EBV vaccine based on vesicular stomatitis virus as well as preparation method and application of EBV vaccine

A virus and vaccine technology, applied in the field of bioengineering, can solve the problems of reducing the incidence of infectious mononucleosis, unable to prevent EBV from infecting cells, and the clinical safety of granulated protein has not been effectively evaluated, so as to reduce the incidence of disease. rate, good immune effect

Pending Publication Date: 2022-03-08
SUN YAT SEN UNIV CANCER CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although vaccines against EBV have been developed for decades, most of them are designed for the target of gp350, and their related clinical trials have also shown that they cannot prevent EBV from infecting cells, but can only reduce the risk of infectious mononucleosis. Morbidity—a self-limiting disease associated with acute EBV infection
Although it has been reported in the previous literature that gHgL was displayed on the surface of granulated protein for the preparation of EBV vaccines, there is no relevant clinical trial, and the clinical safety of granulated protein, an exogenous substance, has not been effectively evaluated.
There have been no reported deaths from vesicular stomatitis virus infection

Method used

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  • EBV vaccine based on vesicular stomatitis virus as well as preparation method and application of EBV vaccine
  • EBV vaccine based on vesicular stomatitis virus as well as preparation method and application of EBV vaccine
  • EBV vaccine based on vesicular stomatitis virus as well as preparation method and application of EBV vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Packaging and amplification of embodiment 1VSV recombinant virus

[0049] 1) Packaging and amplification of VSV recombinant virus see figure 1 . Among them such as figure 1 In A, the whole genome sequence of VSV was integrated into pBS plasmid vector (pBS-VSV) driven by T7 promoter. When the pBS-VSV plasmid is transfected into the cell, it starts to transcribe and synthesize the RNA chain under the action of T7 RNA polymerase, and is processed by the delta-ribozyme at the end of the RNA chain to obtain the complementary strand of the VSV genome, which is then used as The template is to synthesize the genomic RNA and mRNA of VSV by the RNA polymerase of VSV, and express each viral protein N, P, M, G, L, etc., and finally assemble the virus particles of VSV.

[0050] Such as figure 1 Shown in B, on the basis of pBS-VSV plasmid vector, remove the nucleotide sequence of the glycoprotein G of VSV, and replace it with the nucleotide sequence (pBS-VSV-△G) of GFP, the VSV v...

Embodiment 2V

[0095] Purification and identification of embodiment 2VSV recombinant virus

[0096] 1. Purification and identification of VSV recombinant virus

[0097] (1) Concentrate the amplified virus liquid VSV-△G-gB, VSV-△G-gB-G and VSV-△G-gHgL respectively in a high-speed centrifuge, and centrifuge at 100,000g for 2 hours at 4°C , and the pellet was resuspended in 1 mL of PBS.

[0098] (2) Prepare a 20%-50% sucrose density gradient with a density gradient preparation apparatus, and ultracentrifuge the concentrated VSV-△G-gB and VSV-△G-gHgL virus liquids at 4°C at 40,000g for 16 hours , using a 20% to 50% sucrose density gradient for separation, you can see an obvious white band in the centrifuge tube, which is the virus sample layer.

[0099] (3) Take out the samples after ultracentrifugation in layers with a density gradient separator, take a small amount of samples respectively, and analyze the distribution of samples in each layer with Coomassie Brilliant Blue staining experiment...

Embodiment 3

[0103] Embodiment 3 mouse immunization

[0104] 1. VSV-ΔG-gB without adjuvant

[0105] 1) Take 40 Balb / c mice, start immunization at the age of 8 weeks, and give a booster immunization in the 3rd and 6th weeks after the initial immunization (see Figure 5 );

[0106] 2) Prepare 200 μL of the following vaccines and administer them by subcutaneous injection in the abdomen: VSV-ΔG-1E6, VSV-ΔG-gB-1E5, VSV-ΔG-gB-1E6, VSV-ΔG-gB-1E7, VSV-ΔG-gB -1E8;

[0107] 3) Before immunization, two weeks after the initial immunization, and two weeks after each booster immunization, blood was collected from the retrocanthus venous plexus, 5-6 drops each time, and collected into 1.5ml EP tubes;

[0108] 4) Incubate at 37°C for 30 minutes; centrifuge at 18,000 rcf at 4°C for 30 minutes;

[0109] 5) Transfer the supernatant to a new EP tube and inactivate at 56°C for 30 minutes;

[0110] 6) Store at -80°C for later use;

[0111] 2. VSV-ΔG-gB-G, with aluminum adjuvant

[0112] 1) Take 20 C57 mi...

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Abstract

The invention discloses an EBV (Epstein-Barr Virus) vaccine based on a vesicular stomatitis virus as well as a preparation method and application of the EBV vaccine. EBV key glycoproteins gB and gHgL are displayed on the surface of VSV for the first time, the modified VSV is used for animal immunization, a new EBV vaccine variety is provided, an organism is expected to be induced to generate strong enough immune response to prevent EBV from infecting host cells, and therefore the morbidity of EBV-related neoplastic and non-neoplastic diseases is reduced. Detection finds that the modified VSV can induce obvious specific antibodies aiming at EBV surface glycoproteins gB and gHgL; in addition, it is proved that the generated specific antibody can inhibit EBV from infecting epithelial cells and B lymphocytes; the invention also shows that the EBV vaccine based on the VSV can be used for preventing and controlling EBV infection of people and related neoplastic and non-neoplastic diseases and has a good immune effect.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to an EBV vaccine based on vesicular stomatitis virus and its preparation method and application. Background technique [0002] EBV is the first identified human oncogenic virus, which is closely related to the occurrence and development of various neoplastic and non-neoplastic diseases. In particular, EBV-related nasopharyngeal carcinoma is also known as "Guangdong cancer" because of its high incidence in Guangdong. Although vaccines against EBV have been developed for decades, most of them are designed for the target of gp350, and their related clinical trials have also shown that they cannot prevent EBV from infecting cells, but can only reduce the risk of infectious mononucleosis. Morbidity - a self-limiting disease associated with acute EBV infection. Although it has been reported in the previous literature that gHgL was displayed on the surface of granulat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/86C07K16/08G01N33/569G01N33/68A61K39/245A61K39/39A61P31/22C12R1/93
CPCC12N7/00C12N15/86C07K14/005C07K16/085G01N33/56994G01N33/6854A61K39/12A61K39/39A61P31/22C12N2760/20021C12N2760/20043C12N2710/16234C12N2710/16222C12N2800/107G01N2333/05G01N2469/10G01N2469/20A61K2039/5256
Inventor 曾木圣孔祥炜胡柱龙卜国龙冯国开
Owner SUN YAT SEN UNIV CANCER CENT
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