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Publication Number
US-9968672-B2
Publication Date
2018-05-15
Expiration Date
Abstract
Described herein are iDNA vectors and vaccines and methods for using the same. The iDNA generates live attenuated vaccines in eukaryotic cells in vitro or in vivo for pathogenic RNA viruses, particularly yellow fever virus and Venezuelan equine encephalitis virus. When iDNA is injected into the vaccine recipient, RNA of live attenuated virus is generated by in vivo transcription in the recipient's tissues. This initiates production of progeny attenuated viruses in the tissues of the vaccine recipient, as well as elicitation of an effective immune response protecting against wild-type, non-attenuated virus.
Core Innovation
The disclosure describes iDNA vaccines in which DNA encoding an infectious RNA molecule is operably linked to a eukaryotic RNA polymerase promoter. The infectious RNA molecule encodes an alphavirus or a flavivirus, including infectious, live attenuated virus generation after transcription in cultured cells or in recipient tissues. The approach is presented as eliciting protective immune responses against wild-type pathogenic viruses.
In the described constructs, a CMV promoter is used to drive expression, with the promoter positioned upstream of the 5′ end of the DNA encoding the infectious RNA molecule. The disclosure further describes downstream regulatory elements associated with the infectious RNA coding region, including poly-A tail and transcription termination/ribozyme features. Additional promoter arrangement is described, including an optional duplication of 26S promoters to express structural proteins from independent promoters.
The disclosure also describes producing and harvesting clonally purified live attenuated virus generated from the iDNA constructs. Multiple examples are described at a conceptual level, including detection of antigen/virus generation, immunization of mice, and assessment of an avirulent phenotype with attenuation stability during virus production from iDNA.
Claims Coverage
The independent claims cover DNA vectors encoding an infectious RNA molecule from alphavirus or flavivirus, together with a eukaryotic RNA polymerase promoter, and CMV promoter positioning constraints. One claim also recites a non-pathogenic alphavirus with a first 26S promoter and a second 26S promoter.
Operably linked infectious RNA encoding alphavirus and eukaryotic RNA polymerase promoter
A vector comprising DNA encoding an infectious RNA molecule that encodes an alphavirus and a eukaryotic RNA polymerase promoter, wherein the DNA encoding the infectious RNA molecule is operably linked to the RNA polymerase promoter.
Venezuelan Equine Encephalitis vector with CMV promoter at 15(±3) nucleic acid residues upstream of 5′ end
A vector comprising DNA encoding an infectious RNA molecule that encodes a Venezuelan Equine Encephalitis (VEE) virus and a CMV promoter located 15(±3) nucleic acid residues upstream of the 5′ end of the DNA encoding the infectious RNA molecule, wherein the DNA encoding the infectious RNA molecule is operably linked to the CMV promoter.
Vaccine comprising a therapeutically effective amount of the VEE vector with CMV promoter linkage
A vaccine comprising a therapeutically effective amount of the vector of claim 16; wherein the encoded VEE virus is non-pathogenic.
Yellow Fever vector with CMV promoter at 15(±3) nucleic acid residues upstream of 5′ end
A vector comprising DNA encoding an infectious RNA molecule that encodes a Yellow Fever virus and a CMV promoter located from 15(±3) nucleic acid residues upstream of the 5′ end of the DNA encoding the infectious RNA molecule, wherein the DNA encoding the infectious RNA molecule is operably linked to the CMV promoter.
Non-pathogenic Yellow Fever vector vaccine with an effective amount
A vaccine comprising an effective amount of the a DNA vector encoding an infectious RNA molecule that encodes a non-pathogenic Yellow Fever virus.
Human vaccine with nonpathogenic alphavirus or nonpathogenic flavivirus encoded by CMV-driven infectious RNA
A human vaccine comprising a therapeutically effective amount of the a DNA vector encoding an infectious RNA molecule that encodes a nonpathogenic alphavirus or a nonpathogenic flavivirus and a CMV promoter located from 15(±3) nucleic acid residues upstream of the 5′ end of the DNA encoding the infectious RNA molecule, wherein the DNA encoding the infectious RNA molecule is operably linked to the CMV promoter, and the vaccine is pharmaceutically acceptable for a human.
Human vaccine with nonpathogenic flavivirus encoded by CMV-driven infectious RNA
A human vaccine comprising an effective amount of a DNA vector encoding an infectious RNA molecule that encodes a nonpathogenic flavivirus and a CMV promoter located from 15(±3) nucleic acid residues upstream of the 5′ end of the DNA encoding the infectious RNA molecule, wherein the DNA encoding the infectious RNA molecule is operably linked to the CMV promoter, and the vaccine is pharmaceutically acceptable for a human.
Non-pathogenic alphavirus vector with first 268 promoter and second 26S promoter plus CMV promoter at about 12 to about 18 upstream of 5′ end
A vector comprising a DNA encoding an infectious RNA molecule, wherein the RNA molecule encodes a non-pathogenic alphavirus and the RNA molecule comprises a first 268 promoter and a second 26S promoter, and a CMV promoter located from about 12 to about 18 nucleic acid residues upstream of the 5′ end of the DNA encoding the infectious RNA molecule.
Across the independent claims, the core claimed coverage is DNA vectors that encode an infectious RNA molecule from alphavirus or flavivirus with a eukaryotic RNA polymerase promoter, including CMV promoter positioning constraints. One claim further includes a promoter architecture with a first 26S promoter and a second 26S promoter for a non-pathogenic alphavirus.
Stated Advantages
Protective immune responses against wild-type pathogenic viruses.
Documented Applications
iDNA vaccination eliciting protective immune responses against wild-type pathogenic viruses.
Vaccine use context for VEE virus, including vaccines using a non-pathogenic encoded VEE virus.
Human vaccines comprising pharmaceutically acceptable amounts of vectors encoding infectious RNA molecules from nonpathogenic alphavirus or nonpathogenic flavivirus.
Human vaccines comprising pharmaceutically acceptable amounts of vectors encoding an infectious RNA molecule that encodes a nonpathogenic flavivirus.
Immunization of mice and evaluation of avirulent phenotype stability during virus production from iDNA.
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