CN113521266B - Coronavirus vaccine - Google Patents
Coronavirus vaccine Download PDFInfo
- Publication number
- CN113521266B CN113521266B CN202010294207.XA CN202010294207A CN113521266B CN 113521266 B CN113521266 B CN 113521266B CN 202010294207 A CN202010294207 A CN 202010294207A CN 113521266 B CN113521266 B CN 113521266B
- Authority
- CN
- China
- Prior art keywords
- cells
- coronavirus
- vaccine
- human
- infection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 229960005486 vaccine Drugs 0.000 title claims abstract description 33
- 241000004176 Alphacoronavirus Species 0.000 title 1
- 241000711573 Coronaviridae Species 0.000 claims abstract description 36
- 239000011859 microparticle Substances 0.000 claims abstract description 27
- 239000000427 antigen Substances 0.000 claims abstract description 23
- 108091007433 antigens Proteins 0.000 claims abstract description 23
- 102000036639 antigens Human genes 0.000 claims abstract description 23
- 230000001640 apoptogenic effect Effects 0.000 claims abstract description 14
- 208000001528 Coronaviridae Infections Diseases 0.000 claims abstract description 9
- 230000003612 virological effect Effects 0.000 claims abstract description 7
- 210000004027 cell Anatomy 0.000 claims description 78
- 241001678559 COVID-19 virus Species 0.000 claims description 17
- 241000700605 Viruses Species 0.000 claims description 15
- 210000005260 human cell Anatomy 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 10
- 230000006907 apoptotic process Effects 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 7
- 208000025721 COVID-19 Diseases 0.000 claims description 6
- 239000003814 drug Substances 0.000 claims description 6
- 239000008194 pharmaceutical composition Substances 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 5
- 201000010099 disease Diseases 0.000 claims description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 5
- 238000012216 screening Methods 0.000 claims description 5
- 238000000338 in vitro Methods 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 230000002265 prevention Effects 0.000 claims description 4
- 101710198474 Spike protein Proteins 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 239000000654 additive Substances 0.000 claims description 2
- 239000002671 adjuvant Substances 0.000 claims description 2
- 101800000904 Spike protein S1 Proteins 0.000 claims 4
- 229940096437 Protein S Drugs 0.000 claims 1
- 208000037847 SARS-CoV-2-infection Diseases 0.000 claims 1
- 239000000463 material Substances 0.000 abstract description 9
- 229940021747 therapeutic vaccine Drugs 0.000 abstract description 6
- 229940021993 prophylactic vaccine Drugs 0.000 abstract description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 20
- 210000004443 dendritic cell Anatomy 0.000 description 20
- 210000002540 macrophage Anatomy 0.000 description 18
- 101000629318 Severe acute respiratory syndrome coronavirus 2 Spike glycoprotein Proteins 0.000 description 11
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 11
- 230000002147 killing effect Effects 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 101001028244 Onchocerca volvulus Fatty-acid and retinol-binding protein 1 Proteins 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 6
- 108700031361 Brachyury Proteins 0.000 description 5
- 102000001398 Granzyme Human genes 0.000 description 5
- 108060005986 Granzyme Proteins 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 5
- 239000012980 RPMI-1640 medium Substances 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- 210000002919 epithelial cell Anatomy 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 230000009385 viral infection Effects 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- 238000008157 ELISA kit Methods 0.000 description 4
- 108010002350 Interleukin-2 Proteins 0.000 description 4
- 102000000588 Interleukin-2 Human genes 0.000 description 4
- 230000006044 T cell activation Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 238000002372 labelling Methods 0.000 description 4
- 210000004072 lung Anatomy 0.000 description 4
- 210000000605 viral structure Anatomy 0.000 description 4
- 108010074328 Interferon-gamma Proteins 0.000 description 3
- 102000004388 Interleukin-4 Human genes 0.000 description 3
- 108090000978 Interleukin-4 Proteins 0.000 description 3
- 241000186781 Listeria Species 0.000 description 3
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 3
- 108700026244 Open Reading Frames Proteins 0.000 description 3
- 230000003213 activating effect Effects 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000001185 bone marrow Anatomy 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 229950010131 puromycin Drugs 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 102100027723 Endogenous retrovirus group K member 6 Rec protein Human genes 0.000 description 2
- 101710091045 Envelope protein Proteins 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 2
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 2
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 description 2
- 102000008070 Interferon-gamma Human genes 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 241001292005 Nidovirales Species 0.000 description 2
- 206010035664 Pneumonia Diseases 0.000 description 2
- 101710188315 Protein X Proteins 0.000 description 2
- 101150027674 S1 gene Proteins 0.000 description 2
- 241000315672 SARS coronavirus Species 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 208000036142 Viral infection Diseases 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 210000003557 bones of lower extremity Anatomy 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 229960003130 interferon gamma Drugs 0.000 description 2
- 210000001930 leg bone Anatomy 0.000 description 2
- 210000003712 lysosome Anatomy 0.000 description 2
- 230000001868 lysosomic effect Effects 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000011325 microbead Substances 0.000 description 2
- 230000003278 mimic effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000002663 nebulization Methods 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 230000003449 preventive effect Effects 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 229960004854 viral vaccine Drugs 0.000 description 2
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 1
- 229940123373 Adenovirus E1A gene Drugs 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 1
- 102100031673 Corneodesmosin Human genes 0.000 description 1
- 101710139375 Corneodesmosin Proteins 0.000 description 1
- 102100031256 Cyclic GMP-AMP synthase Human genes 0.000 description 1
- 101710118064 Cyclic GMP-AMP synthase Proteins 0.000 description 1
- 108010041986 DNA Vaccines Proteins 0.000 description 1
- 229940021995 DNA vaccine Drugs 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 101000746373 Homo sapiens Granulocyte-macrophage colony-stimulating factor Proteins 0.000 description 1
- 101001009603 Homo sapiens Granzyme B Proteins 0.000 description 1
- 101000599940 Homo sapiens Interferon gamma Proteins 0.000 description 1
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 1
- 235000003332 Ilex aquifolium Nutrition 0.000 description 1
- 235000002296 Ilex sandwicensis Nutrition 0.000 description 1
- 235000002294 Ilex volkensiana Nutrition 0.000 description 1
- 102000002227 Interferon Type I Human genes 0.000 description 1
- 108010014726 Interferon Type I Proteins 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 101710128836 Large T antigen Proteins 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108020005196 Mitochondrial DNA Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 101000746372 Mus musculus Granulocyte-macrophage colony-stimulating factor Proteins 0.000 description 1
- 101001009604 Mus musculus Granzyme B(G,H) Proteins 0.000 description 1
- 101001044384 Mus musculus Interferon gamma Proteins 0.000 description 1
- 102000004722 NADPH Oxidases Human genes 0.000 description 1
- 108010002998 NADPH Oxidases Proteins 0.000 description 1
- 101710151619 Replicase polyprotein 1ab Proteins 0.000 description 1
- 101710196623 Stimulator of interferon genes protein Proteins 0.000 description 1
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 208000009956 adenocarcinoma Diseases 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229940028617 conventional vaccine Drugs 0.000 description 1
- 230000000139 costimulatory effect Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 244000309457 enveloped RNA virus Species 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000005338 frosted glass Substances 0.000 description 1
- 102000046157 human CSF2 Human genes 0.000 description 1
- 102000043557 human IFNG Human genes 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 210000001985 kidney epithelial cell Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 201000005249 lung adenocarcinoma Diseases 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 108700021021 mRNA Vaccine Proteins 0.000 description 1
- 229940126582 mRNA vaccine Drugs 0.000 description 1
- 238000002826 magnetic-activated cell sorting Methods 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 229940023143 protein vaccine Drugs 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
Classifications
-
- 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
-
- 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
-
- 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
-
- 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
-
- 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 present invention relates to a coronavirus vaccine comprising apoptotic cell released microparticles comprising a vesicle-like structural material released by apoptotic cells of human origin and a coronavirus-derived viral antigen entrapped in said vesicle-like structural material. The coronavirus vaccine of the present invention can be used as a prophylactic vaccine for preventing coronavirus infection, and also as a therapeutic vaccine.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a vaccine for coronaviruses.
Background
New coronavirus (SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), or 2019-nCoV) infections have been globally prevalent, and development of effective vaccines has been hampered. The preparation of vaccines using conventional strategies includes inactivated viral vaccines, DNA vaccines, mRNA vaccines or protein vaccines, etc., with the goal of activating B cells and generating protective neutralizing antibodies. However, conventional vaccines are basically prophylactic vaccines, and it is difficult to exert the effect of therapeutic vaccines. In the face of hundreds of thousands of infected patients, it is urgent to develop therapeutic vaccines. The vaccine with both preventive and therapeutic effects is prepared, and a novel therapeutic method is provided for preventing and treating the new coronaries pneumonia (COVID-19 (Corona Virus Disease 2019)).
The body can produce various antibodies against viral proteins, however, only surface antibodies are effective, so that traditional component vaccines are developed against Spike proteins on the surface of coronavirus particles, and the antibodies cannot effectively clear viruses hidden in cells. Therefore, the final elimination of the virus also depends on specific T cells, which are key factors for virus infection immunity, and the time for activating the specific T cells is far less than the time required for the B cells to generate specific antibodies, and a large number of T cells are amplified out from 24 to 48 hours and enter the infection sites, and the infected cells are killed efficiently by recognizing MHC-virus antigen peptide complexes on the surfaces of the virus-infected cells, so that the virus is eliminated fundamentally. T cells are more suitable for use as therapeutic vaccines due to their faster production rate.
It has been demonstrated that Microparticles (MP) released by apoptotic cells can activate specific T cell immune responses as efficient vaccines. MP is a vesicle-shaped structural substance with a size of hundreds of nanometers, which is formed by wrapping cell contents by cell membranes, and has two key points of dominant vaccines: the antigen carrying capacity is strong, and the costimulatory signals are proper and effective. For example, a listeria-infected macrophage-derived MP comprising a listeria antigen, activating listeria-specific T cells via DC presentation [1] The method comprises the steps of carrying out a first treatment on the surface of the Whereas OVA-B16 tumor cell derived MP, containing tumor antigen, activates specific CD8+ T cells [2,3] . The co-stimulatory signal of T-MP (T stands for tumor cell) is due to its pocket cavity containing mitochondrial DNA fragment which can effectively activate the cGAS-STING pathway, inducing the production of type I interferon [2] . In addition, T-MP enters DC lysosomes, activates NADPH oxidase system, on one hand increases the pH value of the lysosomes, promotes more efficient production of tumor antigen peptides, and on the other hand upregulates the expression of CD80, CD86 and IL-12 by 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 diseases associated with coronavirus infection.
It is a second object of the present invention to provide pharmaceutical compositions containing said coronavirus vaccines.
A third object of the present invention is to provide the use of said vaccine and/or pharmaceutical composition for the preparation of a medicament for the prevention and treatment of diseases associated with coronavirus infection.
According to one aspect of the invention, a coronavirus vaccine comprises apoptotic cell released microparticles comprising an apoptotic cell released vesicle structure substance and an antigen of coronavirus origin entrapped in said vesicle structure substance.
The coronavirus vaccine of the present invention is a human-derived cell strain which can be cultured by contacting cells derived from a human with any one selected from the group consisting of a drug, radiation and/or ultraviolet rays to cause apoptosis and release of a vesicle-like structure substance.
The coronavirus vaccine of the present invention, wherein the antigen derived from coronavirus may preferably be coronavirus component protein which activates specific T cells; 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 present invention, the particle size of the microparticles released by apoptotic cells is 50 to 1000nm, more preferably 50 to 500nm.
By applying the concepts and methods of the present invention, vaccines against different coronaviruses, e.g., against SARS-CoV-2 virus, can be prepared by selecting different viral antigens.
Microparticles for encapsulation of viral antigens may be obtained using any method capable of apoptosis of human cells including, but not limited to: human cells are contacted with various medicaments, radioactive rays and ultraviolet rays to cause apoptosis of the cells and form vesicle-shaped structural substances. And then contacting the required virus antigen with the apoptotic cells, so that the virus is wrapped in a vesicle-like structural material, thereby obtaining the virus vaccine of the invention. Or co-culturing the human cells and an expression vector for expressing the virus antigen, transfecting the virus antigen into the human cells, and then apoptosis the human cells to obtain the vesicles coated with the virus antigen.
In a preferred embodiment, the coronavirus vaccine of the present invention can be prepared by the following method:
constructing a humanized cell over-expressing viral component proteins: transfecting the cultured human cell strain with virus component protein, and screening to obtain human cells over-expressing the virus component;
culturing a large amount of human cells over-expressing the viral component in vitro, and apoptosis by ultraviolet UVB irradiation;
and collecting microparticles released by the apoptotic cells to obtain the coronavirus vaccine.
In the embodiment of the present invention, the human cells are preferably induced to apoptosis by ultraviolet irradiation, and the vesicle-like structure material (cell vesicles) can be collected and separated under low temperature or room temperature conditions using a conventional centrifuge and a refrigerated high-speed centrifuge. Preferably, the cell vesicles are collected by a high-speed centrifuge under low temperature conditions (around 4 ℃) at a centrifugal force of 500-50,0000 g. Likewise, collection of microparticles encapsulating viral antigens can also be performed at low temperature using an ultracentrifuge. Preferably, microparticles encapsulating the viral antigen are collected by a centrifuge under low temperature conditions (around 4 ℃) at a centrifugal force of 500-50,0000 g. A preferred method of collecting microparticles is density gradient centrifugation, which collects microparticles at a centrifugal force of 1000-50,0000 g.
For the collected microparticles encapsulating the viral antigen, pharmaceutical formulations may be prepared according to conventional methods, including, but not limited to, injections such as subcutaneous and/or intravenous injections, oral administration, nebulization for nebulization therapy, and the like.
The person skilled in the art can select a suitable method for inducing apoptosis of human cells according to the above-described method for preparing coronavirus vaccines based on human cell sources, according to the type of coronavirus infection to be prevented and/or treated and the type of virus antigen used, and conditions such as a method for collecting cell vesicles encapsulating the virus antigen and a suitable ratio of the amount of cell vesicles to the amount of the virus antigen, so long as the coronavirus component protein encapsulated as an antigen component in the finally obtained vesicle structural material 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 a viral vaccine according to the present invention, which can be prepared by adding various pharmaceutically and/or physiologically acceptable adjuvants and/or additives to a coronavirus vaccine according to the present invention.
The coronavirus vaccine of the present invention can be used as a prophylactic vaccine for preventing coronavirus infection, and also as a therapeutic vaccine. Can be used as therapeutic vaccine for treating diseases caused by SARS-CoV-2 virus infection, including but not limited to new coronapneumonia (COVID-19), and can also be used for treating patients with complex yang and asymptomatic after virus infection treatment.
Drawings
FIG. 1 is a flow cytometer screening for detection of cells stably over-expressing SARS-CoV-2Spike (S1);
FIG. 2 flow cytometer detecting expression of SARS-CoV-2Spike (S1) on MPs surface;
FIG. 3 shows ELISA for detecting IFN-. Gamma.and Granzyme B expression in MPs overexpressing Spike S1 in mouse T cells;
FIG. 4 shows ELISA for detecting IFN-. Gamma.and Granzyme B expression in MPs over-expressing Spike S1 in human T cells;
FIG. 5 shows the killing effect of mouse CTL on SARS-CoV-2 infected cells;
FIG. 6 shows the killing effect of human CTL on SARS-CoV-2 infected cells.
Detailed Description
For the purpose of illustrating the technical aspects of the present invention, the present invention is further described below with reference to the accompanying drawings and examples, which are not to be construed as limiting the present invention in any way.
The term "Microparticles (MP)" as used herein is produced by apoptotic cells, having a vesicle-like structure in which the contents are encapsulated; "cell vesicles" are produced by apoptotic cells without encapsulating the contents; "coronaviruses" are a group of viruses of the genus Coronaviras (Coronaviridae) of the order Nidovirales (Nidovirales) Coronaviridae, which are a group of enveloped RNA viruses whose genome is linear single-stranded plus strand. SARS-CoV-2 (2019 novel coronavirus, or 2019-nCoV) is one of them, and may cause novel coronavirus pneumonia COVID-19 after infection.
The various tumor cells, drugs, experimental animals, and instruments used in the examples below are not particularly specified and commercially available.
Example 1: construction of cells overexpressing the 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 Yiqiao Shenzhou;
SARS-CoV Spike Antibody antibody, cat:40150-D001, which cross-reacts with SARS-CoV-2 (2019 nCoV) Spike S1 protein, purchased from Beijing Yiqiao Shenzhou;
lipofectamine3000 was purchased from Invitrogen;
g418 Genetisin antibiotics are available from Gibco under the accession number 11811-031;
the endotoxin-free plasmid miniprep kit was purchased from TIANGEN, catalog number DP118;
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 basic medical institute of China medical sciences.
2. The experimental steps are as follows:
1) Amplification of SARS-CoV-2Spike (S1) plasmid: plasmid transformation competent bacteria were cultured overnight with ampicillin-resistant LB plates; plating culture, monoclonal amplification culture, and plasmid extraction by using plasmid extraction kit.
2) The Lipofectamine3000 is adopted to transfect LLC, A549, BEAS-2B and 293T cells with SARS-CoV-2Spike (S1) plasmid respectively;
3) Screening monoclonal cell strains, and constructing stable cell strains: the infected cells after 48 hours of culture were inoculated into 96-well plates at 0.5 cells/well, and subjected to screening culture in RPMI1640 medium containing 5. Mu.g/ml puromycin and 10% FBS. The monoclonal growing cells were transferred to 24-well plates for expansion culture.
4) SARS-CoV-2Spike antibody is added into the cell, then PE fluorescent secondary antibody is added, monoclonal cell strain which over-expresses Spike S protein is screened out, and the stable screened cell strain is expanded and cultured.
3. Experimental results: cells that stably overexpress SARS-CoV-2Spike (S1), LLC-hS1, A549-hS1, BEAS-2B-hS1 and 293T-hS1 were selected. As in fig. 1.
Example 2: microparticles overexpressing the Spike S1 protein were prepared and their surface Spike S1 protein expression was examined.
1. Experimental equipment and materials:
the UVB ultraviolet lamp tube is Philips UVB 311nm TL 100w/01 ultraviolet lamp tube;
the high-speed refrigerated centrifuge is Shanghai Lu Xiang, model GL-21MS;
BD FACSCanto II flow cytometer;
SARS-CoV Spike Antibody antibody, cat:40150-D001, which cross-reacts with SARS-CoV-2 (2019 nCoV) Spike S1 protein, purchased from Beijing Yiqiao Shenzhou;
g418 Genetisin antibiotics are available from Gibco under the accession number 11811-031;
the endotoxin-free plasmid miniprep kit was purchased from TIANGEN, catalog number DP118;
four cells LLC-hS1, A549-hS1, BEAS-2B-hS1 and 293T-hS1 (prepared as described in example 1) overexpressing SARS-CoV-2Spike (S1).
2. The experimental steps are as follows:
1) Preparation of microparticles MP: taking MP from LLC-hS1 cells as an example, the other three cell lines were prepared in the same way. LLC-hS1 cells were cultured in vitro in large quantities with a cell count of 1X 10 per dish 8 . The culture is carried out by adopting Gibco RPMI-1640 culture medium and adding 10% holly embryo calf serum, and the culture is carried out by 20 ml/dish. Culturing in carbon dioxide incubator at 37deg.C under 5% CO 2 . Ultraviolet UVB irradiation with intensity of 300J/m 2 The irradiation time is 1 hour, and the mixture is placed at 37 ℃ and 5 percent CO 2 The culture time was 24 hours in a conditioned carbon dioxide incubator. Cells were collected in 50ml high speed centrifuge tubes and diluted with an equal volume of PBS buffer. The centrifugation steps are as follows: 500g×8min, collecting supernatant; transferring to a new centrifuge tube, and collecting supernatantCentrifuging for 3000g×5min, and collecting supernatant; transferring to a new centrifuge tube at 14000g×2min and 4deg.C, centrifuging the supernatant, and at 14000g×60min and 4deg.C; pouring out the supernatant, washing the inner surface of the centrifuge tube for 2 times by precooling PBS, and adding 1ml of new precooled PBS to resuspend the vesicle sediment at the bottom of the tube to obtain LLC-hS1-MPs. Similarly, A549-hS1-MPs, BEAS-2B-hS1-MPs and 293T-hS1-MPs were prepared.
2) Detecting the expression of SARS-CoV-2Spike (S1) on LLC-hS1-MPs surface: 100 mu l of PBS resuspended LLC-hS1-MPs are added into a flow detection tube, then 1 mu l of SARS-CoV-2Spike antibody is added, 3ml of PBS is added after 30min, and excess antibody is centrifugally washed out after 14000g multiplied by 60min; after 100 mu l PBS is resuspended, PE fluorescent secondary antibody is added, the mixture is incubated for 30min in a dark ice bath, 3ml PBS is added, and then 14000g multiplied by 60min;0.5ml PBS was used to resuspend MPs and flow cytometry was used to detect SARS-CoV-2Spike (S1) expression on the MPs surface.
3. Experimental results: MPs prepared by LLC-hS1 express SARS-CoV-2Spike (S1) protein on surface. The results were the same for A549-hS1-MPs, BEAS-2B-hS1-MPs and 293T-hS1-MPs. As shown in fig. 2.
Example 3: in vitro validation of activation of T cells by MPs overexpressing Spike S1.
1. Experimental equipment and materials:
NEST 6 well cell culture plate (non-TC treated) accession number 703011;
c57BL/6 mice were purchased from Hubei province medical laboratory animal research center subordinate to Hubei province disease prevention control center;
human blood is from normal volunteers;
recombinant mouse and human GM-CSF, IL-4, and IL-2 were purchased from peprotech;
the Biotin anti-mouse CD3 anti-ibody and the Biotin anti-human CD3 anti-ibody are purchased from eBioscience company;
streptavidin microbeads (2 ml) was purchased from Meitian and gentle;
mouse interferon-gamma (INF-gamma) ELISA kit, human interferon-gamma (INF-gamma) ELISA kit and 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 cells) cells: taking the limb bones of a mouse under the aseptic condition, flushing for 2 times by PBS, placing the limb bones into a new plate, placing 10ml of RPMI1640 culture medium with 1% FBS into the plate, shearing off two end parts of the leg bones, flushing the bone marrow cavity by using a 2ml syringe to suck the culture medium until red bone marrow is blown out and the leg bones are white. The red bone marrow was repeatedly blown, filtered through a 200 mesh screen, and centrifuged at 1300rpm X8 min. The supernatant was discarded, 2ml of erythrocyte lysate was added and incubated at room temperature for 5 minutes, 5ml 1%FBS RPMI1640 broth was added for neutralization, the discarded supernatant was centrifuged at 1300rpm×8 minutes, 10ml of 1% FBS PBS was added for washing 2 times by pipetting, 10% FBS RPMI1640 medium was resuspended, and counted. The 6-well plate was cultured with suspension cells, 2X 105 cells per well, and 3ml of 10% FBS RPM 1640 culture medium supplemented with 20ng/ml GM-CSF and 10ng/ml IL-4 was added. 37 ℃,5% co2, half a day change and cytokine supplementation. After 6 days of culture, the suspended and semi-adherent immature DCs were collected. Mice were treated with LLC-hS1-MPs at a ratio of immature DC MPs to DC of 1:100, and after 24 hours the treated DC cells were collected by centrifugation and counted.
2) Isolation of mouse T cells: the spleen of the mouse is taken, ground and blown by a frosted glass slide, and filtered by a 200-mesh filter screen to prepare a cell suspension. Centrifugation was performed at 400 g.times.8 min, and after discarding the supernatant, 5ml of red blood cell lysate was added to incubate for 5min to lyse the red blood, neutralization was performed with PBS containing 1% FBS, centrifugation was performed at 400 g.times.8 min, and washing was performed 3 times. The supernatant was discarded, the pellet was resuspended in 500. Mu.l PBS, biotin anti-mouse CD3 (1. Mu.l, 1X 107 cells) was added and incubated on ice for 15min. Labeling buffer was washed once, centrifuged at 1300rpm X8 min, the supernatant discarded, the cells resuspended in 450. Mu. l Labeling buffer, mixed well at streptavidin microbeads with 50. Mu.l MACS added and incubated at 4℃for 15min. The sorting column was pre-rinsed, after which 500. Mu.l of the cell suspension was passed through the column and washed three more times with 500. Mu. l Labeling buffer. The column was removed, placed on a 15ml centrifuge tube, and 1ml Labeling buffer was added to push out the cells adsorbed in the column, i.e., cells positive for CD 3. The sample was washed twice with 1% FBS PBS, centrifuged at 500 g.times.8 min, and counted.
3) Mouse T cell activation assay: t cells and MPs treated DC cells were mixed in round bottom 96 well plates at 10:1T cells/DC and IL-2 (30U/ml) was added. After 4 days, ELISA was used to detect IFN-. Gamma.and Granzyme B expression in each group.
Human T cell activation assay
1) Isolation and culture of human DC cells: isolation of DC cells. Venous blood from healthy volunteers was taken in 20ml in anticoagulation tubes, and PBMCs were isolated using lymphocyte separation liquid Ficoll after dilution with PBS. And (3) re-suspending the obtained PBMC in a GT551 serum-free culture medium, adding the culture medium into a 6-hole plate, and culturing for 2-4h, wherein the adherent cells are precursor cells of DC, and the 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, and cytokine was supplemented by half-cell changes every other day, induced to culture DC cells for 5 days, and suspension and half-adherent immature DC 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 the treated DCs were collected by centrifugation after 24 hours and counted.
2) The suspended T cells were collected and then added to a new 6-well plate, and T cells were cultured using 551 medium containing IL-2 for 5 days, and the counts were collected.
3) Human T cell activation assay: t cells and MPs treated DC cells were mixed in round bottom 96 well plates at 10:1T cells/DC and IL-2 (30U/ml) was added. After 4 days, ELISA was used to detect IFN-. Gamma.and Granzyme B expression in each group.
3. Experimental results: MPs treated DCs that highly express Spike S1 activate specific T cells to highly express IFN-gamma and Granzyme B. As shown in fig. 3 and 4.
4. Example 4: in vitro assay to detect expression of CTL (cytotoxic T lymphocyte) cells to Spike
Killing of macrophages, dendritic cells and lung epithelial cells of S1
1. Experimental equipment and materials:
the model of the sorting type flow cytometer is BD FACSAria III;
ABI7500 fluorescent quantitative PCR instrument;
FITC anti-mouse CD3 anti-body and APC anti-mouse CD8 anti-body are purchased from Biolegend;
2. the experimental steps are as follows:
macrophage assay for mouse CTL killing Spike S1 expression
1) Activated mouse T cells were prepared as in example 3 and cd8+ CTLs were isolated for killer cells by flow sorting.
2) Mouse macrophages overexpressing the Spike S1 protein were prepared to mimic viral infection: regulating macrophage concentration to 5×10 4 Per ml, 200. Mu.l/well (1X 10) 4 ). Adenovirus expressing Spike S1 gene was added to the cell well, moi=5.0, after 12 hours of culture, fresh medium was changed, puromycin was screened, and Spike S1 expression in macrophages was detected after 48 hours.
3) The killing effect of mouse CTL on macrophages overexpressing Spike S1 protein was examined: the cell concentration is adjusted to be 1 multiplied by 10 by taking the over-expressed Spike S1 macrophage as a target cell 6 Per ml, 100. Mu.l/well (1X 10) 5 ). The prepared CTL cells and target cells are added into a 96-well plate according to the ratio of 40/1, 20/1 and 10/1, three wells are multiplexed, and blank control and normal macrophage are used as control. After 4 hours of culture, the supernatant of each well was collected, and the killing efficiency of each well was detected by LDH detection kit.
Human CTL killing of lung epithelial cells and macrophages expressing Spike S1 experiments
1) Activated human T cells were prepared as in example 3 and cd8+ CTLs were isolated for killer cells by flow sorting.
2) Human lung epithelial cells BEAS-2B or macrophages overexpressing the Spike S1 protein were prepared to mimic viral infection: adjusting BEAS-2B or macrophage concentration to 5×10 4 Per ml, 200. Mu.l/well (1X 10) 4 ). Adenovirus expressing Spike S1 gene was added to the cell wells, moi=5.0, after 12 hours of incubation, fresh medium was changed, puromycin was screened, and expression of Spike S1 in bees-2B or macrophages was detected after 48 hours.
3) The killing effect of human CTL on BEAS-2B or macrophages over-expressing Spike S1 protein was examined: the cell concentration is adjusted to 1X 10 by using the over-expressed Spike S1BEAS-2B or macrophage as target cells 6 Per ml, 100. Mu.l/well (1X 10) 5 ). The CTL cells prepared above and target cells were added to a 96-well plate at 40/1, 20/1, 10/1, and three wells were multiplexed, and normal BEAS-2B or macrophages were used as controls. After 4 hours of culture, the supernatant of each well was collected, and the killing efficiency of each well was detected by LDH detection kit.
3. Experimental results: the MPs expressing Spike S1 can induce T cells to kill macrophages expressing Spike S1 effectively without obvious killing effect on normal cells, and as shown in FIG. 5 and FIG. 6, the MPs expressing Spike S1 can induce specific CTL effectively to kill cells expressing Spike S1.
The virus Spike S2 protein, envelope protein (E), membrane protein (Membrane glycoprotein, M), nucleocapsid protein (Nucleocapsid protein, N) and open reading frame 1ab protein (Orf 1ab polyprotein) can also be used to prepare over-expressed MPs of the corresponding proteins according to this method, as well as vaccines, alone or in combination for the prevention and treatment of new coronapneumonitis.
Reference is made to:
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 (4)
1. a coronavirus vaccine comprising microparticles released by apoptotic cells, said microparticles released by apoptotic cells comprising a vesicle-like structure substance released by apoptotic cells derived from a human and a coronavirus-derived viral antigen entrapped in said vesicle-like structure substance; the virus antigen of coronavirus source is Spike protein S1 subunit of SARS-CoV-2;
wherein the vaccine is prepared by the following method:
constructing a humanized cell over-expressing the Spike protein S1 subunit: transfecting the Spike protein S1 subunit into a cultured human cell strain, and screening to obtain a human cell over-expressing the Spike protein S1 subunit;
culturing said human cells in vitro in bulk, wherein said cells overexpress the S1 subunit of said Spike protein, and apoptosis is caused by irradiation with ultraviolet UVB;
collecting the microparticles released by the apoptotic cells to obtain the coronavirus vaccine;
the human cells are 293T cells.
2. The coronavirus vaccine of claim 1, wherein the microparticles have a particle size of 50-1000nm.
3. A pharmaceutical composition comprising the coronavirus vaccine of any one of claims 1-2 and pharmaceutically or physiologically acceptable adjuvants and/or additives.
4. Use of a coronavirus vaccine according to any one of claims 1-2 or a pharmaceutical composition according to claim 3 for the manufacture of a medicament for the prevention and/or treatment of diseases associated with coronavirus infection; wherein the disease associated with coronavirus infection comprises COVID-19 caused by novel coronavirus SARS-CoV-2 infection, post-novel coronavirus infection complex positive and/or post-novel coronavirus infection asymptomatic patients.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010294207.XA CN113521266B (en) | 2020-04-15 | 2020-04-15 | Coronavirus vaccine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010294207.XA CN113521266B (en) | 2020-04-15 | 2020-04-15 | Coronavirus vaccine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113521266A CN113521266A (en) | 2021-10-22 |
| CN113521266B true CN113521266B (en) | 2024-01-30 |
Family
ID=78088194
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010294207.XA Active CN113521266B (en) | 2020-04-15 | 2020-04-15 | Coronavirus vaccine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113521266B (en) |
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 |
-
2020
- 2020-04-15 CN CN202010294207.XA patent/CN113521266B/en active Active
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 |
|---|
| Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV;Xiuyuan Ou等;NATURE COMMUNICATIONS;第11卷(第1期);第2页右栏第1段至第10页右栏第4段 * |
| Exosomal vaccines containing the S protein of the SARS coronavirus induce high levels of neutralizing antibodies;Seraphin Kuate等;Virology;第362卷;摘要，第26页右栏倒数第1段，第33页左栏倒数第1段和第34页左栏倒数第1-2段 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113521266A (en) | 2021-10-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| ES2535835T3 (en) | Compositions for the preparation of mature dendritic cells | |
| US20090263421A1 (en) | Cellular vaccine | |
| Eyrich et al. | Development and validation of a fully GMP-compliant production process of autologous, tumor-lysate-pulsed dendritic cells | |
| KR101415039B1 (en) | Medium Composition and Method for Massive Culture of Autologous Activated Lymphocyte | |
| JPH11290068A (en) | Activation of natural killer (nk) cell and its procedure | |
| Ojima et al. | The boosting effect of co-transduction with cytokine genes on cancer vaccine therapy using genetically modified dendritic cells expressing tumor-associated antigen | |
| Zhang et al. | Interleukin-12-anchored exosomes increase cytotoxicity of T lymphocytes by reversing the JAK/STAT pathway impaired by tumor-derived exosomes | |
| KR101568323B1 (en) | Composition for inducing maturation of dendritic cell comprising gelsolin and method of inducing the same | |
| KR20110036570A (en) | Respiratory syncytial virus renders dendritic cells tolerogenic | |
| CN113521266B (en) | Coronavirus vaccine | |
| Huang et al. | Maturation of dendritic cells for enhanced activation of anti-HIV-1 CD8+ T cell immunity | |
| Pai et al. | Nasopharyngeal carcinoma‐associated Epstein–Barr virus‐encoded oncogene latent membrane protein 1 potentiates regulatory T‐cell function | |
| CN111978376A (en) | Pharmaceutical composition for preventing and/or treating coronavirus infection, and preparation method and application thereof | |
| WO2023005486A1 (en) | Ebv composite antigen, dendritic cell vaccine and use thereof | |
| Konduri et al. | Modeling dendritic cell vaccination for influenza prophylaxis: potential applications for niche populations | |
| US20110300179A1 (en) | Novel Compositions and Uses Thereof | |
| CN108251370B (en) | DC-CIK cell sensitized by polypeptide from non-cell source, and construction method and application thereof | |
| Kostinova et al. | The Effect of Influenza Vaccines on Maturation of Dendritic Cells Generated from Bone Marrow | |
| EP2527428A1 (en) | Tolerogenic dendritic cells and their use in cell therapy | |
| Thothathri | Transfer of dc Instruction to t Cells via Extracellular Vesicles | |
| Xue et al. | Dendritic cells modified with 6Ckine/IFNγ fusion gene induce specific cytotoxic T lymphocytes in vitro | |
| Takeuchi et al. | Perforin is expressed in CTL populations generated in vivo | |
| CN117487748A (en) | Preparation method and application of artificial antigen presenting cell | |
| Fukui et al. | Antitumor activity of a streptococcus pyogenes preparation (OK-432). II. Analysis of the cytotoxic lymphocytes induced by OK-432 injection into tumor-bearing F344 rats | |
| EP2829600A1 (en) | Method for preparing dendritic cells to be used as antigen-presenting cells in immunotherapy |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |