Multi plasmid system for the production of influenza virus

a technology of influenza virus and plasmid system, which is applied in the field of multi-plasmid system for the production of influenza virus, can solve the problems of limiting flexibility, delays and shortages in production and distribution, virus does not replicate in the lungs of infected ferrets, etc., and achieves enhanced replication ability, modulating ha receptor binding activity and/or na neuraminidase activity, and improving replication

Inactive Publication Date: 2005-07-21
MEDIMMUNE LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0045] The present invention also relates to the identification and manipulation of amino acid residues in HA and NA which affect influenza virus replication in cells and embryonated chicken eggs. The present invention further relates to the use of reverse genetics technology to generate HA and NA influenza virus vaccine variants with improved replication in embryonated chicken eggs and/or cells. The invention further relates to methods for modulating HA receptor binding activity and/or NA neuraminidase activity. Additionally, the invention provides influenza viruses with enhanced ability to replicate in embryonated chicken eggs and/or cells.
[0046] In one embodiment the invention provides methods for manipulating the amino acid residues of HA and/or NA to increase the ability of an influenza viru...

Problems solved by technology

Additionally, this virus does not replicate in the lungs of infected ferrets (att, attenuation).
Supplies of eggs must be organized, and strains for vaccine production selected months in advance of the next flue season, limiting the flexibility of this approach, and often resulting in delays and shortages in production and distribution.
Unfortunately, some influenza vaccine strains, such as the prototype A/Fujian/411/02 strain that circulated during the 2003-04 season, do not replicate well in embryonated chicken eggs, and have to be isolated by cell culture a costly and time consuming procedure.
While eliminating many of the difficulties related to vaccine production in hen's egg...

Method used

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  • Multi plasmid system for the production of influenza virus
  • Multi plasmid system for the production of influenza virus
  • Multi plasmid system for the production of influenza virus

Examples

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example 1

Construction of pAD3000

[0174] The plasmid pHW2000 (Hoffmann et al. (2000) A DNA transfection system for generation of influenza A virus from eight plasmids Proc Natl Acad Sci USA 97:6108-6113) was modified to replace the bovine growth hormone (BGH) polyadenylation signals with a polyadenylation signal sequences derived from Simian virus 40 (SV40).

[0175] Sequences derived from SV40 were amplified with Taq MasterMix (Qiagen) using the following oligonucleotides, designated in the 5′ to 3′ direction:

(SEQ ID NO:1)polyA.1:AACAATTGAGATCTCGGTCACCTCAGACATGATAAGATACATTGATGAGT(SEQ ID NO:2)polyA.2:TATAACTGCAGACTAGTGATATCCTTGTTTATTGCAGCTTATAATGGTTA

[0176] The plasmid pSV2His was used as a template. A fragment consistent with the predicted 175 bp product was obtained and cloned into pcDNA3.1, using a Topo TA cloning vector (Invitrogen) according to the manufacturer's directions. The desired 138 bp fragment containing the SV40 polyadenylation signals was excised from the resulting plasmid with...

example 2

Eight Plasmid System for Production of MDV-A

[0177] A cold-adapted influenza virus type A strain A / AA / 6 / 60 variant has commonly been used as a master donor virus for the production of nasally administered Influenza A vaccines. This strain is an exemplary Master Donor Virus (MDV) in the context of the present invention. For simplicity, this strain A / AA / 6 / 60 variant is designated herein MDV-A. MDV-A viral RNA was extracted using the RNeasy mini kit (Qiagen) and the eight corresponding cDNA fragments were amplified by RT-PCR using the primers listed in Table 1.

TABLE 1Sequence of the primers used for cloningMDV-A eight segmentsSEQID.PrimerSequence (5′-3′)MDV-A FORWARD PRIMERSSEQ IDAarI PB2 longCAC TTA TAT TCA CCT GCC TCANO:3GGG AGC GAA AGC AGG TCSEQ IDBsmBI-PBITAT TCG TCT CAG GGA GCG AAANO:4GCA GGC AAASEQ IDBsmBI-PATAT TCG TCT CAG GGA GCG AAANO:5GCA GGT ACTSEQ IDBsmBI-NPTAT TCG TCT CAG GGA GCA AAANO:6GCA GGG TAG ASEQ IDAarI HA-longCAC TTA TAT TCA CCT GCC TCANO:7GGG AGC AAA AGC AGG GGSE...

example 3

Generation of Infectious Recombinant MDV-A and Reassorted Influenze Virus

[0181] Madin-Darby canine kidney (MDCK) cells and human COS7 cells were maintained in modified Eagle Medium (MEM) containing 10% fetal bovine serum (FBS). Human embryonic kidney cells (293T) were maintained in Opti-MEM I (Life Technologies) containing 5% FBS. MDCK and either COS7 or 293T cells were co-cultured in 6-well plates at a ratio of 1:1 and the cells were used for transfection at a confluency of approximately 80%. 293T and COS7 cells have a high transfection efficiency, but are not permissive for influenza virus replication. Co-culture with MDCK cells ensures efficient replication of the recombinant viruses. Prior to transfection, serum-containing media were replaced with serum free medium (Opti-MEM I) and incubated for 4-6 hours. Plasmid DNA transfection was performed using TransIT-LT1 (Mirus) by mixing 1 μg of each of the 8 plasmid DNAs (PB2, PB1, PA, NP, M, NS, HA and NA) with 20 μl of TransIT-LT1 d...

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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.

Description

[0001] This application claims the benefit under 35 U.S.C § 119 (e) of U.S. Provisional Application No. 60 / 532,164 filed Dec. 23, 2003, which is incorporated by reference herein.BACKGROUND OF THE INVENTION [0002] Influenza viruses are made up of an internal ribonucleoprotein core containing a segmented single-stranded RNA genome and an outer lipoprotein envelope lined by a matrix protein. Influenza A and B viruses each contain eight segments of single stranded RNA with negative polarity. The influenza A genome encodes at least eleven polypeptides. Segments 1-3 encode the three polypeptides, making up the viral RNA-dependent RNA polymerase. Segment 1 encodes the polymerase complex protein PB2. The remaining polymerase proteins PB1 and PA are encoded by segment 2 and segment 3, respectively. In addition, segment 1 of some influenza A strains encodes a small protein, PB1-F2, produced from an alternative reading frame within the PB1 coding region. Segment 4 encodes the hemagglutinin (HA...

Claims

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Application Information

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IPC IPC(8): A61K35/76A61K39/00A61K39/12A61K39/145C12N7/02C12N15/85
CPCA61K2039/5254C12N7/00C12N15/85C12N2760/16151C12N2760/16251C12N2840/20C12N2760/16234C12N2760/16261A61K39/145C12N2760/16152C12N2760/16252A61K2039/543C12N2760/16134C12N2760/16162A61K39/12A61P31/16
Inventor KEMBLE, GEORGEDUKE, GREGORY
Owner MEDIMMUNE LLC
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