Universal influenza vaccine and preparation method thereof

A kind of influenza vaccine, universal technology, applied in the field of nanovesicle mimicking influenza virus, can solve the problems of chicken embryo death, secondary pollution virus, long production cycle, etc., to achieve large-scale vaccine production, enhance Immunogenicity, the effect of avoiding the production of dangerous viruses

Active Publication Date: 2015-12-16
XIAMEN UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0003] At present, most influenza vaccines are produced in chicken embryos, and the production cycle is relatively long. At the same time, the production of avian influenza virus or other influenza viruses in chicken embryos is likely to cause the death of chicken embryos, which reduces the vaccine production.
In addition, the production process of highly pathogenic influenza viruses requires a high-level GMP environment, which is likely to cause secondary pollution and virus leakage

Method used

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  • Universal influenza vaccine and preparation method thereof
  • Universal influenza vaccine and preparation method thereof
  • Universal influenza vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1V

[0051] The preparation method of embodiment 1 VMV, comprises the following steps:

[0052] (1) 24 hours before transfection, digest the 293T cells in the logarithmic growth phase with 0.25% trypsin at 37°C for 2-3 minutes, and adjust the cell density to 1.2x10 with the medium containing 10% serum. 7 Cells / 20ml, reseeded in 15cm cell culture dish, 37℃, 5%CO 2 Cultured in an incubator. After 24 hours, when the cell density reaches 70%-80%, it can be used for transfection. Cell state is critical for virus packaging, so good cell state and low passage times need to be guaranteed. The cell culture medium was replaced with serum-free medium 2 h before transfection. The HA gene used comes from the H1N1 influenza virus (A / California / 04 / 2009). It is directly obtained by whole gene synthesis.

[0053] (2) Mix the diluted lentiviral packaging plasmid (PLV-HA, PRRE, REV, VSVG, according to the mass ratio 1:1:1:1) and the diluted Lipofectamine2000 (1:2.5), after vigorous shaking, Incu...

Embodiment 2

[0059] Example 2 Cellular localization of influenza HA protein, figure 1 :

[0060] Confocal laser microscopy was used to determine the localization of HA protein in eukaryotic cells, while a surfactant was used to promote the production of larger cellular vesicles. Specifically, after the cells were sliced ​​overnight, the PLV-HA plasmid was mixed with liposome2000, then added to the serum-free medium, and replaced with normal medium after 6 hours, and after 48 hours of transfection, the cells were taken out and fixed. , after staining nuclei with DAPI, wash four times with PBS, then add anti-HA mouse monoclonal antibody to incubate for half an hour, after washing, add FITC-labeled goat anti-mouse secondary antibody for immunofluorescence, the final 90% The sections were mounted in glycerol and observed using a Zeiss LSM-780 laser confocal microscope. It can be seen that most of the HA protein is present on the cell membrane, and under the influence of the surfactant (sodiu...

Embodiment 3

[0063] Example 3. In vitro receptor binding function evaluation (coagulation test) of influenza VMV particles:

[0064] In order to test the in vitro receptor-binding ability of influenza VMV particles, we derived 5×10 5 The VMV of 293T cells was diluted and mixed with 0.5% chicken red blood cells. After standing at room temperature for 40 minutes, the erythrocyte agglutination was observed. After that, there was a significant increase in hemagglutination titer, and this increase has a positive correlation with time and cell number. These data indicate that influenza mimic virus vesicles can bind to cell membrane receptors like normal live viruses, and promote chicken red blood cells. cross-linking ( image 3 ).

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Abstract

The invention relates to universal influenza vaccine and a preparation method thereof. After an influenza virus HA is subjected to gene transfection to eukaryocyte, influenza haemagglutinin (HA) protein subjected to eukaryotic expression modification is inserted into a host plasma membrane, then vesicles largely displaying influenza virus envelope glycoprotein is produced with a method that a surface active agent induces a cell to bud, and the vesicles are changed into controllable and nanometer mimicry virus vesicles with uniform dimensions after purification and re-adding of the surface active agent for treatment. At the same time, by the aid of establishment of stably expressing an HA cell strain and usage of a bioreactor, the method is also applicable to large scale influenza vaccine production. The method is simple and feasible, hemagglutinin of most influenza virus (including avian influenza and swine influenza) can be displayed, and the method has excellent universality, has a great potential and can be used for designing emerging highly lethal influenza virus vaccines.

Description

technical field [0001] The invention relates to a nanovesicle (VMV) mimicking influenza virus, which can display various subtypes of influenza HA and other antigens. It is used to prevent the outbreak of newly emerging unknown influenza virus, and belongs to the field of biomedical materials, nanomedicine and preventive medicine. Background technique [0002] Influenza virus is referred to as influenza virus for short. The envelope glycoprotein of influenza virus is composed of HA (haemmagglutinin) and NA (neuraminidase). Due to the variability of RNA viruses, HA and NA are highly variable, which causes influenza to spread every year around the world. There are small epidemics of different scales or a worldwide pandemic once every few years. Influenza not only brings great harm to human health but also to the development of the world economy. The World Health Organization warns that a global pandemic influenza will cause 2-7 million deaths, especially, after highly pathogen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/145A61P31/16C12N15/867C12N5/10
Inventor 刘刚张鹏飞陈小元陈毅歆
Owner XIAMEN UNIV
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