Pseudoinfectious
View Patent ↗The present invention discloses a replication-deficient pseudoinfective virus belonging to the Flaviviridae family that lack the capsid gene, where the replication-deficient pseudoinfective virus propagates only in cells expressing the capsid or capsid, prM and envelope protein of the flavivirus. The present also discloses the method of producing such viruses on a large scale and the use of these pseudoinfective viruses as vaccines for preventing diseases caused by infections of humans or animals by the viruses belonging to this family.
1. A replication-deficient pseudoinfectious virus comprising:
a West Nile or Yellow Fever deletion mutant genome comprising a deletion within the nucleotide sequence encoding amino acids 26 to 100 of the capsid protein, wherein the deletion mutant genome cannot produce capsid-containing viral particles in a cell that does not express a capsid protein, wherein the deletion does not disrupt the maturation of the prM protein or the RNA sequence required for genome cyclization, and a complementary capsid protein.
2. The replication-deficient pseudoinfectious virus of claim 1 , wherein said virus is a chimeric virus comprising a heterologous prM-E cassette.
3. The replication-deficient pseudoinfectious virus of claim 2 , wherein the heterologous prM-E cassette is from a yellow fever virus, as West Nile virus, a dengue virus, a tick-borne encephalitis virus, a Saint Louis encephalitis virus, a Japanese encephalitis virus, or a Murray Valley encephalitis virus.
4. The replication-deficient pseudoinfectious virus of claim 1 , wherein the deletion is of amino acids 26 to 93, 31-93, 31-100, or 26 to 100 of the capsid protein.
5. The replication-deficient pseudoinfectious virus of claim 1 , wherein said deletion mutant genome further encodes a marker protein or an antigen.
6. The replication-deficient pseudoinfectious virus of claim 5 , wherein the marker protein is a green fluorescent protein.
7. A cell culture system comprising:
a West Nile or Yellow Fever deletion mutant genome comprising a deletion within the nucleotide sequence encoding amino acids 26 to 100 of the capsid protein, wherein the deletion mutant genome cannot produce capsid-containing viral particles in a cell that does not express a capsid protein, and wherein the deletion does not disrupt the maturation of the prM protein or the RNA sequence required for genome cyclization; the deletion mutant genome being inside a cell that expresses a complementary capsid protein, wherein the cell does not express prM or envelope proteins.
8. The cell culture system of claim 7 , wherein the cell comprises a replicon encoding a codon-optimized version of the complementary capsid protein.
9. The cell culture system of claim 7 , wherein the cell comprises an alphavirus replicon encoding the complementary capsid protein.
10. The cell culture system of claim 9 , wherein the alphavirus is Venezuelan Equine Encephalitis Virus.
11. A method of producing a replication-deficient pseudoinfectious virus comprising:
providing a West Nile or Yellow Fever deletion mutant genome comprising a deletion within the nucleotide sequence encoding amino acids 26 to 100 of the capsid protein, wherein the deletion mutant genome cannot produce capsid-containing viral particles in a cell that does not express a capsid protein, wherein the deletion does not disrupt the maturation of the prM protein or the RNA sequence required for genome cyclization;
providing a cell that expresses a complementary capsid protein;
inserting the deletion mutant genome into the cell; and
culturing the cell to produce a replication-deficient pseudoinfectious virus.
12. The method of claim 11 , wherein the deletion mutant genome comprises a heterologous prM-E cassette.
13. The method of claim 12 , wherein the heterologous prM-E cassette is from a yellow fever virus, a West Nile virus, a dengue virus, a tick-borne encephalitis virus, a Saint Louis encephalitis virus, a Japanese encephalitis virus, or a Murray Valley encephalitis virus.
14. The method of claim 11 , wherein the cell comprises a genetically engineered replicon derived from a viral vector.
15. The method of claim 14 , wherein the replicon encodes a codon-optimized version of the complementary capsid protein.
16. The method of claim 14 , wherein the replicon comprises the unnatural cyclization sequence of SEQ ID NO:3.
17. The method of claim 11 , wherein the deletion mutant genome comprises one or both of altered C-prM junction sequences SEQ ID NO:4 and SEQ ID NO:5.
18. The method of claim 11 , wherein inserting the deletion mutant genome into the cell comprises transfecting with in vitro synthesized replicon RNAs, transfecting with plasmid DNAs designed to synthesize functional alphaviral replicons from cellular RNA-polymerase II-specific promoter, or by infecting with alphaviral replicons packaged inside alphaviral structural proteins.
19. The method of claim 14 , wherein the replicon is an alphavirus replicon.
20. The method of claim 19 , wherein the alphavirus is Venezuelan Equine Encephalitis Virus, Sindbis virus, Eastern Equine Encephalitis virus, Western Equine Encephalitis virus, or Ross River virus.
21. A pharmaceutical composition, comprising the replication-deficient pseudoinfectious virus of claim 1 .
22. The replication-deficient pseudoinfetious virus of claim 1 , wherein the deletion mutant genome comprises one or both of altered C-prM junction sequences SEQ ID NO:4 and SEQ ID NO:5.
23. The method of claim 11 , wherein the cell does not express prM or envelope proteins.
24. The cell culture system of claim 8 , wherein the replicon comprises the unnatural cyclization sequence of SEQ ID NO3.
25. The cell culture system of claim 7 , wherein the cell further expresses a marker gene.