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Vero (Rabies Purified Vaccine for Human Use) cell cold-adapted strain of influenza A virus and application thereof

An influenza A virus, cold-adapted technology, applied in the field of live virus vaccine or live attenuated influenza, can solve the problems of continuous passage variation, mismatch of vaccine epidemic strains, and reduced vaccine efficacy, and achieves easy standardization and avoids blood clotting. vegetative antigens are prone to changing effects

Active Publication Date: 2014-07-02
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are following disadvantages in using chicken embryos as the culture substrate: (1) Potential contamination may cause chicken infection causes to be vaccinated and disperse; Reduce vaccine efficacy; (3) Because the culture period is too long, it is difficult to produce large-scale vaccines that can meet the sudden demand of influenza pandemic; (4) The purification process is complicated; (5) It is easy to cause allergic reactions
Some domestic and foreign manufacturers directly inoculate influenza vaccine production virus seeds provided by WHO. Vero cells have low or unstable toxin production, which restricts the use of Vero cells to culture influenza viruses to prepare influenza vaccines.

Method used

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  • Vero (Rabies Purified Vaccine for Human Use) cell cold-adapted strain of influenza A virus and application thereof
  • Vero (Rabies Purified Vaccine for Human Use) cell cold-adapted strain of influenza A virus and application thereof
  • Vero (Rabies Purified Vaccine for Human Use) cell cold-adapted strain of influenza A virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Influenza A virus Vero cell cold-adapted strain A / Yunnan / 1 / 2005Vca (H 3 N 2 ) preparation method:

[0049] 1. On the Vero cells grown into a dense monolayer within 24 hours, inoculate the influenza A virus A / kunming / 1 / 2005Va (H3N2) with the preservation number of CCTCC NO.V200514 with a hemagglutination titer of 1:1024 at 1 MOI;

[0050]2. Maintain the composition of mother liquor as MEM, penicillin-streptomycin 20,000 U / ml, glutamine solution 0.6mg / ml, bovine serum albumin 1mg / ml, TPCK-trypsin concentration 1.0ug / ml, pH7.2 , under the condition of 30±1°C, after culturing the influenza A virus in step 1 for 72 hours, harvest the virus liquid;

[0051] 3. Freeze and thaw the virus liquid harvested in step 2 repeatedly 3 times, and then inoculate it on Vero cells that have grown into a dense monolayer for 24 hours, and then repeat steps 2 and 3, so that the Vero cells are continuously passed on for 20 generations, and the seeds are maintained. The hemagglutination tite...

Embodiment 2

[0062] Embodiment 1 gained influenza A virus A / Yunnan / 1 / 2005Vca (H 3 N 2 ) was continuously passaged at 25°C on Vero cells, and still maintained a stable and high yield with Ca, Ts Phenotype and Att characteristic:

[0063] Inoculate the Influenza A virus of Example 1 of 1MOI on Vero cells growing into a compact monolayer in 24 hours, maintain the mother liquor composition as MEM, penicillin-streptomycin 20,000 U / ml, glutamine solution 0.3mg / ml, Bovine serum albumin 1mg / ml, TPCK-pancreatin concentration 1.0ug / ml, pH 7.2, cultured at 25±1°C for 168h, harvested virus liquid, cell harvest liquid was repeatedly frozen and thawed 3 times, harvested virus liquid was again Inoculated on Vero cells, the method is the same as above, and the continuous passage on Vero cells at 25°C, the hemagglutination titer of the harvested liquid of toxic cells can be maintained at 1:512-1:1024.

[0064] Influenza A virus Vero cell cold-adapted strain A / Yunnan / 1 / 2005Vca (H 3 N 2 ),have Ca, Ts...

Embodiment 3

[0070] Embodiment 1 gained influenza A virus A / Yunnan / 1 / 2005Vca (H 3 N 2 ) maintains a stable and high yield in continuous passage at 25°C on serum-free Vero cells:

[0071] Inoculate the influenza A virus of Example 1 at 1MOI to Vero cells that have grown into a dense monolayer and cultured without serum in 24 hours, and maintain the mother liquor composition as SFM (Hyclone), penicillin-streptomycin 20,000 U / ml, gluten Aminoamide solution 0.6mg / ml, bovine serum albumin 1mg / ml, TPCK-trypsin concentration 0.5ug / ml, pH 7.0-7.2, cultured at 25±1°C for 120-168h, harvested the virus liquid and harvested the cells The solution was repeatedly frozen and thawed 3 times, and the harvested virus solution was inoculated on Vero cells again. The method was the same as above. After such continuous passage on Vero cells, the hemagglutination titer of the virus cell harvest solution could be maintained at 1:512.

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Abstract

The invention provides a Vero cell cold-adapted strain of influenza A virus and an application thereof. The Vero cell cold-adapted strain is named as A / Yunnan / 1 / 2005Vca (H3N2), the microbial preservation number is CCTCC NO. V201253. The strain can be subjected to a continuous passage culture on a Vero cell, the hemagglutination titer is maintained at 1:512-1:1024, and the infectious titer is maintained at 7.0-8.5log10TCID50 / ml, and has phenotypes of Ca and Ts and characteristics of Att by identification. The strain as a female parent strain and an epidemic strain is subjected to a genetic reassortment, or six internal genes of the strain and two surface protein genes of the epidemic strain are subjected to a gene reassortment by a reverse genetics technology to obtain the Vero cell cold-adapted strain containing surface antigens of the epidemic strain. The Vero cell cold-adapted strain can be used in the preparation of Vero cell live-attenuated influenza vaccines or live-attenuated influenza vaccines, and can be also used for the production of inactivated influenza vaccines.

Description

technical field [0001] The present invention relates to a cold-adapted Vero cell strain of influenza A virus, and involves reassortment of the adapted strain as a parental strain with other circulating strains, thereby obtaining a Vero cell cold-adapted strain containing the surface antigen of the circulating strain, to The invention belongs to the technical field of biological products as the preparation of Vero cell influenza live attenuated vaccine or influenza live attenuated vaccine and virus seeds of influenza inactivated vaccine. Background technique [0002] Influenza virus belongs to the Orthomyxoviridae family, and its virus particles are spherical or filamentous, and the nucleocapsid is helically symmetrical. The viral genome is a single-strand negative-strand segmented RNA with an envelope. According to the antigenicity of viral nucleoprotein (NP) and matrix protein (MP), influenza viruses can be divided into three types: A (A), B (B), and C (C). Contains 8 RNA ...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K39/145A61P31/16C12R1/93
Inventor 廖国阳李卫东杨景晖周芳烨马磊周健蔡玮寸怡娜高菁霞张新文戴宗祥姜述德
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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