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US 8,309,099 B2Granted
DNA transfection system for the generation of negative strand RNA virus
AssigneeSt. Jude Children's Research Hospital
InventorErich Hoffmann
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AssigneeSt. Jude Children's Research Hospital
InventorErich Hoffmann
Estimating the term…
The present invention is based on the development of a dual promoter system (preferably a RNA pol I-pol II system) for the efficient intracellular synthesis of viral RNA. The resultant minimal plasmid-based system may be used to synthesize any RNA virus, preferably viruses with a negative single stranded RNA genome. The viral product of the system is produced when the plasmids of the system are introduced into a suitable host cell. One application of the system is production of attenuated, reassortant influenza viruses for use as antigens in vaccines. The reassortant viruses generated by cotransfection of plasmids may comprise genes encoding the surface glycoproteins hemagglutinin and neuraminidase from an influenza virus currently infecting the population and the internal genes from an attenuated influenza virus. An advantageous property of the present invention is its versatility; the system may be quickly and easily adapted to synthesize an attenuated version of any RNA virus. Attenuated or inactivated RNA viruses produced by the present invention may be administered to a patient in need of vaccination by any of several routes including intranasally or intramuscularly.
A method of preparing a vaccine comprising a segmented negative strand RNA virus virion, which method comprises 1) culturing a host cell comprising a minimum plasmid-based system, wherein the minimum plasmid-based system comprises a set of plasmids, each plasmid of said set comprises one viral genomic segment of said negative strand RNA virus, and wherein the viral cDNA corresponding to the viral genomic segment is inserted between an RNA polymerase I (pol I) promoter and a regulatory element, which results in expression of vRNA or cRNA with an exact 3′ end, which are in turn inserted between an RNA polymerase II (pol II) promoter and a polyadenylation signal, which results in expression of viral mRNA and a corresponding viral protein, wherein the expression of vRNAs or cRNAs and viral proteins from said set of plasmids results in a segmented negative strand RNA virus virion, wherein the total number of the plasmids in the minimum plasmid-based system does not exceed the total number of the gene segments of the negative strand RNA virus, 2) purifying the negative strand RNA virus virion produced in the host cell, and 3) producing a vaccine comprising the purified virion.
Dependencies are inferred from the claim text.