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Recombinant influenza viruses for vaccines and gene therapy

a technology vaccines, applied in the field of recombinant influenza viruses for vaccines and gene therapy, can solve the problems of generating segmented negative-sense rna viruses from cloned cdnas, limiting the progress of negative-sense rna viruses, and limiting the number of positive-sense rna viruses, so as to enhance the efficiency of virus generation and enhance the effect of viruses as vaccin

Inactive Publication Date: 2006-03-16
KAWAOKA YOSHIHIRO +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0016] Moreover, the same approach may be employed for other viruses to generate nonsegmented negative strand RNA viruses (i.e., Paramyxoviridae, Rhabdoviridae, and Filoviridae), or other segmented negative strand RNA viruses, e.g., Arenaviridae and Bunyaviridae, entirely from cloned cDNA. Further, the expression of cRNA in cells instead of vRNA may improve the efficiency of virus generation.
[0017] The method of the invention allows easy manipulation of influenza viruses, e.g., by the introduction of attenuating mutations into the viral genome. Further, because influenza viruses induce strong humoral and cellular immunity, the invention greatly enhances these viruses as vaccine vectors, particularly in view of the availability of natural variants of the virus, which may be employed sequentially, allowing repetitive use for gene therapy.

Problems solved by technology

However, this progress had been relatively limited for negative-sense as compared with positive-sense RNA viruses, because neither the genomic viral RNA (vRNA) nor the antigenomic complementary RNA (cRNA) of negative-sense RNA viruses can serve as a direct template for protein synthesis.
Thus, generating segmented negative-sense RNA viruses from cloned cDNAs poses a formidable challenge, as one must produce a separate vRNA for each gene segment.
However, the efficiency of virus recovery was low.
With both methods, however, transfectants must be selected from a vast background of helper viruses, which requires a strong selection system and complicates the generation of growth-defective viruses.
However, the efficiency of the system is low: in 25% of the experiments, the investigators failed to detect reporter gene expression.

Method used

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  • Recombinant influenza viruses for vaccines and gene therapy
  • Recombinant influenza viruses for vaccines and gene therapy
  • Recombinant influenza viruses for vaccines and gene therapy

Examples

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example 1

Materials and Methods

[0056] Cells and Viruses.

[0057] 293T human embryonic kidney cells and Madin-Darby canine kidney cells (MDCK) were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum and in modified Eagle's medium (MEM) containing 5% newborn calf serum, respectively. All cells were maintained at 37° C. in 5% CO2. Influenza viruses A / WSN / 33 (H1N1) and A / PR / 8 / 34 (H1N1) were propagated in 10-day-old eggs.

[0058] Construction of Plasmids.

[0059] To generate RNA polymerase I constructs, cloned cDNAs derived from A / WSN / 33 or A / PR / 8 / 34 viral RNA were introduced between the promoter and terminator sequences of RNA polymerase I. Briefly, the cloned cDNAs were amplified by PCR with primers containing BsmBI sites, digested with BsmBI, and cloned into the BsmBI sites of the pHH21 vector which contains the human RNA polymerase I promoter and the mouse RNA polymerase I terminator, separated by BsmBI sites (FIG. 2). The PB2, PB1, PA, HA, NP, NA, M, a...

example 2

[0081] Expression of the Influenza Virus Proteins PB2, PB1, PA, and NP Leads to Replication and Transcription of an Artificial Viral RNA.

[0082] To generate influenza VLPs, the RNA polymerase I system for the intracellular synthesis of influenza viral RNAs in vivo was employed (FIG. 7). In this system, a cDNA encoding a reporter gene in antisense orientation is flanked by the 5′ and 3′ noncoding regions of an influenza viral RNA. This cassette is inserted between an RNA polymerase I promoter and terminator. Transfection of such constructs into eukaryotic cells leads to transcription of the reporter gene by cellular RNA polymerase I, thereby generating influenza virus-like RNAs (Neumann et al., 1994). Upon influenza virus infection, the artificial vRNAs are replicated and transcribed by the viral polymerase complex, resulting in the expression of the reporter gene.

[0083] To determine whether expression of the PB2, PB1, PA, and NP proteins leads to expression of the reporter gene enc...

example 3

[0100] By using the Cre-loxP system, one can generate packaging cell lines for the production of replication-defective viruses. For example, a protein expression vector is prepared that contains a transcription stop cassette (e.g., pBS302 of Life Technologies, Bethesda, Md.; and Sauer et al., 1993; Lasko et al., 1992; Pichel et al., 1993; Bolivar et al., 1977; Stuhl et al., 1981; Stuhl, 1985; Fiers et al., 1978), flanked by two loxP sites, and one of the viral genes. Transcription, initiated at the promoter sequence, is blocked at the transcription stop sites. Thus, the viral gene is not transcribed and translated. A cell that is stably transfected with such a vector is infected with an influenza virus that lacks the vRNA encoding the gene cloned into the loxP system. This virus also contains an additional vRNA encoding the Cre protein. This virus is not viable in normal cells, because it lacks one of its vRNAs. However, in the packaging cell line, the Cre protein which is expressed...

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Abstract

The invention provides a composition useful to prepare influenza A viruses, e.g., in the absence of helper virus.

Description

CROSS REFERENCE TO RELATED APPLICATION [0001] This application is a continuation of U.S. application Ser. No. 09 / 971,372 filed Oct. 4, 2001, which is a continuation of International Patent Application No. PCT / US00 / 09021, filed on 5 Apr. 2000, which in turn is an international filing of U.S. Provisional Patent Application No. 60 / 127,912, filed Apr. 6, 1999 and U.S. Provisional Patent Application No. 60 / 132,839, filed May 6, 1999, all of which are incorporated herein by reference.STATEMENT OF GOVERNMENT RIGHTS [0002] This invention was made with a grant from the Government of the United States of America (grant AI-29599 from the National Institute of Allergy and Infectious Diseases Public Health Service). The Government may have certain rights in the invention.BACKGROUND OF THE INVENTION [0003] The ability to generate infectious RNA viruses from cloned cDNAs has contributed greatly to the biological understanding of these pathogens and hence to improved methods of disease control (Pal...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K48/00C12N5/08C12N15/86A61K39/00C07K14/11C12N7/04
CPCA61K39/00C12N15/85A61K48/00A61K48/0091A61K2039/525A61K2039/5258C07K14/005C12N7/00C12N15/86C12N2760/16122C12N2760/16123C12N2760/16134C12N2760/16143C12N2760/16151C12N2800/30C12N2810/6081A61K39/145A61K39/12C12N2760/16121C12N2760/16152
Inventor KAWAOKA, YOSHIHIRONEUMANN, GABRIELE
Owner KAWAOKA YOSHIHIRO
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