Patent Document

SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 29, 2016, is named 12775.0001_SL.txt and is 33,087 bytes in size. 
     The present invention refers to recombinant  Mycobacterium  strains that encode the mutant  Escherichia coli  heat-labile toxin LT or the A subunit of the mutant  Escherichia coli  heat-labile toxin 
     This invention also refers to strains of  Mycobacterium  that encode the mutant heat-labile toxin LT or the A subunit of the heat-labile toxin LT of  Escherichia coli  in position 63. 
     Specifically, this invention refers to strains of  Mycobacterium  that encode the mutant heat-labile toxin LT or the A subunit of the heat-labile toxin LT of  Escherichia coli  from serine to lysine at position 63. 
     This invention also provides immunogenic compositions that include the strains of the present invention. 
     This invention also provides for the use of the above mentioned strains and immunological compositions in the production of a vaccine for prevention against  tuberculosis  and infections caused by  Mycobacterium tuberculosis.    
     Finally, the present invention refers to methods for preventing or treating  tuberculosis  in animals. 
     GROUNDS OF THE INVENTION 
     Mycobacteria 
     Mycobacteria compose the genus  Mycobacterium  of the family Mycobacteriacae of the order Actinomycetales of the class Actinomycetes. The mycobacteria that form this genus are shaped like slightly curved rods, measuring from 1 to 10 μm in length and 0.2 to 0.6 μm in diameter. These bacteria cannot be classified as either Gram-positive or Gram-negative. The most striking feature of this  bacillus  is the complex nature of its cell envelope, containing a relatively high percentage of lipids, which include the long chains of mycolic acid. This envelope confers strong hydrophobicity, making it resistant to lysis and relatively impermeable to antibiotics and other chemical agents. The bacilli are labeled acid-alcohol resistant, i.e., they are resistant to discoloration caused by weak acids after staining with Fuchsine or similar dyes. The genomic DNA contains a high content of guanosine/cytosine, between 58-79%, which impairs the use of the bacterium  Escherichia coli  as a genetic host. These aspects are considered basic characteristics for identifying the  bacillus  as a member of the genus  Mycobacterium  [ORME, I. (1995) Medical Intelligent Unit: Immunity to Mycobacteria, Austin: R. G. Lands Company, p. 5; JAWETZ, S. MELNICK, J. L. ADELBERG, E. A. (1995) Medical Microbiology. 21 st  Ed. Appleton &amp; Lange, Stamford, Conn.; SHINNICK, T. M. &amp; GOOD, R. C. (1994) Mycobacterial taxonomy. Eur J Clin Microbiol Infect M Dis 11: 884-901]. 
     Mycobacteria have been classified in two main groups: slow-growing bacteria, which have a generation time of around 13 hours and can take from 3 weeks to 3 months of culturing to provide visible colonies; and the fast-growing bacteria, which have a generation time of around 2-5 hours. The slow-growing mycobacteria include many of the greatest animal and human pathogens, such as  M. tuberculosis, M. bovis, M. paratuberculosis, M. avium, M. leprae , etc., while the fast-growing ones include non pathogenic species, such as  M. smegmatis, M. aurum, M. vaccae , etc. Four of the five pathogenic species of mycobacteria are grouped in the  M. tuberculosis  complex—a group of four species that can cause the disease tuberculosis ( M. tuberculosis, M. bovis, Mycobacterium microtti  and  M. africanum ); the fifth is  M. leprae , the agent that causes Hansen&#39;s disease (informally known as leprosy) [SHINNICK, T. M. &amp; GOOD, R. C. (1994) Mycobacterial taxonomy. Eur J Clin Microbiol Infect Dis 11: 804-901; ORME, I. (1995) Medical Intelligent Unit: Immunity to Mycobacteria. Austin: R. G. Lands Company, p. 5; CONNELL, N. D. (2001) Expression systems for use in Actinomycetes and related organisms.  Current Opinion in Biotechnology  12: 446-449]. 
       M. tuberculosis  (MTB) is transmitted primarily through the respiratory tract and although it can cause diseases in several organs, pulmonary  tuberculosis  is the most common. It is estimated that a third of the world population is infected with the  bacillus  and that 200 million will present symptoms of  tuberculosis , of whom 35 million may die by 2020 if control and prevention measures are not taken (WHO Annual Publication, 2000). 
     Prophylaxis of  Tuberculosis  and Use of  Bacillus  Calmette-Guérin (BCG) 
     At the beginning of the last century, Albert Calmette and Camille Guérin attenuated a virulent strain of  Mycobacterium bovis . This strain is currently known as  Bacillus  Calmette-Guérin (BCG), and is the only  tuberculosis  vaccine currently used successfully, having already been administered to more than three billion individuals worldwide. 
     Nevertheless, its efficacy against the adult form of  tuberculosis  has given rise to controversy, as it can range from 0-80%, depending on the study [Andersen, P. and Doherty, T. M. (2005) The success and failure of BCG—implications for a novel  tuberculosis  vaccine. Nat Rev Microbiol. 3: 656-662]. 
     Hence, several efforts have been undertaken in the development of new vaccines, based on a) recombinant proteins or dominant MTB antigens; b) the expression of these antigens in various vectors, such as viral vectors, bacterial vectors or recombinant BCG-based vectors; c) other mycobacteria that do not cause  tuberculosis  such as  Mycobacterium smegmatis ; or d) the actual attenuated  M. tuberculosis  [Sweeney, K. A, Dao, G. M., Goldberg, P. F., Hsu, T., Venkataswamy, M. M., Henao-Tamayo, M., Ordway, D., Sellers, R. S., Jain, P., Chen, B., Chen, M., Kim, J., Lukose, R., Chan, J., Orme, I. M., Porcelli, S. A. and Jacobs, W. R Jr. (2011) A recombinant  Mycobacterium smegmatis  induces potent bactericidal immunity against  Mycobacterium tuberculosis . Nat Med. 4:1261-1268; Kaufmann, S. H. (2010) Future vaccination strategies against  tuberculosis : thinking outside the box. Immunity 33: 567-577. 
     In general, the idea behind these vaccines is to seek a way of imitating what happens in a real situation, i.e., to present the entire pathogen in dead or live form, yet attenuated and that does not provoke infection [Kaufmann, S. H. (2010) Future vaccination strategies against  tuberculosis : thinking outside the box.  Immunity  33: 567-577], or using another species of the same genus, also dead or attenuated, such as BCG, which has important antigens in common with the pathogen of interest. Another variant of these vaccines would be their use as live vehicles for presentation or expression of heterologous antigens, in this case MTB molecules being expressed in BCG or  M. smegmatis  [Sweeney, K. A, Dao, D. N., Goldberg, M. F., Hsu, T., Venkataswamy, M. M., Henao-Tamayo, M., Ordway, D., Sellers, R. S., Jain, P., Chen, B., Chen, M., Kim, J., Lukose, R., Chan, J., Orme, I. M., Porcelli, S. A. and Jacobs, W. R Jr. (2011) A recombinant  Mycobacterium smegmatis  induces potent bactericidal immunity against  Mycobacterium tuberculosis . Nat Med. 41261-1268; Kaufmann, S. H. (2010) Future vaccination strategies against  tuberculosis : thinking outside the box. Immunity 33: 567-577]. 
     Despite efforts, the results in an animal model, when compared with the BCG vaccine, suggest a reduction in the bacillary load in the lungs of only 1.0 log, although following an immunization system based on prime-boost. In this immunization schedule, the first dose is administered using one of the above mentioned formulations followed by a second dose in another formulation, i.e., two immunizations with different formulations or presentations are required to achieve a better result than the BCG vaccine used at present, which is administered as a single dose [Sweeney, K. A, Dao, D. N., Goldberg, M. F., Hsu, T., Venkataswamy, M. M., Henao-Tamayo, M., Ordway, D., Sellers, R. S., Jain, P., Chen, B., Chen, M., Kim, J., Lukose, R., Chan, J., Orme, I. M., Porcelli, S. A. and Jacobs, W. R Jr. (2011) A recombinant  Mycobacterium smegmatis  induces potent bactericidal immunity against  Mycobacterium tuberculosis . Nat Med. 4:1261-1268; Kaufmann, S. H. (2010) Future vaccination strategies against  tuberculosis : thinking outside the box. Immunity 33: 567-577]. 
     Recombinant BCG 
     BCG is the most widely used vaccine in the world as it has a long-lasting immune response and a very low frequency of serious adverse effects. This and other characteristics make BCG an excellent candidate as a vehicle for presentation of heterologous antigens through BCG-based recombinant vaccines (U.S. Pat. No. 6,673,353). In this regard, the expression of immunogenic domains of bacteria, viruses and parasites has been used successfully in BCG, generating recombinant strains (rBCG), which produce an immune response not only against the tubercle  bacillus , but also against the pathogens whose proteins would be expressed in this rBCG (U.S. Pat. No. 5,504,005). However, it is worth emphasizing that in the rBCGs described to date, the addition of a heterologous immunogenic domain confers the expected specific immunological protection against the pathogens whose proteins would be expressed in the aforesaid rBCG. Reactions differing from those known habitually, i.e., in which rBCG confers different immunological protection from that expected, have not yet been described. 
       Escherichia Coli  Heat-Labile Toxin LT and its Immunomodulator Properties 
     Adjuvant properties have been attributed to several bacterial toxins. For example, it is widely known that the tetanus (TT), diphtheria (DT) and cholera (CT) toxins as well as the  Escherichia coli  ( E. coli ) heat-labile toxin (LT) act as adjuvants that direct the immune response to Th2 when coadministered with other antigens [Ryan, E. J. et al. (2000) Modulation of innate and acquired immune responses by  Escherichia coli  heat-labile toxin: distinct pro and anti-inflammatory effects of the nontoxic AB complex and the enzyme activity.  J Immunol.  165:5750-5759; Miyaji, E. N. et al. (2001) Induction of neutralizing antibodies against diphtheria toxin by priming with recombinant  Mycobacterium bovis  BCG expressing CRM197, a mutant diphtheria toxin.  Infect Immun.  69:869-874]. 
     In particular, the  E. coli  LT toxin is among the most potent adjuvants described so far [Lycke, N. et al. (199 The adjuvant effect of  Vibrio cholerae  and  Escherichia coli  heat-labile enterotoxins is linked to their ADP-ribosyltransferase activity.  Eur J Immunol.  22: 2277-2281; Pizza, M. et al. (2001) Mucosal vaccines: nontoxic derivatives of LT and CT as mucosal adjuvants.  Vaccine,  19:2534-2541]. 
     The  E. coli  LT toxin is formed by a single A subunit molecule (LTA, 27 kDa) (SEQ ID NO: 11), with ADP-ribosyltransferase activity, bound to a B subunit pentamer (LTB, 11.6 kDa each (SEQ ID NO: 9) which binds to the ganlioside GM1 receptor of mammal cells. The LT-B subunit is a potent signaling molecule able to modulate the immune response. The immune-stimulatory effect of LTB appears to be related to its ability to increase the presentation of antigen via the class I (MHC-I) and class II (MHC-II) major histocompatibility complex, among other factors. The adjuvant effect of LTB, in turn, has been directly related to the activity of binding to GM1, through the activation of B cells and CD4 +  T cells by means of the interaction of the B subunit pentamers and GM1 receptors of these cells. Moreover, LTB increases antigen presentation through the activation of dendritic cells (DCs) and other antigen-presenting cells (APCs). The LTB binding to ganglioside GM1 allows the toxic A subunit to enter the cell [Spangler, B. D. (1992) Structure and function of cholera toxin, and the related  Escherichia coli  heat-labile enterotoxin. Microbiol Rev. 56: 622-647]. 
     The use of LT as an adjuvant is not recommended due to the toxicity of the A subunit. As the B subunit is not toxic, it has been used more frequently as an adjuvant [de Haan, L., Verweij, W. R., Feil, I. K., Holtrop, M., Hol, W. G., Agsteribbe, E. and Wilschut, J. (1998) Role of GM1 binding in the mucosal immunogenicity and adjuvant activity of the  Escherichia coli  heat-labile enterotoxin and its B subunit.  Immunology,  94: 424-430]. 
     The immunomodulatory properties that lead to the increase in the immunogenicity and protective efficacy of LT have been widely studied. Although the mechanisms that lead to this effect are not well characterized, some aspects such as the increase in inflammatory cytokines and production of chemokines, as well as transient recruitment of effector cells of the immune system to the site of inflammation have been established as important factors. LT can also influence dendritic cell maturation, antigen presentation and T-cell activation, besides promoting the induction of response by antigen-specific cytotoxic T-cells in animal model. The use of LT as an adjuvant commonly leads to a balance of cytokines, involving the production of a response by cytokines with both Th1 and Th2 characteristics and some antibody classes in mice and humans. 
     This toxin is able to activate an immune response against another antigen when they are presented simultaneously on mucosal surface or by oral, intranasal or parenteral administration [Holmgren, J., Lycke, N. and Czerkinsky, C. (1993) Cholera toxin and cholera B subunit as oral-mucosal adjuvant and antigen vector systems. Vaccine, 11: 1179-1184]. 
     As a consequence of these properties, LT has been employed extensively as an adjuvant in animal models. However, in spite of the immunomodulatory action of LT, this substance is highly toxic and inappropriate for clinical use in humans. Thus to avoid the toxicity associated with the use of the native toxin, while maintaining its adjuvant properties, different strategies have been used which include the isolation of the B subunit (nontoxic) and the construction of nontoxic mutants of LT, containing specific site-directed mutations. Using computational modeling studies of the structure of the LT protein, we have been able to identify some amino acids potentially involved in the enzymatic activity of this protein, which could be studied through their exchange by site-directed mutation [Magagnoli, O. et al., (1996) Infect Immun. 64: 5434-5438]. Some of these mutants, such as LTR192G (substitution of glycine for arginine at position 192), have mutation in a handle region that is protease-sensitive, making this region insensitive to the action of proteases, an essential, stage for the activation of enzymatic activity and consequently of its toxicity. Other mutants exhibit a mutation in the enzymatically active region of the A subunit, such as LTR72 (substitution of arginine for alanine at position 72), which retains only 1% of the ADP-ribosyltransferase activity. 
     Another important mutant is LTK63 (substitution of lysine for serine at position 63). This mutation eliminates the ADP-ribosyltransferase activity associated with toxicity while eliminating the latter. However, it maintains all the other biological properties, including adjuvant properties of the native LT [Pizza, M., et al., (2001) Mucosal vaccines: nontoxic derivatives of LT and CT as mucosal adjuvants.  Vaccine,  19:2534-2541]. 
     LTK63 has been shown to act as a potent adjuvant when administered parenterally or through the mucosa. De Haan and collaborators [De Haan, L., Holtrop, M., Verweij, W. R., Agsteribbe, E. and Wilschut, J. (1999) Mucosal immunogenicity and adjuvant activity of the recombinant A subunit of the  Escherichia coli  heat-labile enterotoxin. Immunology, 97: 706-7013] suggest that adjuvants using nontoxic LTA in association with the LTB pentamer may be more potent than adjuvants using the B subunit alone, as the complex could stimulate the immune system more strongly than each molecule individually. Data that indicate an important function of the A subunit enzymatically inactive in the induction of an immune response, as well as in immunomodulatory activities, such as effects on antigen processing and presentation, support this view. 
     Expression of LT and its Variants 
     Recombinant LTB (SEQ ID NO: 9) and LTK63 (SEQ ID NO: 8) have been expressed typically in  E. coli . However, other expression systems have been used. The LTB molecule was expressed in  Mycocabtrium bovis  BCG,  Lactobacillus casei, Saccharomyces cerevisiae, Pichia pastoris , and plants that: include  Oryza sativa  (rice),  Lactuca sativa  (lettuce) and  Peperomia pellucid . LTK63 has also been expressed in tobacco and chloroplasts, as well as attenuated  Salmonella enterica  serovar  Typhimurium  [da Hora, V. P. et al. (2011) Non-toxic derivatives of LT as potent adjuvants. Vaccine, 29: 1538-15441. 
     However, the goal of all these studies was to use the LT molecule or one of its subunits or variants to produce vaccines against  E. coli . The use of LT in  tuberculosis  vaccines was not described or suggested in the existing literature. 
     OBJECTIVES OF THE INVENTION 
     One objective of this invention is to provide recombinant  Mycobacterium  strains that encode the mutant  Escherichia coli  heat-labile toxin LT or the A subunit of the mutant  Escherichia coli  heat-labile toxin LT 
     In particular, one of the goals of this invention is to provide recombinant strains of  Mycobacterium bovis Bacillus  Calmette Guerin (BCG), which encode the mutant  Escherichia coli  heat-labile toxin LT or the A subunit of the mutant  Escherichia coli  heat-labile toxin LT 
     More specifically, one of the goals of this invention is to provide strains of  Mycobacterium , in particular  Mycobacterium bovis Bacillus  Calmette Guerin (BCG), which encode the mutant  Escherichia coli  heat-labile toxin LT or A subunit at position 63. 
     More specifically, one of the goals of this invention is to provide strains of  Mycobacterium , in particular  Mycobacterium bovis Bacillus  Calmette Guerin (BCG), which encode the mutant  Escherichia coli  heat-labile toxin LT or A subunit at position 63 from serine to lysine, respectively rBCG-LTK63 and rBCG-LTAK63. 
     The present invention is also aimed at providing immunogenic compositions that involve the strains of this invention. 
     Another objective of the invention is to provide the use of the above mentioned strains and immunological compositions in the production of vaccines for prevention against  tuberculosis  and/or infections caused by  Mycobacterium tuberculosis  or by other mycobacteria. 
     In particular, the present invention has the objective of providing improved BCG vaccines against  tuberculosis  (i.e. that induce better protection than the conventional BCG strain) which involve the recombinant strains of  Mycobacterium bovis Bacillus  Calmette Guerin (BCG) of this invention. 
     This invention is also designed to provide methods for preventing or treating  tuberculosis  in animals, more particularly humans. 
     DEFINITIONS 
     Abbreviations are used several times within the scope of this patent application. Their definitions as used in this request are summarized below: 
     BCG refers to attenuated  Mycobacterium bovis, Bacillus  Calmette-Guérin; 
     LTK63 refers to  Escherichia coli  heat-labile toxin containing the A subunit modified by site-directed mutagenesis. 
     LTAK63 (SEQ ID NO: 8) refers to the A subunit of the  Escherichia coli  heat-labile toxin, modified by site-directed mutagenesis; and 
     rBCG-LTK63 (SEQ ID NO: 1) or rBCG-LTAK63 (SEQ ID NO: 2) refers to the recombinant BCG expressing LTK63 (SEQ ID NO: 10) or LTAK63 (SEQ ID NO:7), respectively. 
    
    
     
       DESCRIPTION OF THE FIGURES 
       The figures below are part of this report and are included here to illustrate certain aspects of the invention. The purpose of the invention can be better understood with reference to one or more of these figures, in combination with the detailed description of the modality of choice presented here. 
         FIG. 1  shows an agarose gel containing the PCR products that confirm the presence of the LTK63 gene in the recombinant BCG (rBCG-LTK63). The PCR products were amplified from plasmid DNA extracted from the rBCG-LTK63 construction using specific initiator oligonucleotides to amplify the LTA subunit or the LTB unit. 1 Kb plus, molecular weight; well 1, complete LTA fragment (˜700 bp); well 2, complete LTB fragment (˜316 bp). 
         FIG. 2  shows the characterization of the LTAK63 expression (˜31.0 kDa) in recombinant BCG. Extract of soluble proteins (˜10 μg) of rBCG transformed with pLNIP-LTAK63 (A), or empty BCG as negative control (B) were used in this immunoassay. Anti-LT 1:1000 (polyclonal) rabbit serum was used. 
         FIGS. 3 and 4  show the comparative analysis of the response of antibodies IgG1 and IgG2a induced against proteins recovered from the BCG culture supernatant—PDS Female BALB/c mice aged four weeks (18-22 g), from the Animal Facility of the School of Veterinary Medicine—USP (São Paulo University), were immunized with BCG or rBCG-LT ( FIG. 3 ) or with BCG or rBCG-LTAK63 ( FIG. 4 ) and the isotypes determined in isolated sera one day before the MTB challenge or 90 days after immunization. The sera of 5 animals per group were analyzed individually by ELISA, using specific mice anti IgG1 and IgG2a antibodies. 
         FIGS. 5 and 6  show the production of IFN-γ by splenocytes cultivated in the presence of PDS. Female BALB/c mice were immunized with 1×10 6  CFU of BCG or rBGC-LT ( FIG. 5 ) or BCG, or rBCG-LTAK63 ( FIG. 6 ) and after four weeks or eight weeks, respectively, the spleens were recovered and single-cell preparations were cultivated in vitro (2×10 cells/well) in the presence of 2.0 μg/mL of PDS. The supernatants of the cultures were recovered after 48 h and the levels of IFN-γ determined by ELISA. 
         FIGS. 7 and 8  show the production of TNF-α by splenocytes cultivated in the presence of PDS. Female BALB/c mice were immunized with 1×10 6  CFU of BCG or rBGC-LT ( FIG. 7 ) or BCG or rBCG-LTAK63 ( FIG. 8 ). After 4 weeks ( FIG. 7 ) or eight weeks ( FIG. 8 ) the spleens were recovered and single-cell preparations were cultured in vitro (2×10 6  cells/well) in the presence of 2.0 μg/mL of PDS. The culture supernatants were recovered after 24 h ( FIG. 7 ) or 48 h ( FIG. 9 ) and the TNF-α levels determined by ELISPOT ( FIG. 7 ) or ELISA ( FIG. 8 , respectively). SFU=spot-forming units. 
         FIGS. 9 and 10  show the profile of CD4 +  T cells that produce the cytokines TNF-γ and TNF-α, respectively. Female BALB/c mice (n=5 per group) were immunized by means of a single subcutaneous injection with 1×10 6  CFU/animal with BCG or rBCG-LTK63. A non-immunized group was used as negative control. Eight weeks after the immunization, the spleens were recovered and single-cell preparations were cultured in vitro (2×10 6  cells/well) in the presence of 2.0 μg/mL of PDS. The profile of CD4 +  T cells that produce the various cytokines were analyzed by Flow Cytometry (FACs) using specific anti-CD4, anti-INF-γ and anti-TNF-α antibodies marked with the appropriate fluorochrome. 
         FIG. 11  shows the result of the protection assays involving intratracheal challenge with the  M. tuberculosis  H37Rv strain. Female BALB/c mice (n=10 per group) A and B were immunized subcutaneously with a single injection of  M. bovis  BCG or with rBCG-LT. A non-immunized group was used as negative control. Eight weeks after the immunization the animals were challenged by the intratracheal route with 1×10 5  CFU of  M. tuberculosis  H37Rv. Error bars represent the standard deviation of the mean. A and B represent two assays conducted at different times under the same conditions. C represents an assay conducted with female C57BL/6 mice (n=10 per group) immunized subcutaneously with a single injection of  M. bovis  BCG or rBCG-LTA. Eight weeks after the immunization the animals were challenged by the intratracheal route with 1×10 5  CFU of  M. tuberculosis  H37Rv. Error bars represent the standard deviation of the mean. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Description of the Strains 
     The present invention refers to recombinant strains of  Mycobacterium  that encode the mutant  Escherichia coli  heat-labile toxin LT 
     This invention also refers to recombinant strains of  Mycobacterium  that encode the A subunit of the mutant  Escherichia coli  heat-labile toxin LT. 
     In particular, this invention refers to strains of  Mycobacterium  that encode the mutant heat-labile toxin LT or A subunit of the  Escherichia coli  heat-labile toxin LT at position 63. 
     More particularly, this invention refers to strains of  Mycobacterium  that encode the mutant heat-labile toxin LT or A subunit of the  Escherichia coli  heat-labile toxin at position 63 from serine to lysine, respectively rBCG-LTK63 and rBCG-LTAK63. 
     The above mentioned mutations allow the  Escherichia coli  heat-labile toxin LT or the A subunit of the  Escherichia coli  heat-labile toxin LT encoded by  Mycobacterium  to maintain the adjuvant properties of the native LT, yet they lose the ADP-ribosyltransferase activity associated with toxicity, thus avoiding toxicity problems in the case of humans or other animals. 
     Strains of  Mycobacterium  that encode the heat-labile toxin LT or the A subunit of the  Escherichia coli  heat-labile toxin LT that exhibit other mutations in their sequence that target an equivalent effect, i.e. maintenance of the adjuvant properties of the native LT and reduction or suppression of toxicity, are also involved in this invention. Such mutations may include, but are not limited to, positions 72 and 192. 
     The strains of this invention are obtained from any strain of the genus  Mycobacterium  as carrier for presentation of one or more antigens of different organisms, obtained and cloned in expression vector in mycobacteria and inserted by means genetic manipulation. 
     The recombinant  Mycobacterium  strains of this invention preferably include strains of the MTB complex,  Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium  microtti and  Mycobacterium africanum  or of fast-growing mycobacteria,  Mycobacterium smegmatis, M. aurum, M. vaccae , etc. 
     The recombinant  Mycobacterium  strains of this invention more preferably include strains of  Mycobacterium bovis Bacillus  Calmette Guerin (BCG). 
     Description of the Immunogenic Compositions 
     The present invention refers to immunogenic compositions involving one or more strains of this invention and one or more within a physiologically acceptable vehicle, excipient, diluent or solvent. 
     The compositions of this invention preferably include strains of  Mycobacterium bovis Bacillus  Calmette Guerin (BCG), which encode the mutant heat-labile toxin LT at position 63 from serine to lysine, i.e., rBCG-LTK63. 
     Alternatively, the compositions of this invention preferably include strains of  Mycobacterium bovis Bacillus  Calmette Guerin (BCG), which encode the A subunit of the mutant heat-labile toxin LT at position 63 from serine to lysine, i.e., rBCG-LTAK63. 
     The compositions of this invention may also involve a mixture of strains of  Mycobacterium bovis Bacillus  Calmette Guerin (BCG), which encode the mutant heat-labile toxin LT at position 63 from serine to lysine, i.e., rBCG-LTK63 and the A subunit of the mutant heat-labile toxin LT at position 63 from serine to lysine, i.e., rBCG-LTAK63. 
     The immunogenic compositions of this invention may also additionally involve one or more antigens, preferably inactivated toxins. Such antigens may be for use in the prevention and/or treatment of  tuberculosis  or of other diseases caused by different pathogens. The inactivated components of the synergic immunogenic compositions of this invention can be obtained using any method known in the technique, such as chemical procedures, such as treatment with formaldehyde or hydrogen peroxide, or even DNA recombination techniques. 
     According to this invention, adjuvants are attenuated or dead molecules, components, macromolecules or microorganisms that potentiate the response to immunizations, reduce the amount of antigen required and direct the type of immune response to be developed, besides sustaining it for a longer period of time, as an immunogen; it is any material or substance that alters the type, speed, intensity or duration of the immune response. 
     The compositions of this invention can also include excipients, such as bactericides, bacteriostatics, antioxidants, preservatives, buffers, stabilizers, pH adjusters, osmolarity adjusters, antifoaming agents and surfactants; and residues of antigen inactivation or fractionating agents, growth media components and solvents commonly used in the production of vaccines; examples of these types of component can be found in the  Epidemiology and Prevention of Vaccine - Preventable Diseases The Pink Book,  11 th  edition, under “ Vaccine Excipient  &amp;  Media Summary ” (Centers for Disease Control and Prevention. Epidemiology and Prevention of Vaccine-Preventable Diseases. Atkinson W, Wolfe S, Hamborsky J, McIntyre L, eds. 11th ed. Washington, D.C.: Public Health Foundation, 2009) incorporated here as a reference. 
     As employed in this invention, the use of the term “pharmaceutically acceptable” means an inert nontoxic solid, semisolid liquid excipient, diluent, ancillary formulation of any kind, or simply a sterile aqueous medium, such as saline solution. Some examples of the materials that can serve as pharmaceutically acceptable vehicles are sugars, such as lactose, glucose and sucrose, the searches, such as corn starch and potato starch, cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate, cyclodextrin; oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol, poly oils, such as glyceringlycol, sorbitol, mannitol and polyethylene; esters, such as ethyl laurate, ethyl oleate, agar; buffering agents, such as aluminum hydroxide and magnesium hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer&#39;s solution; buffer solutions of ethyl alcohol and phosphate, as well as other compatible nontoxic substances used in pharmaceutical formulations. 
     A range of routes for administration of the immunotherapy compositions and vaccines described in this invention is available. The particular method selected will depend on the particular active ingredient selected, the necessary dosage for therapeutic efficacy and on the patient to whom the composition is to be administered. The methods of the present invention can generally be practiced using any biologically acceptable means of administration, i.e., any method that produces effective levels of immune response without causing clinically undesirable adverse effects. Such methods of administration include the intradermal, oral, rectal, sublingual, topical, nasal, transdermal or parenteral routes. The term “parenteral” includes subcutaneous, intravenous, epidural, irrigation, intramuscular, release or infusion pumps. In this invention particularly, the intradermal, oral, parenteral and nasal routes are preferred for administering the compositions advocated here. 
     For parenteral administration, the active ingredients can be dissolved in a pharmaceutical vehicle and administered as a solution, or emulsion, including micro- and nanoemulsions, or suspension. Examples of appropriate vehicles are water, saline, dextrose solutions, fructose solutions or oils of animal, vegetable or synthetic origin. 
     For nasal administration, the active ingredients can be dissolved in a pharmaceutical vehicle and administered as a solution, or emulsion, including micro- and nanoemulsions, or suspension. Examples of appropriate vehicles are water and saline solution or solid suspensions, such as spray, lactose, fructose or chitosan flakes. Other vehicles can also contain other ingredients, e.g., preservatives, suspensor agents, solubilizing agents, buffers and alike. 
     The immunogenic compositions of this invention are preferably for intradermal, oral and parenteral administration. 
     Properties of the Strains and of the Immunogenic Compositions of this Invention 
     The strains and the immunogenic compositions of this invention exhibit an unexpected synergic effect on the immune response against  Mycobacterium . The recombinant antigens of LTK63 or LTAK63 exhibit the unexpected technical effect of inducing an adjuvant effect against the proteins of the actual  Mycobacterium.    
     As can be seen in the examples below, the strains and the immunogenic compositions of this invention exhibit the unexpected technical effect of producing a more intense anti-TB immune response than vaccines involving traditional BCG. In other words, the addition of an immunogenic domain, such as the nontoxic subunit LTAK63 of the  E. coli  heat-labile toxin, which is known to have no bond with any type of mycobacteria, in a recombinant  mycobacterium , in particular BCG, brings about an increase in the protective response against  tuberculosis.    
     In particular the strains and the immunogenic compositions of this invention promote a decrease in the bacillary load in the lungs in animal models of  tuberculosis.    
     Moreover, the strains and the immunogenic compositions of this invention lead to a significant increase in the expression of IFN-γ in relation to the control group, a cytokine considered essential in the response against  tuberculosis ; this fact indicates a Th1 polarized response. 
     The strains and immunogenic compositions of this invention also lead to a significant increase in TNF-α. Since TNF-α is related as a signaler of proinflammatory action, characterizing the acute phase of the inflammatory process triggered by the bacilli, then the vaccines of this invention promote direct activation of the immune system. 
     Finally, we can conclude that the strains and the immunogenic compositions of this invention, in combining mycobacteria, in particular BCG, and toxin derivatives of other pathogens, preferably of  Escherichia coli , generating a recombinant mycobacterial strain, constitute a new vaccine that is more potent and effective than conventional BCG for prophylaxis or immunization against  tuberculosis.    
     Use of the Strains and of the Immunogenic Compositions of this Invention. 
     Considering the properties of the recombinant strains of  Mycobacterium  and of the immunogenic compositions of this invention, another aspect of this invention is the use of immunogenic compositions to prevent infections caused by mycobacteria, in particular  Mycobacterium tuberculosis  in animals, more particularly humans. 
     Another aspect of this invention is the use of one or more recombinant strains of  Mycobacterium  or of one or more immunogenic compositions of this invention in the production of a vaccine. 
     More particularly, an aspect of this invention is the use of one or more Recombinant  Mycobacterium  strain or of one or more immunogenic compositions of this invention in the production of a vaccine for the prevention and/or treatment of  tuberculosis  and/or infections caused by mycobacteria. 
     Yet another aspect of this invention is the methods used to prevent or treat  tuberculosis  in animals, more particularly humans. 
     EXAMPLES 
     To allow a better understanding of this invention and to clearly demonstrate the technical advances obtained, we present below, as examples, the results of the different assays carried out in relation to this invention. 
     In Example 1 we describe the obtainment of the vaccines of this invention. The other examples (2 to 3) serve to illustrate the properties and the use or the vaccine of this invention. These examples are presented merely for illustration and should in no way be considered as limiting the scope and sphere of this invention. 
     Example 1 
     Obtainment of the rBCG-LTK63 and rBCG-LTAK63 Vaccines 
     This example describes the obtainment of the rBCG-LTK63 and rBGC-LTAK63 vaccines. 
     a) Preparation of competent BCG stock batch: to prepare stocks of competent BCG, one or more BCG colonies were cultured in Middlebrook 7H9 plus Tween-80 liquid medium and supplemented with 10% albumin dextrose catalase (MB7H9/Tw/ADC) until the exponential phase. Composition of the Middlebrook 7H9 medium according to the manufacturer (Difco-ED) Ammonium Sulfate, L-Glutanic Acid, Sodium Citrate, Pyridoxine, Biotin, Disodium Phosphate, Mono-Potassium Phosphate, Ferric Ammonium Citrate, Magnesium Sulfate, Calcium Chloride, Zinc Sulfate and Copper Sulfate. After this the culture was sedimented by centrifugation at 4000 rpm and washed twice with 10% glycerol at 4° C., then finally resuspended in 5% of the original volume, with 10% glycerol and stored at −70° C. until its transformation by electroporation with pNL12-LTK63 plasmid or with pNL12-LTAK63 plasmid. 
     b) Construction of the expression vector of the LTK63 gene in recombinant BCG here called pNL12-LTK63: the mycobacterial expression vector used for expression of LTK63 in recombinant BCG is called pMIP12 and was described by Le Dantec [Le Dantec et al. 2001 J Bacteriol. 183: 2157-2164]. The LTK63 gene was kindly contributed by Dr. Rino Rappuoli (Novartis). The expression vector in recombinant BCG of the LTK63 gene was constructed using conventional molecular biology methods. The LTK63 gene was amplified by PCR using the following initiator oligonucleotides: N-terminus 
                             (SEQ ID NO: 3)           (5′-TAG GGTACC CAAAAATATAACTTCATTTTTTTTATTTT-3′            
containing the Kpn I restriction site (underlined) and C-terminus
 
                             (SEQ ID NO: 4)           (5′-TAG CTGCAG CTAGTTTTTCATACTGATTGCCC-3′,            
containing the Pst I restriction site (underlined). The PCR product corresponding to the LTK63 gene was generated using the following PCR conditions: 94° C. for 4 minutes; 25 cycles of 94° C. for 1 min, 50° C. for 1 min and 72° C. for 30 s; and 4° C. final. This PCR product was then digested with the restriction enzymes Kpn I and Pst I and then cloned in the expression vector pMIP12, which was also previously digested with the same enzymes Kpn I and Pst I, thus generating the expression vector called pMIP12/LTK63.
 
     Gene Sequence of Plasmid pNP12-LTK63 (SEQ ID NO: 1) 
     
       
         
               
               
             
           
               
                 Gene sequence of plasmid pNP12-LTK63 (SEQ ID NO: 1) 
                   
               
               
                 GCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAA 
               
               
                   
               
               
                 AGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGC 
               
               
                   
               
               
                 TCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTCGA 
               
               
                   
               
               
                 CTTAATTAACTCCGGGTGGTCACTGAGTACATCCCGTCGGTGCGTTCGGCATCGGTCGGGGTGTGGGTGGGTGTCGGCTCC 
               
               
                   
               
               
                 CGCGACGAAGGACGAAGCGTCGCGGGTGCCGCCCACTTCTTGGAGCATCTGCTGTTCAAGGCCACCCCGACGCGCACGCGG 
               
               
                   
               
               
                 TCGACATCGCGCAGGCTGTCGATGCCGTCGGCGGTGAGCTGAACGCGTTCACCACGCGCGAGCACACCTGTTACTACGCGC 
               
               
                   
               
               
                 ATGTGCTCGACTCCGACCTGGAGCTCGCGGTCGACCTGGTGCGCCGATGTCGTGTTGCGTGGGCGTTGTGCCACCGAGGAT 
               
               
                   
               
               
                 GTCGAAGTGGAGCGCGACGTCGTCCTCGAGGAGATCGCCATGCGTGACGACGATCCCGAGGACAGCCTCGGCGACGTGTTC 
               
               
                   
               
               
                 CTCTCGGCGATGTTCGGCGATCACCCGGTGGGACGTCCGGTGATCGGCAGCGTCGAGTCGATCGAGACCATGACGCGTGCA 
               
               
                   
               
               
                 CAGCTGCATTCGTTCCACGTCCGGCGTTACACACCCGAACGGATGATCGTGGCGGTGGCCGGCAACGTCGACCACGACGTG 
               
               
                   
               
               
                 TGGTGTCGTTGGTCCGAGAGCATTTCGGCCCCCGGCTGGAGGCCGACGTTCCGCGGTGGCTCCCCGTAAGGCTCGGGACGG 
               
               
                   
               
               
                 GTCGGTGGTAAGCCATCGCTGCTCGTGGTCGACCGCGACGGGGAACAGTCCCATGTCTCGCTGGGCGTTCGCACGCCCGGC 
               
               
                   
               
               
                 CGGCACTGGGAGCACCGGTGGGCCCTGTCGGTGTTGAACACCGCGCTGGGAGGCGGGCTCAGTTCTCGTCTGTTCCAACAG 
               
               
                   
               
               
                 ATTCGCGAGTCCCGCGGCCTGGCCTACCTCGGTGTACTCGACCGTGGACCACTTCGCGACAGCGGGGCTCTGTCGGTGTAT 
               
               
                   
               
               
                 GCGGGATGTCAGCCGGAACGTTTCGACGAAGTGGTGCGGGTGACCACCGAAGTTTTGGAAGGTGTTGCCAGAGACGGGATC 
               
               
                   
               
               
                 ACCGCCGACGAATGCCGGATCGCCAAAGGCTCGTTGCGCGGTGGGCTGGTGCTCGGCCTGGAGGATTCCGGATCACGTATG 
               
               
                   
               
               
                 CACCGGATCGGCCGTAGCGAGCTCAATTACGGTGgAGCACCGGACCATCGACCACACGCTGGCCCAGATCGAGGCAGTCAC 
               
               
                   
               
               
                 TCTAGAAGAGGTCAACGCCGTCGCTCACCAGTTGCTGTCGCGGGACTACGGTGCCGCCGTACTCGGTCCCTATAGTTCGAA 
               
               
                   
               
               
                 AAAGGCGCTGCCACAACAGCTTCAAACTATCGCCGGCTGACCCGCTACACTGGGTCCAATGGATTAGAAGGAGAAGTACCG 
               
               
                   
               
               
                 ATGGGATCCGGTACCAATGGCGACAGATTATACCGTGCTGACTCTAGACCCCCAGATGAAATAAAACGTTCCGGAGGTCTT 
               
               
                   
               
               
                 ATGCCCAGAGGGCATAATGAGTACTTCGATAGAGGAACTCAAATGAATATTAATCTTTATGATCACGCGAGAGGAACACAA 
               
               
                   
               
               
                 ACCGGCTTTGTCAGATATGATGACGGATATGTTTCCACTAAGCTTAGTTTGAGAAGTGCTCACTTAGCAGGACAGTCTATA 
               
               
                   
               
               
                 TTATCAGGATATTCCACTTACTATATATATGTTATAGCGACAGCACCAAATATGTTTAATGTTAATGATGTATTAGGCGTA 
               
               
                   
               
               
                 TACAGCCCTCACCCATATGAACAGGAGGTTTCTGCGTTAGGTGGAATACCATATTCTCAGATATATGGATGGTATCGTGTT 
               
               
                   
               
               
                 AATTTTGGTGTGATTGATGAACGATTACATCGTAACAGGGAATATAGAGACCGGTATTACAGAAATCTGAATATAGCTCCG 
               
               
                   
               
               
                 GCAGAGGATGGTTACAGATTAGCAGGTTTCCCACCGGATCACCAAGCTTGGAGAGAAGAACCCTGGATTCATCATGCACCA 
               
               
                   
               
               
                 CAAGGTTGTGGAAATTCATCAAGAACAATCACAGGTGATACTTGTAATGAGGAGACCCAGAATCTGAGCACAATATATCTC 
               
               
                   
               
               
                 AGGGAATATCAATCAAAAGTTAAGAGGCAGATATTTTCAGACTATCAGTCAGAGGTTGACATATATAACAGAATTCGGGAT 
               
               
                   
               
               
                 GAATTATGAGGATTAGGAGAAGTACCGGAATTCATGAATAAAGTAAAATGTTATGTTTTATTTACGGCGTTACTATCCTCT 
               
               
                   
               
               
                 CTATGTGCATACGGAGCTCCCCAGTCTATTACAGAACTATGTTCGGAATATCGCAACACACAAATATATACGATAAATGAC 
               
               
                   
               
               
                 AAGATACTATCATATACGGAATCGATGGCAGGCAAAAGAGAAATGGTTATCATTACATTTAAGAGCGGCGCAACATTTCAG 
               
               
                   
               
               
                 GTCGAAGTCCCGGGCAGTCAACATATAGACTCCCAAAAAAAAGCCATTGAAAGGATGAAGGACACATTAAGAATCACATAT 
               
               
                   
               
               
                 CTGACCGAGACCAAAATTGATAAATTATGTGTATGGAATAATAAAACCCCCAATTCAATTGCGGCAATCAGTATGGAAAAC 
               
               
                   
               
               
                 TAGCTGCAGCATCACCATCACCATCACTAGTGAAATAGCGAAACACGGGATCGGGCGAGTTCGACCTTCCGTCGGTCTCGC 
               
               
                   
               
               
                 CCTATTAATAGTGGCATGCAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAAC 
               
               
                   
               
               
                 TTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGT 
               
               
                   
               
               
                 TGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGCA 
               
               
                   
               
               
                 GGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCATACCAGGAACGAGGACAGTCGCACGACGAA 
               
               
                   
               
               
                 GTTCTTCTGGATCGCGCCCGTGCTGGAAGCACTCAACCTCGAAGCGTGTGGTTGCGGAGCCATCTAGCAACCACACGAAAC 
               
               
                   
               
               
                 ATGCGCAACGAACCGCGCAACGAACAACGCCTAGAACTGGCCCTAGATGAGCTGACTCGTATCGTTGGTAAACCTAGTTTG 
               
               
                   
               
               
                 ACCAGCATGTTTTAACTACGTTCGGTGAGCTGTCAACGGGGCCTGTAACGGCACAACGAACCGTGCAACGAGAGTGGCCAC 
               
               
                   
               
               
                 GGATGCCACCACAGGCACTACAACGGAGTTCGCCACGTACATCACCACAACCACCGATTCTGGCGGTGAGCTCCCCGATAT 
               
               
                   
               
               
                 TCAGCGGAAATGGCTTGGTATCGACCAAGATTCGTAGAACCCCGTCTCGTCTGGCTGGTATTCAAAACGGGCGCAACGAAA 
               
               
                   
               
               
                 CACGCAACGAGACAGGCATGGCCCAAACCAGAAAACTAGCGTCTACCAGGACTTTTACCTGTCCGACCCGTTGCAACGGAA 
               
               
                   
               
               
                 CCCCCCACGGAACCCCCGCGACACCCGCTCCCCAATTGCGTTAGAACAGCGGTGGATTGTCGGCTTCGTTGTGGGCCTTTT 
               
               
                   
               
               
                 GAGCCGCTTCCTGTTCTGCCGCACGCTCTTTCCTCGCCCGATAGCCGAGTCGCTTAACGGTGTCCAGATGCAGCCCGAAAT 
               
               
                   
               
               
                 GTTTGGCCGTTTGCGGCCAAGAGTGGCCCTCGTCGTCGTGATAGGCGCGGATGCGTTCGCGGCGTGCAGCCTGCTCGGCGA 
               
               
                   
               
               
                 GCCACTCGCTGCGTTCCTGCGCCACGAGCCGGACGACGTGGCGTTCGGATAGTCCGGTGATTCGAGCGCCTTCGGCGGCGG 
               
               
                   
               
               
                 TCACGCGCCGCTTTTTGCGGACAGTCGGCTGCCGGTTGTAGCCGTCGCTGTAGCCGTCGCTCATAGCAATGCCTCCATGGC 
               
               
                   
               
               
                 TGACGCGGACTTTGCGCGCCGCGCAACTGTGCTCGCCGCCGTGCGCGCTGCTGCGCCCTTCCGCGAGATGGCCGACTGGCG 
               
               
                   
               
               
                 CGCACTGAGTGTGGCCTCGTAGACCACGATCCCGTCCGCCCAAATGCGCGACTTGGTTGTGATCCAACGCCAAATGCTGTT 
               
               
                   
               
               
                 GGCGATGGCGCGGACCTCGCTGTCCGGTAGCGGTCCGGGACACACGTCGTTGCACGGGAATTCGGCGTTTCGCGCGTGGCA 
               
               
                   
               
               
                 CTCGGCATAGATCGCGCGGCCGAGTCCGTCCACGTTCCGGGTCGGCAGGTAGATCCGCATGAGGGCGGGACGATAGGCCCA 
               
               
                   
               
               
                 CAACCTGACGGAATCGAACAGTGCGCAATTCCGCCCTAGCGGCGTCGGAGCCGCTTTGTACGTGGTCTGCTGACGCCAGCG 
               
               
                   
               
               
                 CGGCGGTGGCATGTTCGCGCCGAGCTCGGCCTCGATGTGGCTGAGTGTGTAGAGATCTGAGTGGAGCCATTCCGTTTCCCA 
               
               
                   
               
               
                 GGCGATGTGGCCGGGGTTTTTGGTCATGAGGCCTGAGTAACTGCGGTCGCCGTCGACGGCGCGCCGAAGGCCTTCGGCGCA 
               
               
                   
               
               
                 CGCCGCCATGTATGCGAGCGGCTTACGCCGCGCGTATTCGGTGCGTGGAACAGGGGCGTTGAGTGCCCACACTGCGTGTGC 
               
               
                   
               
               
                 GTGGCCGTTGGCGCGATTGCCCACGATCGCGTTGGGCAGCGGATGGGACCCCCGGGCGCTGAGCGCTCGGAGCGCTGCGTC 
               
               
                   
               
               
                 TGGATGGTCTACGTCCACGACCAGCAGGTTTGCCAGCGCTGTTGGGTTCGCCTCGATGTACCGGCGGCCTAGGGCCGACGC 
               
               
                   
               
               
                 GCGGCTTTGGCGGTAGATCCCCTCGAGCAGATCGTCGCTTGCCAGCGGCCAGTACGGCAGCCAGAGCTGCTCAAATTCGTC 
               
               
                   
               
               
                 GGCGACGTGGCTCACGCTTGGTAGTAGACCACGATTAATCACCGGTGTATGGTCCGACACGAGCTCCAAGTCAGATATTTC 
               
               
                   
               
               
                 GCTGAGGGGCCACCCCACAACTGCACACTCCCCCGCTCTCCCGTCGAGCCCTGGTGGTGGAACACCAGCGACAGCCGAGCA 
               
               
                   
               
               
                 CCCCCAACCACCTGTACCAACCAGGAGGAACACATGCGTCGTTTCGAGGACGTTTCCGGGCCGCTGAGAGCCGCTGTGGCG 
               
               
                   
               
               
                 GCCGTACACGCCGCCTTAGACCCGTTAGACCCCCTGCCGCCTGAATGCGCGGGTACGAGCCACACAGCGCCCGAACTTACG 
               
               
                   
               
               
                 GAGCTGGTGGGCTCACCTGGCTTTATGGCGTACGAATCGGCTGTGTGCGACCTGTTGGGCGAGGTGAGGTACGCGCTACTC 
               
               
                   
               
               
                 ACGCTGGCAAGGGCGACACAGCCGCCCCACCGAGCCCGCACGGCCGCGCGCGGTGTCAACAACCGGGTGAGTCGTGCACAC 
               
               
                   
               
               
                 CAGCAGGTGTTCGAGGCTTGGCTCGAAGTGCAGGACATCGTGGCGAACGCCGCCCGATGAGCCGCGCCTTACGCTGGCTGC 
               
               
                   
               
               
                 CAGCCGTTCGCGGGCTGGTTGGTGCAGCGCGTCGAGCGGTTAGAGGCCCTGCGGTGTTCCACCACCGCAGGGCCTCGCCCT 
               
               
                   
               
               
                 TTTTAAGGCTGAATTTGCTTGTCTCCGAATCCAACTGGCTTGTCCAAGGGTGTATCTACGCTTAATCCAAAGTTCAAACGA 
               
               
                   
               
               
                 GGGGATTACACATGACCAACTTCGATAACGTTCTCGGCTCGATCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAG 
               
               
                   
               
               
                 CCACGGTTGATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTG 
               
               
                   
               
               
                 TCGGGAAGATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCG 
               
               
                   
               
               
                 TAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTAT 
               
               
                   
               
               
                 TCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATA 
               
               
                   
               
               
                 GGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAA 
               
               
                   
               
               
                 TAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGA 
               
               
                   
               
               
                 CTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCT 
               
               
                   
               
               
                 GAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCA 
               
               
                   
               
               
                 GCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGA 
               
               
                   
               
               
                 GTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGA 
               
               
                   
               
               
                 CCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACA 
               
               
                   
               
               
                 ATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAAT 
               
               
                   
               
               
                 TTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACA 
               
               
                   
               
               
                 GTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTCCCCCC 
               
               
                   
               
               
                 CCCCCCTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAAC 
               
               
                   
               
               
                 TATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTC 
               
               
                   
               
               
                 ATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGAC 
               
               
                   
               
               
                 CAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTT 
               
               
                   
               
               
                 TTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACC 
               
               
                   
               
               
                 AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCA 
               
               
                   
               
               
                 CCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAA 
               
               
                   
               
               
                 GTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCAC 
               
               
                   
               
               
                 ACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGA 
               
               
                   
               
               
                 AGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGC 
               
               
                   
               
               
                 CTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAG 
               
               
                   
               
               
                 CCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGC 
               
               
                   
               
               
                 GTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCG 
               
               
                   
               
               
                 CAGCGAGTCAGTGAGCGAGGAAGCGGAAGA 
               
             
          
         
       
     
     c) Construction of the expression vector of the LTAK63 gene in recombinant BCG here called pLN12-LTAK63: the expression vector in recombinant BCG of the LTAK63 gene was constructed using conventional molecular biology methods. The LTAK63 gene was amplified by PCR from the LTK63 gene using the following initiator oligonucleotides: N-terminus 
                             (SEQ ID NO: 5)           (5′- TAG     GGATCC     AATGGCGACAGATTATACCGTTG -3′),            
containing the BamH I restriction site (underlined) and C-terminus
 
                             (SEQ ID NO: 6)           (5′- TAG     GGTACC     TAATTCATCCCGAATTCTGTTATA -3′)            
containing the Kpn I restriction site (underlined). The PCR product corresponding to the LTAK63 gene was generated using following PCR conditions: 94° C. for 4 minutes; 25 cycles of 94° C. for 1 min, 50° C. for 1 min and 72° C. for 30 s; and 4° C. final. This PCR product was then digested with the restriction enzymes BamH I and Kpn I and then cloned in the expression vector pMIP12, which was also previously digested with the same enzymes BamH I and Kpn I, thus generating the expression vector called pLNIP/LTAK63.
 
     Gene Sequence of Plasmid pNL12-LTAK63 (SEQ ID NO: 2) 
     
       
         
               
               
             
           
               
                 Gene sequence of plasmid pNL12-LTAK63 (SEQ ID NO: 2) 
                   
               
               
                 GCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGA 
               
               
                   
               
               
                 AAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCG 
               
               
                   
               
               
                 GCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAGCT 
               
               
                   
               
               
                 CGACTTAATTAACTCCGGGTGGTCACTGAGTACATCCCGTCGGTGCGTTCGGCATCGGTCGGGGTGTGGGTGGGTGTCGG 
               
               
                   
               
               
                 CTCCCGCGACGAAGGACGAAGCGTCGCGGGTGCCGCCCACTTCTTGGAGCATCTGCTGTTCAAGGCCACCCCGACGCGCA 
               
               
                   
               
               
                 CGCGGTCGACATCGCGCAGGCTGTCGATGCCGTCGGCGGTGAGCTGAACGCGTTCACCACGCGCGAGCACACCTGTTACT 
               
               
                   
               
               
                 ACGCGCATGTGCTCGACTCCGACCTGGAGCTCGCGGTCGACCTGGTGCGCCGATGTCGTGTTGCGTGGGCGTTGTGCCAC 
               
               
                   
               
               
                 CGAGGATGTCGAAGTGGAGCGCGACGTCGTCCTCGAGGAGATCGCCATGCGTGACGACGATCCCGAGGACAGCCTCGGCG 
               
               
                   
               
               
                 ACGTGTTCCTCTCGGCGATGTTCGGCGATCACCCGGTGGGACGTCCGGTGATCGGCAGCGTCGAGTCGATCGAGACCATG 
               
               
                   
               
               
                 ACGCGTGCACAGCTGCATTCGTTCCACGTCCGGCGTTACACACCCGAACGGATGATCGTGGCGGTGGCCGGCAACGTCGA 
               
               
                   
               
               
                 CCACGACGTGTGGTGTCGTTGGTCCGAGAGCATTTCGGCCCCCGGCTGGAGGCCGACGTTCCGCGGTGGCTCCCCGTAAG 
               
               
                   
               
               
                 GCTCGGGACGGGTCGGTGGTAAGCCATCGCTGCTCGTGGTCGACCGCGACGGGGAACAGTCCCATGTCTCGCTGGGCGTT 
               
               
                   
               
               
                 CGCACGCCCGGCCGGCACTGGGAGCACCGGTGGGCCCTGTCGGTGTTGAACACCGCGCTGGGAGGCGGGCTCAGTTCTCG 
               
               
                   
               
               
                 TCTGTTCCAACAGATTCGCGAGTCCCGCGGCCTGGCCTACCTCGGTGTACTCGACCGTGGACCACTTCGCGACAGCGGGG 
               
               
                   
               
               
                 CTCTGTCGGTGTATGCGGGATGTCAGCCGGAACGTTTCGACGAAGTGGTGCGGGTGACCACCGAAGTTTTGGAAGGTGTT 
               
               
                   
               
               
                 GCCAGAGACGGGATCACCGCCGACGAATGCCGGATCGCCAAAGGCTCGTTGCGCGGTGGGCTGGTGCTCGGCCTGGAGGA 
               
               
                   
               
               
                 TTCCGGATCACGTATGCACCGGATCGGCCGTAGCGAGCTCAATTACGGTGgAGCACCGGACCATCGACCACACGCTGGCC 
               
               
                   
               
               
                 CAGATCGAGGCAGTCACTCTAGAAGAGGTCAACGCCGTCGCTCACCAGTTGCTGTCGCGGGACTACGGTGCCGCCGTACT 
               
               
                   
               
               
                 CGGTCCCTATAGTTCGAAAAAGGCGCTGCCACAACAGCTTCAAACTATCGCCGGCTGACCCGCTACACTGGGTCCAATGG 
               
               
                   
               
               
                 ATTAGAAGGAGAAGTACCGATGGGATCCGGTACCAATGGCGACAGATTATACCGTGCTGACTCTAGACCCCCAGATGAAA 
               
               
                   
               
               
                 TAAAACGTTCCGGAGGTCTTATGCCCAGAGGGCATAATGAGTACTTCGATAGAGGAACTCAAATGAATATTAATCTTTAT 
               
               
                   
               
               
                 GATCACGCGAGAGGAACACAAACCGGCTTTGTCAGATATGATGACGGATATGTTTCCACTAAGCTTAGTTTGAGAAGTGC 
               
               
                   
               
               
                 TCACTTAGCAGGACAGTCTATATTATCAGGATATTCCACTTACTATATATATGTTATAGCGACAGCACCAAATATGTTTA 
               
               
                   
               
               
                 ATGTTAATGATGTATTAGGCGTATACAGCCCTCACCCATATGAACAGGAGGTTTCTGCGTTAGGTGGAATACCATATTCT 
               
               
                   
               
               
                 CAGATATATGGATGGTATCGTGTTAATTTTGGTGTGATTGATGAACGATTACATCGTAACAGGGAATATAGAGACCGGTA 
               
               
                   
               
               
                 TTACAGAAATCTGAATATAGCTCCGGCAGAGGATGGTTACAGATTAGCAGGTTTCCCACCGGATCACCAAGCTTGGAGAG 
               
               
                   
               
               
                 AAGAACCCTGGATTCATCATGCACCACAAGGTTGTGGAAATTCATCAAGAACAATCACAGGTGATACTTGTAATGAGGAG 
               
               
                   
               
               
                 ACCCAGAATCTGAGCACAATATATCTCAGGGAATATCAATCAAAAGTTAAGAGGCAGATATTTTCAGACTATCAGTCAGA 
               
               
                   
               
               
                 GGTTGACATATATAACAGAATTCGGGATGAATTATGACTGCAGCATCACCATCACCATCACTAGTGAAATAGCGAAACAC 
               
               
                   
               
               
                 GGGATCGGGCGAGTTCGACCTTCCGTCGGTCTCGCCCTATTAATAGTGGCATGCAAGCTTGGCACTGGCCGTCGTTTTAC 
               
               
                   
               
               
                 AACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAAT 
               
               
                   
               
               
                 AGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCT 
               
               
                   
               
               
                 CCTTACGCATCTGTGCGGTATTTCACACCGCATATGCAGGGGGGGGGGGGCGCTGAGGTCTGCCTCGTGAAGAAGGTGTT 
               
               
                   
               
               
                 GCTGACTCATACCAGGAACGAGGACAGTCGCACGACGAAGTTCTTCTGGATCGCGCCCGTGCTGGAAGCACTCAACCTCG 
               
               
                   
               
               
                 AAGCGTGTGGTTGCGGAGCCATCTAGCAACCACACGAAACATGCGCAACGAACCGCGCAACGAACAACGCCTAGAACTGG 
               
               
                   
               
               
                 CCCTAGATGAGCTGACTCGTATCGTTGGTAAACCTAGTTTGACCAGCATGTTTTAACTACGTTCGGTGAGCTGTCAACGG 
               
               
                   
               
               
                 GGCCTGTAACGGCACAACGAACCGTGCAACGAGAGTGGCCACGGATGCCACCACAGGCACTACAACGGAGTTCGCCACGT 
               
               
                   
               
               
                 ACATCACCACAACCACCGATTCTGGCGGTGAGCTCCCCGATATTCAGCGGAAATGGCTTGGTATCGACCAAGATTCGTAG 
               
               
                   
               
               
                 AACCCCGTCTCGTCTGGCTGGTATTCAAAACGGGCGCAACGAAACACGCAACGAGACAGGCATGGCCCAAACCAGAAAAC 
               
               
                   
               
               
                 TAGCGTCTACCAGGACTTTTACCTGTCCGACCCGTTGCAACGGAACCCCCCACGGAACCCCCGCGACACCCGCTCCCCAA 
               
               
                   
               
               
                 TTGCGTTAGAACAGCGGTGGATTGTCGGCTTCGTTGTGGGCCTTTTGAGCCGCTTCCTGTTCTGCCGCACGCTCTTTCCT 
               
               
                   
               
               
                 CGCCCGATAGCCGAGTCGCTTAACGGTGTCCAGATGCAGCCCGAAATGTTTGGCCGTTTGCGGCCAAGAGTGGCCCTCGT 
               
               
                   
               
               
                 CGTCGTGATAGGCGCGGATGCGTTCGCGGCGTGCAGCCTGCTCGGCGAGCCACTCGCTGCGTTCCTGCGCCACGAGCCGG 
               
               
                   
               
               
                 ACGACGTGGCGTTCGGATAGTCCGGTGATTCGAGCGCCTTCGGCGGCGGTCACGCGCCGCTTTTTGCGGACAGTCGGCTG 
               
               
                   
               
               
                 CCGGTTGTAGCCGTCGCTGTAGCCGTCGCTCATAGCAATGCCTCCATGGCTGACGCGGACTTTGCGCGCCGCGCAACTGT 
               
               
                   
               
               
                 GCTCGCCGCCGTGCGCGCTGCTGCGCCCTTCCGCGAGATGGCCGACTGGCGCGCACTGAGTGTGGCCTCGTAGACCACGA 
               
               
                   
               
               
                 TCCCGTCCGCCCAAATGCGCGACTTGGTTGTGATCCAACGCCAAATGCTGTTGGCGATGGCGCGGACCTCGCTGTCCGGT 
               
               
                   
               
               
                 AGCGGTCCGGGACACACGTCGTTGCACGGGAATTCGGCGTTTCGCGCGTGGCACTCGGCATAGATCGCGCGGCCGAGTCC 
               
               
                   
               
               
                 GTCCACGTTCCGGGTCGGCAGGTAGATCCGCATGAGGGCGGGACGATAGGCCCACAACCTGACGGAATCGAACAGTGCGC 
               
               
                   
               
               
                 AATTCCGCCCTAGCGGCGTCGGAGCCGCTTTGTACGTGGTCTGCTGACGCCAGCGCGGCGGTGGCATGTTCGCGCCGAGC 
               
               
                   
               
               
                 TCGGCCTCGATGTGGCTGAGTGTGTAGAGATCTGAGTGGAGCCATTCCGTTTCCCAGGCGATGTGGCCGGGGTTTTTGGT 
               
               
                   
               
               
                 CATGAGGCCTGAGTAACTGCGGTCGCCGTCGACGGCGCGCCGAAGGCCTTCGGCGCACGCCGCCATGTATGCGAGCGGCT 
               
               
                   
               
               
                 TACGCCGCGCGTATTCGGTGCGTGGAACAGGGGCGTTGAGTGCCCACACTGCGTGTGCGTGGCCGTTGGCGCGATTGCCC 
               
               
                   
               
               
                 ACGATCGCGTTGGGCAGCGGATGGGACCCCCGGGCGCTGAGCGCTCGGAGCGCTGCGTCTGGATGGTCTACGTCCACGAC 
               
               
                   
               
               
                 CAGCAGGTTTGCCAGCGCTGTTGGGTTCGCCTCGATGTACCGGCGGCCTAGGGCCGACGCGCGGCTTTGGCGGTAGATCC 
               
               
                   
               
               
                 CCTCGAGCAGATCGTCGCTTGCCAGCGGCCAGTACGGCAGCCAGAGCTGCTCAAATTCGTCGGCGACGTGGCTCACGCTT 
               
               
                   
               
               
                 GGTAGTAGACCACGATTAATCACCGGTGTATGGTCCGACACGAGCTCCAAGTCAGATATTTCGCTGAGGGGCCACCCCAC 
               
               
                   
               
               
                 AACTGCACACTCCCCCGCTCTCCCGTCGAGCCCTGGTGGTGGAACACCAGCGACAGCCGAGCACCCCCAACCACCTGTAC 
               
               
                   
               
               
                 CAACCAGGAGGAACACATGCGTCGTTTCGAGGACGTTTCCGGGCCGCTGAGAGCCGCTGTGGCGGCCGTACACGCCGCCT 
               
               
                   
               
               
                 TAGACCCGTTAGACCCCCTGCCGCCTGAATGCGCGGGTACGAGCCACACAGCGCCCGAACTTACGGAGCTGGTGGGCTCA 
               
               
                   
               
               
                 CCTGGCTTTATGGCGTACGAATCGGCTGTGTGCGACCTGTTGGGCGAGGTGAGGTACGCGCTACTCACGCTGGCAAGGGC 
               
               
                   
               
               
                 GACACAGCCGCCCCACCGAGCCCGCACGGCCGCGCGCGGTGTCAACAACCGGGTGAGTCGTGCACACCAGCAGGTGTTCG 
               
               
                   
               
               
                 AGGCTTGGCTCGAAGTGCAGGACATCGTGGCGAACGCCGCCCGATGAGCCGCGCCTTACGCTGGCTGCCAGCCGTTCGCG 
               
               
                   
               
               
                 GGCTGGTTGGTGCAGCGCGTCGAGCGGTTAGAGGCCCTGCGGTGTTCCACCACCGCAGGGCCTCGCCCTTTTTAAGGCTG 
               
               
                   
               
               
                 AATTTGCTTGTCTCCGAATCCAACTGGCTTGTCCAAGGGTGTATCTACGCTTAATCCAAAGTTCAAACGAGGGGATTACA 
               
               
                   
               
               
                 CATGACCAACTTCGATAACGTTCTCGGCTCGATCCTGAATCGCCCCATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTG 
               
               
                   
               
               
                 ATGAGAGCTTTGTTGTAGGTGGACCAGTTGGTGATTTTGAACTTTTGCTTTGCCACGGAACGGTCTGCGTTGTCGGGAAG 
               
               
                   
               
               
                 ATGCGTGATCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCGCCGTCCCGTCAAGTCAGCGTAATGCT 
               
               
                   
               
               
                 CTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATAT 
               
               
                   
               
               
                 CAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATG 
               
               
                   
               
               
                 GCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAG 
               
               
                   
               
               
                 GTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTT 
               
               
                   
               
               
                 GTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGA 
               
               
                   
               
               
                 GCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAG 
               
               
                   
               
               
                 CGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGA 
               
               
                   
               
               
                 GTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTG 
               
               
                   
               
               
                 ACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATA 
               
               
                   
               
               
                 CAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGG 
               
               
                   
               
               
                 AATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCA 
               
               
                   
               
               
                 GACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGCTTTC 
               
               
                   
               
               
                 CCCCCCCCCCCTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCA 
               
               
                   
               
               
                 GGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAG 
               
               
                   
               
               
                 TTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAAT 
               
               
                   
               
               
                 CTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTG 
               
               
                   
               
               
                 AGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATC 
               
               
                   
               
               
                 AAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCG 
               
               
                   
               
               
                 TAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGC 
               
               
                   
               
               
                 CAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGG 
               
               
                   
               
               
                 GGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGC 
               
               
                   
               
               
                 GCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCT 
               
               
                   
               
               
                 TCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCT 
               
               
                   
               
               
                 CGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCT 
               
               
                   
               
               
                 CACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCG 
               
               
                   
               
               
                 CAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGA 
               
             
          
         
       
     
     e) Obtainment of the strain of recombinant BCG expressing LTK63, rBCG-LTK63 to prepare rBCG-LTK63, a stock aliquot of 50-200 μl of competent BCG was mixed with 0.1-1 μg of pNL12-LTK63, in 2 mm electroporation cuvettes and this was submitted to pulsations of 2.5 kV, 25 μF and 1000Ω, in an electroporation system (GenePulser, BioRad, Hemel Hempstead, UK). After electroporation, the content of the cuvettes was recovered in 2 mL of medium (MB7H9/Tw/ADC) without antibiotic and incubated at 37° C. for 20 h before being seeded in Middlebrook 7H10 (Difco) solid medium, supplemented with 10% oleic acid-albumin-dextrose-catalase (MB7H10/OADC) plus kanamycin (20 μg/mL) for selection of the transformants. Approximate composition of the Middlebrook 7H10 solid medium according to the manufacturer (Difco-BD): Ammonium Sulfate, Potassium Monophosphate, Potassium Biphosphate, Sodium Citrate, Magnesium Sulfate, Calcium Chloride, Zinc Sulfate, Copper Sulfate, L-Glutamic Acid, Ferric Ammonium Citrate, Pyridoxine Hydrochloride, Biotin, Malachite Green and Agar. The selected rBCG colonies were then transferred to 5 mL of liquid medium of MB7H9/Tw/ADC culture with kanamycin (20 μg/mL). 
     e) Obtainment of the strain of recombinant BCG expressing the A subunit LTAK63, rBCG-LTAK63: to prepare rBCG-LTAK63, a stock aliquot of 50-200 μl of competent BCG was mixed with 0.1-1 μg of pLN12-LTAK63, in 2 mm electroporation cuvettes and this was submitted to pulsations of 2.5 kV, 25 μF and 1000Ω, in an electroporation system (GenePulser, BioRad, Hemel Hempstead, UK). After electroporation, the content of the cuvettes was recovered in 2 mL of medium (MB7H9/Tw/ADC) without antibiotic and incubated at 37° C. for 20 h before being seeded in Middlebrook 7H10 solid medium (Difco) supplemented with 10% oleic acid-albumin-dextrose-catalase (MB7H10/OADC) plus kanamycin (20 μg/mL) for selection of the transformants. Approximate composition of the Middlebrook 7H10 solid medium according to the manufacturer (Difco-BD): Ammonium Sulfate, Potassium Monophosphate, Potassium Biphosphate, Sodium Citrate, Magnesium Sulfate, Calcium Chloride, Zinc Sulfate, Copper Sulfate, L-Glutamic Acid, Ferric Ammonium Citrate, Pyridoxine Hydrochloride, Biotin, Malachite Green and Agar. The selected rBCG colonies were then transferred to 5 mL of liquid medium of MB7H9/Tw/ADC culture with kanamycin (20 μg/mL). 
     g) Preparation of the batches of rBCG-LTK63 and rBCG-LTAK63: Next the clones of these two strains were expanded to 50 ml of ME7H9/Tw/ADC liquid medium or in any medium described for the culture of mycobacteria plus kanamycin (20 μg/mL). After 2-3 weeks, when the culture reaches DO 600  0.6-0.8, the samples were centrifuged, washed twice with distilled H 2 O, resuspended in 1 mL of 10% glycerol and aliguoted at a volume of 50 μl, then stored at −80° C., for subsequent use in the immunization assays. 
     h) Evaluation of the batches of rBCG-LTK63 and rBCG-LTAK63: the viability of the batches of rBCG-LTK63 and rBCG-LTAK63 was assessed by counting the number of colony-forming units (CPU). The CFU number was determined as follows: one or more aliquots of the batch frozen at were thawed and several successive dilutions (1×10 2 , 1×10 4 , 1×10 5  and 1×10 6 ) performed. Then the 1×10 5  and 1×10 6  dilutions were cultured in MB7H10/OADC medium plus kanamycin (20 μg/mL) and incubated at 37° C. After 3-4 weeks the number of colonies on the plate is counted. 
     i) Characterization of the Expression of LTK63 in Recombinant BCG 
     As it was not possible to determine the expression of the LTK63 gene using the classic immunoassay method, the characterization of the LTK63 expression was performed indirectly by confirming the presence of the LTK63 gene in the plasmid inside the recombinant BGC. This characterization was performed through PCR assay using specific initiators, the same already described above and used to amplify and clone the LTK63 gene in the pMIP12 vector ( FIG. 1 ). 
     j) Characterization of the LTAK63 Expression in Recombinant BCG 
     To verify the expression of the LTAK63 gene, the participants conducted an immunoassay (Western blot) using a polyclonal anti-LT serum and taking the following steps: one or more stock aliquots of rBCG-LTAK63 were sonicated for 1.5 min on ice at a constant amplitude corresponding to half the maximum value (Soniprep 150 MSE, UK) and centrifuged for precipitation of solids. The supernatants were recovered and the protein concentration dosed using the BIO-RAD Protein Assay (Bio-Rad) kit and employing bovine albumin as standard. Samples containing 10 μg of total proteins were applied in 10% polyacrylamide gel in presence of sodium dodecyl sulfate (SDS-PAGE 1.0%). Electrophoresis was performed at room temperature at 120 V until the dye reached the end of the gel. After electrophoresis, the proteins were transferred to PDVF membrane with 0.45 μm pores (GE Healthcare), using a semi-dry transfer system. The gel containing LT, as positive control, was glued on the PDVF membrane, immersed in transfer buffer (Tris 0.25 M Tris, pH 8.3, 0.129 M glycine and 20% methanol) and placed between five filter sheets also immersed in the same buffer. The set was placed between two plates and submitted to a current of 120 mA for 1.5 h at room temperature. At the end of the electrotransfer, the membrane was removed from the system and incubated in blocking solution (PBS containing 5% milk—milk/PBS) at 4° C. overnight. At the end of the blocking, the membrane was incubated at room temperature for 2 h with serum anti-LT diluted in milk/PBS (dilution 1:1000). After this incubation period, the PVDF membrane was washed three times under slight agitation, with 0.1% PBS/Tween20 (PBS-T) at 10-min intervals. The membrane was then incubated for 2 h under the same conditions in milk/PBS containing HRP-conjugated anti-IgG antibody (Sigma, Chem Co, St. Louis) and once again washed 3 times with PBS-T. The development was carried out by chemoluminescence using the ECL kit (Amersham) through exposure in a photo documentation apparatus (ImageQuant LAS4000—GE) ( FIG. 2 ). 
     Example 2 
     Humoral and Cellular Immune Response Assay of Animals Immunized with rBCG-LTK63 
     The study subjects were adult female BALB/c mice ( Mus musculus , Rodentia, Mammalia) aged 6-8 weeks originating from and maintained under the standard conditions of the Central Animal Facility of the School of Medicine of the University of São Paulo. 
     The animals were split into four groups (n=30): 
     BCG Group: animals immunized subcutaneously (s.c.) with 0.1 mL of BCG suspension (1×10 6  CFU/mouse suspended in PBS 1×); 
     rBCG-LTK63 Group: animals immunized subcutaneously (s.c.) with 0.1 mL of rBCG-LTK63 suspension (1×10 6  CFU/mouse suspended in PBS 1×); 
     rBCG-LTKA63 Group: animals immunized subcutaneously (s.c.) with 0.1 mL of rBCG-LTAK63 suspension (1×10 6  CFU/mouse suspended in PBS 1×); 
     Control Group: animals inoculated with 0.1 mL of saline (PBS 1×). 
     a) Humoral response: for this procedure, the animals immunized or inoculated with the control, with BCG or rBCG-LT ( FIG. 3 ) or with BCG or rBCG-LTAK63 ( FIG. 4 ), underwent retro-orbital bleeding one day before the challenge and had their serum separated and aliquoted for subsequent characterization by the ELISA method. The isotypes were determined in serums isolated one day before the challenge against MTB or 90 days after immunization. The serums of 5 animals per group were analyzed individually by ELISA, using specific mouse anti IgG1 and IgG2a antibodies. The humoral response analysis was conducted comparing the concentration of the antibodies and/or specific subtypes IgG1 and IgG2a against PDS in the different immunization groups. The standard curve of these antibodies detected by specific monoclonal antibodies against the respective immunoglobulins was used for this purpose. The comparative analysis of the response of antibodies IgG1 and IgG2a induced against mycobacteria proteins (PDS) is important, as it can provide data on the profile or behavior of the immune response against these proteins. Emphasizing that a profile that shows a Th1-shifted response, and therefore more suitable for fighting an infection caused by MTB, can be identified through the IgG1/IgG2a ratio ( FIGS. 3 and 4 ). 
     b) Cellular response of rBCG-LTK63: the evaluation of the cellular response of the Saline, BCG and rBCG-LTK63 groups was carried out using the ELISA and ELISPOT methods. Initially the spleens of the mice immunized as described above were removed 30 days after the first and only immunization, macerated and strained through screens with porosity of 70 μm (Cell strainer-BD Falcon, Bedford, Mass.). The splenocytes thus obtained from each group (Saline, BCG and rBCG-LTK63) were counted in a Neubauer chamber and had their viability assessed through Trypan blue staining. Cell dilution was then performed in RPMI complete medium [RPMI 1610 (GIBCO Life Technologies), penicillin (100 U/ml), streptomycin (100 μg/ml) plus 10% of bovine fetal serum], in the concentration of 2×10 6  cells/mL in the ELISA assay and 1×10 5  cells/mL in the ELISPOT assay, followed by seeding on a 24-well cell culture plate under PDS stimulus (2 μg/mL) and incubation for 24 hours and 48 hours, respectively, at 37° C. with 5% CO 2  atmosphere. In the group treated with rBCG-LTK63 a significant increase was observed in the levels of INF-γ and TNF-α in relation to the BCG Group. This fact indicates a Th1 polarized response ( FIGS. 5 and 6 ). 
     c) Cellular response of rBCG-LTAK63 the evaluation of the cellular response of the Saline, BCG and rBCG-LTAK63 groups was carried out using the ELISA and Flow Cytometry (FACS) methods. Initially the spleens of the mice immunized as described above, were removed 60 days after the first and only immunization, macerated in Pyrex tissue homogenizers (Fischer Scientific—USA) and had the splenocytes recovered. The splenocytes thus obtained from each group (Saline, BCG and rBCG-LTAK63), were counted in a Neubauer chamber and had their viability assessed through Trypan blue staining. This stage was followed by cell dilution in RPMI complete medium [RPMI 1640 (GIBCO Life Technologies), penicillin (100 U/ml), streptomycin (100 μg/ml) plus 10% of bovine fetal serum], in the concentration of 2×10 6  cells/mL for the ELISA and FACS assays. In the ELISA assay the cells were seeded on a 24-well cell culture plate under PDS stimulus (2 μg/mL) and incubated for 48 hours, at 37° C. with 5% CO 2  atmosphere. In the FACS assay, a volume of 500 μL of cells was seeded on a 96-well plate under PDS stimulus (5 μg/mL) and incubated for 5 hours at 37° C. with 5% CO 2  atmosphere. Each sample was aliquoted and marked in three different tubes (200 μl/tube) following protocol of intracellular permeabilization and marking to separately analyze the intracellular expression of INF-γ and TNF-α within the subpopulation of CD4 +  T cells. A standard protocol provided by the manufacturer was used for the surface marking (anti-CD4-PerCP, BD Pharmingen, San Diego, Calif. USA) while the monoclonal (mAb) anti-IFN-γ (clone 4S.B3; ED Pharmingen, San Diego, Calif., USA) and anti-TNF-α (clone MAb11; BD Pharmingen, San Diego, Calif., USA) antibodies were used for intracellular marking. In the group treated with rBCG-LTAK63 a significant increase was observed in the levels of INF-γ and TNF-α in relation to the BCG group, both in the ELISA assays and in the flow cytometry assay. This fact indicates a Th1 polarized response ( FIGS. 7 and 8 , ELISA;  FIGS. 9 and 10 , FACS). 
     Example 3 
     Development of the Animal  Tuberculosis  Challenge Model—Assays of Protection Against an Intratracheal Challenge with the Strain of  Tuberculosis  H37Rv 
     The study subjects were adult female C57BL/6 or BALB/c mice ( Mus musculus , Rodentia, Mammalia) aged from 6 to 8 weeks and originating from and maintained under the standard conditions of the Central Animal Facility of the School of Medicine of the University of São Paulo. 
     The strain of  Mycobacterium tuberculosis  (MTB) 37HRv was used for the challenge assays. This strain was cultured in MB7H9/Tw/ADC liquid medium in an oven at 37° C. and 5% CO 2 . The bacillary suspension of MTB was cultured in MB7B9/Tw/ADC medium for two weeks and only suspensions with at least 80% of viable bacilli were used for the challenge by the intratracheal route. The bacillary culture was centrifuged at 4000 rpm and washed twice with an equal volume of the culture with PBS 1×. Then the sediment was resuspended in 1 mL of PBS 1× and the number of bacilli estimated using the Macfarland scale. 
     The intratracheal challenge followed the method previously described [Pelizon et al. (2010) Neonatal BUG immunization followed by DNAhsp65 boosters: highly immunogenic but not protective against Rodentia  tuberculosis —a paradoxical effect of the vector? Scand J Immunol. 71: 63-69. The mice were infected with 1×10 5  CPU of viable MTB or inoculated with 100 μl of PBS (Phosphate Buffered Saline) via intratracheal route under anesthesia (200 μL of a mixture of Ketamine/Xylazine). 
     The animals were followed up for four weeks. After this period they were all sacrificed in a CO 2  chamber and their lungs were removed for processing. One of the lobules of each lung was separated and macerated in a total volume of 1 mL of PBS 1× solution. Then this material was serially diluted and seeded in petri dishes containing MB7H10/OADC solid medium. Thirty days later, the number of CFU was determined in the plates corresponding to each group of immunized animals ( FIG. 11 ).

Technology Category: 1