Live-attenuated yellow fever virus strain adapted to grow on Vero cells and vaccine composition comprising the same
AssigneeSanofi Pasteur Limited
InventorsManuel Vangelisti, Nathalie Mantel, Yves Girerd-Chambaz, Fabienne Piras
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Abstract
The invention relates to a live-attenuated yellow fever virus strain adapted to grow on Vero cells 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.
Claims12
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.
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.
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.
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 (log10 MLD50/mL) of the injected clones;
- and
- i) selecting the injected clones that have a log10 MLD50/mL that is smaller than the parent live-attenuated yellow fever virus strain.
Dependencies are inferred from the claim text.