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Method for preparing nasal-spray type influenza virus vaccine containing CpG ODN and poly I:G adjuvant

A technology for preparation of influenza virus and vaccine, applied in the field of preparation of nasal spray influenza vaccine, to achieve the effect of increasing production capacity, enhancing mucosal immunity and ensuring public health safety

Inactive Publication Date: 2010-06-23
云南沃森生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Preparation of nasal spray influenza vaccine with CpG ODN and PolyI:C two adjuvants has not been reported in the literature at home and abroad

Method used

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  • Method for preparing nasal-spray type influenza virus vaccine containing CpG ODN and poly I:G adjuvant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] 1) Propagating influenza virus

[0020] Inoculate the H1N1, H3N2, and B influenza virus working strains into the allantoic cavity of 1,000 10-day-old healthy chicken embryos, and culture them at 34°C for 72 hours;

[0021] 2) Harvest influenza virus and concentrate by ultrafiltration

[0022] The chicken embryos were placed in a 4°C cold storage overnight, and the allantoic fluid was collected and combined to obtain 12,500 ml of influenza virus harvest fluid. Add 12.5ml of formaldehyde solution to the harvested influenza virus and inactivate at 2-8°C with shaking for 24 hours. The inactivated virus solution was centrifuged at 4000g for 30 minutes, clarified and filtered by a 0.8 micron hollow fiber system, and then concentrated to 150 ml using an ultrafiltration membrane with a molecular weight cut-off of 100kD.

[0023] 3) Sepharose 4FF column purification

[0024] Load the influenza virus sample after ultrafiltration concentration on a Sepharose 4FF column (XK50 / 100 column, c...

Embodiment 2

[0031] 1) Propagating influenza virus

[0032] Inoculate the H1N1, H3N2, and B influenza virus working strains into the allantoic cavity of 1,000 10-day-old healthy chicken embryos, and culture them at 34°C for 72 hours;

[0033] 2) Harvest influenza virus and concentrate by ultrafiltration

[0034] The chicken embryos were placed in a 4°C cold storage overnight, and the allantoic fluid was collected and combined to obtain 12,500 ml of influenza virus harvest fluid. Add 12.5ml of formaldehyde solution to the harvested influenza virus and inactivate at 2-8°C with shaking for 24 hours. The inactivated virus solution was centrifuged at 4000g for 30 minutes, clarified and filtered by a 0.8 micron hollow fiber system, and concentrated to 148 ml using an ultrafiltration membrane with a molecular weight cut-off of 300kD.

[0035] 3) Sepharose 4FF column purification

[0036] Load the influenza virus sample after ultrafiltration concentration on a Sepharose 4FF column (XK50 / 100 column, column...

Embodiment 3

[0043] 1) Propagating influenza virus

[0044] Inoculate the H1N1, H3N2, and B influenza virus working strains into the allantoic cavity of 1,000 10-day-old healthy chicken embryos, and culture them at 34°C for 72 hours;

[0045] 2) Harvest influenza virus and concentrate by ultrafiltration

[0046] The chicken embryos were placed in a 4°C cold storage overnight, and the allantoic fluid was collected and combined to obtain 12,500 ml of influenza virus harvest fluid. Add 12.5ml of formaldehyde solution to the harvested influenza virus and inactivate at 2-8°C with shaking for 24 hours. The inactivated virus solution was centrifuged at 4000g for 30 minutes, clarified and filtered through a 0.8 micron hollow fiber system, and then concentrated to 145 ml using an ultrafiltration membrane with a molecular weight cut-off of 750kD.

[0047] 3) Sepharose 4FF column purification

[0048] Load the influenza virus sample after ultrafiltration concentration on a Sepharose 4FF column (XK50 / 100 colu...

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Abstract

The invention provides a method for preparing nasal-spray type influenza virus vaccine containing CpG ODN and poly I:G adjuvant, which belongs to the technical field of biology. The method comprises the following steps: adopting chicken embryo to culture proliferative influenza virus; obtaining purified influenza virus through ultrafiltration concentration and column chromatography; obtaining influenza vaccine stock solution through lysis; and proportioning the influenza vaccine stock solution of a required type to CpG ODN adjuvant or I:G adjuvant. The invention provides the method for preparing novel nasal-spray type influenza virus vaccine, which can reduce the amount of influenza virus antigen, provides convenience for immunization and vaccination, and is suitable for rapidly improving the capacity of supplying influenza vaccine under threat of global pandemic influenza.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and more specifically, relates to a method for preparing a nasal spray influenza vaccine containing CpG ODN and poly I:C adjuvant. Background technique: [0002] Influenza is a global respiratory disease caused by influenza viruses. Influenza vaccination is an effective way to prevent influenza. [0003] Traditional influenza virus vaccines include inactivated influenza virus vaccines, influenza virus split vaccines and influenza subunit vaccines. These influenza vaccines are injected intramuscularly. In Russia and the United States, a live attenuated influenza vaccine has been approved. The vaccine uses a cold-adapted recombinant influenza strain to be administered via nasal spray, but the vaccine is not approved for use in infants and children under 3 years of age. [0004] CpG ODN (oligodeoxynucleotides, ODN) is a single-stranded oligonucleotide containing unmethylated CpG dinucleotide sequence. CpG ODN ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/39A61K39/145A61K9/12A61P31/16C12N7/02
Inventor 张翊向左云代云波
Owner 云南沃森生物技术股份有限公司
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