Antigen Imitating Extracellular Areas of Membrane Proteins of Type III Produced from Intracellular Pathogenic Micro-Organisms, Derived Conformational Antibodies and the Use Thereof

• US 20080075717A1
• Filed: 01/28/2004
• Published: 03/27/2008
• Est. Priority Date: 01/28/2003
• Status: Active Grant

• Current Assignee: Centre National De La Recherche Scientifique, Université de Liège, Universite de Rouen
• Original Assignee: Centre National De La Recherche Scientifique, Université de Liège, Universite de Rouen
• Inventors: Tranchand-Bunel, Denis
• Granted Patent: US 7,803,378 B2
• Time in Patent Office: Days
• US Class Current: 424/133.1
• CPC Class Codes: A61K 2039/525 Virus
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/20 for DNA viruses
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 16/085 Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
• CPC Class Codes: C07K 2317/732 Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: C12N 2710/16222 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2710/16622 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Abstract

The inventive antigen derived from an intracellular pathogenic micro-organism is characterised in that it comprises at least on peptidic fragment which essentially consists of the concatenation of sequences of at least two extracellular adjacent areas in the native structure of a membrane protein of type III of said intracellular pathogenic micro-organism, derived conformational antibodies and the application thereof.

Claims

1. A chimeric antigen derived from an intracellular pathogenic microorganism, comprising at least one peptide fragment consisting essentially of the concatenation of the sequences of at least two adjacent extracellular domains in the native structure of a type III membrane protein of said intracellular pathogenic microorganism, and wherein the antigen induces the production, in individuals immunized with said antigen, of conformational antibodies that recognize said type type III membrane protein in the native form.

2. The antigen as claimed in claim 1, wherein said type III membrane protein has 2n transmembrane domains.

3. The antigen as claimed in claim 1, said peptide fragment comprises at least one heterologous linking sequence of 1 to 5 amino acids that are identical to or different from one another, preceding the sequence of one of said extracellular domains.

4. The antigen as claimed in claim 3, wherein said amino acids are different from those present in the sequences of said extracellular domains.

5. The antigen as claimed in claim 3, wherein all the sequences of the adjacent extracellular domains, with the exception of those located at the N and/or C-terminal ends of said peptide fragment, are preceded by a heterologous linking sequence.

6. The antigen as claimed in claim 3, wherein all the sequences of the adjacent extracellular domains of said peptide fragment are preceded by a heterologous linking sequence.

7. The antigen as claimed in claim 3, wherein said amino acids are chosen from cysteine (C) and/or lysine (K).

8. The antigen as claimed in claim 1, wherein said peptide fragment also includes, between the heterologous linking sequences and the sequences of the extracellular domains and on either side of said sequences of the extracellular domains, a sequence of at least one amino acid, preferably of 1 to 7 amino acids, preferably 2 amino acids, corresponding to that of the transmembrane domain flanking said extracellular domain in the structure of said type III membrane protein.

9. The antigen as claimed in claim 1, derived from a type III membrane protein selected from the group consisting of:

the EBV LMP1 and LMP2A proteins (Swissprot accession number, respectively P03230 and P13285, with reference to the sequence of the EBV strain of B95.8), the KSHV LAMP K15-P and LAMP K15-M proteins (Genbank accession numbers, respectively AAD45297 and AAD45296), the HCV p7, NS2 and NS4B proteins (EMBL accession number AF009606), the Chlamydia trachomatis MOMP protein (NCBI accession number AF352789) and the Mycobacterium tuberculosis Mmpl 1 to 12 proteins (EBML accession number Z87425.1).

10. The antigen as claimed in claim 1, wherein said peptide fragment consists essentially of the concatenation of the sequences of 2 to 6 adjacent extracellular domains, preferably 2 or 3 adjacent extracellular domains, in the native structure of said protein.

11. The antigen as claimed in claim 1, wherein it is selected from the sequences SEQ ID Nos 10, 11 and 13 to 23.

12. The antigen as claimed in claim 1, wherein it comprises at least two peptide fragments as defined in claim 1, derived from different type III membrane proteins.

13. The antigen as claimed in claim 3, wherein at least one of the amino acids of said heterologous linking sequence is covalently attached to at least one carrier protein or at least one lipid.

14. The antigen as claimed in claim 13, wherein at least two amino acids, each derived from the unique linking sequence of different peptide fragments, are attached to a carrier protein.

15. The antigen as claimed in claim 13, wherein a cysteine or a lysine of a heterologous linking sequence is attached to a carrier protein, respectively via a thioether bond and an amide bond.

16. The antigen as claimed in claim 13, wherein said carrier protein is KLH.

17. The antigen as claimed in claim 13, wherein in that the lipid(s) is (are) branched via ether, thioether, ester and thioester bonds, preferably via thioester bonds.

18. The antigen as claimed in claim 16, wherein at least two amino acids derived from one or more heterologous linking sequence(s) of a single peptide fragment are each attached to a lipid.

19. The antigen as claimed in claim 13, wherein said lipid is selected from cholesterol and palmitic acid.

20. The antigen as claimed in claim 19, comprising at least one cholesterol attached to a cysteine, aspartic acid or glutamic acid residue, or else palmitic acid attached to a serum or a threonine.

21. The antigen as claimed in claim 17, wherein it is in the form of lipopeptide vesicles or micelles.

22. An immunogenic composition, comprising an antigen as claimed in claim 1, combined with at least one pharmaceutically acceptable vehicle.

23. The use of an antigen as claimed in claim 1, for producing a vaccine intended for the prevention or treatment of a latent or chronic infection with an intracellular pathogenic microorganism or of associated pathologies.

24. An antibody directed against a type III membrane protein as defined in claim 1, wherein it is produced by immunization of an appropriate animal with said antigen.

25. The antibody as claimed in claim 24, wherein it is selected from monoclonal antibodies and humanized antibodies.

26. A reagent for diagnosing a latent or chronic infection with an intracellular pathogenic microorganism, and associated pathologies, wherein it comprises at least one antibody as claimed in claim 24.

27. The use of an antibody as claimed in claim 24, for preparing a diagnostic reagent intended for detecting a latent or chronic infection with an intracellular pathogenic microorganism, and the associated pathologies.

28. A kit for diagnosing a latent or chronic infection with an intracellular pathogenic microorganism, and the associated pathologies, comprising at least one antibody as claimed in claim 24.

29. A pharmaceutical composition, comprising at least one antibody as claimed in claim 24, combined with at least one pharmaceutically acceptable vehicle.

30. The use of an antibody as claimed in claim 24, for preparing a medicinal product intended for the treatment of a latent or chronic infection with an intracellular pathogenic microorganism and of the associated pathologies.

32. A probe and/or a primer for obtaining a sequence coding for an antigen as claimed in claim 1, comprising a sequence of approximately 10 to 30 nucleotides corresponding to that located at the junction of the sequences encoding a transmembrane domain and of the sequences encoding an extracellular domain adjacent to the above domain, of a type III membrane protein as defined in claim 1.

31. An isolated nucleic acid molecule, comprising a sequence encoding an antigen as claimed.

33. A eukaryotic or prokaryotic cell recombinant vector, comprising an insert consisting of a nucleic acid molecule as claimed in claim 31.

34. A eukaryotic or prokaryotic cell modified with a recombinant vector as claimed in claim 33.