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Vaccine for hand-foot-and-mouth disease viruses

A hand, foot and mouth disease virus and vaccine technology, applied in the field of human vaccines, can solve the problems of lack of vaccine supply, achieve good immune effect, good immunogenicity, and ensure safety

Active Publication Date: 2010-12-01
BEIJING LUZHU BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Although hand, foot and mouth disease is frequently epidemic in many provinces of our country and Asian countries, there is still no vaccine available to prevent the disease so far

Method used

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  • Vaccine for hand-foot-and-mouth disease viruses
  • Vaccine for hand-foot-and-mouth disease viruses
  • Vaccine for hand-foot-and-mouth disease viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Isolation and determination of EV71 virus

[0045] Collect about 1 gram of fecal samples from patients with hand, foot and mouth disease, add 9ml of sterile normal saline solution, 10mg each of penicillin sodium and streptomycin, and 0.1ml of chloroform, stir with a glass rod for 10 minutes to make a suspension, and then incubate at 4°C. , Centrifuge at 10,000 rpm for 20 minutes, carefully draw 5ml of the supernatant in the centrifuge tube, and filter and sterilize it with a 0.2μ disposable sterile filter membrane.

[0046] Take 1ml of the sterile filtrate obtained by the above method, add it to the VERO cell culture bottle that has been cultured for 2 days, tighten the bottle cap, and incubate at 36°C, suck out the liquid in the cell culture bottle with a sterile pipette after 1 hour, and then add The 199 culture solution containing 3% newborn bovine serum was placed in a 36°C incubator to continue culturing. After 15 hours, the cells began to show pathological changes...

Embodiment 2

[0051] Virus multiplication

[0052] 1. Spinning bottle cell culture and proliferation virus: take VERO cells (or MRC-5 cells, WI-38 cells) grown into a single layer, suck out the culture medium in the culture bottle, add 0.25% trypsin to digest the cells, and suck out the trypsin solution , add 199 cell culture medium, blow repeatedly to disperse the cells, and then subculture according to the ratio of 1:8-1:10, and inoculate EV71 virus after 5-7 days of 36°C spinner bottle culture, the amount of virus added is 36-36 after inoculation. It is advisable to cause 75-90% of the total number of VERO cells to have lesions within 48 hours. Then place it at 4°C for more than 12 hours, but not more than 48 hours, and harvest the virus. Centrifuge at 4°C (7000-12000 rpm) for 30 minutes to remove cell debris, then filter with a 0.45μ filter, collect the filtrate, and place it at -20°C. The titer of the harvested virus liquid should be determined, and the titer should not be lower than...

Embodiment 3

[0056] Virus inactivation and purification

[0057] Take out the harvested virus liquid from 4°C or -20°C, add 1 / 2500 to 1 / 4000 formaldehyde (or β-propiolactone), inactivate at 35-37°C for 72 hours (3 days), take a 3ml sample and fill it In a dialysis bag sterilized by high pressure steam, dialyze 200 times the volume of phosphate buffered saline (40mM PBS, pH7.4) for 24 hours, change the dialysate twice during the period, inoculate VERO cells with the virus sample after removing formaldehyde, and culture for 7 There was no cytopathic effect in the first day, which proved that the EV71 virus could be effectively inactivated under the above conditions.

[0058] The above-mentioned inactivated EV71 virus liquid is concentrated to 1 / 5 of the original volume through a 300KD cut-off molecular weight ultrafiltration membrane produced by Pall (or Millipore), and then recovered with 40mM PBS (containing 0.3-1.0M NaCl, pH 7.4) To the original volume, continue ultrafiltration to 1 / 5 of...

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Abstract

The invention relates to preparation of a vaccine for hand-foot-and-mouth disease viruses and an application method thereof, belonging to a novel vaccine for preventing infectious diseases. The vaccine of the invention mainly comprises the components of high-purified inactivated human enteropathogenic virus 71 (EV 71) and an aluminum adjuvant. The vaccine, which is prepared according to the method of the invention, has excellent immunogenicity, and after immunity, organisms can selectively generate a high titer serum neutralization antibody, thereby preventing infectious diseases caused by the human EV 71.

Description

technical field [0001] The invention belongs to the technical field of human vaccines. The invention discloses a preparation and application method of an inactivated vaccine capable of preventing hand, foot and mouth disease virus EV71 infection. technical background [0002] Hand-foot-mouth disease (HFMD) is one of the common childhood infectious diseases caused by enteroviruses, which mostly occurs in infants under the age of 5 and can cause fever, rashes and ulcers on the hands, feet, mouth and other parts , Individual patients can cause fatal complications such as myocarditis, pulmonary edema, and encephalitis. There are more than 20 enteroviruses that can cause HFMD, and Coxsackie virus A16 (CoxA16) and EV71 are the two most common. [0003] It is known that there are many types of viruses that cause hand, foot, and mouth disease (HFMD), all of which belong to the genus Human Enterovirus of the Picorna family (Picorna), and the types include CoxA5, A10, A16, A19, EV71...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/125A61P31/14C12N7/00C12R1/93
Inventor 孔健周朋蒋先敏许磊涛杨秀华毕海超邹强
Owner BEIJING LUZHU BIOTECH
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