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MVSV virus vector and its virus vector vaccine, a novel coronavirus pneumonia vaccine based on MVSV

A virus vector and virus technology, applied in the field of genetic engineering, can solve the problems of inability to respond to mucosal immune response and low response, and achieve the effect of enhancing specific mucosal immune response, high antigen loading capacity and low toxicity

Active Publication Date: 2020-06-23
FANTASIA BIOPHARMA ZHEJIANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, traditional antigens inoculated in the mucosa cannot effectively stay in the local mucosa because they are rapidly cleared and degraded, so that they cannot be presented by APCs enough to induce an effective mucosal immune response.
Even when induced, the level of response is very low

Method used

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  • MVSV virus vector and its virus vector vaccine, a novel coronavirus pneumonia vaccine based on MVSV
  • MVSV virus vector and its virus vector vaccine, a novel coronavirus pneumonia vaccine based on MVSV
  • MVSV virus vector and its virus vector vaccine, a novel coronavirus pneumonia vaccine based on MVSV

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 The toxicity of the three-site mutation in the Indiana strain of VSV is greatly reduced.

[0050]It is known that the main pathogenic gene of VSV wild strain is M, and at the same time, M protein can induce the apoptosis of host cells, which is the main factor for the disease of artiodactyls infected by VSV wild strain. Based on this, synthetic biology technology can reduce The best way of VSV wild strain is to carry out genetic engineering mutation in the M gene. Existing studies have shown that after the non-synonymous mutation of the 51st amino acid of the M protein, the neurotoxicity of wild VSV will be reduced. Therefore, in the implementation of the technology of the present invention In , the mutation comparison of a single site was first carried out, and methionine was mutated into phenylalanine, alanine, leucine and arginine (control) at the 51st position, such as Figure 7 As shown in A, some results show that mVSV-M51F has weaker cytotoxicity in nor...

Embodiment 2

[0051] Example 2 Construction and identification of two chimeric vaccines based on VSV viral vectors

[0052] According to the codon-optimized sequence of the S gene of the COVID-19 virus released by NCBI to facilitate its expression in cells (named antigen gene A), a new combination of multiple potential antigenic epitopes predicted from the sequence of the COVID-19 virus was synthesized For the antigen gene (named as antigen gene B), the sequences of antigen gene A and antigen gene B were synthesized by Nanjing GenScript Biotech into pCDNA3.1 and pUC57 vectors, respectively, and the target gene was amplified by PCR and recovered by fragment purification kit Purify the target band, digest the fragment and the pmVSV-GFP vector with restriction endonucleases, MCS1 (Xhol) and MCS2 (Nhel) at 37°C for 3 hours, recover the vector and the target fragment from the gel, perform a ligation reaction, and then transfect To the competent cells, positive clones were screened by bacterial l...

Embodiment 3

[0074] Example 3 The immune response effects of two chimeric vaccines based on VSV viral vectors under different immunization schemes.

[0075] Detect the specific IgA and IgG antibody levels in mice after different immunization schemes by indirect ELISA: After coating the microtiter plate with the recombinant RBD protein of COVID-19 virus S virus, immunization will be carried out by three immunization methods: intramuscular, intravenous, and intranasal 21 days after the first time, the mouse serum was diluted 1:200 and added to the corresponding wells. After incubation for 2 hours, different types of (IgA, IgG) secondary antibodies were diluted 1:10000 to detect the specific antibody level ( figure 2 ), the specific steps are as follows:

[0076] 1) Dilute the coated antigen (S-RBD) with coating buffer to a final concentration of 5 μg / ml, take the microplate plate, add samples to the wells in sequence (100 μl / well), and then place it at 4 degrees Celsius for coating overnig...

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Abstract

mVSV virus vector, that is, the attenuated mVSV obtained after multiple modifications and mutations at the amino acid sites of the M protein of the wild Indiana strain VSV, and the preferred heterologous antigen gene is preferentially integrated into the double cloning site region of the mVSV packaging core plasmid pmVSV‑Core A kind of mVSV virus vector vaccine, including the heterologous antigen gene of fusion or chimeric target virus in the mVSV vector envelope G and L gene, the antigen gene comprises the antigen gene encoding the chimeric target virus envelope, Chimeric combined antigen gene or fused antigen gene; mVSV viral vector chimerized or fused the dominant antigen of the spike protein S of the COVID‑19 virus pathogen, and the dominant antigen is preferably from the receptor binding structure of the spike protein S The domain is RBD, forming a new crown pneumonia vaccine based on mVSV, which has a good preventive or therapeutic effect on patients infected with the new crown pneumonia virus.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to mVSV virus vector and its virus vector vaccine, and a novel coronavirus pneumonia vaccine mediated by mVSV. Background technique [0002] Coronavirus (Coronavirus) belongs to the order Nidovirals (order Nidovirals), Coronaviridae (family Coronavirade), and members of the genus Coronavirus (genus Coronavirus) in virological classification. The genome is single-stranded, positive-strand RNA, and the genome is complete. The length is between 26 and 32 kb, and it is the RNA virus with the largest genome known so far. The infection of coronaviruses in nature is generally very widespread, and common mammals such as dogs, cats, mice, pigs, cattle, and poultry are susceptible. In recent years, various types of coronaviruses have been isolated from beluga whales, camels, and especially bats. There are currently six known human coronaviruses, human coronavirus 229E (HCoV-229E...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/86A61K39/215A61P31/14A61P11/00
CPCA61K39/12A61K2039/5256A61K2039/53A61P11/00A61P31/14C12N15/86C12N2760/20243C12N2770/20034C12N2800/107C12N2800/22
Inventor 秦晓峰吴飞韦治明权海峰龙丽梅
Owner FANTASIA BIOPHARMA ZHEJIANG CO LTD
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