Live-attenuated yellow fever virus strain adapted to grow on Vero cells and vaccine composition comprising the same

1. A live-attenuated yellow fever virus 17D substrain adapted to grow on Vero cells, obtained from a parent yellow fever virus 17D substrain that is not adapted to grow on Vero cells, wherein said live-attenuated yellow fever virus strain is less neurovirulent than said parent yellow fever virus 17D substrain in a mouse lethal dose 50 (MLD50) test,

wherein the parent yellow fever virus 17D substrain comprises an RNA sequence of SEQ ID NO. 2.

2. A live-attenuated yellow fever virus 17D substrain comprising a nucleic acid comprising

a mutation in the codon for the amino acid at position 480 of the envelope protein (E) which results in an amino acid change from valine to leucine.

3. The live-attenuated yellow fever virus 17D substrain according to claim 2 , wherein the nucleic acid further comprises

a mutation in the codon for the amino acid at position 65 of the non-structural protein 2A (NS2a) which results in an amino acid change from methionine to valine.

4. The live-attenuated yellow fever virus 17D substrain according to claim 3 , wherein the nucleic acid further comprises a mutation in the codon for the amino acid at position 19 of the non-structural protein 4A (NS4a) which results in a codon change from AAA to AAG.

5. The live-attenuated yellow fever virus 17D substrain according to claim 4 , wherein the nucleic acid further comprises a mutation in the codon for the amino acid at position 145 of the envelope protein (E) which results in a codon change from GUA to GUU.

6. A live-attenuated yellow fever virus 17D substrain comprising a nucleic acid comprising a mutation in the codon for the amino acid at position 65 of the non-structural protein 2A (NS2a) which results in an amino acid change from methionine to valine in SEQ ID NO. 16, wherein the NS2a protein comprises a sequence that is at least 90%, 95%, 98% or 100% identical to sequence of SEQ ID NO. 16.

7. A method for obtaining a live-attenuated yellow fever virus strain adapted to grow on Vero cells, the method comprising the steps of:

a) purifying viral genomic RNA from a parent live-attenuated yellow fever virus strain that is not adapted to grow on Vero cells;

b) transfecting first Vero cells with the purified viral genomic RNA purified;

c) growing the transfected first Vero cells in a culture medium, whereby a first population of yellow fever virus is obtained and recovered;

d) amplifying the recovered first population of yellow fever virus two or more times on second Vero cells, whereby a second population of yellow fever virus is obtained;

e) cloning the second population of yellow fever virus by two or more successive plaque purifications on third Vero cells, whereby a plurality of yellow fever virus clones is obtained;

f) individually amplifying each of the recovered yellow fever virus clones two or more times on fourth Vero cells, whereby a plurality of yellow fever virus strains is obtained and recovered;

g) injecting individual clones of the recovered plurality of yellow fever virus strains via intra-cerebral administration into mice;

h) determining the mouse lethal dose 50 (log 10 MLD 50 /mL) of the injected clones; and

i) selecting the injected clones that have a log 10 MLD 50 /mL that is smaller than the parent live-attenuated yellow fever virus strain.

8. The method of claim 7 , wherein the parent live-attenuated yellow fever virus strain is adapted to grow on eggs.

9. The method of claim 8 , wherein the eggs are embryonated hen eggs.

10. The method of claim 7 , wherein the culture medium is serum-free.

11. The method of claim 7 , wherein the culture medium is free of any human or animal-derived substances.

12. A live-attenuated yellow fever virus strain obtained by the method of claim 7 .