High yield yellow fever virus strain with increased propagation in cells
Inventors
Lee, Cynthia K. • Monath, Thomas P. • Guertin, Patrick M. • Hayman, Edward G.
Assignees
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Publication Number
US-8741312-B2
Publication Date
2014-06-03
Expiration Date
Abstract
The invention provides a an inactive, non-replicating vaccine comprising whole virion, chemically inactivated Yellow Fever virus which is inactivated using a method that ensures preservation of critical, neutralizing epitopes. The Yellow Fever virus has been adapted to propagate in cells to higher yields than the unadapted virus. The invention also provides methods for preventing Yellow Fever viral infection.
Core Innovation
The invention relates to an inactivated, non-replicating Yellow Fever vaccine comprising chemically inactivated whole virions produced from Vero-cell cultures. The modified Yellow Fever virus strain results in increased propagation in cells and a higher yield in the conditioned medium of a cell culture relative to Yellow Fever virus.
The modified Yellow Fever virus strain comprises a nucleic acid molecule encoding an envelope protein with a mutation in the codon for the amino acid at position 160. The codon-position 160 change is in the molecular hinge region, and the mutation changes AAG to AGG, AGA, CGC, CGA, CGG or CGU.
The codon-position 160 envelope mutation can be optionally combined with additional mutations including NS1 (Thr317→Ile), NS2A (Phe170→Leu), and NS4B (Ile113→Met). The partial content reports increased titers between serial passages and characterization of propagation and yield in conditioned medium using growth curves and plaque assay results.
Inactivated formulations using beta-propiolactone (BPL) and alum elicit robust neutralizing antibody responses in mice as measured by PRNT, with higher neutralizing antibody titers using alum versus Freund’s and no-adjuvant groups compared to a live 17D baseline response.
Claims Coverage
The partial claim set includes one independent claim directed to a modified Yellow Fever virus strain with increased propagation and conditioned-medium yield due to an envelope codon-position 160 mutation. Dependent claims refine the mutation context, specify an encoded envelope sequence by reference to SEQ ID NO: 4, and narrow to inactivated vaccine compositions including specified inactivation and adjuvant choices.
Increased propagation via envelope codon-position 160 mutation
A modified Yellow Fever virus strain results in increased propagation in cells and a higher yield in the conditioned medium of a cell culture relative to Yellow Fever virus, wherein the modified Yellow Fever virus strain comprises a nucleic acid molecule encoding an envelope protein with a mutation in the codon for the amino acid at position 160 that changes AAG to AGG, AGA, CGC, CGA, CGG or CGU.
Mutation context with distance and side-chain pKa increase
The modified Yellow Fever virus strain includes a nucleic acid molecule encoding an envelope protein with a mutated codon within 10 Angstroms or twenty amino acids from amino acid 160, where the mutated position has a side-chain pKa higher than the original amino acid.
Envelope sequence specified as SEQ ID NO: 4
The modified Yellow Fever virus strain includes a nucleic acid molecule whose nucleotide sequence encodes a modified Yellow Fever virus envelope protein sequence specified as SEQ ID NO. 4.
Molecular hinge region mutation with increased side-chain pKa
A modified Yellow Fever virus strain includes a nucleic acid molecule encoding an envelope protein with a mutated codon in the molecular hinge region between Domain I and II, where the mutation increases the side-chain pKa compared to the original amino acid.
Inactivated modified Yellow Fever virus as a vaccine
A vaccine includes an inactivated modified Yellow Fever virus strain as defined in the modified Yellow Fever virus strain that results in increased propagation and higher conditioned-medium yield via an envelope codon-position 160 mutation.
Beta-propiolactone inactivation of the modified virus
An inactivated modified Yellow Fever virus is inactivated using beta-propiolactone.
Across the independent claim and its dependents, the core claim is directed to a modified Yellow Fever virus strain with increased propagation and conditioned-medium yield enabled by an envelope codon-position 160 mutation (AAG to AGG/AGA/CGC/CGA/CGG/CGU). Dependent claims further narrow the mutation’s location and context, specify an encoded envelope sequence by SEQ ID NO: 4, and restrict vaccine embodiments to inactivated forms including beta-propiolactone inactivation.
Stated Advantages
Increased propagation in cells.
Higher yield in the conditioned medium of a cell culture relative to Yellow Fever virus.
Robust neutralizing antibody responses in mice as measured by PRNT.
Higher neutralizing antibody titers using alum versus Freund’s and no-adjuvant groups compared to a live 17D baseline response.
Documented Applications
An inactivated, non-replicating Yellow Fever vaccine comprising chemically inactivated whole virions produced from Vero-cell cultures.
Mouse immunogenicity study reporting neutralizing antibody responses measured by PRNT, including comparisons of alum, Freund’s, and no-adjuvant groups relative to a live 17D baseline.
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