WO2018006939A1

WO2018006939A1 - Inactivated tuberculosis vaccine

Info

Publication number: WO2018006939A1
Application number: PCT/EP2016/065799
Authority: WO
Inventors: Carlos MARTIN MONTAÑÉS; Juan Ignacio AGUILÓ; Santiago URANGA MAÍZ; Esteban RODRIGUEZ SANCHEZ; Eugenia PUENTES COLORADO; Concepción FDEZ. ALVAREZ-SATUALLO
Original assignee: Biofabri SL; Universidad de Zaragoza
Current assignee: Biofabri SL; Universidad de Zaragoza
Priority date: 2016-07-05
Filing date: 2016-07-05
Publication date: 2018-01-11
Anticipated expiration: 2019-01-05

Abstract

The present invention refers to aninactivated isolated microorganism belonging to the Mycobacterium tuberculosis complex that comprises the deletion or previous disruption of the phoP gene; and a second gene to prevent DIM production. Also it refers to its use as vaccine for the prevention or treatment of tuberculosis and/or bladder cancer.

Description

Inactivated tuberculosis vaccine

The present invention refers to an inactivated isolated microorganism belonging to the Mycobacterium tuberculosis complex that comprises the deletion or previous disruption of the phoP gene; and a second gene to prevent DIM production. Also it refers to its use as vaccine for the prevention or treatment of tuberculosis and/or bladder cancer. Therefore the present invention belongs to the medical field. BACKGROUND ART

Tuberculosis (TB) disease causes one and a half million deaths per year, and is one of the leading infectious diseases affecting mainly developing and underdeveloped countries. The rising spread of multidrug resistant strains with the increasing globalization makes TB an alarming global health problem (Marinova, D., et al., Expert Rev Vaccines, 2013. 12(12): p. 1431 -48). Therefore, there is an urgent need for new effective TB vaccines.

Vaccination through the natural route of infection represents an attractive approach in vaccinology for priming the natural host immunity. In the case of TB, pulmonary mucosal tissue is the primary site for establishment of infection. It is been well described in different preclinical models that vaccination with BCG by the pulmonary route confers a substantially improved protection in comparison to subcutaneous or intradermal immunization (Aguilo, N., et al., J Infect Dis, 2016. 213(5): p. 831 -9; Lagranderie, M., et al., Tuber Lung Dis, 1993. 74(1 ): p. 38-46 . Indeed in the last few years, it has raised a special interest in exploring new vaccination approaches delivered through pulmonary routes of administration. These strategies include live attenuated M. tuberculosis (Kaushal, D., et al., Nat Commun, 2015. 6: p. 8533), as well as subunit vaccines formulated with adjuvants or non-replicative virus (White, A.D., et al., Clin Vaccine Immunol, 2013. 20(5): p. 663-72). In 2013, it was reported the first clinical trial of an aerosol TB vaccine (Satti, I., et al., Lancet Infect Dis, 2014. 14(10): p. 939- 46).

MTBVAC is a live rationally-attenuated derivative of the M. tuberculosis isolate MT103, which belongs to the Lineage 4 (Euro-American) which is one of the most widespread lineages of M. tuberculosis. MTBVAC contains two independent stable deletion mutations in the virulence genes phoP and fadD26 without antibiotic resistance marker, in accordance to the established by the Second Geneva Consensus (Arbues, A., et al., Vaccine, 2013. 31 (42): p. 4867-73). PhoP is a transcription factor that controls 2% of the genome of M. tuberculosis including production of immunomodulatory cell-wall lipids and ESAT-6 secretion (Gonzalo-Asensio, J., et al., PLoS One, 2008. 3(10): p. e3496). Deletion of fadD26 leads to complete synthesis abrogation of the virulence surface lipids phtioceroldimycocerosates (DIM) (Camacho, L.R., et al., Mol Microbiol, 1999. 34(2): p. 257-67). MTBVAC has been the first and only live attenuated M. tuberculosis vaccine approved to enter in clinical trials. First-in-human MTBVAC clinical trial was conducted successfully in healthy adults in Lausanne (Switzerland) (Spertini, F., et al., Lancet Respir Med, 2015. 3(12): p. 953-62).

It is assumed that inactivated whole-cell TB vaccines lose most of their immunogenic and protective potential, in comparison to BCG or other live attenuated vaccines (Collins, D.M., New tuberculosis vaccines based on attenuated strains of the Mycobacterium tuberculosis complex. Immunol Cell Biol, 2000. 78(4): p. 342-8). Nevertheless, likely due to some safety concerns described for BCG under some situations (i.e, immunodeficiencies), different authors have explored diverse inactivated vaccine approaches for TB. To overcome the loss of immunogenicity, different strategies have been conducted with these vaccine approaches, including multiple immunizations or the use of inactivated whole-cell vaccines as booster for BCG (see for example in WO2008128065A2 or WO0047225A2). Vaccination through the natural route of infection represents an attractive approach in vaccinology for priming the natural host immunity. In the case of TB, pulmonary mucosal tissue is the primary site for establishment of infection. In this regard, vaccines delivered by pulmonary route represent an unique approach in the TB vaccine field as they trigger induction of pulmonary mucosal immune response directly in the target organ of TB infection. In the particular case of mucosal immunoglobulins type A (IgA), IgA-deficient mice are more sensitive to pulmonary challenge with BCG (Rodriguez, A., et al., Vaccine, 2005. 23(20): p. 2565-72), suggesting a role for IgAs in mycobacterial infections, which could result an advantage for IgA- inducing vaccines.

Nevertheless the achievements complied by the actual vaccination strategies, an improved immunization is needed due to the high incidence of TB around the world.

SUMMARY OF THE INVENTION

The present invention discloses a novel vaccine that comprises an inactivated isolated microorganism belonging to the Mycobacterium tuberculosis complex. Surprisingly the inventors have found that the inactivated (dead) isolated microorganism belonging to the M. tuberculosis complex that comprises the deletion or previous disruption of the phoP gene and the fadD26 gene when administered after Bacille Calmette-Guerin (BCG) confers more protection than the BCG alone or the alive isolated microorganism against TB. Moreover, the administration of MTBVAC+ by intranasal route presents better results than the subcutaneous administration.

The results obtained with the inactivated M. tuberculosis of the present invention support that this vaccine approach confers superior protection in comparison to other whole inactivated mycobacteria that contain the genes phoP and fadD26. The vaccine of the present invention is exemplified by an inactivated version of the live attenuated M. tuberculosis vaccine MTBVAC (Arbues A, et al. Vaccine 2013 31 (42):4867-73; Kamath AT, et al. Vaccine 2005 23(29):3753- 61 ), called MTBVAC+. This vaccine shows great efficacy when given by intranasal route in mice previously vaccinated with subcutaneous BCG. The inventors have demonstrated in addition that phoP and fadD26 deletions in MTBVAC+ are crucial for protection, as the parental strain MT103 (which contains phoP and fadD26 genes) inactivated in the same conditions as MTBVAC+, and administered to mice similarly to MTBVAC+, results unable to improve BCG-induced protection. In addition, in the present invention is described that MTBVAC+ given by the intranasal route as BCG booster induces specifically the generation of specific mucosal immunoglobulins type A (IgA), therefore, it is demonstrated the role of the vaccine of the present invention in protective efficacy.

Therefore, a first aspect of the present invention an inactivated isolated microorganism belonging to the M. tuberculosis complex that comprises the deletion or previous disruption of:

a) the phoP gene; and

b) a second gene to prevent DIM production, preferably wherein this second gene is selected from the list that consists of: fadD22, fadD26, fadD28, ddrC and mmpL7 gene; more preferably is the fadD26 gene. The inactivated isolated microorganism of the first aspect of the invention is also referred to as "the microorganism of the invention" or "the microorganism of the present invention".

The present invention has been exemplified by the M. tuberculosis microorganism and the results can be extrapolated to other members of the M. tuberculosis complex. One skilled in the art knows that members of the M. tuberculosis complex, including M. tuberculosis (the main causative agent of TB in humans), M. bovis (responsible for TB in cattle and including BCG strain) M. africanum (the main cause of TB in Africa), M. microti, M. caprae, M. pinnipedii, and M. Canetti, represent a single species because of the similarities between them (Cole ST, et al. Nature 1998 393: 537-544; Imaeda T, International Journal of Systematic Bacteriology 1985 35 (2): 147-150; Van Soolingen D, et al. International Journal of Systematic Bacteriology 199 747 (4): 1236-1245; Brosch R, et al. PNAS. 2002 99 (6): 3684-3689). Moreover, as is well known to those skilled in the art, between the various members of the M. tuberculosis complex, it is known the existence of the phoP gene and several genes that produce DIM (including fadD26 gene) and also the great similarity between them (Zahrt TC and Deretic V, PNAS 2001 98 (22): 12706- 1271 1 ; Soto CY, et al. J. Clin Microbiol 2004 42 (1 ): 212-219; Camacho LR, et al. Mol Microbiol 1999 34 (2):. 257-267, Camacho LR, et al. 2001 J Biol Chem 276 (23): 19845-19854; Cox JS, et al. Nature 1999 402: 79-83).

Preferably the microorganism of the present invention is M. tuberculosis isolate MT103.

In the present invention the term "disruption" is the interruption of the gene expression by any method known to those skilled in the art, such as by inserting a marker or antibiotic resistance gene. In the present invention the term "deletion" refers to the removal of a part of a gene or an entire gene that causes that the expression of said gene and the deleted strain phenotype is lost.

In the present invention the term gene "phoP' refers to the gene known as Rv0757 by the skilled artisan. For example, in the case of M. tuberculosis H37Rv strain, it refers to the gene with GenBank reference number NC_000962.3; in the case of strain M. bovis AF2122/9, to the gene with GenBank reference number NC_002945.3, gene identification 1092447 (GenBank); and in the case of M. africanum GM041 182 strain, with the gene GenBank reference number NC_015758.1 and gene identification 1095513 (GenBank).

In the present invention, genes that prevent (avoid) the production of DIM (dimycocerosates of tiocerol) are known to the skilled person (for example, those described in Camacho LR, et al. J Biol Chem. 2001 276 (23): 19845- 54). These genes can be selected from the list comprising: fadD26, fadD28, ddrC and mmpL7. In a particular embodiment fadD26 gene is the preferred one (known by the skilled person as Rv2930). For example, the fadD26 gene of M. tuberculosis H37Rv strain of reference number GenBank NC_000962.3, in the case of strain M. bovis, AF2122/97 gene with GenBank reference number NC_002945.3 1 ,092,151 gene identification (GenBank) and in the case of M. africanum GM041 182 strain with the gene GenBank reference number NC_015758.1 and 10956394 gene identification number (GenBank).

The microorganism of the present invention can have also other genes deleted or inactivated, for example the erp gene. It is known by the expert in the field that the erp gene is present in various members of the M. tuberculosis complex and it shares similarities among them (Berthet FX, et al. Science 1998 October 23; 282 (5389): 759-62; Bigi F, et al. Tuberculosis (Edinb) 2005 Jul; 85 (4): 221 -6). In the present invention the term gene "erp" is the gene known for the skilled person as Rv3810, also known as PIRGS. For example, in the case of M. tuberculosis H37Rv strain, it refers to gene GenBank reference number L38851 .1 ; NC_002755.2 in case of M. tuberculosis CDC1551 strain; in the case of M. bovis strain AF2122 / 97, to the gene with GenBank reference number NC_002945.3, gene identification 1093509 (GenBank); and in the case of M. africanum GM041 182 strain, with the gene GenBank reference number NC_015758.1 , 10957490 identification gene (GenBank). The microorganism of the invention may or may not include antibiotic resistance markers, such as kanamycin resistance marker. In general, any type of inactivation procedure can be used for inactivating the microorganism of the present invention as long as the treatment leaves the population of bacteria unable to produce a productive infection at the host, while at the same time preserving antigenic structures necessarily for eliciting a productive response to the corresponding disease-causing mycobacterium. The mycobacterium preparation is typically incapacitated. By "incapacitated" in the context of an incapacitated bacterial cell produced according to the invention, is meant that the bacterial cell is in a state of irreversible bacteriostasis. While the bacterium retains its structure-and thus retains, for example, the immunogenicity, antigenicity, and/or receptor-ligand interactions associated with a wild-type bacterium-it is not capable of replicating. In some embodiments, it is incapable of replication due to the presence of an infecting phage with in the bacterial cell.

A preferred embodiment of the first aspect of the present invention refers to the inactivated isolated microorganism wherein is inactivated by heat, fixation (for example by formalin) or radiation; preferably by heat. The microorganism can also be inactivated by osmotic pressure via salts, freezing or drying process. A preferred type of inactivation is gamma-irradiation. Other types of inactivation known in the art include, e.g. ultra-violet irradiation.

Another preferred embodiment of the first aspect of the present invention refers to the inactivated isolated microorganism wherein the inactivated isolated microorganism is a whole cell, a cell lysate or cell fragments, or combination of them. Example of cell fragments can be: suitable components include whole cell lysate, culture filtrate proteins, cell wall fraction, cell membrane fraction, cytosol fraction, soluble cell wall proteins, and soluble protein pool. In the present invention the inactivated isolated microorganism is selected from the following list: M. tuberculosis, M. bovis, M. africanum, M. caprae, M. pinnipedii, M. canetti or M. microti, preferably is M. tuberculosis.

The present invention also relates to the use of the microorganism of the first aspect of the invention and a pharmaceutical composition that comprises it for the same indications that one skilled in the art knows to BCG. Therefore, the microorganism of the invention and the pharmaceutical composition that comprises it can be used for the treatment or prevention of TB , for the treatment against bladder cancer (Lamm DL, et al. The Journal of Urology 2000; 163: 1 124-1 129; Saint F, et al. European Urology 2003; 43 351 -361 ), and as an adjuvant or vector (Stover CK, et al. Nature 1991 ; 351 (6.): 456- 460 and Bastos RG, et al. Vaccine 2009; 27: 6495-6503).

In some embodiments, at least 90% of the M. tuberculosis complex cells are inactivated, e.g., 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the M. tuberculosis complex cells (preferably M. tuberculosis). When the subject is a human, 100% of the cells are preferably inactivated.

A second aspect of the present invention refers to the inactivated isolated microorganism of the first aspect of the present invention for use as a medicament.

Therefore, the present invention refers to the use of the inactivated isolated microorganism for the manufacture of a medicament. A third aspect of the present invention refers to the inactivated isolated microorganism for use in the prevention or treatment of TB in a subject, preferably a mammal, more preferably a human. Therefore, the present invention refers to the use of the inactivated isolated microorganism for the manufacture of a medicament for the prevention or treatment of TB in a subject, preferably a mammal, more preferably a human.

In the present invention the term "subject" comprises any mammal, preferably a human being, man or woman of any age. The subject can also be an individual at risk of immunosuppression, carrier of HIV, or has impaired cellular immunity (by solid organ transplantation, lymphomas, chronic renal failure, for example) or that has other causes of immunosuppression as solid tumors and leukemias. In another preferred embodiment the individual is a newborn with or without risk of immunosuppression and may also be a HIV carrier newborn. The subject can be also a pregnant animal prior to birth to increase production of hyper immune colostrum.

The present invention also refers to the uses of the inactivated isolated microorganism of the first aspect of the invention and the pharmaceutical composition that comprises it for veterinary uses, therefore the subject can be any other mammal. More preferably the mammal includes, but is not limited to, a pet, a primate or a ruminant; in the context of the present invention, the individual is preferably a goat, cow, bull, sheep, reindeer, horse, deer, cat or dog.

A fourth aspect of the present invention refers to the inactivated isolated microorganism for use in the prevention or treatment of bladder cancer in a subject, preferably a mammal, more preferably a human.

Therefore, the present invention refers to the use of the inactivated isolated microorganism for the manufacture of a medicament for the prevention or treatment of bladder cancer in a subject, preferably mammal, more preferably a human.

A fifth aspect of the present invention refers to the inactivated isolated microorganism for use as a vector or as adjuvant.

In this specification, the term "vector" refers to that the inactivated microorganism of the invention may comprise other vaccine antigens from other infectious diseases such as diphtheria, tetanus, pertussis, or degenerative diseases such as amyotrophic lateral sclerosis and others.

In this specification, the term "adjuvant" refers to an agent, which can stimulate the immune system by increasing its response to a vaccine. The microorganism of the present invention can be used as an adjuvant to other vaccine antigens from other infectious diseases such as diphtheria, tetanus, pertussis, or degenerative diseases such as amyotrophic lateral sclerosis and others.

A sixth aspect of the present invention refers to a pharmaceutical composition that comprises the inactivated isolated microorganism of the first aspect of the present invention wherein said composition is formulated for intranasal, mucosal, oral, aerosol or intrapulmonary delivery to a subject, preferably a mammal, more preferably a human. The term "pharmaceutical composition" herein refers to any substance used for prevention, diagnosis, alleviation, treatment or cure of any disease. In the context of the present invention relates to a composition comprising the inactivated isolated microorganism of the invention. The pharmaceutical composition of the invention can be used either alone or in combination with other pharmaceutical compositions, including vaccines and antiviral. The combination of said pharmaceutical composition with vaccines or antivirals could make more effective the immune response generated, thus acting as an adjuvant. In the present invention the terms "composition", "pharmaceutical composition", "drug" and "medicament" are used interchangeably. In the context of the present invention the term "vaccine" refers to an antigenic preparation used to elicit an immune response to disease caused by a bacterium or a virus. It is a preparation of antigens that once inside the body, causes the immune response by antibody production and generates immunological memory producing permanent or temporary immunity. The pharmaceutical composition of the present invention is therefore a vaccine.

In the present invention the term "antiviral" refers to any substance that does not permit replication, assembly or virus release, such as interferon or ribavirin.

A "disorder" is any condition that would benefit from treatment with the composition of the invention, as described herein.

In a preferred embodiment, the pharmaceutical composition of the invention may further comprise a pharmaceutically acceptable carrier or excipient.

In another further preferred embodiment, the pharmaceutical composition of the invention may further comprise an adjuvant. In the present invention, when the inactivated microorganism is accompanied by an adjuvant this could be for example, known to those skilled in the art. For example, but not limited to, aluminum. Adjuvants may include but are not limited to salts, emulsions (including oil/water compositions), saponins, liposomal formulations, virus particles, polypeptides, pathogen-associated molecular patterns (PAMPS), nucleic acid-based compounds. For example, the adjuvant may be aluminum phosphate, aluminum hydroxide, Freud's adjuvant, squalene, vegetable oils and alum; and being particularly preferred Freund's adjuvant. Other adjuvants include agents such as immune- stimulating complexes (ISCOMs), synthetic polymers of sugars (CARBOPOL®), aggregation of the protein in the vaccine by heat treatment, aggregation by reactivating with pepsin treated (Fab) antibodies to albumin, mixture with bacterial cells such as C. parvum or endotoxins or lipopolysaccharide components of gram-negative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA®) used as a block substitute may also be employed.

The inactivated microorganism of the present invention may be contained in a mucosal bacterial toxin adjuvant such as the Escherichia coli labile toxi (LT) and cholera toxin (CT) or in CpG oligodeoxynucleotide (CpG ODN), derivatives from LPS such as Monophosphoryl lipid A (MPL), Eurocine L3™, endogenous and pharmaceutically accepted lipids, immune modulating substances such as cytokines or synthetic IFN-[gamma] inducers such as poly l:C alone or in combination with the above-mentioned adjuvants.

Other adjuvants that may be used include micropartides or beads of biocompatible matrix materials. The micropartides may be composed of any biocompatible matrix materials as are conventional in the art, including but not limited to, agar and polyacrylates, Chitosan or any bioadhesive delivery system which may be used are known in the art. The term "excipient" refers to a substance that helps the absorption of the pharmaceutical composition comprising the inactivated isolated microorganism of the invention, and also that helps stabilizing or supporting it in its preparation in the sense of giving consistency, shape, flavor or other functional specific characteristics. Thus, the excipients may have the function of holding the ingredients together such as starches, sugars or celluloses, sweeten function dye function, protection function of the drug such as to isolate it from the air and/or humidity; function of filling in a tablet, capsule or any other form of presentation such as dibasic calcium phosphate; disintegrating function to facilitate dissolution of the components and their absorption in the intestine; without excluding other excipients not listed in this paragraph.

A "pharmacologically acceptable carrier" refers to those substances or combination of substances known in the pharmaceutical industry, used in the manufacture of pharmaceutical dosage forms and includes, but not limited to, solids, liquids, solvents or surfactants. The vehicle can be inert or have analogous action to any of the compounds of the present invention substance and whose function is to facilitate the incorporation of the drug as well as other compounds, allow better dosage and administration or give consistency and shape the composition pharmaceutical. When the presentation is liquid, the vehicle is the diluent. The term "pharmacologically acceptable" means that the compound referenced permitted and evaluated so that no harm to the organisms to which it is administered.

In another preferred embodiment, the composition of the invention may further comprise another active ingredient, such vaccine antigens from other infectious diseases such as diphtheria, tetanus, pertussis, or degenerative diseases such as amyotrophic lateral sclerosis and others.

As used herein, the term "active ingredient" ("active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient") means any component that potentially provides pharmacological activity or other different effect in the diagnosis, cure, mitigation, treatment or prevention of a disease, which affects the structure or function of the body of man or other animals. The term includes those components that promote a chemical change in the development of the drug and are present in a modified form that provides the specific activity or effect. The pharmaceutical or drug composition provided by this invention can be provided by any route of administration, for which said composition is formulated in the suitable to the chosen route of administration dosage form. In a preferred embodiment said composition is formulated for intranasal, mucosal, oral, aerosol or intrapulmonary delivery to a subject.

A preferred embodiment of the sixth aspect of the present invention refers to the pharmaceutical composition wherein composition is lyophilized, solid or liquid form.

The inactivated microorganism of the present invention may be contained in a dry powder formulation such as but not limited to a sugar carrier system, preferably that could include lactose, mannitol, and/or glucose. If desired, the inactivated microorganism of the present invention may be contained in a liposomal formulation.

The inactivated microorganism of the present invention may be incorporated into microparticles or microcapsules to prolong the exposure of the antigenic material to the subject animal and hence protect the animal against infection for long periods of time. The microparticles and capsules may be formed from a variety of well-known inert, biocompatible matrix materials using techniques conventional in the art. Suitable matrix materials include for example natural or synthetic polymers such as alginates, poly(lactic acid), poly(lactic/glycolic acid), poly(caprolactone), polycarbonates, polyamides, polyanhydrides, polyortho esters, polyacetals, polycyanoacrylates, polyurethanes, ethytlene- vinyl acetate copolymers, polystyrenes, polyvinyl chloride, polyvinyl fluoride, polyvinyl imidazole), chlorosulphonated, polyethylene oxide agar and polyacrylates. Examples of techniques for incorporation of materials into microparticles or encapsulation are known by the expert in the field. The inactivated microorganism of the present invention may be contained also in small particles suspended in the water or saline.

In another preferred embodiment, the pharmaceutical composition of the invention may be administered either in a single dose or repeated dose chosen by the skilled artisan.

The subject may be vaccinated at any time. In one embodiment, the vaccine may be administered in at least two doses 1 -12 months apart.

Another aspect described herein relates to the use of the pharmaceutical composition described above for stimulating and/or inducing the immune response in a subject. A preferred embodiment refers to the pharmaceutical composition of the invention for use as a vaccine, as a vector or adjuvant, preferably for the treatment or prevention of TB , and/or for the treatment of bladder cancer. Thus, the pharmaceutical composition of the invention is used for treatment or prevention of TB and/or for the treatment of bladder cancer.

In the pharmaceutical composition of the invention the inactivated microorganism is present in a therapeutically effective dose.

In the present invention the term "therapeutically effective dose" (or "therapeutically effective amount") refers to the amount of the agent or compound capable of producing a desired effect and will generally be determined by the compounds' characteristics, the route and frequency of administration form thereof and other factors, including age, condition of the patient and the severity of the alteration or disorder.

The vaccine can be administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to mount an immune response, and the degree of protection desired. Suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination with a preferred range from about 0.1 μg to 1000 μg, such as in the range from about 1 μg to 300 μg, and especially in the range from about 10 μg to 50 μg.

Another preferred embodiment of the sixth aspect of the present invention refers to the pharmaceutical composition wherein the microorganism is in a dose from the equivalent to 10³-10⁹ (10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹) CFU (colony forming units) (as is inactivated, is the equivalent as the CFU before its inactivation), preferably 10⁷ CFU. A preferred embodiment of the second, third, fourth and fifth aspect of the present invention refers to the inactivated isolated microorganism for use for simultaneous or sequential use with Bacille Calmette-Guerin (BCG). The inactivated microorganism and the pharmaceutical composition of the present invention act then as a boosting TB vaccine in patients who have already received BCG or another subunit TB immunostimulant. The BCG used is 10³-10⁹ CFU (10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹), preferably 2.5x10⁵ for newborns or 5x10⁵ CFU for adults and is administered subcutaneously. For newborns the time between the BCG and the inactivated microorganism of the present invention can be from 3 months to 2 years, preferably form 6 months to 1 year (6, 7, 8, 9, 10, 1 1 or 12 months). In adults previously vaccinated with BCG, the inactivated microorganism of the present invention can be administered at any age, preferably from 6 years old, more preferably from 7-10 years old (7, 8, 9 years old), preferably by intranasal, oral, mucosal or intrapulmonary delivery. A seventh aspect of the present invention refers to a kit or a device that comprises the inactivated isolated microorganism of the first aspect of the present invention or the pharmaceutical composition of the sixth aspect of the present invention.

The microorganism of the invention or the pharmaceutical composition can be provided as an aerosol or spray package. The microorganism of the invention or the pharmaceutical composition can be provided as a nose-drop or as an eye-drop package.

An eighth aspect of the present invention refers to the use of the kit or device of the seventh aspect as a medicament, as a medicament for the prevention or treatment of TB or bladder cancer, or as a vector or adjuvant. A ninth aspect of the present invention refers to a method to inactivate an isolated microorganism belonging to the M. tuberculosis complex that comprises the deletion or inactivation of:

a) the phoP gene; and

b) a second gene to prevent DIM production, preferably wherein this second gene is selected from the list that consists of: fadD22, fadD26, fadD28, ddrC and mmpL7 gene; more preferably is the fadD26 gene; wherein the isolated microorganism is inactivated by heat, fixation or irradiation; and wherein the inactivation is by heat is performed by heating at 80-120 °C, preferably at 100°C, during 20-50 minutes, preferably during 30 minutes; when the inactivation is performed by fixation by incubation in 0.05- 0.5% formalin during 48 hours; and wherein the inactivation is performed by irradiation is performed by γ-irradiation at 25-35 kGy.

In another aspect, the invention provides a method of vaccinating a mammal against TB and/or bladder cancer, wherein the method includes administering to the mammal, preferably a human, the inactivated microorganism of the first aspect of the invention, the pharmaceutical composition of the sixth aspect of the invention or the kit o device of the seventh aspect of the present invention; wherein the vaccination of the subject, preferably mammal, more preferably human, is mucosal, preferably intranasal, aerosol or oral, or intrapulmonary; and wherein the composition comprises an immunologically protective dose when delivered to the host. Preferably in the method the administration of the inactivated microorganism of the first aspect of the invention, the pharmaceutical composition of the sixth aspect of the invention or the kit o device of the seventh aspect of the present invention is preformed after BCG or another TB stimulant has been administered to the same subject, preferably it was administered BCG subcutaneously. In a more preferred embodiment the concentration of the BCG is 10³-10⁹ (10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹), preferably 2.5x10⁵ for newborns or 5x10⁵ CFU for adults; the vaccine of the present invention is administered at is 10³-10⁹ (10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹), preferably 10⁷ CFU. In a preferred embodiment, for newborns the time between the BCG and the inactivated microorganism of the present invention can be from 3 months to 2 years, preferably form 6 months to 1 year. In adults previously vaccinated with BCG, the inactivated microorganism of the present invention can be administered at any age, preferably from 6 years old, more preferably from 7-10 years old.

The present invention refers also to a method for testing the vaccine of the present invention in a non-human animal model of TB The animal model can be, e.g., a mouse, guinea pig, rabbit, bovine, or non-human primate.

The present invention also refers to a method to determine specific immunity against the inactivated microorganism of the first aspect of the invention in a subject previously administered with the inactivated microorganism of the first aspect of the invention, the pharmaceutical composition of the sixth aspect of the invention or the kit o device of the seventh aspect of the present invention; wherein the administration has been mucosal, preferably intranasal, aerosol or oral, or intrapulmonary; that comprises the detection and/or quantification of IgA in a biological sample of said subject and wherein the detection and/or quantification of IgA in the biological sample of said subject is indicative of specific immunity against the inactivated microorganism of the first aspect of the invention. In a preferred embodiment the method comprises the comparison with a control (positive or negative) in order to find a significant difference. From now on is called "the detection method of the invention".

In a preferred embodiment the biological sample can be, for example, without limitation, blood, serum, plasma, saliva, urine, sputum, bronchoalveolar lavage (BAL) and bronchial aspirates (BAS).

The term "detection method" in the present invention refers to a method for establishing whether a given sample comprises or includes IgA specific to the inactivated microorganism of the present invention with adequate sensitivity and specificity. Typical intervals detection sensitivity can be between about 20% and about 90% of the maximum signal. The specificity a method is given by the probability of detection is negative in a sample not containing the compound to be detected.

The detection and/or quantification is preferably performed by an immunoassay.

The term "immunoassay" or "immunochemical analysis technique" is an immunochemical assay method in which an antibody that specifically binds to an antigen is used (in the present case an antigen of the IgA to be detected).

The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.

Immunoassays include, without limitation, immunological techniques such as ELISA (Enzyme-linked immunosorbent assay), Western blot, RIA

(radioimmunoassay), competitive EIA (immunoassay competitive enzyme),

DAS-EL ISA (Double Antibody Sandwich-ELISA), immunoaffinity chromatography test strip or lateral flow (lateral-flow immunoassay), immunoprecipitation, dot-blot, radioimmunoassay, flow cytometry, immunocytochemical and immunohistochemical techniques based on the use of biochips biomarker techniques, biosensor or microarray that include specific antibodies or colloidal precipitation-based formats such as "dipsticks" assays. Within other immunoassays also are the transduction principle immunosensors which can be optical, electrochemical, thermometric or massic. Similarly, immunosorbent or immunoaffinity extraction systems, allowing selective extraction of the analyte within a complex mixture, are also included. These systems often are biohybrid materials resulting from stable binding of the antibody to a solid support (polymer, inorganic material, metal particles, etc.) and used for the separation or extraction of the analyte from other components of the matrix. Such formats may be heterogeneous or homogeneous, sequential or simultaneous, competitive or noncompetitive.

Preferably the immunochemical technique is ELISA. They are known different types of ELISA such as direct ELISA, indirect ELISA or sandwich ELISA, competitive or non-competitive. A role in protection of mucosal IgA or other immunoglobulins induced by MTBVAC+ vaccination is demonstrated, therefore, measurement of these immunoglobulins could be used to determine the potency of MTBVAC+ vaccination, which could be crucial to assess potency lot during vaccine production, or to potentially differentiate individuals protected or not in clinic. Therefore, another aspect of the present invention is a method to determine the potency of the vaccination with the microorganism of the present invention that comprises the detection and/or quantification of immunoglobulins induced. The detection and/or quantification of the immunoglobulins can be performed by the immunodetection methods herein described. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. Throughout the description and claims the word "comprise" and its variations are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1. Groups of six C57BL/6 mice were vaccinated by the subcutaneous route with BCG Danish vaccine 10⁶ CFU for adults (A, B), or 2.5x10⁵ for neonates (C, D). Four weeks later (eight weeks in the case of newborns) groups of mice were intranasally immunized with 10⁷ MTBVAC+ bacteria (except indicated group in panel B with 10⁴). One month later, animals were intranasally challenged with 100 CFU of H37Rv (A, B, C) or 10⁴ CFU (D). In panels A, B and C, lung bacterial burden was determined one month post challenge. A representative experiment of at least two independent is shown in each panel. Data in the graphs are represented as mean ± SEM. Mann- Witney tests were performed to calculate statistical significance. For survival experiments, groups of ten animals were vaccinated and challenged. Animal survival was determined according to pre-established endpoint criteria approved by ethical committee. Data from one experiment are represented in a Kaplan-Meier survival curve and statistical significance calculated by a Logrank test. ^* p<0.05; ^** p<0.01 ; ^*** p<0.001 ; ^**** p<0.0001 . "sc", subcutaneous route; "in", intranasal; "unvacc", unvaccinated; "LogCFUs", logarithm of colony forming units. Fig. 2. Groups of six C57BL/6 mice were vaccinated by the subcutaneous route with BCG Danish vaccine 10⁶ CFU. Four weeks later mice were intranasally immunized with 10⁷ HK Mt103 or MTBVAC+ bacteria. One month later, animals were intranasally challenged with 100 CFU of H37Rv, and lung bacterial burden was determined one month post challenge. Results from one experiment are shown. Data in the graphs are represented as mean ± SEM. One-way ANOVA with Dunnett's multiple comparison tests were performed to calculate statistical significance. ^* p<0.05; ^** p<0.01 ; ^*** p<0.001 ; ^**** p<0.0001 . "sc", subcutaneous route; "in", intranasal; "unvacc", unvaccinated; "LogCFUs", logarithm of colony forming units; ΉΚ", heat- killed.

Fig. 3. Groups of six C57BL/6 mice were vaccinated by the subcutaneous route with BCG Danish vaccine 10⁶ CFU. Four weeks later, mice were intranasally immunized with 10⁷ MTBVAC+ bacteria. One month later, animals were euthanized and PPD-specific IgA levels measured in BAL by ELISA as described in materials and methods section. A representative experiment of two independent is shown in each panel. Data in the graphs are represented as mean ± SEM. One-way ANOVA tests with Bonferroni post analysis were performed to calculate statistical significance. ^* p<0.05.. "sc", subcutaneous route; "in", intranasal; "unvacc", unvaccinated; "LogCFUs", logarithm of colony forming units.

Fig. 4. Groups of six C57BL/6 mice were vaccinated by the subcutaneous route with BCG Danish vaccine 10⁶ CFU. Four weeks later mice were intranasally immunized with 10⁷ MTBVAC bacteria inactivated by heat (A), γ- irradiation (B), or formalin (C). One month later, animals were intranasally challenged with 100 CFU of H37Rv, and lung bacterial burden was determined one month post challenge. Results from one experiment are shown. Data in the graphs are represented as mean ± SEM. One-way ANOVA with bonferroni multiple comparison tests were performed to calculate statistical significance. ^** p<0.01 ; ^*** p<0.001 ; ^**** p<0.0001 . "sc", subcutaneous route; "in", intranasal; "unvacc", unvaccinated; "LogCFUs", logarithm of colony forming units; ΉΚ", heat-killed; "γ-'RR". γ-irradiated.

EXAMPLES

MATERIAL AND METHODS Bacteria: BCG and H37Rv strains were grown at 37°C in Middlebrook 7H9 broth (BD Biosciences) supplemented with ADC (BD Biosciences) and 0.05% (v/v) Tween-80. BCG and H37Rv suspensions for vaccination or infection were prepared in PBS from glycerol stocks previously quantified. For MTBVAC+ elaboration, a MTBVAC (Arbues, A., et al., Vaccine, 2013. 31 (42): p. 4867- 73) liquid culture adjusted to a cellular density of about 2x10⁸ CFU per mililiter was inactivated by incubation at 100 degrees during 30 minutes. Heat-killed Mt103 was inactivated following the same conditions.

Mice:

All mice were kept under controlled conditions and observed for any sign of disease. Experimental work was conducted in agreement with European and national directives for protection of experimental animals and with approval from the competent local ethics committees.

Groups of eight to ten weeks-old female C57/BL6 mice (Janvier Biolabs) were vaccinated subcutaneously (100 μΙ) with 10⁶ CFU of BCG Danish 1331 in PBS. Four weeks post-vaccination, in the corresponding groups mice were intranasally vaccinated with the indicated dose of MTBVAC+ or heat-killed Mt103 in 40 μΙ of PBS. One month later, animals were challenged intranasally with 100 CFU of H37Rv virulent strain in 40 μΙ of PBS. Bacterial load from lungs was determined four weeks post-challenge by plating lung homogenates on 7H1 1 +ADC solid agar medium. In indicated experiments, neonatal mice were subcutaneously vaccinated up to three days post birth with 2.5x10⁵ CFU of BCG Danish in 50 μΙ of PBS. Eight weeks later, indicated animals were intranasally immunized with MTBVAC+ as described above. For survival experiments, animals were intranasally challenged with 10⁴ H37Rv CFU. Disease-associated symptoms (including weight, aspect and individual/social behaviour) were monitored twice a week, and mice were humanely euthanized according to pre-established endpoint criteria.

For bronchoalveolar lavage (BAL) collection, mice were euthanized by cervical dislocation. Trachea was cannulated and BAL performed following inoculation of 0.8 ml of ice-cold PBS. Supernatant was separated from cells by centrifugation and frozen at -80°C for further IgA detection analysis.

Enzyme-linked immunoassay (ELISA):

For IgAs determination in BAL, maxisorp ELISA plates (NUNC) were coated with 10 μg ml of purified-protein derivative (PPD) (Statens Serum Institut, Copenhagen) and incubated overnight at 4°C. After a washing step with PBS-Tween20 0.05% (v/v) buffer, the plate was blocked with Bovine Serum Albumin 1 % (w/v) in washing buffer for 1 hour at 37°C. Then, PPD-coated plates were incubated with 100 μΙ of BAL during 90 minutes at 37°C. Following washing step, plates were incubated for 1 hour at 37°C with Horseradish Peroxidase (HRP)-conjugated goat anti-mouse IgA diluted 1 :10000 (Sigma). Finally, enzyme-substrate reaction was developed during 30 minutes using 3,3',5,5'-Tetramethylbenzidine (TMB) (Sigma) as substrate, and reaction was stopped with H2SO₄ 0.1 N. Optical density was measured at 450 nm. Example 1 : Intranasal immunization with heat-killed MTBVAC (MTBVAC+) enhances protection conferred by subcutaneous BCG

Eight to ten weeks old female C57BL/6 mice were subcutaneously immunized with 10⁶ CFU of BCG Danish. Four weeks later, two groups of mice were vaccinated with 10⁷ MTBVAC HK bacteria (MTBVAC+) inoculated intranasally or subcutaneously, respectively. To elaborate MTBVAC+, a MTBVAC liquid culture was inactivated following incubation at 100 degrees during 30 minutes. Four weeks after MTBVAC+ administration, animals were challenged intranasally with 100 CFU of the virulent M. tuberculosis strain H37Rv, and one month later mice were sacrificed and lung bacterial load was determined by plating lung homogenates in solid agar medium.

Our data revealed that MTBVAC HK intranasal administration reduced approximately one additional log challenge bacterial load in lungs, when compared to BCG alone, whereas no effect was observed following subcutaneous boosting. Our data also showed that intranasal MTBVAC HK had a protective effect only when combined with BCG, but not when administered in na^'fve mice (Figure 1A). Finally, we only observed improved protection with MTBVAC HK following vaccination with a high dose (10⁷ bacteria), while no additional protection was obtained using a low dose (10⁴ bacteria), suggesting a dose-response protection profile induced by intranasal MTBVAC HK (Figure 1 B). As BCG is given in neonatal population, we assessed ability of MTBVAC HK to enhance BCG-conferred protection following vaccination at birth (Figure 1 C). Bacterial reduction four weeks post challenge under this situation was comparable to the observed in adult mice immunized with BCG. In addition, we evaluated survival in a high-dose challenge model (challenge with 10⁴ CFU of H37Rv), finding that MTBVAC HK intranasal boost extended mouse survival in comparison to BCG sc group (Figure 1 D). Example 2: Heat-inactivated Mt103, the parental strain of MTBVAC, does not confer additional protection to BCG.

Absent genes in MTBVAC, phoP and fadD26, regulate intracellular processes, including the synthesis of surface lipid as PAT, DAT or phthiocerol dimycocerosate (PDIM), described to be important in immunomodulation. Thus, we next explored whether a heat-killed version of Mt103, the parental strain of MTBVAC that contains PhoP and FadD26, and subsequently expresses the mentioned surface lipids, was protective in comparison to MTBVAC+.

As shown in figure 2, our results revealed that intranasal boost with 10⁷ Mt103 heat-killed bacteria, inactivated in similar conditions as MTBVAC+, did not improve protection observed in the BCG only group. Conversely, intranasal MTBVAC+ boost protected almost one log compared to other immunized groups.

Example 3: MTBVAC+ intranasal boost specifically induces generation of mucosal IgA in respiratory airways.

We next performed bronchoalveolar lavages (BAL) from immunized mice to assess the presence of IgA in respiratory airways following vaccination. IgAs have been found important in the immune response against different respiratory pathogens, and indeed we previously reported that pulmonary but not subcutaneous live BCG triggered specifically IgA production in respiratory airways (Aguilo, N., et al., J Infect Dis, 2016. 213(5): p. 831 -9). Our data obtained by ELISA revealed a strong and specific induction of M. tuberculosis- specific IgA in the BCG-vaccinated group boosted with intranasal MTBVAC+ (Figure 3). Noteworthy, immunization only subcutaneous BCG did not elicit specific IgA production. Altogether, these data show a correlation between IgA induction and protection. Indeed, experiments are on course in IgA-deficient to decipher the role of mucosal immunoglobulins in protective efficacy conferred by MTBVAC+. In case these data reveal a protective function of IgA, this could represent a parameter to be exploited as a correlates of protection marker for the MTBVAC+ vaccine.

Claims

1 .- An inactivated isolated microorganism belonging to the Mycobacterium tuberculosis complex that comprises the deletion or previous disruption of: a) the phoP gene; and

b) a second gene to prevent DIM production, preferably wherein this second gene is selected from the list that consists of: fadD22, fadD26, fadD28, ddrC and mmpL7 gene; more preferably is the fadD26 gene.

2.- The inactivated isolated microorganism according to claim 1 , wherein is inactivated by heat, fixation or radiation, preferably by heat.

3. - The inactivated isolated microorganism according to any one of claims 1 or 2, wherein the inactivated isolated microorganism is a whole cell, a cell lysate or cell fragments.

4. - The inactivated isolated microorganism according to any one of claims 1 to 3, wherein the inactivated isolated microorganism is selected from the following list: M. tuberculosis, M. bovis, M. africanum, M. caprae, M. pinnipedii, M. canetti or M. microti, preferably is M. tuberculosis.

5. - The inactivated isolated microorganism according to any one of claims 1 to 4 for use as a medicament.

6.- The inactivated isolated microorganism according to any one of claims 1 to 4 for use in the prevention or treatment of tuberculosis in a subject, preferably a mammal, more preferably a human.

7.- The inactivated isolated microorganism according to any one of claims 1 to 4 for use in the prevention or treatment of bladder cancer in a subject, preferably a human.

8. - The inactivated isolated microorganism according to any one of claims 1 to 4 for use as a vector or as adjuvant.

9. - The inactivated isolated microorganism for use according to claims 5 to 8 for simultaneous or sequential use with Bacille Calmette-Guerin (BCG), wherein preferably the dose of BCG is 10³-10⁹ CFU, preferably 2.5x10⁵ for newborns or 5x10⁵ CFU for adults and the BCG is formulated for subcutaneous administration; and preferably wherein if administered sequentially more preferably for newborns the time between the BCG and the inactivated microorganism according to claims 1 to 4 or the pharmaceutical composition according to claims 10 to 12 can be from 3 months to 2 years, preferably form 6 months to 1 year, or in adults previously vaccinated with BCG, the inactivated microorganism of the present invention can be administered at any age, preferably from 6 years old, more preferably from 7- 10 years old.

10. - A pharmaceutical composition that comprises the inactivated isolated microorganism according to any one of claims 1 to 4 wherein said composition is formulated for intranasal, mucosal, oral or intrapulmonary delivery to a subject, preferably a mammal, more preferably a human.

1 1 . - The pharmaceutical composition according to claim 10 wherein composition is lyophilized, solid or liquid form.

12.- The pharmaceutical composition according to any one of claims 10 or 1 1 wherein the microorganism is in a dose from 10³-10⁹ CFUs.

13.- A kit or a device that comprises the inactivated isolated microorganism according to claims 1 to 4 or the pharmaceutical composition according to claims 10 to 12.

14. - Use of the kit according to claim 13 for the prevention or treatment of tuberculosis or bladder cancer or as a vector or adjuvant.

15. - Method to inactivate an isolated microorganism belonging to the M. tuberculosis complex that comprises the deletion or inactivation of:

a) the phoP gene; and

b) a second gene to prevent DIM production, preferably wherein this second gene is selected from the list that consists of: fadD22, fadD26, fadD28, ddrC and mmpL7 gene; more preferably is the fadD26 gene;

wherein the isolated microorganism is inactivated by heat, fixation or irradiation; and wherein the inactivation is by heat is performed by heating at 80-120 °C, preferably at 100°C, during 20-50 minutes, preferably during 30 minutes; when the inactivation is performed by fixation by incubation in 0.05- 0.5% formalin during 24-72 hours, preferably 48 hours; and wherein the inactivation is performed by irradiation is performed by γ-irradiation at 25-35 kGy.