Influenza virus reassortment
View Patent ↗Methods for producing reassortant viruses are provided wherein the transcription and/or translation of the hemagglutinin and/or neuraminidase genes are suppressed.
1. A method of preparing a reassortant influenza virus comprising the steps of
(i) contacting a culture host infected with a vaccine strain and a backbone strain with a nucleic acid inhibitory agent wherein said inhibitory agent preferentially reduces the transcription and/or translation of the hemagglutinin and/or neuraminidase genes of the backbone strain; and,
(ii) culturing the culture host in order to produce the reassortant virus.
2. The method of claim 1 , further comprising the step (iii) of purifying the reassortant virus obtained in step (ii).
3. A method of preparing a reassortant influenza virus comprising the steps of
(i) introducing into a culture host that has been infected with a first influenza virus strain having at least one target segment one or more expression construct(s) encoding the target segment(s) from a second influenza virus strain;
(ii) contacting the culture host with a nucleic acid inhibitory agent which preferentially reduces the transcription and/or translation of the first influenza strain's target segment(s);
(iii) culturing the culture host in order to produce reassortant virus; and optionally
(iv) purifying the virus obtained in step (iii).
4. The method of claim 1 , further comprising the steps of (a) infecting a culture host with the virus obtained in step (ii) of claim 1 ; (b) culturing the host from step (a) to produce further virus; and (c) purifying the virus obtained in step (b).
5. The method of claim 3 , further comprising the steps of (a) infecting a culture host with a reassortant influenza virus obtained in step (iii) or (iv) of claim 3 ; (b) culturing the host from step (a) to produce the virus; and optionally (c) purifying the virus obtained in step (b).
6. A method of preparing a vaccine, comprising steps of (a) preparing a reassortant virus by the method of claim 1 and (b) preparing a vaccine from the reassortant virus.
7. The method of claim 1 , wherein the culture host is an embryonated hen egg.
8. The method of claim 1 , wherein the culture host is a mammalian cell.
9. The method of claim 8 , wherein the cell is an MDCK, Vero or PerC6 cell.
10. The method of claim 8 , wherein the cell grows adherently.
11. The method of claim 8 , wherein the cell grows in suspension.
12. The method of claim 11 , wherein the cell is of the cell line MDCK 33016 (DSM ACC2219).
13. The method of claim 4 , wherein step (c) involves inactivating the virus.
14. The method of claim 6 , wherein the vaccine is a whole virion vaccine.
15. The method of claim 6 , wherein the vaccine is a split virion vaccine.
16. The method of claim 6 , wherein the vaccine is a surface antigen vaccine.
17. The method of claim 6 , wherein the vaccine is a virosomal vaccine.
18. The method of claim 6 , wherein the vaccine contains less than 10 ng of residual host cell DNA per dose.
19. The method of claim 1 wherein at least one of the influenza strains is of the H1, H2, H5, H7 or H9 subtype.
20. The method of claim 19 , wherein at least one of the strains is a H1N1, H5N1, H5N3, H9N2, H2N2, H7N1 or H7N7 strain.
21. The method of claim 1 , wherein one of the influenza strains is a high-growth strain.
22. The method of claim 19 , wherein at least one influenza strain is selected from the group consisting of A/Puerto Rico/8/34, A/Ann Arbor/6/60, B/Ann Arbor/1/66, A/Chile/1/83 and A/California/04/09.
23. A cell for preparing a reassortant influenza virus comprising expression construct(s) encoding:
(i) all eight viral segments of a first influenza A or B virus genome;
(ii) at least one target segment of a second influenza A or B virus genome, wherein the second influenza strain's target segment(s) differs in sequence from the target segment of the first influenza strain; and
(iii) a nucleic acid inhibitory agent wherein said inhibitory agent preferentially reduces transcription and/or translation of the target segment(s) of the first influenza strain.
24. The method of claim 1 , wherein the nucleic acid inhibitory agent is an antisense oligonucleotide.
25. The method of claim 1 , wherein the nucleic acid inhibitory agent is a synthetic antisense oligomer.
26. The method of claim 1 , wherein the nucleic acid inhibitory agent is a phosphorothioate oligomer (PMO).
27. The method of claim 1 , wherein the nucleic acid inhibitory agent is a phosphorodiamidate morpholino oligomer (PSO).
28. The method of claim 1 , wherein the nucleic acid inhibitory agent is selected from the group consisting of short interfering RNAs (siRNA), double-stranded RNAs (dsRNA), micro-RNAs (miRNA), short hairpin RNAs (shRNA), and small interfering DNAs (siDNA).