Zika virus vaccine and preparation method thereof

A Zika virus and vaccine technology, applied in the field of virus vaccines, can solve the problems of DNA vaccine safety, RNA vaccine stability and application limitation, cross-linking virus structure damage, complicated preparation process, etc., so as to retain antigenicity and immunogenicity , good antigenicity and immunogenicity, the effect of simple preparation method

Pending Publication Date: 2019-07-19
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The balance between immunogenicity and safety of live attenuated vaccines is difficult to grasp, and its safety for pregnant women has certain problems
Traditional subunit vaccines, vector vaccines and virus-like particle vaccines adopt different methods to simulate the natural structure of the virus surface envelope protein, the preparation process is complicated and the cost is high
The safety of DNA vaccines and the stability of RNA vaccines also limit their application
Inactivated viruses have received widespread attention because of their safety and effectiveness, but traditional inactivated viruses use formaldehyde to inactivate viruses. Although the viruses after formaldehyde inactivation retain the surface envelope protein to a certain extent Part of the structure, but its cross-linking effect also caused great damage to the structure of the virus itself, limiting the effect of inactivated vaccines
[0005] The inventor discovered a polypeptide that can inactivate Zika virus——Z2 in the previous research. The research shows that Z2 can specifically bind to the envelope glycoprotein of Zika virus, causing the virus to burst and release genomic RNA, which in turn leads to the loss of the virus. live

Method used

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  • Zika virus vaccine and preparation method thereof
  • Zika virus vaccine and preparation method thereof
  • Zika virus vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0062] Example 2 Plaque method to detect infectivity of Zika virus inactivated by polypeptide Z2

[0063] In order to further detect whether the Z2 inactivated ZIKV can continue to infect cells and proliferate in cells, preferably, a plaque formation assay is used to detect the infectivity of the inactivated virus.

[0064] (1) 8×10 3 PFU Zika virus was mixed with 50 μM Z2, and incubated in a 37°C incubator for 24 hours;

[0065] (2) Simultaneously set ZIKV treated with DMSO and PBS as a control, and incubated in a 37°C incubator for 24h;

[0066] (3) ZIKV with different treatments was diluted 10 times to reduce the concentration of the polypeptide, and 1ml of the diluted solution was taken to infect the monolayer BHK-21 cells spread in a 6-well plate, at 37°C, 5% CO 2 Under the condition of adsorption for 2h;

[0067] (4) Discard the supernatant and replace it with DMEM medium containing 1% ultrapure low-melting point agarose (ThermoFisher, America) and 2% FBS;

[0068] (...

Embodiment 3

[0072] The distribution of embodiment 3 polypeptide Z2 on virus surface

[0073] In order to detect the direct interaction between Z2 and ZIKV, as well as its mode of action, preferably, stimulated emission depletion fluorescence microscopy (STED) is used to observe the interaction between Z2 and virus particles and the distribution state of Z2. In order to carry out fluorescent labeling to the virus, preferably, a general labeling method for enveloped viruses is adopted in the present invention, and biotin-labeled phosphatidylethanolamine (Biotin Cap PE, Avanti, America) is added to the cell culture medium to make the outer membrane of the cells and the inner membrane system are biotin-labeled; then use the labeled cells to culture ZIKV, and use the process of virus budding from the endoplasmic reticulum to obtain the envelope to label the virus with biotin; then use Cy5-labeled streptavidin (SA-Cy3 , ThermoFisher, America) Cy3-labeled ZIKV. During synthesis, polypeptide Z2 ...

Embodiment 4

[0083] The preparation of embodiment 4 vaccine

[0084] Through research on the mechanism of Z2 inactivation of ZIKV, the inventors found that ZIKV after Z2 inactivation releases genomic RNA, but retains the natural conformation of the viral envelope protein, making the inactivated ZIKV structurally similar to virus-like particles. The method for preparing Zika virus inactivated vaccine by using Z2 inactivated ZIKV will be further elaborated below in conjunction with specific examples.

[0085] Preparation of virus stock solution

[0086] (1) The day before inoculation, the Vero-E6 cells were subcultured so that they could grow to a monolayer the next day;

[0087] (2) Discard the cell supernatant, replace it with serum-free DMEM medium, add ZIKV stock solution, mix gently, and place at 37°C, 5% CO 2 Adsorption in the incubator for 2h;

[0088] (3) Add DMEM virus maintenance solution containing 2% FBS, place at 37°C, 5% CO 2 Cultivate in an incubator for 5-6 days;

[0089] ...

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Abstract

The invention relates to the field of virus vaccines, in particular to a Zika virus vaccine and a preparation method and applications thereof. The Zika virus vaccine comprises Zika virus inactivated by polypeptide Z2, wherein the sequence of the polypeptide Z2 is MAVLGDTAWDFGSVGGALNS LGKGIHQIFGAAF. The surface envelope protein of virus inactivated by Z2 still maintains the original conformation, and has natural antigenicity and immunogenicity. The Zika virus inactivated vaccine prepared by using the inactivated virus has obvious protection effect on mice infected with the Zika virus. The invention provides a new strategy for preparing inactivated virus vaccines. Compared with a subunit vaccine, a DNA vaccine or an attenuated live vaccine, the Zika virus inactivated vaccine has the advantages of simple preparation method, high safety and obvious protection effect, and has better application value.

Description

technical field [0001] The invention relates to the field of virus vaccines, in particular to a Zika virus vaccine and a preparation method thereof. Background technique [0002] Zika virus belongs to the family Flaviviridae, and it is an arbovirus that is mainly transmitted through the bite of Aedes mosquitoes, but studies have shown that its transmission methods also include vertical mother-to-child transmission, sexual transmission and blood transmission. Zika virus was isolated from rhesus monkeys in Uganda's Zika forest in 1947. Before 2007, only sporadic infections were reported in local areas. In 2015, a major outbreak of Zika virus infection occurred in Brazil. So far, it has affected 86 countries and regions around the world. [0003] About 80% of people infected with Zika virus have no obvious clinical symptoms or only mild symptoms such as rash, fever, conjunctivitis, muscle and joint pain, which usually last for 2-7 days and usually improve on their own. Howev...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/12A61P31/14C07K14/18C12N15/40
CPCA61K39/12A61P31/14C07K14/005C12N2770/24134A61K2039/5252C12N2770/24122Y02A50/30
Inventor 陆路陈振国孙蕾司露露姜世勃
Owner FUDAN UNIV
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