Chimeric polynucleotides and polypeptides enabling secretion of a polypeptide of interest in association with exosomes and use thereof for the production of immunogenic compositions
The present invention concerns a chimeric polypeptide comprising several polypeptide domains, which is capable of being secreted in association with membrane vesicles and in particular exosomes. The present invention also concerns a polypeptide constituted by one of said domains for the secretion of a peptide or polypeptide of interest in association with membrane vesicles and in particular exosomes. The invention also concerns the use of polypeptides of the invention and of polynucleotides coding for said polypeptides for the production of immunogenic compositions based on exosomes or based on DNA or for screening protein interactions. The present invention also concerns exploiting the properties of exosomes comprising a polypeptide of the invention and of immunogenic compositions of the invention for the prophylaxis and/or treatment of an infection by a pathogenic agent, a pathogenic organism, a tumour antigen or a cytoplasmic antigen, in particular to elicit or promote, in vivo, in a host, in particular in a human or non-human mammal or a bird, a humoral and/or cellular response against a virus, bacterium, parasite or tumour.
1. A membrane vesicle comprising a chimeric polypeptide, wherein said chimeric polypeptide comprises or consists of the following domains:
(i) a peptide or polypeptide of interest;
(ii) a transmembrane domain having the capacity to anchor in the lipid bilayer of a cell membrane; and
(iii) a mutated cytoplasmic domain (CD) of the transmembrane protein (TM) of the Bovine Leukaemia Virus (BLV), wherein a sequence of the mutated cytoplasmic domain comprises or consists of the sequence SEQ ID NO: 12, wherein said mutated cytoplasmic domain has the capacity of addressing of said chimeric polypeptide to membrane vesicles and/or to the cell compartment(s) involved in the formation of said membrane vesicles, and wherein the sequence of said mutated cytoplasmic domain is a 45-80 amino acid sequence.
2. The membrane vesicle of claim 1 , which is an exosome.
3. The membrane vesicle of claim 1 , wherein the peptide or polypeptide of interest is exposed, partly or completely, on the outside of said membrane vesicle.
4. An immunogenic composition, which comprises one or several membrane vesicle(s) of claim 1 .
5. The immunogenic composition of claim 4 , which is a drug.
6. The immunogenic composition of claim 4 or 5 , which is a drug for the prophylaxis and/or treatment of a bacterial, viral or parasitic infection, or of a tumour, by induction or stimulation of a humoral and/or cellular response against said tumour, virus, bacterium or parasite.
7. A recombinant exosome-producing cell, which comprises or has absorbed a membrane vesicle according to claim 1 .
8. A method for in vitro production of membrane vesicles, which comprises bringing an exosome-producing cell into contact with one or several membrane vesicle(s) according to claim 1 ;
a) growing said exosome-producing cell; and
b) recovering the membrane vesicles produced by said exosome-producing cell.
9. The method of claim 8 , which further comprises an intermediate step between steps a) and b), during which said cell is selected and/or stimulated to induce and/or increase the secretion of exosomes or to induce a specificity in the composition of the exosomes in certain cellular proteins.
10. A method for preparing a polyclonal serum directed against one or several antigenic peptide(s) or polypeptide(s) of interest expressed on the surface of membrane vesicles, comprising the following steps:
a) administering to a non-human animal, the membrane vesicle of claim 1 , or the immunogenic composition of claim 4 , associated or not with an adjuvant, to induce the production of antibodies by said non-human animal; and
b) recovering from said non-human animal the antibodies, which bind to said antigenic peptide(s) or polypeptide(s) of interest.
11. A method for preparing monoclonal antibodies directed against one or several antigenic peptide(s) or polypeptide(s) expressed on the surface of membrane vesicles, comprising the following steps:
a) fusing, with myeloma cells, spleen cells previously obtained from a host to which the membrane vesicle of claim 1 , or the immunogenic composition of claim 4 has been administered to produce hybridomas;
b) growing said hybridomas under conditions allowing the production of antibodies;
c) recovering the monoclonal antibodies, which are directed against said antigenic peptide(s) or polypeptide(s) of interest.
12. The membrane vesicle of claim 1 , wherein the sequence of the mutated cytoplasmic domain is a 45-60 amino acid sequence.
13. The membrane vesicle of claim 1 , wherein the sequence of the mutated cytoplasmic domain is devoid of the sequence KCLTSRLLKLLRQ (SEQ ID NO: 126).
14. The membrane vesicle of claim 1 , wherein said peptide or polypeptide of interest (i) comprises or consists of
one or several domain(s) of an extracellular protein; or
one or several cytoplasmic domain(s) of a transmembrane protein; or
one or several domain(s) of a cytosolic protein.
15. The membrane vesicle of claim 1 , wherein said peptide or polypeptide of interest (i) is a viral peptide or polypeptide, or a bacterial peptide or polypeptide, or a tumor antigen.
16. The membrane vesicle of claim 1 , wherein said peptide or polypeptide of interest (i) is
the ectodomain of the haemagglutinin (HA) of an influenza virus, or a fragment comprising or consisting of one or several epitope(s) of said ectodomain; or
the ectodomain of the HA protein of the H5N1 avian influenza virus, or a fragment comprising or consisting of one or several epitope(s) of said ectodomain; or
the ectodomain of the Spike protein of a coronavirus of severe acute respiratory syndrome (SARS) or a fragment comprising or consisting of one or several epitope(s) of said ectodomain.
17. The membrane vesicle of claim 1 , wherein said transmembrane domain (ii) is the transmembrane domain of the membrane protein of a virus, a bacterium or a tumor; or is a mutated derivative of said transmembrane domain, wherein said mutated derivative of transmembrane domain is defined by substitution, deletion and/or insertion of one or several residue(s) in the sequence of said transmembrane domain and by the capacity to anchor in the lipid bilayer of a cell membrane, and wherein the sequence of said mutated derivative of transmembrane domain consists of 10-50 amino acids and is at least 80% identical to the sequence of said transmembrane domain.
18. The membrane vesicle of claim 1 , wherein said peptide or the polypeptide of interest (i) and said transmembrane domain (ii) are not fragments of the same protein.
19. The membrane vesicle of claim 1 , wherein said chimeric polypeptide further comprises at least one linker linking two of said domains (i), (ii) and (iii).
20. The recombinant exosome-producing cell of claim 7 , which is a cell of the immune system.
21. The recombinant exosome-producing cell of claim 7 , which is a lymphocyte.