WO2012038486A1

WO2012038486A1 - Method for obtaining a purified hbha polypeptide or fragment thereof

Info

Publication number: WO2012038486A1
Application number: PCT/EP2011/066461
Authority: WO
Inventors: Camille Locht; Jérôme SEGERS
Original assignee: Inserm (Institut National De La Sante Et De La Recherche Medicale); Institut Pasteur De Lille; Centre National De La Recherche Scientifique (Cnrs)
Priority date: 2010-09-21
Filing date: 2011-09-21
Publication date: 2012-03-29

Abstract

The present invention relates to a method for discriminating between an antigenic or immunogenic HBHA polypeptide or fragment thereof and a non antigenic/non immunogenic HBHA polypeptide or fragment thereof, wherein said method comprises a step of assessing whether one or both lysine residues in position 160 and/or 161 of SEQ ID N°1 are mono- or di-methylated and wherein the presence of at least one methyl group on one lysine residues selected from lysine residues 160 and 161 is indicative of the antigenicity/immunogenicity of said HBHA polypeptide or fragment thereof.

Description

METHOD FOR OBTAINING A PURIFIED HBHA POLYPEPTIDE OR FRAGMENT

THEREOF

FIELD OF THE INVENTION:

The present invention relates to the general field of vaccines against infections by Mycobacterium tuberculosis in mammals, namely tuberculosis as well as to diagnostic tools for detecting infections by Mycobacterium tuberculosis.

The present invention namely relates to vaccines and diagnostic tools based on methylated immunogenic peptide sequences comprising mycobacterial heparin-binding hemagglutinin.

The invention specifically concerns a method for discriminating between a HBHA polypeptide or fragment thereof which is antigenic in infected subj ects and/or induces protective immunity in subjects and a HBHA polypeptide or fragment thereof which is not antigenic in infected subjects or does not induce protective immunity in subjects, wherein said method comprises a step of assessing whether one or both lysine residues in position 160 and/or 161 of SEQ ID N° l are mono- or dimethylated and wherein the presence of at least one methyl group on one lysine residues selected from lysine residues 160 and 161 is indicative of the antigenicity/immunogenicity of said HBHA polypeptide or fragment thereof.

The invention further concerns a method for obtaining purified peptide sequences of HBHA having specific antigenic and/or immunogenic properties as defined above.

BACKGROUND OF THE INVENTION:

Tuberculosis (TB) is still a major global public health problem, with an estimated 1.5 to 2 million annual deaths and 8 to 10 million new cases each year. In addition, roughly 2 billion subjects are currently infected with the causative agent Mycobacterium tuberculosis although most of them will never develop overt disease and will evolve towards latency, in which the bacilli remain dormant; approximately 5 to 10 % of the latently infected individuals eventually develop the disease. Except for obvious immunodepression, the reasons for this reactivation are not yet fully understood, but may be related to the induction of regulatory T cells with a CD4⁺CD25⁺FoxP3⁺ phenotype.

Current tools to control TB include the bacillus Calmette and Guerin (BCG) as the only available vaccine, skin test reactions to tuberculin and, more recently, INF-γ release assays for diagnostics, and a handful of drugs for therapy. Clearly insufficient to control the disease or the M. tuberculosis infection, novel tools are desperately needed, including novel vaccines and diagnostics with improved sensitivity and specificity, especially for the detection of latent infection.

Heparin-binding hemagglutinin (HBHA) is produced by all the members of the

Mycobacterium tuberculosis complex, with identical amino-acid sequence in all strains analysed so far. It is a surface-associated protein involved in adherence of M. tuberculosis to non-phagocytic cells, such as epithelial cells, fibroblasts and endothelial cells. During latent infection, M. tuberculosis is largely present in these cell types, and the HBHA-encoding gene is strongly upregulated when M. tuberculosis has invaded epithelial cells, but not macrophages. Mouse infection experiments have indicated that HBHA is an important virulence factor involved in extrapulmonary dissemination, and HBHA-coated beads are able to transcytose through epithelial and endothelial cell layers.

As an important virulence factor, being strongly upregulated when M. tuberculosis invades epithelial cells, where the organisms reside during latent infection, HBHA is a good candidate as a latency antigen. Studies in humans have shown that the protein is antigenic during infection. Anti-HBHA serum antibodies are generated in infected humans. More importantly, HBHA is a strong T cell antigen in humans, as peripheral blood monocytes isolated from M tuberculosis-m^' iected individuals produce high levels of IFN-γ upon stimulation with HBHA. Interestingly, this T cell response is much stronger in infected healthy individuals (i.e. during latency), than in patients with active TB. However, upon successful treatment, tuberculosis patients recover their ability to mount a HBHA-specific T cell response. In latently infected subjects the T cell response to HBHA is characterised by CD8⁺ T cell mediated perforin-dependent cytotoxicity and microbicidal activity, in addition to CD4⁺ T cell- and CD8⁺ T cell-mediated HBHA-specific IFN-γ responses. These observations strongly suggest that HBHA induces protective immunity in latently infected human subjects. Importantly, this T cell response strongly depends on the appropriate methylation pattern of HBHA.

Unlike the peripheral blood cells, however, local lymphocytes of TB patients react strongly to HBHA, indicating that HBHA can be used both for the detection of latently infected individuals when peripheral blood lymphocytes are used in IFN-γ release assays, and for the detection of active disease, both pulmonary and extra-pulmonary tuberculosis, when local lymphocytes are analysed. HBHA- mediated protection has been assessed in mice and guinea pigs. Balb/c mice immunised three times at two weeks intervals with 5 μg of purified HBHA in the presence of monophosphoryl lipid A and dimethyldioctadecylammonium are protected against intravenous challenge as well as mice vaccinated with BCG, as evidenced by the bacterial load in the lungs, spleen and liver. HBHA also protected in the mouse aerosol challenge model. In both models, only the properly methylated form provided protection.

These and additional observations indicate that HBHA is a strong protective antigen, perhaps particularly useful for boosting after BCG priming.

HBHA is a 198 amino-acid long protein and undergoes post-translational methylation. The methylation occurs at in the C-terminal domain of the protein, which contains several lysine-rich repeats important for the adhesin function of the protein to epithelial cells. The methylation pattern is very complex and consists of several mono- and several di-methylated lysines, arranged in a precise fashion. The methylation protects the protein against proteolytic degradation and also plays an important role in antigenicity in humans, and protective immunogenicity in rodents

Methylation of HBHA in the C-terminal lysine-rich region of the antigen is immunogenically and antigenically important.

Despite many attempts to obtain reliable diagnostic tools, namely for discriminating between acute and latent tuberculosis and many efforts to produce a new, better performing vaccines, there is still a need for more efficient and reliable tools.

HBHA has become a primary candidate as a diagnostic and vaccine tool. However the difficulties encountered in obtaining HBHA polypeptides that are highly immunoprotective and immunogenic in latently infected subj ects are still to be solved, namely there is an unsatisfied need in providing HBHA forms with a proper methylation pattern, which is a critical feature for the activity of HBHA for diagnosis as well as for vaccine applications.

SUMMARY OF THE INVENTION:

The invention is based on the discovery that a fine-tuned methylation pattern of HBHA is critical for both protective immunogenicity and for antigenicity in M. tuberculosis- infected subjects and furthermore that the current mass-spectrometry technology is not able to distinguish between antigenic/immunogenic and non-antigenic/immunogenic forms of methylated HBHA.

Thus, the invention relates to a method for discriminating between a HBHA polypeptide or fragment thereof which is antigenic in infected subj ects and/or induces protective immunity in subjects and a HBHA polypeptide or fragment thereof which is not antigenic in infected subjects or does not induce protective immunity in subjects, wherein said method comprises a step of assessing whether one or both lysine residues in position 160 and/or 161 of SEQ ID N° l are mono- or dimethylated and wherein the presence of at least one methyl group on one lysine residues selected from lysine residues 160and 161 is indicative of the antigenicity/immunogenicity of said HBHA polypeptide or fragment thereof.

The invention also concerns a method for obtaining a purified HBHA polypeptide or fragment thereof which is antigenic in infected subjects and/or induces protective immunity in subjects comprising the steps of : a. Growing a Mycobacterium strain or a recombinant strain transformed with a nucleic acid encoding the HBHA polypeptide or a fragment thereof in a suitable culture medium in conditions allowing to obtaining an HBHA polypeptide or fragment thereof, b. Submitting said HBHA polypeptide or fragment thereof to a chemical or enzymatic methylation step, if necessary, c. Selecting the HBHA polypeptides or fragment thereof wherein one or both lysine residues in position 160 and/or 161 of SEQ ID N°l are mono- or dimethylated, and and/or discarding the HBHA polypeptides or fragment thereof that do not bear a methyl group in position 160 and:or 161 of SEQ ID N°2. d. Optionally, isolating said HBHA polypeptides or fragment thereof.

The invention further concerns a diagnostic or therapeutic composition comprising a purified polypeptide fragment of HBHA consisting of SEQ ID N°2 wherein at least one lysine residue in position 10 or 1 1 bears one or two methyl groups, wherein the polypeptide fragment is not the HBHA fragment naturally produced by Mycobacterim tuberculosis or Mycobacterium bovis BCG.

The invention also concerns the use of an antibody directed against an epitope of sequence SEQ ID N°2 ID N°7 for quality control or purification of an HBHA polypeptide produced by a Mycobacterium strain or a recombinant HBHA, or fragments thereof.

DETAILED DESCRIPTION OF THE INVENTION: Definitions:

Throughout the specification, several terms are employed and are defined in the following paragraphs.

Within the context of the present invention, the term "native HBHA" (nHBHA) designates the heparin-binding hemagglutinin polypeptide obtained from Mycobacterium bovis BCG.

The term "rHBHA" designates the recombinant heparin-binding hemagglutinin polypeptide obtained in E. coli

The term "SMrHBHA" designates the recombinant heparin-binding hemagglutinin polypeptide obtained in Mycobacterium smegmatis

The term "peptide sequence" designates all or a portion of the sequence for the HBHA protein, provided that said "peptide sequence" contains at least the lysine-rich carboxy- terminal region which ensures heparin binding. The sequence for said carboxy-terminal region is as follows: KKAAPAKKAAPAKKAAPAKKAAAKKAP AKKAAAKKVTQK (SEQ ID NO:3). This sequence was disclosed in the International patent publication with publication number W097/44463.

The sequence of the HBHA protein is represented as sequence SEQ ID N° l and described in Menozzi et al., 1998. (Molecular characterization of the mycobacterial heparin- binding hemagglutinin, a mycobacterial adhesin. Proc. Natl. Acad. Sci. USA 95, 12625- 12630.)

The term "protein", "HBHA protein" or "HBHA" as used in the present invention means all or a portion of the peptide sequence for HBHA, provided that it includes at least the C-terminal region of said HBHA. When the sequence under consideration comprises at most the C-terminal region of the HBHA, the term "peptide" will advantageously be used. The term "peptides" will be used to designate products from the enzymatic digestion of HBHA. However, the use of the term "peptide" is not limited to this instance, "peptide" also being synonymous with "protein" within the context of the invention.

The term "purified" employed herein means that the HBHA peptide contains less than 5% other components from the host namely (poly)peptides contaminants.

A "recombinant" peptide sequence in accordance with the invention corresponds to a peptide sequence obtained by expression, in a heterologous cell host, of a nucleotide sequence encoding said peptide sequence. In particular, said heterologous cell host can be a bacterium that does not belong to the Mycobacterium genus, for example Escherichia coli, or other organisms such as yeasts or animal or plant cells.

The expression "nucleotide sequence" designates any DNA sequence encoding a peptide sequence as defined in the context of the present invention.

In accordance with accepted use, an "antigen" designates any peptide sequence of the present invention having an immunogenic power.

By « antigenic in infected subjects" is meant that the HBHA polypeptide or fragment thereof is recognized by lymphocytes of infected subjects (latent tuberculosis, in particular in peripheral blood as well as acute tuberculosis, in particular at the site of infection) and further that after pooling of these lymphocytes and HBHA or a fragment thereof, these lymphocytes produce and secrete cytokines, in particular IFN-γ.

According to the invention, the subject is a mammal and preferably a human being.

The invention relates to a method for discriminating between a HBHA polypeptide or fragment thereof which is antigenic in infected subjects and/or induces protective immunity in subjects and a HBHA polypeptide or fragment thereof which is not antigenic in infected subjects or does not induce protective immunity in subjects, wherein said method comprises a step of assessing whether one or both lysine residues in position 160 and/or 161 of SEQ ID N°l are mono- or dimethylated and wherein the presence of at least one methyl group on one ly sine residues sel ected from ly sine resi dues 1 60and 16 1 i s indi cative of the antigenicity/immunogenicity of said HBHA polypeptide or fragment thereof.

In one embodiment the lysine in position 161 is mono- or di-methylated, the lysine is position 160 being non- methylated.

In a second embodiment, both lysine residues in position 160 and 161 are mono-or dimethylated

In a third embodiment, the lysine residue one of the lysine residue in position 160 or

161 is mono-methylated, the second being di-methylated.

Other methyl groups may be present in various position of the HBHA polypeptide namely as described in WO 03/044048.

The methylation pattern in positions 160 and 161 is determined through any means available in the art;

According to a first embodiment the presence and number of methyl group in positions 160 and 161 is determined by amino acid sequencing of the relevant region of the HBHA protein. The determination may also be performed by NMR, preferably after digestion of the protein in peptide fragments.

The method may be performed on any kind of HBHA protein.

The method may thus be used for the quality control of a methylated HBHA protein obtained by cultivating a Mycobacterium strain expressing a methylated HBHA protein, preferably Mycobacterium bovis BCG in a suitable medium followed by various purification steps in order to determine its antigenic/immunogenic potency.

A method for obtaining a purified methylated HBHA from Mycobacterium bovis BCG is for example described in Menozzi et al . 1996( Identification of a heparin-binding hemagglutinin present in mycobacteria. J. Exp. Med. 184, 993-1001), and is based on heparin-Sepharose chromatography.

The methylated HBHA may further be purified according to the method described in Masungi et al. 2002. (Differential T and B cell responses against Mycobacterium tuberculosis heparin-binding hemagglutinin adhesin in infected healthy individuals and patients with tuberculosis. J. Infect. Dis. 185, 513-520), that is based on a heparin-Sepharose chromatography.

At the end of the purification it is advantageous to control the antigenic/immunogenic potency of the protein by the method described above.

The methylated protein may be a recombinant protein expressed by a host capable of expressing HBHA in the methylated form.

The method may further be used for assessing the antigenic/immunogenic potency of a recombinant HBHA protein expressed in a non methylated form, the protein thus obtained being submitted to an enzymatic or chemical methylation step.

The chemical methylation is derived from the literature (Means G E, 1977 Meth Enzymol 47: 469-478). In particular, the chemical methylation reaction is carried out in a solution comprising formaldehyde and NaBH4.

The enzymatic methylation methods of the invention can be carried out using one or more mycobacterial methyltransferases. Said methyltransferases catalyze the transfer of methyl groups from a donor to an acceptor, in this instance the peptide sequence for the previously purified recombinant HBHA. The methyl donor can be S-adenosylmethionine (AdoMet), which is well known to the skilled person.

More particularly, the methyltransferase or methyltransferases are present and active in extracts from total mycobacterial proteins such as M. bovis BCG. The method of the invention may advantageously be carried out by reacting the purified HBHA polypeptide with an antibody specifically recognizing the epitope of SEQ ID N°2 wherein at least one lysine residues in position 10 or 11 bears one or two methyl groups.

In a preferred embodiment, the antibody is selected so as to specifically recognize the epitope of SEQ ID N°2 wherein one lysine residue in position 10 and 11 bears one or two methyl groups such as described above.

In a particularly preferred embodiment, the antibody is the monoclonal antibody 4057D2.

The antibody is described in Pethe et al. 2002. (Mycobacterial heparin-binding hemagglutinin and laminin-binding protein share antigenic methyllysines that confer resistance to proteolysis. Proc. Natl. Acad. Sci. USA 99, 10759-10764)

The invention further relates to a a method for obtaining a purified HBHA polypeptide or fragment thereof which is antigenic in infected subj ects and/or induces protective immunity in subjectscomprising the steps of : a. Growing a Mycobacterium strain or a recombinant strain transformed with a nucleic acid encoding the HBHA polypeptide or a fragment thereof in a suitable culture medium in conditions allowing to obtaining an HBHA polypeptide or fragment thereof, b. Submitting said HBHA polypeptide or fragment thereof to a chemical or enzymatic methylation step, if necessary, c. Selecting the HBHA polypeptides or fragment thereof wherein one or both lysine residues in position 160 and/or 161 of SEQ ID N°l are mono- or dimethylated, and and/or discarding the HBHA polypeptides or fragment thereof that do not bear a methyl group in position 160 and/or 161 of SEQ ID

N°2, d. optionally, isolating said HBHA polypeptides or fragment thereof

In a preferred embodiment, step c) is performed by contacting HBHA polypeptides or fragment thereof with an antibody specially recognizing the epitope of SEQ ID N°2 wherein at least one lysine residues in position 10 or 11 bears one or two methyl groups The antibody is for example the antibody 4057D2.

The invention also concerns a diagnostic or vaccine composition comprising a purified polypeptide fragment of HBHA consisting of SEQ ID N°2 wherein at least one lysine residues in position 10 or 1 1 bears one or two methyl groups and wherein the polypeptide fragment is not the HBHA fragment naturally produced by Mycobacterium tuberculosis or Mycobacterium bovis BCG.

The invention also concerns the use of an antibody directed against an epitope of sequence SEQ ID N°2 for quality control or purification of an HBHA polypeptide produced by a Mycobacterium strain or a recombinant HBHA, or fragments thereof. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1 illustrates the results obtained by ELISA on standardized nHBHA (N), rHBHA (R) and SMrHBHA (RN) using monoclonal antibodies 3921E4 (E4) and 4057D2 (D2).

Figure 2 illustrates Peptide scan analysis of HBHA methylated in situ. The indicated peptides were spotted onto glass slides, methylated in situ and probed with monoclonal antibody 4057D2 at a 1 :80 and 1 :320 dilution.

EXAMPLE: Material & Methods

Preparation of native methylated HBHA fromM bovis BCG

M. bovis BCG was grown in static cultures at 37°C using 175 cm3 Roux flasks containing approximately 200 ml of Sauton medium. At stationary phase (when the OD reached approximately 1.5), the cultures were centrifuged (10 000 x g for 20 min), heat inactivated and lysed by sonication. The sonicate was centrifuged at 13,500 x g for 20 min, and the supernatant was applied onto a heparin-Sepharose CL-B column ( 1 X5 cm) equilibrated with DPBS. The column was then washed with 100ml DPBS, and the bound material was eluted by a 0-500mM NaCl gradient in 100ml DPBS. The eluted 1-ml fractions were identified by SDS-PAGE using a 12% gel followed by Coomassie Brilliant Blue R-250 staining.

The HBHA-containing fractions were pooled and applied onto a reverse-phase HPLC Nucleosil C18 column (TSK gel super ODS; Interchim) equilibrated in 0, 05% trifluoroacetic acid. Bound material was eluted with a linear 0-80% acetonitrile gradient. HBHA-containing fractions were pooled, and the solvent was eliminated by evaporation. The pH of the final fraction of 1 ml was adjusted to pH8 by using 1 M Tris-HCl (pH9). The final product was stored at - 80°C. Yields were between 500 μg and 1 mg per liter of culture.

Preparation of recombinant HBHA from E. coli and Mycobacterium smegmatis

Recombinant HBHAs (rHBHA and MSrHBHA) were respectively obtained from E. coli and M. smegmatis as described in Pethe et al. 2002. Mycobacterial heparin-binding hemagglutinin and laminin-binding protein share antigenic methyllysines that confer resistance to proteolysis. Proc. Natl. Acad. Sci. USA 99, 10759-10764.

Antibodies

The antibodies 4057D2 and 3921E4 have been obtained from the FDA (Rouse et al.

1991. Immunological characterization of recombinant antigens isolated from a Mycobacterium avium gtl l expression library by using monoclonal antibody probes. Infect. Immun. 59, 2595-2600).

Reaction of different forms of HBHA with monoclonal antibodies 4057D2 and 3921E4

The concentrations of all three forms of HBHA were measured and adjusted in order to coat overnight at 4°C the same quantities (1 μg) of each form on ELISA plates. The plates were then washed with phosphate buffered saline (PBS)/Tween (PBST), followed by blocking for 2 hrs at room temperature with PBST containing 3% BSA. After washing again three times with PBST, the plates were incubated for lh30 at room temperature with serial fourfold dilutions of monoclonal antibodies 3921E4 or 4057D2 in PBST + BSA. The latter two preferentially recognize the methylated forms of HBHA (Pethe et al. 2002. Mycobacterial heparin-binding hemagglutinin and laminin-binding protein share antigenic methyllysines that confer resistance to proteolysis. Proc. Natl. Acad. Sci. USA 99, 10759-10764). The plates were then washed 10 times with PBST and incubated for 1 h at room temperature with anti- mouse antibodies linked to peroxidase. After 10 additional washings with BPST, 100 μΐ of peroxidase substrate TMB was added to each well, and the colour reaction was stopped by the addition of 50 μΐ H₃PO₄. Finally the adsorbancy at 450 nm was read in each well using a standard ELISA plate reader.

Epitope analysis

A set of 31 18-amino-acid long peptides with a 6-residue overlap, corresponding to the entire HBHA sequence was synthesized, purified by HPLC and characterized by mass spectrometry using standard techniques. The peptides were printed as a peptide array onto glass slides using standard techniques. The peptides were then in situ methylated using 50 μΐ of a 9/10 solution of 0.1 M Hepes (pH 7.5), 47 mg/ml NiCl₂, 15 mg/ml NaC BH₃/0.1 M Hepes (pH 7.5), 36 mg/ml formaldehyde. After 2 min incubation at room temperature, the chemical reaction was stopped by rinsing with distilled water. The slides were then blocked for 15 min. at 37°C with PBST + 5 % BSA. After washing with PBST, the 4057D2 antibody was added, and the slides were incubated at room temperature for 1 h. After 5 washes with PBST, FITC-linked anti-mouse antibodies were added and incubated for 1 h at room temperature. After 5 washes with PBST, the plates were read using a microarray fluorescence reader

RESULTS

One of the characteristics of FIBHA that are immunogenically and antigenically important is its complex methylation pattern in the C-terminal lysine-rich region of the antigen (see above). The methylation can be analyzed by mass-spectrometry or by the use of monoclonal antibodies that are specific for methylated FIBHA. Mass-spectrometry analysis shows a typical pattern of nHBHA in its methylated form, and this pattern is different when non-methylated rHBHA is analysed and mass-spectrometry analyses indicated that the M smegmatis-produced rHBHA is methylated in its C-terminal lysine-rich domain, with a methylation pattern very similar to that of nHBHA produced by BCG, as evidenced by mass spectrometry. However, this recombinant form of HBHA is very poorly recognized by peripheral blood mononuclear cells fromM tuberculosis-infected individuals. It also does not induce protection in mouse models. These results show that a fine-tuned methylation pattern of HBHA is critical for both protective immunogenicity and for antigenicity in M. tuberculosis-infected subjects. They also indicate that the current mass-spectrometry technology is not able to distinguish between antigenic/immunogenic and non- antigenic/immunogenic forms of methylated HBHA. The different forms, nHBHA and MSrHBHA, were analyzed by ELISA using the monoclonal antibodies as described above. As shown in figure 1, E. co/z^'-produced rHBHA (R) did not react with 3921E4, or with 4057D 2. In contrast, both nHBHA (N) and SMrHBHA (RN) reacted equally well with 3921E4 .However, SMrHBHA (RN) reacted approximately 100-fold less well with 4057D2, as compared to nHBHA (N) .

This ELISA thus represents a quality control tool to distinguish methylated antigenic/immunogenic HBHA from methylated non-antigenic/immunogenic HBHA after standardizing the different HBHA forms.

The 4057D2 epitope was then mapped using overlapping, methylated peptides of HBHA spotted on glass slides as described above. The results indicated the strongest reaction with peptide P26 (Fig. 2).

The 4057D2 antibody can therefore be used for the quality control of the production of nHBHA or rHBHA to be used either for vaccine development or as a diagnostic reagent.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. A method for discriminating between a HBHA polypeptide or fragment thereof which is antigenic in infected subjects and/or induces protective immunity in subjects and a a HBHA polypeptide or fragment thereof which is not antigenic in infected subjects or does not induce protective immunity in subjects, wherein said method comprises a step of assessing whether one or both lysine residues in position 160 and/or 161 of SEQ ID N°l are mono- or dimethylated and wherein the presence of at least one methyl group on one lysine residues selected from lysine residues 160and 161 is indicative of the antigenicity/immunogenicity of said HBHA polypeptide or fragment thereof.

2. The method of Claim 1 wherein the lysine in position 161 is mono-or di-methylated.

3. The method of Claim 1 or 2 wherein the HBHA polypeptide is a naturally occurring polypeptide produced by a Mycobacterium strain.

4. The method of Claim 3 wherein the HBHA polypeptide is a naturally occurring polypeptide produced by a Mycobacterium strain able to methylate the HBHA polypeptide.

5. The method of Claim 1 or 2 wherein the HBHA polypeptide is a recombinant polypeptide.

6. The method of Claim 1 or 2 wherein the HBHA polypeptide naturally occurring or recombinant is submitted to an enzymatic or chemical methylation step

7. The method of anyone of Claims 1 to 6, wherein the presence of one or two methyl groups on the lysine residues in position 160 and/or 161 of SEQ ID N°l is performed by reacting said polypeptide with an antibody specifically recognizing the epitope of SEQ ID N°2 wherein at least one lysine residue in position 10 or 11 bears one or two methyl groups

8. The method of claim 7 wherein the antibody specifically recognizes the epitope of SEQ ID N°2 wherein the lysine residue in position 11 bears one or two methyl groups.

9. The method of claim 8 wherein said antibody is the monoclonal antibody 4057D2.

10. A method for obtaining a purified HBHA polypeptide or fragment thereof which is antigenic in infected subjects and/or induces protective immunity in subjects, comprising the steps of : a. Growing a Mycobacterium strain or a recombinant strain transformed with a nucleic acid encoding the HBHA polypeptide or a fragment thereof in a suitable culture medium in conditions allowing to obtaining an HBHA polypeptide or fragment thereof, b. Submitting said HBHA polypeptide or fragment thereof to a chemical or enzymatic methylation step, if necessary, c. Selecting the HBHA polypeptides or fragment thereof wherein one or both lysine residues in position 160 and/or 161 of SEQ ID N°l are mono- or dimethylated and/or discarding the HBHA polypeptides or fragment thereof that do not bear a methyl group in position 160 and:or 161 of SEQ ID N°2. d. Optionally, isolating said HBHA polypeptides or fragment thereof.

11. The method of claim 10 wherein step c) is performed by contacting HBHA polypeptides or fragment thereof with an antibody specially recognizing the epitope of SEQ ID N°2 wherein at least one lysine residues in position 10 or 11 bears one or two methyl groups.

12. The method of claim 11 wherein said antibody is the antibody 4057D2.

13. A diagnostic or therapeutic composition comprising a purified polypeptide fragment of HBHA consisting of SEQ ID N°2 wherein at least one lysine residues in position 10 or 11 bears one or two methyl groups , wherein the polypeptide fragment is not the HBHA fragment naturally produced by Mycobacterim tuberculosis or Mycobacterium bovis BCG.

14. The use of an antibody directed against an epitope of sequence SEQ ID N°2 for quality control or purification of a HBHA polypeptide or fragment thereof which is antigenic in infected subjects and/or induces protective immunity in subjects produced by a Mycobacterium strain or a recombinant HBHA, or fragments thereof.