Method for rapid generation of an attenuated RNA virus

Patent metadata

Field	Value
Publication number	US-10619137-B2
Application number	US-201515315687-A
Country	US
Kind code	B2
Filing date	Jun 19, 2015
Priority date	Jun 20, 2014
Publication date	Apr 14, 2020
Grant date	Apr 14, 2020

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.

First claim

The invention claimed is:

1. A method for generating an attenuated RNA virus comprising:
A) 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:
i) 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
ii) the regions of said viral genome which are involved with RNA secondary structure are not modified;

B) generating an attenuated RNA virus by:
i) introducing a promoter of DNA-dependent RNA polymerase in position 5′ and optionally a terminator and a RNA polyadenylation sequence in position 3′ of the re-encoded viral genome as obtained in step A;
ii) amplifying the re-encoded viral genome as prepared in sub-step B) i) including said promoter and optionally said terminator and RNA polyadenylation sequence, in at least 2 overlapping cDNA fragments;
iii) transfecting said cDNA fragments into a host cell;
iv) incubating said host cell of sub-step B) iii); and
v) recovering the attenuated RNA virus from said incubated host cell.

2. The method of claim 1 , wherein in step A, 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.

3. The method of claim 1 , wherein step A 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 of table 1, 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.

4. The method of claim 1 , wherein said virus is a single stranded positive RNA virus.

5. The method of claim 1 , wherein, in step B) i),
said promoter of DNA-dependent RNA polymerase in position 5′ is the human cytomegalovirus promoter (pCMV); and/or
said optional terminator and RNA polyadenylation sequence is respectively the hepatitis delta ribozyme and the simian virus 40 polyadenylation signal (HDR/SV40pA).

6. The method of claim 1 , wherein:
step B) iii) is a step of direct transfection of the cDNA fragments obtained in step B) ii), and
said step B) iii) occurs directly after step B) ii).

7. The method of claim to 1 , wherein step B) iii) is a step of transfection of plasmids or vectors comprising a cDNA fragment obtained in step B) ii), wherein each cDNA fragment is in individual and separate plasmid or vector.

8. The method of claim 1 , wherein the transfected cDNA fragments of step B) iii) spontaneously recombine in the host cells during the incubation step B) iv).

9. The method of claim 1 , wherein said virus is Chikungunya virus and said step A of re-encoding is performed:
in the region coding for the non-structural protein nsP1, wherein the re-encoded cassette is depicted in SEQ ID NO: 63;
in the region coding for the non-structural protein nsP4, wherein the re-encoded cassette is depicted in SEQ ID NO: 64; and
in the region coding for the region overlapping the structural protein E2 and E1, wherein the re-encoded cassette is depicted in SEQ ID NO: 65.

10. The method of claim 1 , wherein said virus is Tick-borne encephalitis virus and said step A of re-encoding step is performed in the NS5 genomic region, wherein the re-encoded cassette is depicted in SEQ ID NO: 66.

11. The method of claim 1 , wherein said virus is Japanese encephalitis virus and said step A is performed in the complete open reading frame (ORF), from the beginning of PrM to the end of NS5 genomic region, wherein at least one re-encoded cassette is 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.

12. The method of claim 1 wherein said method produces a live attenuated vaccine.

13. The method of claim 4 , wherein said virus is a virus selected from the group consisting of flavivirus, alphavirus and enterovirus.

Assignees

• Univ Aix Marseille
• Inst Nat Sante Rech Med

Inventors

• Nougairéde Antoine
• De Fabritus Lauriane
• Aubry Fabien
• De Lamballerie Xavier
• Gould Ernest Andrew

Classifications

• A61K2039/525

  Virus · CPC title

• C12N7/00 Primary

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title

• C12N2770/24151

  Methods of production or purification of viral material · CPC title

• C12N2770/24062

  by genetic engineering · CPC title

• C12N2770/24162

  by genetic engineering · CPC title