mRNA rabies vaccine

A rabies virus and nucleic acid vaccine technology, applied in DNA/RNA vaccination, antisense single-stranded RNA virus, vaccines, etc., can solve problems such as poor translation efficiency and low antigenic activity, and achieve the effect of reducing production costs

Active Publication Date: 2020-01-21
珠海丽凡达生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The first purpose of the present invention is to provide an optimized RVG mRNA to alleviate the technical problems of low antigenic activity, poor translation efficiency and stability for nucleic acid vaccines in the prior art

Method used

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preparation example Construction

[0090] The invention provides a method for preparing the above-mentioned rabies virus nucleic acid vaccine. The vaccine carrier and the optimized RVG mRNA provided by the invention are mixed to obtain the rabies virus nucleic acid vaccine. The method is simple and easy to operate, and the production cost is greatly reduced.

[0091] In a preferred embodiment, the vaccine carrier is the cationic lipid nanoparticle in the rabies virus nucleic acid vaccine provided by the invention, and the preparation method of the rabies virus nucleic acid vaccine comprises:

[0092] (a) dissolving protonatable cationic lipids, structural lipids, auxiliary lipids and surfactants in an organic solution according to the formula ratio to obtain an organic phase;

[0093] (b) dissolving the optimized RVG mRNA in PBS or citrate solution to obtain an aqueous phase;

[0094] (c) mixing the organic phase obtained in (a) and the aqueous phase obtained in (b) to obtain a mixed solution, and replacing th...

Embodiment 1

[0106] The preparation method of embodiment 1 rabies virus nucleic acid vaccine

[0107] The mRNA of SEQ ID NO.2 was dissolved in citrate buffer at pH 4, and the concentration was adjusted to 0.1 mg / ml to obtain an aqueous phase.

[0108] The formula ratio of Dlin-MC3-DMA, cholesterol, DSPC and PEG-DMG was dissolved in absolute ethanol, and the total lipid concentration was adjusted to 6 mg / ml to obtain an organic phase.

[0109] The organic phase and the aqueous phase were mixed at a volume ratio of 1:3 by means of microfluidic equipment mixing. The flow rate is 12.0ml / min when using the microfluidic equipment for mixing.

[0110] The mixture was immediately diluted 50-100 times with a PBS solution of pH 7.4, and the ethanol component in the solution was removed by tangential flow filtration (TFF) and concentrated to an mRNA concentration of about 100 μg / ml to obtain lipid nanoparticles encapsulating mRNA, namely For the rabies virus nucleic acid vaccine.

Embodiment 2

[0111] Embodiment 2 freeze-dried preparation

[0112] The rabies virus nucleic acid vaccine in Example 1 is prepared into a freeze-dried preparation, and the freeze-drying protective agent sucrose and the surfactant Tween 20 are added. The process includes pre-freezing, primary freeze-drying and secondary freeze-drying: the pre-freezing temperature is -50 °C, and the temperature was maintained for 5 hours. The first freeze-drying temperature was -40°C for 24 hours, the second freeze-drying temperature was 10°C and kept for 17 hours, and the vacuum degree during the freeze-drying process was 40 μbar.

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Abstract

The present invention relates to the field of nucleic acid vaccines and particularly provides an mRNA rabies vaccine. A nucleotide sequence for transcribing the provided optimized RVG mRNA is shown asSEQ ID NO.1. A coding region of a rabies virus CTN-1 strain G protein (RVG) is optimized and an obtained nucleotide sequence is the optimized RVG mRNA sequence shown in the SEQ ID NO.1. The nucleotide sequence enables structure of the transcribed mRNA to be more stable and the target protein is more efficiently translated in mammals and humans. The provided rabies virus nucleic acid vaccine comprises a vaccine vector and the optimized RVG mRNA, achieves sufficient protection effects by using an extreme small dose, and is superior to existing rabies vaccine technologies in terms of safety andeffectiveness.

Description

technical field [0001] The invention relates to the field of nucleic acid vaccines, in particular to an mRNA rabies vaccine. Background technique [0002] Rabies is one of the oldest diseases affecting human health in the world. The earliest record was found 4300 years ago. The disease is a viral animal infectious disease caused by Rabies Virus (RV). The transmission route of the virus is mainly through animals such as dogs and cats that have not been vaccinated or have failed inoculation. At present, in the veterinary rabies vaccine, the Vero cell inactivated vaccine is mainly used. Although the emergence of these vaccines can greatly reduce the incidence of rabies, However, there are still many technical difficulties in the production process of this kind of vaccine, such as large-scale suspension culture technology of cells, expanded production of virus, etc. Therefore, new rabies vaccines are being widely developed worldwide. [0003] Currently commercially available ra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/47A61K39/205A61K9/19A61P31/14
CPCA61K9/0019A61K9/19A61K39/12A61K2039/53A61K2039/552A61P31/14C07K14/005C12N2760/20122C12N2760/20134C12N2800/22
Inventor 刘隽彭育才向晟楠苏晓晔刘琪雷奕欣李爽
Owner 珠海丽凡达生物技术有限公司
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