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Title:  Identification and cloning of a mycobacterial antigen corresponding to a heparin-binding haemagglutinin

United States Patent:  6,949,345

Issued:  September 27, 2005

Inventors:  Menozzi; Franco (Mons-Hyon, BE); Locht; Camille (Wannehain, FR)

Assignee:  Institut National de la Sante et de la Recherche Medicale (Paris, FR); Institut Pasteur de Lille (Lille, FR)

Appl. No.:  192579

Filed:  November 17, 1998

Abstract

The present invention relates to peptide sequences enabling mycobacteria to adhere to host cells (e.g., epithelial cells). More particularly, the invention relates to a mycobacterial heparin-binding haemagglutinin type antigen from M. bovis ECG or M. tuberculosis. The invention also relates to a recombinant peptide sequence enabling mycobacteria to adhere to host cells. The polypeptides can be used to prepare vaccines against mycobacterial infections and for serological diagnosis of mycobacterial infections.

Description of the Invention

The invention relates to peptide sequences enabling mycobacteria to adhere to host cells, in particular to epithelial cells. More particularly, the invention relates to a mycobacterial heparin-binding haemagglutinin (HBHA) type antigen obtained from Mycobacterium bovis BCG or Mycobacterium tuberculosis. The invention also relates to a recombinant peptide sequence enabling mycobacteria to adhere to host cells. In particular, the invention relates to the expression product of an Escherichia coli strain transformed with a nucleotide sequence coding for a protein enabling mycobacteria to adhere to host cells. These polypeptides can be used in immunogenic compositions, to prepare vaccines against mycobacterial infections, and for serological diagnosis of mycobacterial infections.

The invention also relates to a nucleotide sequence coding for a peptide sequence enabling mycobacteria to adhere to host cells, and in particular a nucleotide sequence coding for a mycobacterial heparin-binding haemagglutinin (HBHA) type antigen. The invention also relates to recombinant vectors comprising said nucleotide sequence and to the use of these vectors in producing recombinant host cells which can be used in therapy, in particular in anti-cancer therapy.

Mycobacteria are among the most important pathogenic micro-organisms which cause disease in both man and in animals. Mycobacterial infections are still among the main causes of death in the world. Human tuberculosis, caused by Mycobacterium tuberculosis, by itself leads to approximately 3 million deaths per annum (1, 2). Mycobacterium bovis causes tuberculosis in cattle, but it is also highly virulent in man. Leprosy, caused by Mycobacterium leprae, remains a major unresolved health problem in developing countries (3).

Infections by members of the Mycobacterium avium intracellulare complex cause disease in birds and in pigs and are among the most frequent opportunistic infections found in patients suffering from acquired immunodeficiency syndrome (AIDS) (4, 5). Further, the recent dramatic re-appearance of tuberculosis in developed countries and the appearance and propagation of drug resistant M. tuberculosis strains (6) underline the difficulty of controlling mycobacterial diseases.

Molecular characterisation of the various steps in the pathogenesis of mycobacterial diseases is fundamental to the development of optimised and rational therapeutic and prophylactic approaches to such diseases. The virulence carriers are often good antigens which can be used as candidates for vaccines. Despite the importance of mycobacterial infections, little is known about the basic molecular mechanisms involved in their pathogenesis (7).

One of the initial and crucial events in bacterial pathogenesis is adhesion of the micro-organism to its target cells. Mycobacteria exhibit tropism for pulmonary macrophages (8). However, since such micro-organisms are readily transmitted by aerosol, the first structures in the host which they encounter during infection are those of the respiratory epithelium. As a result, interactions with epithelial cells or with the extracellular matrix (ECM) during the initial and subsequent steps of pathogenesis can be important (9), although they have not yet been studied to any great extent.

Within the context of the present invention, the inventors have obtained a novel mycobacterial antigen involved in adhesion of mycobacteria to epithelial type host cells. It is a 28 kDa heparin binding haemagglutinin (HBHA) which has been obtained from culture supernatants prepared from cell walls of Mycobacterium bovis BCG and Mycobacterium tuberculosis. Immunoblot analysis using polyclonal and monoclonal antibodies have indicated that HBHA differs from proteins of the antigen 85 complex and represents a novel antigen. From this basic protein, the inventors have been able to evaluate and propose the development of a series of polypeptides which can be used for diagnosis, therapy and prophylaxis.

The invention thus provides a peptide sequence enabling mycobacteria to adhere to host cells, in particular epithelial cells. More particularly, the peptide sequence of the invention is characterized in that it is a mycobacterial heparin-binding haemagglutinin (HBHA) type antigen, in particular an antigen obtained from Mycobacterium bovis BCG or Mycobacterium tuberculosis.

In a preferred embodiment of the present invention, the peptide sequence is characterized in that it comprises the sequence corresponding to the sequence shown in FIG. 10 (see Original Patent) (SEQ ID No. 19), or any variant of that sequence which enables mycobacteria to adhere to host cells and obtained by addition, substitution or deletion of one or more amino acids of the sequence of FIG. 10 (SEQ ID No. 19).

Throughout the text, the term "polypeptide sequence" or "polypeptide" represents all or part of the sequence of FIG. 10, which itself represents the DNA and the HBHA protein as it is. The term "protein" designates the modified or non modified polypeptide sequence.

Preferably, the invention more particularly provides a peptide sequence comprising a region involved in interactions with sulphated glycoconjugates and in heparin binding. This peptide sequence is as follows:

bulletKKAAPAKKAAPAKKAAPAKKAAAKKAPAKKAAAKKKVTQK (SEQ ID No. 1).

The invention thus concerns a peptide sequence comprising the C-terminal portion of the sequence of FIG. 10 (see Original Patent) and more particularly the sequences comprising approximately the last 30 to 50 amino acids of the C-terminal portion of the sequence of FIG. 10 (see Original Patent) . This region of the sequence of FIG. 10 is involved in the interaction of the protein with heparin, although a shorter sequence, in particular of about 10 to 20 amino acids, may be sufficient.

The inventors have also expressed the nucleotide sequence coding for the peptide sequence of the invention in E. coli. The polypeptide obtained has a lower molecular weight than that of the purified protein from M. bovis BCG or M. tuberculosis. These differences are the result of post-translational modifications which do not occur in E. coli.

The invention thus also provides a recombinant peptide sequence, characterized in that it enables mycobacteria to adhere to host cells. More particularly, the recombinant sequence of the present invention is the expression product of a nucleotide sequence coding for a peptide sequence enabling mycobacteria to adhere to host cells, in particular an antigenic sequence obtained from M. bovis BCG or M. tuberculosis, said recombinant sequence being, for example, the expression product of an E. coli strain transformed with a suitable nucleotide sequence.

The invention also relates to the use of one of the peptide sequences described above, whether recombinant or non recombinant, and more particularly in its native form for serological diagnosis of the presence of mycobacteria. The invention also provides an immunogen composition characterized in that it comprises one of the peptide sequences described above and to the use of that peptide sequence to prepare vaccines against mycobacterial infections, particularly infections caused by M. bovis or M. tuberculosis.

The inventors have also discovered that adhesion of mycobacteria to epithelial cells can be specifically inhibited by sulphated glucides. The invention thus concerns the use of a sulphated glucide to inhibit adhesion of mycobacteria to epithelial cells. Sulphated glucides of particular interest include heparin, chondroitin sulphate and dextran sulphate as well as their synthetic derivatives.

The inventors have also isolated the whole of the gene coding for HBHA from the DNA of M. bovis BCG. The invention thus provides a nucleotide sequence, characterized in that it codes for a peptide sequence enabling mycobacteria to adhere to host cells. More particularly, the nucleotide sequence of the invention codes for a mycobacterial heparin-binding haemagglutinin (HBHA) type antigen, in particular the peptide sequence of FIG. 10 or any portion of that peptide sequence enabling mycobacteria to adhere to host cells and obtained by addition, substitution or deletion of one or more amino acids from said peptide sequence.

The invention also provides a recombinant host cell, characterized in that it comprises one of the nucleotide sequences described above in its genome. In one preferred embodiment of the invention, the recombinant host cell is BCG, but not exclusively, for which expression vectors directly usable for developing recombinant BCG for use in man or animal have been developed.

BCG is used in therapy, more particularly in anti-cancer therapy, in particular against superficial cancers of the bladder. In this type of therapeutic application, a correlation between the adhesion ability of the BCG and its anti-tumoral power appears to exist. Within the context of the present invention, identification of the HBHA and cloning of its gene renders possible an increase in adhesion capacity via overexpression of the gene coding for HBHA.
 

Claim 1 of 3 Claims

1. A kit for serological diagnosis of mycobacterial infections comprising at least:

a) a reactant consisting of:

(i) an HBHA protein purified from a preparation of mycobacterium cell walls, or a fragment thereof, determined by epitope mapping; or

(ii) a fragment comprised in the last 30 to 50 amino acids in a C-terminal portion of said HBHA protein or in the last 50 C-terminal amino acids of SEQ ID NO. 19; or

(iii) a recombinant peptide sequence which is obtainable by expression in a host cell of a polynucleotide sequence of SEQ ID NO. 19, and wherein said recombinant peptide sequence is an HBHA mycobacterial antigen enabling the adhesion of mycobacteria to the sulphated glucides of epithelial cells;

said reactant being coupled to or adsorbed on a support;

b) an anti-antibody antibody, modified such that a detection signal can be coupled thereto.

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

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