REVERSE GENETICS SYSTEMS

Abstract

The invention provides various reverse genetics systems for producing segmented RNA viruses, wherein the systems do not require bacteria for propagation of all of their expression constructs.

Claims

1-16. (canceled)

17. A reverse genetics system comprising a combination of at least one plasmid bacterial expression construct and at least one non-bacterial expression construct, wherein each construct comprises coding sequences for one or more genome segments of a segmented RNA virus.

18. The reverse genetics system of claim 17, wherein the RNA virus genome segments from the plasmid and the at least one non-bacterial expression construct are different.

19. The reverse genetics system of claim 17, wherein the at least one non-bacterial expression construct is linear.

20. The reverse genetics system of claim 17, wherein the at least one non-bacterial expression construct is circular.

21. A eukaryotic host cell comprising the reverse genetics system of claim 17.

22. A bacterial plasmid comprising coding sequences for eight different genome segments of an influenza virus, wherein expression of each segment is controlled by a mammalian pol-I promoter or a bacteriophage polymerase promoter.

23. A eukaryotic cell comprising the bacterial plasmid of claim 22.

24. A bacterial cell comprising the bacterial plasmid of claim 22.

25. A method of preparing a eukaryotic host cell, comprising inserting the plasmid and the at least one non-bacterial expression construct of claim 17 into the eukaryotic host cell.

26. A method of preparing a segmented RNA virus, comprising culturing a host cell under conditions that enable expression of segments of the RNA virus from a reverse genetics system comprising a combination of at least one plasmid bacterial expression construct and at least one non-bacterial expression construct, wherein each construct comprises coding sequences for one or more genome segments of the segmented RNA virus.

27. A segmented RNA virus produced by the method of claim 26.

28. A method of preparing a viral vaccine, comprising: infecting a culture host with a segmented RNA virus produced by culturing a host cell under conditions that enable expression of segments of the RNA virus from a reverse genetics system comprising a combination of at least one plasmid bacterial expression construct and at least one non-bacterial expression construct, wherein each construct comprises coding sequences for one or more genome segments of the segmented RNA virus; growing the segmented RNA virus in the infected culture host; and preparing a vaccine from the grown virus.

29. The method of claim 28, wherein the culture host is cells.

30. The method of claim 28, wherein the culture host is eggs.

31. The method of claim 25, wherein the plasmid and the at least one non-bacterial expression construct are inserted into the eukaryotic host cell by any suitable transfection method.

32. The eukaryotic cell of claim 23, wherein the bacterial plasmid is inserted into the eukaryotic cell by any suitable transfection method.

33. The reverse genetics system of claim 17, wherein the plasmid and the at least one non-bacterial expression construct encode for both viral protein and viral RNA expression.

34. The reverse genetics system of claim 33, wherein the plasmid and the at least one non-bacterial expression construct each comprise a bi-directional expression construct.

35. The reverse genetics system of claim 17, wherein the segmented RNA virus is an influenza virus.

36. The method of claim 26, wherein the segmented RNA virus is an influenza virus.

37. The method of claim 28, wherein the segmented RNA virus is an influenza virus.

Description

MODES FOR CARRYING OUT THE INVENTION

[0150] Source influenza viruses are S-OIV strain A/California/4/09 for HA and NA segments and PR/8/34 for the remaining six backbone segments. DNA sequences encoding these eight segments are prepared, with each segment being flanked by a human pol-I promoter at one end and a pol-I terminator at the other end. These pol-I elements are surrounded by a pol-II promoter from CMV and a pol-II terminator sequence and polA signal. The pol-I promoter drives transcription of a negative sense viral RNA segment with faithful wild-type vRNA termini. The pol-II promoter drives transcription of a mRNA encoding the viral protein. DNA segments for each segment are joined to give a single linear DNA molecule, 24 kbp, encoding the whole reassortant influenza virus genome. The overall synthesis of this molecule follows the general methods disclosed by Gibson et al. in reference 8.

[0151] The linear DNA construct is transfected into a culture of MDCK 33016 cells. This cell line has been found to recognise the human pol-I promoter for influenza virus reverse genetics rescue. Incubation of the transfected cells soon leads to the appearance of reassortant influenza virus in the culture medium. This strain (RG-lin-CA-1) can be purified by conventional methods and used as a seed for vaccine manufacture.

[0152] It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

REFERENCES

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