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, 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 a 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,
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 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 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 position 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.