of an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 1 1 ; comprises or consists of a PE13 antigen comprising or consisting of an amino acid sequence encoded by a nucleic acid sequence at least 80% identical to the nucleic acid sequence of SEQ ID NQ:10.

12. The vaccine composition of claim 6, wherein the one or more additional mycobacterial antigen comprises or consists of at least one nucleic acid encoding PE13antigen or a functional variant thereof, wherein the nucleic acid comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 10, or variants thereof that are at least 80% identical thereto.

13. The vaccine composition of claim 6, wherein the one or more additional mycobacterial antigen comprises or consists of a PPE15 antigen comprising or consisting of an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 1 1 ; comprises or consists of a PPE15 antigen comprising or consisting of an amino acid sequence encoded by a nucleic acid sequence at least 80% identical to the nucleic acid sequence of SEQ ID NO:12.

14. The vaccine composition of claim 6, wherein the one or more additional mycobacterial antigen comprises or consists of at least one nucleic acid encoding a PPE15 antigen or a functional variant thereof, wherein the nucleic acid comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 12, or variants thereof that are at least 80% identical thereto.

15. The vaccine composition of claim 6, wherein the one or more additional mycobacterial antigen comprises or consists of a MPT83 antigen comprising or consisting of an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 15; comprises or consists of a MPT83 antigen comprising or consisting of an amino acid sequence encoded by a nucleic acid sequence at least 80% identical to the nucleic acid sequence of SEQ ID NO:14.

16. The vaccine composition of claim 6, wherein the one or more additional mycobacterial antigen comprises or consists of at least one nucleic acid encoding a MPT83 antigen or a functional variant thereof, wherein the nucleic acid comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 14, or variants thereof that are at least 80% identical thereto.

17. The vaccine composition of any one of claims 1 to 16, wherein the immunogen comprises or consists of a fusion polypeptide comprising of one or more SAP components; or at least one nucleic acid encoding a fusion polypeptide comprising of one or more SAP components.

18. The vaccine composition of claim 17, wherein the fusion polypeptide comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:2, or an amino acid sequence encoded by a nucleic acid sequence at least 80% identical to the nucleic acid sequence of SEQ ID NO:1 ; and one or more additional mycobacterial antigen or functional variant thereof.

19. The vaccine composition of claim 17 or 18, wherein the fusion polypeptide is CysVac2, or a functional fragment or variant thereof.

20. The vaccine composition of any one of claims 17 to 19, wherein the fusion polypeptide comprises or consists of an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 5; or comprises or consists of an amino acid sequence encoded by a nucleic acid sequence at least 80% identical to the nucleic acid sequence of SEQ ID NO:6.21 . The vaccine composition of any one of claims 17 to 20, wherein the immunogen comprises or consists of a nucleic acid sequence set forth in SEQ ID NO: 6, or a nucleic acid sequence at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO:6.

22. The vaccine composition of any one of claims 1 to 21 , wherein chimeric CD40L polypeptide or functional equivalent thereof comprises or consists of an ISF35 amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 7.

23. The vaccine composition of any one of claims 1 to 22, wherein the chimeric CD40L polypeptide or functional equivalent thereof is encoded by a nucleic acid sequence that comprises or consists of a nucleic acid sequence at least 80% identical to the nucleic sequence as set forth in SEQ ID NO: 8.

24. The vaccine composition of any one of claims 1 to 23, wherein the composition comprises an expression vector comprising a nucleic acid sequence that encodes the chimeric CD40L or functional equivalent thereof; preferably an adenoviral vector most preferably the expression vector is MemVax.

25. The vaccine composition of claim 24, wherein the expression vector comprises or consists of a nucleic acid sequence set forth in SEQ ID NO: 9, or a nucleic acid sequence at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO:9.

26. The vaccine composition of any one of claims 1 to 25, wherein the vaccine composition comprises a single expression vector encoding (i) the immunogen and (ii) the chimeric CD40L polypeptide or functional equivalent thereof.

27. The vaccine composition of any one of claims 1 to 26, wherein the composition comprises an expression vector encoding the chimeric CD40L polypeptide or functional equivalent thereof; and the immunogen in the form of polypeptide or functional equivalent thereof, optionally a fusion polypeptide, preferably a recombinant, purified fusion polypeptide.

28. The vaccine composition of any one of claims 1 to 27, wherein the composition further comprises:- an additional adjuvant;- a pharmaceutically acceptable excipient, diluent, stabiliser and / or carrier, optionally a buffer; and / or- a delivery system.

29. A method for inducing an immune response to a mycobacterial SAP component in a subject in need thereof, the method comprising administering an effective amount of the vaccine composition of any one of claims 1 to 28 to the subject, thereby inducing an immune response to a SAP protein from a Mycobacterium species in the subject.

30. A method for inducing an immune response to M. tuberculosis in a subject in need thereof, the method comprising administering an effective amount of the vaccine composition of any one of claims 1 to 28 to the subject, thereby inducing an immune response to M. tuberculosis in the subject.31 . The method of any one of claims 29 to 30, wherein the immune response is a protective immune response to a mycobacterial infection, preferably an infection with M. tuberculosis (Mtb).

32. A method for treating or preventing a Mycobacterium infection in a subject, the method comprising an effective amount of the vaccine composition of any one of claims 1 to 28 to the subject, thereby inducing an immune response to the Mycobacterium in the subject.

33. The vaccine composition of any one of claims 1 to 28, wherein the composition is formulated for intramuscular or mucosal administration, preferably intranasal administration; or the method of any one of claims 29 to 32, wherein the vaccine composition is administered mucosally (ie via the mucosa), preferably, intranasally (ie via the nasal mucosa).

34. A kit comprising a vaccine composition of any one of claims 1 to 28, or 33.

35. An expression vector encoding the vaccine composition of any one of claims 1 to 28 or 33; preferably an adenoviral vector, more preferably an Ad5 vector.

36. Use of the vaccine composition of any one of claims 1 to 28 or 33 in the manufacture of a medicament inducing an immune response to a mycobacterial SAP component in a subject.

37. Use of the vaccine composition of any one of claims 1 to 28 or 33 in the manufacture of a medicament inducing an immune response to M. tuberculosis in a subject.

38. Use of the vaccine composition of any one of claims 1 to 28 or 33 in the manufacture of a medicament for treating or preventing a Mycobacterium infection in a subject.

39. The vaccine composition of any one of claims 1 to 28 or 33 for use in inducing an immune response to a mycobacterial SAP component in a subject.

40. The vaccine composition of any one of claims 1 to 28 or 33 for use in inducing an immune response to M. tuberculosis in a subject.

41. The vaccine composition of any one of claims 1 to 28 or 33 for use in for or preventing a Mycobacterium infection in a subject.

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