Recombinant influenza viruses for vaccines and gene therapy

Abstract

A method for producing influenza virus particles for preparation of a vaccine. The method comprises growing reassortant influenza viruses to produce virus particles and recovering the produced virus particles. The reassortant influenza viruses are produced in cultured cells in the absence of helper virus by introducing into the cultured cells expression vectors sufficient to produce genomic vRNA or antigenomic vRNA (cRNA) segments in the cells of reassortant influenza viruses. The method produces a nucleoprotein and an RNA dependent RNA polymerase in the cells so that RNP complexes containing the genomic vRNA segments of the reassortant influenza viruses are formed. The method uses expression vectors containing HA, NA, non-HA and non-NA vRNA segments in which the HA and NA vRNA segments are HA and NA vRNA segments of a viral strain different from a strain that contains all of the non-HA and non-NA vRNA segments.

Description

STATEMENT OF GOVERNMENT RIGHTS[0001]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[0002]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 (Palese et al., 1996). 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. Rather, the vRNA, after its encapsidation by viral nucleoprotein (NP), must be transcribed into positive-sense mRNA by the viral RNA polymerase complex. Thus, the minimal replicat...

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Benefits of technology

[0015]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.

[0016]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.

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