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mVSV virus vector and virus vector vaccine, and COVID-19 vaccine based on mVSV mediation

A viral vector and virus technology, applied in the field of genetic engineering, can solve the problems of low response degree and inability to respond to mucosal immune response, and achieve the effects of high antigen loading capacity, enhancement of specific mucosal immune response, and stability of the viral genome

Active Publication Date: 2020-05-01
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 virus vector vaccine, and COVID-19 vaccine based on mVSV mediation
  • mVSV virus vector and virus vector vaccine, and COVID-19 vaccine based on mVSV mediation
  • mVSV virus vector and virus vector vaccine, and COVID-19 vaccine based on mVSV mediation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 The toxicity of the three-site mutation in the Indiana strain of VSV is greatly reduced

[0049] 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 7As shown in A, some results show that mVSV-M51F has weaker cytotoxicity in norm...

Embodiment 2

[0050] Example 2 Construction and identification of two chimeric vaccines based on VSV viral vectors

[0051] According to the S gene sequence of SARS-CoV-2 released by NCBI, the codon is optimized to facilitate its expression in cells (named as antigen gene A), and multiple potential antigenic epitopes predicted from the sequence of SARS-CoV-2 are synthesized The new antigen gene (named as antigen gene B), the antigen gene A and antigen gene B sequences were handed over to Nanjing GenScript to synthesize them on the pCDNA3.1 and pUC57 vectors respectively, after the target gene was amplified by PCR, it was purified by fragment purification reagents Recover and purify the target band from the cassette, 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, and perform a ligation reaction. Then transfer to competent cells, positive clones were screened...

Embodiment 3

[0073] Example 3 Immune response effects of two chimeric vaccines based on VSV viral vectors under different immunization schemes

[0074] 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 SARS-CoV-2S virus, it will be immunized 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:

[0075] 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 overnight;

[0076] 2) Pour o...

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Abstract

The invention provides an mVSV virus vector, i.e., attenuated mVSV obtained after multiple modification mutations occur to an M protein amino acid site of a wild Indiana strain VSV, and an optimized heterologous antigen gene is preferentially integrated to a double cloning site area of an mVSV packaging core plasmid pmVSV-Core at the same time. The mVSV virus vector vaccine comprises a heterologous antigen gene which fuses or embeds a target virus between G and L genes of an mVSV vector envelope, wherein the antigen gene comprises an enveloped and embedded antigen gene encoding the target virus, an embedded combination antigen gene or a fused antigen gene; the mVSV virus vector is embedded or fused with a dominant antigen of spike protein S of an SARS-CoV-2 pathogen; the dominant antigen is preferably selected from a receptor binding domain of spike protein S, namely RBD; and a COVID-19 vaccine based on mVSV mediation is formed. The vaccine has good prevention or treatment effect on COVID-19 infected people.

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 Applications(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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