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-9655960-B2
Publication Date
2017-05-23
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 whole-virion Yellow Fever vaccine made from chemically inactivated virus. The vaccine is produced using a modified Yellow Fever 17D virus that preserves neutralizing epitopes and is associated with increased propagation in Vero cells and higher yield in conditioned medium relative to unmodified Yellow Fever virus.
The modified virus is associated with specific codon/amino-acid mutations, including an envelope protein mutation at amino acid position 160 (Lys→Arg, E160 Lys→Arg). Additional mutations are described in NS1 at amino acid 317 (Thr317→Ile), NS2A at amino acid 170 (Phe170→Leu), and NS4B at amino acid 113 (Ile113→Met), with NS1 and NS2A mutations included and NS4B mutations optionally included.
The description provides a rationale linking E160, within the envelope protein molecular hinge region between domains I and II, to pKa/proton affinity and enhanced membrane fusion/infectivity, and correspondingly higher titers. A manufacturing context is described that uses serum-free Vero processes, purification, and β-propiolactone inactivation for the inactivated whole-virion vaccine.
Claims Coverage
The independent claim describes a modified Yellow Fever virus strain with specific codon-level mutations and performance attributes in Vero cells, and the dependent claims extend to an inactivated vaccine, immune-response administration to a subject, and a vaccine-making workflow from infected-cell culture to live virus bulk purification and inactivation. The inventive features center on mutation combinations and the resulting increased propagation and yield.
Codon-defined NS1, NS2A, and envelope E160 mutations for increased Vero propagation and conditioned-medium yield
A modified Yellow Fever virus strain comprising nucleic acid sequence mutations encoding NS1 at codon for amino acid position 317 with a threonine to isoleucine change and NS2A at codon for amino acid position 170 with a phenylalanine to leucine change, optionally including an NS4B codon change at amino acid position 113 (isoleucine to methionine), and further including an envelope protein codon mutation at amino acid position 160 (lysine to arginine), wherein the modified strain has increased propagation in Vero cells and a higher yield in the conditioned medium relative to unmodified Yellow Fever virus.
Inactivated form of the modified Yellow Fever virus strain
An inactivated Yellow Fever virus comprising the modified Yellow Fever virus strain as defined in the modified Yellow Fever virus strain feature.
Vaccine comprising the inactivated virus
A vaccine comprising the inactivated virus as defined in the inactivated-form feature.
Inducing an immune response to Yellow Fever virus by administering the vaccine
A method that induces an immune response to Yellow Fever virus in a subject by administering the vaccine as defined in the vaccine-comprising feature.
Administration to a subject at risk but not yet infected
The method of inducing an immune response applies to a subject who is at risk of developing a Yellow Fever virus infection but does not yet have a Yellow Fever virus infection.
Vaccine production by culturing infected cells, purifying to a live virus bulk, and inactivating
A method for making a vaccine by culturing cells infected with the modified Yellow Fever virus strain, purifying the infected virus to form a live virus bulk, and then inactivating said live virus bulk.
Across the independent claim and its dependents, the core coverage is a mutation-defined Yellow Fever virus strain characterized by increased Vero-cell propagation and conditioned-medium yield, followed by making an inactivated whole-virion vaccine from that modified strain, administering it to induce immune response, and producing it via culture of infected cells, purification to a live virus bulk, and inactivation.
Stated Advantages
Increased propagation in Vero cells.
Higher yield in the conditioned medium relative to unmodified Yellow Fever virus.
The described rationale links the E160 hinge region pKa/proton affinity to enhanced membrane fusion/infectivity and correspondingly higher titers.
Robust neutralizing-antibody responses with alum-adjuvanted inactivated vaccine versus limited response with live YF Vax.
Documented Applications
Inducing an immune response to Yellow Fever virus in a subject by administering a vaccine comprising an inactivated Yellow Fever virus with the modified strain.
Use of the method for subjects at risk of developing Yellow Fever virus infection but who do not yet have a Yellow Fever virus infection.
Vaccine manufacturing using a workflow including culturing cells infected with the modified Yellow Fever virus strain, purifying the infected virus to form a live virus bulk, and inactivating the live virus bulk.
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