Vaccinia virus MVA-E3L-knockout-mutants and use thereof
View Patent ↗The present invention relates to mutant MVA vaccinia viruses, which are used for the generation of recombinant MVA viruses, as well as host cells, which have been infected with these mutant MVA viruses. The present invention further relates to DNA-vector constructs, and a method for the generation of recombinant MVA by using the mutant MVA viruses and the DNA-vector constructs. The mutant MVA vaccinia viruses of the present invention are characterized in that the MVA ORF 050L gene or a functional part thereof has been inactivated in the viral genome.
1. MVA-knock-out-mutant, wherein the MVA ORF 050L gene or a functional part thereof has been inactivated in the viral genome.
2. MVA-knock-out-mutant of claim 1 , in which the MVA ORF 050L gene or a functional part thereof has been inactivated by deletion from the viral genome.
3. An isolated host cell, wherein the cell has been infected with the MVA of claim 1 .
4. The isolated host cell of claim 3 , which is a eucaryotic cell.
5. The isolated host cell of claim 4 , which is a BHK-21 cell, a BS-C-1 cell, a MA104 cell, or a CV-1 cell.
6. A method for generating a recombinant MVA, the method comprising:
(a) infecting a host cell with an MVA, wherein an MVA ORF 050L gene or a functional part thereof has been inactivated in the MVA;
(b) transfecting the host cell with a DNA-vector construct comprising an MVA ORF 050L gene or encoding a functionally equivalent MVA ORF 050L derived polypeptide; and
(c) selecting restored MVA by growth on CEF cells or chicken embryo derived LSCC-H32 cells or avian cells, thereby generating a recombinant MVA.
7. The method of claim 6 , wherein the DNA-vector construct further comprises a nucleic acid sequence encoding a foreign protein.
8. The method of claim 7 , wherein the foreign protein is a heterologous protein selected from the group consisting of a therapeutic polypeptide, an antigen encoded by a pathogenic agent, and an immunogenic fragment of said antigen.
9. The method of claim 8 , wherein the therapeutic polypeptide is selected from the group consisting of t-PA and interferon.
10. The method of claim 8 , wherein the pathogenic agent is selected from the group consisting of a virus, a bacterium, a protozoan, a parasite, and a tumor cell.
11. The method of claim 10 , wherein the virus is selected from the group consisting of an influenza virus, a measles virus, a respiratory syncytial virus, a dengue virus, a human immunodeficiency virus, a human hepatitis virus, a herpes virus, and a papilloma virus.
12. The method of claim 10 , wherein the protozoan is Plasmodium falciparum.
13. The method of claim 10 , wherein the bacterium is a tuberculosis-causing Mycobacterium.
14. The method of claim 8 , wherein the foreign protein is a tumor-associated antigen or immunogenic fragment thereof, selected from the group consisting of a melanoma-associated differentiation antigen, a cancer-testes antigen, and a non-mutated shared antigen overexpressed in a tumor.
15. The method of claim 14 , wherein the melanoma-associated differentiation antigen is selected from the group consisting of a tyrosinase, a tyrosinase-related protein 1, and a tyrosinase-related protein 2.
16. The method of claim 14 , wherein the cancer-testes antigen is selected from the group consisting of MAGE-1, MAGE-2, MAGE-3 and BAGE.
17. The method of claim 14 , wherein the non-mutated shared antigen overexpressed in a tumor is selected from the group consisting of Her-2/neu, MUC -1, and p53.
18. The method of claim 7 , wherein the MVA ORF 050L gene and the nucleic acid sequence encoding the foreign protein are both flanked by a DNA sequence flanking a non-essential site within an MVA genome.
19. The method of claim 18 , wherein one or both of the MVA ORF 050L gene and the nucleic acid sequence encoding the foreign protein is flanked by a site of deletion III in an MVA genome.
20. The method of claim 6 , wherein the DNA vector construct comprises a plasmid.
21. The method of claim 6 , wherein the avian cell is selected from the group consisting of a quail QT6 fibroblast and a quail QT35 fibroblast.
22. The method of claim 6 , wherein the isolated host cell is a eucaryotic cell.
23. The method of claim 22 , wherein the isolated host cell is selected from the group consisting of a BHK-21 cell, a BS-C-1 cell, a MA104 cell, and a CV-1 cell.