Mutated parvovirus structural proteins as vaccines
The present invention is related to a method for identifying a parvovirus mutated structural protein capable of specifically binding to a binder for an antigen, a parvovirus mutated structural protein which comprises at least one B-cell epitope heterologous to the parvovirus, a multimeric structure comprising the protein, a nucleic acid encoding the protein, a virus or cell comprising the protein, a method of preparing the protein, a medicament comprising the protein, nucleic acid or multimeric structure and its use.
1. A method for identifying a parvovirus mutated structural protein capable of specifically binding to a binder for an antigen, wherein the binder comprises a therapeutic antibody, a therapeutic single chain antibody, or an antibody fragment of a therapeutic antibody, the method comprising the steps of:
a) providing a library of parvovirus virions expressing at least one mutated parvovirus structural protein,
b) providing a binder for an antigen,
c) selecting at least one parvovirus virion specifically binding to the binder, and
d) identifying
i) the parvovirus mutated structural protein or a mutated part thereof, or
ii) the gene or a mutated part thereof encoding the parvovirus mutated structural protein of the parvovirus virion selected in step c).
2. The method of claim 1 wherein the at least one parvovirus virion selected in step c) is amplified by viral replication and subsequent packaging in a production cell under suitable conditions, and wherein at least steps b) to c) are repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.
3. The method of claim 1 , wherein the selecting step is performed using a binder immobilized on a carrier.
4. The method of claim 1 , wherein the selecting step is performed using the binder in suspension.
5. The method of claim 1 , wherein said selecting at least one parvovirus virion further comprises selecting for non-binding to a second binder.
6. The method of claim 1 , wherein said method further comprises the steps of
e) randomizing the gene encoding the parvovirus mutated structural protein by inserting a randomly or partially randomly generated sequence into the coding region of the parvoviral structural gene,
f) packaging the randomized genes into a further library of parvoviruses, and
g) repeating the steps a)-d).
7. The method of claim 1 , wherein the parvovirus mutated structural protein further comprises at least one random mutation compared to the respective parvovirus wild-type structural protein.
8. The method of claim 7 , wherein the parvovirus is selected from the group consisting of adeno-associated virus (AAV), bovine AAV (b-AAV), canine AAV (CAAV), canine parvovirus (CPV), mouse parvovirus, minute virus of mice (MVM), B19, H1, avian AAV (AAAV), feline panleukopenia virus (FPV), and goose parvovirus (GPV).
9. The method of claim 8 , wherein the AAV is AAV-1, AAV-2, AAV-3b, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11 or AAV-12.
10. The method of claim 1 , wherein the library has a multiplicity of parvoviral mutants of greater than 10 5 .
11. The method of claim 1 , wherein the parvovirus mutant structural protein comprises at least one insertion of 4-30 amino acids.
12. The method of claim 11 , wherein said insertion is of 5-20 amino acids.
13. The method of claim 12 , wherein said insertion is 5-15 amino acids.
14. The method of claim 11 , wherein the insertion comprises two cysteines capable of forming a disulfide bond to form a loop consisting of the inserted amino acids.
15. The method of claim 11 , wherein
a) the insertion is inserted into one or more positions selected from the group consisting of the positions before and/or after amino acids I-1, I-34, I-138, I-139, I-161, I-261, I-266, I-381, I-447, I-448, I-453, I-459, I-471, I-534, I-570, I-573, I-584, I-587, I-588, I-591, I-657, I-664, I-713, and I-716; or
b) the insertion is inserted into two positions selected from the group consisting of the positions before and/or after amino acids I-261, I-453, I-534, I-570, I-573, and I-587.
16. The method of claim 15 , wherein the insertion is inserted at the positions before and/or after amino acids I-261, I-453, I-534, I-570, I-573, or I-587.
17. The method of claim 15 , wherein said two positions are I-261 in combination with I-587 or I-261 in combination with I-453.
18. The method of claim 1 , wherein the parvovirus mutated structural protein comprises at least one further mutation selected from the group consisting of a point mutation, an internal or terminal deletion, a second insertion, and a substitution.
19. The method of claim 18 , wherein said further mutation is a second insertion and said second insertion is internal or a N- or C-terminal fusion, and has a length of 4 to 40, 5 to 30, or 7 to 20 amino acids.
20. The method of claim 19 , wherein the second insertion is a tag useful for binding to a ligand.
21. The method of claim 1 , wherein said parvovirus mutated structural protein comprises at least one B-cell epitope heterologous to the parvovirus, wherein the B-cell epitope is located on the surface of the virus.
22. The method of claim 21 , wherein said B-cell epitope is a tolerogen-derived epitope.
23. The method of claim 1 , wherein the therapeutic antibody is a monoclonal antibody or a fragment thereof.
24. The method of claim 1 , wherein the binder consists of a therapeutic antibody, a therapeutic single chain antibody, or an antibody fragment of a therapeutic antibody.