Mutated parvovirus structural proteins as vaccines

Abstract

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 - 33 . (canceled) 
     
     
         34 . 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).   
     
     
         35 . The method of  claim 34  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. 
     
     
         36 . The method according to  claim 34 , wherein the selecting step is performed using a binder immobilized on a carrier. 
     
     
         37 . The method according to  claim 34 , wherein the selection step is performed using a binder in suspension. 
     
     
         38 . The method according to  claim 34 , wherein selected parvovirus virion is further selected for non-binding to a second binder. 
     
     
         39 . The method according to  claim 34 , wherein the 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).   
     
     
         40 . The method according to  claim 34 , wherein the parvovirus mutated structural protein further comprises at least one random mutation compared to the respective parvovirus wild type structural protein. 
     
     
         41 . The method according to  claim 40 , 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). 
     
     
         42 . The method according to  claim 40 , 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. 
     
     
         43 . The method according to  claim 34 , wherein the library of parvovirus virions is produced by transfecting a plasmid library into production cells under suitable conditions wherein a low copy number of viral genomes equal to or less than 100 genomes per cell is used, resulting in a geno-/phenotypically coupled library. 
     
     
         44 . The method according to  claim 34 , wherein the library of parvovirus is produced by transducing the library into production cells under suitable conditions at a ratio of genomes per cell of 5 to 5,000 and selecting transduction conditions to be independent from infection pathways resulting in a geno-/phenotypically coupled library. 
     
     
         45 . The method according to  claim 44 , wherein the transduction of the parvovirus virion library is performed using production cells seeded on immobilized parvovirus virions. 
     
     
         46 . The method according to  claim 43  or  44 , wherein the library has a genotype/phenotype coupling of at least 5%. 
     
     
         47 . The method according to  claim 34 , wherein the parvovirus mutant structural protein comprises at least one insertion of 4-30 amino acids. 
     
     
         48 . The method according to  claim 34 , wherein the insertion comprises two cysteins capable of forming a disulfide bond to form a loop consisting of inserted amino acids. 
     
     
         49 . The method according to  claim 34 , wherein the parvovirus mutated structural protein comprises at least one further mutation selected from a point mutation, an internal or terminal deletion, a second insertion and a substitution. 
     
     
         50 . The method according to  claim 49 , wherein the second insertion is a tag useful for binding to a ligand. 
     
     
         51 . The method according to  claim 47  or  49 , 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.   
     
     
         52 . A parvovirus mutated structural protein obtained by the method of  claim 34 . 
     
     
         53 . A parvovirus mutated structural protein which comprises at least one B-cell epitope heterologous to the parvovirus wherein the B-cell epitope is located on the surface of the virus. 
     
     
         54 . The parvovirus mutated structural protein according to  claim 53  wherein the B-cell epitope is a tolerogen-derived epitope. 
     
     
         55 . The parvovirus mutated structural protein according to  claim 53  wherein the B-cell epitope is a part of a protein selected from the group consisting of a tumor antigen, a misfolded protein, a serum protein, a membrane protein, a TNF-family member, an interleukin, CETP, CD20, acetylcholine receptors, IL13R, EGFR, IgE, Melan A, HMW MAA, CA125, Her2/NEU, L1 cell adhesion molecule, VEGF, EGFR, CD20, TNF-α, IL-6, IL9, IL-13, IL-17, and β-amyloid. 
     
     
         56 . The parvovirus mutated structural protein according to  claim 53 , wherein the parvovirus mutated structural protein is capable of inducing an immunoglobulin capable of binding to a target antigen. 
     
     
         57 . The parvovirus mutated structural protein according to  claim 53  wherein the B-cell epitope comprises an anti-idiotypic epi-/mimotope of an anti-IgE antibody, and/or an IgE tolerogen-derived epi-/mimotope. 
     
     
         58 . The parvovirus mutated structural protein according to  claim 53  wherein the B-cell epitope it is inserted into (i) I-453 or (ii) I-587 or (iii) I-453 and I-587. 
     
     
         59 . The parvovirus mutated structural protein according to  claim 53 , wherein the protein is fused to a second protein or peptide. 
     
     
         60 . A multimeric structure comprising a parvovirus mutated structural protein of  claim 53 . 
     
     
         61 . A nucleic acid coding for a parvovirus mutated structural protein according to  claim 53 . 
     
     
         62 . A virus comprising a parvovirus mutated structural protein according to  claim 53  or a nucleic acid according to  claim 61 . 
     
     
         63 . A method of preparing a structural protein according to  claim 52 , the method comprising the steps of:
 a) expressing a nucleic acid coding for a parvovirus mutated structural protein which comprises at least one B-cell epitope heterologous to the parvovirus wherein the B-cell epitope is located on the surface of the virus by cultivating a cell comprising the nucleic acid under suitable conditions, and   b) isolating the expressed parvovirus mutated structural protein of step a).   
     
     
         64 . A medicament comprising at least one parvovirus mutated structural protein according to any of  claims 53  or a nucleic acid according to  claim 61 . 
     
     
         65 . The medicament according to  claim 64 , wherein the medicament is a vaccine. 
     
     
         66 . A method of preventing or treating an autoimmune disease, a tumor disease, an allergic disease, a metabolic disease, an inflammatory disease, a neurological disease or to be used in ophthalmology, comprising the step of administering to a subject need thereof a therapeutically effective amount of the medicament of  claim 64 .