WO2008020108A1

WO2008020108A1 - Peptides which are immunogenic in relation to the genuses listeria and mycobacterium, antibodies and uses of these

Info

Publication number: WO2008020108A1
Application number: PCT/ES2007/070144
Authority: WO
Inventors: Carmen ÁLVAREZ DOMÍNGUEZ; Eugenio CARRASCO MARÍN
Original assignee: Fundación Marqués De Valdecilla
Priority date: 2006-08-09
Filing date: 2007-07-31
Publication date: 2008-02-21
Also published as: ES2298055A1

Abstract

The present invention relates to the field of biotechnology, and more particularly to peptides which can immunise in relation to infections produced by bacteria belonging the genuses Listeria and Mycobacterium, as well as to those antibodies or fragments of them which are specific in relation to these peptides.

Description

IMMUNOGENIC PEPTIDES AGAINST LISTERIA AND MYCOBACTERIUM GENDERS, ANTIBODIES AND USES OF THE SAME.

The present invention relates to the field of biotechnology and more particularly, to peptides capable of immunizing against infections produced by bacteria belonging to the Listeria and Mycobacterium genera as well as those antibodies or fragments thereof specific against said peptides.

BACKGROUND OF THE INVENTION

At present there are bacterial species that, for different reasons, are becoming increasingly important within the medical and pharmaceutical field, and among which are those belonging to the genera Listería and Mycobacterium.

Both the genus Listeria and the genus Mycobacterium, as well as other groups of bacteria that have pathogenic species, have in common that, as a result of the evolution, many of the proteins or enzymes, which in principle were destined to the realization of metabolic activities, they have evolved to be able to interact with the host cells and, thus, favor their growth inside the cell. One of the metabolic enzymes whose activity has evolved into a virulence factor in certain pathogenic bacteria, such as Streptococus pyogenes, is Ia GAPDH (Gliceraldehyde-3-phosphate) (Int J. Med. Microbiol. 293, 391-401.) . This enzyme involved in the final part of glycolysis, catalyzing the oxidation of glyceraldehyde-3-phosphate to glycerol-1,3-diphosphate, has never been implicated in different enzymatic processes either in Listeria or in the Mycobacterium genus.

Although proteomic analyzes of bacterial proteins have described that Listeria GAPDH, more specifically of Listeria monocytogenes (GAPDH-LM), is a protein associated with the membrane of the bacterium (Proteomics 2004, 4: 2991-06) and capable of If it is secreted, it is unknown if it has another enzymatic function other than its implication in glycolysis or as a plasminogen binding protein (Proteomics 2005, 5: 1544-57). The GAPDH of other gram-positive bacteria, pathogenic but not intracellular but extracellular, have, in addition to their glycolytic activity, other enzymatic activities that favor their activity as virulence factors. However, no activity other than the oxidation of glyceraldehyde-3-phosphate has been described for the GAPDH of the genera Listería and Mycobacteríum, both intracellular pathogens.

One of the enzymatic activities that sometimes show virulence factors is ADP-ribosylation. This activity involves a covalent modification limited only to a few bacterial toxins secreted by non-intracellular, gram-negative or gram-positive pathogenic bacteria, such as diphtheria toxin, Pseudomonas aeruginosa ExoS protein, pertussis toxin and cholera toxin (Infeci Immun. 2001, 69: 5329-34; PNAS USA. 2004, 101: 6182-87). In general, ADP-ribosylation inactivates cellular target proteins such as intracellular signaling proteins or regulators of intracellular traffic.

Virulence factors are commonly used to develop vaccines directed against those pathogens that carry them, through the complete inoculation of said factor or with fragments thereof in individuals. This strategy, which a priori should be simple, once a virulence factor has been detected, is not so much because (i) the inoculation of complete virulence factors is very toxic, (ii) its generation by recombinant DNA techniques gives rise to a conformational structure different from that which the factor has in its natural state and, therefore, the humoral and / or cellular response is not efficient and (iii) the post-translational modifications that the virulence factor undergoes in its natural state cause that the inoculation with the recombinant protein or peptides of this one produces an immune response incapable of acting efficiently against the pathogen.

These circumstances make it necessary to detect certain regions of the virulence factors capable of giving rise to an adequate immune response and allow efficient immunization against the pathogen. So far, no GAPDH of the genera Listería and Mycobacteríum capable of producing a good immune response has been detected. It is also not known if these proteins, in addition to their enzymatic activity carried out in glycolysis, catalyze reactions related to the virulence of the pathogen, if they can interfere with the function of certain proteins involved in the processes of endosome-endosome fusion and traffic regulation of these cellular elements or if they are capable in general of giving rise to a good immune response. Confirmation of these characteristics would make it possible for these proteins to be used with therapeutic targets or for vaccine development.

DESCRIPTION OF THE INVENTION

To carry out the present invention, the intracellular interference of the human pathogen and gram-positive bacteria was analyzed: Listería monocytogenes (LM), and it was observed that the intracellular target of this pathogen was the phagosomal traffic regulatory factor, GTPase Rabδa, decreasing its activation, that is, the exchange between the inactive form or GDP and the active form or GTP, thereby blocking its function. Surprisingly, it was observed that the bacterial protein of Rabδa binding and inhibition was Ia GAPDH-LM (LM glyceraldehyde-3-phosphate dehydrogenase), which, through its enzymatic action of ADP-ribosylation, decreased the interaction of Rabδa with its factor of activation, or GDP / GTP exchanger, the region of the GAPDH-LM being responsible for said inhibition the N-terminal region of the protein, in particular the first 22 amino acids (22-mer). Throughout the description, this amino acid end of 22 amino acids of GAPDH-LM will also be referred to as SEQ ID No. 1.

From this structural and functional analysis, a detailed search was carried out in databases of the domain corresponding to the 22-mer N-terminal peptide, and it was found that it had an 86% homology with the amino terminal end of 22 amino acids of the GAPDH of Mycobacteríum tuberculosis, TB, (GAPDH-TB) (Fig. 1a, underlined amino terminal end); while the C-terminal domain sparsely It had a homology of 59% and an identity of 27% (Fig. 1a, carboxyl end terminal underlined). When the homologous domain of the GAPDH-TB was analyzed in more detail, an almost exact amino acid sequence was observed in the first 15-amino acids (15-mer), raising the homology percentage up to 99% and differing only in one amino acid with the same load (K in LM and R in TB. See Figure 1A). As the binding of peptides to MHC-I or MHC-II molecules has a molecular restriction of non-reamers and dodecamers, respectively; it is very likely that in those 15-mer there could be both MHC-I and MHC-II epitopes that could thus be shared by both pathogens. Throughout the description, the amino terminus of 15 amino acids of GAPDH-LM and GAPDH-TB may be referred to respectively as SEQ ID N ⁰ 2 and SEQ ID N ⁰ 3.

Continuing with this structural study, the different short N-terminals (15-mer) of the GAPDH of different species within the Mycobacterium genus, both human pathogens and pathogens of important animals in the livestock / food industry and different controls of other bacteria were analyzed. gram-positive as gram-negative, as well as the sequence of human and murine GAPDH. This study, shown in Figure 2, highlights that only the N-terminal of the Listeria GAPDH and of the Mycobacterium genus have the highest possible homology of 98% (bold amino acids in Fig. 2); while this homology decreases to 60% or 40% when studying the negative controls corresponding to human or mouse GAPDH, respectively.

Within the genus Mycobacterium, that is, tuberculosis, avium, leprae (Ml) and bovis, if the structural study is extended to the first 30 amino acids, it can be seen that they are 100% homologous for M. tuberculosis, M. avium and M. Bovis

When the study extends well to other gram-positives whose GAPDH has been described as having ADP-ribosylation activity, such as Streptococcus pyogenes or Staphylococcus aureus (Pancholi, V. And

Fishetti, VA (1993) Proc. Nati Acad. Sci. USA 90: 8154-8158. Molinari, G., Rohde, M., Just, L, Aktorias, K. and Chatwal, GS (2006). I infected. Immun 74: 3673-36677), the homology of the 15-mer peptide is reduced to 75-80%, but when it is analyzed that of other gram-negatives such as Pseudomonas aeruginosa, which has a specific toxin with ADP-ribosylation activity called ExoS (Bette-Bobillo, P., Giro, D., Sainte-Marie, J. and Vidal, M. (1998). Biochem. Biophys. Common Res. 244: 3366-341), the N-terminal region of Ia GAPDH, specifically the 15-mer peptide, was barely 60% homologous to that of the GAPDH-LM (see Fig. 2).

This suggested that the GAPDH of Pseudomonas would not have ADP-ribosylation capacity on Rabδa and, therefore, the functional comparison of different protein extracts and their capacity of ADP-ribosilar Rabδa was analyzed. As shown in Figure 3, both the LM extract, as a murine cell extract of macrophages infected with LM (run E1, Fig. 3), as the TB extract are capable of ADP-ribosilar Rabδa, and to a lesser extent the Ml extract, whose ADP-ribosylation capacity on Rabδa was similar to that of the 22-mer N-terminal peptide of the GAPDH-LM (run - for extract and + for peptide in Fig. 3). Meanwhile, extracts of Escherichia coli (Ec race, Fig. 3), a murine cell extract of macrophages not infected but containing murine GAPDH (run E, Fig. 3) or a murine cell extract of macrophages infected with heat-killed LM ( HKLM) prior to infection (run E2 in Fig. 3) were unable to ADP-ribosilar Rabδa.

All these high similar structural data between the genus Listeria and the genus Mycobacteríum indicate not only that the possible interference with Rabδa is common to both genders, but surprisingly this similarity also extends to their immune response, as shown in the examples that are part of the memory of the present invention.

Therefore, the inventors of the present invention have discovered that GAPDH-LM and GAPDH-TB are capable of performing two relevant actions not known to date. The first action resides in its ADP-ribosylation capacity where, in addition, said capacity is used to inactivate the phagosomal traffic regulatory cellular protein, Rabδa. As we show in the present invention, said inactivation of Rabδa by Ia GAPDH, is mediated by the N-terminal region of the protein, specifically by the peptide that contains the first 22 amino acids.

On the other hand, the second interesting action of GAPDH-LM and GAPDH-TB can be inferred from its location as a protein associated with the outer membrane of the bacterial wall of both pathogens. Where the N-terminal peptide would be exposed which would cause it to be easily extracted or that could be accessible for recognition by antibodies. Thus, and since LM does not induce a humoral response, this protein could be the only one capable of inducing an adequate humoral and cellular immune response. In fact, the immunization with SEQ ID N ⁰ 1 -added but without adjuvant, that is, only with the adjuvant excipient, mineral oil or IFA, is capable of generating a good immune response in rabbits generating antibodies, which is synonymous of a good humoral and cellular response.

In addition, the direct analysis of the ability of the peptide of SEQ ID N ⁰ 1 to induce a good T-cell response was investigated after immunization of mice in the plantar bearings and analysis of the capacity of specific response to said peptide by the T cells, isolated from the popliteal nodes of said mice. This study indicated that only 0.1 μg / ml of the peptide was necessary to induce a good immune response by said T cells at least 10 times higher compared to the control without peptide.

The versatility of the peptide in inducing a good immune response is also reflected by the antibody's own versatility to recognize several pathogens, which indicated that it could be a good candidate to test as a vaccine.

In short, GAPDH-LM constitutes an interesting antigen both in the field of bacterial pathogenesis as it is the same virulence factor shared by two intracellular bacteria; but it is also a peculiar and promising antigen in the area of immunology, since the same antigen from two different bacteria would share amino acid regions large enough (15-mer) to contain epitopes recognized by B cells, epitopes recognized by CD4 + T cells (MHC-II) and by CD8 + T cells (MHC-I); Which can be inferred, initially, since the rabbit antibody generated anti-SEQ ID N ⁰ 1, obtained by exclusive immunization with the peptide without adjuvant, is capable of recognizing the protein in a complex structure such as the entire bacterium, as in protein extracts of both genera of bacteria, Listeria and Mycobacterium, where said peptide is exposed (see examples and Figure 6). The same conclusion can be drawn from the fact that the T response generated by the peptide is very good (Table 1), that CD4 / CD8 T cells have the ability to transfer immunity against two different pathogens. (Table 2) and its ability to be used as a vaccine.

Therefore, a first aspect of the present invention refers to an isolated immunogenic polypeptide (hereinafter polypeptide of the invention) against any of the Listeria and Mycobacterium genera selected from the group comprising:

a) Polypeptide whose sequence comprises SEQ ID N ° 1. b) Polypeptide whose sequence comprises fragments of at least 8 consecutive amino acids of SEQ ID N ° 1 that maintain the immunogenic capacity against the genera Listería and Mycobacterium c) Polypeptide whose sequence has at least 85% homology with any of the polypeptides of a) and b). In a preferred embodiment homology is at least 90% and more preferably at least 95%.

In a preferred embodiment, the polypeptide of the invention has the sequence SEQ ID N ⁰ 1.

In a preferred embodiment of this aspect of the invention, the polypeptide of the invention is chosen from the group:

a.- Polypeptide whose sequence comprises the SEQ ID N ⁰ 2. b.- Polypeptide whose sequence consists of the SEQ ID N ⁰ 2. In a preferred embodiment of the present invention, the polypeptide is chosen from the group:

a.- Polypeptide whose sequence comprises SEQ ID N ⁰ 3. b.- Polypeptide whose sequence consists of SEQ ID N ⁰ 3.

In an even more preferred embodiment, the polypeptide of the invention is a derived polypeptide that has undergone post-translational modifications or made by methods well known in the state of the art.

A second aspect of the invention relates to an isolated polynucleotide (hereinafter polynucleotide of the invention) capable of coding for any of the polypeptides of the invention.

A third aspect of the invention refers to an isolated antibody or fragment thereof (hereinafter antibody of the invention) capable of recognizing any of the polypeptides or polypeptides derived from the invention.

A fourth aspect of the invention refers to a polypeptide of the invention fused or chemically bound to an additional peptide, polypeptide or protein.

A fifth aspect of the invention refers to an expression vector or expression system (hereinafter vector or expression system of the invention) comprising any of the polynucleotides of the invention.

A sixth aspect of the invention refers to an isolated host cell, prokaryotic or eukaryotic, (hereinafter host cell of the invention), transformed, or transfected with any of the polynucleotides of the invention or with any of the vectors or expression systems of the invention. A seventh aspect of the invention relates to a method for preparing the polypeptide of the invention (hereinafter method of the invention), by recombinant DNA techniques or chemical synthesis.

In a preferred embodiment, the method of the invention comprises culturing a host cell of the invention under conditions such that any of the polynucleotides of the invention is expressed, and isolating said expressed polypeptide.

An eighth aspect of the invention relates to a polypeptide of the invention for use as a medicine.

A ninth aspect of the invention relates to a vaccine comprising any of the polypeptides of the invention.

A preferred embodiment of the present invention comprises the use of a polypeptide of the invention for the elaboration of a vaccine capable of immunizing against an infection produced by bacteria belonging to the genus Listeria and / or Mycobacterium, more particularly Listeria monocytogenes and / or Mycobacterium tuberculosis .

An even more preferred embodiment of the present invention comprises the use of an antibody of the invention, for the preparation of a pharmaceutical composition for the treatment of an infection produced by bacteria belonging to the genus Listeria and / or Mycobacterium, more particularly Listeria monocytogenes and / or Mycobacterium tuberculosis.

A tenth aspect of the present invention relates to molecules modulating the activity of GAPDH of the genera Listeria and / or Mycobacterium, activators or inhibitors, specifically designed to interact with any of the peptides of the invention.

A eleventh aspect of the present invention relates to molecules specifically designed from the ADP-ribosylation domain of the GAPDH of the Listeria and / or Mycobacterium genera and capable of mimicking the activity of said domain. A twelfth aspect of the invention refers to the use of the ADP-ribosylation domain of the GAPDH of the genera Listeria and / or Mycobacteríum for the rational design of drugs.

A thirteenth aspect of the invention refers to the use of any of the polypeptides, polypeptides derived from the invention or fragments thereof of the invention for its ability to ADP-ribosylation.

Next, the definition of a series of terms that help to correctly interpret the present invention is facilitated.

The term "polypeptide or peptide" is used to designate a linear sequence of amino acids connected to each other by amide bonds between the alpha-amino group of an amino acid and an alpha-carboxy group of the adjacent amino acid. From said polypeptides, smaller fragments of at least 8 amino acids can be derived, preferably at least 15 amino acids and preferably at least 22. Said polypeptides and fragments include any post-translational modification or carried out by methods well known in the state of the art. . These modified polypeptides and fragments will be referred to as "derived polypeptides or peptides."

The term "polynucleotide" is used to designate a linear sequence of nucleotides or ribonucleotides. This term refers exclusively to the primary structure of the molecule including single and double stranded DNA, as well as single and double stranded RNA. Similarly, the modified forms of these molecules are also included, for example, by methylation.

The term "epitope" refers to an antigenic determinant of a polypeptide; An epitope may comprise 3 amino acids in a spatial conformation that is exclusive to the epitope, generally consisting of at least 5 amino acids, and more usually consisting of at least 8-10 amino acids. The methods for determining the spatial conformation of amino acids are known in the state of the art, and include, for example, X-ray crystallography and magnetic resonance imaging nuclear in 2 dimensions.

An "immunogenic polypeptide or peptide" refers to a polypeptide capable of generating specific antibodies, as well as promoting both a humoral and cellular response.

The term "vaccine" refers to antigenic preparations or cells previously treated with said antigenic preparations and capable of promoting a humoral and / or cellular response or expression vectors or other forms of vaccine vectors. This response will lead to the generation of an immunological memory producing, in most cases, permanent or transient immunity against a specific pathogen.

The term "antibody" refers to both monoclonal and polyclonal antibodies capable of interacting with the polypeptides defined in the present invention.

The term "antibody fragment" refers to all those antibody fragments capable of interacting with the peptides of the invention and fragments thereof, where said fragments comprise the variable regions Vh and / or Vl, as well as their conserved regions.

The term "domain" of a protein refers to the three-dimensional structure that acquires a certain region or polypeptides of said protein and that can carry out purely structural or enzymatic functions. More specifically, this term will refer to the three-dimensional structure adopted by the polypeptides, derived polypeptides or fragments thereof of the invention, corresponding to the 22 amino acid end of the amino terminal region of the GAPDH of the Listeria and Mycobacterium genera.

Unless defined otherwise, all the technical and scientific terms used in this document have the same meaning as usually understood by a specialist in the technique to which This invention belongs. In the practice of the present invention methods and materials similar or equivalent to those described herein can be used. Throughout the entire description and the claims, the word "comprises" and its variations are not intended to exclude other technical characteristics, additives, components or stages. Other objects, advantages and characteristics of the invention will be apparent to those skilled in the art after examining the description or can be learned by the practice of the invention. The following examples and figures are provided by way of illustration and are not intended to limit the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Figure 1a shows the amioacidic sequence of GAPDH-TB and GAPDH-LM. Top: amino terminal end of 22 amino acids of GAPDH-TB (underlined) 99% homologous in its first 15 amino acids with the amino terminal end of 15 amino acids of GADPH-LM. Bottom: amino and carboxyl ends (underlined) of GAPDH-LM. Figure 1b shows the 3D structure of the GAPDH-LM. The substrate binding domain refers to the domain with ADP-ribosilant activity and the NAD binding domain corresponds to the domain that catalyzes the oxidation of glyceraldehyde-3-phosphate to glycerol-1,3-diphosphate in glycolysis.

Figure 2.- Structural comparison between the N-terminals, 15-mer, of different GAPDH. In bold, the 15-mer peptide of the N-terminal end of those bacteria in which it has more than 90% homology stands out; underlined, those bacteria of the genus Mycobacterium whose extended N-terminal (30-mer) is 100% homologous and in lowercase, the only different amino acid between Listeria and Mycobacterium.

Figure 3.- Capacity of ADP-ribosylation on Rabδa of the different bacterial and cellular extracts. ADP-ribosylation of the GST-Rab5a fusion protein: wt in the presence of 30 μg of the different extracts: TB, Mycobacterium tuberculosis extract: Ml, Mycobacterium leprae extract: Ec, Escheríchia coli extract; LM, Listería monocytogenes extract, E1; extract of mouse macrophage-like cells infected with LM; E2, extract of mouse macrophage-like cells infected with heat-killed LM; E, extract of uninfected mouse macrophage cells.

Figure 4.- A. Proteins recognized by the anti-SEQ ID N ⁰ 1 antibody. PNS (LM), post-nuclear fraction of cells infected with LM; Phages, purified phagosomes of the PNS fraction (LM); PNS (Nl), post-nuclear fraction of cells not infected with LM; LM-lysate, LM lysate containing wall proteins; LM-supernatant, LM supernatant containing secreted proteins; Nt-E, crude extract of LM; R5t-eluted or R5t-E, eluted from affinity column-Rabδa incubated with crude LM extract. BE. E-clone cells infected with live LM for 15 min (B) or 60 min (CD) and incubated with anti-SEQ ID N ⁰ 1 antibodies (B, C and E) or with preimmune serum (D) from the same animal to from which the antibodies against SEQ ID N ⁰ 1 are obtained.

Figure 5.- Proteins recognized in different mutants of LM by the anti-SEQ antibody N ⁰ 1 of the N-terminal of the GAPDH-LM. Different used of different Lm mutants: (i) mutant lacking listeriolysin O-LM- / lysate and LM- / sup- (runs 3 and 4); (ii) mutant lacking phospholipase C-pIcA / B-lysate or supernadanis of those pIcA / B-sup - (races 5 and 6); (ii) wild phenoipo - LM + / lysate and LM + / sup (runs 1 and 2).

Figure 6.- Proieins recognized by the ani-22mer antibody of the GAPDH-LM or anti-SEQ ID N ⁰ 1 from 30 μg of different bacterial and cellular extracts: Lm, Listeria monocytogenes; TB, M. tuberculosis; Ml, M. leprae and cel, cell extract.

Figure 7: Panel A. Naked phagosomes (30μg per run) incubated with 3 μg of GST-Rab5a: wt pre-treated and pretreated with different concentrations of SEQ ID N ⁰ 1 (N1 and N2) and SEQ ID N ⁰ 4 (C1 and C2 ) (N1 and N2 correspond respectively with 100 and 10 ng / ml of the SEQ ID N ⁰ 1. C1 and C2 correspond respectively with 100 and 10 ng / ml of the SEQ ID N ⁰ 4) and the presence (runs +) or absence (runs -) of 5 μg of LM extracts (Nt-E). Panel B. 5 μg of the recombinant GST-Rabδa protein: wt was ADP-ribosylated using 50 μM of NAD-biotinylated as a substrate in the presence or absence of Nt-E and in the presence of N1, N2 and C1. Panel C. Naked phagosomes incubated with GST-Rab5a: wt pre-piled and ADP-ribosylated (ADP-r + runs), as was the case in panel B, using NAD instead of NAD-biotinylated and N1 as a source of ADP-ribosylation proteins in the presence or absence of LM extracts (runs Nt-E + and Nt-E -). Panel D. Naked phagosomes incubated with GST-Rab5a: wt pre-stacked and ADP-ribosylated (ADP-r + runs) or not (ADP-r runs) in the presence of His-EEA1 or His-Vps9.

EXAMPLES OF REALIZATION

EXAMPLE 1: Immune study of the GAPDH of the genera Listeria and Mycobacteríum.

To obtain antibodies, a concentration of the peptide of SEQ ID N ⁰ 1 (22-mer of the GAPDH-LM) of 10 mM was used, which is oxidized after resuspension in water and vacuum drying as insoluble material. This treatment produced molecular aggregates of the peptide that turned out to be highly immunogenic as it did not require its emulsion in Freund's complete adjuvant (CFA) to obtain a good immune response, but only with incomplete adjuvant containing the excipient, or mineral oil (IFA) It was enough to generate an immune response. The insoluble aggregate was injected into rabbits in the ears or in the peritoneum of mice. After 14 days, a first booster dose was inoculated with 10 mM of the peptide perfectly dissolved in saline buffer, and 7 days later with another booster dose equal to the previous one. Finally, the animal ad libitum was bled, the serum was collected with anti-SEQ ID N ⁰ 1 antibodies that were tested on the ELISA plate peptide.

With these antibodies the different bacterial extracts of

LM: (i) used bacterial LM containing the wall proteins, therefore, structural proteins; (ii) growth supernatants of

LM containing the proteins secreted by LM (Figures 4 and 5); (iii) crude extracts of LM, which contains most of the proteins of both the wall and the bacterial cytosol (Nt-E strokes in Fig. 4 and LM in Fig. 6); (iv) as well as an eluate from an affinity column with Rabδa, which contained all the LM proteins that bind to Rabδa (run R5t-eluted in Fig. 4).

On the other hand, with said antibodies different murine cell fractions of macrophages infected or not with the pathogen, different bacterial fractions of LM, both structural and used of bacteria, or total bacterial extracts or secreted to the medium as supernatants of their growth were analyzed, as well as extracts of the genus Mycobacteríum and murine cell extracts (Figures 4-6).

All this analysis has reflected the following results performed with the anti-SEQ ID N ⁰ 1 antibody of the N-terminal of the GAPDH-LM:

one . The antibody recognizes both the GAPDH-LM of the structural wall, and the protein secreted to the pathogenic LM medium (LM-lysate and LM-supernatant strokes in Fig. 4 and LM + / lysate and LM + / sup strokes in Fig. 5) , as of different mutants of LM in phagosomal virulence factors: the listeriolysin O, LLO, (LM- / lysate and LM- / sup in Fig. 5) or phospholipase C (plcA / B- / lysate and plcA strokes / B- / sup in Fig. 5) as of a total crude extract of LM (Nt-E strokes in Fig. 4 and LM in Fig. 6), as well as eluted from the affinity columns with Rabδa incubated with said extract LM crude (run R5t-eluted or R5t-E in Fig. 4).

2. The antibody also recognizes the GAPDH-LM of cells infected with LM (PNS LM race in Fig. 4), as well as phagosomes containing the live bacteria (LM phage race in Fig. 4).

3. The antibody also recognizes the GAPDH of the genus Mycobacteríum, both tuberculosis and leprosy of crude extracts of said pathogens with which it shares more than 86% structural homology (runs TB and Ml in Fig. 6). 4. The antibody does not however recognize the murine GAPDH present in the post-nuclear macrophage cells not infected with LM (PNS race (Nl) in Fig. 4), nor in a total cell extract thereof (cel race in Fig. 6), with which it barely shares a 40% structural homology.

5. In addition, the antibody is capable of recognizing the entire bacterial structure of LM, once it has infected E-clone cells (Álvarez-

Dominguez, C, Barbieri, AM, Beron, W., Wandinger-Ness, A. and Stahl, PD (1996). J. BhI. Chem. 271: 13834-13843) for a period of between 15 and 60 minutes (Figure 4b and 4c). However, the preimmune serum, that is, before the infection of the animal with SEQ ID N ⁰ 1, it can be seen that it is not specific for LM (Figure 4d).

6. The antibody does not recognize any band in PNS fractions of cells not infected with LM (PNS race (Nl), Figure 4) or E clone infected with HKLM (Heat killed LM) (PNS race (HKLM), Figure 4), that is, LM killed with heat by incubation for 60 minutes at 7O ⁰ C.

Thus and since the structural and immune studies are equivalent, it can be inferred that the antibodies generated against SEQ ID N ⁰ 1 are structural antibodies that recognize similar sequences with more than 85% sequence homology in highly related bacteria and, by So much, and more specifically of Mycobacterium tuberculosis (SEQ ID N ⁰ 3). This suggests that the same antigen, GAPDH-LM, would contain both B-cell and T-cell epitopes.

Example 2. Analysis of the response T generated by SEQ ID N ⁰ 1

Mice were immunized with SEQ ID N ⁰ 1 in the plantar bearings and after 7 days of immunization the popliteal ganglia were isolated, where mainly T and dendritic cells specific for said antigen are collected. With the cells isolated from these ganglia their ability to generate a T response against SEQ ID N ⁰ 1 was analyzed with a dose-response kinetics (Table 1).

Table 1.- T response capacity generated by the 22-mer N-terminal peptide of the GAPDH-LM. After homogenization of the popliteal nodes, the cells are cultured in the presence of different concentrations of the peptide for 24 hours, after which the last 8 hours 3H-thymidine (tritiated thymidine) is added which will be incorporated into the DNA of the proliferating cells.

Given the good cellular response generated by the peptide of SEQ ID N ⁰ 1 of the GAPDH-LM, it was analyzed whether it was able to confer protection against an infection by Listeria monocytogenes. To this end, a vaccination protocol was designed with dendritic cells that had been pretreated with the peptide in vitro (10 μg / ml dose). In this way, the dendritic cells will process the peptide, and the appropriate epitope will bind both MHC-I and MHC-II molecules, as appropriate, that after washing the excess peptide they will be used as a specific vaccine vector for said peptide and inoculated into mice of the same congenital strain (in our case, CBA / J).

By intraperitoneally injecting 2 x 10 ⁶ specific dendritic peptide cells per mouse, after 7 days another similar booster dose is inoculated with the same number of specific dendritic peptide cells. As a control, the same number of dendritic cells is inoculated in another group of animals but not treated with said peptide, then not specific for it. After 14 days in total, said mice are inoculated with a sub-lethal dose of Listeria monocytogenes (in the case of CBA / J 5 x 10 ⁴ bacteria / mouse are inoculated). After 3 days post-infection, the mice are bled to obtain serum and are sacrificed for clinical analysis and recovery of spleens and livers where a viable count, CFU, will be performed, which will determine the number of LM recovered in each case. will indicate Ia efficiency of said vaccination protocol to confer protection against the pathogen. The data obtained from this study are collected in Table 2.

Table 2.- Protection of specific dendritic-peptide cells against infection with sub-lethal dose of LM. After the vaccination protocol, blood is extracted, as well as LM-specific immune organs: spleen and liver, to perform the CFU count and analyze the general physical condition of the immunized animals.

As observed in this vaccination protocol, the specific dendritic cells for the peptide were able to confer a very good protection, greater than 80% in the liver and 50% in the spleen, against a sub-lethal dose of the pathogen.

Example 3. Inhibitory effect of GAPDH-LM on Rabδa.

First, Rabδa recruitment of phagosomes was analyzed including different concentrations of the peptides designed from the amino and carboxyl ends of GAPDH-LM (Figure 1a). As shown in Figure 7A, the preincubation of the GST-Rabδa fusion protein with 100 ng / ml of SEQ ID N ⁰ 1 (N1) and SEQ ID N ⁰ 4 (C1) and 10 ng / ml SEQ ID N ⁰ 1 (N2) and SEQ ID N ⁰ 4 (C2) revealed that N1 or N2 was able to compete with the Nt-E extract (LM crude extract) by decreasing approximately 10 times the effect of Rabδa binding on naked phagosomes of HKLM This indicates that the amino terminal end of GAPDH-LM contains the Rabδa binding domain while the carboxyl terminal end did not participate in this interaction. Understanding that ADP-ribosylation could be involved in the Rabδa inhibition process, it was investigated whether the Nt-E extract showed ADP-ribosylating activity in the presence or absence of different concentrations of the amino and carboxyl end peptides of GAPDH-LM (Fig. 1a), following the protocol developed by Zhang (Zhang J. (1997). Methods Enzymol. 280: 255-65) that uses NAD-biotin as a substrate and the recombinant GST-Rab5a fusion protein: wt .

Thus, the recombinant protein (GST-Rab5a: wt) where said action is to be tested, was incubated with N1 in the following buffer: 25 mM Tris-CIH, pH 7.6, 15 Mm ATP, 100 mM MgCI2, 10 mM NAD, 2.5 mM ADP-ribose and 50 μM of NAD-biotin, in the presence or absence of 5 μg of cytosolic proteins (cell cytosol extract from interferon-gamma activated macrophages as a source of enzymatic co-factors), for 30 minutes at 37 ⁰ C and the problem protein was recovered by means of the fusogenic part attached to affinity columns and / or diluting it 10X on ice with PBS to stop the reaction. The ADP-ribosylation reaction is well observed by its apparent increase in molecular weight when running a sample of the ADP-ribosylated problem protein and comparing it with the untreated problem protein in 10% SDS-PAGE gels, or after transfer of said proteins separated in the SDS-PAGE gels to nitrocellulose membranes where western blots will be revealed with peroxidase-conjugated streptoavidin, which binds to the biotinylated NAD incorporated into the ADP-ribosylated protein as a product of the enzymatic reaction.

As shown in Figure 7B, the GST-Rab5a: wt was ADP-ribosylated in the presence of an enzymatic source: Nt-E, N1 or a cell lysate obtained from E-clone cells infected with live LM (E1 and E2). Meanwhile, ADP-ribosylation was not observed in the absence of an enzymatic source, high concentrations of SEQ ID N ⁰ 4 (C1) and a lysate of E-clone cells infected with HKLM and not infected. The single addition of N1 showed a low but significant ADP-ribosylation signal. The highest ADP-ribosylation signal was detected in the Nt-E extracts after the addition of any of the concentrations of SEQ ID N ⁰ 1 (N1 and N2). Once the ADP-ribosylation capacity of SEQ ID N ⁰ 1 was verified, it was checked whether the ribosylated Rabδa was able to bind naked phagosomes of HKLM.

As shown in Figure 7C, ribosylated Rabδa (Rab5a-ADP) using N1 is capable of more efficiently binding to the naked phagosomes of HKLM than non-ribosilar Rabδa. This suggests that ADP-ribosylation is responsible for the retention of Rabδa in phagosomes.

To verify how ADP-ribosylation was also capable of affecting the activation of Rabδa, the effect of this modification on the interactions with two different effectors involved in the activation of Rabδa was examined: the Vps9 factor and the GTP binding protein ( EEA1).

Using the His-Vps9 and His-EEA1 recombinant proteins, a binding assay was proposed in the presence of the GST-Rab5a: wt ribosylated and ribosylated recombinant protein. Next, a HKLM naked phagosome recruitment trial was performed followed by immunoprecipitation of His-Vps9 and His-EEA1 proteins. The result of this test is shown in Figure 7D, where it can be seen that ADP-ribosylation does not affect the His-EEA1 / Rab5a junction, although it significantly reduces the interaction with His-Vps9 / Rab5a. This implies a lower interaction with effectors of the endosome-endosome fusion and, consequently, a possible decrease in this activity.

From these tests it can be deduced that the SEQ ID N ⁰ 1 peptide of the GAPDH-LM is responsible for: (i) recruitment of Rabδa to phagosomes while SEQ ID N ⁰ 4 is not involved, (ii) of its ADP-ribosylation in the presence or not of the Listeria extract, (iii) its retention in these phagosomal fractions, (iv) of the blockage in the union with its His-Vps9 activator that exchanges the inactive GDP form, for the active, GTP form, and in consequence of responsible for its inactivation.

Since the activation of Rabδa is necessary for endosome-endosome fusion, an assay was carried out in which it was possible to verify how the presence of HKLM's phagosomes affected the presence of GST-Rab5a: wt pretreated or not with Nt-E, R5t-E, N1 and C1 (see example 4). As shown in Table 3 GST-Rab5a: wt pretreated with Nt-E or N1 blocks the fusion reaction, while pretreatment R5t-E or C2 does not affect this process at all. In this way, it is demonstrated that ADP-ribosylation carried out by the amino end of GAPDH-LM affects the activation of Rabδa by inactivating the endosome-endosome fusion and favoring the survival of LM inside the cell.

Table 3. Fusion of phagosomes containing HKLM and endosomes containing no Rab and Rabδa dependent.

The ss-NHS-biotin / phagosome and endosome complexes of HKLM with streptoavidin-HRP were treated with GDI (from English, guanidine-dissociation-inhibitor) to remove the different types of Rab proteins, in their molecular form GDP, which were found in the membranes of phagosomes and endosomes and thus get these naked. Next, these sub-cellular fractions were resuspended in fusion buffer supplemented with cytosolic proteins (0.5 mg / ml) and with 5 μg of GST-Rabδa: wt pre-treated and pretreated or not with different reagents.

The fusion reaction was performed at 37 ⁰ C for 45 min and being stopped by immersion in homogenization buffer (HBE) and ice and washed twice before lysis of subcellular fractions with a low composition detergent lysis buffer. The intact bacteria that with this low concentration detergent buffer are not lysed because they are gram-positive bacteria, were recovered by centrifugation.

One of the fundamental functions of Rabδa is to be the regulator of interactions, that is, fusions between phagosomes and endosomes. A covalent modification such as ADP-ribosylation could affect this function. To establish a fusion system between phagosomes and endosomes that only depends on Rabδa, all possible Rabs are eliminated from them, by treatment with GDI and subsequent incubation with Rabδa pre-treated or not with different reagents that may or may not ADP-ribosylate it, as the Listeria extract, Listeria extract lacking Rabδa binding proteins, then lacking GAPDH-LM, the N1-terminal peptide N1 of the GAPDH-LM or the C-terminal C1 peptide of the GAPDH-LM. Those treatments for which it has been proven that they are capable of ADP-ribosilar to Rabδa, such as with the Listeria extract containing the GAPDH-LM or the N1 peptide of the GAPDH-LM block this fusion reaction between phagosomal and endosomal fractions ; Therefore, it can be concluded that Rabδa ADP-ribosylation also affects the interactions between phagosomes and endosomes dependent on said GTPase.

Example 4. Phagosome-endosome fusion assay.

Phagosomes or phagosomal fractions were obtained from E-clone cells infected with ss-NHS-biotin / LM for 15 minutes. Endosomal fractions were obtained after 10 minutes of incubation with streptoavidin-HRP (Álvarez-Domínguez, C, Peña-Macarro, C and Prada-Delgado, A. (2003). In Intracellular pathogens in membrane interactions and vacuole biogenesis. Ed. JP ).

Nude phagosomal and endosomal fractions, that is, free of Rab proteins (Yang C, Slepnev Vl, Goud B. (1994). J Biol Chem. 269 (50): 31891-9. Rubino M, Miaczynska, M, Lippe, R and Zerial, M. (2000). J. Biol. Chem. 275: 3745-3748) were contacted with a fusion buffer (250 mM sucrose, 0.5 mM EGTA, 20 mM HEPES-KOH, pH 7.2, 1 mM dithiothreitol or DTT, 1.5 mM MgCI2, 100 mM KCI, 1 mM ATP, 8 mM creatine phosphate, 31 units / ml creatine phosphokinase, and 0.25 mg / ml avidin as a blocker) supplemented with gel-filtered cytosol (0.5 mg / ml) and 5 μg / ml GST-Rab5a: recombinant wt pre-stacked and ADP-ribosylated or not. ADP-ribosylation was carried out with GST-Rab5a: wt pre-incubated and pre-incubated with 10 μg / ml of: Nt-E, R5t-E, N1 or C1 for 30 min at 37 ⁰ C to allow the binding of said recombinant protein to phagosomal and endosomal fractions, which will favor the union between the two that is called fusion. The fusion reaction was incubated for 45 min at 37 ⁰ C and the reaction was stopped by immersion in ice. The streptoavidin-HRP / biotin-bacterium complexes were recovered by centrifugation (10,000 xg, 6 min, at 4 ⁰ C) after solubilization of phagosomal and endosomal membranes with lysis buffer solution with low detergent concentration (PBS, 0.05 % Triton X-100 containing 0.25 mg / ml avidin as a blocker.

Finally, the fusion was quantified by arbitrary absorbance units per protein amount (absorbance units / mg) as described in Álvarez-Domínguez, C, Barbieri, AM, Beron, W., Wandinger-Ness, A. and Stahl, PD (1996). J. Biol. Chem. 271: 13834-13843.

Example 5. Search for compounds with modulating activity

A large number of molecules that affect the GAPDH activity of the Listeria and Mycobacterium genera for PSAPS can be created randomly by combinatorial chemistry, or specifically designed to subsequently be analyzed by high throughput screening (HTS) techniques. The use of HTS allows many compounds to be analyzed simultaneously in parallel, so that the analysis can be performed very quickly (Houston JG, Manks M., The chemical-biological interface: Developments in automated and miniaturized screening technology. Current Opinion in Biotechnology , VoI. 8, 1997). Generally, these types of techniques usually use the well-known 96-well plates, from English 96-well microtiter plates that usually require sample volumes of between 50 and 500 μl per well to carry Out experiments. Additionally, the rest of instruments, devices to mechanize the procedure of loading and reading of plates, etc. They are widely disseminated and commercially available.

In the state of the art we can find a large number of assays for the detection of protein-protein interactions, which allow us to detect compounds that interact with GAPDH and that may affect their activity. Some examples of these studies can be found in Beutel et al. U.S. Patent 5,976,813, Jayawickreme et al., Proc. Nati Acad. Sci. U. S. A.19, 1614-18 (1994), among others. Some of the most commonly used trials are mentioned below:

Protein-protein interaction assays (binding assay). In this type of assay, a small peptide is generally tested to verify the inhibition of the activity of a specific protein. These types of tests can also be carried out using methodologies, such as real-time Bimolecular Interaction

Analysis (BIA) (Sjolander & Urbaniczky, Anal. Chem. 63,2338-2345, 1991, and Szabo et al., Curr. Opin. Struct. Biol. 5, 699-705,1995).

- Double-hybrid screening (Two-hybrid screening). These types of techniques are also used for the identification of protein-protein interactions (Szabo et al., (1995); Zervos et al. (1993); Madura et al. 1993). - Competitive ligand binding assay (Timothy Zacharewski, Dept. of Biochemistry, Michigan State University, Lansing, Ml, USA. October 2002. Protocol for the competitive Ligand Binding Assay).

Example 6. Rational drug design

One of the greatest successes of the rational design of drugs consists in being able to achieve biologically active structural analogues, such as polypeptides or small molecules that mimic them, which can act as agonist, antagonists, or inhibitors of certain target molecules or proteins. An example of these techniques Ia can be found in Zhaowen Lao et al. J. Chem. Inf. Comput. Sci. 1996. 36, 1187-1194, "A new method for structure-based drug design").

In a first approach, the three-dimensional structure of the proteins of interest is determined by X-ray crystallography, computer modeling and more commonly by combination of both techniques. The structural information obtained from these studies will allow the development of other molecules that inhibit, activate or mimic the activity of the target protein. Once these new agents are produced, they are studied by different techniques that will determine their activity.

Specifically, the amino terminus of Listeria and Mycobacterium GAPDH has a predictable three-dimensional structure, the first portion of 1-8 amino acids being a beta-lamina structure and the next 12 to 22 amino acids an alpha-helix. These data together with the fact that ADP-ribosilant activity resides in this region of the enzyme can be of great help for the design of drugs or substances modulating this activity, as is done in Suresh, S., Bressi, JC , Kennedy, KJ, Verlinde, CL, GeIb, MH and HoI ₁ W. G, "Conformational changes in Leishmania mexicana glyceraldehyde-3-phosphate dehydrogenase induced by designed", inhibitorsJ. Mol. Biol. 309 (2), 423-435 (2001) and Kim, H., FeilJ.K., Verlinde, CL, Petra, PH and Hol, WG, "Crystal structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase from Leishmania mexicana : implications for structure-based drug design and a new position for the inorganic phosphate binding site ", Biochemistry 34 (46), 14975-14986 (1995) for the NAD domain of GAPDH of Mexican Leishmania.

Claims

1. Isolated immunogenic polypeptide against the Listeria and Mycobacterium genera selected from the group comprising: a) Polypeptide whose sequence comprises SEQ ID N ⁰ 1. b) Polypeptide whose sequence comprises fragments of at least 8 consecutive amino acids of SEQ ID N ⁰ 1. c) Polypeptide whose sequence has at least 85% homology with any of the polypeptides described in a) or b).

2. Isolated immunogenic polypeptide according to the preceding claim whose sequence has at least 90% homology with any of the polypeptides of claim 1.

3. Isolated immunogenic polypeptide according to the claim whose sequence has at least 95% homology with any of the polypeptides of claim 2.

4. Immunogenic polypeptide according to any of claims 1 to 3 whose sequence is SEQ ID N ⁰ 1.

5. Isolated immunogenic polypeptide according to any of claims 1 to 3 whose sequence is SEQ ID N ⁰ 2.

6. Immunogenic polypeptide according to any of claims 1-3 whose sequence is SEQ ID N ⁰ 3.

7. Polypeptide derived from any of the polypeptides of claims 1-6.

8. Isolated polynucleotide capable of encoding any of the polypeptides of any of claims 1-6.

9. An isolated antibody or fragments thereof capable of recognizing any of the polypeptides of claims 1-7.

10. Polypeptide according to any one of claims 1-7, fused or chemically bound to an additional peptide, polypeptide or protein.

eleven . An expression vector or expression system comprising a polynucleotide according to claim 8.

12. An isolated, prokaryotic or eukaryotic host cell, transformed, or transfected with a polynucleotide of claim 8 or with an expression vector or system of claim 1.

13. Method for making a polypeptide according to any one of claims 1-7 by recombinant DNA techniques or chemical synthesis

14. Method for making a polypeptide according to any one of claims 1-7 comprising: a. Cultivate a host cell according to claim 12 under conditions such that: b. Any of the polynucleotides according to claim 8 is expressed. C. Isolate the polypeptide obtained in the previous step.

15. A polypeptide or fragment thereof according to any one of claims 1-7 for use as a medicament.

16. Vaccine comprising a polypeptide or fragment thereof according to any one of claims 1-7.

17. Modulating molecule of the activity of the GAPDH of any of the Listeria or Mycobacterium genera specifically designed to interact with any of the polypeptides according to any of claims 1-7.

18. A molecule specifically designed from the ADP-ribosylation domain of the GAPDH of any of the Listeria and Mycobacteríum genera, defined according to any of the polypeptides of claims 1-7, and capable of mimicking the activity of said domain.

19. Use of a polypeptide or fragment thereof according to any of claims 1-7 for the preparation of a vaccine capable of immunizing against bacteria belonging to the genus Listeria.

20. Use of a polypeptide or fragment thereof according to the preceding claim for the preparation of a vaccine capable of immunizing against Listeria Monocytogenes.

twenty-one . Use of a polypeptide or fragment thereof according to claim 21 for the preparation of a vaccine capable of immunizing against bacteria belonging to the Mycobacteríum genus.

22. Use of a polypeptide or fragment thereof according to any one of claims 1-7 for the preparation of a vaccine capable of immunizing against Mycobacterium Tuberculosis.

23. Use of the ADP-ribosylation domain of GAPDH of any of the genera Listeria and Mycobacteríum, defined according to any of the polypeptides of claims 1-7, for the rational design of drugs.

24. Use of an isolated antibody or fragment thereof according to claim 8 for the preparation of a pharmaceutical composition for the treatment of an infection caused by bacteria belonging to any of the genera Listeria and Mycobacteríum.

25. Use of a polypeptide or fragment thereof according to any of claims 1-7 for its ability to ADP-ribosylation.