CN113521266A - Coronavirus vaccine - Google Patents
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- CN113521266A CN113521266A CN202010294207.XA CN202010294207A CN113521266A CN 113521266 A CN113521266 A CN 113521266A CN 202010294207 A CN202010294207 A CN 202010294207A CN 113521266 A CN113521266 A CN 113521266A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20051—Methods of production or purification of viral material
Abstract
The invention relates to a coronavirus vaccine, which comprises microparticles released by apoptotic cells, wherein the microparticles released by the apoptotic cells comprise vesicular structural substances released by apoptotic cells derived from human beings and coronavirus-derived viral antigens packaged in the vesicular structural substances. The coronavirus vaccine of the present invention can be used as a prophylactic vaccine to prevent coronavirus infection and also as a therapeutic vaccine.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a vaccine aiming at coronavirus.
Background
Infection with a novel Coronavirus (SARS-CoV-2(Severe acid response Syndrome Coronavir 2), or 2019-nCoV) has been prevalent worldwide and the development of effective vaccines has been slow. Vaccines prepared using conventional strategies include inactivated virus vaccines, DNA vaccines, mRNA vaccines or protein vaccines, among others, with the goal of activating B cells to produce protective neutralizing antibodies. However, conventional vaccines are basically prophylactic vaccines and are difficult to achieve the effect of therapeutic vaccines. In the face of hundreds of thousands of infected patients, the development of therapeutic vaccines is imperative. The invention prepares a vaccine with both preventive property and therapeutic property, and provides a new treatment method for preventing and treating new coronary pneumonia (COVID-19(Corona Virus Disease 2019)).
The body can generate a plurality of antibodies against virus proteins, however, only surface antibodies are effective, so that the traditional component vaccines are developed against the surface Spike proteins of the coronavirus particles, and the antibodies can not effectively remove viruses hidden in cells. Therefore, the final elimination of the virus also depends on specific T cells which are key factors of virus infection immunity, the activation time of the specific T cells is far shorter than the time required by B cells to generate specific antibodies, a large number of T cells are amplified after 24 to 48 hours and enter infected parts, and the infected cells are efficiently killed by recognizing MHC-virus antigen peptide complexes on the surfaces of the virus-infected cells, so that the virus is fundamentally eliminated. Because of the faster production rates, T cells are more suitable for use as therapeutic vaccines.
It is proved that Microparticles (MP) released from apoptotic cells can be used as a highly effective vaccine to activate specific T cell immune responses. MP is a vesicular structure substance with the size of hundreds of nanometers and formed by wrapping cell contents by cell membranes, and has two key points of an advantageous vaccine: one is the strong antigen carrying ability and the other is the proper and effective co-stimulation signal. For example, Listeria-infected macrophage-derived MP, comprising Listeria antigen, are DC-presented to activate Listeria-specific T cells[1](ii) a Whereas, OVA-B16 tumor cell-derived MP, containing tumor antigen, activates specific CD8+ T cells[2,3]. The costimulatory signal of T-MP (T stands for tumor cell) is due to the fact that the capsule cavity contains a mitochondrial DNA fragment which can effectively activate cGAS-STING pathway and induce the production of type I interferon[2]. In addition, T-MP enters into DC lysosome to activate NADPH oxidase system, on one hand, the pH value of lysosome is increased, the more efficient generation of tumor antigen peptide is promoted, and on the other hand, the expression of CD80, CD86 and IL-12 is up-regulated through ROS signals[3]。
Disclosure of Invention
The present invention aims to provide a vaccine against coronavirus as a vaccine and/or medicament for the prevention and treatment of related diseases caused by coronavirus infection.
It is a second object of the present invention to provide a pharmaceutical composition comprising said coronavirus vaccine.
The third purpose of the invention is to provide the application of the vaccine and/or the pharmaceutical composition in the preparation of medicines for preventing and treating diseases related to coronavirus infection.
According to one aspect of the invention, a coronavirus vaccine comprises apoptotic cell-releasing microparticles comprising vesicular structure material released by apoptotic cells and a coronavirus-derived antigen encapsulated in the vesicular structure material.
The coronavirus vaccine of the present invention is characterized in that human-derived cells are contacted with any one selected from the group consisting of a drug, radiation and ultraviolet rays to cause apoptosis of the cells and release a vesicular structure substance, and the preferred human-derived cells are human-derived cell lines which can be cultured.
The coronavirus vaccine of the present invention, wherein the coronavirus-derived antigen may preferably be a coronavirus component protein capable of activating a specific T cell; preferred coronavirus component proteins may be selected from the group consisting of Spike proteins, nucleocapsid proteins, envelope proteins and open reading frame 1ab proteins of coronaviruses.
As a preferred embodiment of the invention, the microparticles released by apoptotic cells have a particle size of 50 to 1000nm, more preferably 50 to 500 nm.
By selecting different viral antigens, vaccines against different coronaviruses, e.g., against SARS-CoV-2 virus, can be prepared using the concepts and methods of the invention.
Microparticles for use as encapsulated viral antigens may be obtained using any method that is capable of apoptosis in human cells including, but not limited to: contacting human cells with various medicaments, radioactive rays and ultraviolet rays to apoptosis and form vesicular structural substances. Then contacting the required virus antigen with the apoptotic cell to enable the virus to be wrapped into the vesicular structure substance, thereby obtaining the virus vaccine. Or co-culturing human cells and expression vectors for expressing the virus antigens, transfecting the virus antigens into the human cells, and then carrying out apoptosis on the human cells to obtain the vesicles coated with the virus antigens.
In a preferred embodiment, the coronavirus vaccine of the present invention can be prepared by the following method:
constructing human cells over-expressing viral component proteins: transfecting the cultured human cell strain with virus component protein, and screening to obtain human cells over-expressing virus components;
culturing the human cells with the over-expressed virus components in vitro in a large quantity, and irradiating the human cells with ultraviolet UVB to make the human cells die;
collecting microparticles released by the apoptotic cells to obtain the coronavirus vaccine.
In the scheme of the invention, ultraviolet irradiation is preferably used for inducing apoptosis of human cells, and for collection of vesicular structural substances (cell vesicles), a conventional centrifuge and a freezing high-speed centrifuge can be used for separation under a low-temperature condition or a room-temperature condition. Preferably, the cell vesicles are collected by a high-speed centrifuge under a low temperature condition (about 4 ℃) with a centrifugal force of 500-. Similarly, the collection of microparticles encapsulating viral antigens can also be performed using an ultracentrifuge at low temperature. Preferably, the microparticles coated with the viral antigen are collected by a centrifuge under a low temperature condition (about 4 ℃) with a centrifugal force of 500-. The preferred method of collecting microparticles is density gradient centrifugation, collecting microparticles with a centrifugal force of 1000-.
The collected microparticles encapsulating the viral antigen can be prepared into pharmaceutical preparations according to conventional methods, including but not limited to injections such as subcutaneous injection and/or intravenous injection, oral preparations, sprays for aerosol therapy, and the like.
Those skilled in the art can select an appropriate method for inducing apoptosis of human-derived cells, a method for collecting vesicles encapsulating viral antigens, and an appropriate ratio of the amount of the vesicles to the viral antigens, according to the above-described method for preparing a human-derived cell-derived coronavirus vaccine, depending on the type of coronavirus infection to be prevented and/or treated and the type of coronavirus antigen to be used, as long as the finally obtained protein of the coronavirus component encapsulated in the vesicular-structure substance is sufficient to exert the desired preventive and/or therapeutic effect.
According to another aspect of the present invention, there is also provided a pharmaceutical composition comprising the virus vaccine of the present invention, which can be prepared by adding various pharmaceutically and/or physiologically acceptable excipients and/or additives to the coronavirus vaccine of the present invention.
The coronavirus vaccine of the present invention can be used as a prophylactic vaccine to prevent coronavirus infection and also as a therapeutic vaccine. When used as a therapeutic vaccine, the vaccine can be used for treating related diseases caused by SARS-CoV-2 virus infection, including but not limited to new crown pneumonia (COVID-19), and can also be used for treating patients with yang recovery and asymptomatic after the virus infection treatment.
Drawings
FIG. 1 is a screening of flow cytometry to detect cells stably overexpressing SARS-CoV-2Spike (S1);
FIG. 2 flow cytometry is used to detect the expression of SARS-CoV-2Spike (S1) on the surface of MPs;
FIG. 3 shows ELISA detection of IFN-. gamma.and Granzyme B expression in MPs overexpressing Spike S1 from mouse T cells;
FIG. 4 shows ELISA detection of IFN-. gamma.and Granzyme B expression in MPs overexpressing Spike S1 from human T cells;
FIG. 5 shows the killing effect of mouse CTL against SARS-CoV-2 infected cells;
FIG. 6 shows the killing effect of human CTL against SARS-CoV-2 infected cells.
Detailed Description
The present invention will be further described below with reference to the accompanying drawings and examples for illustrating the technical solutions of the present invention, and the following examples should not be construed as limiting the present invention in any way.
The term "Microparticle (MP)" as used herein is produced by apoptotic cells, and has a vesicle-like structure in which the contents are encapsulated; "cell vesicles" are produced by apoptotic cells without encapsulating the contents; a "Coronavirus" is a virus of the genus Coronavir (Coronavir) of the family Coronaviridae (Nidovirales) of the order of the nested viruses, which is a type of enveloped, single-stranded, positive-stranded RNA virus with a linear genome. SARS-CoV-2(2019 new coronavirus, or 2019-nCoV) is one of them, and may cause new coronavirus pneumonia COVID-19 after infection.
The tumor cells, drugs, experimental animals, and instruments used in the examples below are not particularly limited and are commercially available.
Example 1: construction of cells overexpressing Spike S1 protein
1. Experimental equipment and materials:
BD FACS Canto II flow cytometer
SARS-CoV-2(2019-nCoV) Spike (S1) open reading frame mammalian expression plasmid was purchased from Beijing Yiqian Shenzhou;
SARS-CoV Spike Antibody, Cat:40150-D001, which has cross reaction with SARS-CoV-2(2019nCoV) Spike S1 protein and is purchased from Beijing Yiqiaoshengzhou;
lipofectamine3000 from Invitrogen;
the G418 GENEICIN antibiotic was purchased from Gibco, cat # 11811-;
endotoxin-free plasmid miniprep medium kit was purchased from TIANGEN under catalog number DP 118;
LLC (Lewis murine lung adenocarcinoma cells), A549 (adenocarcinoma human alveolar basal epithelial cells), BEAS-2B (human normal lung epithelial cells) and 293T cells (human kidney epithelial cell line transfected with adenovirus E1A gene, expressing SV40 large T antigen) were purchased from the cell center of the institute of basic medicine of Chinese medical academy of sciences.
2. The experimental steps are as follows:
1) amplification of SARS-CoV-2Spike (S1) plasmid: plasmid transformation competent bacteria, use LB culture dish containing ampicillin resistance to culture overnight; plate coating culture, selecting single clone to enlarge culture, and extracting plasmid by using plasmid extraction kit.
2) Transfecting the SARS-CoV-2Spike (S1) plasmid with a Lipofectamine3000 to LLC, A549, BEAS-2B and 293T cells respectively;
3) screening monoclonal cell strains, constructing stable cell strains: after 48 hours of culture, the infected cells were seeded at 0.5 cell/well in a 96-well plate, and selection culture was carried out using RPMI1640 medium containing 5. mu.g/ml puromycin and 10% FBS. Cells grown from the single clone were transferred to a 24-well plate for expansion.
4) Adding SARS-CoV-2Spike antibody into the cell, adding PE fluorescent secondary antibody, screening out monoclonal cell strain over expressing Spike S protein, and expanding culture of the stably screened cell strain.
3. The experimental results are as follows: cells stably overexpressing SARS-CoV-2Spike (S1), LLC-hS1, A549-hS1, BEAS-2B-hS1 and 293T-hS1 were selected. As in fig. 1.
Example 2: microparticles over-expressing Spike S1 protein were prepared and tested for surface Spike S1 protein expression.
1. Experimental equipment and materials:
the UVB ultraviolet lamp tube is a Philips UVB 311nm TL 100w/01 ultraviolet lamp tube;
the high-speed refrigerated centrifuge is Shanghai Luxiang, model GL-21 MS;
BD facscan II flow cytometer;
SARS-CoV Spike Antibody, Cat:40150-D001, which has cross reaction with SARS-CoV-2(2019nCoV) Spike S1 protein and is purchased from Beijing Yiqiaoshengzhou;
the G418 GENEICIN antibiotic was purchased from Gibco, cat # 11811-;
endotoxin-free plasmid miniprep medium kit was purchased from TIANGEN under catalog number DP 118;
four cells LLC-hS1, A549-hS1, BEAS-2B-hS1 and 293T-hS1 (prepared as in example 1) overexpressing SARS-CoV-2Spike (S1).
2. The experimental steps are as follows:
1) preparation of microparticle MP: for the preparation of MP derived from LLC-hS1 cells, the same procedure is used for the other three cell lines. LLC-hS1 cells were cultured in vitro in large quantities with a cell count of 1X 10 per dish8. Adopting Gibco RPMI-1640 culture medium, adding 10% Chinese holly leafFetal bovine serum was cultured at 20 ml/dish. Culturing in carbon dioxide incubator at 37 deg.C and 5% CO2. Irradiating with ultraviolet UVB at intensity of 300J/m2Irradiating for 1 hr at 37 deg.C with 5% CO2In a carbon dioxide incubator under the conditions, the incubation time is 24 hours. Cells were collected in 50ml high speed centrifuge tubes and diluted with an equal volume of PBS buffer. The centrifugation steps were as follows: 500g multiplied by 8min, and taking supernatant; changing a new centrifuge tube, centrifuging the supernatant again, taking the supernatant again after 3000g is multiplied by 5 min; 14000g multiplied by 2min, 4 ℃, changing a new centrifuge tube, and centrifuging the supernatant again, 14000g multiplied by 60min, 4 ℃; and pouring out the supernatant, washing the inner surface of the centrifuge tube for 2 times by using precooled PBS, and adding 1ml of fresh precooled PBS to resuspend the vesicle sediment at the bottom of the centrifuge tube to obtain the LLC-hS 1-MPs. Similarly, A549-hS1-MPs, BEAS-2B-hS1-MPs and 293T-hS1-MPs were prepared.
2) Detecting the expression of surface SARS-CoV-2Spike (S1) of LLC-hS 1-MPs: adding 100 μ l of LLC-hS1-MPs resuspended in PBS into the flow detection tube, then adding 1 μ l of SARS-CoV-2Spike antibody, adding 3ml of PBS after 30min, centrifuging 14000g × 60min to wash off excessive antibody; adding PE fluorescent secondary antibody after 100 mu l of PBS is resuspended, incubating for 30min in ice bath in the dark, adding 3ml of PBS, and then 14000g multiplied by 60 min; 0.5ml PBS was resuspended in MPs and the expression of SARS-CoV-2Spike (S1) on the surface of MPs was examined by flow cytometry.
3. The experimental results are as follows: the surface of MPs prepared by LLC-hS1 highly expresses SARS-CoV-2Spike (S1) protein. The results for A549-hS1-MPs, BEAS-2B-hS1-MPs and 293T-hS1-MPs were the same. As shown in fig. 2.
Example 3: the T cell activation by MPs overexpressing Spike S1 was verified in vitro.
1. Experimental equipment and materials:
NEST 6 well cell culture plates (non-TC treated) cat No. 703011;
the C57BL/6 mouse is purchased from the research center of Hubei provincial medical experimental animals under the Hubei provincial disease prevention and control center;
human blood was from normal volunteers;
recombinant mouse and human GM-CSF, IL-4 and IL-2 were purchased from peprotech;
biotin anti-mouse CD3antibody and Biotin anti-human CD3antibody were purchased from eBioscience;
streptavidin microbeads (2ml) were purchased from America, whirlpool;
mouse interferon-gamma (INF-gamma) ELISA kit, human interferon-gamma (INF-gamma) ELISA kit, mouse Granzyme B ELISA kit and human Granzyme B ELISA kit were purchased from R & D company.
2. The experimental steps are as follows:
mouse T cell activation assay
1) Isolation and culture of mouse DC (dendritic cell) cells: taking the limb bones of the mouse under the aseptic condition, washing the limb bones for 2 times by PBS, putting the limbs bones into a new plate, sucking 10ml of 1% FBS RPMI1640 culture medium, putting the plate into the plate, cutting off the parts at the two ends of the leg bones, sucking the culture medium by a 2ml syringe, and washing the marrow cavity until all red marrow is blown out and the leg bones are white. Repeatedly beating red bone marrow, filtering with 200 mesh screen, and centrifuging at 1300rpm × 8 min. Discarding the supernatant, adding 2ml of erythrocyte lysate, incubating for 5 minutes at normal temperature, adding 5ml of 1% FBS RPMI1640 culture solution for neutralization, centrifuging at 1300rpm × 8min, discarding the supernatant, adding 10ml of 1% FBS PBS, blowing and washing for 2 times, resuspending the 10% FBS RPMI1640 culture medium, and counting. 6 well plates were cultured with suspension cells at 2X 105 cells per well, and 3ml of 10% FBS RPMI1640 medium supplemented with 20ng/ml GM-CSF and 10ng/ml IL-4 was added. At 37 deg.C, 5% CO2, half an exchange every other day, and supplemented with cytokines. Suspended and semi-adherent immature DCs were collected after 6 days of culture. Mice were treated with LLC-hS1-MPs at a ratio of immature DC MPs to DC of 1:100, and after 24 hours, treated DC cells were collected by centrifugation and counted.
2) Isolation of mouse T cells: taking the spleen of a mouse, grinding and blowing the spleen by using a frosted glass slide, and filtering the spleen by using a 200-mesh filter screen to prepare cell suspension. Centrifuging at 400g × 8min, discarding the supernatant, adding 5ml of erythrocyte lysate, incubating for 5min, neutralizing with 1% FBS PBS, centrifuging at 400g × 8min, and washing for 3 times. The supernatant was discarded, 500. mu.l of PBS was resuspended in the pellet, and Biotin anti-mouse CD3 (1. mu.l, 1X 107cells) was added and incubated on ice for 15 min. The cells were resuspended in 450. mu.l of Labeling buffer after one Labeling buffer wash at 1300 rpm. times.8 min, the supernatant discarded, 50. mu.l of streptavidin microbeads from MACS were added and mixed well, and incubated at 4 ℃ for 15 min. The column was rinsed beforehand, and then 500. mu.l of the cell suspension was passed through the column, followed by washing three times with 500. mu.l of the Labeling buffer. The column was removed and placed in a 15ml centrifuge tube, and 1ml of labelling buffer was added to push out the cells adsorbed in the column, i.e., CD3 positive cells. Wash twice with 1% FBS PBS, centrifuge at 500g × 8min, and count.
3) Mouse T cell activation assay: t cells and MPs treated DC cells were mixed in a round bottom 96 well plate with a T cell/DC ratio of 10:1 and IL-2(30U/ml) was added. After 4 days, the expression of IFN-. gamma.and Granzyme B was measured for each group by ELISA.
Human T cell activation assay
1) Isolation and culture of human DC cells: and (4) separating the DC cells. 20ml of venous blood of healthy volunteers was taken in an anticoagulation tube, and PBMC was isolated using a lymphocyte separation medium Ficoll after dilution with PBS. And (3) resuspending the obtained PBMC in a GT551 serum-free culture medium, adding the PBMC into a 6-well plate, and culturing for 2-4h, wherein adherent cells are precursor cells of the DC, and suspension cells are T cells. Two cells were isolated, adherent DC precursor cells were cultured using GT551 medium containing 20ng/ml GM-CSF and 10ng/ml IL-4, cytokine supplementation with half-exchange medium every other day, DC cells were induced for 5 days, and suspended and semi-adherent immature DCs were collected on day 6. Human immature DCs were treated with 293T-hS1-MPs at a ratio of MPs to DCs of 1:100, and after 24 hours, the treated DCs were collected by centrifugation and counted.
2) The suspended T cells were collected, added to a new 6-well plate, cultured for 5 days using 551 medium containing IL-2, and counted.
3) Human T cell activation assay: t cells and MPs treated DC cells were mixed in a round bottom 96 well plate with a T cell/DC ratio of 10:1 and IL-2(30U/ml) was added. After 4 days, the expression of IFN-. gamma.and Granzyme B was measured for each group by ELISA.
3. The experimental results are as follows: MPs-treated DCs that highly express Spike S1 can activate specific T cells, allowing them to highly express IFN- γ and Granzyme B. As shown in fig. 3 and 4.
4. Example 4: in vitro assay for expression of Spike by CTL (cytotoxic T lymphocytes) cells
Killing of macrophages, dendritic cells and lung epithelial cells of S1
1. Experimental equipment and materials:
the sorting flow cytometer is BD FACSAria III;
ABI7500 fluorescent quantitative PCR instrument;
FITC anti-mouse CD3Antibody and APC anti-mouse CD8 Antibody were purchased from Biolegend;
2. the experimental steps are as follows:
mouse CTL killing macrophage experiment expressing Spike S1
1) Activated mouse T cells were prepared as in example 3 and CD8+ CTL was isolated by flow sorting for killing.
2) Mouse macrophages overexpressing Spike S1 protein were prepared to mimic viral infection: adjusting macrophage concentration to 5 × 104Perml, added to a 96-well plate at 200. mu.l per well (1X 10)4). Adding adenovirus expressing Spike S1 gene into the cell hole, culturing for 12 hr at MOI of 5.0, replacing fresh culture medium, puromycin screening, and detecting Spike S1 expression in macrophage after 48 hr.
3) The killing effect of mouse CTLs on macrophages overexpressing Spike S1 protein was examined: the macrophage over-expressing Spike S1 was used as a target cell to adjust the cell concentration to 1X 106Perml, added to a 96-well plate at 100. mu.l per well (1X 10)5). The CTL cells and target cells prepared above were added to a 96-well plate according to 40/1, 20/1 and 10/1, and three wells were replanted, with blank control and normal macrophages as controls. After culturing for 4 hours, collecting the supernatant of each well, and detecting the killing efficiency of each well by using an LDH detection kit.
Human CTL killing experiment on lung epithelial cells and macrophages expressing Spike S1
1) Activated human T cells were prepared as in example 3 and CD8+ CTL was isolated by flow sorting for killing.
2) Human lung epithelial cells BEAS-2B or macrophages overexpressing Spike S1 protein were prepared to mimic viral infection: adjusting the concentration of BEAS-2B or macrophages to 5X 104Adding into 96-well plate at 200 μ l per well (1 extracted)104). Adding adenovirus expressing Spike S1 gene into the cell hole, culturing at MOI of 5.0 for 12 hr, replacing fresh culture medium, puromycin screening, and detecting the expression of Spike S1 in BEAS-2B or macrophage after 48 hr.
3) The killing effect of human CTL on BEAS-2B or macrophage over-expressing Spike S1 protein was tested: using over-expressed Spike S1BEAS-2B or macrophage as target cell, adjusting cell concentration to 1 × 106Perml, added to a 96-well plate at 100. mu.l per well (1X 10)5). The CTL cells and target cells prepared above were added to a 96-well plate according to 40/1, 20/1 and 10/1, and multiple three wells were plated with normal BEAS-2B or macrophage as a control. After culturing for 4 hours, collecting the supernatant of each well, and detecting the killing efficiency of each well by using an LDH detection kit.
3. The experimental results are as follows: the MPs-induced T cells highly expressing Spike S1 can efficiently kill macrophages expressing Spike S1 without significant killing effect on normal cells, as shown in FIG. 5 and FIG. 6, which indicates that MPs of Spike S1 can effectively induce specific CTL, thereby killing cells expressing Spike S1.
The virus Spike S2 protein, Envelope protein (E), Membrane protein (M), Nucleocapsid protein (N) and open reading frame 1ab protein (Orf1ab polyprotein) can also be used for preparing the over-expressed MPs of the corresponding protein according to the method, and also can be prepared into vaccines for preventing and treating new coronary pneumonia by single use or combined use.
Reference documents:
1.Zhang Y,Zhang R,Zhang H,Liu J,Yang Z,Xu P,Cai W,Lu G, Cui M,Schwendener RA,Shi HZ,Xiong H,Huang B*.Microparticles released by Listeria monocytogenes-infected macrophages are required for dendritic cell-elicited protective immunity.Cell Mol Immunol.2012 Nov；9(6):489-96.
2.Zhang H,Tang K,Zhang Y,Ma R,Ma J,Li Y,Luo S,Liang X,Ji T,Gu Z,Lu J,He W,Cao X,Wan Y,Huang B*.Cell-free tumor microparticle vaccines stimulate dendritic cells via cGAS/STING signaling. Cancer Immunol Res.2015Feb；3(2):196-205.
3.Ma J,Wei K,Zhang H,Tang K,Li F,Zhang T,Liu J,Xu P,Yu Y,Sun W, Zhu L,Chen J,Zhou L,Liang X,Lv J,Fiskesund R,Liu Y,Huang B*.Mechanisms by Which Dendritic Cells Present Tumor Microparticle Antigens to CD8(+)T Cells. Cancer Immunol Res.2018Sep；6(9):1057-1068。
Claims (10)
1. a coronavirus vaccine comprising apoptotic cell-releasing microparticles comprising vesicular fabric released by apoptotic cells derived from a human and coronavirus-derived viral antigen encapsulated in the vesicular fabric.
2. The coronavirus vaccine according to claim 1, wherein the apoptosis is caused by contacting a human-derived cell with any one selected from a drug, radiation and/or ultraviolet rays to release a vesicular-structure substance.
3. The coronavirus vaccine of claim 1, wherein the coronavirus-derived antigen is a coronavirus component protein that activates a specific T cell.
4. The coronavirus vaccine according to any one of claims 1-3, wherein the human-derived cells are human-derived cell strains, and the coronavirus component proteins are selected from the group consisting of Spike protein, nucleocapsid protein, envelope protein and open reading frame 1ab protein (ORF 1ab) of coronavirus.
5. The coronavirus vaccine of any one of claims 1-4, wherein the microparticles have a particle size of 50-1000 nm.
6. The coronavirus vaccine of any one of claims 1-5, wherein the coronavirus is SARS-CoV-2.
7. The coronavirus vaccine of any one of claims 1-6, wherein the vaccine is prepared by the method of:
constructing human cells over-expressing viral component proteins: transfecting the cultured human cell strain with virus component protein, and screening to obtain human cells over-expressing virus components;
culturing the human cells overexpressing the viral components in vitro in a large amount, and irradiating the human cells with ultraviolet UVB to cause the human cells to be apoptotic;
collecting microparticles released by the apoptotic cells to obtain the coronavirus vaccine.
8. A pharmaceutical composition comprising a coronavirus vaccine according to any one of claims 1-7 and a pharmaceutically or physiologically acceptable adjuvant and/or additive.
9. Use of a coronavirus vaccine according to any one of claims 1-7 or a pharmaceutical composition according to claim 8 for the preparation of a medicament for the prevention and/or treatment of a disease associated with a coronavirus infection.
10. The use of claim 9, wherein the disease associated with coronavirus infection comprises covi-19 caused by infection with novel coronavirus SARS-CoV-2, a relapse after infection with novel coronavirus, and/or an asymptomatic patient after infection with novel coronavirus.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103446580A (en) * | 2012-05-31 | 2013-12-18 | 湖北盛齐安生物科技有限公司 | Tumor vaccine and preparation method thereof |
| CN109893515A (en) * | 2019-02-26 | 2019-06-18 | 华中科技大学 | A kind of macrophage carries medicine microparticle preparation and preparation method thereof |
| CN111875680A (en) * | 2020-08-08 | 2020-11-03 | 武汉圣润生物科技有限公司 | Preparation method and application of novel coronavirus prevention microparticles |
| CN112111513A (en) * | 2020-07-17 | 2020-12-22 | 深圳市第二人民医院 | Preparation method of novel coronavirus vaccine based on exosome platform |
-
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103446580A (en) * | 2012-05-31 | 2013-12-18 | 湖北盛齐安生物科技有限公司 | Tumor vaccine and preparation method thereof |
| CN109893515A (en) * | 2019-02-26 | 2019-06-18 | 华中科技大学 | A kind of macrophage carries medicine microparticle preparation and preparation method thereof |
| CN112111513A (en) * | 2020-07-17 | 2020-12-22 | 深圳市第二人民医院 | Preparation method of novel coronavirus vaccine based on exosome platform |
| CN111875680A (en) * | 2020-08-08 | 2020-11-03 | 武汉圣润生物科技有限公司 | Preparation method and application of novel coronavirus prevention microparticles |
| WO2022032496A1 (en) * | 2020-08-08 | 2022-02-17 | 武汉圣润生物科技有限公司 | Preparation method and application for micro-particles for preventing novel coronavirus |
Non-Patent Citations (2)
| Title |
|---|
| SERAPHIN KUATE等: "Exosomal vaccines containing the S protein of the SARS coronavirus induce high levels of neutralizing antibodies", VIROLOGY, vol. 362, pages 1 - 2 * |
| XIUYUAN OU等: "Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV", NATURE COMMUNICATIONS, vol. 11, no. 1, pages 2 * |
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