WO2022170394A1

WO2022170394A1 - Recombinant strains of mycobacterium bovis bcg

Info

Publication number: WO2022170394A1
Application number: PCT/AU2022/050086
Authority: WO
Inventors: Gopinath Krishnamoorthy; Aliabbas A. HUSAIN; Andreas KUPZ
Original assignee: James Cook University
Current assignee: James Cook University
Priority date: 2021-02-10
Filing date: 2022-02-10
Publication date: 2022-08-18
Anticipated expiration: 2023-08-10

Abstract

The present invention provides recombinant strains of M. bovis BCG comprising a heterologous nucleic acid encoding a fusion protein, wherein the fusion protein comprises or consists of: a) a polypeptide that is at least about 80% identical to an ESAT-6 protein and b) a secretion peptide for enabling secretion of the polypeptide via an ESX-5 secretion system of M. bovis BCG.

Description

Recombinant strains of Mycobacterium bovis BCG

Field of the invention

[0001] The present invention relates to recombinant strains of Mycobacterium bovis bacilli Calmette-Guerin (M. bovis BCG) comprising a heterologous nucleic acid sequence encoding a fusion protein.

Related application

[0002] This application claims priority from Australian provisional application AU 2021900320, the contents of which are hereby incorporated by reference in their entirety.

Background of the invention

[0003] Tuberculosis (TB) is a significant global health problem with approximately 10 million new cases and 1.5 million deaths annually. In addition, two billion people are latently infected with Mycobacterium tuberculosis ( Mtb ), the causative agent of TB, and potential reactivation of latent TB infection (LTBI) into destructive pulmonary disease could severely compromise any serious attempts aimed 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 reduce the LTBI burden, a foremost step in achieving TB control globally.

[0005] Bacille Calmette-Guerin (BCG) the only licensed TB vaccine, which prevents TB in children, has varied efficacy against pulmonary TB in adults and is contraindicated in people with impaired immunity.

[0006] 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. Most new vaccine strategies aim to broaden the immune responses by increasing the antigenic repertoire of cellular and by humoral immunity to Mtb infection. In particular, the activation of Mtb- specific CD4+ T cells via immunodominant Mtb antigens is a major strategy for many new subunit TB vaccines under development. Despite these global efforts, most new anti-TB vaccine candidates employing this strategy have not shown superiority over BCG. In addition, the gradual decline of CD4⁺ T cells, the hallmark of HIV infection, is a major contributing factor in LTBI reactivation, and poses an additional challenge for vaccines that solely rely on boosting CD4⁺ T cell immunity.

[0007] There remains, therefore, a need for new vaccines for the prevention of TB, for reducing the severity of TB disease and/or for reducing the likelihood and occurrence of LTBI.

[0008] 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 invention

[0009] The invention provides a recombinant strain of M. bovis BCG comprising a heterologous nucleic acid encoding a fusion protein, wherein the fusion protein comprises or consists of: a) a polypeptide that is at least about 80% identical to the amino acid sequence of an ESAT-6 protein, or a functional variant or homolog thereof; and b) a secretion peptide for enabling secretion of the polypeptide via an ESX-5 secretion system of M. bovis BCG.

[0010] The amino acid sequence of the ESAT-6 protein may be an amino acid sequence from any species of Mycobacterium. In certain embodiments, the ESAT-6 protein is an ESAT-6 protein from M. tuberculosis ( Mtb ). In further embodiments, the ESAT-6 protein is from M. marinum. Preferably, the polypeptide is at least about 80%, 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 or 100% to the amino acid sequence of an ESAT-6 protein from Mtb. [0011] Typically, the ESAT-6 protein corresponds to the ESAT-6 protein encoded by the gene esxA from the ESX-1 secretion system, as further defined herein. However, it will be appreciated that the ESAT-6 protein may be any homolog of the protein encoded by esxA, such as a homolog selected from: a protein encoded by esxC, esxH, esxT or esxN.

[0012] Preferably, the fusion protein comprises a polypeptide 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: 5. The fusion protein may comprise a polypeptide that consists of the amino acid sequence set forth in SEQ ID NO: 5.

[0013] The architecture of the fusion protein may be varied such that the secretion peptide may be located N-terminally or C-terminally to the polypeptide that is at least 80% identical to a ESAT-6 polypeptide. In a preferred embodiment, the secretion peptide is located C-terminally to the ESAT-6 polypeptide in the fusion protein.

[0014] In any embodiment, the secretion peptide for enabling secretion of the polypeptide via an ESX-5 secretion system, comprises or consists of the amino acid sequence YXXXD/E, wherein Y is a tyrosine residue, X is any amino acid residue, D is an aspartic acid residue and E is a glutamic acid residue. In certain embodiments, the secretion peptide comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 6, 8 or 9, or variants or fragments thereof that retain the YXXXD/E motif. In a particularly preferred embodiment, the peptide consists of the amino acid sequence set forth in SEQ ID NO: 6, or a variant or fragment thereof that comprises the YXXXD/E motif.

[0015] In any embodiment, the fusion protein encoded by the heterologous nucleic acid comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7, or variants thereof that are at least 80% identical thereto. Preferably, the fusion protein 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: 7. In certain embodiments, the fusion protein consists of the amino acid sequence set forth in SEQ ID NO: 7.

[0016] In any embodiment, the recombinant strain of M. bovis BCG may be sensitive to one or more antibiotics. In any embodiment, the recombinant strain of M. bovis BCG may be resistant to one or more antibiotics. In any embodiment, the recombinant strain of M. bovis BCG may be derived from a strain which was resistant to an antibiotic and which has been modified to remove the gene responsible for conferring resistance, such that the recombinant strain is no longer resistant to the antibiotic.

[0017] In any embodiment, the recombinant strain of M. bovis BCG may be an auxotrophic strain.

[0018] The present invention also provides a heterologous nucleic acid encoding a fusion protein, wherein the fusion protein comprises a) a polypeptide that is at least about 80% identical to an ESAT-6 protein or functional variant or homolog thereof and b) a secretion peptide for enabling secretion of the polypeptide via the ESX-5 secretion system of M. bovis BCG.

[0019] In preferred embodiments, the nucleic acid comprises or consists of the nucleic acid sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, or a sequence at least about 80%, 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 thereto. More preferably, the nucleic acid comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 3 or 4.

[0020] Preferably, the nucleic acid is at least about 80%, 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: 3 or 4.

[0021] Preferably the nucleic acid encodes a fusion protein as herein described, being a fusion protein that comprises the amino acid sequence of the ESAT-6 protein from any species of Mycobacterium. In certain embodiments, the ESAT-6 protein is an ESAT-6 protein from M. tuberculosis ( Mtb ). In further embodiments, the ESAT-6 protein is from M. marinum. Preferably, the nucleic acid encodes a fusion protein comprising an ESAT-6 protein that is at least about 80%, 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 or 100% to the amino acid sequence of an ESAT-6 protein from Mtb.

[0022] Preferably, the nucleic acid encodes a fusion protein that comprises a. ESAT- 6 protein 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: 5. The fusion protein may comprise a polypeptide that consists of the amino acid sequence set forth in SEQ ID NO: 5.

[0023] The heterologous nucleic acid encodes a fusion protein wherein the secretion peptide of the fusion protein is located N-terminally or C-terminally to the polypeptide that is at least 80% identical to a ESAT-6 polypeptide. In a preferred embodiment, the heterologous nucleic acid encodes a fusion protein wherein the secretion peptide is located C-terminally to the ESAT-6 polypeptide in the fusion protein.

[0024] The heterologous nucleic acid preferably encodes a fusion protein as herein described, wherein the secretion peptide for enabling secretion of the polypeptide via an ESX-5 secretion system, comprises or consists of the amino acid sequence YXXXD/E, wherein Y is a tyrosine residue, X is any amino acid residue, D is an aspartic acid residue and E is a glutamic acid residue. In certain embodiments, heterologous nucleic acid encodes a fusion protein comprising a secretion peptide that comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 6, 8 or 9, or variants or fragments thereof that retain the YXXXD/E motif.

[0025] In any embodiment, the heterologous nucleic acid encodes a fusion protein that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7, or variants thereof that are at least 80% identical thereto. Preferably, the fusion protein encoded by the heterologous nucleic acid 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: 7. In certain embodiments, the fusion protein encoded by the nucleic acid consists of the amino acid sequence set forth in SEQ ID NO: 7.

[0026] Further still, the invention provides a vector comprising a nucleic acid molecule of the invention and as described herein. The invention also provides a host cell comprising the vector. In preferred embodiments, the host cell is M. bovis BCG.

[0027] The present invention further provides vaccines and immune stimulating compositions comprising a recombinant M. bovis BCG strain as described herein, optionally in combination with a pharmaceutically acceptable carrier or excipient. The vaccine or immune stimulating compositions may further comprise one or more adjuvants for potentiating the immune response to the M. bovis BCG strain. Further still, the vaccine or immune stimulating compositions may also comprise one or more subunit vaccines, or additional antigens of a Mycobacterium species for further potentiating the immune response.

[0028] The present invention also provides a method for inducing an immune response to an ESAT-6 protein in a subject in need thereof, the method comprising administering an effective amount of the recombinant M. bovis BCG strain, vaccine or immune stimulating composition of the invention to the subject, thereby inducing an immune response to an ESAT-6 protein from a Mycobacterium species in the subject. Preferably, the ESAT-6 protein is an ESAT-6 protein from Mtb. [0029] The present invention also provides a method for inducing an immune response to Mtb in a subject in need thereof, the method comprising administering an effective amount of the recombinant M. bovis BCG strain, vaccine or immune stimulating composition as described herein, to the subject, thereby inducing an immune response to Mtb in the subject.

[0030] Further, the present invention provides for the use of a recombinant strain of M. bovis BCG as described herein, in the manufacture of a vaccine or immune stimulating composition for inducing an immune response to a mycobacterial ESAT-6 protein in a subject.

[0031] Further, the present invention provides for the use of a recombinant strain of M. bovis BCG as described herein, in the manufacture of a vaccine or immune stimulating composition for inducing an immune response to Mtb in a subject.

[0032] The invention provides for a recombinant strain of M. bovis BCG as described herein, or a vaccine or immune stimulating composition described herein, for use in stimulating an immune response to an ESAT-6 protein, preferably an ESAT-6 protein from a Mycobacterium in a subject

[0033] The invention provides for a recombinant strain of M. bovis BCG, or a vaccine or immune stimulating composition as described herein, for use in stimulating an immune response to Mtb in a subject.

[0034] In any of these embodiments, the immune response may be a protective immune response. The immune response may comprise a Th1 immune response.

[0035] The present invention also provides a method for treating or reducing the severity of a mycobacterial infection in a subject in need thereof, the method comprising administering an effective amount of the recombinant M. bovis BCG strain, vaccine or immune stimulating composition of the invention to the subject, thereby treating or reducing the severity of the mycobacterial infection in the subject.

[0036] Further still, the invention provides for a recombinant strain of M. bovis BCG, or a vaccine or immune stimulating composition of the invention, for use in treating or reducing the severity of a mycobacterial infection in a subject. [0037] Further, the present invention provides for the use of a recombinant strain of M. bovis BCG of the invention, in the manufacture of a vaccine or immune stimulating composition for treating or reducing the severity of a mycobacterial infection in a subject.

[0038] The mycobacterial infection is preferably an infection with M. tuberculosis (Mtb). In certain embodiments, the mycobacterial infection may be 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.

[0039] In any embodiment, the inducing of an immune response or the treating or reducing the severity of a mycobacterial infection may be in a neonate, adolescent, adult or geriatric subject, preferably a human subject. The subject may or may not have symptomatic infection with M. tuberculosis.

[0040] In still further embodiments, the invention provides a method for stimulating an immune response to an antigen in a subject, the method comprising:

- providing an antigen to which an immune response is desired;

- administering to a subject in need thereof, an amount of the antigen and an amount of the recombinant M. bovis BCG strain, vaccine or immune stimulating composition of the present invention sufficient to potentiate an immune response to the antigen, thereby stimulating an immune response to the antigen in the subject. The antigen and the BCG strain may be administered concomitantly or sequentially.

[0041] The antigen may be any antigen from an infectious disease or toxin. In certain embodiments, the antigen is a non-mycobacterial antigen. Thus, in any embodiment, the antigen may be an antigen from a virus (for example, HIV, hepatitis virus, herpes virus, influenza virus, coronavirus such as SARS, SARS-CoV-2), a parasite (for example, a Plasmodium, Trypanosome, trematode worm, tick) or a bacterium (for example, a Salmonella, Escherichia, Enterobaceter, Corneybacerium diptheriae, Clostridium, Bacillus, Streptomyces, sp.), or toxin therefrom. [0042] In still further embodiments, the invention provides a method for reducing the susceptibility of subject to a viral, bacterial or parasitic infection, the method comprising:

- administering to a subject in need thereof, an amount of the recombinant M. bovis BCG strain, vaccine or immune stimulating composition of the present invention, sufficient to stimulate a non-specific immune response in the subject, thereby reducing the susceptibility of subject to a viral, bacterial or parasitic infection.

[0043] The viral, bacterial or parasitic infection may be an infection with HIV, hepatitis, herpes virus, influenza virus, coronavirus (such as SARS, SARS-CoV-2), Salmonella, Escherichia, Enterobaceter, Corneybacerium diptheriae, Clostridium, Bacillus, Streptomyces, sp., a Plasmodium, Trypanosome, trematode worm, tick or toxin therefrom.

[0044] The invention also provides a kit comprising a recombinant strain of M. bovis BCG, vaccine or immune stimulating composition of the invention. The kit may optionally comprise written instructions for the use of the kit in a method as described herein.

[0045] 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.

[0046] Further aspects of the present invention 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

[0047] Figure 1: Genomic and molecular characterisation of BCG::ESAT6- PE25SS. (A) The pMV306hsp plasmid containing the esxA- pe25SS gene cassette was amplified in E. coli and electroporated into BCG SSI. (B) Midiprep plasmid isolation (1 pg/lane). (C) Restriction digestion of isolated plasmid with EcoR1, Hindi 11 or both (1 pg/lane). (D) 1% agarose gel electrophoresis of duplex PCR detecting esxA and the M. bovis IS6110 insertion element. (E) Western blot stained with monoclonal anti-ESAT-6 antibody. (F) ELISA using monoclonal ESAT-6 antibody against cell filtrates of BCG, BCG::RD1and BCG::ESAT6-PE25SS. (G) Growth curve of BCG, BCG::RD1 and BCG::ESAT6-PE25SS in 7H9 broth. Results are presented as representative images (B, C, D, E) and data mean ± SEM (F) from triplicates of 2 independent experiments.

[0048] Figure 2. Immune responses in C57BL/6 mice after i.t. vaccination with BCG::ESAT-6-PE25SS. C57BL/6 mice were infected i.v. with 1x10⁸ CFU (A) or were vaccinated i.t. with 2x10^s CFU (B-F) of BCG::pYUB (black), BCG::RD1 (red) or BCG::ESAT6-PE25SS (blue). (A) Serum IL-18 levels at 24 hours after i.v. infection. (B- F) ESAT-6-specific T-cell induction (B, C, D) and frequencies of CD4⁺ and CD8⁺ T-cells in BALF and Lung parenchyma (E, F) were assessed 20 and 60 days post-vaccination. Gating strategy of live single CD3⁺ T cells and representative plots are shown in B. Results are presented as individual data points ± SEM (C, D), representative dot plots (B), and data mean ± SEM (A, E, F) from 2-3 pooled independent experiments (n = 7- 15 mice per group). Statistical analyses: One-way ANOVA followed by Tukey's multiple comparison test; significant differences are indicated by asterisks: * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.00005.

[0049] Figure 3. Vaccination with BCG::ESAT6-PE25SS results in lower bacterial burden and lung pathology. C57BL/6 mice were vaccinated i.t. with 2x10^s CFU of either BCG::pYUB (black), BCG::RD1 (red) or BCG::ESAT6-PE25SS (blue). CFU in lung tissue (A), tissue infiltration (B), and cytokine levels in serum (C, D) were assessed 20 and 60 days post-vaccination. Results are presented as individual data points ± SEM (A, B), representative images (B) and data mean ± SEM (C, D) from 2 pooled independent experiments (n = 8 mice per group). Statistical analyses: One-way ANOVA followed by Tukey's multiple comparison test; significant differences are indicated by asterisks: * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.00005.

[0050] Figure 4. BCG::ESAT6-PE25SS is safer in immunocompromised Rag2r IL2rgf mice. Rag2/IL2rg/ mice were infected i.v. with 1^c10⁶ CFU of BCG::pYUB (black), BCG::RD1 (red) or BCG::ESAT6-PE25SS (blue). (A) Percent of starting weight at 30 days after infection. (B, C) CFU in lung (B) and spleen (C), and cytokine levels in serum (D) 30 days after infection. Results are presented as individual data points ± SEM (A, B, C) and data means ± SEM (A, B, C, D) from 2 pooled independent experiments (n = 6-12 mice per group). Statistical analyses: One-way ANOVA followed by Tukey’s multiple comparison test; significant differences are indicated by asterisks: * p < 0.05, ** p < 0.005, *** p < 0.0005, **** p < 0.00005. [0051] Figure 5. BCG::ESAT6-PE25SS and BCG::RD1 are equally protective against Mtb challenge in mice. C57BL/6 mice were vaccinated i.t. (full circle) or s.c. (empty circle) with either BCG::pYUB (black), BCG::RD1 (red), BCG::ESAT6-PE25SS (blue) or left unvaccinated (grey) and aerosol infected with 50 CFU of Mtb H37Rv 60 days later. Lung infiltration (A, B), CFU in lung and spleen (C), and cytokines in serum (D, E) were assessed 60 days after Mtb challenge. Results are presented as individual data points ± SEM (B, C), representative images (A), and data mean ± SEM (B-E) from 2 independent pooled experiments (n = 5-7 mice per group). Statistical analyses: One way ANOVA followed by Tukey's multiple comparison test; significant differences are indicated by asterisks: * p < 0.05, ** p < 0.005, **** p < 0.00005.

Sequence information

[0052] Table 1: sequences of the invention

Detailed description of the embodiments

[0053] 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.

[0054] Reference will now be made in detail to certain embodiments of the invention. 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 invention 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.

[0055] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. 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.

[0056] All of the patents and publications referred to herein are incorporated by reference in their entirety.

[0057] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0058] 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.

[0059] 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).

[0060] Tuberculosis (TB) is the leading infectious cause of death globally. The only licensed TB vaccine, Bacille Calmette-Guerin (BCG), has low efficacy against TB in adults and is not recommended in people with impaired immunity. The absence of a 9.5 kbp genomic region across all BCG strains, termed the Region of Difference 1 (RD1) is understood to be the principal molecular determinant underlying BCG attenuation. RD1 harbors genes required for the paradigm type VII secretion system (T7SS) ESX-1 (also referred to as the ESAT-6 secretion system), that is dedicated to the export of proteins that play key roles in host-pathogen interactions and pathogenic potential.

[0061] In Mtb, ESAT-6 is secreted as a heterodimer with CFP-10 via the ESX-1 secretion system. In Mtb infection, ESX-1 -mediated ESAT-6 secretion acts as a key virulence factor and plays a critical role in evasion of the host immune response. One of the major functions of ESX-1 is the induction of phagosomal rupture allowing Mtb and its products to initiate complex cytosolic immune signalling cascades of the host’s innate and adaptive immune system. The absence of this critical function in BCG due to the deletion of the ESX-1 region broadly limits its efficacy to induce such immune responses

[0062] The incorporation of the Mtb ESX-1 secretion system into BCG improves immunogenicity and protection against TB in animal models. However, the resulting strain, BCG::ESX1^Mtb, has been deemed unsafe as a human vaccine, due to prolonged persistence and increased virulence in immunocompromised mice.

[0063] However, the present inventors have developed an alternative approach, generating a new recombinant BCG strain that uncouples the beneficial aspects of ESAT-6 secretion from the detrimental ESX-1 effects on virulence and persistence.

[0064] Mycobacteria rely on a novel secretion apparatus known as Type VII Secretion Systems (T7SS). These systems were identified in mycobacteria following the detection of unknown secreted antigens in Mtb culture filtrates. Strikingly, these proteins are deficient of the conventional N-terminal signal sequence of about 20 amino acids, such as the well characterized secreted T-cell Antigen 85A, that would direct them to one of the known secretion systems (SS). Additionally, the genome sequence of the paradigm reference strain Mtb H37Rv predicted the flanking genes of these detected secreted antigens to be trans -membrane proteins, conceivably forming chaperones and channels for their secretion.

[0065] The T7SS are dedicated to the secretion of low-molecular-weight proteins, notably the "6-kD Early Secreted Antigenic Target" ESAT-6, or EsxA, and its protein partner "10-kD Culture Filtrate Protein" CFP-10, or EsxB. In the genome of Mtb homologues with sequence similarities to the ESAT-6/CFP-10 family can be found. In five cases these genes are encompassed by genes encoding integral inner-membrane proteins, ATP-binding proteins and cell-wall-associated mycosins i.e. components of a putative secretion machinery, leading to the postulation that in Mtb, five T7SSs can be distinguished. Subsequently, these have been named ESX- 1-5 (Early Secreted Antigen 6 kD System) whereby the ESX-1 represents the paradigm T7SS.

[0066] Phylogenetically, the ESX loci are of ancient origin and have derived from gene duplication. The most ancient putative T7SS is ESX-4. It contains the smallest set of genes and its role in infection has not yet been established, questioning its functionality. The ESX-1 locus has been most extensively studied and considered the paradigm T7SS as it is partly absent in the TB vaccine strain M. bovis BCG contributing to its attenuation. The role of ESX-2 which encodes all core genes has not yet been described. Mutants for ESX-2 genes were shown to be viable suggesting that ESX-2 is not necessary for survival of Mtb. ESX-3, like ESX-1 contains all the basic core components and is conserved in all mycobacterial genomes available today. Its substrates and ESAT-6/CFP-10 homologues can be detected in culture supernatants underlining the functioning of this ESX system. The ESX-5 locus is believed to be the most recently evolved T7SS in mycobacteria and is only found in slow-growing mycobacteria such as M. bovis and M. leprae.

[0067] The ESX systems are comprised of genes that encode: (i) the structural components of the secretion system such as the putative channel protein (EccD), conserved membrane proteins (EccB and EccC), and AAA+ ATPase (EccA), (ii) Mycosin (MycP) which has homology to subtilisin-like proteases, and (iii) two secreted Esx proteins. Additionally, all ESX systems, except ESX-4, also contain genes encoding members of the PE/PPE protein family which derives its name from the Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs found in the N-terminus of these proteins.

[0068] The present inventors have taken a novel approach to uncoupling the beneficial aspects of ESAT-6 secretion from the detrimental ESX-1effects on virulence and persistence. More specifically, the inventors have developed a new recombinant strain of BCG in which the Mtb ESAT-6-encoding gene esxA is fused to the general secretion signal for the mycobacterial type VII secretion pathway protein PE25. This new strain, BCG::ESAT6-PE25SS, secretes full-length ESAT-6 via the ESX-5 secretion system, which in contrast to ESX-1 is also present in BCG. In vivo testing reveals that ESX-5-mediated ESAT-6 export, induces cytosolic contact, generates ESAT-6-specific T cells and enhances the protective efficacy against TB disease, but is associated with low virulence and reduced persistence in immunocompetent and immunocompromised mice.

[0069] Additionally, compared to BCG::ESX1^Mtb and parental BCG, mucosal administration of BCG::ESAT6-PE25SS is associated with more rapid clearance from the lung. These results indicate that BCG::ESAT6-PE25SS is a live attenuated vaccine candidate for treatment and prevention, or for reducing the severity of TB infection.

Recombinant strains of M. bovis BCG

[0070] Accordingly, the present invention provides recombinant strains of M. bovis BCG comprising a heterologous nucleic acid encoding a polypeptide that is at least about 80% identical to an ESAT-6 protein, wherein the nucleic acid encodes a secretion peptide for enabling secretion of the polypeptide via the ESX-5 secretion system.

[0071] It will be understood that the heterologous nucleic acid encodes a fusion protein, such that the present invention can also be said to relate to a recombinant strain of M. bovis BCG, comprising a heterologous nucleic acid encoding a fusion protein, wherein the fusion protein comprises a polypeptide that is at least about 80% identical to an ESAT-6 protein and a secretion peptide for enabling secretion of the polypeptide via the ESX-5 secretion system.

[0072] As used herein, “M. bovis BCG” refers to the live, attenuated strains of Mycobacterium bovis Bacille Calmette-Guerin that are currently in used as a vaccine against tuberculosis. The skilled person will be familiar with various sources of M. bovis BCG for use in accordance with the present invention and which are further described herein.

[0073] As used herein, ESAT-6 (also termed Rv3875) typically refers to the "6-kDa Early Secreted Antigenic Target" encoded by a T7SS of mycobacteria. In preferred embodiments, ESAT-6 refers to the protein encoded by the ESX-1 T7SS from mycobacterium.

[0074] ESAT-6 is a secreted effector protein, encoded by the gene esxA which is located in the ESX-1 locus of Mtb. The amino acid sequence of Mtb ESAT-6 is provided herein as SEQ ID NO: 5. An exemplary nucleic acid sequence encoding Mtb ESAT-6 is provided in SEQ ID NO: 1. [0075] In accordance with the present invention, it will be understood that the ESAT-6 protein to which the polypeptide in the fusion protein is identical or at least 80% homologous thereto, may be an ESAT-6 protein from any species of Mycobacterium, optionally from any species selected from the group consisting of: M. tuberculosis ( Mtb ), M. marinum, virulent M. bovis, M. kansaii, M. africanum , and M. szulgai. In certain preferred embodiments, the ESAT-6 protein is an ESAT-6 protein from M. tuberculosis (Mtb). In further embodiments, the ESAT-6 protein is from M. marinum. Preferably, the polypeptide is at least about 80% identical to the amino acid sequence of an ESAT-6 protein from Mtb.

[0076] Moreover, as indicated above, ESAT-6 protein may correspond to the ESAT-6 protein from the ESX-1 secretion system. However, the skilled person will appreciate that the term ESAT-6 may refer to any 6-kDa Early Secreted Antigenic Target homolog, and may accordingly correspond to or have sequence homology to any one or more of the proteins encoded by a gene from a related secretion systems: esxC, esxH, esxT and esxN.

[0077] Further still, the invention provides a nucleic acid encoding a fusion protein, wherein the fusion protein comprises a polypeptide that is at least about 80% identical to an ESAT-6 protein and a secretion peptide for enabling secretion of the polypeptide via the ESX-5 secretion system.

[0078] In preferred embodiments, the architecture of the fusion protein is such that the secretion peptide is located C-terminal to the polypeptide that is homologous to ESAT-6. The polypeptide and the peptide may be contiguous, or separated by a linker sequence.

[0079] Preferably, the heterologous nucleic acid encodes a polypeptide that is at least 80% identical to an ESAT-6 from a strain of M. tuberculosis (Mtb), or a functional homolog or derivative thereof. The ESAT-6 may be the ESAT-6 encoded by the ESX-1 secretion system of Mtb, or a homolog of ESAT-6 encoded by an alternative secretion system of Mtb (such as esxC, esxH, esxT and esxN).

[0080] Alternatively, the nucleic acid encodes a polypeptide that is at least 80% identical to an ESAT-6 from another species of mycobacterium, such as M. marinum. [0081] The heterologous nucleic acid may encode a polypeptide that is an amino acid sequence variant of ESAT-6. Such variants can be substitutional, insertional, or deletion variants. A variation in a polypeptide of the invention may affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more non contiguous or contiguous amino acids of the polypeptide, as compared to wild-type. A variant can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substitute amino acids.

[0082] The heterologous nucleic acid may comprise or consist of a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 1 , 3 or 4.

[0083] The heterologous nucleic acid may encode a polypeptide that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 5.

[0084] The heterologous nucleic acid may encode a polypeptide that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 7.

[0085] It will also be understood that amino acid and nucleic acid sequences of the invention may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity (e.g., immunogenicity) where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region. [0086] As used herein, "recombinant" may refer to a biomolecule, e.g., a gene or protein, or to an organism. The term "recombinant" may be used in reference to cloned DNA isolates, chemically synthesized polynucleotides, or polynucleotides that are biologically synthesized by heterologous systems, as well as proteins or polypeptides and/or R As encoded by such nucleic acids. A "recombinant" nucleic acid may be a nucleic acid linked to a nucleotide or polynucleotide to which it is not linked in nature. A "recombinant" protein or polypeptide may be (1) a protein or polypetide linked to an amino acid or polypeptide to which it is not linked in nature; and/or (2) a protein or polypeptide made by transcription and/or translation of a recombinant nucleic acid. Thus, a protein synthesized by a microorganism is recombinant, for example, if it is synthesized from an mRNA synthesized from a recombinant nucleic acid present in the cell. A "recombinant" organism is an organism comprising a "recombinant" biomolecule. For example, a "recombinant" strain of M. bovis BCG is a strain of M. bovis BCG that comprises a "recombinant" nucleic acid.

[0087] The term "polynucleotide", "nucleic acid molecule", "nucleic acid", or "nucleic acid sequence" refers to a polymeric form of nucleotides of at least 10 bases in length. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native internucleoside bonds, or both. The nucleic acid can be in any topological conformation. For instance, the nucleic acid can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or in a padlocked conformation. The nucleic acid (also referred to as polynucleotides) may include both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. They may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphorami dates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.) Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as the modifications found in "locked" nucleic acids.

[0088] The nucleic acids of the invention may also be termed “synthetic” “recombinant” or “isolated.” A "synthetic" RNA, DNA or a mixed polymer is one created outside of a cell, for example one synthesized chemically.

[0089] As used herein, the term "isolated" refers to a substance or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In some embodiments, isolated agents are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components.

[0090] The "isolated" products of this invention, including isolated nucleic acids, proteins, polypeptides, and antibodies are not products of nature (i.e., "non-naturally occurring"). Rather, the "isolated" nucleic acids, proteins, polypeptides, and antibodies of this invention are "man-made" products. The "isolated" products of this invention can be "markedly different" or "significantly different" from products of nature. By way of non-limiting example, the isolated nucleic acids may be purified, recombinant, synthetic, labeled, and/or attached to a solid substrate. Such nucleic acids can be markedly different or significantly different than nucleic acids that occur in nature. By way of further non-limiting example, the "isolated" proteins, polypeptides, and antibodies of this invention may be purified, recombinant, synthetic, labeled, and/or attached to a solid substrate. Such proteins, polypeptides, and antibodies can be markedly different or significantly different from proteins, polypeptides, and antibodies that occur in nature. [0091] As used herein, an endogenous nucleic acid sequence in the genome of an organism (or the encoded protein product of that sequence) is deemed "recombinant" herein if a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence. In this context, a heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, whether or not the heterologous sequence is itself endogenous (originating from the same host cell or progeny thereof) or exogenous (originating from a different host cell or progeny thereof). By way of example, a promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of a host cell, such that this gene has an altered expression pattern. This gene would now become "recombinant" because it is separated from at least some of the sequences that naturally flank it.

[0092] A nucleic acid is also considered "recombinant" if it contains any modifications that do not naturally occur to the corresponding nucleic acid in a genome. For instance, an endogenous coding sequence is considered "recombinant" if it contains an insertion, deletion or a point mutation introduced artificially, e.g., by human intervention. A "recombinant nucleic acid" also includes a nucleic acid integrated into a host cell chromosome at a heterologous site and a nucleic acid construct present as an episome.

[0093] As used herein, the phrase "degenerate variant" of a reference nucleic acid sequence encompasses nucleic acid sequences that can be translated, according to the standard genetic code, to provide an amino acid sequence identical to that translated from the reference nucleic acid sequence. The term "degenerate oligonucleotide" or "degenerate primer" is used to signify an oligonucleotide capable of hybridizing with target nucleic acid sequences that are not necessarily identical in sequence but that are homologous to one another within one or more particular segments.

[0094] The term "percent sequence identity" or "identical" in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32, and even more typically at least about 36 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Ws. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63- 98 (1990). For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOP AM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, herein incorporated by reference. Alternatively, sequences can be compared using the computer program, BLAST (Altschul et al., J. Mol. Biol. 215:403- 410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al, Meth. Enzymol. 266: 131 -141 (1996); Altschul et al, Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al, Nucleic Acids Res. 25:3389-3402 (1997)).

[0095] In preferred embodiments, the nucleic acid constructs of the invention comprise a sequence, such as a promoter sequence, for enabling expression of the nucleic acid in an organism, preferably, for enabling expression in M. bovis BCG. Such promoter sequences may also be termed “expression control sequences”.

[0096] As used herein, an "expression control sequence" refers to polynucleotide sequences which affect the expression of coding sequences to which they are operatively linked. Expression control sequences are sequences which control the transcription, post-transcriptional events and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence. The term "control sequences" is intended to encompass, at a minimum, any component whose presence is essential for expression, and can also encompass an additional component whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0097] As used herein, "operatively linked" or "operably linked" expression control sequences refers to a linkage in which the expression control sequence is contiguous with the gene of interest to control the gene of interest, as well as expression control sequences that act in trans or at a distance to control the gene of interest.

[0098] As used herein, the term "peptide" refers to a short polypeptide that contains at least 2 amino acids and typically contains less than about 50 amino acids and more typically less than about 30 amino acids. In some embodiments a peptide consists of from 2 to 50, from 2 to 20, from 2 to 10, from 5 to 10, from 5 to 15, from 5 tro 20, from 10 to 20, from 10 to 30, from 10 to 40, from 10 to 50, from 20 to 40, or from 20 to 50 amino acids. The term as used herein encompasses analogs and mimetics that mimic structural and thus biological function.

[0099] The term "polypeptide" encompasses both naturally-occurring and non- naturally occurring proteins, and fragments, mutants, derivatives and analogs thereof. A polypeptide may be monomeric or polymeric. Further, a polypeptide may comprise a number of different domains each of which has one or more distinct activities. For the avoidance of doubt, a "polypeptide" may be any length greater two amino acids. Accordingly, a "polypeptide" may be a protein or a peptide.

[0100] The term "protein" refers to a polypeptide that comprises at least 50 amino acids. A "protein" may have the amino acid sequence of a naturally occuring protein or may be a modified derivative or mutein thereof.

[0101] The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) exists in a purity not found in nature, where purity can be adjudged with respect to the presence of other cellular material (e.g., is free of other proteins from the same species) (3) is expressed by a cell from a different species, or (4) does not occur in nature (e.g., it is a fragment of a polypeptide found in nature or it includes amino acid analogs or derivatives not found in nature or linkages other than standard peptide bonds). Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be "isolated" from its naturally associated components. A polypeptide or protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art. As thus defined, "isolated" does not necessarily require that the protein, polypeptide, peptide or oligopeptide so described has been physically removed from a cell in which it was synthesized.

[0102] The protein or polypeptide can be purified. Preferably, the purified protein or polypeptide is more than 50%, 75%, 85%, 90%, 95%, 97%, 98%, or 99% pure. Within the context of this invention, a purified protein that is more than 50% (etc.) pure means a purified protein sample containing less than 50% (etc.) other proteins. For example, a sample of a protein comprising can be 99% pure if it contains less than 1% contaminating host cell proteins.

[0103] The term "polypeptide fragment" as used herein refers to a polypeptide that has a deletion, e.g., an amino -terminal and/or carboxy-terminal deletion compared to a full-length polypeptide, such as a naturally occurring protein. In an embodiment, the polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to the corresponding positions in the naturally-occurring sequence. Fragments typically are at least 5, 6, 7, 8, 9 or 10 amino acids long, or at least 12, 14, 16 or 18 amino acids long, or at least 20 amino acids long, or at least 25, 30, 35, 40 or 45, amino acids, or at least 50 or 60 amino acids long, or at least 70 amino acids long, or at least 100 amino acids long.

[0104] The term "fusion protein" refers to a polypeptide comprising a polypeptide or fragment coupled to heterologous amino acid sequences. Fusion proteins are useful because they can be constructed to contain two or more desired functional elements that can be from two or more different proteins. A fusion protein comprises at least 10 contiguous amino acids from a polypeptide of interest, or at least 20 or 30 amino acids, or at least 40, 50 or 60 amino acids, or at least 75, 100 or 125 amino acids. The heterologous polypeptide included within the fusion protein is usually at least 6 amino acids in length, or at least 8 amino acids in length, or at least 15, 20, or 25 amino acids in length. Fusions that include larger polypeptides, such as an IgG Fc region, and even entire proteins, such as the green fluorescent protein ("GFP") chromophore-containing proteins, have particular utility. Fusion proteins can be produced recombinantly by constructing a nucleic acid sequence which encodes the polypeptide or a fragment thereof in frame with a nucleic acid sequence encoding a different protein or peptide and then expressing the fusion protein. Alternatively, a fusion protein can be produced chemically by crosslinking the polypeptide or a fragment thereof to another protein. [0105] As used herein, a protein has "homology" or is "homologous" to a second protein if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein. Alternatively, a protein has homology to a second protein if the two proteins have similar amino acid sequences (Thus, the term "homologous proteins" is defined to mean that the two proteins have similar amino acid sequences). As used herein, homology between two regions of amino acid sequence (especially with respect to predicted structural similarities) is interpreted as implying similarity in function.

[0106] When "homologous" or “similar” is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson, 1994, Methods Mol. Biol. 24:307-31 and 25:365-89.

[0107] The following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine, Threonine; 2) Aspartic Acid, Glutamic Acid; 3) Asparagine, Glutamine; 4) Arginine, Lysine; 5) Isoleucine, Leucine, Methionine, Alanine, Valine, and 6) Phenylalanine, Tyrosine, Tryptophan.

[0108] Sequence homology or similarity for polypeptides, which is also referred to as percent sequence identity, is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using a measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as "Gap" and "Bestfit" which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1.

[0109] An exemplary algorithm when comparing a particular polypeptide sequence to a database containing a large number of sequences from different organisms is the computer program BLAST (Altschul et al, J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al, Meth. Enzymol. 266:131-141 (1996); Altschul et al, Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al, Nucleic Acids Res. 25:3389-3402 (1997)).

[0110] Exemplary parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 1 1 (default); Cost to extend a gap: 1 (default); Max. alignments: 100 (default); Word size: 1 1 (default); No. of descriptions: 100 (default); Penalty Matrix: BLOWSUM62. The length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, or at least about 20 residues, or at least about 24 residues, or at least about 28 residues, or more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it may be useful to compare amino acid sequences. Database searching using amino acid sequences can be measured by algorithms other than blastp known in the art. For instance, polypeptide sequences can be compared using FASTA, a program in GCG Version 6.1. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990). For example, percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (a word size of 2 and the PAM250 scoring matrix), as provided in GCG Version 6.1, herein incorporated by reference.

[0111] As used herein, "polypeptide mutant" or "mutein" refers to a polypeptide whose sequence contains an insertion, duplication, deletion, rearrangement or substitution of one or more amino acids compared to the amino acid sequence of a reference protein or polypeptide, such as a native or wild-type protein. A mutein may have one or more amino acid point substitutions, in which a single amino acid at a position has been changed to another amino acid, one or more insertions and/or deletions, in which one or more amino acids are inserted or deleted, respectively, in the sequence of the reference protein, and/or truncations of the amino acid sequence at either or both the amino or carboxy termini. A mutein may have the same or a different biological activity compared to the reference protein.

[0112] In some embodiments, a mutein has, for example, at least 85% overall sequence homology or similarity to its counterpart reference protein. In some embodiments, a mutein has at least 90% overall sequence homology or similarity to the wild-type protein. In other embodiments, a mutein exhibits at least 95% sequence identity, or 98%, or 99%, or 99.5% or 99.9% overall sequence identity.

PE/PPE secretion signal

[0113] The peptide for enabling secretion of the ESAT-6 polypeptide (or functional derivative of or homolog thereof), may be any peptide that is recognised by the ESX-5 secretion system of BCG, and which facilitates secretion of the ESAT-6 from BCG cells. Such peptides may generally be referred to as PE/PPE secretion signals.

[0114] The peptide may be any peptide that comprises or consists of the consensus sequence YXXXD/E, wherein Y is a tyrosine residue, X is any amino acid residue and D/E refers to aspartic acid or glutamic acid.

[0115] In certain embodiments, the peptide comprises the consensus sequence YXXXD/E and additional amino acid sequences. In one example, the peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6, or a peptide that is at least 80% identical thereto, provided that the peptide retains the YXXXD/E consensus sequence.

[0116] The peptide may be of any amino acid length, but preferably a length of at least about 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 11 amino acids, at least 12 amino acids, at least 13 amino acids, at least 14 amino acids, at least 15 amino acids, at least 16 amino acids, at least 17 amino acids or more amino acids, and which comprises the consensus sequence YXXXD/E, wherein Y is a tyrosine residue, X is any amino acid residue and D/E refers to aspartic acid or glutamic acid. Preferably, the peptide for enabling secretion of the ESAT-6 polypeptide (or functional derivative of or homolog thereof), is no more than about 20, no more than about 21, no more than about 22, no more than about 23, no more than about 24, no more than about 25, no more than about 26, no more than about 27, no more than about 28, no more than about 29, no more than about 30, amino acids in length, or more, and which comprises the consensus sequence YXXXD/E, wherein Y is a tyrosine residue, X is any amino acid residue and D/E refers to aspartic acid or glutamic acid.

[0117] Further examples of suitable peptides which enable secretion of the ESAT-6 polypeptide via the ESX-5 secretion system are set forth in any one of SEQ ID NOs: 8 to 13.

Methods for making recombinant strains of M. bovis BCG

[0118] The invention also encompasses methods of making a recombinant strain of M. bovis BCG of the invention. In some embodiments the methods comprise providing a vector comprising a nucleic acid sequence as described herein, introducing the vector into M. bovis BCG cells, and selecting M. bovis BCG cells that stably maintain the heterologous nucleic acid sequence. The nucleic acid sequence may be any heterologous nucleic acid sequence described herein or any nucleic acid sequence encoding a fusion protein as described herein.

[0119] In some embodiments the vector is an integrating vector and the method further comprises selecting M. bovis BCG cells in which the heterologous nucleic acid sequence comprising the plurality of open reading frames has integrated into the host cell chromosome.

[0120] Those of skill in the art will recognize that several suitable BCG strains exist which are suitable for use in the practice of the invention. M. bovis BCG cells used to make a recombinant strain of M. bovis BCG of the invention are preferably from a commercially available BCG strain which has been approved for use in humans such as Pasteur, Frappier, Connaught (Toronto), Tice (Chicago), RIVM, Danish 1331, Glaxo- 1077, Tokyo-172 (Japan), Evans, Prague, Russia, China, Sweden, Birkhaugh, Moreau and Phipps.

[0121] Other suitable strains include but are not limited to those designated ATCC® Number: 27289 Mycobacterium bovis BCG, Chicago; 27291 M. bovis; 35731 M. bovis TMC 1002 [BCG Birkhaug]; 35732 M. bovis TMC 1009 [BCG Swedish]; 35735 M. bovis TMC 1012 [BCG Montreal; CIP 105920]; 35736 M. bovis TMC 1013 [BCG Brazilian]; 35737 M. bovis TMC 1019 [BCG Japanese]; 35738 M. bovis TMC 1020 [BCG Mexican]; 35739 M. bovis TMC 1021 [BCG Australian]; 35741 M. bovis [BCG Glaxo]; 35742 M. bovis TMC 1025 [BCG Prague]; 35744 TMC 1029 [BCG Phipps]; 35745 M. bovis TMC 1030 [BCG Connaught]; 35746 M. bovis TMC 1101 [BCG Montreal, SM-R]; 35747 M. bovis TMC 1103 [BCG Montreal, INH-R; CIP 105919]; 35748 M. bovis TMC 1108 [BCG Pasteur SM-R]; 27290 M. bovis [BCG Copenhagen H]; 19274 M. bovis deposited as tuberculosis subspecies bovis 50 [BCG]; 19015 Mycobacterium sp. M. bovis Karlson and Lessel BCG; 35733 M. bovis TMC 1010 [BCG Danish, SSI 1331] and 35734 M. bovis TMC 1011 [BCG Pasteur], etc. In any embodiment, the BCG strain may be an auxotrophic strain.

[0122] In any embodiment of the invention, the BCG strain may be further modified to introduce various selection markers, including antibiotic resistance or antibiotic sensitivity. In certain examples, the BCG strain may be kanamycin-resistant or kanamycin-sensitive. Preferably, the BCG strain of the invention is kanamycin-sensitive.

[0123] Methods for determining antibiotic sensitivity or resistance are known in the art and may include simple determination of the absence or presence a gene encoding a resistance-conferring protein, by PCR. Other approaches can include growing the strains on selective media to determine resistance or sensitivity. Methods for removing antibiotic-resistance markers from existing strains are also known in the art and are described for example in Gopinath et al. , (2015) in Parish and Roberts (eds), Mycobacteria Protocols, Methods in Molecular Biology, vol 1285, pp131-149, incorporated herein by reference.

[0124] In some embodiments the heterologous nucleic acid sequence is present on a plasmid or vector. In some embodiments the heterologous nucleic acid sequence is integrated into the M. bovis BCG chromosome. In some embodiments the recombinant M. bovis BCG strain comprises a single copy of the heterologous nucleic acid sequence integrated on its chromosome. In some embodiments the recombinant M. bovis BCG strain comprises multiple copies of the heterologous nucleic acid sequence integrated on its chromosome. [0125] It will be appreciated that the nucleic acid of the invention described herein, may be integrated at any site in the genome of M. bovis BCG. In a non-limiting example, the nucleic acid of the invention (encoding the fusion protein comprising a) a polypeptide that is at least about 80% identical to the amino acid sequence of an ESAT- 6 protein, or a functional variant or homolog thereof; and b) a secretion peptide for enabling secretion of the polypeptide via an ESX-5 secretion system of M. bovis BCG) is integrated into the attB site of the M. bovis BCG chromosome.

[0126] The recombinant M. bovis BCG strains of the invention may be made by any suitable method known in the art. In some embodiments an integrating shuttle vector is electroporated into a strain of M. bovis BCG and recombinant M. bovis BCG cells comprising the heterologous nucleic acid sequence integrated into the host cell chromosome are identified. In some embodiments the integrating shuttle vector is a cosmid. In some embodiments the integrating shuttle vector is pMV306hsp or PYUB412.

[0127] Methods for determining successful introduction of the heterologous nucleic acid sequence, or integration thereof into the genome of M. bovis BCG will be familiar to the skilled person. For examples, standard sequence techniques can be used to determine insertion of the nucleic acid sequence. Furthermore, protein detection methods, such as western blot and ELISA techniques can be used to determine expression of the fusion protein in the host strain.

[0128] As used herein, a "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which generally refers to a circular double stranded DNA loop into which additional DNA segments may be ligated, but also includes linear double- stranded molecules such as those resulting from amplification by the polymerase chain reaction (PCR) or from treatment of a circular plasmid with a restriction enzyme. Other vectors include cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC). Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome (discussed in more detail below). Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). The integrating cosmid vector pYUB412 is an example of a "vector".

[0129] The term "recombinant host cell" (or simply "recombinant cell" or "host cell"), as used herein, is intended to refer to a cell into which a recombinant nucleic acid such as a recombinant vector has been introduced. In some instances the word "cell" is replaced by a name specifying a type of cell. For example, a "recombinant microorganism" is a recombinant host cell that is a microorganism host cell. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "recombinant host cell," "recombinant cell," and "host cell", as used herein. A recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism.

Compositions

[0130] The invention provides vaccines and immune stimulating compositions comprising a recombinant strain of M. bovis BCG according to the present invention and optionally, a pharmaceutically-acceptable carrier. Such compositions may be, for example, for use for inducing a protective immune response against M. tuberculosis in a subject and/or for use for treating an tuberculosis infection in a subject.

[0131] In addition to the strain of recombinant BCG, these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the living vaccine into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). In some embodiments the pharmaceutical composition is suitable for oral, subcutaneous, intradermal, intramuscular, intravenous, nasal, mucosal and/or intravesicular administration. [0132] The vaccines and immune stimulating compositions of the invention may also comprise or be administered in combination with an adjuvant for potentiating an immune response to the recombinant BCG strain. There are many examples of adjuvants known in the art (see Allison (1998, Dev. Biol. Stand., 92:3-11; incorporated herein by reference), Unkeless et al. (1998, Annu. Rev. Immunol., 6:251-281), and Phillips et al. (1992, Vaccine, 10:151-158). Exemplary adjuvants that can be utilized in accordance with the invention include, but are not limited to, cytokines, aluminium salts (e.g., aluminium hydroxide, aluminium phosphate, etc.; Baylor et al., Vaccine, 20:S18, 2002), gel-type adjuvants (e.g., calcium phosphate, etc.); microbial adjuvants (e.g., immunomodulatory DNA sequences that include CpG motifs; endotoxins such as monophosphoryl lipid A (Ribi et al., 1986, Immunology and Immunopharmacology of bacterial endotoxins, Plenum Publ. Corp., NY, p407, 1986); exotoxins such as cholera toxin, E. coli heat labile toxin, and pertussis toxin; muramyl dipeptide, etc.); oil-emulsion and emulsifier-based adjuvants (e.g., Freund's Adjuvant, MF59 [Novartis], SAF, etc.); particulate adjuvants (e.g., liposomes, biodegradable microspheres, etc.); synthetic adjuvants (e.g., nonionic block copolymers, muramyl peptide analogues, polyphosphazene, synthetic polynucleotides, etc.); and/or combinations thereof. Other exemplary adjuvants include some polymers (e.g., polyphosphazenes; described in U.S. Patent 5,500,161), Q57, saponins (e.g., QS21, Ghochikyan et al., Vaccine, 24:2275, 2006), squalene, tetrachlorodecaoxide, CPG 7909 (Cooper et al., Vaccine, 22:3136, 2004), poly[di(carboxylatophenoxy)phosphazene] (PCCP; Payne et al., Vaccine, 16:92, 1998), interferon-g (Cao et al. , Vaccine, 10:238, 1992), block copolymer P1205 (CRL1005; Katz et al., Vaccine,. 18:2177, 2000), interleukin-2 (IL-2; Mbwuike et al., Vaccine, 8:347, 1990), polymethyl methacrylate (PMMA; Kreuter et al., J. Pharm. ScL, 70:367, 1981), dimethyloctadecylammonium bromide (DDA), IC31 D (Vann Dissel, Vaccine, 29:2100, 2011), etc.

[0133] These compositions may also include diluents, excipients and carriers enabling administration of the composition, as known in the art.

[0134] The determination of an effective dose for inducing an immune response or for treatment of a condition or infection as described herein, is well within the capability of those skilled in the art. An effective dose refers to that amount of active ingredient, i.e the number of cells administered, which induces an immune response against M. tuberculosis and/or ameliorates the symptoms of M. tuberculosis infection. Efficacy and toxicity may be determined by standard pharmaceutical procedures in experimental animals, e.g., ED50 (the dose effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. Of course, ED50 is to be modulated according to the mammal to be treated or vaccinated. In this regard, the invention contemplates a composition suitable for human administration as well as a veterinary composition.

[0135] The invention also encompasses a vaccine comprising a recombinant strain of M. bovis BCG according to this disclosure and a suitable carrier. This vaccine is especially useful for preventing tuberculosis.

Methods for inducing an immune response

[0136] The invention also encompasses methods or uses of the strains and compositions of the invention.

[0137] In preferred examples, the methods and uses are for inducing an immune response to a Mycobacterium species, preferably to M. tuberculosis in a subject. Accordingly, the invention provides methods for: (i) prophylaxis; (ii) treatment; and (iii) boosting immunity to an infection with a Mycobacterium species. It is in these contexts that the methods of the invention 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.

[0138] It will be understood that the immune response induced may be specifically against the ESAT-6 protein or functional variant or homolog thereof encoded by the heterologous nucleic acid sequence comprised in the BCG strain of the invention. In one embodiment, 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.

[0139] Accordingly in one embodiment there is provided a method for minimising the likelihood of development of a Mycobacterium infection, preferably a Mycobacterium tuberculosis infection, in a subject including: - forming an immune response in a subject in need thereof to an ESAT-6 protein, or functional variant or homolog thereof,

- by administering to the subject a composition or strain as described herein, thereby minimising the likelihood of a Mycobacterium infection from developing in the individual.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] Typically the immune response is predominantly a Th1 response. This response is determined by detecting cellular proliferation after administration of the vaccine as measured by ³H thymidine incorporation, or using cellular assays in which IFN-g production is assessed, such as flow cytometry and/or ELISA. The immune response can also be measured by detecting specific antibodies (at a titer in the range of 1 to 1 x 10⁶, preferably 1 x 10³, more preferable in the range of about 1 x 10³ to about 1 x 10⁶, and most preferably greater than 1 x 10⁶).

[0144] An in vitro cellular response is determined by release of a relevant cytokine such as IFN-gamma, from lymphocytes withdrawn from an animal or human currently or previously infected with virulent mycobacteria, or by detection of proliferation of these T cells. The induction is performed by addition of the polypeptide or the immunogenic portion to a suspension comprising from 1x10^s cells to 3x10^s cells per well. The cells are isolated from either blood, the spleen, the liver or the lung and the addition of the polypeptide or the immunogenic portion of the polypeptide result in a concentration of not more than 20 ug per ml suspension and the stimulation is performed from two to five days. For monitoring cell proliferation the cells are pulsed with radioactive labelled thymidine and after 16-22 hours of incubation the proliferation is detected by liquid scintillation counting. A positive response is a response more than background plus two standard deviations. The release of IFN-gamma can be determined by the ELISA method, which is well known to a person skilled in the art. A positive response is a response more than background plus two standard deviations. Other cytokines than IFN-gamma could be relevant when monitoring an immunological response to the polypeptide, such as IL-12, TNF-alpha, IL-4, IL-5, IL-10, IL-6, TGF-beta. Another and more sensitive method for determining the presence of a cytokine (e.g. IFN-gamma) is the ELISPOT method where the cells isolated from either the blood, the spleen, the liver or the lung are diluted to a concentration of preferable of 1 to 4 x 10⁶ cells/ml and incubated for 18-22 hrs in the presence of the polypeptide or the immunogenic portion of the polypeptide resulting in a concentration of not more than 20 ug per ml. The cell suspensions are hereafter diluted to 1 to 2 x 10⁶/ml and transferred to Maxisorp plates coated with anti-IFN-gamma and incubated for preferably 4 to 16 hours. The IFN- gamma producing cells are determined by the use of labelled secondary anti-IFN- antibody and a relevant substrate giving rise to spots, which can be enumerated using a dissection microscope. It is also a possibility to determine the presence of mRNA coding for the relevant cytokine by the use of the PCR technique. Usually one or more cytokines will be measured utilizing for example the PCR, ELISPOT or ELISA. It will be appreciated by a person skilled in the art that a significant increase or decrease in the amount of any of these cytokines induced by a specific polypeptide can be used in evaluation of the immunological activity of the polypeptide.

[0145] The present invention also includes the implementation of serological assays to evaluate whether and to what extent an immune response is induced or evoked by compositions of the invention. There are many types of immunoassays that can be implemented. Immunoassays encompassed by the present invention 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.

[0146] In some embodiments of the invention, the compositions of the invention confer protective immunity to a subject. 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.

[0147] The immune response may be formed by providing a BCG strain of the invention to a subject in conditions for enabling formation of an immune response to said strain in said individual. Generally the methods comprise administering an effective dose of a composition of the invention to a subject and inducing an immune response in the subject that is protective against M. tuberculosis. In some embodiments, the methods further comprise administering at least one isolated recombinant protein or peptide antigen of a mycobacterium to the subject. In some embodiments, the methods further comprise administering at least one mycobacterium subunit vaccine to the subject. In some embodiments the methods further comprise administering at least one vaccine selected from M72-AS01, MTBVAC, MVA85A, rBCG30, AERAS-402, AdAg85A, M72, H1-IC31, H1-CAF01, H4-IC31 (AERAS-404), rBCGdeltaUreC:Hly (VPM1002), RUTI, and M. vaccae to the subject.

[0148] It will be appreciated that similarly to other strains of BCG, the recombinant strains of BCG, and compositions comprising the same, of the present invention, may also be useful for forming non-specific immune responses in a subject. For example, the strains and compositions of the invention may be administered for the purposes of providing a non-specific immune response and therefore protection against a subsequent infection with an infectious agent. In other words, the strains and compositions of the invention may be utilised similarly to related BCG strains, for reducing the likelihood of a subject succumbing to an infectious agent at a later time.

[0149] Similarly, the strains and compositions of the invention may be useful for potentiating an immune response in a subject to an antigen derived from an infectious agent. In such circumstances, the recombinant BCG strain of the invention may be administered prior to, at the same time, or subsequent to administration of the antigen. [0150] The infectious agent may be any viral, bacterial or parasitic agent. Examples of viruses for which protection may be provided include but are not limited to HIV, herpes viruses, hepatitis viruses, influenza virus, coronaviruses (such as SARS and SARS-CoV-2). Examples of bacteria for which protection from infection may be provided include, but are not limited to Salmonella, Escherichia, Enterobaceter, Corneybacerium diptheriae, Clostridium, Bacillus, Streptomyces, sp. Examples of infectious parasites include but are not limited to Plasmodium, Trypanosome, trematode worm, tick) or toxin therefrom.

[0151] In methods comprising administering a composition of this disclosure and a second agent to the subject, the composition of this disclosure and the second agent may be administered simultaneously or at separate times. In embodiments in which the pharmaceutical composition of this disclosure and the second agent are administered at separate times the order of administration may be (1) that the pharmaceutical composition of this disclosure is administered first and the second agent is administered at a later point in time; or (2) that the second agent is administered first and the pharmaceutical composition of this disclosure is administered at a later point in time. In some embodiments the second agent is administered second and is used to boost an immune response of the subject that was raised against the recombinant strains of M. bovis BCG present in the pharmaceutical composition of this disclosure. In some embodiments one or both of the pharmaceutical composition of this disclosure and the second agent is administered at multiple timepoints.

[0152] The recombinant strains, immune stimulating compositions and vaccines of the invention may be administered orally, subcutaneously, intranasally, intradermally, intravenously, by inhalation (including mucosal delivery into the respiratory tract) and/or intravesicularly.

[0153] Prior to administration to humans as a vaccine, the BCG strains 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 preparations to elicit an immune response is likewise typically tested in suitable animal models, e.g. mice, guinea pigs, etc. In addition, protection studies involving vaccination, boosting, and subsequent challenge with live 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 preparations. For details, see, for example, United States patent application 20060121054 (Sun et al.) published June 8, 2006, and references cited therein.

Methods of treatment

[0154] The invention also encompasses methods and uses of the strains and compositions of the invention, for treating an M. tuberculosis infection in a subject.

[0155] In some embodiments the subject has an active tuberculosis infection. In some embodiments the subject has a latent tuberculosis infection.

[0156] The subject may be a neonate, infant, paediatric, adolescent, adult or geriatric subject, preferably a human subject.

[0157] Generally the methods comprise administering an effective dose of a pharmaceutical composition of this disclosure to a subject infected with M. tuberculosis and inducing an immune response in the subject that ameliorates one or more signs or symptoms of the M. tuberculosis infection. In some embodiments, the M. tuberculosis infection is cured and the subject becomes free of M. tuberculosis. In some embodiments, the methods further comprise administering at least one isolated recombinant protein or peptide antigen of a mycobacterium to the subject. In some embodiments, said isolated recombinant protein or peptide antigen is selected from CFP-10 protein, ESAT-6 protein, and peptides thereof. In some embodiments, the methods further comprise administering at least one mycobacterium subunit vaccine to the subject.

[0158] In some embodiments, the methods further comprise administering at least one vaccine selected from M72-AS01, MTBVAC, MVA85A, rBCG30, AERAS-402, AdAg85A, M72, H1-IC31, H1-CAF01, H4-IC31 (AERAS-404), rBCGdeltaUreC:Hly (VPM1002), RUTI, and M. vaccae to the subject.

[0159] In methods comprising administering the pharmaceutical composition of this disclosure and a second agent to the subject, the pharmaceutical composition of this disclosure and the second agent may be administered simultaneously or at separate times. In embodiments in which the pharmaceutical composition of this disclosure and the second agent are administered at separate times the order of administration may be (1) that the pharmaceutical composition of this disclosure is administered first and the second agent is administered at a later point in time; or (2) that the second agent is administered first and the pharmaceutical composition of this disclosure is administered at a later point in time. In some embodiments the second agent is administered second and is used to boost an immune response of the subject that was raised against the recombinant strains of M. bovis BCG present in the pharmaceutical composition of this disclosure. In some embodiments one or both of the pharmaceutical composition of this disclosure and the second agent is administered at multiple timepoints.

[0160] It will be appreciated that the pharmaceutical composition of the invention may also find utility in treating one or more conditions caused by a Mycobacterium.

[0161] Further still, it will be appreciated that the compositions of the invention may be useful for treating other pathologies. In one embodiment, the compositions of the invention may be useful for treating bladder cancer, as has been postulated for related BCG strains.

[0162] Further, the compositions of the invention may be useful for treating 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.

[0163] In any embodiment the strains and compositions described herein may be administered by an oral, intramuscular, subcutaneous, intradermal, intravenous, mucosal, nasal, and/or intravesicular mode.

[0164] In any method or use described herein, the subject may be any mammal. As used herein, the term "mammal" refers to any member of the taxonomic class mammalia, including placental mammals and marsupial mammals. Thus, "mammal" includes humans, primates, livestock, and laboratory mammals. 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. In some embodiments, the mammal is at least one of a transgenic mammal, a genetically-engineered mammal, and a cloned mammal. Preferably, the mammal is a human.

Kits

[0165] The invention further encompasses a kit for use for inducing a protective immune response against M. tuberculosis in a subject and/or for treating an M. tuberculosis infection in a subject. Said kit comprises a recombinant strain of M. bovis BCG according to this disclosure and a container.

[0166] The kit may also comprise written instructions for use of the kit in accordance with any method of treatment or for inducing an immune response, as described herein.

[0167] In some embodiments, said kit further comprises at least one isolated recombinant protein or peptide antigen of a mycobacterium. In some embodiments, said mycobacterium is a strain selected from M. bovis BCG, M. marinum, and M. tuberculosis. In some embodiments, said kit further comprises at least one subunit vaccine. In some embodiments, said kit further comprises at least one vaccine selected from M72-AS01, MTBVAC, MVA85A, rBCG30, AERAS-402, AdAg85A, M72, H1-IC31, H1-CAF01, H4-IC31 (AERAS-404), rBCGdeltaUreC:Hly (VPM1002), RUTI, and M. vaccae.

Examples

Example 1: Materials and Methods

Bacterial cultures

[0168] BCG strains SSI (Danish), Pasteur, BCG::pYUB (BCG carrying an empty vector), BCG::ESX1^Mtb (referred to as BCG::RD1), BCG::ESAT6-PE25SS and Mtb H37Rv were cultured in Middlebrook 7H9 (BD Difco) broth supplemented with ADC (BD BBL), 0.05% Tween80 (Sigma), 0.4% glycerol (Sigma) and appropriate antibiotics at 37°C with continuous shaking. Bacteria were cultured until log phase at an Oϋeoo of 0.8, either used directly or harvested and stored at -80°C for subsequent use.

Design, synthesis and cloning of esxA-PE25SS

[0169] The mycobacterial integrating plasmid pMV306hsp³¹ was a gift from Brian Robertson & Siouxsie Wiles (Addgene plasmid # 26155; http://n2t.net/addgene:26155; RRID:Addgene_26155). Nucleotide sequences of Mtb esxA and pe25 were obtained from the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/). The recombinant esxA-PE25 signal sequence (SS) fusion construct was designed using Clone Manager version 10 (Sci-Ed Software, CO, USA). Briefly, 51 -nucleotide bases encoding the signal sequence of PE25 ESX-5 was fused to the 3’ end of the esxA gene (just before the stop codon) with flanking EcoR\ and Hind\\\ restriction sites (Figure 1A). The synthesised esxA- PE25SS nucleotide sequence (GenScript, USA) was cloned into the pMV306hsp plasmid and putative Escherichia coli DH5a transformants were selected with kanamycin resistance. Plasmid was extracted using Monarch Plasmid Miniprep kit (NEB) and verified by restriction digestion mapping and Sanger sequencing. pMV306hsp::esxA-PE25SS was transformed into BCG SSI to generate kanamycin-resistant recombinant BCG::ESAT6- PE25SS strains, which were screened and genotyped using PCR and genome sequencing using an lllumina MiSeq system (AGRF Australia), as described previously (Gopinath et al., (2015) Methods in Molecular Biology, 1285: 131-149).

[0170] The PCR primers used in this study are as follows:

Validation of insertion by sequencing

[0171] A hybrid sequence assembly was creating consisting of the BCG Danish 1331 WHO reference strain and the vector/insert construct sequence generated by GenScript (esxA- PE25SS cloned into the pMV306hsp vector) lllumina reads were aligned to the hybrid assembly using BWA³⁴ and a sorted BAM file generated using SAMTools (Li et al., (2009) Bioinformatics 25: 2078-2079). Reads pairs aligning to both the reference genome and the vector/insert construct were identified and reads with clipped bases used to identify the exact insertion sites.

Protein isolation

[0172] To study the expression of ESAT-6 in cloned BCG, culture filtrates of BCG::ESAT6-PE25SS, BCG Pasteur and BCG::RD1 (used as ESAT-6 control) were prepared by harvesting early log phase cultures of respective BCG strains in Middlebrook ADC media at 4500 rpm for 20 min. Cells were supplemented with glycine at a concentration of 1.5% one day before harvesting. Cell pellets were washed three times and bacteria were lysed in lysis buffer (1% NaCI, 2% Triton X-100, 2% SDS, 50mM Tris, 1 mM EDTA, 5M Urea, 2M Thiourea) supplemented with a proteinase inhibitor cocktail (Sigma, p8340) at 37°C with continuous shaking for approximately 2 hours. After lysis, BCG strains were sonicated on ice at 90-100% amplitude for 30 cycles (15 sec off/ 15 sec on) with a sonicator (Qsonica) to release proteins. Supernatant collected after centrifugation underwent another round of sonification. The supernatants were pooled and centrifuged one more time to clear any cell debris. The supernatant was filtered through a 0.22 pm filter (Millipore) and concentrated using acetone precipitation followed by a 3 KDa cut-off column (Pall life sciences, OD003C45). The protein concentration in cell filtrates was quantified using a Nanodrop spectrophotometer and cell filtrates were stored at -30°C until further use.

Western blot

[0173] Culture filtrates of respective BCG strains were separated on 12-15% SDS page gels. One gel was stained with coomassie blue to confirm the presence of protein. Another gel was transferred to a nitrocellulose membrane (GE Health, 10600004) and blocked for 1 hour at RT with 2% BSA (Sigma) in 0.05% PBST solution. Following three washes with 0.05% PBST, the membrane was blotted with a primary mouse anti-ESAT- 6 monoclonal antibody (Abeam, Ab26246) for 2.5 hours at RT. Subsequently, the membrane was washed three times and incubated with a secondary goat anti-Mouse- HRP polyclonal antibody (Thermo Fischer Scientific, 32230) for one hour at RT. Antibodies were diluted in PBST (0.05% Tween80) at concentrations of 1:500 and 1:10,000, respectively. After the final wash, the blot was visualized with a detection agent (GE health, RPW22336) and visualized with a VersaDoc MP4000 (Bio-Rad). ESAT-6 ELISA

[0174] To quantify ESAT-6 expression, an in-house developed indirect ELISA protocol was used. Briefly, plates (Maxisorb, type 96F; corning) were coated with culture filtrates (1pg/well, in phosphate buffered saline (PBS), pH 7.4) of BCG::ESAT6- PE25SS, BCG Pasteur and BCG::RD1 (used as ESAT-6 control) for 3 hrs at 37°C. After incubation, plates were washed once with PBS-T (PBS; 0.05% Tween-20, pH 7.4) and free binding sites were blocked by 1% bovine serum albumin-PBST and incubated for 2 hrs. After blocking, plates were washed three times with PBS-T and incubated with 100 pi of monoclonal ESAT-6 antibody (1:3200) (Abeam) for 45min. After incubation, the plates were washed three times with PBS-T and then incubated for another 45 with rabbit anti-mouse IgG conjugated with horseradish peroxidase (HRP, 1:5000 diluted). After five washes with PBS-T, the enzyme activity was assayed by incubation for 15 min at room temperature with 100 mI of mixtures of tetramethylbenzidine (TMB) per well. The reaction was terminated by adding of 50 mI of stop solution (1N sulphuric acid) and the OD was determined at 450nm. Each assay was repeated in triplicates.

Mice

[0175] Sex- and age-matched C57BL/6 mice were purchased from the Animal Resource Centre (ARC; Perth, Australia). Rag2^~/~IL2rg ^~/~ mice were bred and maintained in-house. Prior to vaccination or challenge, all mice were acclimatized for at least one week and kept in biosafety level 2 or 3 facilities, respectively, under specific pathogen- free (SPF) conditions at the Australian Institute of Tropical Health and Medicine (AITHM), James Cook University, Australia. All experiments were conducted in accordance with the National Health and Medical Research Council (NHMRC) animal care guidelines and were approved by the animal ethics committee (A2346) of James Cook University, Australia.

Vaccinations and infections

[0176] Prior to vaccination, frozen vials of bacterial stocks were thawed, washed and diluted in PBS. C57BL/6 mice were vaccinated with the BCG::ESAT6-PE25SS, BCG::pYUB or BCG::RD1 intratracheally (i.t.) or subcutaneously (s.c) with 5x10^s CFU in a volume of 20 mI or 100 mI, respectively. For i.t. vaccination, mice were anesthetized using isoflurane, the tongue was pulled out of the oral cavity and bacterial suspension was administered into the oropharynx with the nostrils covered until the inoculum was inhaled. To determine safety in immunocompromised hosts, Rag2^~/~IL2rg mice were intravenously infected with 1x10⁶ CFU of BCG::pYUB, BCG::RD1 or BCG::ESAT6- PE25SS.

Mtb challenge

[0177] Mtb challenge was performed in a BSL3 laboratory 60 days after BCG vaccination. Frozen Mtb aliquots were thawed, diluted to the appropriate concentration, and treated in an ultrasound water bath to disrupt bacterial clumps. Mice were infected with 20-50 CFU Mtb H37Rv using a Glas-Col inhalation exposure system. The initial infectious dose was determined by plating lung tissues collected from 5 mice one day after aerosol infection on 10% OADC enriched 7H11 agar plates.

Sample collection

[0178] At distinct time-points after vaccination or infection, mice were sacrificed by carbon dioxide asphyxiation. Blood was collected into Z-gel tubes (Sarstedt) and serum was prepared by centrifugation of clotted blood, filtered using 0.2 pm SpinX columns (Sigma) and stored at -20°C. For collection of bronchoalveolar lavage fluid (BALF) a small incision was made below the exposed larynx. Using an 18-gaugue blunted needle, 1 ml_ PBS was flushed into the lungs via the incision and aspirated. This was repeated three times to obtain a total of 3 ml_ BALF per mouse. Subsequently, lungs were perfused with PBS through the left ventricle. Lungs and spleen were aseptically removed. The upper right lobe was used for CFU enumeration, the left lung lobe was used for histopathology, and the remaining section of lung tissue was used for flow cytometry. Spleens were used for CFU enumeration.

CFU enumeration

[0179] Lung and spleen tissues were homogenized in sterile sample bags containing 1ml of sterile PBS/ 0.05% Tween 80. CFU were determined after plating tenfold dilutions of organ homogenates onto Middlebrook 7H11 agar plates supplemented with 0.2% glycerol, 0.05% Tween80, and 10% oleic albumin dextrose catalase (OADC) enrichment (BD) containing 50 pg/ml hygromycin for (BCG::pYUB, BCG::RD1) and kanamycin for BCG::ESAT6-PE25SS. For Mtb culture, thiophene-2-carboxylic acid hydrazide (TCH, 2 pg/ml; Sigma) was added to 7H11 agar plates to restrict the growth of BCG strains. Agar plates were sealed and incubated aerobically for 4-5 weeks at 37°C. Colonies were counted and the total CFU per organ was calculated based on dilution factors and organ size.

Histopathology

[0180] Lung lobes from vaccinated and challenged C57BL/6 mice were harvested and fixed overnight with 4% w/v paraformaldehyde, transferred to 70% ethanol and embedded in paraffin. Processed lungs were sectioned with a microtome into 4 pm slices, transferred to glass slides, dewaxed and stained with H&E. Stained slides were scanned with an Aperio CS2 (Leica) followed by analysis with Image Scope (Leica) and ImageJ software (NIH). To calculate the percentage of lung damage, the total surface area was measured followed by the areas of dense cell infiltration, as described previously (Sathkumara et al., (2019) Frontiers in Immunology, 10: 532).

Cell isolation

[0181] Lung lobes designated for flow cytometry were mechanically disrupted and digested for 30 mins at 37°C with sterile RPMI 1640 medium supplemented with 10% heat inactivated fetal bovine serum (FBS) (Gibco), 100 U/ml penicillin, 100 pg/ml streptomycin (Gibco), 7.5 pg/ml Collagenase D (Sigma), 1.75 pg/ml Collagenase VIII (Sigma) and 200 pg/ml DNasel (Sigma). Single cell suspensions were prepared by passing digested organs through a 70 pm cell strainer (Biologix) followed by red cell lysis. The resulting cell pellet was resuspended in FACS buffer (PBS / 5% FBS and 0.1% NaNs).

Flow Cytometry

[0182] Identification of T cells was performed by using the following antibodies and fluorochromes against CD3 (A700; clone 500 A2), NKp46 (BV711; clone 29A1.4), CD4 (BUV395; clone GK1.5), CD8 (BV510; clone), CD44 (BV421; clone IM7), CD62L (PE- Cy7; clone MEL-14), CD103 (APC; clone M290); CD11a (PerCP-Cy5.5; clone 2D7) and CD69 (PE-CF594; clone H1.2F3); all purchased from BD Biosciences. The l-A(b) Mtb ESAT6 4-17 QQWNFAGIEAAASA tetramer (PE) was provided by the NIH Tetramer Core Facility, USA. Fixable Viability stain 780 (BD Biosciences) was used to exclude dead cells. Cells were incubated with viability stain for 10 minutes at room temperature and washed with FACS buffer solution. Samples were incubated with the tetramer (1:50 dilution) for 1 hour at room temperature, and washed with FACS buffer solution. Cells were then incubated for 30 minutes with antibodies (1:200 dilution) on ice, and washed with FACS buffer solution. Samples were resuspended in 150 pi of blank calibration particles (BD Biosciences) diluted in FACS buffer solution (1:74). Samples were analysed using a FortessaX20 analyser (BD Biosciences). Data was analysed using FlowJo software version 10 (Treestar, CA). Cells were enumerated using calibration particle counts.

Cytokine measurements

[0183] To enumerate cytokine levels, frozen serum samples were thawed and prepared according to the Bio-Plex Pro Mouse Cytokine 23-Plex assay (BioRad) specifications. Data was acquired on a MagPix (Luminex) instrument and analysed with MilliPlex (luminex) software. Visualizations of cytokine levels were done using Prism version 8.3.0 (GraphPad).

In vivo inflammasome activation

[0184] Systemic IL-18 levels were measured as a proxy of in vivo inflammasome activation as previously reported (Kupz et al., (2016) The Journal of Clinical Investigation, 126). C57BL/6 mice were intravenously injected with BCG::pYUB, BCG::RD1 or BCG::ESAT6-PE25SS and blood was collected 24 hours later. Serum was obtained by centrifugation and stored at -80°C until further use. IL-18 was measured by ELISA (ELISAkit, EK-0048) according to the manufacturer's protocol. Briefly, serum samples were diluted 1:3 with assay diluent. Standards, samples and zero standard controls were incubated in the ELISA plate for 2 hours at room temperature. The plate was washed 4 times and incubated with a biotin-labelled detection antibody for one hour at room temperature. After four washes, the streptavidin-HRP conjugate was added and incubated 45 min at room temperature followed by five washes. TMB substrate was added and incubated for 15 min at room temperature in the dark. The reaction was stopped and the OD was measured with a microplate reader (PolarSTAR Omega, BMG Labtech) at a wavelength of 450nm.

Peroxidase immunostaining

[0185] To visualize ESAT-6 in lung tissue, tissue slides were dewaxed with xylene and decreasing alcohol concentration. Slides were blocked with blocking buffer (10% FCS, 10% Normal goat serum in TBS) for 20 min at RT followed by staining with the primary detection antibody (1:200, mouse anti-ESAT6 polyclonal) for 1 hour at RT. Slides were washed with TBS, blocked with 3% H2O2 for 15 min and washed again. A secondary biotinylated antibody (1:200, goat anti-mouse) was added and slides were incubated for 30 min at RT. Following an additional washing step, slides were stained with HRP in TBS (1:300, Life technologies) for 30 min at RT, washed again and developed with DAB (Abeam) for 5 min at RT. Washed slides were counterstained with Mayers Heamatoxylin for 2 min at RT, washed with running tap water, and mounted with a glycerine based mounting media.

Statistics

[0186] Statistical analysis was performed, and graphs were generated using Prism version 8.3.0 (GraphPad). Multiple parametric group analyses were carried out using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison tests. P< 0.05 was considered significant, unless otherwise stated.

Example 2: A recombinant BCG strain exporting ESAT-6 via the ESX-5 secretion system

[0187] To uncouple the detrimental effects (increased persistence and increased virulence) associated with incorporation of the entire RD1 region into BCG from the beneficial immunogenic properties of full-length ESAT-6 secretion via ESX-1 (cytosolic contact, ESAT-6-specific T cell responses), a new recombinant BCG (rBCG) was designed. We reasoned that fusion of the esxA gene to the general secretion signal (SS) for the mycobacterial type VII secretion pathway³⁰, may enable secretion of full- length ESAT-6 via the ESX-5 secretion system, which in contrast to ESX-1 is also present in BCG. To achieve this, the nucleotides encoding the 17-amino acids long signal sequence at the C-terminus of PE25 were linked as a tag at the end of the EsxA coding sequence. The mycobacterial chromosome integrating plasmid pMV306hsp with the BCG hsp60 promoter was used to achieve cellular expression of the resulting ESAT-6-PE25SS fusion protein (Figure 1A). Successful chromosomal integration of the gene cassette was confirmed by restriction digestion (Figure 1 B, C) and by PCR with primers spanning the esxA gene and the pe25 signal sequence, as well as the M. bovis insertion sequence IS6110 (Figure 1D). Most notably, whole-genome sequencing of the resulting rBCG strain, hereafter called BCG::ESAT6-PE25SS, confirmed the attB site specific insertion of the gene cassette without any additional off-site mutations. ESAT-6 expression in BCG::ESAT6-PE25SS was confirmed by western blot. While both BCG::RD1 and BCG::ESAT6-PE25SS expressed full-length ESAT-6, no band was detected in the parental BCG. The correct molecular weight for ESAT-6 of 10 kDa in BCG::RD1 and of 11.6 kDa in BCG::ESAT6-PE25SS, respectively, were confirmed (Figure 1E), with the 1.6 kDa difference being attributed to the PE25SS in BCG::ESAT6-PE25SS. In addition, the presence of ESAT-6 was validated by ELISA using culture filtrate protein of BCG, BCG::ESAT6-PE25SS and BCG: RD1 (Figure 1F). In line with western blot results, optimal ESAT-6 secretion was found in around 1pg of cell lysates of BCG::ESAT6-PE25SS. All strains showed similar growth kinetics with BCG::ESAT-6 growing slightly slower than BCG::RD1 (Figure 1G). Collectively, these results provide molecular and genetic confirmation of full-length ESAT-6 expression and secretion by BCG::ESAT6-PE25SS.

Example 3: In vivo activity of BCG::ESAT6-PE25SS

[0188] The BCG::ESAT6-PE25SS strain was designed to achieve cytosolic contact via secretion of full-length ESAT-6. It has previously been shown that only full-length, but not truncated ESAT-6 leads to cytosolic access and inflammasome-mediated IL-18 secretion in vivo. To first determine the effect of the strain on the NLRP3 inflammasome pathway, C57BL/6 mice were infected intravenously with a high dose of BCG::pYUB, BCG::RD1 or BCG::ESAT6-PE25SS. In vivo inflammasome activation was assessed by measuring IL-18 levels in serum 24 hours post-infection. BCG::pYUB did not induce IL- 18 production because it lacks ESAT-6 (Figure 2A). In contrast both BCG::RD1 and BCG::ESAT6-PE25SS showed increased levels of IL-18, although these increases did just miss our cut off for statistical significance (Figure 2A).

[0189] To characterize if BCG::ESAT6-PE25SS also shows ESAT-6-dependent T cell responses, C57BL/6 mice were vaccinated intratracheally with BCG::pYUB, BCG::RD1 or BCG::ESAT6-PE25SS and immune responses in the lung were determined via flow cytometry and immunohistochemistry. BCG::pYUB, a strain carrying an empty vector, was used as a control strain to account for in vivo fitness-related effects of a vector insertion. ESAT-6 specific T cells were enumerated using the l-A(b) ESAT-6 QQWNFAGIEAAASA MHC-II tetramer in BALF and lung parenchyma at 20 and 60 days after vaccination (Figure 2B-D). No ESAT-6-specific T cells were induced by vaccination with BCG::pYUB, while mice vaccinated with BCG::RD1 yielded significant numbers of tetramer specific T cells in both BALF and lung parenchyma on day 20 and day 60. On day 20 after vaccination, ESAT-6-specific T cells in BCG::RD1 vaccinated mice accounted for 4.784 ± 0.898% of CD4⁺ T cells in the BALF and 5.551 ± 0.743% of CD4⁺ T cells in the lung parenchyma (Figure 2C). In comparison, BCG::ESAT6- PE25SS induced 1.982 ± 0.633% of tetramer-specific CD4⁺ T cells in the BALF and 3.513 ± 1.101% of tetramer-specific CD4⁺ T cells in the lung parenchyma, albeit with a significant variation between mice (Figure 2C). Sixty days post-vaccination BCG::ESAT6-PE25SS and BCG::pYUB vaccinated animals showed no tetramer- specific CD4⁺ T cells, while BCG::RD1-mediated ESAT-6-specific T cell induction was still significantly elevated (Figure 2D). We also determined the overall frequencies of CD4⁺ and CD8⁺ T cells in BALF and lung parenchyma. While T cell frequencies were not significantly different between BALF and lung parenchyma at either time point, both BCG::RD1 and BCG::ESAT6-PE25SS showed similar trends of increased CD8⁺ T cells and reduced CD4⁺ T cell frequencies relative to BCG::pYUB (Figure 2E, F). These data suggest that BCG::ESAT6-PE25SS and BCG::RD1 have a similar impact on T cell distribution and that BCG::ESAT6-PE25SS induces ESAT-6-specific T cells early after vaccination, but fails to maintain the level of ESAT-6-specific T cell induction mediated by BCG::RD1 long-term.

Example 4: Decreased bacterial burden and lung pathology after intratracheal vaccination with BCG::ESAT6-PE25SS.

[0190] BCG::RD1 is associated with prolonged persistence after vaccination in pre- clinical animal models. To determine if clearance of BCG::ESAT6-PE25SS is faster than that of BCG::RD1, C57BL/6 mice were i.t. vaccinated as above and bacterial burden and lung pathology were assessed 20 and 60 days later (Figure 3A). BCG::ESAT6- PE25SS showed significantly lower CFU counts on both day 20 and day 60 after vaccination compared to BCG::pYUB and BCG::RD1. Some mice even showed no detectable bacteria as early as 20 days after i.t. inoculation with BCG::ESAT6-PE25SS. BCG::pYUB numbers had significantly decreased by day 60 while the numbers of BCG::RD1 bacilli remained high (Figure 3A). Histological analysis of lung tissue infiltration further confirmed the apparent hastened clearance of BCG::ESAT6-PE25SS (Figure 3B). While BCG::pYUB vaccinated animals showed 33.39 ± 5.019% and 9.852 ± 2.255% of lung infiltration at 20 and 60 days post-vaccination, BCG::RD1 showed 44.69 ± 11.500% and 27.65 ± 4.522% and BCG::ESAT6-PE25SS showed 21.60 ± 5.478% and 9.244 ± 1.962% infiltration, respectively. Overall, lung pathology was significantly higher in animals that had received BCG::RD1 compared to animals that had received the other strains, particularly at 60 days after vaccination.

[0191] The inventors also analysed serum cytokine levels to assess systemic inflammation. While most cytokines showed no significant differences on either day 20 or 60, the levels of TNF-a, IL12p40, CCL5 (RANTES), G-CSF, GM-CSF, IL-13 and MIP- 1b remained higher in BCG::RD1 vaccinated animals at day 60 after vaccination compared to both other BCG strains (Figure 3C, D).

Example 5: BCG::ESAT6-PE25SS shows increased safety in Raa2^~/~IL2ra^~/~ mice

[0192] Incorporation of RD1 into BCG is also associated with increased virulence in immunocompromised animals. To assess whether BCG::ESAT6-PE25SS is associated with lower virulence compared to BCG::RD1, Rag2^~/~IL2rg^~/~ mice were intravenously injected with BCG::pYUB, BCG::RD1 or BCG::ESAT6-PE25SS. Mice were monitored for signs of diseases, including weight-loss and survival, and 30 days post-infection bacterial burden was determined in lung and spleen. While BCG::RD1 infected mice lost a significant amount of weight, with one mouse having to be culled for ethical reasons before day 30, neither BCG::pYUB nor BCG::ESAT6-PE25SS vaccinated animals showed signs of morbidity, such as weight loss (Figure 4A). Similarly, BCG::pYUB and BCG::RD1 showed similar CFU counts in both lung and spleen parenchyma that were significantly higher than those of mice infected with BCG::ESAT6-PE25SS (Figure 4B). Furthermore, while most serum cytokine levels in BCG::pYUB and BCG::ESAT6- PE25SS were similar, KC, MCP-1, G-CSF and IL-6 were elevated in BCG::RD1- infected mice (Figure 4C). Collectively, these data show that BCG::ESAT6-PE25SS is safer in immunocompromised mice compared to BCG::RD1.

[0193] BCG::RD1 has also been reported to induce mycobacterial meningoencephalitis in mice. As such, in a related study, the safety of BCG::ESAT6- PE25SS, was compared to BCG::pYUB and BCG::RD1 in Rag2 ^/ IL2rg ^/_ mice (an immunocomprised mouse breed).

[0194] Mice were infected with BCG::pYUB, BCG::RD1 or BCG::ESAT6-PE25SS. Several weeks after infection, mice were culled and brains collected for histopathological examination. Distinct differences were observed in the brains from mice infected with BCG::RD1 compared to mice infected with BCG::pYUB (control) or BCG::ESAT6-PE25SS (strain of the invention). In particular, marked multifocal lesions were observed in the brains of mice infected with BCG::RD1, often with extensive necrosis. These results further demonstrate the increased safety of BCG::ESAT6- PE25SS compared to BCG::RD1.

Example 6: BCG::ESAT6-PE25SS and BCG::RD1 protect equally against Mtb challenge.

[0195] To determine if BCG::ESAT-6-PE25SS also offers improved protection against Mtb challenge, C57BL/6 mice were vaccinated with either BCG::pYUB, BCG::RD1 or BCG::ESAT6-PE25SS via i.t. or s.c. injection followed by aerosol challenge with 20-50 CFU of Mtb H37Rv 60 days later. Mice were sacrificed 45 days after challenge and assessed for readouts of TB disease. Relative to unvaccinated mice, significantly decreased pathology was observed in the lungs of mice vaccinated with BCG::RD1 or BCG: : ESAT6-PE25SS (Figure 5A, B). Tissue infiltration was 57.92 ± 8.645% in unvaccinated mice, 56.69 ± 9.621% in i.t. BCG::pYUB vaccinated mice; 38.10 ± 3.821% in i.t. BCG::RD1 vaccinated mice; and 26.43 ± 3.334% in i.t. BCG::ESAT6-PE25SS vaccinated mice (Figure 5B). Tissue infiltration in s.c. vaccinated mice was 60.27 ± 4.367%, 30.61 ± 5.606% and 34.70 ± 3.105% for BCG::pYUB, BCG::RD1 and BCG::ESAT-6-PE25SS, respectively (Figure 5B). Unvaccinated animals had higher tissue infiltration compared to both s.c. BCG::RD1 and s.c. BCG::ESAT6-PE25SS groups. Furthermore, BCG::pYUB mice showed higher infiltration levels than BCG::RD1 and BCG::ESAT6-PE25SS groups (Figure 5A, B). The pattern of difference was similar when vaccination routes were compared individually or pooled (Figure 5B). Unvaccinated animals had significantly higher tissue infiltration compared to 34.36 ± 5.396% for BCG::RD1 vaccination and 30.57 ± 2.470% for BCG::ESAT6-PE25SS vaccination. BCG::pYUB vaccination showed similar infiltration to unvaccinated animals, 58.28 ± 3.361%, and was significantly higher compared to BCG::RD1 and BCG::ESAT6-PE25SS (Figure 5A, B). Altogether these results show similar levels of tissue infiltration after BCG::ESAT6-PE25SS vaccination compared to BCG::RD1 vaccination, indicating a similar level of protective capacity for both strains. Bacterial burden in the lung was variable and showed no significant difference between vaccination groups, with logio CFUs of 4.246 ± 0.142, 3.199 ± 0.321, 2.685 ± 0.473 and 3.002 ± 0.433 for unvaccinated, BCG::pYUB, BCG::RD1 and BCG::ESAT6-PE25SS vaccinated animals, respectively (Figure 5C). Both BCG::ESAT6-PE25SS and BCG::RD1 groups showed increased protection compared to BCG::pYUB (Figure 5C). In the spleen, CFU numbers for all vaccinated mice were lower compared to unvaccinated mice, albeit not significant, except for BCG::RD1. Vaccinated mice showed CFUs of 1.623 ± 0.491, 1.035 ± 0.385 and 2.195 ± 0.409 for BCG::pYUB, BCG::RD1 and BCG::ESAT6-PE25SS, respectively (Figure 5C). Taken together these results show that BCG::RD1 and BCG::ESAT6-PE25SS induce similar protection against Mtb challenge in the lung. Serum cytokine levels at 45 days after Mtb challenge were similar between all strains and vaccine delivery routes, with the exception of MCP- 1 being significantly increased in BCG::ESAT6-PE25SS vaccinated mice compared to unvaccinated and BCG::pYUB vaccinated animals (Figure 5D, E).

[0196] 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.

Claims

1. A recombinant strain of M. bovis BCG comprising a heterologous nucleic acid encoding a fusion protein, wherein the fusion protein comprises or consists of: a) a polypeptide that is at least about 80% identical to an ESAT-6 protein and b) a secretion peptide for enabling secretion of the polypeptide via an ESX-5 secretion system of M. bovis BCG.

2. The recombinant strain of claim 1 , wherein the polypeptide is at least about 80% identical to the amino acid sequence of an ESAT-6 protein from Mtb.

3. The recombinant strain of claim 1 or 2, wherein the polypeptide is at least about 80%, 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.

4. The recombinant strain of any one of claims 1 to 3, wherein the secretion peptide comprises or consists of the amino acid sequence YXXXD/E.

5. The recombinant strain of claim 4, wherein the secretion peptide comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 6, 8 or 9, or variants or fragments thereof comprising the YXXXD/E motif.

6. The recombinant strain of claim 4, wherein the peptide consists of the amino acid sequence set forth in SEQ ID NO: 6, or a variant or fragment thereof that comprises the YXXXD/E motif.

7. The recombinant strain of any one of claims 1 to 6, wherein the secretion peptide is located C-terminal to the ESAT-6 polypeptide in the fusion protein.

8. The recombinant strain of any one of claims 1 to 7, wherein the fusion protein encoded by the heterologous nucleic acid comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7, or variants thereof that are at least 80% identical thereto.

9. A nucleic acid encoding a fusion protein, wherein the fusion protein comprises a) a polypeptide that is at least about 80% identical to an ESAT-6 protein and b) a secretion peptide for enabling secretion of the polypeptide via the ESX-5 secretion system of M. bovis BCG.

10. The nucleic acid of claim 9, wherein the polypeptide is at least about 80% identical to the amino acid sequence of an ESAT-6 protein from Mtb.

11. The nucleic acid of claim 9 or 10, wherein the polypeptide is at least about 80%, 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.

12. The nucleic acid of any one of claims 9 to 11, wherein the nucleic acid comprises or consists of the nucleic acid sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, or sequences at least 80% identical thereto.

13. The nucleic acid of any one of claims 9 to 12, wherein the nucleic acid comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 3 or 4, or sequences at least 80% thereto.

14. A vector comprising a nucleic acid molecule of any one of claims 9 to 13.

15. A host cell comprising a vector of claim 14.

16. The host cell of claim 15, wherein the host cell is M. bovis BCG.

17. A vaccine or immune stimulating composition comprising a recombinant M. bovis BCG strain of any one of claims 1 to 8 and optionally, a pharmaceutically acceptable carrier or excipient.

18. The vaccine or immune stimulating composition of claim 17, further comprising an adjuvant for potentiating the immune response to the M. bovis BCG strain.

19. The vaccine or immune stimulating composition of claim 18, further comprising a subunit vaccine, or antigens of Mtb for further potentiating the immune response.

20. A method for inducing an immune response to Mtb in a subject in need thereof, the method comprising administering an effective amount of the recombinant M. bovis BCG strain of any one of claims 1 to 8, or a vaccine or immune stimulating composition of any one of claims 17 to 19, thereby inducing an immune response to Mtb in the subject.

21. Use of a recombinant strain of M. bovis BCG of any one of claims 1 to 8, in the manufacture of a vaccine or immune stimulating composition for inducing an immune response to Mtb in a subject.

22. A recombinant strain of M. bovis BCG of any one of claims 1 to 8, or a vaccine or immune stimulating composition of any one of claims 17 to 19, for use in stimulating an immune response to Mtb in a subject.

23. The method or use of claim 20 or 21 , or the recombinant strain for use of claim 22, wherein the immune response is a protective immune response.

24. The method or use of claim 20 or 21 , or the recombinant strain for use of claim 22, wherein the immune response is a Th1 immune response.

25. A method for treating or reducing the severity of an Mtb infection in a subject in need thereof, the method comprising administering an effective amount of the recombinant M. bovis BCG strain of any one of claims 1 to 8, or the vaccine or immune stimulating composition any one of claims 17 to 19, to the subject, thereby treating or reducing the severity of the Mtb infection in the subject.

26. Use of a recombinant strain of M. bovis BCG of any one of claims 1 to 8, in the manufacture of a vaccine or immune stimulating composition for treating or reducing the severity of an Mtb infection in a subject.

27. A recombinant strain of M. bovis BCG of any one of claims 1 to 8, or a vaccine or immune stimulating composition of any one of claims 19 to 17, for use in treating or reducing the severity of an Mtb infection in a subject.

28. A kit comprising a recombinant strain of M. bovis BCG of any one of claims 1 to 8, or a vaccine or immune stimulating composition of any one of claims 17 to 19, optionally comprising written instructions for the use of the kit in a method of claims 20 or 25.