Acquisition method and adaptive sites of influenza A virus vaccine mammalian cell adaptive strain

A type of influenza A virus, influenza virus technology, applied in the fields of biotechnology and immunology

Inactive Publication Date: 2014-10-01
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 virus strains for mammalian cell production platforms, and even develop corresponding optimal virus strains for each different cultured cells to achieve the best results. good production effect

Method used

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  • Acquisition method and adaptive sites of influenza A virus vaccine mammalian cell adaptive strain
  • Acquisition method and adaptive sites of influenza A virus vaccine mammalian cell adaptive strain
  • Acquisition method and adaptive sites of influenza A virus vaccine mammalian cell adaptive strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0183] Example 1, PR8 was continuously passaged in Vero cells to obtain a greatly accelerated adaptive strain PR8-p20

[0184] The growth rate of PR8 in Vero cells was slow, and only small plaques could be formed 48h after infection ( figure 1 A). In the presence of 1 μg / μl TPCK-trypsin, inoculated at MOI=0.001, after 24 hours, the virus titer was only 10 4.5 PFU / ml, it takes at least 48h for the virus titer to reach a peak value of about 10 6.5 PFU / ml( figure 1 B). Such a growth rate is difficult to meet the needs of vaccine production.

[0185] Therefore, the inventors obtained a Vero-adapted strain of PR8 virus with increased growth rate by continuous passage on Vero cells. After Vero cells were uploaded for 10 passages, the plaques formed by PR8 became significantly larger. After 20 passages, PR8 can form larger plaques. Viral RNA was extracted from the strain after 20 passages, and the viral cDNA was obtained by reverse transcription, which was cloned into the reve...

Embodiment 2

[0194] Embodiment 2, internal virus gene is sufficient to support the rapid growth characteristic of PR8-p20

[0195] In order to find out the mutation sites that make PR8-p20 adaptable, the inventors sequenced the whole genome of PR8 and PR8-p20, and found a total of 13 amino acid site mutations, distributed in HA, NA, NS1, NP, PA , PB1 and PB2 seven proteins (Table 1). Considering the need to obtain a high-yielding PR8 backbone, the present inventors therefore investigated whether external or internal viral genes contributed to the rapid growth properties of PR8-p20.

[0196] The inventor constructed two strains of recombinant viruses, one containing the external gene HANA of PR8-p20, and the internal six genes (NP, M, NS, PA, PB1, PB2) were from PR8, named PR8-p20HA / NA .

[0197] The HA and NA of another strain virus come from PR8, and the internal 6 genes (NP, M, NS, PA, PB1, PB2) come from PR8-p20, named as PR8-p20 backbone.

[0198] Plaque experiments showed that ( ...

Embodiment 3

[0200] Example 3. Four point mutations on the polymerase and NS1 proteins determine the rapid growth characteristics of the PR8-p20 backbone

[0201] In order to further determine the point mutations that contribute to the rapid growth characteristics of the PR8-p20 backbone, the inventors constructed single-gene recombinant viruses for the five genes carrying the mutations, named PR8-p20NS, PR8-p20NP, PR8-p20PA, PR8 - p20PB1 and PR8-p20PB2; and single point mutant viruses PR8-NP S377N and PR8-NP N309H.

[0202] Through plaques on Vero cells ( image 3 A) and growth curve experiment ( image 3 B) Evaluation of the growth characteristics of these viruses. The results showed that PR8-p20NP and PR8-p20PB1 could form significantly larger and clearer plaques than PR8, and the growth rate was also significantly accelerated. The growth rate of PR8-p20NS and PR8-p20PA was slightly faster than that of PR8. However, the growth rate of PR8-p20PB2 was not increased compared with PR8. ...

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Abstract

The invention relates to an acquisition method and adaptive sites of an influenza A virus vaccine mammalian cell adaptive strain. According to the invention, a conventional influenza A vaccine skeleton strain is subjected to continuous passage in mammalian cells so as to obtain the mammalian cell adaptive strain; four amino acid sites on the viral protein coded by a virogene in the adaptive strain undergo mutation, which contributes to adaptability to the skeleton strain. The adaptive strain does not has enhanced pathogenicity on living bodies like chick embryos and mice, but has substantially improved adaptability to 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. Influenza viruses belong to the Orthomyxoviridae family (Orthomyxoviridae). According to the antigenicity of their internal nucleoprotein (NP) and matrix protein (M), they are divided into three types: A\B\C (A\B\C). Among them, type A (type A) is the main type of influenza that causes humans and various animals. It has a wide range of hosts (including wild birds, poultry, various mammals, and humans), and it mutates quickly (can change antigenicity and Evade the attack of the host immune system, or change its own drug target characteristics to obtain drug resistance). These characteristics d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N7/01C07K14/11C12N9/12C12N15/44C12N15/54C12R1/93
Inventor 孙兵徐可胡伟斌张宏
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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