WO2015076656A1

WO2015076656A1 - A recombinant mycobacterium bovis bcg strain lacking the bcg1419c gene, with enhanced biofilm-formation capacity

Info

Publication number: WO2015076656A1
Application number: PCT/MX2014/000168
Authority: WO
Inventors: Mario Alberto FLORES VALDEZ; Rogelio HERNÁNDEZ PANDO; César PEDROZA ROLDÁN; Michel de Jesús ACEVES SÁNCHEZ
Original assignee: Centro De Investigación Y Asistencia En Tecnología Y Diseño Del Estado De Jalisco A.C.; Instituto Nacional De Ciencias Médicas Y Nutrición Salvador Zubirán
Priority date: 2013-11-25
Filing date: 2014-10-28
Publication date: 2015-05-28
Also published as: MX2013013771A; MX363576B

Abstract

The present invention relates to a Mycobacterium bovis BCG strain lacking the BCG1419c gene, with registration number PTA- 120572, in which nucleotides 222 to 924 of the open reading frame (ORF) are eliminated from a total of 924 base pairs that encode the protein with second messenger c-di-GMP phosphodiesterase activity (strain BCGABCG1419c), in addition to 47 base pairs downstream of the stop codon of the gene. The BCGABCG1419c strain is capable of forming from 3 to 15% more biofilm in vitro as compared with unmodified BCG, and it is capable of halting weight loss in animals vaccinated therewith at levels similar to those achieved with unmodified BCG but by means of the use of lower doses. The BCGABCG1419c strain mimics combined aspects of a chronic infection and could therefore be used as a vaccine against latent tuberculosis.

Description

A strain of Mycobacterium bovis BCG RECOMBINANT CARENTE GENE BCG1419C, WITH INCREASED CAPACITY OF FORMING BIOPELLICLES

TECHNICAL FIELD OF THE INVENTION The present invention belongs to the field of biotechnology and refers to the Mycobacterium bovis BCG strain, to which nucleotides 222 to 924 were removed from the open reading frame (ORF, by its name and acronym in English)] of 924 total base pairs that code for the phosphodiesterase activity protein of the second messenger di-GMPc, in addition to 47 base pairs downstream of the stop codon of the BCG1419c gene, which forms in vitro biofilms in greater proportion than the unmodified strain, and its use in the treatment, prevention or delay of chronic or latent tuberculosis.

BACKGROUND OF THE INVENTION

Currently, tuberculosis (TB) is a major public health problem (Comas & Gagneux, 2009, WHO, 2009). It is the disease of bacterial origin with the highest morbidity and mortality rate (1.8 million people die annually from tuberculosis) worldwide (WHO, 2009). The World Health Organization (WHO) estimated that during 1990 there were 8 million new cases of tuberculosis and 3 million deaths worldwide, of which 95% occurred in underdeveloped countries (Raviglione, 2007). This situation forced WHO in 1993 to declare tuberculosis as a global public health emergency and to propose strategies for its control.

Although many of the cases can be treated with drug therapy, in recent years the appearance of drug-resistant strains has increased in all parts of the world, with intractable cases in India (Loewenberg, 2012, Shah et al, 2011, Donald & van Helden, 2009). The main route of infection in tuberculosis is the aerial route, due to exposure to the tubercle bacillus, usually when in contact with the respiratory secretions that people with pulmonary tuberculosis leave, when they cough, talk or sneeze. It is estimated that 1 to 3 bacteria is a sufficient dose to cause infection with M. tuberculosis in humans (Van Rhijn et al, 2008).

It has been considered that a third of the population is latent infected with Mycobacterium tuberculosis, the causative agent of tuberculosis (Dye et al, 1999). Mycobacterium bovis Bacillus Calmette-Guérin (BCG) is the only vaccine currently available and approved by WHO to prevent and reduce the incidence of human disease through a prophylactic immunization strategy (WHO, 2009). BCG is a live attenuated vaccine, originally derived from 230 serial passages in vitro of a virulent strain of Mycobacterium bovis, developed by Albert Calmette and Camille Guérin between 1908 and 1921. During the under-cultivation genetic alterations accumulated, including deletions (loss of genes) and duplications, which resulted in the loss of virulence factors, which resulted in Attenuation The BCG strain has been used as a vaccine in humans since 1921, and to date more than 3 billion doses have been administered with about 115 million additional doses applied annually to about 80% of children in the world ( Doherty & Andersen, 2005, Skeiky & Sadoff, 2006].

BCG is a safe vaccine, side effects are well tolerated and only contraindicated for immunocompromised individuals, such as people with AIDS, in whom it can cause serious disseminated disease and even death (WHO, 2009, Tullius et al, 2008). However, its effectiveness is still a controversial issue. On the one hand, it reduces the risk for disseminated forms of TB in early childhood, including miliary disease and meningeal tuberculosis, providing greater than 80% efficacy against these severe forms of the disease (WHO, 2009, Colditz et al, 1995, Rodrigues et al, 1993, Trunz et al, 2006, McShane & Williams, 2011, Meena & Rajni, 2010, McShane, 2011). However, the efficacy of protection against pulmonary TB, the predominant form in adults, is variable, it is estimated from randomized clinical studies in a range of 0 to 80% (Brewer, 2000). In addition, this vaccine does not prevent primary infection and, more importantly, does not prevent the reactivation of latent pulmonary infection, the main source of bacillary dissemination in the community (WHO, 2009). On the other hand, it should be considered that the period of immunity conferred by BCG after neonatal vaccination gradually decreases, extending to no more than 10 to 20 years (Doherty & Andersen, 2005), so it has a small effect on TB rate in adults and which is also not effective in populations previously sensitized by mycobacterial antigens (Trunz et al., 2006). Likewise, different vaccine strains have mutations in transcriptional regulators, such as PhoP and Crp (Brosch et al, 2007), which control the expression of multiple genes and therefore, their capacity for multiplication in the host, degree of virulence and presentation of antigens varies, with consequent changes in protection efficacy in experimental models (Castillo-Rodal et al, 2006) and in human population (Mahomed et al, 2006). Currently the use of the bacterium Mycobacterium bovis Bacillus Calmette-Guérin (BCG) is to immunize healthy people who have never been in contact with Mycobacterium tuberculosis. In the development of new vaccines, most strategies focus on preventing active or progressive disease, through the use of attenuated variants of Mycobacterium tuberculosis. As examples we can cite auxotrophs for amino acids (Sampson et l, 2004), for nucleotide metabolism (Sambandamurthy et al, 2006, Sambandamurthy et al, 2005, Brown et al, 2005), lacking transcriptional regulators, such as PhoP (Martin et al, 2006) or unable to produce some lipids, such as the mutant in fadD26 (Infante et al, 2005).

Other attempts have been based on the production of recombinant BCG strains expressing Mycobacterium tuberculosis antigens, such as Ag85A and Ag85C, ESAT-6, Ag 38KDa, Ag 19KDa; or even those that produce cytokines such as IL-18 and TGF-β (Hernández-Pando et al, 2007). It should be noted that except for two recent cases, on the one hand a subunit vaccine based on the mixture of a protein expressed during acute infection, and another implicated in chronic infection in mice (Aagaard et al, 2011), and on the other a modified BCG strain To overexpress antigens of the DosR region (Flores Valdez & Schoolnik, 2010), the efforts reported to date to obtain a latent tuberculosis vaccine are scarce. In US Patent 7,935,354B2, a modified BCG strain was constructed, capable of constitutively expressing the DosR transcriptional regulator, and thus inducing the transcription of most of the 40 genes that make up the DosR region, whose antigens are found proteins preferentially recognized by people with latent tuberculosis. Variants based on DosR itself or members of its region have been notified in patent applications AU20110203012 and US20070945680 20071127.

Another option for vaccines against latent tuberculosis is that reported in patent MX2011011186 (A), based on seeking response against constitutively expressed antigens, including ESAT6, CFP10 and other member antigens of the ESX-1 secretion system and that does not have no relation to the change we made in our BCG strain lacking the BCG1419c gene.

Finally, it can also be mentioned the patent MX2012008790 (A), which through the use of modified Rv3616c polypeptides or the polynucleotides that encode them, in the field of latent mycobacterial infections, again, where no use of particular components is cited associated to the change by the elimination of the BCG1419c gene that we carry out.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates in particular to a strain of Mycobacterium bovis BCG lacking the BCG1419c gene (or its genetic counterparts in other mycobacteria), which has accession number PTA-120572, capable of forming 3-15% more biofilms in vitro compared with unmodified BCG, and able to stop the weight loss of animals vaccinated with it, at levels comparable with BCG but applied in lower doses.

In the present invention it has been found that the elimination of the BCG1419c gene gives Mycobacterium bovis BCG (strain BCGABCG1419c) an increased ability to form in vitro biofilms, a phenomenon that is associated with better replication in vivo in the lungs and spleen of immunocompetent animals.

The BCGABCG1419c strain mimics combined aspects of a chronic infection, such as increased persistence in the host, and changes in the expression of proteins that are necessary to respond to nitrooxidative stress. Consequently, strain BCGABCG1419c could serve as a vaccine against latent tuberculosis.

In its broadest aspect, the present invention provides a modified Mycobacterium bovis BCG strain, where the BCG1419c gene has been removed, so that the protein with phosphodiesterase activity of the second cyclic diguanilate messenger (cyclic diguanosine monophosphate messenger) cannot be expressed therein , di-GMPc, c-di-GMP-by its acronym in English-).

In one aspect of the invention, a modified Mycobacterium bovis BCG strain is provided, where nucleotides 222 through 924 were removed from the open reading frame (ORF) of 924 pairs of total bases encoding the phosphodiesterase activity protein of the second messenger di-GMPc, strain BCGABCG1419c) in addition to 47 base pairs downstream of the stop codon of the gene. This leads to the BCGABCG1419c strain being able, in theory, to produce a peptide that includes only amino acids 1 to 73 (of a total of 307 that has the complete protein), and that lacks amino acid residues 89, 90 and 91 (EAL , glutamic acid, alanine, leucine) necessary for the enzymatic degradation activity of di-cGMP. The BCG1419c gene and its counterparts from other bacteria of the Mycobacterium tuberculosis complex, code for a protein with predicted EAL domain. This domain is associated with the degradation activity (phosphodiesterase) of the second di-cGMP messenger, same activity that was demonstrated for the protein encoded by the Rvl357c gene (Gupta et al., 2010). Di-cGMP has been implicated in the regulation of the expression of microbial virulence factors and biofilm production in various microorganisms (Hengge, 2009), and recently it was reported that a homologous mutant (with elimination of the Rvl357c gene, in M. tuberculosis strain H37Rv), was less able to multiply in lungs and spleens of immunocompetent mice (Hong et al, 2013), findings opposite to what we obtained.

With respect to unmodified BCG, the BCGABCG1419c strain increases in vitro biofilm production from 3 to 15%, modifies the expression of at least 9 proteins during biofilm formation, increases persistence in lungs and spleen of 3 to 6 times immunocompetent BALB / c mice, and similarly stops the weight loss of vaccinated mice and then infected with Mycobacterium tuberculosis H37Rv, despite being administered at a lower dose. Then, when the modified BCG strain of the present invention, or all, some or some of the proteins whose expression is regulated differentially during biofilm formation by the elimination of the BCG1419c gene or its homologs in other mycobacterial species, is administered to a mammalian host, an immune response will be induced towards some, some, or all of the proteins, or different components (carbohydrates, lipids, or combinations of these two, or combinations with proteins of one of the two or both) that are expressed in a manner different from unmodified BCG. Therefore the mycobacterium that then infects this immunized mammal will be unable to establish a latent infection in the host; When proteins, carbohydrates, lipids or combinations thereof, required to establish a latent infection, are expressed they will be recognized by the immune system and consequently the mycobacterium will be destroyed. Alternatively, or additionally, individuals where the latent infection is already established, the ability of mycobacteria to reactivate and cause an active infection will be attenuated. In this scenario, the latent mycobacterium may be eradicated by the immune system, or it may be unable to emerge from the latent (chronic, persistent) state and confined to a state of inactivity (dormancy). In any case, the active and contagious infection is prevented and with it the transmission to other people.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Amplification of the Rvl356c and Rvl358c genes of the Mycobacterium tuberculosis H37Rv strain. An aliquot of the PCR reaction was subjected to 1% agarose gel electrophoresis. Lane 1 molecular weight marker 1 Kb plus (Invitrogen), lane 2 PCR Rvl356c (65.4 bp), lane 3 PCR Rvl358c (549 bp).

Figure 2. Quadruple digestion gel (Xba I, Kpn I, Hind III and Bgl II) for pMarl357c, where it contains the fragments, of pYUB854, Rvl356C, Rvl358c. In lane 1. Marker 1 Kb plus, 2. empty, 3. pYUB854, 4.pYUB854-Rvl356c, 5.pYUB854-Rvl358c, 6.pYUB854-Rvl356c + Rvl358c el, 7. pYUB854-Rvl356c + Rvl358c c2, 8. pYUB854-Rvl356c + Rvl358c c3, 9pYUB854- Rvl356c + Rvl358c c4, 10 pYUB854-Rvl356c + Rvl358c c5, 11. pYUB854-Rvl356c + Rvl358c c6, 12. pvUB355 pYUB854- Rvl356c + Rvl358c c9, 15. pYUB854-Rvl356c + Rvl358c clO. Positive clones to both inserts were sequenced and clone 3 was selected and named pMarRvl357c. Figure 3 Alignment of the sequence obtained from clone 3 with oligonucleotide Rvl356c-F to the Mycobacterium genome. tuberculosis H37Rv.

Figure 4. Alignment of the sequence obtained from clone 3 with oligonucleotide Rvl358-R to the Mycobacterium genome. tuberculosis H37Rv. Figure 5. From the pMar construction ARvl357c the allelic exchange substrate was extracted using the restriction enzymes Xbal and Nhel generating two bands of 3.1 and 1.9 kb. The 3.1 band corresponding to the sequence with the deletion was cut from the gel and purified to be subsequently transformed into the recombination strain. Figure 6. Graphical representation of plasmid pJV53, which contains the viral recombinase gene Che9c-60-61 under the control of the acetamide-inducible promoter (Taken from Van Kessel, Marinelli and Hatfull, Nature Reviews Microbiology, 2008],

Figure 7. Expected result of double homologous recombination events after the elimination of plasmid pJV53, to generate strain BCGABCG1419c, at genomic level Figure 8. Expected result of double homologous recombination events after plasmid pJV53 removal, to generate BCGABCG1419c strain at the level of the predicted amino acid sequence.

Figure 9. Possible results of homologous recombination events after double recombination and elimination of plasmid pJV53 and its characterization with oligonucleotides for polymerase chain reaction (PCR).

Figure 10. Amplification of the outward ends of the hygromycin cassette. The image confirms with this new combination of oligonucleotides that two of the mutant candidates (C5 and CIO) are illegitimate recombinants and that all strains are resistant to hygromycin (hyg) by the presence of the hyg gene on the chromosome. Figure 11. Possible results of homologous recombination events after double recombination and elimination of plasmid pJV53, verifiable with the indicated oligonucleotides to determine presence or absence of transcript covering the open reading frame of the BCG1419c gene.

Figure 12. Amplification of the open reading frame of the BCG1419c gene, with oligonucleotides RV1357c-ORF-F and Rvl357c-ORF-R. In cases where gene expression was analyzed by RT-PCR, (-) indicates reaction without reverse transcriptase and (+) with reverse transcriptase, as a control of amplification by transcript and not by contaminating genomic DNA.

Figure 13. Amplification of the constituent gene rrs. In cases where the expression of the gene was analyzed by RT-PCR, (-) indicates reaction without reverse transcriptase and (+) with reverse transcriptase, as a control of amplification by transcript and not by contaminating genomic DNA. Figure 14. Prediction of domains present in the protein encoded by the BCG1419c gene. The EAL domain (amino acids 89, 90 and 91] has c-di-GMP phosphodiesterase activity in vitro.

Figure 15. Photographs of biofilms formed by strains of Mycobacterium bovis BCG. Figure 16. Quantification of the formation of biofilms formed in vitro by BCG Wild Pasteur (Wt) and BCGABCG1419c (Delta). Here it is observed that the BCGABCG1419c strain produces more biofilm than the wild BCG strain.

Figure 17. Comparison of the protein profile of the soluble fraction differentially expressed by Mycobacterium bovis BCG strains in biofilms. On the right, the number of proteins that differed in presence or quantity between strains is indicated, after comparison and statistical analysis performed with the Quantity One software (BioRad Laboratories), as well as the relationship between the BCGABCG1419c strain and the wild strain (WT) .

Figure 18. Survival of strains of Mycobacterium bovis BCG Pasteur in lungs of male BALB / c mice aged 9-10 weeks infected intravenously Figure 19. Survival of strains of Mycobacterium bovis BCG Pasteur in spleens of male BALB / c mice of 9 -10 weeks old infected intravenously

Figure 20. Survival of strains of Mycobacterium bovis BCG Pasteur in lungs of female BALB / c mice aged 9-10 weeks infected intravenously

Figure 21. Survival of strains of Mycobacterium bovis BCG Pasteur in spleens of female BALB / c mice aged 9-10 weeks infected intravenously

Figure 22. Average weights of groups of mice vaccinated with wild BCG (WT), BCGABCG1419c (Delta) or control (SS), in grams, with respect to week 2 post-infection with M. tuberculosis H37Rv.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a strain of Mycobacterium bovis BCG (BCGABCG1419c), lacking the BCG1419c gene, characterized in that it has the accession number PTA-120572, where the BCG1419c gene was exchanged for a hygromycin resistance gene by recombination homologue With respect to unmodified BCG, the BCGABCG1419c strain described here, increases in vitro biofilm production from 3 to 15%, modifies the expression of at least 9 proteins during biofilm formation, increases persistence in lungs 3 to 6 times and spleen of immunocompetent BALB / c mice, and stops at the level Similar weight loss of vaccinated mice and then infected with M. tuberculosis H37Rv, despite being administered in smaller doses.

The available scientific literature indicates that regularly, inactivation of genes encoding proteins with di-cGMP phosphodiesterase enzymatic activity results in cells with greater capacity to form biofilms in vitro. Although there is no formal evidence today that biofilms of M. tuberculosis in vivo similar in physical appearance to those formed in vitro are formed, contrary to what occurs with other microorganisms, during infection in animal models with M. tuberculosis, they find microcolonies that are hypothesized to be a kind of biofilm (Lenaerts et al, 2007). When the modified BCG strain object of the present invention, or all, some or some of the proteins whose expression is regulated differentially during the formation of biofilms by the elimination of the BCG1419c gene or its homologs in other mycobacterial species, administered to a mammalian host, an immune response will be induced towards some, some, or all proteins, or different components (carbohydrates, lipids, combinations of these two, or combinations with proteins of one of the two or both) that are expressed differently from unmodified BCG. or leads us to think that the mycobacterium that then infects this immunized mammal will be unable to establish a latent infection in the host; When proteins, carbohydrates, lipids or combinations thereof, required to establish a latent infection, are expressed they will be recognized by the immune system and consequently the mycobacterium will be destroyed.

Alternatively, or additionally, individuals where the latent infection is already established, the ability of mycobacteria to reactivate and cause an active infection will be attenuated. In this scenario, the latent mycobacterium may be eradicated by the immune system, or it may be unable to emerge from the latent (chronic, persistent) state and confined to a state of inactivity (dormancy). In any case, the active and contagious infection is prevented and with it the transmission to other people.

An example sequence of nucleotides comprising the BCG1419c gene (SEQ ID NO: 1) and an example sequence of amino acids encoded by such a nucleotide sequence (SEQ ID NO: 2) are included. These sequences represent BCG1419c, of the genome of BCG Pasteur 1173P2, available at the BCGList network site of the Pasteur Institute. In general, for the purposes of the present invention "BCGABCG1419c" or "BCG lacking the BCG1419c gene" or "BCG Delta BCG1419c" refers to the BCG strain where the region from nucleotide 221 was removed from the beginning of translation and up to 38 nucleotides downstream of the translation stop codon of the BCG1419c gene (Figure 7) to originate a truncated sequence protein without the phosphodiesterase domain of di-cGMP (Figure 8). However, "BCGABCG1419c" also we It allows to cover different variants, analogs and derivatives of the aforementioned strain if they have an equivalent or similar activity so as not to be able to produce a phosphodiesterase of the second messenger di-GMPc.

All these variants are considered within the applications of the present invention. For example, those with experience and mastery of the art will recognize that many changes can be made in the BCG1419c gene sequence, such as non-conservative amino acid changes in the EAL domain responsible for the degradation enzymatic activity of the second messenger di-GMPc, or either directed removal of the EAL domain only, or changes in other regions that affect the conformation of the protein making it non-functional, or changes in the region that regulates transcription or translation to eliminate both and not give rise to any protein, or gene substitutions BCG1419c by genes other than antibiotic resistance (hygromycin) used in the present invention, or even without any antibiotic or other screening that facilitates the identification of the change that leads to the inactivation or decrease of di-GMPc phosphodiesterase activity . Another case covered by the present invention is the variants of BCG or other mycobacteria that can be isolated from nature or generated in the laboratory, which contain insertions or removals (deletions) of nucleotides that result in non-production, protein production truncated or non-functional in its enzymatic activity of phosphodiesterease from di-cGMP. All the variants described will share with the sequence present in our invention the null or decreased ability to degrade the second messenger di-cGMP produced by mycobacterial strains used as vaccines or candidates for vaccines against tuberculosis. Methods for determining or predicting the loss of phosphodiesterase activity of di-GMPC are known. Likewise, there is knowledge of procedures necessary to measure the enzymatic activity of proteins, and to determine the acceptable levels of low degradation activity of the second messenger di-GMPc. To cite examples, variants with null, low or decreased activity of di-cGMP phosphodiesterase can be evaluated in bacterial growth in biofilms or as isolated (planktonic) cells. In general, all variants of BCG1419c would, with respect to the activity of the wild protein, show a decrease in the ability to degrade in vitro or in vivo the second messenger di-cGMP in a range of about 20-30%, or 30 -50%, or preferably 50-70%) or better yet 70-90% and in optimal cases 90-100%.

Alternatively, the inactivation or decrease of the phosphodiesterase activity of the second di-cGMP messenger can be performed transiently, contrary to the stable form object of the present invention, if expression control sequences are placed to respond to environmental stimuli. In these cases, for example, the decrease in phosphodiesterase activity of the second di-cGMP messenger can occur if proteins that negatively regulate the activity of BCG1419c are expressed, placing the coding region for such negative regulators under the control of inducible macrophage promoters [resulting in selective induction of genes within the macrophage phagosome, (Schnappinger et al, 2003)] or tetracycline inducible (Blokpoel et al, 2005), among others. In addition, promoters of other species could be used, examples of which we have included in, but not limited to, several viral promoters, after similar strategies to "gene therapy" (eg cococulation of mycobacteria and a virus modified by genetic engineering), the antigens expressed by the mycobacterium where the phosphodiesterase activity of the second messenger di-GMPc was reduced or eliminated, are expressed in selected tissues as a result of inactivation achieved by the co-administered virus.

There are different alternatives to introduce and exchange for recombination or insertion, nucleic acid sequences within a host mycobacterium, to achieve the decrease or loss of di-cGMP phosphodiesterase activity. For example, the sequences can be included within vectors that are then introduced into the mycobacterium.

There are many vectors that serve these purposes, which include but are not limited to several extrachromosomal elements such as plasmids, eg those that share the origin of replication pAL500, or other plasmids that have or will have different origins of replication, or extrachromosomal elements that do not replicate or integrate into the mycobacterial genome. The introduction of such sequences to achieve the decrease or elimination of phosphodiesterase activity of the second di-cGMP messenger can be accomplished by means of several methods developed for exchange or inactivation vectors, including but not limited to mycobacteriophage-mediated electroporation and transduction. Many vectors can be used to enter the mycobacteria, so that the resulting strain reduces or eliminates the phosphodiesterase activity of di-cGMP in vitro and / or in vivo. In its preferred presentation, the vector is a nucleotide sequence that is introduced by electroporation. In other presentations of the invention, the decrease or loss of di-cGMP phosphodiesterase activity can be achieved by means of extrachromosomal elements.

There are several molecular biology strategies to generate a mycobacterial strain with this property. For example, vectors can be introduced that code for negative transcription regulators of the BCG1419c gene, which are able to increase constitutively or inducibly before a specific stimulus, the production of such a negative regulator to "turn off" the gene that produces the enzyme with activity degradative di-cGMP. Another option would be for such a vector to produce proteins with RNAse enzymatic activity that preferentially degrades the BCG1419c transcript, constitutively or inducibly before a specific stimulus, or even with an antisense sequence that hybridizes with the BCG1419c messenger RNA and prevents its translation. (Antisense RNA). A further example would be that such vectors lead to the production of a molecule with EAL active site blocking activity responsible for the phosphodiesterase activity of di-cGMP, or a protease that specifically degrades the enzyme of interest. Those skilled in the art will recognize that there are several BCG strains that may be useful in the practice of the invention, but not limited to those shown in Table 1.

ATCC Number Description Designation Strain

19274 M. bovis deposited as strain 50 [BCG]

27289 Mycobacterium bovis BCG, Chicago 1 [B, BCGT, Tice]

35731 M. bovis TMC1002 [BCG Birkhaug]

35732 M. bovis TMC 1009 [BCG Swedish]

35733 M. bovis TMC1010 [BCG Danish]

35734 M. bovis TMC 1011 [BCG Pasteur]

35735 M. bovis TMC 1012 [BCG Montreal, CIP 105920]

35736 M. bovis TMC 1013 [BCG Brazilian]

35737 M. bovis TMC 1019 [BCG Japanese]

35738 M. bovis TMC 1020 [BCG Mexican]

35739 M. bovis TMC 1021 [BCG Australian]

35740 M. bovis TMC 1022 [BCG ussian]

35741 M. bovis TMC1024 [BCG Glaxo]

35742 M. bovis TMC 1025 [BCG Prague]

35743 M. bovis TMC 1028 [BCG Tice]

35744 M. bovis TMC 1029 [BCG Phipps]

35745 M. bovis TMC 1030 [BCG Connaught]

35746 M. bovis TMC 1101 [BCG Montreal, SM-R]

35747 M. bovis TMC 1103 [BCG Montreal, 1NH-R, CIP 105919]

35748 M. bovis TMC 1108 [BCG Pasteur SM-R]

27290 M. bovis BCG, Copenhagen [H], etc.

Table 1

In addition, the recombinant mycobacterial strain of the invention can be a modified BCG, including but not limited to: BCG expressing perfringolysin (Sun et al, 2009], BCG expressing listeriolysin (Grbde et al, 2005, Desel et al, 2011) , BCG expressing DosR (Flores Valdez & Schoolnik, 2010), among others.In addition, recombinant mycobacteria should not necessarily be confined to BCG strains. Media experts will recognize that other strains of Mycobacterium could also be used to eliminate or decrease the phosphodiesterase activity of the second di-cGMP messenger by manipulating its BCG1419c homologous genes from BCG Pasteur 1173P2, which include but are not limited to: M. tuberculosis strain CDC1551 (Fleischmann et ai, 2002), M. tuberculosis strain Beijing (van Soolingen et al, 1995), M. tuberculosis strain H37Rv (Colé et al, 1998), M. tuberculosis auxotrophic strain for pantothenate (Sambandamurthy et al., 2006) or other attenuated strains or re combinations derived from M. tuberculosis. Other candidate bacteria include members of the M. tuberculosis complex, other mycobacteria (eg M. africanum or members of the M. aviurri complex) or other species of mycobacteria. Any mycobacterium containing a homologue of the BCG1419c gene can be used in the practice of the invention. In his preferred presentation, the Recombinant bacterium where di-GMPc phosphodiesterase activity was eliminated or reduced, is a BCG mycobacterium, and in particular M. bovis BCG Pasteur 1173P2, although any BCG can be used as listed as examples in Table 1, without being limiting of these BCG strains. Before the strain of the invention can be administered as a vaccine to humans, the recombinant mycobacteria of the present invention should be evaluated following methods well known to those skilled in the art. For example, toxicity, safety, etc. tests should be carried out in animal models such as mice, guinea pigs, etc., some of which must be immunocompromised and others not. The ability of vaccine preparations to induce an immune response on a regular basis is analyzed in animal models, eg mice, non-human primates, etc. Also, vaccination, booster and challenge experiments with virulent M. tuberculosis can be carried out using animal models that include mice, rabbits, guinea pigs, nonhuman primates. Finally, those skilled in the art will have knowledge to carry out clinical studies in human beings who consent to this, in order to prove the efficacy of the vaccine.

The present invention provides a strain to induce an immune response to one or more antigens that are differentially expressed during biofilm production, between the wild BCG Pasteur 1173P2 strain and its isogenic derivative BCGABCG1419c, among which are proteins present in the fraction soluble (Figure 17). Referring to "inducing an immune response to one or more antigens" we refer to the fact that the administration of the vaccine of the present invention promotes the synthesis of specific antibodies (in titers in the range of 1 to 10 ³ , or ^{3 3} , or better in the range of lxlO ³ to lxlO ⁶ , and optimally greater than lxlO ⁶ ) and / or measured cell proliferation, eg by means of cell assays in which IFN-γ production is evaluated, by incorporation of thymidine H ³ , etc. In its preferred presentation, the immune response is a protective response, that is, it protects the vaccinated individual from future infection with virulent M. tuberculosis.

Additionally, the recombinant strain BCGABCG1419c may have modified content of carbohydrates, lipids or proteins, or combinations of 2 or more molecules thereof, soluble or insoluble, with respect to the current BCG vaccine. In the preferred presentation of the invention, differentially expressed antigens induce a strong IFN-γ response, are potent immunogens for B or T cells, and possess high sequence identity with their M. tuberculosis counterparts. In particular, this would include bacterial components accessible to cells of the immune system that induce the release of IFN-γ with an effect on anti-inflammatory molecules that is not able to stop the response necessary for protection but which can be beneficial in regulating possible damage from exacerbated inflammation. EXAMPLE

Construction of a BCG strain lacking the BCG1419c gene (BCGABCG1419c), characterization in biofilm formation and as a vaccine against chronic tuberculosis in murine model

To carry out the homologous recombination, the allelic exchange vector was first constructed, where in the plasmid pYUB854 the segments of DNA obtained by polymerase chain reaction (PCR) were cloned next to the hygromycin resistance gene. in English) of 654 base pairs of the Rvl356c gene (obtained with oligonucleotides Rvl356cF 5'- CCAAGCTTTAGAACAGTATGCCC-3 ^' , Hind III site underlined; and Rvl356c-R 5'- TGGCGTCGAGATCTAAGGTACACT-3', site Bgl II underline) and site Bgl II underlined base pairs of the Rvl358 gene (obtained with oligonucleotides Rvl358-F 5 ^' -GCATCTAGACGAGGAAGAATTCACC-3', Xba I site underlined and Rvl358-R 5'-TGGGTACCTCAGATGCGGG-3 ^' Kpn I site underlined from DNA genomic amplified to DNA Mycobacterium tuberculosis H37Rv (Figure 1), to originate plasmid pMarl357c. The plasmids where these fragments were cloned were characterized by quadruple enzymatic digestion with the endonucleases Xba I, Kpn I, Hind III and Bgl II (Figure 2). The identity and fidelity of the cloned sequences was verified by sequencing (Figures 3 and 4). Then, by enzymatic digestion with the Xba I and Nhe I endonucleases of pMarl357c, the 3.1 kilobase (kb) and 1.9 kb fragments derived from this plasmid were separated by agarose gel electrophoresis (Figure 5), and the fragment was purified from 3.1 kb to serve as an allelic exchange substrate for electroporation to transform it into the Mycobacterium bovis BCG Pasteur 1173P2 strain containing plasmid pJV53 (holder of a gene encoding a viral recombinase, under the control of the inducible acetamidase promoter, Figure 6). The exchange of the BCG1419c gene (homologue in BCG Pasteur 1173P2 of the Rvl357c gene of Mycobacterium tuberculosis H37Rv and of genes with other names in other strains of the Mycobacterium tuberculosis complex, for example MT1400 in CDC1551, and Mbl392c in M. bovis AF2122 / 97, to cite some examples) by the hygromycin resistance gene was verified by PCR, to confirm whether after the plasmid pJV53 was removed the exchange was achieved or returned to the original (wild) version of the gene (Figures 7-9). PCR analysis with oligonucleotides Rvl357c-3R (5'-CGGGGTAATCGAATGGATCA-3 ') and Hyg-cH3 (5 ^' - CCCCAGGTGGCTAAAATGTATCC-3 ') showed that most of the selected strains did have a double homologous event with replacement of the BCG1419c gene by that of hygromycin (Figures 8 and 9), thus generating several candidate strains that may or may not have the replacement of the BCG1419c gene by that of hygromycin (Figures 10 and 11), until obtaining the BCGABCG1419c strain. The inability of the strain obtained to produce transcript was verified by RT-PCR using random hexamers for reverse transcription and oligonucleotides Rvl357c-ORF5 (5 ^' - CAAAGCTTGGATGGATCGTTGT-3'. Hind III site underlined) and Rvl357c-ORF3 (5 ^' - 5 ^' CCATCGATCTACACCCACGC-3 Cía site underlined) for PCR (Figure 12). The absence of transcript in the BCGABCG1419c strains was confirmed and was not a consequence of RNA degradation, since the rrs gene control transcript was amplified, as well as the PCR product observed in each case of reverse transcription reaction (lanes with + sign) comes from RNA and not contaminating genomic DNA, since without the reverse transcription treatment (lanes with sign -) no PCR product was observed (Figure 13).

The BCG1419c gene and its counterparts from other bacteria of the Mycobacterium tuberculosis complex, give rise to a protein with predicted EAL domain (Figure 14). This domain is associated with the degradation activity (phosphodiesterase) of the second c-di-GMP messenger (cyclic diguanosine monophosphate, cyclic diguanylate), which was recently demonstrated for the protein encoded by the Rvl357c gene. The c-di-GMP has been implicated in the regulation of the expression of microbial virulence factors and biofilm production in various microorganisms, without reports of the association of this gene with biofilms and virulence in bacteria of the Mycobacterium complex today. tuberculosis. The available scientific literature indicates that regularly, the inactivation of genes encoding proteins with c-di-GMP phosphodiesterase enzymatic activity results in cells with greater capacity to form biofilms in vitro.

Based on evidence published between 2008 and 2012 that demonstrate that bacteria of the Mycobacterium tuberculosis complex form biofilms in vitro, we find common characteristics among biofilms and infections caused by various microorganisms, such as: (1) in vitro, bacteria in biofilms are more tolerant to Antibiotics compared to bacteria present in liquid culture as individual (planktonic) cells, and (2) in vivo, bacteria in biofilms are associated with chronic infections. Two relevant issues should be noted: (1) in the disease (tuberculosis) there are two cases: active disease and chronic, latent - asymptomatic - infection, of the latter, the World Health Organization estimates that one third of the world's population is in this state; and (2) although there is no formal evidence that biofilms of Mycobacterium tuberculosis are formed in vivo similar in physical appearance to those formed in vitro as with other microorganisms, microcolonies are found to be hypothetized during infection in animal models with Mycobacterium tuberculosis. They could be a kind of biofilm. Thus, adding the knowledge that the inactivation of genes encoding enzymes with phosphodiesterase activity of c-di-GMP increases the ability to form biofilms in vitro, where biofilms increase antibiotic tolerance and chronicity of infections, both common aspects During tuberculosis, we assess whether the Mycobacterium bovis strain BCGABCG1419c:

1. Produces or not biofilms other than the current wild BCG vaccine strain; 2. It differs in its antigenic content with respect to the wild strain

3. More time persists in the lungs and spleen of immunocompetent mice and

4. If used as a vaccine in immunocompetent mice BALB / c shows differences or not in the weight loss of animals and in the replication of Mycobacterium tuberculosis H37Rv in lungs of vaccinated and then infected mice, during the chronic phase (26 weeks) of the disease, compared to the current vaccine without modification.

Regarding the ability to form biofilms, it was found that the BCGABCG1419c strain produces these in vitro with a more rugged appearance than those produced by the wild BCG strain

/

(Figure 15), an effect that is reversed once the Rvl357c gene is reintroduced into the strain lacking the BCG1419c gene (supplemented), confirming that it is a specific effect of the gene and not due to a polar effect mutation. Additionally, at the time of quantifying the biofilm formed by the wild and BCGABCG1419c strains, the second form was found on average between 3 to 15% more biofilm than the first (Figure 16). This confirms that the elimination of the BCG1419c gene results in a bacterium with increased ability to form biofilms in vitro.

Next, the protein profile was compared by double-dimension electrophoresis of the wild BCG and BCGABCG1419c strains, during in vitro biofilm growth, in order to determine if it was modified as a result of the BCG1419c gene elimination, and if this could in turn impact the presentation of antigens of a protein nature to animals immunized with both strains. As can be seen in Figure 17, after two-dimensional analysis and Coomasie blue staining, 9 proteins were found in the soluble fraction that differed significantly before the removal of the BCG1419c gene, which indicates that, in effect, there are differences in the production of potential antigens that modify the humoral and cellular immune responses to immunization. Additionally, after intravenously infecting BALB / c immunocompetent mice, we found that we recovered between 3-6 times more Colony Forming Units of strain BCGABCG1419c, depending on the time evaluated, and regardless of the sex of the model animal used (Figures 19- twenty-one).

Later, we proceeded to immunize with approximately 5 x 10 ³ Colony Forming Units (CFU) of the strain BCGABCG1419c (Delta) and approximately 15x10 ³ CFU of the wild BCG strain (WT), via caudal vein, groups of female and male mice, 9-10 weeks of age, immunocompetent BALB / c strain, or sterile saline (SS) as a control, to leave mice at rest for 8 weeks, at which time approximately 2xl0 ⁵ CFU of Mycobacterium tuberculosis H37Rv was administered intratracheally , and after 2 weeks post-infection and every 2 weeks, until week 26, check the weights and signs of disease of the animals, then sacrifice them at the time of 26 weeks and determine CFU in the lungs of the 3 groups of mice. Figure 22 shows the course of the average weight loss of the group, relative to week 2 post-infection, of the control group and immunized groups. It is appreciated that from the chronic phase of the infection (week 16) differences begin to be detected, which become significant in both vaccinated groups with respect to the control (SS), which remains until the time of 26 weeks, despite having used an approximately 3 times lower dose of the BCGABCG1419c vaccine compared to the unmodified wild BCG vaccine.

This example demonstrates that the recombinant strain BCGABCG1419c has properties of greater biofilm production than the current vaccine, differential protein production compared to the current vaccine, greater persistence in immunocompetent mice and protection against weight loss in immunized mice and then challenged with M. tuberculosis, comparable to the current BCG vaccine strain despite being administered in a lower dose.

, POSSIBILITIES OF USE / APPLICATIONS: 1. Specific vaccine against chronic or latent pulmonary tuberculosis.

2. Model for testing drugs against mycobacteria with increased ability to form biofilms and, consequently, tolerant to antibiotics in concentrations greater than cells in individual or planktonic forms.

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Claims

1. A strain of Mycobacterium bovís BCG characterized in that it has the accession number PTA-120572.

2. The strain of Mycobacterium bovis. BCG according to claim 1, characterized in that it comprises the elimination of nucleotide sequences encoding the BCG1419c protein, with phosphodiesterase activity of the second messenger di-GMPc, which results in the differential production of biofilm proteins in said mycobacterium with respect to the current BCG vaccine.

3. The Mycobacterium bovis BCG strain according to claim 1 and 2 characterized in that it forms 3 to 15% more biofilm in vitro than the strain without this modification.

4. The strain of Mycobacterium bovis BCG according to claim 1, characterized in that the decrease or loss of phosphodiesterase activity of di-GMPc is a product of the inactivation of the EAL catalytic site by change in the amino acid sequence of this domain.

5. The Mycobacterium bovis BCG strain according to claim 1, characterized in that the decrease or loss of di-GMPc phosphodiesterase activity is a product of the inactivation of the EAL catalytic site by change in the amino acid sequence in other domains different from the EAL but affecting the union and degradation of the second messenger.

6. The strain of Mycobacterium bovis BCG according to claim 1, characterized in that the decrease or loss of phosphodiesterase activity of di-GMPc is a product of the inactivation of the EAL catalytic site by change in the nucleotide sequence by insertion or deletion of nucleotides that alter the aforementioned EAL sequence.

7. The strain of Mycobacterium bovís BCG according to claim 1, characterized in that the decrease or loss of phosphodiesterase activity of di-GMPc is the product of changes in the region that regulates transcription or translation to eliminate both and not give Origin to no protein.

8. The strain of Mycobacterium bovis BCG according to claim 1, characterized in that the decrease or loss of phosphodiesterase activity of di-GMPc is a product of substitutions of the BCG1419c gene by other genes than the antibiotic resistance (hygromycin) used in the present invention, or even without no antibiotic or other marking that facilitates the identification of the change that leads to the inactivation or decrease of the phosphodiesterase activity of di-cGMP.

9. The strain of Mycobacteríum bovis BCG according to claim 1, characterized in that the decrease or loss of phosphodiesterase activity of di-GMPc is the product of the inactivation or decrease of enzymatic activity in a transient manner, contrary to the stable object of the present invention, if expression control sequences are placed to respond to environmental stimuli.

10. The strain of Mycobacteríum bovis BCG according to claim \, characterized in that the decrease or loss of phosphodiesterase activity of di-GMPc is the product of the activity of a subunit fraction (cell fraction, proteins, lipids, carbohydrates or combinations between them] or of DNA, if for example only the production of protein that partially or totally inhibits the phosphodiesterase activity of di-cGMP is sought.

11. The strain of Mycobacteríum bovis BCG according to claim 1, characterized in that it produces an infection that results in 3-6 times more in number of

Colony forming units in immunocompetent mice, being able to serve as a model for chronic mycobacterial infections.

12. The strain of Mycobacteríum bovis BCG according to claim 1, characterized in that it is used for the treatment of chronic or latent pulmonary tuberculosis.