Therapeutic efficacy of pulmonary delivery of live attenuated mycobacteria

Abstract

The present invention relates to a live-attenuated Mycobacterium tuberculosis composition for use in therapy in a human subject in need thereof, comprising an isolated microorganism belonging to the M. tuberculosis MTBVAC strain having: i) a PhoP phenotype due to inactivation by genetic deletion of the Rv0757 gene, wherein the phoP open reading frame (ORF) sequence consists of SEQ ID NO: 4; and ii) a deletion of a second gene, Rv2930 (fadD26), that prevents PDIM production (PDIM phenotype), wherein the fadD26 open reading frame (ORF) sequence consists of SEQ ID NO: 2; wherein the composition is administered to the subject by pulmonary delivery.

Description

[Technical Field]

[0001] The present invention relates to pharmaceutical compositions, such as vaccines, and methods of making and using such compositions. [Background technology]

[0002] According to the World Health Organization (WHO), asthma has reached epidemic proportions, with over 200 million asthma sufferers worldwide. While particularly prevalent in developed countries, the incidence is also rapidly increasing in low- and middle-income settings. One of the most accepted explanations for the increase in asthma over the past few decades is called the "hygiene hypothesis," which suggests that one of the causes behind this dramatic increase is the result of children's reduced exposure to certain environmental factors (Non-Patent Document 1). In this regard, exposure to certain microorganisms and mites (such as those present on farms) during early development appears to educate the immune system, leading to the acquisition of greater tolerance to allergens (Non-Patent Document 2).

[0003] Asthma is a heterogeneous disease characterized by chronic airway inflammation and remodeling. While asthma can be associated with various types of inflammatory responses, type 2 inflammation is observed in over 80% of pediatric asthma cases. T helper (Th) lymphocytes with a Th2 profile are present in most patients and produce cytokines such as IL-4, IL-5, or IL-13, which are responsible for some of the characteristic clinical symptomatology. IL-5 plays a central role in the survival and recruitment of eosinophils, one of the key players in asthma, and its presence in sputum is one of the most accepted biomarkers for diagnosing the disease. Furthermore, IL-4 and IL-13 cause airway remodeling by inducing airway epithelial cell proliferation and exacerbating mucus production (Non-Patent Document 3).

[0004] In addition to adaptive responses, various studies over the past few years have demonstrated the crucial importance of innate lung populations in the induction of asthma. Indeed, allergen presentation via MHC-II molecules from antigen-presenting cells (APCs) is essential for the consequent induction of allergen-specific T cells (Non-Patent Document 4). Asthma is associated with pathological macrophage polarization toward the M2 phenotype (Non-Patent Document 5). M2 (i.e., alternatively activated) macrophages adopt regulatory capabilities and induce an immunoregulatory environment that impairs Th1 responses and promotes the expansion of Th2 cells (Non-Patent Document 6). Consequently, exacerbated levels of M2 macrophages have been found in animal asthma models and samples from patients. Therapies targeting M2 macrophages have been shown to alleviate allergic responses (Non-Patent Document 7).

[0005] BCG, the current tuberculosis vaccine, is the most widely administered vaccine in history. Because BCG has classically been considered a Th1 response-promoting vaccine, the benefits of intradermal BCG vaccination for asthma have been debated for many years. The conclusions regarding this question remain controversial, with various epidemiological studies showing opposing results.

[0006] Both live and inactivated BCG vaccines and various mycobacterial components have been widely proven to be effective against asthma in various animal models (Non-Patent Documents 8, 9, and 10). However, most of these results were obtained by delivering BCG before or simultaneously with allergen sensitization. As a result, the ability of BCG to reverse established asthma has not been addressed, which would be important for elucidating the therapeutic potential of this vaccine as an asthma treatment. Furthermore, previous studies have primarily focused on the Th1 / Th2 response balance, without paying attention to other arms of the immune system involved in asthma, such as alveolar macrophages, which appear to play a key role during asthma pathogenesis.

[0007] MTBVAC is a live vaccine based on attenuated Mycobacterium tuberculosis (NPL 11), which has been demonstrated to be more immunogenic and protective against tuberculosis in various animal models (NPL 12), and has been shown to be safe and immunogenic in human adult and neonatal populations (NPL 13) (NPL 14). MTBVAC is currently being evaluated in two Phase IIa trials to define a dose for efficacy testing as a tuberculosis vaccine. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Strachan DP. Hay fever, hygiene, and household size. BMJ 1989; 299:1259-60. [Non-patent document 2] Stein MM, Hrusch CL, Gozdz J, Igartua C, Pivniouk V, Murray SE, et al. Innate Immunity and Asthma Risk in Amish and Hutterite Farm Children. N Engl J Med 2016; 375:411-21. [Non-patent document 3] Holgate ST, Wenzel S, Postma DS, Weiss ST, Renz H, Sly PD. Asthma. Nat Rev Dis Primers 2015; 1:15025. [Non-patent document 4] Holgate ST. Innate and adaptive immune responses in asthma. Nat Med 2012; 18:673-83. [Non-Patent Document 5] Girodet PO, Nguyen D, Mancini JD, Hundal M, Zhou X, Israel E, et al. Alternative Macrophage Activation Is Increased in Asthma. Am J Respir Cell Mol Biol 2016; 55:467-7

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Summary of the Invention

[0009] In this study, we evaluated the therapeutic efficacy of the live-attenuated vaccines MTBVAC and BCG administered intranasally to allergen-sensitized mice in various models of acute asthma. We hypothesized that vaccines may modulate the immune environment associated with asthma through direct interactions between bacteria and the lung compartment. In this context, our results revealed that both vaccines were able to reeducate M2 macrophages induced by allergen challenge to an M1 phenotype and convert allergen-specific Th2 lymphocytes to Th1. Importantly, our data demonstrated the potent therapeutic efficacy of both BCG and MTBVAC given to allergen-challenged mice in the setting of established disease, demonstrating the potential of live-attenuated tuberculosis vaccines as a treatment for asthma. We also demonstrated optimal protection of intranasal MTBVAC against challenge with M. tuberculosis, suggesting that the pulmonary route of administration may be advantageous not only for treating asthma but also for preventing tuberculosis. [Brief explanation of the drawings]

[0010] [Figure 1]Figure 1 shows that intranasal BCG and MTBVAC prevent allergic airway responses in allergen-sensitized mice. Eosinophils were determined by flow cytometry in an OVA-driven acute asthma model. Mice were intranasally treated with 10 CFU of BCG or 10 CFU of MTBVAC one week after the second sensitization. (A) Eosinophils were determined by flow cytometry as SSChighSiglecF+CD11b+CD11b- cells. Representative images are shown in Figure (B), showing the percentage of eosinophils in the BAL and lungs of mice treated with MTBVAC (C) or BCG (D). Total numbers of leukocytes, eosinophils, neutrophils, and alveolar macrophages (AM) in the BAL were determined by flow cytometry in mice treated with MTBVAC (E) or BCG (F). (E) Representative images of PAS-stained fixed lungs from untreated (1, 2) or BCG-treated (3, 4) OVA-challenged mice. Mucosal material and goblet cells are stained purple (G). Data shown are representative means ± SEM from three independent experiments. A minimum of six mice per group and experiment was used. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 by two-way ANOVA with Bonferroni post-hoc analysis. [Figure 2]Figure 2 shows that the therapeutic effects of BCG and MTBVAC on pulmonary eosinophilia are dose-dependent. The dose-response profile of the vaccines was assessed in the acute model of OVA-induced asthma described in Figure 1. Vaccination with different doses of MTBVAC and BCG was compared to an untreated OVA positive control. A dose-response effect was observed when comparing different doses of vaccination, demonstrating that for both MTBVAC and BCG, eosinophilia decreased with increasing vaccination dose. Graphed data are mean ± SEM from one independent experiment (n = 6 mice per group). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by two-way ANOVA with Dunn's post-hoc test comparing each group to OVA-challenged mice. Note that both BCG and MTBVAC dose-dependently reduced eosinophilia in the BAL (Figure 2A) and lungs (Figure 2B). In the BAL, a dose of 106 CFU resulted in a complete reduction in both cases, whereas in the lungs, a reduction in eosinophils to the negative control level was achieved at the highest doses tested (107 CFU for MTBVAC and 106 CFU for BCG). [Figure 3]Figure 1 shows that intranasal BCG infects lung-resident macrophages and induces their classical activation. (A) Groups of mice were immunized with 10 CFU of GFP-expressing BCG. One month later, infected cells were monitored and characterized by flow cytometry. Expression of the M1 polarization markers iNOS and CD86 was analyzed. Representative figures are shown in the figure. (B) Percentage of iNOS-, CD86-, and MHC-positive alveolar macrophages in OVA-challenged, untreated, or BCG-treated alveolar macrophages. (C) Surface expression of the M2 activation marker CD206. Representative overlay histograms showing CD206 surface expression in the indicated experimental groups. The graph shows a comparison of mean fluorescence intensity (MFI) corresponding to CD206 expression levels. (D) M1 and M2 activation markers measured by qRT-PCR in lungs from OVA-challenged, untreated, or BCG-treated mice. Data are representative means ± SEM from two independent experiments (B, C) or one experiment (D). A minimum of 6 mice were used per group and experiment. *p<0.05; **p<0.01; ***p<0.001 by independent single-sample (B, C) or multiple (D) Student's t-test. [Figure 4]Figure 1 shows that intranasal BCG reshapes allergen-specific responses toward a Th1 profile. (A) Th1 and Th2 activation markers measured by qRT-PCR in the lungs of OVA-challenged mice, either untreated or treated with BCG, 1 week after sensitization. (B) Determination of IL-5 in BAL. (C) Determination of IL-4 and IFNγ in lung explants. (D-G) Allergen-specific IL-4, IL-5, IL-13, and IFNγ produced by mediastinal lymph node cells after ex vivo stimulation with OVA. Each point corresponds to the subtraction value obtained in the presence of OVA minus the value without allergen. (H, I) IL-5- and IFNγ-producing cells visualized by intracellular staining and flow cytometry after ex vivo stimulation with anti-CD3 / CD28 or OVA. Representative figures are shown. Data are pooled means ± SEM from three independent experiments (B-E, G) or one experiment (A, I). A minimum of six mice per group and experiment was used. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 by multiple t-Student's test (A), one-way ANOVA with Bonferroni post-hoc test (B, D-G), and two-way ANOVA with Bonferroni post-hoc test (C, I). [Figure 5] Figure 1 shows modulation of immune responses by MTBVAC. Th2 versus Th1 responses were analyzed in a model of acute allergic asthma. IL-5 and IFNγ were analyzed in bronchoalveolar lavage (BAL) (A) and lymph nodes (B) as cytokines representative of Th2 and Th1 responses, respectively. (C) iNOS and Arg1 were analyzed in lung macrophages as molecules representative of M2 and M1 macrophage-polarized responses, respectively. Intranasal treatment with MTBVAC reverted Th2 and M2 asthma-associated responses to Th1 and M1. Data shown are means ± SEM from one independent experiment (n = 6 mice per group). *p < 0.05; **p < 0.01; ***p < 0.001 by one-way ANOVA with Bonferroni post-hoc test (A) and two-way ANOVA with Bonferroni post-hoc test (B). [Figure 6]Figure 1 shows that intranasal BCG and MTBVAC reverse established allergic airway responses. (A) Experimental scheme based on the OVA-induced chronic model. Mice were challenged with 10 μg of OVA intranasally twice weekly from week 3 to week 13. The vaccine was delivered at week 9, halfway through the challenge phase. (B) Total eosinophil counts in BAL at week 8, one week before vaccination. This demonstrated the presence of established eosinophilia before vaccination. (C) Total eosinophil counts in BAL at week 13, four weeks after treatment with MTBVAC or BCG. (E) Representative images of PAS-stained fixed lungs from untreated or MTBVAC-treated OVA-challenged mice. Data shown are means ± SEM from a representative experiment of at least two independent experiments. A minimum of six mice per group and experiment was used. *p<0.05; **p<0.01; ***p<0.001 by t-Student's test (B, C), one-way ANOVA with Bonferroni post-hoc test (D). [Figure 7]MTBVAC reverses asthma-related phenotypes in a model of established asthma induced by the relevant allergen house dust mite (HDM). (A) Schematic of the HDM-induced chronic model. Mice were challenged intranasally with 10 μg of HDM twice a week for three consecutive weeks. The vaccine was delivered at week 4, and one month later, animals were challenged intranasally with 10 μg of HDM for three consecutive days. The following day, animals were sacrificed. (B) Eosinophils were determined in the BAL of an additional group sacrificed at week 3 to confirm the presence of eosinophilia at the time of vaccine administration. (C) A graph shows a comparison of the percentage of eosinophils in the BAL of untreated or BCG-treated or MTBVAC-treated mice from weeks 3 to 7. Lung remodeling and mucus secretion were assessed by PAS staining and histological evaluation. Representative images are shown in (D, E). (F, G) The Th2 cytokines IL-5, IL-4, and IL-13 were analyzed in lymph nodes after ex vivo stimulation with BAL and HDM. (F, G) The Th1 cytokine IFNγ was analyzed in lymph nodes after ex vivo stimulation with BAL and HDM. Data shown are means ± SEM from one independent experiment (n = 6 mice per group). *p < 0.05; **p < 0.01; ***p < 0.001 by one-way ANOVA with Bonferroni post-hoc test (A, F, G) and two-way ANOVA with Bonferroni post-hoc test (D, E). [Figure 8] Figure 1 shows that MTBVAC is more efficiently nebulized than BCG in a clinical nebulizer. MTBVAC and BCG were nebulized using the clinical device OMRON U100 (A) and recovered in a known volume of sterile water in a gas-washed flask (B). (C, D) CFU loss in the nebulized fraction compared to the reservoir for MTBVAC (C) and BCG (D) in two independent experiments. (E) Nebulization efficacy index calculated as the % of bacteria recovered in the nebulized fraction compared to that initially available in the GMP formulation. [Figure 9] The size of MTBVAC is smaller than that of BCG. Figure 1 shows representative electron microscope images of BCG (A) and MTBVAC (B). [Figure 10]Figure 1 shows that MTBVAC reduces eosinophilia in the esophagus. Eosinophils in the esophagus were determined in an acute model of OVA-induced asthma in MTBVAC-treated groups. Treatment with intranasal MTBVAC significantly reduced eosinophilia. Graphs show mean ± SEM from pooled data from two independent experiments (n = 6 mice per group and experiment). *p < 0.05 by one-way ANOVA with Bonferroni post-hoc test. [Figure 11] Figure 1 shows that intranasal MTBVAC improves protection against tuberculosis compared with BCG delivered by the standard subcutaneous route. Mice were vaccinated with BCG by the subcutaneous route (equivalent to the clinically used intradermal route) or MTBVAC intranasally. Two months later, mice were challenged intranasally with a low dose of M. tuberculosis strain H37Rv, and bacterial burden in the lungs was measured 4 weeks later. Graphs show mean ± SEM from pooled data from four independent experiments (n = 6 mice per group and experiment). ****p < 0.00001 by one-way ANOVA with Bonferroni post-hoc test. [Figure 12] A, OMRON U100 nebulizer used for the experiments performed for this figure. B, MTBVAC and BCG ONCOTICE CFU determined in the nebulized and reservoir fractions. C, Nebulization efficacy index calculated as the percentage of bacteria recovered in the nebulized fraction compared to those contained in the reservoir. Each dot corresponds to the results obtained with a different filter. Data on the graph represent the mean ± SEM from six independent nebulizations with MTBVAC and three independent nebulizations with BCG ONCOTICE. *p<0.05 by Student's t-test. DETAILED DESCRIPTION OF THE INVENTION

[0011] definition The term "MTBVAC strain" is used to refer to an isolated M. tuberculosis strain that lacks the Rv0757 gene of the M. tuberculosis Mt103 clinical strain and also contains a deletion of the Rv2930 (fadD26) gene. Thus, the strain presents two independent mutations derived from M. tuberculosis, and the independent phoP deletions do not affect the vaccine properties resulting from the inactivation of the genes. Therefore, the "MTBVAC strain" is characterized by the deletion of the Rv2930 (fadD26) gene, which inactivates PDIM production, and therefore the strain is characterized by the deletion of the Rv2930 and Rv0757 genes.

[0012] Therefore, the MTBVAC strain was constructed to contain two independent non-reverting deletion mutations without an antibiotic marker, and it is important to note that it meets the first Geneva consensus safety requirements for advancing a live mycobacterial vaccine into Phase I clinical evaluation. The MTBVAC strain was genetically engineered to resemble the prototype SO2 strain phenotypically and functionally. SO2 is an Mt103 phoP mutant (Mt103phoP::kmr) marked by the insertion of a kanamycin resistance cassette (kmr). In addition to the engineered PhoP-deficient phenotype, SO2 spontaneously loses PDIM biosynthesis, a process described as common in M. tuberculosis, as a result of repeated subculture and manipulation in the laboratory (see Figure 2 in Dessislava Marinova, Jesus Gonzalo-Asensio, Nacho Aguilo & Carlos Martin (2017) MTBVAC: from discovery to clinical trials in tuberculosis-endemic countries, Expert Review of Vaccines, 16:6, 565-576, DOI:10.1080 / 14760584.2017.1324303).

[0013] The MTBVAC strain was constructed following a stepwise approach. First, an unmarked deletion of fadD26 was introduced into SO2, resulting in SO2ΔfadD26. Consequently, the MTBVAC strain was generated by an unmarked deletion in phoP of SO2ΔfadD26. Construction of MTBVAC used a suicide plasmid carrying the deleted fadD26 and phoP genes, and the deleted region was transcribed with a hygromycin resistance marker (hyg) flanked on both sides by res sites. r )(res::hyg r The γδ-resolvase from E. coli catalyzes the excision of the antibiotic resistance cassette following recognition of the res site, leaving a copy of the residual res "scar" at the deletion site (Malaga, et al. 2003), which does not contain any exogenous coding sequence. The final construct, SO2ΔfadD26::ΔphoP, was designated the MTBVAC strain. In the MTBVAC strain, an unmarked deletion in fadD26 ensures the genetically stable abolition of PDIM biosynthesis. The resulting deletion in the gene fadD26 contains 1511 bp, resulting in the complete inactivation of this essential gene in PDIM biosynthesis. The wild-type gene is 1752 bp (583 amino acids). The residual res scar is responsible for the hyg gene expression by the γδ-resolvase. r This deletion resulted in a reduction in the transcription levels of the following five genes in the PDIM locus (fadD26-ppsE), completely eliminating PDIM biosynthesis in MTBVAC (Ainhoa Arbues PhD Thesis). The PDIM locus of M. tuberculosis contains 13 genes clustered on a 50 kb fragment of the chromosome. This region is the largest operon in the M. tuberculosis genome (Camacho, et al. 2001; Camacho, et al. 1999; Cox, et al. 1999; Trivedi, et al. 2005).

[0014] In M. tuberculosis, phoP (744 bp) maps upstream of phoR (1458 bp), and both genes are transcribed in the same direction. Replacement of a 94-bp deletion in the phoP gene with the remaining res site requires the presence of multiple stop codons, while resulting in the loss of translation of the DNA-binding domain of PhoP (corresponding to 92 amino acids) in MTBVAC.

[0015] The deletion of the phoP and fadD26 genes in MTBVAC can be detected / localized using a presence / absence approach of RT-PCR, in which fluorescent PCR reagents (primers and probes) are used to indicate the presence of res sites in the ΔphoP and ΔfadD26 genes and the absence of the wild-type phoP and fadD26 genes.

[0016] Below, we provide the open reading frame (ORF) sequences of the fadD26 gene in Mt103a) and MTBVAC(ΔfadD26)b) and the ORF sequences of the phoP gene in Mt103c) and MTBVAC(ΔphoP)d). Nucleotide sequences corresponding to the deleted gene regions in fadD26 (a) and phoP (c) are shown in lowercase, with the remaining res sites highlighted in gray. For fluorescent PCR detection, primers for each target are underlined, and TaqMan probes are shown in bold.

[0017] a) Wild-type fadD26 gene in Mt103 SEQ ID NO: 1 TIFF0007720097000001.tif116170

[0018] b) ΔfadD26 in MTBVAC SEQ ID NO: 2 TIFF0007720097000002.tif32170

[0019] c) Wild-type phoP gene in Mt103 SEQ ID NO: 3 TIFF0007720097000003.tif60170

[0020] d) ΔphoP in MTBVAC SEQ ID NO:4 TIFF0007720097000004.tif70170

[0021] SO2 has a thorough and complete preclinical history demonstrating a robust safety and attenuation profile and promising efficacy compared to BCG in relevant animal models. Fortunately, most of these preclinical studies were replicated with MTBVAC to confirm the functional profile and biological activity of the doubly attenuated PhoP-PDIM phenotype. Lipid profile analysis demonstrated that MTBVAC and its prototype SO2 are phenotypically equivalent and lack DATs, PATs, and PDIMs.

[0022] However, in the context of the present invention, BCG will be used hereinafter to refer to the current vaccine used against tuberculosis since 1921. This vaccine is a live attenuated vaccine derived from an M. bovis strain that has lost its pathogenicity after passage in the laboratory and is now known to have deleted more than 100 genes. Behr, MA BCG—different strains, different vaccines Lancet Infect Dis 2002, 2(2), 86-92.

[0023] In the context of the present invention, H37Rv will hereinafter be used to refer to the sequenced pathogenic M. tuberculosis strain, and Cole et al. refer to these genes as Rv (see Cole et al 1998 Deciphering the biology of M. tuberculosis from the complete genome sequence. Nature 393:537-544).

[0024] In the context of the present invention, Mt103 will be used hereinafter to refer to a clinical isolate of M. tuberculosis. Camacho et al. 1999 Identification of a virulence gene cluster of M. tuberculosis by signature-tagged transposon mutagenesis. Mol Microbiol 34:257-267. In the context of the present invention, PDIM-strain will be used hereinafter to refer to a strain of the M. tuberculosis complex that is unable to synthesize phthiocerol dimycocerosate, a key lipid associated with the virulence of M. tuberculosis.

[0025] In the context of the present invention, hereinafter, M. tuberculosis phoP- will be used to refer to an M. tuberculosis strain that has been inactivated by deletion of the Rv0757 gene between the EcoRV-BspEI sites, and whose phenotype is PhoP-PDIM+.

[0026] In the context of the present invention, Rv2930 (fadD26) will be used hereinafter to refer to the gene at the beginning of the operon responsible for the synthesis of phthiocerol dimycocerosate (PDIM) (Camacho et al.), and elimination of this gene in M. tuberculosis confers a stable PDIM- phenotype.

[0027] explanation Available data in the literature attest to the primary role of innate lung cells in the pathogenesis of asthma. Specifically, alternatively activated macrophages, or type M2, are elevated in the lungs of asthmatic patients, both in animal models and in humans. The reason behind this pathological macrophage polarization is unclear. A plausible explanation is that allergens can directly damage the alveolar epithelium, and macrophages are alternatively activated in response to the injury, inducing a wound-healing response (Murray PJ, Wynn TA. Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol 2011;11:723-37). M2 is a simplified term that encompasses various subsets of macrophages with regulatory capabilities. Accordingly, three distinct types of M2 macrophages have been defined: M2a, M2b, and M2c, each with its own unique characteristics. In the specific case of M2a macrophages, their presence is associated with the induction of Th2 adaptive responses (Saradna A, Do DC, Kumar S, Fu QL, Gao P. Macrophage polarization and allergic asthma. Transl Res 2018;191:1-14). In the context of allergic asthma, M2a macrophages may contribute to the induction of allergen-specific T cell responses in at least two distinct ways. First, once activated, M2a macrophages express high levels of MHC-II molecules, allowing them to present allergen-derived peptides to T lymphocytes. Second, they secrete cytokines such as IL-4, which drive the polarization of T cell responses toward a Th2 profile (von Bubnoff D, Geiger E, Bieber T. Antigen-presenting cells in allergy. J Allergy Clin Immunol 2001;108:329-39).Therefore, as exacerbated M2 macrophage activation appears to be important in the inflammatory response in asthma, this represents a very attractive opportunity to design novel immunomodulatory treatments that target this imbalance in lung macrophage populations.

[0028] In this study, we evaluated the therapeutic potential of a live-attenuated tuberculosis vaccine delivered by the intranasal route in various preclinical models of asthma. Our results are highly robust. Intranasal BCG and MTBVAC reverse asthma-related responses in all settings tested, including both short-term and long-term acute models induced by various allergens, as well as in established asthma settings (Figures 1, 2, 6, and 7). Our data clearly demonstrate that BCG and MTBVAC impair M2 macrophages associated with allergen exposure (Figures 3 and 5). A possible mechanism behind this inhibition may rely on the vaccine's ability to activate macrophages in a classical manner (or M1 phenotype), presumably due to their natural intracellular state. Thus, BCG and MTBVAC are efficiently internalized by alveolar macrophages upon immunization, leading to the expression of markers such as iNOS and the secretion of cytokines such as IL-1β, TNFα, and IL-12, which otherwise produce antagonists of the inflammatory response produced by activated macrophages. Importantly, our analysis using GFP-expressing BCG bacteria showed that not only did infected macrophages adopt an M1 phenotype, but uninfected cells also adopted an M1 phenotype. These findings suggest that initially infected macrophages generate a series of signals that also activate bystander neighbors, supporting an expanded response that counteracts the asthma-related environment.

[0029] The benefits of BCG for asthma patients have been studied extensively in clinics, both in observational studies comparing asthma prevalence between BCG-vaccinated populations (Sarinho E, Schor D, Veloso M, Lima M. BCG scar diameter and asthma: a case-control study. J Allergy Clin Immunol 2000; 106:1199-200) and in interventional studies in which two arms were vaccinated with BCG or placebo and asthma symptoms were reported and compared between the two groups (Choi IS, Koh YI. Therapeutic effects of BCG vaccination in adult asthmatic patients: a randomized, controlled trial. Ann Allergy Asthma Immunol 2002; 88:584-91). The data are controversial, and studies have shown opposing results. A few years ago, a meta-analysis comparing various published studies suggested no clear association between intradermal BCG vaccination and a lower risk of developing asthma (Arnoldussen DL, Linehan M, Sheikh A. BCG vaccination and allergy: a systematic review and meta-analysis. J Allergy Clin Immunol 2011;127:246-53, 53 e1-21). A plausible hypothesis to explain this apparent discrepancy between the clinic and our data is that the beneficial effects of BCG are organ-dependent, and therefore BCG must be physically present in the lungs to induce an efficient antiasthmatic response. Indeed, subcutaneous BCG does not prevent asthma in mice (Non-Patent Document 9), a result confirmed by the inventors in the MTBVAC study. This should clearly be expected given our observation that BCG must interact with lung-resident macrophages to reshape their activated phenotype. A literature search reveals intriguing data that may support this hypothesis.Unlike those reported for BCG, data showing a lower prevalence of asthma in people infected with tuberculosis appear more robust. In this regard, a significant correlation has been found between the decrease in asthma prevalence and notifiable TB cases (von Mutius E, Pearce N, Beasley R, Cheng S, von Ehrenstein O, Bjorksten B, et al. International patterns of tuberculosis and the prevalence of symptoms of asthma, rhinitis, and eczema. Thorax 2000; 55:449-53.). Studies conducted in individuals with subclinical TB infection (LTBI) have demonstrated a strong association between a positive tuberculin skin test (TST) and a decreased prevalence of various types of allergies. Interestingly, in the case of asthma, the authors found a significant reduction in the incidence of asthma in the group with the highest TST levels, suggesting a correlation between the magnitude of the TB-specific immune response and the degree of nonspecific protection (Obihara CC, Kimpen JL, Gie RP, Lill SW, Hoekstra MO, Marais BJ, et al. M. tuberculosis infection may protect against allergy in a tuberculosis endemic area. Clin Exp Allergy 2006;36:70-6.). Notably, the inventors recently demonstrated protection against asthma in mice infected with tuberculosis (Tarancon R, Uranga S, Martin C, Aguilo N. Mycobacterium tuberculosis infection prevents asthma and abrogates eosinophilopoiesis in an experimental model of Allergy. 2019 May 22. doi:10.1111 / all.13923).Because M. tuberculosis infection is usually acquired by the respiratory route (in contrast to BCG, which is administered intradermally), we could conclude that our results with a live attenuated vaccine delivered to the lung may reflect a natural nonspecific protection that already occurs naturally.

[0030] Allergen-specific CD4+ T cells are thought to play a central role in asthmatic inflammation. The use of allergen-MHC-II tetramers enabled the characterization of allergen-specific CD4+ T cells in asthmatic patients, identifying tetramer-positive T cell clones expressing central memory markers (Kwok WW, Roti M, Delong JH, Tan V, Wambre E, James EA, et al. Direct ex vivo analysis of allergen-specific CD4+ T cells. J Allergy Clin Immunol 2010;125:1407-9 e1). These long-lived T cells ultimately contribute to the perpetuation of asthma throughout life. Upon allergen exposure, specific memory CD4+ T cells migrate to the lung, efficiently recognize allergen-derived peptides presented by APCs, and rapidly respond by secreting Th2-associated cytokines, triggering the asthmatic inflammatory response. Therefore, therapeutic strategies seeking asthma relief must focus on disrupting allergen-specific Th2 memory cells. For example, immunotherapy based on low-level allergen epitope exposure works by inducing anergy in allergen-specific Th2 T cells (O'Hehir RE, Prickett SR, Rolland JM. T Cell Epitope Peptide Therapy for Allergic Diseases. Curr Allergy Asthma Rep 2016;16:14). Our results analyzing OVA-specific T cells in draining lymph nodes show that intranasal BCG and MTBVAC strongly influence OVA-specific Th2 cells (Figures 4 and 5). Intracellular staining and flow cytometry, which allow direct visualization of cytokine-producing cells, showed that the population of IL-5-producing T cells in the OVA group was clearly reduced by BCG treatment, while the population of OVA-specific IFNγ-producing cells emerged (Figures 4H and 4I). This suggests that live mycobacteria may reshape the phenotype of T cells involved in asthmatic responses.

[0031] Over the past few years, novel experimental immunotherapies for asthma, primarily based on blocking specific pathways that contribute to asthma-induced inflammation, such as IL-4, IL-5, IL-13, or IgE, have emerged and often show positive results. However, these studies also demonstrate that the efficacy achieved by inhibiting a single pathway generally leads to partial results. For example, IL-5-specific therapy substantially suppresses eosinophilia, but results in other measures, such as lung function, are less favorable (Pavord ID, Korn S, Howarth P, Bleecker ER, Buhl R, Keene ON, et al. Mepolizumab for severe eosinophilic asthma (DREAM): a multicenter, double-blind, placebo-controlled trial. Lancet 2012;380:651-9.). Because asthma is a highly complex disease, we speculate that the more global approach proposed in this study may more efficiently address various aspects of the pathology.

[0032] MTBVAC is a novel tuberculosis vaccine that has been demonstrated to be safe in human neonates and adults via the intradermal route. The results presented herein demonstrate the intranasal ability of MTBVAC to reverse asthma-associated inflammatory responses. Thus, pulmonary MTBVAC is an attractive approach for allergic asthma and offers several advantages over BCG, which are briefly summarized below.

[0033] First, data from clinical trials indicate a lower reactogenicity of intradermal MTBVAC compared with BCG (13), which may be related to the more efficient clearance observed in the present study. Notably, MTBVAC has been demonstrated to be fully susceptible to current tuberculosis antibiotics, unlike BCG, which is resistant to isoniazid and pyrazinamide.

[0034] Second, as already discussed above, a plausible hypothesis to explain our findings is that the beneficial effects of BCG and MTVBAC are organ-dependent and therefore require both to be physically present in the lungs to induce an efficient antiasthmatic response. Therefore, therapeutic efficacy against asthma must be induced by pulmonary delivery. Through the pulmonary route, drugs can be administered via two main modes: first, intranasal administration, which has anatomical limitations such as narrower airway lumen, and second, oral inhalation. Oral inhalation administration allows for the administration of very small particles with only a 20% concentration loss compared to 85% via the nasal route, which promises much better results. Oral inhalation administration can be further classified as intratracheal instillation and intratracheal inhalation. The most common method used in laboratories is intratracheal instillation. Intratracheal instillation involves the delivery of a small amount of drug solution or dispersion to the lungs using a specialized syringe. This provides a fast and quantifiable method of pulmonary drug delivery. Local drug deposition is achieved within a relatively small absorption area. Therefore, the injection process is very simple, inexpensive, and the drug distribution is non-uniform. In preclinical animal studies, intratracheal injection has been frequently used to evaluate pulmonary absorption and systemic bioavailability, especially with regard to the precise administration and efficacy associated with this method. However, intratracheal injection is not a physiological route for application, and the results obtained from these studies may not be transferable to aerosol application in humans. In contrast, the inhalation method uses aerosol technology, which allows us to obtain high penetration and more uniform distribution.

[0035] Therefore, in this study, we evaluated the nebulization efficacy of BCG and MTBVAC using the OMRON U100 aerosol nebulizer device (Figure 8A), which is used in clinics for pulmonary drug delivery. This should be the optimal method for administering live attenuated vaccines in the clinic that can not only prevent but also reverse the inflammatory response of asthma, and therefore may have potential therapeutic applications when administered to human patients. Figure 8 shows that MTBVAC is nebulized more efficiently than BCG with this clinical nebulizer. The GMP lots of BCG and MTBVAC tested in this study each had a nebulization efficiency of 1.7 × 10 7 CFU / ml and 1.1 × 10 7 CFU / ml. Five minutes after spraying, the amount of bacteria recovered was 1.98 × 10 for MTBVAC and 1.98 × 10 for VAC. 6 CFU (Fig. 8C ), and 3.3 × 10 for BCG. 4 CFU (Figure 8D). The average nebulization efficacy index from two independent experiments, calculated as the % of bacteria recovered in the nebulized fraction compared to those initially available in the reservoir, was 15% and 0.15% for MTBVAC and BCG, respectively (Figure 8E).

[0036] Importantly, our data shown in Figure 2 indicate that the therapeutic effects of BCG and MTBVAC on allergen-induced eosinophilia are strongly dose-dependent, and therefore it is important that a certain critical dose of vaccine reaches the lung to reduce eosinophil infiltration. 6 CFU could be considered the optimal therapeutic dose for both vaccines.

[0037] Considering the data obtained from nebulization studies under established experimental conditions, this dose was only obtainable with MTBVAC, whereas with the BCG formulation, we nebulized only approximately 3% of this optimal dose of bacteria. Therefore, we can conclude that therapeutic doses of MTBVAC, but not BCG, can be achieved using aerosol inhalation with clinical devices.

[0038] These differences may be explained by the size differences between BCG and MTBVAC observed in electron microscope images (Figure 9). MTBVAC bacilli are shorter than 1 micrometer, while BCG are longer than 2 μm. Furthermore, our laboratory observations suggest that BCG is more likely to form aggregates than MTBVAC when the vaccine is cultured in the absence of detergents (as in industrial production). Because the average aerosol particle size of clinical nebulizers is generally approximately 5 micrometers, we believe that two factors, bacterial size and aggregation, may explain the difference in nebulization efficacy between BCG and MTBVAC.

[0039] Our results also showed that in the OVA-acute asthma model, there was also eosinophil infiltration into the esophagus, a condition that was prevented by intranasal administration of MTBVAC (Figure 10). These data suggest that live-attenuated mycobacteria may be used to treat other types of pathogenic eosinophilia, such as eosinophilic esophagitis (EoE), in addition to allergic asthma.

[0040] The present invention describes for the first time the therapeutic efficacy against asthma induced by pulmonary delivery of live attenuated MTBVAC mycobacteria by disrupting the lung immune environment, preferably when MTBVAC is administered via the pulmonary route by inhalation using aerosol technology. Furthermore, the present invention further illustrates in Figure 11 that pulmonary delivery of MTBVAC improves protection against tuberculosis compared to BCG delivery via the standard subcutaneous route.

[0041] Accordingly, a first aspect of the present invention relates to a live-attenuated M. tuberculosis composition (composition of the present invention) for use in therapy in a human subject in need thereof, the live-attenuated M. tuberculosis composition comprising an isolated microorganism belonging to the M. tuberculosis MTBVAC strain having: i) a PhoP phenotype due to inactivation by genetic deletion of the Rv0757 gene, wherein the phoP open reading frame (ORF) sequence consists of SEQ ID NO: 4; and ii) a deletion of a second gene, Rv2930 (fadD26), that prevents PDIM production (PDIM phenotype), wherein the fadD26 open reading frame (ORF) sequence consists of SEQ ID NO: 2, the live-attenuated M. tuberculosis composition comprising: a microorganism belonging to the MTBVAC strain having: i) a PhoP phenotype due to inactivation by genetic deletion of the Rv0757 gene, wherein the phoP open reading frame (ORF) sequence consists of SEQ ID NO: 4; and ii) a deletion of a second gene, Rv2930 (fadD26), that prevents PDIM production (PDIM phenotype), wherein the second gene, Rv2930 (fadD26), has a deletion of the second gene, Rv2930 (fadD26), whose open reading frame (ORF) sequence consists of SEQ ID NO: 2; the live-attenuated M. tuberculosis composition is administered to the subject by pulmonary delivery. Preferably, the composition is administered to the subject via pulmonary delivery by inhalation using aerosol technology.

[0042] Preferably, the composition of the first aspect of the invention is a lyophilized composition further comprising certain components (e.g., certain stabilizers, bulking agents, and buffers) that have been found to be advantageous in the preparation of lyophilized MTBVAC strain vaccines. The present invention also relates to reconstituted vaccines, as well as prophylactic and therapeutic methods using the compositions described in the first aspect of the invention, insofar as the compositions are administered to the subject via pulmonary delivery, preferably by inhalation using aerosol technology. The compositions and methods of the invention are further described as follows.

[0043] In a preferred embodiment of the first aspect of the present invention, or any of its preferred embodiments, the composition comprises at least 10 5 cfu~10 6 More preferably, the composition comprises 10 cfu of the isolated microbial strain. 6 cfu~10 7 cfu of isolated microbial strains.

[0044] In a second aspect of the invention, the vaccine compositions of the first aspect of the invention can be administered via the pulmonary route, preferably by inhalation using aerosol technology, as a primary prophylactic agent to individuals at risk of infection with M. tuberculosis or developing tuberculosis disease, or can be used as a secondary agent to treat infected patients. Because the strains in these compositions are attenuated, they are particularly suitable for administration to "at-risk individuals," such as neonates, children, adolescents, adults, and the elderly. Such vaccines can also be used in veterinary settings.

[0045] In a third aspect of the present invention, the vaccine composition of the first aspect of the present invention can be administered via the pulmonary route, preferably by inhalation using aerosol technology, as a prophylactic to individuals at risk of suffering from an allergic reaction, preferably targeting M2 macrophages to reduce allergic reactivity, more preferably to individuals at risk of suffering from or at risk of developing asthma, particularly allergic asthma, or as a medicament for treating patients with an allergic reaction, preferably by targeting M2 macrophages to reduce allergic reactivity, more preferably to treat asthma, particularly allergic asthma. Because the strains of these compositions are attenuated, they are particularly suitable for administration to "at-risk individuals," such as neonates, children, adolescents, adults, and the elderly. Such vaccines can also be used in veterinary settings.

[0046] A preferred embodiment of the second aspect of the invention relates to the composition of the first aspect of the invention for immunizing an individual against symptoms caused by tuberculosis. It is noted that the vaccine may also be suitable for the treatment of bladder cancer, as well as for the treatment or prevention of TB, or as a vector or adjuvant, preferably for immunizing an individual against symptoms caused by TB.

[0047] In another preferred embodiment of the second aspect of the invention, the composition of the first aspect is administered for the prophylaxis against infection caused by the M. tuberculosis complex, preferably M. tuberculosis, in newborns at risk of becoming infected with M. tuberculosis or at risk of developing TB disease.

[0048] In another preferred embodiment of the second aspect of the invention, the composition of the first aspect is administered for the prevention or inhibition (including booster vaccination) of infection caused by the M. tuberculosis complex, preferably M. tuberculosis, in non-neonatal humans, such as children, adolescents and adults, who are at risk of infection by M. tuberculosis.

[0049] In another preferred embodiment of the second aspect of the invention, the composition of the first aspect is administered to prevent or inhibit the development of clinical symptomatology associated with active disease caused by the M. tuberculosis complex, preferably M. tuberculosis, in non-neonatal humans, such as children, adolescents and adults, who are at risk of developing TB disease and who have latent tuberculosis infection.

[0050] In another preferred embodiment of the second aspect of the invention, the composition of the first aspect is administered for use as a second line agent to treat patients with latent and / or active TB infection in neonates and non-neonatal humans such as children, adolescents and adults.

[0051] In another preferred embodiment of the second aspect of the invention, the composition of the first aspect is administered for booster vaccination or dosing in the prophylactic or preventative treatment of infection caused by the M. tuberculosis complex, preferably M. tuberculosis, in non-neonatal humans, such as children, adolescents, and adults at risk of infection with M. tuberculosis. In this sense, it is noted that a booster injection or dosing after initial immunization is a re-exposure to the immunizing antigen. Re-exposure is intended to increase immunity to that antigen back to a protective level after memory against that antigen has waned over time.

[0052] Throughout the specification and claims, the word "comprises" and variations thereof do not imply the exclusion of other technical features, additives, ingredients, or steps. For those skilled in the art, other objects, advantages, and properties of the present invention will arise partly from the specification and partly from the practice of the invention. The following examples and figures are provided by way of non-limiting illustration of the present invention. [Example]

[0053] method bacteria BCG Danish SSI strain (Pfizer), BCG Pasteur strain (1173P2, Institut Pasteur, Paris, France), and MTBVAC strain (University of Zaragoza) were grown at 37°C in Middlebrook 7H9 broth (Difco) supplemented with 10% ADC (Difco) and 0.05% (vol / vol) Tween-80 (Sigma) or on solid Middlebrook 7H11 (Difco) supplemented with 10% ADC. BCG Pasteur and MTBVAC were transformed with the replicating pJKD6 plasmid encoding green fluorescent protein (GFP) (kindly gifted by Luciana Leite, Butantan Institute, Brazil). Bacterial suspensions for vaccination were prepared in PBS from previously quantified glycerol stocks by plating serial dilutions. For bacterial quantification, serial dilutions of the bacterial suspension were plated on 7H10 agar medium supplemented with ADC.

[0054] Animal experiments All mice were housed under controlled conditions and observed for signs of disease. Experimental work was carried out in accordance with the European and national directives for the protection of laboratory animals and with the approval of the Ethics Committee of the University of Zaragoza (protocol PI22 / 15).

[0055] For induction of OVA-specific asthma, 8- to 10-week-old female C57BL / 6 mice (Janvier Biolabs) were sensitized twice, one week apart, by intraperitoneal injection of 50 μg of chicken egg albumin (freeze-dried powder, ≥98% (Sigma)) containing 2 mg of aluminum hydroxide (Sigma, St. Louis, MO). One week later, mice were immunized with 10 μg of OVA in 40 μl of PBS. 6Mice were immunized intranasally with CFU of the designated vaccine. Four weeks after vaccination, in the acute model, animals were challenged intranasally with 100 μg OVA in sterile PBS for three consecutive days and humanely sacrificed the following day. In the chronic model, three weeks after immunization, mice were challenged intranasally with 10 μg OVA twice weekly for eight weeks. In this case, the vaccine was administered during the ninth week of the procedure, halfway through the challenge phase. For HDM-induced chronic asthma, mice were challenged intranasally with 10 μg HDM twice weekly for three consecutive weeks. The vaccine was delivered at week four, and one month later, mice were challenged intranasally with 10 μg HDM for three consecutive days. The following day, animals were sacrificed.

[0056] For bronchoalveolar lavage (BAL) collection, the trachea was cannulated and BAL was performed with 0.8 ml of ice-cold PBS. The supernatant was separated from the cells by centrifugation at 4500 x g for 5 minutes.

[0057] Lungs were removed aseptically. To obtain a cell suspension, the lungs were added to HEPES buffer (10 mM HEPES; 0.15 M NaCl; 5 mM KCl; 1 mM MgCl2; 1.8 mM CaCl2, pH 7.4) containing 100 mg / ml collagenase D (Roche) and 400 IU DNase I (AppliChem), incubated at 37°C for 30 min, and homogenized using a GentleMACS (Miltenyi Biotech) dissociator with a lung-specific program according to the manufacturer's instructions. Residual red blood cells were then lysed using erythrocyte lysis buffer (Sigma). For histological analysis, lungs were fixed in 40% formaldehyde for 24 h before the staining procedure.

[0058] Mediastinal lymph nodes were aseptically removed and mechanically disrupted for cell collection before being filtered to remove tissue debris.

[0059] The esophagus was aseptically removed and transversely cut. Then, the tissue was homogenized in 2 ml of HEPES buffer (10 mM HEPES; 0.15 M NaCl; 5 mM KCl; 1 mM MgCl; 1.8 mM CaCl, pH 7.4) containing 100 mg / ml collagenase D (Roche) and 400 IU DNase I (AppliChem) at 37°C for 30 minutes. The homogenate was then filtered to remove tissue debris.

[0060] Flow cytometry analysis 10 6 Lung or BAL cells were incubated with Fc receptor blocking reagent (Miltenyi Biotech) for 15 minutes at 4°C. The presence of eosinophils, neutrophils, and macrophages was then determined by extracellular staining with the following antibodies: CD45-FITC, siglecF-APC, Ly-6G-Vioblue (Miltenyi Biotech); and CD11c-PE, CD11b-PerCP / Cy5.5 (BD Biosciences). Eosinophils were identified using SSC. high CD45 + CD11b + SiglecF + CD11c - cells; neutrophils are CD45 + Ly6G + CD11b + CD11c - cells; and alveolar macrophages are CD45 + SiglecF + CD11c + CD11b dim was defined as a cell.

[0061] For intracellular staining (ICS), membrane proteins were labeled with the above antibodies, in addition to MHCII-Vioblue (Miltenyi), and CD206-APC and CD86-PE (BD Biosciences), according to the manufacturer's instructions. Cells were then fixed and permeabilized with a FoxP3 staining set (Miltenyi Biotech). As intracellular antibodies, we used iNOS-APC and iNOS-PE (Miltenyi), and Arg1-APC (eBiosciences). Cells were acquired using a Gallios flow cytometer (Beckman Coulter) and analyzed with Weasel software.

[0062] Cytokine analysis Quantification of IL-5, IL-4, IL-13, and IFN-γ was performed using specific commercially available ELISA kits according to the manufacturer's instructions (Mabtech Biotech). Cytokine determination in lungs was performed from organ explants. These were prepared by mincing the lungs and incubating them overnight at 37°C in 0.5 ml of culture medium.

[0063] To analyze OVA- or HDM-specific responses, mediastinal lymph nodes were aseptically removed and mechanically disrupted for cell collection. 6Cells were incubated with or without 1 mg / ml OVA or 10 μg / ml HDM for 96 hours. Supernatants were then collected for cytokine concentration determination. The OVA-specific response for each cytokine was calculated as the difference between the cytokine concentration obtained after OVA stimulation and that obtained in the unstimulated control. For ICS, cells were incubated with 1 mg / ml OVA or 1 μg / ml anti-CD3 / CD28 (BD Biosciences) for 24 hours, with 10 μg / ml Brefeldin A (Sigma) added for the final 6 hours. For surface staining, cells were labeled with anti-CD4-FITC (BD Biosciences) and anti-CD3-PerCPVio700 (Miltenyi Biotec) in culture medium containing 10% FCS. Cells were then fixed and permeabilized with Cytofix / Cytoperm Fixation / Permeabilization Kit (BD Biosciences) according to the manufacturer's instructions and stained with anti-IFNγ-APC (BD Biosciences) and anti-IL5-PE (Miltenyi Biotech).

[0064] qRT-PCR For RNA extraction, lungs were immersed in TRIzol reagent (Invitrogen) immediately after collection and immediately frozen on dry ice. After thawing, lungs were homogenized using the GentleMACS using the RNA 0.2 protocol. 200 μl of chloroform was added per ml of TRIZOL, and after vigorously vortexing, the tube was centrifuged at 18,000 × g for 1 hour at 4°C. The aqueous upper phase containing eukaryotic RNA was collected, added to 700 μl of isopropanol, and centrifuged at 18,000 × g for 10 minutes at 4°C. The resulting pellet was washed with 70% EtOH and stored at -20°C. Residual DNA was removed by DNase treatment, and RNA was purified by phenol-acid-chloroform-based extraction and precipitated in PBS using isopropanol and sodium acetate at -20°C. A cDNA library was constructed for gene expression analysis by RT-qPCR. The primer pairs used in this study were as follows: [Table 1]

[0065] spray research MTBVAC and BCG produced under GMP conditions were resuspended at 1 ml per vial and placed in the reservoir of a clinical nebulizer U100 (OMRON). The nebulizer was connected to a gas-washing flask containing 5 ml of sterile water via plastic tubing and connected to a vacuum pump to collect the nebulized fraction. Bacteria were nebulized for 5 minutes, and both the nebulized and reservoir fractions were plated on solid agar 7H10 medium supplemented with ADC. The nebulization efficacy index was calculated as follows:

number

[0066] statistics Before starting the experimental procedures, commercially available mice were randomly distributed into groups of six animals per cage. Results were not blinded for analysis. No statistical methods were used to calculate sample size in animal experiments. GraphPrism software was used for statistical analysis. The statistical tests used for each experiment are indicated in the figure legends. All statistical tests used were two-sided. Grubb's test was applied to all data sets to determine outliers, which were discarded from the final statistical analysis. Differences were considered significant when p-values were <0.05.

[0067] Example 1 In an acute model of OVA-driven asthma (Figure 1A) and determination of lung eosinophilia by flow cytometry (Figure 1B), intranasally administered MTBVAC vaccine (Figure 1C) and BCG vaccine (Figure 1D) to sensitized mice dramatically reduced the percentage of allergen-induced eosinophilia to levels comparable to negative controls in both BAL and lung. This reduction was also observed when analyzing the total number of eosinophils, without significantly affecting other cell populations (Figure 1E, Figure 1F). The effect of vaccine treatment was also observed when analyzing mucus secretion and lung epithelial remodeling, as observed by staining with PAS technology. BCG-treated mice showed significantly lower levels of goblet cells and mucus material, as well as a lower rate of epithelial proliferation (Figure 1G).

[0068] Example 2 Both BCG and MTBVAC reduced eosinophilia in BAL (Fig. 2A) and lung (Fig. 2B) in a dose-dependent manner. 6 CFU dose, but in the lungs, the highest dose tested (10 7 CFU, 10 for BCG 6 CFU) to achieve a reduction in eosinophils to negative control levels.

[0069] Example 3 BCG primarily infects alveolar macrophages. Internalization of BCG resulted in the expression of M1 polarization markers, such as iNOS or CD86 (Figure 3A). Furthermore, BCG infection up-regulates M1 markers (iNOS or CD86) and down-regulates M2 markers (CD206) across the entire lung macrophage population (Figures 3B and 3C). The BCG-induced M1 repolarization of macrophages was confirmed by examining the pulmonary gene expression of M1 markers iNOS and IL1b (higher in the BCG group) and M2 markers Ym1, Arg1, and retnla (lower in the BCG group) (Figure 3D).

[0070] Example 4 Intranasal administration of BCG rebalances allergen-induced Th2 responses to a Th1 phenotype. BCG vaccination induces Th1-related genes, such as Ifng and Il12a, or the transcription factor Tbet. Conversely, genes encoding typical Th2 cytokines and chemokines, such as IL-5, IL-4, IL-13, and CCL-1, are downregulated by BCG (Figure 4A). Consistent with these results, IL-5 and IL-4 cytokines are elevated in the BAL and lungs, respectively, of the OVA group, whereas IFNγ is increased after BCG vaccination (Figures 4B and 4C).

[0071] These changes were recapitulated when allergen-specific T cells were studied after ex vivo OVA stimulation of lymphocytes harvested from mediastinal lymph nodes. The data show higher IL-4, IL-5, and IL-13 levels and lower OVA-specific IFNγ production in the OVA group compared with BCG-vaccinated mice (Figures 4D-G). Using intracellular staining and flow cytometry, we directly visualized cytokine-producing CD4+CD44+ T cells, which correspond to a memory phenotype. After stimulation with either CD3 / CD28 or OVA, T cell response polarization was found to be opposite between the two groups. Notably, OVA-specific IL-5-producing cells were reduced in BCG-treated mice (Figures 4H and 4I).

[0072] Example 5 MTBVAC treatment resulted in a decrease in the Th2 cytokine IL-5 and an increase in the Th1 marker IFNγ in BAL from OVA-challenged mice (Figure 5A) and in lymph node cells stimulated with OVA ex vivo (Figure 5B). MTBVAC administration abolished the expression of the M2 marker Arg1 in lung macrophages but induced the expression of the M1 marker iNOS (Figure 5C).

[0073] Example 6 MTBVAC and BCG abolished established eosinophilia in a chronic model of OVA-induced asthma (Figure 6A). Administration of the vaccine to OVA-challenged mice, in the setting of already infiltrating eosinophils in the airways (Figure 6B), reduced eosinophilia to levels comparable to those of negative controls (Figures 6C and 6D). The effect of vaccine treatment was also observed when analyzing mucus secretion and lung epithelial remodeling, as observed by staining with PAS technology. BCG-treated mice showed significantly lower levels of goblet cells and mucus material, as well as a lower rate of epithelial proliferation (Figure 6E).

[0074] Example 7 MTBVAC and BCG abolished established eosinophilia in a chronic model of asthma induced by the physiological allergen house dust mite (HDM) (Figure 7A). Administration of the vaccine to HDM-challenged mice, in the setting of already infiltrated airways with eosinophils, dramatically reduced eosinophilia to levels comparable to those of negative controls (Figure 7B). The efficacy of vaccine treatment was also observed when analyzing mucus secretion and lung epithelial remodeling, as observed by staining with PAS technology. BCG-treated mice showed significantly lower levels of goblet cells and mucus material, as well as a lower rate of epithelial proliferation (Figure 7C).

[0075] Intranasal administration of MTBVAC and BCG rebalanced the HDM-induced Th2 response to a Th1 phenotype. The Th2 cytokines IL-5, IL-4, and IL-13 were reduced in both allergen-stimulated BAL cells and lymph node cells in the vaccine-treated group (Figures 7D and 7E). Conversely, MTBVAC and BCG increased the levels of the Th1 cytokine IFNγ (Figures 7F and 7G).

[0076] Example 8 Vaccine nebulization with the OMRON U100 clinical aerosolization device (Figures 8A and 8B) demonstrates substantially higher nebulization efficacy for MTBVAC compared with BCG. Approximately 15% of the MTBVAC bacteria initially placed in the nebulizer reservoir are aerosolized, whereas this percentage drops to 0.15% for BCG (Figures 8C-8E). This indicates that using aerosols as a pulmonary route of vaccine administration makes it more feasible to reach a therapeutic dose in the lung with MTBVAC than with BCG. Electron microscopy images of BCG and MTBVAC demonstrate the smaller size of MTBVAC (Figures 9A and 9B), which may explain the observed difference in nebulization efficacy.

[0077] Example 9 Intranasal MTBVAC reduced esophageal eosinophilia induced by OVA challenge (Figure 10). This result suggests that live attenuated vaccines may be used to treat other types of eosinophilia, such as eosinophilic esophagitis (EoE), in addition to allergic asthma.

[0078] Example 10 Pulmonary delivery of MTBVAC improves the protective efficacy of the vaccine against challenge with TB compared to BCG administered by the standard subcutaneous route (Figure 11).

[0079] Example 11 GMP vials of MTBVAC and ONCOTICE were resuspended at 1 ml / vial to a concentration of 10 7Two milliliters of each vaccine preparation, normalized by CFU / ml, was placed in the reservoir of a clinical nebulizer U100 (OMRON). There are strong regulatory safety concerns regarding intranasal vaccine delivery in humans. However, aerosol administration is an accepted method for reaching the pulmonary compartment in the clinic, and several commercially available nebulizers approved for human use are available. From a practical perspective, a key step toward bringing pulmonary administration of live mycobacteria into clinical development is the feasibility of delivering therapeutic vaccine doses via the aerosol route. To assess this, we measured the nebulization efficacy of GMP formulations of MTBVAC and BCG (ONCOTICE) adapted for human use with the clinical nebulizer OMRON U100 (Figure 12A). We loaded the reservoir with MTBVAC or BCG resuspended in the corresponding eluent, placed the nebulizer in a horizontal position, and connected it via plastic tubing to a vacuum pump-connected gas-washing flask containing 5 ml of sterile water, where the nebulized fraction was collected. The bacterial density in the reservoir was approximately 1.5 × 10 for both vaccines. 7 CFU / ml. One minute after nebulization, the average amount of bacteria recovered in the nebulized fraction was 1.85 × 10 for MTBVAC. 6 CFU, 5 × 10 for BCG 5 CFU (Figure 12B). The mean efficacy of nebulization with respect to the initial bacterial load in the reservoir was 11.7% for MTBVAC and 3.1% for ONCOTICE (Figure 12C).

[0080] The inventors identified certain properties of BCG and MTBVAC that may explain the observed differences in nebulization efficacy. Electron microscopy images revealed shorter bacterial lengths in MTBVAC (<1 μm) compared to BCG (>2 μm). Considering that the average particle size emitted by clinical nebulizers is approximately 5 μm, it is realistic to speculate that aggregation and bacterial size may critically affect the nebulization efficacy of various live vaccines. These differences in the physical properties of MTBVAC make it more advantageous to administer it using a clinical nebulizer.

Claims

1. For the prevention or treatment of allergic diseases in human subjects, i) a PhoP-phenotype due to inactivation by gene deletion of the Rv0757 gene, wherein the open reading frame (ORF) sequence of phoP consists of SEQ ID NO: 4; ii) A deletion of a second gene Rv2930 (fadD26) that prevents PDIM production (PDIM-phenotype), wherein the open reading frame (ORF) sequence of fadD26 consists of SEQ ID NO:

2.

1. A live-attenuated M. tuberculosis composition comprising an isolated microorganism belonging to the M. tuberculosis MTBVAC strain, comprising: A live-attenuated M. tuberculosis composition, wherein the composition is administered to the subject by inhalation using aerosol technology.

2. 10. The live-attenuated M. tuberculosis composition of claim 1, wherein the composition is a lyophilized composition further comprising a specific stabilizer, bulking agent, or buffer.

3. 3. The live-attenuated M. tuberculosis composition of claim 1 or 2, wherein the composition is for the prevention of allergic diseases by targeting M2 macrophages to alleviate allergic responses.

4. 3. The live-attenuated M. tuberculosis composition of claim 1 or 2, wherein the composition is for use in preventing allergic disease in a person at risk of suffering from or developing allergic asthma.

5. 3. The live-attenuated M. tuberculosis composition of claim 1 or 2, wherein the composition is for the treatment of an allergic disease by targeting M2 macrophages to reduce allergic responses.

6. 3. The live-attenuated M. tuberculosis composition of claim 1 or 2, wherein the composition is for the treatment of allergic asthma or other types of eosinophilia, such as eosinophilic esophagitis (EoE).

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