Recombinant bicistronic
This invention relates to bicistronic flavivirus vectors, methods of using such vectors in the prevention and treatment of disease, and methods of making such vectors.
1. A chimeric flavivirus comprising a yellow fever virus in which one or more structural proteins of the yellow fever virus have been replaced with corresponding structural proteins of a second, different flavivirus, wherein the genome of said chimeric flavivirus comprises an internal ribosome entry site and a transgene located in the 3′-untranslated region of the flavivirus.
2. The chimeric flavivirus of claim 1 , wherein the chimeric flavivirus comprises membrane and envelope proteins of said second flavivirus.
3. The chimeric flavivirus of claim 1 , wherein the second flavivirus is selected from the group consisting of Japanese encephalitis, Dengue-1, Dengue-2, Dengue-3, Dengue-4, Murray Valley encephalitis, St. Louis encephalitis, West Nile, Kunjin, Rocio encephalitis, Ilheus, Tick-borne encephalitis, Central European encephalitis, Siberian encephalitis, Russian Spring-Summer encephalitis, Kyasanur Forest Disease, Omsk Hemorrhagic fever, Louping ill, Powassan, Negishi, Absettarov, Hansalova, Apoi, and Hypr viruses.
4. The chimeric flavivirus of claim 1 , wherein the second flavivirus is a Japanese encephalitis virus.
5. The chimeric flavivirus of claim 1 , wherein the flavivirus comprises a deletion within the 3′-untranslated region of the flavivirus.
6. The chimeric flavivirus of claim 1 , wherein the transgene encodes a vaccine antigen or an immunogen.
7. The chimeric flavivirus of claim 6 , wherein the vaccine antigen or the immunogen is derived from an infectious agent.
8. The chimeric flavivirus of claim 7 , wherein the infectious agent is an influenza virus.
9. The chimeric flavivirus of claim 8 , wherein the immunogen is selected from the group consisting of hemagglutinin, neuraminidase, or M2, or an immunogenic fragment thereof.
10. The chimeric flavivirus of claim 9 , wherein the M2 immunogen comprises the M2e region of the M2 protein.
11. The chimeric flavivirus of claim 6 , wherein the vaccine antigen is a tumor-associated antigen.
12. A pharmaceutical composition comprising the chimeric flavivirus of claim 1 .
13. A method of producing the chimeric flavivirus of claim 1 , comprising culturing Vero cells into which RNA corresponding to the virus has been introduced at a temperature below 37° C.
14. The method of claim 13 , wherein the temperature is 31° C.-36° C.
15. The method of claim 14 , wherein the temperature is 34° C.
16. A method of propagating the chimeric flavivirus of claim 1 , comprising incubating cells infected with said flavivirus at a temperature below 37° C.
17. The method of claim 16 , wherein the temperature is 31° C.-36° C.