WO2011130878A9 - Tuberculosis vaccines including recombinant bcg strains overexpressing phop, and/or phop regulon protein(s) - Google Patents

Abstract

A live recombinant Mycobacterium bovis-BCG strain comprising a nucleic acid encoding PhoP protein and/or one or more phoP regulon proteins is provided, in which the nucleic acid is capable of being overexpressed. A vaccine or immunogenic composition comprising the live recombinant Mycobacterium bovis-BCG strain and a method for treatment or prophylaxis of a mammal against challenge by Mycobacterium tuberculosis or Mycobacterium bovis are also provided.

Description

Tuberculosis Vaccines Including Recombinant BCG Strains Overexpressing

PhoP, and/or PhoP Regulon protein(s)

Field of the Invention

This invention relates to tuberculosis (TB) vaccines. In particular, the invention provides a recombinant BCG strain that overexpresses the transcription factor PhoP at a level sufficient to cause induction of the phoP regulon.

Background of the Invention

Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tb), remains a global health emergency. The latest surveillance data by the World Health Organization (WHO) reveals that in 2006, there were 9.2 million new cases and 1.7 million deaths from TB. The challenges of TB prevention, case detection, and treatment are in stark contrast to the ease of M, tb transmission. Additional threats to TB control include the spread of multidrug-resistant TB (MDR-TB), the appearance of extensively drug-resistant TB (XDR-TB), and the destructive impact of TB/HIV coinfection. Because of these situations, effective approaches alternative to antibiotics are urgently needed for the control of TB. According to the Global Plan to Stop TB (2006-2015), the introduction of new, effective TB vaccines will be an essential component of any strategy to eliminate TB by 2050.

Bacille Calmette-Guerin (BCG), an attenuated strain of Mycobacterium bovis, is currently the only available vaccine for the prevention of TB. Since 1974, BCG vaccination has been included in the WHO Expanded Program on Immunization [1]. It is estimated that more than 3 billion individuals have been immunized with BCG and over 100 million doses of BCG are administered annually, making it the most widely used vaccine in humans [1]. In animal models of infection, BCG vaccinations have been demonstrated to induce protective immunity against M. tb challenges [2], In humans, BCG vaccination has demonstrated consistent protection against the childhood forms of TB, including tuberculous meningitis and miliary TB [3]. However, BCG vaccination is controversial due to variations in its efficacy for protecting adults from pulmonary TB [4-6]. Trials conducted in the 1940s and 1950s in developed countries such as the United Kingdom, Denmark and North America demonstrated the vaccine to be highly efficient (70-80%). However, in the single largest clinical trial, which took place in India in 1970s and involved more than 265,000 persons, BCG vaccination provided no detectable protection against pulmonary TB. Several explanations have been suggested for the variation in protective efficacy of BCG [7], including differences among the vaccine strains used in clinical studies, exposure of trial populations to environmental mycobacteria, nutritional or genetic differences in human populations, differences in trial methods, and variations among clinical M. tb strains [5,8-11]. These explanations are not mutually exclusive and all may contribute to the heterogeneity in vaccine efficacy.

A key aspect of this issue concerns the immunogenicity of BCG vaccine. Numerous BCG strains are currently used as commercial vaccines. They are all descendants of the original M. bovis isolate that Calmette and Guerin passaged through 230 cycles during 1909-1921. Subsequent in vitro passages under different laboratory conditions around the world continued until 1960s, when the frozen seed lots were established. Because of the excessive in vitro passages (more than 1600 times for certain strains), it is thought that current BCG strains may have been over attenuated thus not immunogenic enough to provide effective protection [12].

It is now clear that BCG is not an ideal vaccine and gives protection for only a limited period of time. The goal to developing a new and effective TB vaccine is to provide long-term protection [13,14]. Existing BCG vaccines impart protection against the manifestations of TB in children, but their efficacy wanes over a period of 10 to 15 years, presumably because the protective immunity induced by BCG is gradually lost [13]. Currently, the consensus in the scientific filed is that the new generation of TB vaccines will work best using a heterologous prime-boost strategy to strengthen the immune response introduced by BCG [15]. This "prime-boost" strategy would include administration of a new recombinant BCG (rBCG), the "prime", followed by a "booster" inoculation with a different vaccine (protein/peptide or DNA) to infants and young children before they are exposed to TB, or as a separate booster to young adults, or as an adjunct to chemotherapy [15].

The first example of recombinant BCG is rBCG30, a recombinant BCG-Tice strain that overexpresses (~ 5 fold) Ag85B [16], which is a secreted protein and belongs to the mycolyl transferase family comprising Ag85A, B and C. A second example of recombinant BCG is r CG::AureC-llo⁺, a urease-deficient strain of BCG-Pasteur that expresses listeriolysin O of Listeria monocytogenes [17]. Urease is deleted as a means of providing the optimal pH for listeriolysin function, which damages the phagosome membrane, allowing BCG leakage into the cytosol and increasing the amount of antigens available for presentation to CD8⁺ T cells. Others have attempted to make new live vaccines by attenuating M. tb, reasoning that this would give the closest simulation of natural immunity occurring after M. tb infection. Examples include the phoP mutant of M. tb [18] and the non-replicating M. tb mutant strain (AlysA ApanCD) that is auxotrophic for lysine and pantothenate [19].

Protection against TB requires a cell-mediated immune response, which is not fully understood but involves multiple components including CD4⁺ and CD8⁺ T cells, unconventional T cells such as γδ T cells and CD 1 -restricted αβ T cells [20,21], A critical role of IFN-γ in the control of TB has been demonstrated in mice and humans [22-25], As such, antigen specific IFN-γ production mostly by CD4⁺ T cells has been used most widely as measure of protective immunity and vaccine efficacy [26], The identification of M. tb antigens that induce strong IFN-γ production has been the main strategy employed to uncover subunit vaccines. This was done by biochemical fractionation of M. tb protein mixtures, particularly the culture filtrate proteins [27-30]. Using this approach, several antigens were identified including small secreted proteins ESAT-6 (EsxA), ESAT-6-like proteins TB9.8 (EsxG) and Mtb9.9A (EsxN), CFP-10 (EsxB), the antigen 85 complex (Ag85A, B, C), and several PE PPE family proteins (e.g., PPE18, PPE14) [31-36]. Based on these studies, three fusion proteins Ag85B-ESAT-6, Ag85B-TB 10.4 (EsxH), and Mtb72f (PPE18-Rv0125) were constructed and are presently the most advanced protein-based subunit vaccines [36-39].

DNA based subunit vaccines have also been exploited which use replication-deficient viral vectors such as adenovirus or vaccinia virus for delivery to stimulate greater CD8 recognition of the expressed antigens. Examples include MAV-85A, a vaccinia virus expressing Ag85A [40], and Aeras-402, an adenovirus-35 expressing Ag85A, Ag85B, and EsxH [41].

Summary of the Invention

The present invention provides tuberculosis vaccines comprising a recombinant mycobacterium strain that overexpresses PhoP, a transcriptional regulator, and thus induces the expression of the PhoP regulon. The invention also encompasses recombinant BCG strains which overexpress one or more genes of the PhoP regulon. The immunogenicity of current BCG vaccine strains is not sufficient to induce the optimal protection in host against tuberculosis. However, a genetically engineered BCG strain that overexpresses PhoP and/or PhoP regulon protein(s) is more immunogenic and will provide better protection. Any genetically engineered mycobacterium that overproduces PhoP at a level sufficient to cause a 2-(or more) fold induction of phoP regulon genes or proteins may be advantageously used in the practice of this invention. When the recombinant mycobacterium of the invention is administered to a mammalian host, an immune response is elicited to the proteins encoded by the induced phoP regulon genes, which will provide protection of the mammalian host against tuberculosis.

The potency of a BCG vaccine is traditionally determined by measuring the tuberculin sensitivity (delayed type hypersensitivity, DTH, or PPD reactivity) induced by the vaccine in children who were tuberculin-negative before vaccination [42]. If the tested vaccine induces less tuberculin sensitivity than that induced by other strains, it is considered weak. The skin lesion or scar at the site of intradermal vaccination is also measured. Traditionally, tuberculin reactivity is considered a surrogate marker for efficacy and has played a major role in the history of BCG, including the choice of BCG strains for national immunization programmes. Tuberculin reactivity continues to be used as an in vivo assay for cell-mediated immune response and as a marker for immunogenicity [16,43]. Supporting this, a strong association was found between tuberculin reactivity and PPD specific IFN-γ levels in BCG-vaccinated infants in the UK [44] and another recent study in a TB endemic area found that both tuberculin reactivity and IFN-γ releases (level and frequency) are non-redundant,

complementary measures of anti-mycobacterial immunity in young persons [45], Differences in the ability to induce tuberculin reactivity among BCG strains have been shown in both guinea pigs and children [46,47]. Of the 12 BCG strains examined, BCG-Prague consistently exhibits the lowest tuberculin reactivity compared to other BCG strains. Because of this, BCG-Prague, which was used in Czechoslovakia between 1951-1980, was replaced by

BCG-Russia in 1981. The molecular factors that contribute the low tuberculin reactivity of BCG-Prague remain unknown. However, a recent work from my laboratory has found that BCG-Prague contains a nonfunctional PhoP protein due to a genetic mutation in its phoP gene [48]. A frame-shift mutation within the phoP gene of BCG-Prague eliminates the majority of C-terminal DNA binding domain, which makes BCG-Prague a natural phoP mutant (Figure 1). PhoP is the response regulator of the two-component regulatory system PhoP-PhoR and is important for the virulence of M. tb [49-51]. I hypothesize that the exceptionally low tuberculin conversion of BCG-Prague could be explained specifically by the phoP mutation. In the phoP deletion mutant of M. tb, more than 40 genes (defined as the 'PhoP regulon') were repressed [49,51,52]. Some of these are immunogenic proteins. It is likely that these proteins contribute to tuberculin reactivity. In BCG-Prague, due to the strain's phoP mutation, these immunogenic proteins would not be expressed in BCG-Prague, thus eliminating the ability to induce tuberculin reactivity. Taken together, I hypothesize that the PhoP function is directly responsible for BCG tuberculin reactivity. As such, a recombinant BCG strain that

overexpresses phoP will lead to the overexpression of phoP regulon and enhanced tuberculin reactivity, which will also lead to better protection of host against M. tb. This hypothesis is now confirmed by experimental evidence (see Table 1 and Figure 4). In addition, PhoP positively regulate more than 40 genes (the phoP regulon). Recombinant BCG strains that overexpress one or more genes of the PhoP regulon will also lead to stronger immunogenicity. Such BCG strains will have better protective efficacy against M. tb.

The PhoP used in the invention may be a naturally-occurring, functional PhoP, e.g., from genus Mycobacterium, preferably from Mycobacterium tuberculosis, or Mycobacterium bovis, or a homolog thereof. An exemplary amino acid sequence of PhoP is presented in Figure 2A -SEQ ID NO: l and an exemplary nucleotide sequence encoding the same is presented in Figure 2B SEQ ID NO:2. These sequences represent PhoP from the M. tb H37Rv phoP gene, as presented in the genome sequence available at the Pasteur Institute's TubercuList Website (http://genolist.pasteur.fr/TubercuList/index.html).

The present invention relates to a recombinant Mycobacterium bovis BCG, which overexpresses DNA encoding PhoP shown in SEQ ID NO: l. Preferably, the DNA comprises or consists of the nucleotide sequence of SEQ ID NO:2.

The present invention relates to a recombinant Mycobacterium bovis BCG comprising a nucleic acid capable of overexpression, the nucleic acid encoding PhoP shown in SEQ ID NO: 1 . Preferably, the nucleic acid comprises the nucleotide sequence of SEQ ID NO:2.

The invention also relates to a live recombinant Mycobacterium bovis- CG strain comprising a nucleic acid capable of overexpression, the nucleic acid encoding at least one protein or polypeptide selected from the group (PhoP regulon) consisting of Rv0440, Rv0904c, Rv0981, Rvl057, Rvl l SO, Rvl l 82, Rvl l 83, Rvl l 84c, Rvl l85c, Rvl l95, Rvl l96, Rvl361c, Rvl639c, Rvl931c, Rv2227, Rv2276, Rv2288, Rv2289, Rv2329c, Rv2332, Rv2375, Rv2376c, Rv2391 , Rv2392, Rv2396, Rv2590, Rv2987c, Rv3135, Rv3136, Rv3197, Rv3312A, Rv3331 , Rv3332, Rv3343c, Rv3477, Rv3478, Rv3479, Rv3486, Rv3487c, Rv3613c, Rv3686c, Rv3689, Rv3767c, Rv3804c, Rv3822, Rv3823c, Rv3824c, and Rv3825c. Their protein sequences are available at the Pasteur Institute's TubercuList Website (http://genolist.pasteur.fr/TubercuList/index.html).

Preferably, the nucleic acid comprises all or part of at least one nucleic acid molecule selected from the group consisting of Rv0440, Rv0904c, Rv0981, Rvl057, Rvl l 80, Rvl l 82, Rvl l 83, Rvl l 84c, Rvl l 85c, Rvl l 95, Rvl l96, Rvl361c, Rvl639c, Rvl931c, Rv2227, Rv2276, Rv2288, Rv2289, Rv2329c, Rv2332, Rv2375, Rv2376c, Rv2391, Rv2392, Rv2396, Rv2590, Rv2987c, Rv3135, Rv3136, Rv3197, Rv3312A, Rv3331 , Rv3332, Rv3343c, Rv3477, Rv3478, v3479, Rv3486, Rv3487c, Rv3613c, Rv3686c, Rv3689, Rv3767c, Rv3804c, Rv3822, Rv3823c, Rv3824c, and Rv3825c. Their nucleotide sequences are available at the Pasteur Institute's TubercuList Website (http://genolist.pasteur.fr/TubercuList/index.html).

In one embodiment, the live recombinant Mycobacterium bovis-BCG strain is selected from existing BCG strains. Those of skill in the art will recognize that several suitable, BCG exists which are suitable for use in the practice of the invention, including but not limited to: Mycobacterium fovw-BCG-Russia (ATCC number: 35740), Mycobacterium bovis-BCG-Morem (ATCC number: 35736), Mycobacterium bovis-BCG- Japan (ATCC number: 35737), Mycobacterium w-BCG-Sweden (ATCC number: 35732), Mycobacterium 0ovw-BCG-Biridiaug (ATCC number: 35731), Mycobacterium fovw-BCG-Prague (ATCC number: 35742), Mycobacterium 6ov«-BCG-Glaxo (ATCC number: 35741), Mycobacterium fovw-BCG-Denmark (ATCC number: 35733), Mycobacterium bovis-BCG-T\ce (ATCC numbers: 35743, 27289), Mycobacterium ^ovw-BCG-Frappier (ATCC: 35746, SM-R; ATCC: 35747, INH-R), Mycobacterium fovw-BCG-Connaught (ATCC: 35745), Mycobacterium fovw-BCG-Phipps (ATCC number: 35744), Mycobacterium fovw-BCG-Pasteur (ATCC number: 35734), BCG-Mexican (ATCC number: 35738) and Mycobacterium jovzs-BCG-China (Shanghai Institute of Biological Product).

In addition, the recombinant mycobacteria of the invention need not be confined to strains of BCG. Those of skill in the art will recognize that other Mycobacterium strains may also be employed including attenuated strains of M. tb.

In yet another embodiment, the vaccine of the invention may be a subunit or DNA-vaccine. In some embodiments, the vaccine would be delivered via lung pathogens. For example, the DNA sequences coding for PhoP and/or PhoP regulon protein(s) could be harbored within the chromosome or extra chromosomal nucleic acid of a lung pathogen such as attenuated Pseudomonas aeruginosa, or other known attenuated fungi or viruses. Alternatively, the nucleic acid encoding PhoP and/or PhoP regulon protein(s) could be delivered by other means known to those of skill in the art, e.g., via liposomes, adenoviral vectors, etc.

Another aspect of the invention is a pharmaceutical composition comprising a live recombinant Mycobacterium bovis-BCG strain comprising a nucleic acid capable of overexpression, the nucleic acid encoding a functional PhoP, such as the PhoP shown in SEQ ID NO: 1. The nucleic acid is for example shown in SEQ ID NO:2. The invention also relates to a live recombinant Mycobacterium bovis-BCG strain comprising a nucleic acid capable of overexpression, the nucleic acid encoding at least one protein or polypeptide selected from the group consisting of Rv0440, Rv0904c, Rv0981 , Rvl057, Rvl l 80, Rvl l 82, Rvl l83, Rvl l 84c, Rvl l 85c, Rvl l95, Rvl l96, Rvl 361c, Rvl639c, Rvl931c, Rv2227, Rv2276, Rv2288, Rv2289, Rv2329c, Rv2332, Rv2375, Rv2376c, Rv2391 , Rv2392, Rv2396, Rv2590, Rv2987c, Rv3135, Rv3136, Rv3197, Rv3312A, Rv3331 , Rv3332, Rv3343c, Rv3477, Rv3478, Rv3479, Rv3486, Rv3487c, Rv3613c, Rv3686c, Rv3689, Rv3767c, Rv3804c, Rv3822, Rv3823c, Rv3824c, and Rv3825c.

In yet another aspect of the invention there is a pharmaceutical composition comprising a live recombinant Mycobacterium bovis-BCG strain comprising a nucleic acid capable of overexpression, the nucleic acid comprises all or part of at least one nucleic acid molecule selected from the group consisting of Rv0440, Rv0904c, Rv0981, Rvl057, Rvl l80, Rvl l 82, Rvl l83, Rvl l84c, Rvl l 85c, Rvl l95, Rvl l96, Rvl361c, Rvl639c, Rvl931c, Rv2227, Rv2276, Rv2288, Rv2289, Rv2329c, Rv2332, Rv2375, Rv2376c, Rv2391 , Rv2392, Rv2396, Rv2590, Rv2987c, Rv3135, Rv3136, Rv3197, Rv3312A, Rv3331, Rv3332, Rv3343c, Rv3477, Rv3478, Rv3479, Rv3486, Rv3487c, Rv3613c, Rv3686c, Rv3689, Rv3767c, Rv3804c, Rv3822, Rv3823c, Rv3824c, and Rv3825c.

In yet another embodiment, the vaccine of the invention may be a subunit or DNA-vaccine. In some embodiments, the vaccine would be delivered via lung pathogens. For example, the DNA sequences coding for PhoP and/or PhoP regulon protein(s) could be harbored within the chromosome or extra chromosomal nucleic acid of a lung pathogen such as attenuated Pseudomonas aeruginosa, or other known attenuated fungi or viruses. Alternatively, the nucleic acid encoding PhoP and/or PhoP regulon protein(s) could be delivered by other means known to those of skill in the art, e.g., via liposomes, adenoviral vectors, etc.

In a further aspect of the invention there is a vaccine or immunogenic composition for treatment or prophylaxis of a mammal against challenge by mycobacteria comprising a live recombinant Mycobacterium bovis-BCG strain comprising a nucleic acid capable of overexpression, the nucleic acid encoding PhoP, e.g., PhoP shown in SEQ ID NO: l . Preferably, the nucleic acid comprises or consists of the nucleotide sequence shown in SEQ ID NO:2.

In another aspect of the invention there is a vaccine or immunogenic composition for treatment or prophylaxis of a mammal against challenge by mycobacteria comprising a live recombinant Mycobacterium bovis-BCG strain comprising a nucleic acid capable of overexpression, the nucleic acid encoding at least one protein or polypeptide selected from the group consisting of Rv0440, Rv0904c, Rv0981, Rvl057, Rvl l 80, Rvl l 82, Rvl l 83, Rvl l 84c, Rvl l 85c, Rvl l95, Rvl l96, Rvl361c, Rvl639c, Rvl 931c, Rv2227, Rv2276, Rv2288, Rv2289, Rv2329c, Rv2332, Rv2375, Rv2376c, Rv2391, Rv2392, Rv2396, Rv2590, Rv2987c, Rv3135, Rv3136, Rv3197, Rv3312A, Rv3331 , Rv3332, Rv3343c, Rv3477, Rv3478, Rv3479, Rv3486, Rv3487c, Rv3613c, Rv3686c, Rv3689, Rv3767c, Rv3804c, Rv3822, Rv3823c, Rv3824c, and Rv3825c.

In yet another aspect of the invention there is a vaccine or immunogenic composition for treatment or prophylaxis of a mammal against challenge by mycobacteria comprising a live recombinant Mycobacterium bovis-BCG strain comprising a nucleic acid capable of overexpression, the nucleic acid comprises all or part of at least one nucleic acid molecule selected from the group consisting of Rv0440, Rv0904c, Rv0981₅ Rvl057, Rvl l 80, Rvl l 82, Rvl l 83, Rvl l 84c, Rvl l 85c, Rvl l95, Rvl l96, Rvl361c, Rvl639c, Rvl931c, Rv2227, Rv2276, Rv2288, Rv2289, Rv2329c, Rv2332, Rv2375, Rv2376c, Rv2391 , Rv2392, Rv2396, Rv2590, Rv2987c, Rv3135, Rv3136, Rv3197, Rv3312A, Rv3331, Rv3332, Rv3343c, Rv3477, Rv3478, Rv3479, Rv3486, Rv3487c, Rv3613c, Rv3686c, Rv3689, Rv3767c, Rv3804c, Rv3822, Rv3823c, Rv3824c, and Rv3825c.

In a preferred embodiment the vaccine or immunogenic composition is for the treatment or prophylaxis of a mammal against challenge by Mycobacterium tuberculosis. In another preferred embodiment the vaccine or immunogenic compositions of the current invention further comprise a pharmaceutically acceptable carrier. In yet another preferred embodiment the vaccine or immunogenic compositions further comprise adjuvants. In another embodiment the vaccine or immunogenic compositions further comprise immunogenic materials front one or more other pathogens.

Another aspect of this invention relates to a method for treatment or prophylaxis of a mammal against challenge by Mycobacterium tuberuclosis or Mycobacterium bovis comprising administering to the mammal a vaccine or immunogenic composition of the instant invention. In one embodiment the mammal is a cow. In another embodiment the mammal is a human. In yet another embodiment the vaccine or immunogenic composition is administered in the presence of an adjuvant.

A further aspect of the invention is a method for the treatment or prophylaxis of a mammal against cancer comprising administering to the mammal a vaccine or immunogenic composition of the current invention. In one embodiment the cancer is bladder cancer. In another embodiment the vaccine or immunogenic composition is administered in the presence of an adjuvant.

Another aspect of the invention is use of the mammal a vaccine or immunogenic composition of the current invention in a medicament for the treatment or prophylaxis of a mammal against cancer. In one embodiment the cancer is bladder cancer.

Brief Description of the Drawings

Figure 1. DNA sequencing chromatograph showing the single nucleotide (G) insertion within the phoP gene in BCG-Prague. This mutation was confirmed by repeating the PCR amplification and DNA sequencing.

Figure 2. A. Amino acid sequence of PhoP of M. tb H37Rv; B, DNA sequence of phoP of M. tb H37Rv.

Figure 3. Construction of the shuttle vector pME.

Figure 4. Cloning of phoP into the shuttle vector pME. A 1028 bp product was amplified from M. tb genomic DNA using forward primer

(5 ' -AA A AAGCTACCGCTTGTTTGGCCATGTCAAC-3 ') and reverse primer (5'-AAAAACTGCAGGCTGCCGATCCGATTAACTAC-3') containing a Kpn\ and a Pst\ restriction site (underlined), respectively. Using these restriction sites, the PCR product was cloned into shuttle vector pME using Kpnl and Pstl sites to generate pME:phoP. Cloned region contains 257 bp upstream of phoP start site.

Figure 5. Overexpression of phoP in BCG enhances the immunogenicity. Examples of BCG strains (BCG-Japan and BCG-Prague) that overexpress phoP increase immunogenicity than the control BCG C57BL/6 mice (4 per group) were immunized with 5 x 10^s CFU of indicated recombinant BCG (BCG-Japan/pME:phoP, BCG-Prague/pME:phoP), the corresponding BCG control (BCG-Japan/pME, BCG-Prague/pME), or PBS (na^'ive mice). Eight weeks post-vaccination, mice were sacrificed and splenocytes prepared. (A) ELISA analysis of IFN-γ production. Splenocytes were incubated with or without PPD (10 μg/ml) for 72 hr before subjected to ELISA analysis. Results are from 4 mice per group (means ± SD), after subtraction of reading from the same sample without antigen stimulation. (B) Intracellular cytokine staining analysis of cytokine responses of CD4⁺ T cells. Splenocytes were incubated with or without PPD for 24 hr followed by staining for surface markers (CD3-Pacific Blue, CD4-PE-Cy-7) and intracellular cytokines (IFN-γ-ΡΕ, TNF-PE, IL-2-PE, individually). Samples were analyzed by a FACS Calibur. The frequency of cytokine producing CD4⁺ T cells was determined and the total number of cells per spleen was calculated and presented. Figure 5 shows elevated immune responses induced by BCG strains overexpressing phoP.

Detailed Description of the Invention

The present invention provides a vaccine or immune stimulating compositions, which includes one or more genetically engineered mycobacteria that over expresses PhoP, a transcriptional regulator (transcription factor), and/or phoP regulon protein(s).

BCG is live, attenuated strain of M. bovis. It has long been known that administration of killed BCG strains results in a weak and transient immune response. However, it is recognized that the immunogenicity of current live BCG strains is also not optimal, which explains the failure of current BCG strains to provide effective protection. At present various strategies have been attempted to improve BCG immunogenicity, for example, by overexpressing antigen 85 (85A or 85B), or by expressing listerolysin in BCG to allow its escape into cytosol of infected macrophages for better antigen presentation [15]. Both of these recombinant BCG strains have now entered clinical trials or undergoing pre-clinical studies for development as new tuberculosis vaccine [15].

However, M. tb contains more than 4,000 genes and many of which are immunogenic proteins. It is clear that the choices of antigens to be expressed in BCG to enhance its immunogenicity are far from complete and very often the choice of antigens for this purpose lacks a clear rationale. As such, researchers in the scientific community continue to search for new antigens or important genes for overexpression in BCG. For example, antigens involved in latency (the 'DosR regulon') and reactivation/resuscitation are now being exploited for such purpose [53].

Tuberculin reactivity continues to serve as a convenient and effective means for assessing the immunogenicity of vaccine candidates [16,43]. Evidence supporting the positive correlation between tuberculin reactivity and protective efficacy include studies of BCG-Tice. Horwitz and co-workers have used BCG-Tice as the host strain to overexpress antigen 85B. This resulted in a recombinant strain termed rBCG30 that exhibits superior protective efficacy over BCG-Tice and is currently being evaluated as a vaccine candidate in human clinical trials [16,28,54,55], The rBCG30 Tice strain showed significantly stronger immune response and better protection against M. tb challenge than the rBCG30 strain based on BCG-Connaught. In a recent comparative study of BCG strains, including BCG-Pasteur, -Frappier, -Moreau, -Danish, -Sweden, -Connaught, -Birkhaug, -Phipps, and -Mexico, BCG-Tice exhibited the highest ability to induce tuberculin reactivity in mice [56], which explains its superior efficacy over BCG-Connaught.

The current invention is based on our recent finding that BCG-Prague contains a disrupted phoP and the link I made between the phoP mutation of BCG-Prague and its reduced ability to induce tuberculin reactivity. BCG vaccine strains have variable abilities to induce tuberculin reactivity in children, which has been well recognized historically. However, the underlying molecular mechanisms remain unknown. Recent studies from my lab found that one BCG strain, BCG-Prague, is a natural phoP mutant [48] (Fig. 1). PhoP is an important virulence factor of M. tb, which positively control the expression of over 40 genes [49,51,57]. As such, the phoP mutation is responsible for the further attenuation of BCG-Prague. However, I hypothesize that the phoP mutation is also responsible for another important clinical property of BCG-Prague, which is its exceptionally low ability to induce tuberculin reactivity. Previous studies showed that of all BCG strains tested, BCG-Prague exhibited the lowest ability to induce tuberculin reactivity in animal models and in children [46,47]. The reasons for its exceptional low tuberculin reactivity are unknown. I suggest, for the first time, that the phoP mutation is responsible for the low tuberculin reactivity of BCG-Prague, and that PhoP function is directly responsible for BCG tuberculin reactivity or immunogenicity. It follows then that overexpression of PhoP and/or PhoP regulon protein(s) in BCG strains will enhance their immunogenicity and improve protective efficacy as a vaccine. These novel concepts are significant, since thus far PhoP has only been considered as a virulence factor and as such, M. tb phoP deletion mutant is being considered as a new vaccine candidate [50]. It is first time that overexpression of PhoP and/or PhoP regulon protein(s) in BCG strains is considered as a novel vaccine strategy. Recombinant BCG strains that overexpress PhoP and/or PhoP regulon protein(s) will induce stronger immune responses and provide for more effective vaccines to prevent TB. This hypothesis is confirmed by our experimental data. Microarray analysis shows that 45 genes are upregulated (>2 fold) in BCG-Prague/pME: ? ;oP, including 7 genes belong to the PhoP regulon (Table 1). The low degree of overlap could be due to different levels of PhoP in these studies; the previous study compared the phoP mutant of M. tb with the wild type strain, whereas our experiments compared BCG-Prague (a phoP mutant) and BCG-Prague overexpressing phoP. An important finding relevant to the current patent application is that overexpression of phoP in BCG increases the expression of not only Ag85A and PPE18, but also 10 ESAT-6/CFP-10-like proteins (Esxl, J, K, L, M, N, O, P, V, W) and 7 other PE/PPE proteins (Table 1), many of which are known potent T cell antigens and several of them have been exploited as components of subunit vaccines (e.g., Ag85A, PPE18, EsxN) [35,38,40,41]. These results support my hypothesis that overexpression of phoP in BCG increases the expression of multiple potent T cell antigens. Our conclusion is further supported by the direct evidence that overexpression of phoP in a BCG strain such as BCG-Japan induced an elevated CD4⁺ T cell response; both the magnitude of PPD induced IFN-γ production (determined by ELISA) and the number of PPD-specific IFN-γ producing CD4⁺ T cells (determined by intracellular cytokine staining and flow cytometry) increased significantly (2 fold, /XO.001) compared to the BCG control (BCG-Japan) (Fig. 5). Since IFN-γ plays a critical role in the control of TB [22-25] and has been the most commonly used biomarker for TB vaccine selection [26], this result suggests that recombinant BCG strains that overexpress PhoP will have better protective efficacy than current BCG strains. A similar result was observed with BCG-Prague overexpressing phoP (Figure 5). These results support my hypothesis that phoP mutation in BCG-Prague is responsible for its low immunogenicity since overexpression of phoP in BCG-Prague restores the level to that of BCG-Japan. The presence of more antigenic proteins in BCG-Japan may explain the higher level of responses induced by BCG-Japan/pME:phoP than did BCG-Prague/pME:phoP, i.e., BCG-Japan is a 'early' BCG strain containing fewer genomic deletions (e.g., RD2) than 'late' strains such as BCG-Prague [58,59]. These results further support the key concept of my invention that overexpression of phoP in a BCG strain or other mycobacterium strains will enhance immunogenicity.

M. bovis BCG is also used in the treatment of bladder cancer. Numerous randomized controlled clinical trials indicate that intravesical administration of BCG can prevent or delay tumor recurrence [60]. The details of how BCG exerts this effect remain to be determined. However, the antitumour response requires an intact T-cell response, and involves increased expression of Thl-type cytokines, including TNF-a and IL-6 [61]. As such, a BCG strain demonstrating increased immunogenicity may provide enhanced antitumour activity.

In summary, we use recombinant BCG strains that overexpress PhoP and/or PhoP regulon protein(s) as vaccines to prevent TB and other mycobacterial infections. These recombinant BCG vaccines will induce better protective immunity against TB.

In the genetically engineered (i.e., recombinant) mycobacterium of the invention, the PhoP protein is over-expressed, i.e., the protein is expressed at a level that exceeds that of a suitable control organism, such as the same mycobacterium that has not been genetically engineered to overexpress PhoP. Those of skill in the art are well acquainted with comparative measurements of protein activity, and with the use of suitable standards and controls for such measurements.

The overexpression of PhoP or PhoP regulon protein(s) in a mycobacterium may be carried out by any suitable method known in the art. Generally, the method will involve linking nucleic acid sequences encoding the PhoP or PhoP regulon protein to expression control sequences that are not, in nature, linked to the phoP gene or a PhoP regulon gene. Those of skill in the art will recognize that many such expression control sequences are known and would be suitable for use in the invention. For example, if constitutive expression of phoP or a PhoP regulon gene is desired, expressioncontrol sequences (e.g., promoters and associated sequences) including but not limited to: mycobacterium optimal promoter: hsp65, ace or msp 12 promoter, T7 promoter, etc. Alternatively, overexpression of phoP may be inducible for example, under the tetracycline inducible promoters.

The proteins, polypeptides or peptides encoded according to the invention include naturally-occurring proteins, polypeptides or peptides, or the homologs thereof which have the same function as naturally-occurring proteins, polypeptides or peptides. Such homologs include proteins, polypeptides or peptides having at least 60%, preferably about 70% or more, 80% or more, and most preferably 90% or more, e.g., 95%, 96%, 97%, 98 or 99% homology to the amino acid sequences of the naturally-occurring proteins, polypeptides or peptides, e.g., to the amino acid sequence shown in SEQ ID NO: 1. Such homologs include proteins, polypeptides or peptides with substitution, addition and deletion of one or more (e.g., 1-50, 1-20, 1-10, 1-5) amino acid residues in the amino acid sequences of the naturally-occurring proteins, polypeptides or peptides (e.g., in the amino acid sequence shown in SEQ ID NO: 1). Such homologs include especially proteins, polypeptides or peptides with conserved amino acid substitution(s).

The term "PhoP" or "PhoP protein", as used therein, refers to the response regulator, PhoP, of the two-component regulatory system PhoP-PhoR, and is also a transcription factor, PhoP encoded according to the invention includes naturally-occurring, functional PhoPs, e.g., from genus Mycobacterium, preferably from Mycobacterium tuberculosis, or Mycobacterium bovis, or the homologs thereof as described above. An exemplary amino acid sequence of PhoP is presented in Figure 2 A SEQ ID NO: 1. The term "PhoP regulon", as used therein, refers to genes positively regulated by PhoP.

The term "overexpress", "overexpressing" or "overexpression", as used therein, refers to the protein level of the target gene is 2 or more fold than the endogenous level of the same protein in a bacterium, which can be carried out by genetic engineering such as the use of a multiple copy plasmids and/or the use of strong promoters.

Variations of Nucleic Acid Molecules

Modifications

Many modifications may be made to the nucleic acid molecule DNA sequences disclosed in this application and these will be apparent to one skilled in the art. The invention includes nucleotide modifications of the sequences disclosed in this application (or fragments thereof) that encode proteins or peptides in bacterial or mammalian cells which have the same function as the proteins or peptides disclosed in this application. Modifications include substitution, insertion or deletion of one or more (e.g., 1-50, 1-20, 1-10, 1-5) nucleotides or altering the relative positions or order of one or more (e.g., 1-50, 1-20, 1-10, 1-5) nucleotides.

Nucleic acid molecules may encode conservative amino acid changes in PhoP or a PhoP regulon protein. The invention includes functionally equivalent nucleic acid molecules that encode conservative amino acid changes and produce silent amino acid changes in PhoP and a protein of the PhoP regulon. Methods for identifying empirically conserved amino acid substitution groups are well known in the art (see for example, Wu, Thomas D. "Discovering Emperically Conserved Amino Acid Substitution Groups in Databases of Protein Families " (http://www.ncbi.nlm.nih. gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=88 77523&dopt=Abstract).

Nucleic acid molecules may encode non-conservative amino acid substitutions, additions or deletions in PhoP and/or a PhoP regulon gene. The invention includes functionally equivalent nucleic acid molecules that make non-conservative amino acid changes within the amino acid sequences in PhoP and/or a PhoP regulon protein. Functionally equivalent nucleic acid molecules include DNA and RNA that encode peptides, peptides and proteins having non-conservative amino acid substitutions (preferably substitution of a chemically similar amino acid), additions, or deletions but which also retain the same or similar function to the PhoP and PhoP regulon protein or peptide disclosed in this application. The invention includes the DNAs or RNAs encoding fragments or variants of PhoP and a PhoP regulon protein.

The fragments are useful as immunogens and in immunogenic compositions.

PhoP and PhoP regulon like-activity of such fragments and variants is identified by assays as described below.

Sequence Identity

The nucleic acid molecules of the invention also include nucleic acid molecules (or a fragment thereof) having at least about: 60% identity, at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity or, most preferred, at least 99% or 99.5% identity to a nucleic acid molecule of the invention and which are capable of expression of nucleic acid molecules in bacterial or mammalian cells. Identity refers to the similarity of two nucleotide sequences that are aligned so that the highest order match is obtained. Identity is calculated according to methods known in the art. For example, if a nucleotide sequence (called "Sequence A") has 90% identity to a portion of SEQ ID NO: 2, then Sequence A will be identical to the referenced portion of SEQ ID NO: 2 except that Sequence A may include up to 10 point mutations (such as substitutions with other nucleotides) per each 100 nucleotides of the referenced portion of SEQ ID NO: 2.

Sequence identity (each construct preferably without a coding nucleic acid molecule insert) is preferably set at least about: 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity or, most preferred, at least 99% or 99.5% identity to the sequences provided in SEQ ID NO: 2 or its complementary sequence). Sequence identity will preferably be calculated with the GCG program from Bioinformatics (University of Wisconsin). Other programs are also available to calculate sequence identity, such as the Clustal W program (preferably using default parameters; Thompson, JD et al, Nucleic Acid Res. 22:4673-4680), BLAST P, BLAST X algorithms, Mycobacterium avium BLASTN at The Institute for Genomic Research (http:tigrblast.tigr.org/), Mycobacterium bovis, M. Bovis BCG (Pastuer), M. marinum, M. leprae, M. tuberculosis BLASTN at the Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/Projects/MiciObes/). M. tuberculosis BLAST searches at Institute Pasterur (Tuberculist) (http://genolist.pasteur.fr/TubercuList/), M. leprae BLAST searches at Institute Pasteur (Leproma) (http :// genolist.pasteur.fr/Leproma/), M. Paratuberculosis BLASTN at Microbial Genome Project, University of Minnesota (http://www.cbc.umn.edu/ResearchProjects/Ptb/ and http://www.cbc.umn.edu/ResearchProiects/AGAC/Mptb/Mptbhome.html), various BLAST searches at the National Center for Biotechnology Information - USA (http://www.ncbi.nlm.nih.gov/BLAST/) and various BLAST searches at GenomeNet (Bioinformatics Center - Institute for Chemical Research) (http://blast.genome.ad.jp/).

Since the genetic code is degenerate, the nucleic acid sequence in SEQ ID N0:2 is not the only sequence which may code for a polypeptide having PhoP activity. This invention includes nucleic acid molecules that have the same essential genetic information as the nucleic acid molecules described in SEQ ID N0:2. Nucleic acid molecules (including RNA) having one or more nucleic acid changes compared to the sequences described in this application and which result in production of the polypeptides shown in SEQ ID N0: 1 are within the scope of the invention.

Other functional equivalent forms of PhoP and PhoP regulon protein(s)-encoding nucleic acids can be isolated using conventional DNA-DNA or DNA-RNA hybridization techniques.

Hybridization

The invention includes DNA that has a sequence with sufficient identity to a nucleic acid molecule described in this application to hybridize under stringent hybridization conditions (hybridization techniques are well known in the art). The present invention also includes nucleic acid molecules that hybridize to one or more of the sequences in [SEQ ID N0:2] or its complementary sequence. Such nucleic acid molecules preferably hybridize under high stringency conditions (see Sambrook et al. Molecular Cloning: A Laboratory Manual, Most Recent Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). High stringency washes have preferably low salt (preferably about 0.2% SSC) and a temperature of about 50-65 °C.

Vaccines

One skilled in the art knows the preparation of live recombinant vaccines. Typically, such vaccines are prepared as injectable, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified, or the protein encapsulated in liposomes. The live immunogenic ingredients are often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants that enhance the effectiveness of the vaccine. Examples of adjuvants which may be effective include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),

N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(r-2'-dipalmitoyl-sn

-glycero-3-hydroxyphosphoryloxy)-ethylamme (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80™ emulsion.

The effectiveness of an adjuvant may be determined by measuring the amount of antibodies directed against an immunogenic polypeptide containing a Mycobacterium tuberculosis antigenic sequence resulting from administration of the live recombinant Mycobacterium bovis- CG vaccines that are also comprised of the various adjuvants.

The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25%-70%.

The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective.

The vaccine may be given in a single dose schedule, or preferably in a multiple dose schedule. A multiple dose schedule is one in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months. The dosage regimen will also, at least in part, be determined by the need of the individual and be dependent upon the judgment of the practitioner.

In addition, the live recombinant Mycobacterium bovis-BCG vaccine administered in conjunction with other immunoregulatory agents, for example, immune globulins.

A subject of the present invention is also a multivalent vaccine formula comprising, as a mixture or to be mixed, a live recombinant Mycobacterium bovis-BCG vaccine as defined above with another vaccine, and in particular another recombinant live recombinant Mycobacterium bovis-BCG vaccine as defined above, these vaccines comprising different inserted sequences.

Pharmaceutical compositions

The pharmaceutical compositions of this invention are used for the treatmentment or prophylaxis of a mammal against challenge by Mycobacterium tuberculosis or Mycobacterium bovis. The pharmaceutical compositions of this invention are also used to treat patients having degenerative diseases, disorders or abnormal physical states such as cancer.

The pharmaceutical compositions can be administered to humans or animals by methods such as tablets, aerosol administration, intratracheal instillation and intravenous injection.

EXAMPLES

EXAMPLE 1. Construction of a BCG strain overexpressing the transcriptional regulator phoP.

A kanamycin resistant shuttle vector which contains a T7 promoter was obtained as follows. The pDrive cloning vector (obtained from Qiagen) was cut with EcoRl and self-ligated to generate pDRJ. The pDRI was digested with Sspl and the 1903 bp fragment product was isolated. The pMD31 shuttle vector (Wu et al, 1993, Molecular Microbiology, 7, 407-417) was digested with Sspl and the 3379 bp fragment was isolated. The two Sspl generated fragments were ligated to generate pME (5282 bp), which contains the original T7 promoter of the pDRIVE (see Figure 3).

The wild-type M. tuberculosis phoP gene was amplified from M. tb H37Rv strain (ATCC 25618) using the forward primer (5'-AAAAAGGJACCGCTTGTTTGGCCATGTCAAC-3') and reverse primer (5'-AAAAACTGCAGGCTGCCGATCCGATTAACTAC-3') containing a kpnl and a pstl restriction site (underlined), respectively. The forward primer was designed so that it contains 257 bp upstream of phoP start codon, which allows the proper expression of phoP gene. The PCR reaction was performed using genomic DNA from wild type M. tb H37Rv as a template. The resulting PCR products were digested with Kpnl and Pstl and the resulting fragment was cloned into vector pME, thereby creating a plasmid designated pME:phoP (Figure 4). The construct was confirmed by DNA sequencing.

Generation of a recombinant BCG was accomplished as follows: Cells of M. bovis BCG-Japan (ATCC 35737) and M. bovis BCG-Prague (ATCC 35742) were transformed with plasmid pME:phoP by electroporation, and electroporated cells were plated onto 7H11 media plates supplemented with 10% OADC (Difco) and 25 μg/ml of kanamycin. After 4 weeks of incubation at 37°C, individual colonies were selected and grown in 7H9 liquid media supplemented with 10% ADC (Difco) plus 25 μg/ml of kanamycin. After culturing for 3 weeks in this media, plasmid DNA from the selected clones was isolated and checked by restriction digestion and gel electrophoresis. All the colonies gave a plasmid DNA of a size and restriction digestion pattern indicative of the transformation of the construct, thus constituting the new recombinant BCG-Japan/pME:phoP and recombinant BCG-Prague/pME:phoP. Frozen stocks of BCG-Japan/pME:phoP and BCG-Prague/pME:phoP were subsequently made by mixing 1 ml of culture with 1 ml of 50% sterile glycerol solution and stored at -80°C. EXAMPLE 2. Determination of gene induction in recombinant BCG-Prague overexpressing phoP.

In order to determine the effect of phoP overexpression on gene expression in BCG, microarray analysis was carried out to compare the transcriptional profiles of BCG-Prague/pME:phoP and wild type BCG-Prague harboring the empty vector pME. The strains were grown in 7H9 liquid media supplemented with 10% ADC (Difco) and 0.05% Tween 80 at 37°C until OD₆₀o reaches 0.4 to 0.5. To isolate total RNA, cells were pelleted and transferred to 2-ml screw cap tubes containing 1 ml RNA protect Bacterial Reagent (Qiagen) and incubated for 5 min at room temperature. Cells were again pelleted and resuspended in 400 μΐ lysis buffer (20 mM NaCH₃COOH, 0.5% SDS, ImM EDTA, pH 4) and 1 ml phenol/chloroform (pH 4.5, Sigma). Cells were disrupted by bead beating with glass beads by three 30-sec pulses. They were then incubated at 65°C for 4 min and then at 4°C for 5 min before being centrifuged at 13,000 rpm for 5 min. The supernatant was then extracted with 300 μΐ of chloroform/isoamyl alcohol (24: 1) and precipitated with isopropanol. Total RNA was then isolated by standard procedures.

For cDNA production 25μg total RNA was reverse transcribed at 42°C overnight using 2 μΐ Superscript II reverse transcriptase (Invitrogen), 25 g 9-mer random primers and 2 ul dNTP mix (0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.25 mM dTTP, 0.25 mM 5-(3-aminoalyl)-dUTP) in a total volume of 100 μΐ (25 mM Tris pH 8.4, 37.5 mM KC1, 3 mM MgCl₂, and 0.1 M DTT). RNA hydrolysis was performed by adding 1/3 volume 1M NaOH and then neutralized with 1/3 volume HC1 after incubating for 20 min at 65°C. The cDNA was purified using a QIAquick column (Qiagen). cDNA were labelled for 1 hr at RT and then quenched with 4 M hydroxylamine. The labelled cDNA was purified and 2 μg per sample were hybridized to a 15,000-feature M. tb H37Rv ORF array with three distinct probes per ORF (Agilent Technologies) and scanned using the Genepix Professional 4200A scanner. Significance Analysis of Microarrays (SAM) was performed to identify genes that are significantly upregulated or downregulated and the results are presented in Table 1.

The results showed that the recombinant BCG strain carrying the pME:phoP plasmid displayed enhanced expression of 45 genes including well known T cell antigens Ag85A, 10 ESAT-6/CFP-10-like proteins (Esxl, J, K, L, M, N, O, P, V, W), PPE18 and 7 other PE/PPE proteins, when compared to the control BCG carrying the empty vector.

EXMPLE 3. Determination of the capacity of the engineered recombinant BCG to induce antigen specific immune response in mice.

The capacity of the recombinant BCG overexpressing phoP and the parental BCG to elicit a cellular immune response was compared in mice. C57BL/6 mice (4 per group) were immunized with 5 x 10⁵ CFU of recombinant BCG (BCG-Japan/pME:phoP, BCG-Prague/pME:phoP), the corresponding BCG control (BCG-Japan/pME, BCG-Prague/pME), or PBS (naive mice). Eight weeks post-vaccination, mice were sacrificed and splenocytes prepared. For ELISA analysis, splenocytes were plated in triplicate on 96-well plates at 2.5 x 10⁵ cells/well and cultured with or without PPD (obtained from Statens Serum Institute) at 10 μg/ml for 72 h. Three days after antigen stimulation, the cell supernatants were harvested and the productions of IFN-γ were determined by ELISA using the OptEIA™ ELISA Kit (BD Biosciences) with appropriate mAbs. To determine the phenotype and frequency of IFN-γ producing T cell population, intracellular cytokine staining (ICCS) and flow cytometry were performed. For ICCS analysis, splenocytes were stimulated with PPD (obtained from Statens Serum Institute) at 10 g/ml for 24 hr, then washed and incubated with CD16/CD32 monoclonal antibody to block Fc binding. Subsequently, cells are stained for appropriate surface markers and intracellular cytokines such as CD3, CD4, CD8, CD44, IFN-γ, TNF, and IL-2 using a BD Cytofix/Cytoperm kit and then subjected for FACS analysis.

The results, presented in Figure 4, indicated that recombinant BCG overexpressing phoP induce elevated cellular immune response: both the magnitude of PPD induced IFN-γ production (determined by ELISA) and the number of PPD-specific IFN-γ producing CD4⁺ T cells (determined by ICCS) increased significantly compared to the BCG control.

These studies provide direct immunological evidence that the phoP overexpressing BCG strain is capable of eliciting a stronger cellular immune response than the parental BCG as evidenced by antigen-specific T cell responses in mice. Since PPD-specific IFN-γ production is the most important biomarker for vaccine selection and an important measure of anti-mycobacterial immunity, the engineered recombinant BCG will provide a greater protection of vaccinated host against M. tb challenge than does the parental BCG.

The present invention has been described in detail and with particular reference to the preferred embodiments and Examples; however, it will be understood by one having ordinary skill in the art that changes can be made without departing from the spirit and scope thereof. For example, where the application refers to proteins, it is clear that peptides and polypeptides may often be used. Likewise, where a gene is described in the application, it is clear that nucleic acids or gene fragments may often be used.

All publications (including Genbank entries), patents and patent applications are incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Reference List

1. Anonymous(2004). BCG vaccine. WHO position paper. Wkly Epidemiol Rec 79, 27-38.

2. Baldwin, S.L., D'Souza, C, Roberts, A.D., Kelly, B.P., Frank, A.A., Lui, M.A., Ulmer, J.B., Huygen, K., McMurray, D.M., and Orme, I.M.(1998). Evaluation of new vaccines in the mouse and guinea pig model of tuberculosis. Infect Immun 66, 2951-2959.

3. Colditz, G.A., Berkey, C.S., Mosteller, F., Brewer, T.F., Wilson, M.E., Burdick, E., and Fineberg, H.V.(1995). The efficacy of bacillus Calmette-Guerin vaccination of newborns and infants in the prevention of tuberculosis: meta-analyses of the published literature. Pediatrics 96, 29-35.

4. Colditz, G.A., Brewer, T.F., Berkey, C.S., Wilson, M.E., Burdick, E., Fineberg, H.V., and Mosteller, F.(1994). Efficacy of BCG vaccine in the prevention of tuberculosis.

Meta-analysis of the published literature. JAMA 271, 698-702.

5. Fine, P.E.(1995). Variation in protection by BCG: implications of and for heterologous immunity. Lancet 346, 1339-1345.

6. Sterne, J.A., Rodrigues, L.C., and Guedes, I.N. (1998). Does the efficacy of BCG decline with time since vaccination? Int J Tuberc Lung Dis 2, 200-207.

7. Andersen, P. (2001). TB vaccines: progress and problems. Trends Immunol 22, 160-168.

8. Behr, M.A.(2002). BCG~different strains, different vaccines? Lancet Infect Dis 2, 86-92.

9. Comstock, G. W.(l 994). Field trials of tuberculosis vaccines: how could we have done them better? Control Clin Trials 15, 247-276.

10. Demangel, C, Garnier, T., Rosenkrands, I., and Cole, S.T.(2005). Differential effects of prior exposure to environmental mycobacteria on vaccination with Mycobacterium bovis BCG or a recombinant BCG strain expressing RD 1 antigens. Infect Immun 73, 2190-2196.

1 1. Tsenova, L., Harbacheuski, R., Sung, N., Ellison, E., Fallows, D., and Kaplan, G.(2007).

BCG vaccination confers poor protection against M. tuberculosis FIN878-induced central nervous system disease. Vaccine 25, 5126-5132.

12. Behr, M.A. and Small, P.M.(1997). Has BCG attenuated to impotence? Nature 389,

133-134.

13. Orme, I.M.(2001). The search for new vaccines against tuberculosis. J Leukoc Biol 70,

1-10.

14. Young, D.B.(2000). Current tuberculosis vaccine development. Clin Infect Dis 30 Suppl 3, S254-S256

15. Skeiky, Y.A. and Sadoff, J. C. (2006). Advances in tuberculosis vaccine strategies. Nat Rev Microbiol 4, 469-476. 16. Horwitz, M.A., Harth, G., Dillon, B.J., and Maslesa-Galic', S.(2000). Recombinant bacillus calmette-guerin (BCG) vaccines expressing the Mycobacterium tuberculosis 30-kDa major secretory protein induce greater protective immunity against tuberculosis than conventional BCG vaccines in a highly susceptible animal model. Proc Natl Acad Sci USA 91, 13853-13858.

17. Grode, L., Seiler, P., Baumann, S., Hess, J., Brinkmann, V., Nasser, E.A., Mann, P.,

Goosmann, C, Bandermann, S., Smith, D., Bancroft, G.J., Reyrat, J.M., van Soolingen, D., Raupach, B., and Kaufmann, S.H.(2005). Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette-Guerin mutants that secrete listeriolysin. J Clin Invest 115, 2472-2479.

18. Martin, C, Williams, A., Hernandez-Pando, R., Cardona, P. J., Gormley, E., Bordat, Y., Soto, C.Y., Clark, S.O., Hatch, G.J., Aguilar, D., Ausina, V., and Gicquel, B.(2006). The live Mycobacterium tuberculosis phoP mutant strain is more attenuated than BCG and confers protective immunity against tuberculosis in mice and guinea pigs. Vaccine 24, 3408-3419.

19. Sambandamurthy, V.K., Derrick, S.C, Jalapathy, K.V., Chen, B., Russell, R.G., Morris, S.L., and Jacobs, W.R.J.(2005). Long-term protection against tuberculosis following vaccination with a severely attenuated double lysine and pantothenate auxotroph of Mycobacterium tuberculosis. Infect Immun 73, 1 196-1203.

20. Cooper, A.M.(2009). Cell-mediated immune responses in tuberculosis. Annu Rev

Immunol 27, 393-422.

21. North, R.J. and Jung, Y.J.(2004). Immunity to tuberculosis. Annu Rev Immunol 22,

599-623.

22. Cooper, A.M., Dalton, D.K., Stewart, T.A., Griffin, J.P., Russell, D.G., and Orme,

I.M.(1993). Disseminated tuberculosis in interferon gamma gene-disrupted mice. J Exp Med 178, 2243-2247.

23. Flynn, J.L., Chan, J., Triebold, K.J., Dalton, D.K., Stewart, T.A., and Bloom, B.R.(1993).

An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection. J Exp Med 178, 2249-2254.

24. Jouanguy, E., Altare, F., Lamhamedi, S., Revy, P., Emile, J.F., Newport, M., Levin, M., Blanche, S., Seboun, E., Fischer, A., and Casanova, J.L.(1996). Interferon-gamma-receptor deficiency in an infant with fatal bacille Calmette-Guerin infection. N Engl J Med 335, 1956-1961.

25. Newport, M.J., Huxley, CM., Huston, S., Hawrylowicz, CM., Oostra, B.A., Williamson, R., and Levin, M.(1996). A mutation in the interferon-gamma-receptor gene and susceptibility to mycobacterial infection. N Engl J Med 335, 1941-1949. 26. Hanekom, W.A., Dockrell, H.M., Ottenhoff, T.H., Doherty, T.M., Fletcher, H., McShane, H., Weichold, F.F., Hoft, D.F., Parida, S.K., and Fruth, U.J.(2008). Immunological outcomes of new tuberculosis vaccine trials: WHO panel recommendations. PLoS Med 5, el45-Hanekom, Willem.

27. Boesen, H., Jensen, B.N., Wilcke, T., and Andersen, P. (1995). Human T-cell responses to secreted antigen fractions of Mycobacterium tuberculosis. Infect Immun 63, 1491-1497. 28. Horwitz, M.A., Lee, B.W., Dillon, B J., and Harth, G.(1995). Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis. Proc Natl Acad Sci USA 92, 1530-1534.

29. Pal, P.G. and Horwitz, M.A.(1992). Immunization with extracellular proteins of

Mycobacterium tuberculosis induces cell-mediated immune responses and substantial protective immunity in a guinea pig model of pulmonary tuberculosis. Infect Immun 60, 4781-4792.

30. Roberts, A.D., Sonnenberg, M.G., Ordway, D.J., Furney, S.K., Brennan, P.J., Belisle, J.T., and Orme, I.M.(1995). Characteristics of protective immunity engendered by vaccination of mice with purified culture filtrate protein antigens of Mycobacterium tuberculosis.

Immunology 85, 502-508.

31. Borremans, M., de Wit, L., Volckaert, G., Ooms, J., de Bruyn, J., Huygen, K., van Vooren, J.P., Stelandre, M., Verhofstadt, R., and Content, J.(1989). Cloning, sequence

determination, and expression of a 32-kilodalton-protein gene of Mycobacterium tuberculosis. Infect Immun 57, 3123-3130.

32. Bassey, E.O., Life, P.F., Catty, D., Gaston, J.S., and Kumararatne, D.S.(1996). T-cell response to mycobacterial proteins: a comparative study of tuberculous and control immunoblots of Mycobacterium tuberculosis and M. bovis BCG. Tuber Lung Dis 77,

146-153.

33. Brandt, L., Elhay, M., Rosenkrands, I., Lindblad, E.B., and Andersen, P.(2000). ESAT-6 subunit vaccination against Mycobacterium tuberculosis. Infect Immun 68, 791-795.

34. Brodin, P., Majlessi, L., Brosch, R., Smith, D., Bancroft, G., Clark, S., Williams, A.,

Leclerc, C, and Cole, S.T.(2004). Enhanced protection against tuberculosis by vaccination with recombinant Mycobacterium microti vaccine that induces T cell immunity against region of difference 1 antigens. J Infect Dis 190, 1 15-122.

35. Dietrich, J., Aagaard, C, Leah, R., Olsen, A.W., Stryhn, A., Doherty, T.M., and Andersen, P.(2005). Exchanging ESAT6 with TB I 0.4 in an Ag85B fusion molecule-based

tuberculosis subunit vaccine: efficient protection and ESAT6-based sensitive monitoring of vaccine efficacy. J Immunol 174, 6332-6339. 36. Coler, R.N., Skeiky, Y.A., Vedvick, T., Bement, T., Ovendale, P., Campos-Neto, A., Alderson, M.R., and Reed, S.G.(1998). Molecular cloning and immunologic reactivity of a novel low molecular mass antigen of Mycobacterium tuberculosis. J Immunol 161 ,

2356-2364.

37. Weinrich, O.A., van Pinxteren, L.A., Meng, O.L., Birk, R.P., and Andersen, P.(2001).

Protection of mice with a tuberculosis subunit vaccine based on a fusion protein of antigen 85b and esat-6. Infect Immun 69, 2773-2778.

38. Skeiky, Y.A., Alderson, M.R., Ovendale, P.J., Guderian, J.A., Brandt, L., Dillon, D.C., Campos-Neto, A., Lobet, Y., Dalemans, W., Orme, I.M., and Reed, S.G.(2004).

Differential immune responses and protective efficacy induced by components of a tuberculosis polyprotein vaccine, Mtb72F, delivered as naked DNA or recombinant protein. J Immunol 172, 7618-7628.

39. Langermans, J.A., Doherty, T.M., Vervenne, R.A., van der Laan, T., Lyashchenko, K., Greenwald, R., Agger, E.M., Aagaard, C, Weiler, H., van Soolingen, D., Dalemans, W., Thomas, A.W., and Andersen, P. (2005). Protection of macaques against Mycobacterium tuberculosis infection by a subunit vaccine based on a fusion protein of antigen 85B and ESAT-6. Vaccine 23, 2740-2750.

40. Verreck, F.A., Vervenne, R.A., Kondova, I., van Kralingen, K.W., Remarque, E.J.,

Braskamp, G., van der Werff, N.M., Kersbergen, A., Ottenhoff, T.H., Heidt, P. J., Gilbert, S.C., Gicquel, B., Hill, A.V., Martin, C, McShane, H, and Thomas, A.W.(2009).

MVA.85A boosting of BCG and an attenuated, phoP deficient M. tuberculosis vaccine both show protective efficacy against tuberculosis in rhesus macaques. PLoS One 4, e5264 41. Magalhaes, I., Sizemore, D.R., Ahmed, R.K., Mueller, S., Wehlin, L., Scanga, C,

Weichold, F._s Schirru, G., Pau, M.G., Goudsmit, J., Kuhlmann-Berenzon, S., Spangberg, M., Andersson, J., Gaines, H., Thorstensson, R., Skeiky, Y.A., Sadoff, J., and Maeurer, M.(2008). rBCG induces strong antigen-specific T cell responses in rhesus macaques in a prime-boost setting with an adenovirus 35 tuberculosis vaccine vector. PLoS One 3, e3790 42. Milstien, J.B. and Gibson, J.J.(1990). Quality control of BCG vaccine by WHO: a review of factors that may influence vaccine effectiveness and safety. Bull World Health Organ 68, 93-108.

43. Castillo-Rodal, A.I., Castanon-Arreola, M., Hernandez-Pando, R., Calva, J.J., Sada-Diaz, E., and Lopez-Vidal, Y.(2006). Mycobacterium bovis BCG substrains confer different levels of protection against Mycobacterium tuberculosis infection in a BALB/c model of progressive pulmonary tuberculosis. Infect Immun 74, 1718-1724.

44. Lalor, M.K., Ben-Smith, A., Gorak-Stolinska, P., Weir, R.E., Floyd, S., Blitz, R., Mvula, H., Newport, M.J., Branson, K., McGrath, N., Crampin, A.C., Fine, P.E., and Dockrell, H.M.(2009). Population differences in immune responses to Bacille Calmette-Guerin vaccination in infancy. J Infect Dis 199, 795-800.

45. Gallant, C.J., Cobat, A., Simkin, L., Black, G.F., Stanley, K., Hughes, J., Doherty, T.M., Hanekom, W.A., Eley, B., Beyers, N., Jais, J.P., van Helden, P., Abel, L., Alcais, A., Hoal, E.G., and Schurr, E.(2009). Tuberculin skin test and in-vitro assays provide

complementary measures of anti-mycobacterial immunity in children and adolescents. Chest

46. Ladefoged, A., Bunch-Christensen, K., and Guld, J.(1976). Tuberculin sensitivity in

guinea-pigs after vaccination with varying doses of BCG of 12 different strains. Bull World Health Organ 53, 435-443.

47. Vallishayee, R.S., Shashidhara, A.N., Bunch-Christensen, K., and Guld, J.(1974).

Tuberculin sensitivity and skin lesions in children after vaccination with 1 1 different BCG strains. Bull World Health Organ 51, 489-494.

48. Leung, A.S., Tran, V., Wu, Z., Yu, X., Alexander, D.C., Gao, G.F., Zhu, B., and Liu, J.(2008). Novel genome polymorphisms in BCG vaccine strains and impact on efficacy. BMC Genomics 9, 413

49. Lee, J.S., Krause, R., Schreiber, J., Mollenkopf, H.J., Kowall, J., Stein, R., Jeon, B.Y., Kwak, J.Y., Song, M.K., Patron, J.P., Jorg, S., Roh, K., Cho, S.N., and Kaufmann, S.H.(2008). Mutation in the transcriptional regulator PhoP contributes to avirulence of Mycobacterium tuberculosis H37Ra strain. Cell Host Microbe 3, 97-103.

50. Martin, C, Williams, A., Hernandez-Pando, R., Cardona, P J., Gormley, E., Bordat, Y., Soto, C.Y., Clark, S.O., Hatch, G.J., Aguilar, D., Ausina, V., and Gicquel, B.(2006). The live Mycobacterium tuberculosis phoP mutant strain is more attenuated than BCG and confers protective immunity against tuberculosis in mice and guinea pigs. Vaccine 24, 3408-3419.

51. Walters, S.B., Dubnau, E., Kolesnikova, I., Laval, F., Daffe, M., and Smith, 1.(2006). The Mycobacterium tuberculosis PhoPR two-component system regulates genes essential for virulence and complex lipid biosynthesis. Mol Microbiol 60, 312-330.

52. Gao, Q., Kripke, K., Arinc, Z., Voskuil, M., and Small, P. (1909). Comparative expression studies of a complex phenotype: cord formation in Mycobacterium tuberculosis.

Tuberculosis (Edinb) 84, 188-196.

53. Lin, M.Y. and Ottenhoff, T.H.(2008). Not to wake a sleeping giant: new insights into host-pathogen interactions identify new targets for vaccination against latent

Mycobacterium tuberculosis infection. Biol Chem 389, 497-51 1. 54. Horwitz, M.A. and Harth, G.(2003). A new vaccine against tuberculosis affords greater survival after challenge than the current vaccine in the guinea pig model of pulmonary tuberculosis. Infect Immun 71, 1672-1679.

55. Horwitz, M.A.(2005). Recombinant BCG expressing Mycobacterium tuberculosis major extracellular proteins. Microbes Infect 7, 947-954.

56. Castillo-Rodal, A.I., Castanon-Arreola, M., Hernandez-Pando, R., Calva, J. J., Sada-Diaz, E., and Lopez-Vidal, Y.(2006). Mycobacterium bovis BCG substrains confer different levels of protection against Mycobacterium tuberculosis infection in a BALB/c model of progressive pulmonary tuberculosis. Infect Immun 74, 1718-1724.

57. Gao, L.Y., Guo, S., McLaughlin, B., Morisaki, H., Engel, J.N., and Brown, E.J.(2004). A mycobacterial virulence gene cluster extending RD 1 is required for cytolysis, bacterial spreading and ESAT-6 secretion. Mol Microbiol 53, 1677-1693.

58. Behr, M.A., Wilson, M.A., Gill, W.P., Salamon, H., Schoolnik, G.K., Rane, S., and Small, P.M.(1999). Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science 284, 1520-1523.

59. Liu, J., Tran, V., Leung, A.S., Alexander, D.C., and Zhu, B.(2009). BCG vaccines: Their mechanisms of attenuation and impact on safety and protective efficacy. Hum Vaccin 5, 2:70-78.

60. Lamm, D.L.(2000). Efficacy and safety of bacille Calmette-Guerin immunotherapy in superficial bladder cancer. Clin Infect Dis 31 Suppl 3, S86-S90

61. Prescott, S., Jackson, A.M., Hawkyard, S.J., Alexandroff, A.B., and James, K.(2000).

Mechanisms of action of intravesical bacille Calmette-Guerin: local immune mechanisms. Clin Infect Dis 31 Suppl 3, S91-S93

Claims

Claims

1. A live recombinant Mycobacterium bovis~ CG strain comprising a nucleic acid capable of overexpression, the nucleic acid encoding PhoP protein.

2. A live recombinant Mycobacterium bovis- CG strain of claim 1 , wherein the PhoP protein is from Mycobacterium tuberculosis or Mycobacterium bovis.

3. A live recombinant Mycobacterium bovis-BCG strain of claim 1, wherein the nucleic acid encodes

(i) the amino acid sequence shown in SEQ ID NO: l ; or

(ii) a PhoP protein with the substitution, addition, or deletion of one or more amino acids in the amino acid sequence shown in SEQ ID NO: l .

4. A live recombinant Mycobacterium bovis-BCG strain of claim 1, wherein the nucleic acid comprises

(i) the nucleotide sequence shown in SEQ ID NO:2;

(ii) a sequence that hybridize to the nucleotide sequence of (i) under a stringent

hybridization condition;

(Hi) a sequence having at least 60% sequence identity to the nucleotide sequence shown in SEQ ID NO:2.

5. A live recombinant Mycobacterium bovis-BCG strain comprising a nucleic acid capable of overexpression, the nucleic acid encoding at least one protein or polypeptide selected from the group consisting of Rv0440, Rv0904c, Rv0981 , Rvl057, Rvl l 80, Rvl l82, Rvl l83, Rvl l 84c, Rvl l 85c, Rvl l95, Rvl l96, Rvl361c, Rvl639c, Rvl931c, Rv2227, Rv2276, Rv2288, Rv2289, Rv2329c, Rv2332, Rv2375, Rv2376c, Rv2391 , Rv2392, Rv2396, Rv2590, Rv2987c, Rv3135, Rv3136, Rv3197, Rv3312A, Rv3331 , Rv3332, Rv3343c, Rv3477, Rv3478, Rv3479, Rv3486, Rv3487c, Rv3613c, Rv3686c, Rv3689, Rv3767c, Rv3804c, Rv3822, Rv3823c, Rv3824c, and Rv3825c.

6. A live recombinant Mycobacterium bovis-BCG strain comprising a nucleic acid capable of overexpression, the nucleic acid comprises all or part of at least one nucleic acid molecule selected from the group consisting of Rv0440, Rv0904c, Rv0981, Rvl057, Rvl l 80, Rvl l 82, Rvl l 83, Rvl l 84c, Rvl l 85c, Rvl l 95, Rvl l 96„Rvl361c, Rvl639c, Rvl931c, Rv2227, Rv2276, Rv2288, Rv2289, Rv2329c, Rv2332, Rv2375, Rv2376c, Rv2391, Rv2392, Rv2396, Rv2590, Rv2987c, Rv3135, Rv3136, Rv3197, Rv3312A, Rv3331, Rv333¾ Rv3343c, Rv3477, Rv3478, Rv3479, Rv3486, Rv3487c, Rv3613c, Rv3686c, Rv3689, Rv3767c, Rv3804c, Rv3822, Rv3823c, Rv3824c, and Rv3825c.

7. A live recombinant Mycobacterium bovis- CG strain comprising a nucleic acid capable of overexpression, the nucleic acid comprises a sequence having at least 60% sequence identity to at least one nucleic acid molecule selected from the group consisting of Rv0440, Rv0904c, Rv0981, Rvl057, Rvl l 80, Rvll82, Rvl l83, Rvl l 84c, Rvl l 85c, Rvl l95, Rvl l96, Rvl361c, Rvl639c, Rvl 931c, Rv2227, Rv2276, Rv2288, Rv2289, Rv2329c, Rv2332, Rv2375, Rv2376c, Rv2391 , Rv2392, Rv2396, Rv2590, Rv2987c, Rv3135, Rv3136, Rv3197, Rv3312A, Rv3331, Rv3332, Rv3343c, Rv3477, Rv3478₅ Rv3479, Rv3486, Rv3487c, Rv3613c, Rv3686c, Rv3689, Rv3767c, Rv3804c, Rv3822, Rv3823c, Rv3824c, and Rv3825c.

8. The live recombinant Mycobacterium bovis-BCG strain of any one of claims 1 to 5 wherein the nucleic acid molecule has undergone modification.

9. The live recombinant Mycobacterium bovis-BCG strain of any one of claims 1 to 5 wherein the Mycobacterium bovis-BCG strain is selected from existing BCG strains, including but not limited to: Mycobacterium 0ovw-BCG-Russia (ATCC number: 35740),

Mycobacterium ^ovw-BCG-Moreau (ATCC number: 35736), Mycobacterium

bovis-BCG- Japan (ATCC number: 35737), Mycobacterium bovis-BCG-Sxveden (ATCC number: 35732), Mycobacterium 6ovw-BCG-Birkhaug (ATCC number: 35731),

Mycobacterium ¾ovw-BCG-Prague (ATCC number: 35742), Mycobacterium

bovis-BCG-Glaxo (ATCC number: 35741), Mycobacterium foviJ-BCG-Denmark (ATCC number: 35733), Mycobacterium ^ovw-BCG-Tice (ATCC numbers: 35743, 27289),

Mycobacterium ftovis-BCG-Frappier (ATCC: 35746, SM-R; ATCC: 35747, INH-R),

Mycobacterium fovw-BCG-Connaught (ATCC: 35745), Mycobacterium fovw-BCG-Phipps (ATCC number: 35744), Mycobacterium 6ovw-BCG-Pasteur (ATCC number: 35734),

BCG-Mexican (ATCC number: 35738) and Mycobacterium δον/^'-f-BCG-China (Shanghai Institute of Biological Product).

10. A pharmaceutical composition comprising the live recombinant Mycobacterium

bovis-BCG strain of any one of claims 1 to 7.

11. A vaccine or immunogenic composition for treatment or prophylaxis of a mammal against challenge by mycobacteria comprising the live recombinant Mycobacterium bovis-BCG strain of any one of claims 1 to 7.

12. The vaccine or immunogenic composition of claim 9 wherein the mycobacteria is Mycobacterium tuberculosis or Mycobacterium bovis.

13. The vaccine or immunogenic composition of claim 8 or 9 further comprising a pharmaceutically acceptable carrier.

14. The vaccine or immunogenic composition of claim 8 or 9 further comprising an adjuvant.

15. The vaccine or immunogenic composition of any one of claims 8 to 12 further comprising immunogenic materials from one or more other pathogens.

16. A method for treatment or prophylaxis of a mammal against challenge by Mycobacterium tuberculosis or Mycobacterium bovis comprising administering to the mammal the live recombinant Mycobacterium bovis-BCG strain of any one of claims 1 to 7.

17. The method of claim 14 wherein the mammal is a cow.

18. The method of claim 14 wherein the mammal is a human.

19. The method of claim 14 wherein the vaccine or immunogenic composition is administered in the presence of an adjuvant.

20. A method for treatment or prophylaxis of a mammal against cancer comprising administering to the mammal the live recombinant Mycobacterium bovis-BCG strain of any one of claims 1 to 7.

21. The method of claim 18 wherein the vaccine or immunogenic composition is administered in the presence of an adjuvant.

22. The method of claim 18 or 19 wherein the cancer is bladder cancer.