Method for rapid generation of an attenuated rna virus

Abstract

The present invention harnesses the power of mutagenesis to produce an attenuated RNA virus in a very short period, i.e. as soon as the complete sequence of the target virus is known and an infectious genome can be produced.

1 - 13 . (canceled) 
     
     
         14 . A vaccine comprising cDNA fragments, wherein said cDNA fragments are overlapping cDNA fragments obtained by:
 introducing a promoter of DNA-dependent RNA polymerase in position 5′ and optionally a terminator and a RNA polyadenylation sequence in position 3′ of a re-encoded viral genome;   amplifying said re-encoded viral genome in at least 2, preferably at least 3, 4, 5 or 6 overlapping cDNA fragments;   wherein said re-encoded viral genome is obtained by re-encoding the viral genome of an infectious RNA virus by randomly substituting a part of the nucleotide codons of the entire viral genome of said infectious RNA virus by another nucleotide codon encoding for the same amino acid,   with the proviso that:   the number and position of rare nucleotide codons present in said viral genome are not modified, said rare nucleotide codons being CGU, CGC, CGA, CGG, UCG, CCG, GCG and ACG; and   the regions of said viral genome which are involved with RNA secondary structure are not modified.   
     
     
         15 . The vaccine according to  claim 14 , wherein about 1 to about 20% of the nucleotide codons of the entire viral genome of said infectious RNA virus are substituted by another nucleotide codon encoding for the same amino acid. 
     
     
         16 . The vaccine according to  claim 14 , wherein the step of re-encoding the viral genome is performed by:
 determining the amino acid sequence encoded by the entire viral genome of the infectious RNA virus, and determining each nucleotide codon encoding each amino acid; and   substituting 1 to 20% of the nucleotide codon of the viral genome encoding an amino acid comprising Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q),Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Phe (F), Pro (P), Ser (S), Thr (T), Tyr (Y), or Val (V) by a different nucleotide codon encoding the same amino acid as specified in the following table:   
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   Amino acid 
                   Nucleotide codon 
                 
                     
                     
                 
                     
                   Ala, A 
                   GCU, GCC, GCA 
                 
                     
                   Arg, R 
                   AGA, AGG 
                 
                     
                   Asn, N 
                   AAU, AAC 
                 
                     
                   Asp, D 
                   GAU, GAC 
                 
                     
                   Cys, C 
                   UGU, UGC 
                 
                     
                   Gln, Q 
                   CAA, CAG 
                 
                     
                   Glu, E 
                   GAA, GAG 
                 
                     
                   Gly, G 
                   GGU, GGC, GGA 
                 
                     
                   His, H 
                   CAU, CAC 
                 
                     
                   Ile, I 
                   AUU, AUC, AUA 
                 
                     
                   Leu, L 
                   UUA, UUG, CUU, CUC, CUA, CUG 
                 
                     
                   Lys, K 
                   AAA, AAG 
                 
                     
                   Phe, F 
                   UUU, UUC 
                 
                     
                   Pro, P 
                   CCU, CCC, CCA 
                 
                     
                   Ser, S 
                   UCU, UCC, UCA, AGU, AGC 
                 
                     
                   Thr, T 
                   ACU, ACC, ACA 
                 
                     
                   Tyr, Y 
                   UAU, UAC 
                 
                     
                   Val, V 
                   GUU, GUC, GUA, GUG 
                 
                     
                     
                 
             
                
                
                
               
               
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         17 . The vaccine according to  claim 14 , wherein said virus is a single stranded positive RNA virus. 
     
     
         18 . The vaccine according to  claim 17 , wherein said single stranded positive RNA virus is selected from the group consisting of flavivirus, alphavirus and enterovirus. 
     
     
         19 . The vaccine according to  claim 14 , wherein said virus is Chikungunya virus and the step of re-encoding is performed:
 in the region coding for the non-structural protein nsP1, by the re-encoded cassette depicted in SEQ ID No: 63;   in the region coding for the non-structural protein nsP4, by the re-encoded depicted in SEQ ID No: 64; and   in the region coding for the region overlapping the structural protein E2 and E1, by the re-encoded cassette depicted in SEQ ID No: 65.   
     
     
         20 . The vaccine according to  claim 14 , wherein said virus is Tick-borne encephalitis virus and said the step of re-encoding is performed in the NS5 genomic region, by the re-encoded cassette depicted in SEQ ID No: 66. 
     
     
         21 . The vaccine according to  claim 14 , wherein said virus is Japanese encephalitis virus and said the step of re-encoding is performed in the complete open reading frame (ORF), from the beginning of PrM to the end of NS5 genomic region by at least one re-encoded cassette selected from the group consisting of SEQ ID No: 67; SEQ ID No: 68; SEQ ID No: 69; SEQ ID No: 70; SEQ ID No: 71; and SEQ ID No: 72.