Papilloma Virus-Like Particles, Fusion Proteins as Well as Processes for Their Production

Abstract

The invention relates to the recombinant production of proteins as well as VLPs which are suitable as a vaccine for therapeutic and prophylactic vaccination. The invention also relates to processes for the production and purification of recombinant papilloma virus proteins and fusion proteins.

1 - 53 . (canceled) 
     
     
         54 . A method for the production of papilloma virus-like particles, wherein said virus-like particles are formed after expression of viral structure proteins L1 or L1 and L2 and wherein one or more sections of the L1 protein are deleted, wherein the ability to form virus-like particles remains, wherein the expression of the L1 and/or L2 proteins is carried out in viral, eukaryotic or prokaryotic vectors. 
     
     
         55 . The method according to  claim 54  wherein the deleted regions in the L1 protein comprise amino acid sequences 311-351, 331-371, 391-431 or bovine papilloma virus type 1. 
     
     
         56 . The method according to  claim 54  wherein the deleted regions in the L1 protein comprise amino acid sequence 306-315 of the human papilloma virus 16. 
     
     
         57 . The method according to  claim 54  wherein the ability to form virus-like particles is increased compared to the native formation of virus-like particles. 
     
     
         58 . The method according to  claim 57  wherein at least one of the deleted regions in the L1 protein comprises C-terminal amino acid sequences. 
     
     
         59 . The method according to  claim 58  wherein the at least one deleted section in the C-terminal region in the L1 protein comprises an amino acid sequence having a length of about 1 to 34 amino acids, preferably 1 to 26 amino acids. 
     
     
         60 . The method according to  claim 57  wherein the production of virus-like particles is increased 10 times to 100 times. 
     
     
         61 . The method according to  claim 57  wherein the deleted regions in the L1 protein comprise 26 C-terminal amino acids of bovine papilloma virus. 
     
     
         62 . The method according to  claim 61  wherein the 26 amino acid C-terminal deletion comprises the amino acids Gly-Ala-Gly-Cys-Ser-Thr-Val-Arg-Lys-Arg-Arg-Ile-Ser-Gln-Lys-Thr-Ser-Ser-Lys-Pro-Ala-Lys-Lys-Lys-Lys-Lys, corresponding to the nucleotide position 7016 to 7093 GGGGCAGGAT GTTCAACTGT GAGAAAACGA AGAATTAGCC AAAAAACTTC CAGTAAGCCT GCAAAAAAAA AAAAAAAA of bovine papilloma virus type 1. 
     
     
         63 . The method according to  claim 57  wherein the deleted regions in the L1 protein concern 34 C-terminal amino acids of human papilloma virus type 16 (HPV 16). 
     
     
         64 . The method according to  claim 63  wherein the 34 amino acid C-terminal deletion comprises the amino acids Ala-Gly-Leu-Lys-Ala-Lys-Pro-Lys-Phe-Thr-Leu-Gly-Lys-Arg-Lys-Ala-Thr-Pro-Thr-Thr-Ser-Ser-Thr-Ser-Thr-Thr -Ala-Lys-Arg-Lys-Lys-Arg-Lys-Leu, corresponding to the nucleotide position 7052 to 7153 GCAGGATTGA AGGCCAAACC AAAATTTACA TTAGGAAAAC GAAAAGCTAC ACCCACCACC TCATCTACCT CTAGAACTGC TAAACGCAAA AAACGTAAGC TG of human papilloma virus type 16 (HPV 16). 
     
     
         65 . The method according to  claim 57  wherein the deletion of the L1 protein comprises the nuclear localization signal (NLS). 
     
     
         66 . The method according to  claim 57  wherein virus-like particle production from the L1 proteins or L1 and L2 proteins takes place in the cytoplasm. 
     
     
         67 . The method according to  claim 57  wherein virus-like particles are secreted in the supernatant. 
     
     
         68 . The method according to  claim 67  wherein 5 to 10% of the particles are secreted in the supernatant. 
     
     
         69 . The method according to  claim 57  wherein the expression of L1 proteins or L1 and L2 proteins takes place in  E. coli.    
     
     
         70 . The method according to  claim 69  wherein 6 histidines have been inserted at the C-terminal deletion in the L1 protein. 
     
     
         71 . The method according to  claim 70  wherein the production of virus-like particles after expression of L1 proteins or L1 and L2 proteins takes place in  E. coli.    
     
     
         72 . The method according to  claim 57  wherein the deleted regions in the L1 protein concern the amino acid sequences 311-351, 331-371, 391-431 of bovine papilloma virus type 1. 
     
     
         73 . The method according to  claim 72  wherein the deleted regions in the L1 protein concern the amino acid sequence 306-315 of human papilloma virus 16. 
     
     
         74 . The method according to  claim 54  wherein the L1 and/or L2 proteins are produced by expression in vaccinia recombinants. 
     
     
         75 . The method according to  claim 54  wherein the expression of the L1 and/or L2 proteins and fusion proteins is carried out in baculo viruses or in yeasts. 
     
     
         76 . The method according to  claim 73  wherein the expression of the L1 and/or L2 proteins is carried out in  E. coli.    
     
     
         77 . The method according to  claim 54  wherein purification of L1 proteins is carried out by Nickel affinity chromatography and renaturation in a buffer comprising 150 mM NaCl, 1 mM CaCl 2 , 0.01% Triton-X 100, 10 mM Hepes pH 7.4. 
     
     
         78 . The method according to  claim 77  wherein the proteins are present at the time of the purification in denaturation buffer. 
     
     
         79 . The method according to  claim 78  wherein the denaturation buffer is 6 M guanidine hydrochloride. 
     
     
         80 . The method according to  claim 79  wherein assembly of the virus-like particles takes place after dialysis. 
     
     
         81 . The method according to  claim 80  wherein the dialysis is carried out against a buffer comprising 150 mM NaCl, 25 mM Ca 2+ , 10 DMSO, 0.1% Triton X 100 and 10 mM tris acetic acid at pH=5.0.