Novel influenza virus A mammalian cell adapted strain and preparation and application thereof

A type of influenza virus, host cell technology, applied in the field of biotechnology and immunology

Inactive Publication Date: 2016-10-05
INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, PR8 is not suitable for amplification in mammalian cells, so it is necessary to develop new backbone strains for mammalian cell production platforms

Method used

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  • Novel influenza virus A mammalian cell adapted strain and preparation and application thereof
  • Novel influenza virus A mammalian cell adapted strain and preparation and application thereof
  • Novel influenza virus A mammalian cell adapted strain and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0162] Example 1. The NS gene derived from the WSN strain can promote the high-speed growth of the PR8 strain on Vero cells.

[0163] Vero cells are a mammalian cell line recommended by WHO for vaccine production, and their safety has been well proven. However, the backbone virus strain PR8 used in the production of influenza vaccines is an adaptation strain of chicken embryos, and the adaptation of the mammalian cell line Vero The performance is not good enough to meet the requirements of production.

[0164] Therefore, the inventors recombined the PR8 strain with other strains WSN that are more suitable for mammalian cell lines, and improved the adaptability of PR8 on Vero cells. The genes of PR8 strain (except HA and NA) were replaced with WSN by using reverse genetic technology. By comparing the size of the plaques, only the NS gene from WSN replaced the NS gene of PR8. Larger plaques were formed on cells than PR8 ( figure 1 A). And the recombinant virus PR8-WSN M (the ...

Embodiment 2

[0166] Example 2, NS2 K86R The mutation can promote the high-speed growth of PR8 on Vero.

[0167] In order to identify the site of NS gene that promotes the rapid growth of PR8 on Vero cells, the NS gene of PR8 strain was compared with the NS gene of WSN strain. The NS gene of PR8 and the NS gene of WSN shared 11 non-synonymous mutations in the nucleotide sequence, located at the 163,301,308,318,512,537,565,660,661,729,784 nucleotides ( figure 2 , A). Since the NS gene undergoes alternative splicing when it is transcribed into mRNA, two different mRNAs are formed, encoding the nonstructural protein NS1 and the nuclear export protein NS2 (NEP), respectively. These 11 nucleotide mutations resulted in 7 amino acid mutations on the NS1 protein and 6 amino acid mutations on the NS2 protein ( figure 2 , A).

[0168] With the method of reverse genetics, the PR8-based strains with all the above-mentioned NS gene non-synonymous mutations were rescued (103F and 106M are known to st...

Embodiment 3

[0170] Embodiment 3, NS2 K86R The mutation does not change the antigenicity of PR8, as well as the pathogenicity to mice and chicken embryos.

[0171] In mammalian cell culture system, PR8 high-producing strain (PR8-NS2 K86R ) has a faster growth rate than the PR8 wild-type virus, and the inventors need to measure whether its pathogenicity to mammals and birds is also increased accordingly, which is of great significance to the safe production of vaccines.

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PUM

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Abstract

The invention discloses a novel influenza virus A mammalian cell adapted strain and preparation and application thereof and particularly provides PR8 virus strain mutant protein and a recombinant PR8 virus strain containing the PR8 virus strain mutant protein, wherein the PR8 virus strain mutant protein can greatly improve the growing ability of an influenza virus A strain PR8 on mammalian cells (particularly Vero cells), after K at the site 86 of NS2 protein of the PR8 virus strain is mutated into R, the growing ability of the obtained virus strain on the Vero cells is improved by 20-100 times compared with that of a wild PR8 virus strain, and the pathogenicity of the adapted strain on a living body is not enhanced. Therefore, the influenza virus A strain containing the mutant protein can serve as a high-yield vaccine seed strain on mammalian cells.

Description

technical field [0001] The invention belongs to the fields of biotechnology and immunology; more specifically, the invention relates to a method for obtaining a mammalian cell-adapted strain of influenza A virus vaccine and an adapted site. Background technique [0002] Influenza is one of the most serious infectious diseases in human history, and its main pathogen is influenza virus. Its host range is broad, including wild birds, poultry, various mammals, and humans. It mutates quickly and can change its antigenicity to evade host immune system attack, or change its own drug target characteristics to gain drug resistance. These characteristics determine that influenza virus can spread widely among humans and animals and is difficult to control. In addition to causing seasonal epidemics (epidemic) every year, it also causes pandemics (pandemic) from time to time, which brings huge losses to human society. In the past century, influenza viruses have had four major pandemic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/11C12N15/44C12N15/85C12N5/10C12N7/00C12R1/93
CPCC07K14/11C12N5/10C12N7/00C12N15/85
Inventor 孙兵徐可张宏
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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