Inactivated Zika virus vaccine

A Zika virus, inactivation technology, applied in the direction of virus, antiviral agent, virus/phage, etc., can solve the problems of vaccine immunogenicity and stability need to be studied, culture process, purification process and preparation process are still blank, etc. , to achieve the effect of avoiding Brazilian microcephaly, reducing operation steps and reducing patients

Active Publication Date: 2016-07-13
SINOVAC RES & DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no Zika vaccine available so far, and the immunogenicity and stability of the vaccine need to be studied
[0008] Due to the sudden outbreak of Zika virus in this epidemic, al

Method used

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  • Inactivated Zika virus vaccine
  • Inactivated Zika virus vaccine
  • Inactivated Zika virus vaccine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The preparation of embodiment 1 cell working seed and the preparation of virus liquid

[0044] 1. Preparation of high-density cell working seeds

[0045] Use 199 culture medium to dilute Vero main cell seed dilution ratio to 60:1, inoculate to 175cm 2 Cells were cultured in square flasks at a temperature of 36.0°C±1°C. After the cells are fully covered with the cell bottle, continue passage for 4 generations according to the ratio of 1:4 to 1:6. The last generation was transferred to five 40-layer cell factories, trypsinized, and centrifuged to remove the supernatant. Use freezing solution containing 10% DMSO to resuspend, and the cell concentration after mixing is 2.5×10 7 pieces / ml. The cell seeds were subjected to gradient cooling in a temperature program cooling device, keeping the temperature down by 1°C per minute. After the temperature drops to -196°C, it is stored in liquid nitrogen, and the working cell seeds are valid for 10 years.

[0046] 2. Vero cell ...

Embodiment 2

[0051] The screening of embodiment 2 culture medium

[0052] Select commercially available MEM, M199, DMEM, DMEM / F12 medium, use the DOE software of Minitab17 for experimental design, carry out virus culture according to the designed medium formula, compare the results of virus titer and virus immunogenicity, and choose the best Medium. The medium formula of the experimental design is shown in Table 1, wherein "1" represents adding the medium, and "-1" represents not adding the medium. If there are several "1" in the recipe, make several equal parts, and each medium is divided into one equal part. The results of virus titer and immunogenicity are shown in Table 2 and Table 3, wherein the medium in which M199 and DMEM / F12 are mixed in equal volume at 1:1 produces significantly higher virus immunogenicity and titer than other mediums.

[0053] Table 1 DOE test medium formula

[0054]

[0055]

[0056] Table 2 Virus titer results

[0057] formula

[0058] Tabl...

Embodiment 3

[0061] Embodiment 3 Zika virus inactivation and purification

[0062] 1. Zika virus inactivation Add formaldehyde at a final concentration of 200 μg / ml to the Zika virus harvest liquid and inactivate at 37°C for 7 days. That is, the inactivation of Zika virus is completed.

[0063] 2. Purification of Zika virus

[0064] (1) Clarification Filter the virus liquid through filter elements with continuous three-stage pore diameters of 3-0.8 μm, 0.8-0.65 μm, and 0.65-0.22 μm or centrifuge (including continuous flow centrifugation) with a speed of 2000-5000 rpm and a centrifugation time of 0.5-4 hours, to obtain virus clarification;

[0065] (2) The concentrated virus clarified liquid was concentrated through a Pall tangential flow membrane filtration system with a molecular weight cut-off of 300KD, and the concentration factor was 100 times.

[0066] (3) The low and high concentrations of the sucrose solution in density gradient centrifugation are 25% and 55% respectively. The d...

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Abstract

The invention provides an inactivated Zika virus vaccine. The inactivated Zika virus vaccine is obtained by: performing ultrafiltration and concentration on Zika virus liquid after inactivation, centrifuging the concentrated virus liquid by adopting a sucrose density zone, performing ion exchange and concentration sterilization on a centrifugal product to obtain a Zika virus vaccine stock solution, diluting the vaccine stock solution until the total protein content is not more than 20mu g/ml, and adding an adjuvant to obtain a vaccine semi-finished product. The method for preparing the vaccine provided by the invention is simple, convenient and easy to operate, the cost is saved, the produced vaccine is suitable for Asian people, a unit dose of the Zika virus liquid is high in immunogenicity, the content of hybrid protein is low, the side effect after injection is small, and the safety is high, so that the vaccine is suitable for vaccination of fertile women before pregnancy, can avoid newborn Brazil microcephaly caused by infection of Zika virus, and is significant in social value and market efficiency.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to an inactivated Zika virus vaccine. Background technique [0002] Zika virus disease (Zika Virus Disease) is a self-limiting acute infectious disease caused by Zika virus (Zika Virus) and transmitted by mosquito vector. Zika virus was first identified in rhesus monkeys in Uganda in 1947, and was isolated in humans in Uganda and Tanzania in 1952. Before 2007, only 14 sporadic cases of Zika virus disease were reported in the world. In 2007, Zika virus outbreak was first discovered in Yap Island, Micronesia, a Pacific island country. There is an increasing trend in countries and regions. In May 2015, Brazil reported its first case of Zika virus disease. As of the end of January 2016, local infection cases had been found in 24 American countries and regions including Brazil. At the same time, imported cases were found in many countries in Europe, North America and other places, an...

Claims

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Application Information

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IPC IPC(8): A61K39/12A61P31/12
CPCA61K39/12C12N2770/24034Y02A50/30
Inventor 王琳吕哲惠增弟高强
Owner SINOVAC RES & DEV
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