Multi plasmid system for the production of influenza virus

• US 20050158342A1
• Filed: 12/22/2004
• Published: 07/21/2005
• Est. Priority Date: 12/23/2003
• Status: Abandoned Application

• Current Assignee: Medimmune LLC (Astrazeneca PLC)
• Original Assignee: Medimmune Incorporated (Astrazeneca PLC)
• Inventors: Duke, Gregory, Kemble, George
• Application Number:
  US11/018,624
• Publication Number:
  US 20050158342A1
• Time in Patent Office: Days
• US Class Current: 424/209.100
• CPC Class Codes: A61K 2039/5254 avirulent or attenuated
• CPC Class Codes: A61K 2039/543 intranasal
• CPC Class Codes: A61K 39/12 Viral antigens
• CPC Class Codes: A61K 39/145 Orthomyxoviridae, e.g. influenza virus
• CPC Class Codes: A61P 31/16 for influenza or rhinoviruses
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2760/16134 Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 2760/16151 Methods of production or purification of viral material
• CPC Class Codes: C12N 2760/16152 relating to complementing cells and packaging systems for producing virus or viral particles
• CPC Class Codes: C12N 2760/16162 by genetic engineering
• CPC Class Codes: C12N 2760/16234 Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 2760/16251 Methods of production or purification of viral material
• CPC Class Codes: C12N 2760/16252 relating to complementing cells and packaging systems for producing virus or viral particles
• CPC Class Codes: C12N 2760/16261 Methods of inactivation or attenuation
• CPC Class Codes: C12N 2840/20 translation of more than one cistron
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00

Abstract

Vectors and methods for the production of influenza viruses suitable as recombinant influenza vaccines in cell culture are provided. Bi-directional expression vectors for use in a multi-plasmid influenza virus expression system are provided. Additionally, the invention provides methods of producing influenza viruses with enhanced ability to replicate in embryonated chicken eggs and/or cells (e.g., Vero and/or MDCK) and further provides influenza viruses with enhanced replication characteristics. In addition, the present invention includes an improved method of rescue, wherein animal cells (e.g., SF Vero cells) are electroporated with plasmids and vectors of the invention.

Claims

1. A method of rescue of influenza virus, wherein animal cells are electroporated with plasmids that encode an influenza RNA polymerase and nucleoprotein and wherein the electroporated animal cells are co-cultivated with another cell type.

2. The method of claim 1, wherein the animal cells are Vero cells.

3. The method of claim 1, wherein the animal cells are SF Vero cells.

4. The method of claim 1, wherein said another cell type is CEK cells.

5. The method of claim 1, wherein the influenza virus is an influenza A virus.

6. The method of claim 1, wherein the influenza virus is an influenza B virus.

7. The method of claim 1, wherein the influenza virus is a cold adapted virus.

8. The method of claim 1, wherein the influenza virus is an attenuated virus.

9. The method of claim 1, wherein the number of plasmids electroporated is eight.

10. The method of claim 1, wherein the number of plasmids electroporated is twelve.

11. The method of claim 1, wherein the efficiency of said rescue of influenza virus is at least 90%.

12. An influenza virus produced by the method of claim 1.

13. The influenza virus of claim 1, wherein said rescued influenza virus comprises vRNA segments derived from A/PR/8/34.

14. The influenza virus of claim 1, wherein said rescued influenza virus comprises vRNA segments derived from MDV-A.

15. A vaccine composition comprising the influenza virus of claim 1.

16. A method for producing influenza viruses in cell culture, the method comprising:

i) introducing a plurality of vectors comprising an influenza virus genome into a population of Vero cells by electroporation;

ii) co-cultivating the population of Vero cells with another cell type under conditions permissive for viral replication; and

,

iii) recovering a plurality of influenza viruses.

17. A method of rescue of influenza virus, wherein (a) animal cells are electroporated with cell expression vectors which direct the expression in said cells of genomic or antigenomic vRNA segments, and a nucleoprotein, and an RNA-dependent polymerase, such that ribonucleoprotein complexes can be formed and viral particles can be assembled;

and (b) culturing said cells wherein viral particles are packaged and rescued.

18. The method of claim 17, wherein assembly does not require a helper virus.

19-25. -25. (canceled)