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Attenuated human rotavirus strain with broad-spectrum immunogenicity and vaccine prepared from the attenuated strain

A human-derived rotavirus, immunogenic technology, applied in the direction of antiviral agents, inactivation/attenuation, virus antigen components, etc., can solve the safety problems of live attenuated vaccines, etc. Stable, high viral titer effect

Active Publication Date: 2016-09-21
吕宏亮 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to prevent rotavirus infection, oral polyvalent or monovalent rotavirus vaccines have been developed worldwide, and the clinical effects have shown that they are safe and effective. However, the diversity of animal and human rotaviruses, the prevalence and characteristics of rotaviruses, There are still potential safety hazards in the use of attenuated live vaccines, such as the mutation and recombination of animal and human rotaviruses, and the cross-species transmission of human and animal viruses. Therefore, it is more urgent to develop a safe and broad-spectrum vaccine.

Method used

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  • Attenuated human rotavirus strain with broad-spectrum immunogenicity and vaccine prepared from the attenuated strain
  • Attenuated human rotavirus strain with broad-spectrum immunogenicity and vaccine prepared from the attenuated strain
  • Attenuated human rotavirus strain with broad-spectrum immunogenicity and vaccine prepared from the attenuated strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039]Example 1. Isolation, passage and screening of human rotavirus

[0040] In 2011, 10 feces samples of children with acute rotavirus diarrhea were collected in a hospital in China, resuspended with 20% PBS (pH=7.3), sterile filtered, centrifuged at 3000g for 20 minutes, and 0.1ml supernatant was added to trypsin (20ug / ml) mixed for 30 minutes, inoculated with PBS washed 3 times monolayer MA-104 cells (T-25 cell culture medium containing MEM, double antibody (100U gentamicin and 100ug streptomycin) and 8-10% fetal bovine serum ), incubate at 37°C for 1 hour, add virus maintenance solution (containing MEN and 0.5ug / ml trypsin) and incubate at 37°C for 10-15 days, freeze and thaw 3 times and centrifuge at 2000g for 20 minutes, take the supernatant and trypsin (20ug / ml / ml) mixed with MEM for 30 minutes and diluted 20 times with MEM to inoculate MA104 cells at 37°C for 8-9 days, and repeated passage for 8-10 generations until the virus incubation period was 2-3 days. The iso...

Embodiment 2

[0045] Identification and typing of embodiment 2 virus

[0046] The enriched virus HRV-LZ-2013 was typed by RT-PCR, and then confirmed by plaque reduction neutralization test. 45-50CCID 50 The virus was activated with 1.5ug of trypsin at 37°C for 1 hour, mixed with different dilutions of G1-G10, P2-P8 monoclonal antibodies or hyper-free serum to inoculate a 6-well plate of MA-104 cells grown into a monolayer, covering the 6-well plate containing 5ug / ml trypsin, 37°C, 5% CO 2 Incubate in the incubator for 4 days, fix with formaldehyde, count staining, and form 60% plaques, indicating the highest dilution of antibody.

[0047] The rotavirus was centrifuged at 36000rpm×3 hours, then the supernatant was removed, and the purified rotavirus HRV-LZ-2013 was obtained by centrifugation at 38000rpm×3 hours, 30% sucrose density, and Freund’s complete adjuvant and incomplete adjuvant were added to Rabbits were primed and boosted to prepare HRV-LZ-2013 hyper-immune serum.

[0048] Pur...

Embodiment 3

[0055] The preparation of embodiment 3 human rotavirus attenuated live vaccines

[0056] Include the following steps:

[0057] (1) Proliferation and culture of virus

[0058] Vero cells were inoculated at 200,000-300,000 cells / ml in spinner bottles, and after 3 days of culture at 37°C, rotavirus HRV-LZ-2013 or its 20-100 generation Vero cell subcultures were inoculated at MOI 0.01-0.1, and cultured at 37°C for 1 Hours later, wash with normal saline and add virus maintenance solution, the virus maintenance solution is MEM containing 0.1% bovine serum, pH 7.2, cultured at 32°C for 3-5 days, repeated freezing and thawing 3 times, centrifuging to remove cell debris, using MA104 cells were used to determine the virus titer, and the titer was ≥5.5logCCID 50 / ml and sterile as a qualified vaccine stock solution;

[0059] (2) Purification of rotavirus HRV-LZ-2013

[0060] Using continuous sucrose density gradient centrifugation, centrifuge at 120,000g in TNE buffer containing 60% ...

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Abstract

The invention discloses a human-derived rotavirus attenuated strain with wide-spectrum immunogenicity and a vaccine prepared from the human-derived rotavirus attenuated strain. The human-derived rotavirus attenuated strain with wide-spectrum immunogenicity is named as HRV-LZ-2013, has a gene type of G10P8, is preserved in the China general microbiological culture collection center and has a strain preservation number of CGMCC N0. 9452. An experiment proves that the rotavirus attenuated strain keeps low pathogenicity to animals. An immunization protection experiment on animals and especially on rotavirus-caused diarrhoea model rabbit and pig proves that the attenuated live vaccine or inactivated vaccine prepared from the human-derived rotavirus attenuated strain has good immunizing protection properties. An immunizing protection test on cattle and sheep proves that the vaccine provided by the invention has a wide immunizing protection range and can be used for preventing cattle, pig, horse, sheep and dog diarrhoea caused by rotaviruses.

Description

technical field [0001] The invention relates to an attenuated strain of rotavirus and a vaccine prepared from the attenuated strain, in particular to a safe and broad-spectrum immunogenic attenuated strain of human rotavirus, and a vaccine prepared from the attenuated strain. The obtained vaccine and its preparation method belong to the technical field of prevention and treatment of rotavirus infection. Background technique [0002] Diarrhea caused by rotavirus (RV) is a serious infectious disease in developing countries, killing about 50,000 children under 5 years old every year. Rotaviruses belong to the Reoviridae family and can be divided into three groups, A, B, and C. They infect both humans and animals. There are 11 non-segmented double-stranded RNAs in the three-layer coat protein of rotavirus. The coat proteins VP7 and VP4 are the main immunogens for virus-induced neutralizing antibodies, and the inner membrane protein VP6 is the basis for the classification of sub...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/04C12N7/06A61K39/15A61P31/14C12R1/93
Inventor 吕宏亮张澍
Owner 吕宏亮
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