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New coronavirus wild type and variant combined vaccine based on mRNA (messenger ribonucleic acid) and preparation method thereof

A combined vaccine and virus mutation technology, which is applied in the field of vaccine development and biomedicine, can solve the problems of poor effect, achieve the effects of reducing costs, flexible cost-effectiveness, and improving production efficiency

Pending Publication Date: 2022-06-28
苏州祥龙生物医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the function of other mutations and whether there are combined effects of these mutations is unknown, leading to great uncertainty about how the virus' behavior and susceptibility will develop
The Omicron variant will not be the last variant of SARS-CoV-2, and although the impact of the Omicron spike mutation on the effectiveness of existing vaccines remains to be studied, there is good evidence that vaccines developed based on wild-type SARS-CoV-2 Less effective at preventing variant infection

Method used

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  • New coronavirus wild type and variant combined vaccine based on mRNA (messenger ribonucleic acid) and preparation method thereof
  • New coronavirus wild type and variant combined vaccine based on mRNA (messenger ribonucleic acid) and preparation method thereof
  • New coronavirus wild type and variant combined vaccine based on mRNA (messenger ribonucleic acid) and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] This example is used to illustrate the correctness and effectiveness of the novel combination vaccine sequence designed by the present invention, and also to verify that the mutant strain has stronger binding ability to receptors and is more infectious.

[0091] First, the pseudovirus produced with this sequence binds to the ACE2 receptor and is verified. For this purpose, pseudotyped lentiviral particles with different spike proteins were made. Briefly, pseudoviruses were generated in 293T cells by PEI transfection of the lentiviral backbone encoding cmv-luciferasi-ires-zsgreen, along with lentiviral helper plasmids and mutant expression plasmids for each spike; collected in 293T-ACE2 cells And after filtration, run quantitatively by flow cytometry titration.

[0092] (1) Method

[0093] Flow cytometry titers were used to determine the infectious units and infection rates of the SARS-CoV-2 pseudovirus.

[0094] 400,000 293T-ACE2 cells were seeded in each well of a 1...

Embodiment 2

[0099] In this example, a variety of novel combination vaccine wild-type and mutant strain antigen expression plasmids are designed and constructed to illustrate the intracellular expression of the novel combination vaccine wild-type and mutant strain antigens of the present invention.

[0100] (1) Method

[0101] SARS-CoV-2 S protein wild type (Swild), S protein mutant (Somicron), RBD wild type (RBDw), RBD mutant (RBDomi), S protein wild type + S protein mutant (Sw+Somi), S Protein wild type + RBD mutant (Sw + RBDomi), S protein mutant + RBD wild type (Somi + RBDw) vaccine proteins, produced in suspension cultured Expi293F cells using Expi293F expression medium (Life Technologies), temperature 33 ℃, humidity 70%, 8% CO 2 , rotating speed 150rpm; use PEI-MAX (Polyscience) to transfect the cultured cells, the cell density is 3 million cells / mL, and culture for 3 days; centrifuge to clarify the supernatant (4000rcf, 5min), add PDADMAC solution to the final concentration of 0.03...

Embodiment 3

[0108] In this example, the expression level and duration in mice after inoculation of the mRNA vaccine of the present invention are studied to illustrate the speed and duration of the rise in immunity in vivo.

[0109] (1) Method

[0110] The above-mentioned wild-type and mutant DNA fragments of the new coronavirus and the plasmid vector encoding the DNA sequence of luciferase were linearized and transcribed in vitro to synthesize mRNA-LNP vaccine. The mRNA vaccine prepared above was injected into mice, and the in vivo fluorescein detection was performed according to the observation time.

[0111] To examine the in vivo distribution and duration of FLuc mRNA-LNPs, 6-week-old female BALB / c mice (n=36) were inoculated with 10 μg of FLuc mRNA- LNP. At 6h, 12h, 24h, and 2 to 7 days after inoculation, animals were injected intraperitoneally with luciferase substrate (Promega). After 3 min of reaction, the fluorescence signal was collected by IVIS spectrometer (PerkinElmer) for ...

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Abstract

The invention discloses a novel coronavirus wild type and variant combined vaccine based on mRNA (messenger ribonucleic acid) and a preparation method thereof. The invention relates to a mixed vaccine of a new coronavirus wild type mRNA sequence and a variant strain mRNA sequence wrapped by a lipid delivery system, or a wild type S protein or an RBD-variant S protein therein or a chimeric antigen combined mRNA vaccine of RBD therein. The invention further provides an efficient liposome nano delivery system. The invention provides a purification method before vaccine finished products and design of an automatic integrated vaccine production line system. The novel combined vaccine designed and produced by the invention accurately aims at mutant epitope antigens of variant strains and wild type shared epitope antigens, realizes accurate neutralization and accurate prevention, improves the production efficiency and reduces the cost.

Description

technical field [0001] The invention relates to the field of vaccine development, application and biomedicine, in particular to an mRNA-based novel coronavirus wild-type and variant-type combined vaccine and a preparation method thereof. Background technique [0002] Over the past 20 years, numerous pandemics caused by viruses have been reported, including the Sars coronavirus in 2002-2003, the H1N1 influenza in 2009, and the MERS-CoV in 2012, severely impacting world health care. In the case of the novel coronavirus (SARS-CoV-2), it has spread to 223 countries, with more than 265 million confirmed cases worldwide and more than 5.2 million deaths. As of December 2021, 5 related variants (VoCs, variants of concern) have been identified since the beginning of the SARS-CoV-2 pandemic: Alpha (B.1.1.7), Beta (B.1.351), Gamma (P. 1), Delta (B.1.617.2) and Omicron (B.1.1.529). According to the WHO's weekly epidemiological report, the Alpha (B.1.1.7) virus has spread to 170 countr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/215A61K39/295A61K9/127A61K9/51A61K47/24A61K47/28A61K47/14A61P31/14
CPCA61K39/12A61K9/127A61K9/5123A61K9/5146A61P31/14A61K2039/53A61K2039/70C12N2770/20034
Inventor 杨水祥
Owner 苏州祥龙生物医药科技有限公司
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