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CV-A6 virus species and human inactivated vaccine thereof

A CV-A6, inactivated vaccine technology, applied in the direction of positive-sense single-stranded RNA viruses, viruses, antiviral agents, etc., can solve problems such as difficulty in control, lack of CV-A6 vaccine research and development, and achieve the effect of increasing production efficiency

Inactive Publication Date: 2019-04-12
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

In order to solve the difficulty of EV71 vaccine in controlling the prevalence of HFMD caused by other enteroviruses such as CV-A16 and CV-A6, and the public acceptance and questioning of EV-A71 vaccine that may be caused by this, it is proposed that EV-A71 and CV-A16 be used as the basis for research and development. Core, increase the necessity of combined HFMD vaccine with enteroviruses such as CV-A6 as the main component
At present, the CV-A16 vaccine is under development, and the research and development of the CV-A6 vaccine is still lacking.

Method used

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  • CV-A6 virus species and human inactivated vaccine thereof
  • CV-A6 virus species and human inactivated vaccine thereof
  • CV-A6 virus species and human inactivated vaccine thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment one: the isolation, adaptation and purification method of the virus strain involved in the present invention

[0044] (1) Obtain virus samples

[0045] Collect excrement samples from hand, foot and mouth disease patients identified as CV-A6 infection by the Pediatric Department of the hospital, take 1g of feces and resuspend in 5mL of normal saline, centrifuge at 3000g for 30 minutes to get the supernatant, and filter it with a 0.45μm filter (purchased from Millipore Company) , stored at -20°C for later use.

[0046] (2) Adaptation of virus on RD cells

[0047] Select the RD cells that have grown into a dense monolayer, discard the old culture medium, wash the cell surface with an appropriate amount of PBS solution, and add an appropriate amount of 0.125% trypsin to digest the cells. Inoculate 2ml of the culture medium into each well of a 24-well plate, inoculate 200μl of the filtered sample on the cell surface, incubate at 37°C for 3 to 4 days, observe the...

Embodiment 2

[0054] Example 2: Cultivation and vaccine preparation method of CV-A6 strain YNKA1205

[0055] (1) The virus was cultured in KMB17 cells

[0056] Select the P28 generation KMB17 cells that have grown into a dense monolayer, discard the old culture medium, wash the cell surface with an appropriate amount of PBS solution, add an appropriate amount of 0.125% trypsin to digest the cells, and gently pat the cell bottle, the cells slide down from the bottle wall like quicksand, Add cell culture medium, press 2×10 5 Inoculate small square flasks with cells / bottle, inoculate 3-4 days at 37°C, inoculate 200 μl of CV-A6 virus on the cell surface, adsorb at 37°C for 1 hour, then add MEM cell maintenance solution without calf serum, incubate at 37°C and Observe the cell lesions and the virus CPE reaches 90%. Harvest the virus and freeze it for later use. attached Figures 1a-1d For the CV-A6 strain to produce pathological changes in KMB17 cells (cultured for 4 days), cytopathic effects...

Embodiment 3

[0063] Embodiment three: the identification method of virus seed

[0064] (1) Sterility inspection and mycoplasma detection experiment

[0065] Take 4 tubes of thioglycolate liquid medium and 2 tubes of tryptone soy liquid medium, and inoculate each tube with 0.5 ml of virus. After inoculation, 2 tubes of thioglycolate fluid medium were cultivated at 30-35°C; 2 tubes of thioglycolate fluid medium and 2 tubes of tryptone soytone liquid medium were cultured at 20-25°C; method as a negative control. The results were judged after 14 days of culture. Take 4 tubes of semi-solid medium and broth medium for mycoplasma inspection, boil and melt the semi-solid medium, and cool to 56°C. Add 2.5ml of incapacitated calf serum and double-antibody mixture mixed at a ratio of 4:1 to each of the two media, shake quickly, and then add 0.5ml of the sample to be tested to each medium for primary culture. Incubate and observe at 35°C. On the 7th day, take out 2 tubes of each of the two primar...

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Abstract

The present invention discloses a CV-A6 virus species and a human inactivated vaccine thereof. A classification naming of the virus species is Coxsackie virus group A type 6, a preservation number isCGMCC No. 16217, and the prepared human CV-A6 inactivated vaccine consists of 100 [mu]g / ml of CV-A6inactivated purified antigen and 1 mg / ml of aluminum hydroxide. The vaccine product has good immunogenicity and safety through animal experiments.

Description

technical field [0001] The invention relates to a CV-A6 virus seed and an inactivated vaccine for human use. Background technique [0002] Hand foot mouth disease (hand foot mouth disease, HFMD) in the world, especially in the Asia-Pacific region, the epidemic scale and threat impact are increasing, and it is worth noting that the disease is increasingly harmful to children. So far, the epidemiological statistics in my country show that since the disease was listed as a Class C infectious disease in 2008, it has become the first in the number of cases of Class C infectious diseases in 2009, and the overall case fatality rate is about 0.05%. The prevention and control of this infectious disease, especially among children, has become an important public health issue. [0003] In recent years, the outbreak or prevalence of HFMD caused by CV-A6 (Coxsackievirus group A type 6) infection has been increasing, and some areas have become the main HFMD cases other than enterovirus 71...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K39/125A61P31/14
CPCA61K39/12A61P31/14C12N7/00A61K2039/5252C12N2770/32351C12N2770/32321C12N2770/32334
Inventor 马绍辉刘红波张名张捷赵义林张海浩杨昭庆黄小琴
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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