Adapted enterovirus D68 that propagates to high titer in vero cells
Inventors
RAYCHOUDHURI, Amit • Ella, Krishna Murthy
Assignees
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Publication Number
US-12290555-B2
Publication Date
2025-05-06
Expiration Date
Abstract
Provided is an Enterovirus D68 adapted to propagate to high titers in Vero cells and method of adaptation thereof. Also provided is a suitable vaccine composition including inactivated Enterovirus D68 antigen.
Core Innovation
The invention relates to an Enterovirus D68 adapted to propagate to high titers in Vero cells and encoded by a cDNA molecule having the nucleotide sequence of SEQ ID NO: 1. The adapted Enterovirus D68 is characterized as a Vero-adapted virus with sequence-defined cDNA/protein and specific polyprotein amino-acid changes.
The adaptation is described as involving propagation in Vero cells to achieve high titer, with controlled adsorption and maintenance media propagation in the range of 32–35 °C. The described workflow includes sequential infection and passaging using specified passage dilution ranges, and harvesting the virus when cytopathic effect reaches at least 90% or within a specified timeframe.
The invention further describes downstream characterization and use of the adapted virus, including plaque assay determination of titer and preparation of an inactivated vaccine antigen. The inactivated antigen is produced from the adapted virus by nuclease treatment and chemical inactivation, followed by purification using gel filtration and ion-exchange and/or size-exclusion/mixed-mode chromatography.
Vaccine or immunogenic compositions can be formulated, including monovalent and combination formats, and evaluated for stability and neutralization. The immunogenic composition is stated to include other enteroviruses such as EV71 and polioviruses, alone or in combination.
Claims Coverage
The independent claim covers an Enterovirus D68 adapted to propagate to high titers in Vero cells and encoded by a cDNA molecule with the nucleotide sequence of SEQ ID NO: 1. The covered inventive features include a sequence-defined Vero-adapted virus and an inactivated vaccine/immunogenic composition with defined stability, with optional inclusion of EV71 and polioviruses.
High-titer propagation in Vero cells encoded by SEQ ID NO: 1 cDNA
An Enterovirus D68 adapted to propagate to high titers in Vero cells encoded by a cDNA molecule having the nucleotide sequence of SEQ ID NO: 1.
Inactivated antigen production and purification
Nuclease treatment, chemical inactivation using formalin, beta-propiolactone, or hydrogen peroxide, and purification of the inactivated antigen using gel filtration and ion-exchange chromatography and/or size-exclusion/mixed-mode chromatography methods.
Immunogenic composition stability constraints at 2–38 °C and 37 °C
An immunogenic composition comprising the enterovirus that is stable for at least 6 months at 2–38 °C and for at least 1 month at 37 °C.
Combination with other enteroviruses
The immunogenic composition further includes other enteroviruses such as EV71 and polioviruses, alone or in combination.
Overall, the claim coverage centers on a Vero-adapted Enterovirus D68 encoded by SEQ ID NO: 1 and adapted to propagate to high titers, with dependent coverage extending to inactivated antigen production, immunogenic composition stability, and optional combination with EV71 and polioviruses.
Stated Advantages
Propagates Enterovirus D68 to high titers in Vero cells.
Provides an inactivated vaccine antigen preparation from the adapted virus.
Supports immunogenic compositions with defined stability at 2–38 °C and at 37 °C.
Enables monovalent and combination immunogenic compositions including EV71 and polioviruses.
Documented Applications
Inactivated vaccine antigen and immunogenic compositions for enterovirus D68, including monovalent formulations and combination formats with EV71 and polioviruses.
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