Live attenuated influenza virus vaccine and preparation method thereof

A technology of live attenuated vaccine and influenza virus, which is applied in the field of live attenuated influenza virus vaccine and its preparation, can solve the problems of low pathogenicity, achieve strong immunogenicity, good gene stability, and expand immune population Effect

Active Publication Date: 2017-05-10
SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The vaccine is extremely limited in its ability to replicate at a normal human body temperature of 37°C, and has no or low pathogenicity to humans, but can effectively replicate at 25°C and 33°C, Has the characteristics of cold adaptation and temperature sensitivity [Ambrose CS, Luke C, Coelingh K.Current status of liveattenuated influenza vaccine in the United States for seasonal and pandemic influenza. Influenza Other Respi Viruses.2008,2(6):193-202.]
However, due to the restriction and protection of patents and intellectual property rights, this type of live attenuated vaccine has not yet been approved for marketing in China

Method used

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  • Live attenuated influenza virus vaccine and preparation method thereof
  • Live attenuated influenza virus vaccine and preparation method thereof
  • Live attenuated influenza virus vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1. Preparation of influenza virus attenuated live vaccine

[0034]Extract virus RNA from the allantoic fluid of chicken embryos inoculated with A / Puerto Rico / 8 / 34 (H1N1) virus, use RT-PCR technology to prepare cDNA, and amplify PB2, PB1, PA, HA, NP of influenza virus , NA, M, NS eight gene segments. Mutations were respectively introduced into the M gene by overlapping PCR, and the sequence of the mutated M gene is shown in SEQ ID NO.1. With the assistance of transfection reagent Lipofectamine2000, the eight plasmids were co-transfected into 293T cells. After 72 hours, the samples were harvested, and 10-day-old chicken embryos were inoculated. After 48-72 hours, the allantoic fluid of the chicken embryos was harvested, and the correctness of the virus sequence was verified by hemagglutination experiments, gene extraction, and sequencing. The strain was named M-PR8.

[0035] M gene sequence of M-PR8 strain (mutated sequence)

[0036] agcgaaagca tgtagatatt g...

Embodiment 2

[0038] The research of embodiment 2.M-PR8 biological characteristic

[0039] 1) Gene stability study: M-PR8 virus strain was inoculated on MDCK cells for 10 consecutive passages, the culture supernatants of the 1st, 5th and 10th passages were collected, total RNA was extracted, reverse transcribed into cDNA, and PCR amplified The gene is constructed on the pMD18-T Vector, sequenced, and the software compares the genes to identify whether the mutant strain can be stably passed on.

[0040] 2) Study on the growth characteristics of the virus strain at the cell level: inoculate the virus at 0.001 MOI, and measure the TCID at 24, 48, 72, and 96 hours after virus infection 50 , used to compare the growth of M-PR8 virus and PR8 virus.

[0041] 3) Study on attenuation properties: mice were intraperitoneally anesthetized with pentobarbital sodium, and each mouse was instilled with 20 μL of 10 5 TCID 50 After 3 days, the virus solution was anesthetized and killed by neck dissection,...

Embodiment 3

[0074]Example 3. M-PR8 candidate strain of influenza virus attenuated live vaccine can provide protection against homologous virus

[0075] Immunization procedures and steps: 6-8 weeks old female BALB / c mice were divided into 4 groups: ① M-PR8 virus strain (1000TCID50), ② M-PR8 virus strain (100TCID50) ③ M-PR8 virus strain (10TCID50) ④ PBS blank control group. After one immunization, 10LD50 PR8 was challenged 21 days later. Three days after the virus challenge, the broncho-alveolar lavage fluid was taken to measure the virus titer, and the rest were observed for body weight change and survival rate.

[0076] Determination of virus titer: The trachea-lung washing fluid was serially diluted 10 times, and each dilution was used to infect MDCK cells cultured in a 96-well plate (the density was about 80% to 90%), and each dilution was inoculated in parallel 4 cell wells and place infected cells in CO 2 in the incubator. After culturing at 37°C for 72 hours, hemagglutination test...

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Abstract

The invention discloses an influenza virus attenuated live vaccine with a preservation number of CCTCCNO: V201302, which is obtained based on mutation of M gene, the vaccine candidate strain has good gene stability, and attenuation characteristic and has strong immunogenicity, can induce a specific serum antibody of influenza virus and a mucous membrane antibody, and can induce different subgroups for generating T cell and quantity of CD8<+>T cells for secreting the IFN-gamma. Under current production capability and technology, immunization population can be effectively enlarged, and the influenza virus attenuated live vaccine has important social meaning and usage value during a flu period.

Description

technical field [0001] The invention relates to the field of biological products, in particular to a live attenuated influenza virus vaccine and a preparation method thereof. Background technique [0002] Influenza virus epidemics can be roughly divided into three types: worldwide pandemic, local epidemic and sporadic. Since April 2009, a new type of influenza virus (type A H1N1) began to spread in Mexico and quickly caused a worldwide influenza pandemic [Mexico flu deaths raise fears of global epidemic. MSNBC. 2009-04-24.]. As of May 26, 2011, a total of 1,353,141 people were infected and 15,934 died worldwide. [0003] The most effective means of preventing influenza virus epidemics is vaccination. [0004] Compared with traditional inactivated vaccines, live attenuated influenza virus vaccines have many advantages. It retains some of the original activity of the virus. After inoculation, the virus can replicate in the upper respiratory tract to simulate natural infectio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K48/00A61K39/145A61P31/16
Inventor 陈则易应磊
Owner SHANGHAI INST OF BIOLOGICAL PROD CO LTD
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