CN1951499A - A cell mediated immunity vaccine and preparation method thereof - Google Patents
A cell mediated immunity vaccine and preparation method thereof Download PDFInfo
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Abstract
本发明公开了一种细胞免疫疫苗及其制备方法。该疫苗是将单核/巨噬细胞或树突状细胞与单一或多种病原体或其部分组分，或病料共培养后得到的免疫细胞。本发明的疫苗具有以下优点：1)免疫性高；2)安全性高；3)广谱性高；4)保护效果显著。此外，单核/巨噬细胞、树突状细胞的分离、纯化及体外培养方法简单，可大量获得，因而该疫苗的制备方法简单、成本低廉，具有工业化生产的可行性。本发明的疫苗及其制备方法将在医学、动物生产领域，特别是各种自体免疫疾病的预防与免疫治疗方面发挥重要作用，应用前景广阔。The invention discloses a cellular immune vaccine and a preparation method thereof. The vaccine is an immune cell obtained by co-culturing monocytes/macrophages or dendritic cells with single or multiple pathogens or their partial components, or disease materials. The vaccine of the invention has the following advantages: 1) high immunity; 2) high safety; 3) high broad-spectrum; 4) remarkable protective effect. In addition, the isolation, purification and in vitro culture of monocytes/macrophages and dendritic cells are simple and can be obtained in large quantities. Therefore, the preparation method of the vaccine is simple and low in cost, and is feasible for industrial production. The vaccine and its preparation method of the invention will play an important role in the fields of medicine and animal production, especially in the prevention and immunotherapy of various autoimmune diseases, and have broad application prospects.
Description
Technical field
The present invention relates to vaccine and preparation method thereof, particularly relate to a kind of vaccine that on cellular level, multiple infectious disease is had the prevention protective effect and preparation method thereof.
Background technology
Animal and human beings'health in various infectious disease serious threats, and enter 21 century, also constantly have new disease to initiate challenge to the mankind.From SARS in 2003, high pathogenic avian influenza till now, all serious threat is to human beings'health and even life.In addition, tumor and autoimmune disease also are that human health is threatened huge pertinacious disease.Therefore, press for a kind of vaccine that infectious disease and/or tumor is had the effect of preventing and/or treating property.
In addition, the attack of terrorism that utilizes high lethal pathogen to implement, can cause huge fear and threat to human society, use such as anthrax, beaten alarm bell to the U.S. and other country, the biological anti-probably measure of national governments' active research for this reason, wherein, novel immunity inoculation and Therapeutic Method will play a significant role in biological anti-terrorism.
Prophylactic immunization is the main method of infection prevention disease, for huge impetus has been played in the development of physianthropy and the raising of the herding level of production, still is disease-resistant important means and method now.Carry out at present vaccine research mainly to as if diseases induced etiology (or source of disease body etc.), but because the mutating speed of pathogen is very fast, the speed that develops vaccine lags behind far away, often causes early stage uncontrollable of disease popularity, will pay painful cost for this reason.In addition, because the appearance of highly virulent strain,, also can cause the propagation of striding species and popular by the adaptation or the orthomutation of pathogen to new host.Press for a kind of new prophylactic immunization method, popular with the sudden change of tackling new source of disease body and disease will attack in the bio-terrorism of meeting an enemy attack, the biological characteristics of the body that finds the cause of disease in advance, surpass aspect such as early prevention inoculation and play a significant role.
As everyone knows, body is mainly realized by three aspects the defence of pathogen: one, prevent pathogen intrusion body by epidermis and mucosa etc.; Two, utilize immunocyte to eliminate the pathogen of invading, be also referred to as nonspecific immunity; Three, utilize the pathogen specific sign to carry out specific immunity aspect two of cell and body fluid, acquired immunity is otherwise known as.Prophylactic immunization mainly is to cause specific immunity, utilizes the Memorability of immunocyte simultaneously, permanently effective purpose after the realization immunity inoculation.
In bringing out immunoreactive process, at first be that pathogen is invaded body, the intravital epithelial cell of machine, Monocytes etc. have the cell of antigen presentation function pathogen are engulfed, digested, under the synergism of MHC I and MHC II, isolated antigen fragment is transported to cell surface, be afterwards and pass T cell, bring out cell and humoral immune reaction.In the antigen presentation process, Monocytes, dendritic cell (dendritic cell, DC) and the B cell to present efficient higher, therefore be also referred to as full-time antigen-presenting cell.
Mononuclear cell is differentiated by the hematopoietic stem cell of bone marrow, enter different tissues after in blood, staying for some time after, or keep mononuclear cell characteristic (invading profit), or be divided into macrophage as mononuclear cell.In-house macrophage also has difference according to different its names of the type of its place tissue, is Langerhans cell in epithelial tissue, and the macrophage in lung tissue is called pulmonary macrophage, and being called as withered at liver denys (Kupffer) cell or the like.It is generally acknowledged that sophisticated macrophage seldom is with or without multiplication capacity except that minority mononuclear cell or PD macrophage, and the cell that is constantly broken up by bone marrow precursor replenishes.
Summary of the invention
The purpose of this invention is to provide a kind of vaccine that on cellular level, infectious disease is had prevention and therapeutical effect.
Cell mediated immunity vaccine provided by the present invention is single or multiple pathogen or its part component with Monocytes or dendritic cell and purification, or the immunocyte that obtains after cultivating altogether of not purified pathological material of disease.
Described pathogen can be pathogenic microorganism (comprising virus), parasite, or the microorganism of deactivation or parasite; The part component of described source of disease body can be the protein or the RNA of pathogen.Described pathological material of disease can be tumor cell etc.
Second purpose of the present invention provides a kind of preparation method of above-mentioned cell mediated immunity vaccine.
The preparation method of cell mediated immunity vaccine provided by the present invention is with Monocytes or dendritic cell and single or multiple pathogen or its part component, or pathological material of disease cultivates 24-72h altogether under 36-38 ℃, cleans cell, obtains vaccine; The ratio of described Monocytes or dendritic cell and pathogen is 1: 1-10000, the ratio of described Monocytes or dendritic cell and pathological material of disease is 1: 1-10000.
In the preparation method of above-mentioned vaccine, described Monocytes or dendritic cell can be directed to peripheral blood, bone marrow, the tissue of animal, or obtain indirectly through external evoked differentiation.
Wherein, the external evoked differentiation method of Monocytes or dendritic cell can be: mononuclearcell is cultivated 1-10d with being added with RPMI1640 or the TCM199 complete culture solution that concentration is 10-100 μ g/mL cytokine down at 37 ℃, obtain Monocytes or dendritic cell; Described cytokine is one or more among GM-CSF, M-CSF, IL-4 and the IL-6.
The source of described mononuclearcell is widely, the animal that promptly has a mononuclearcell all can, comprise mammals such as people, cattle, pig, dog, cat, deer, and birds such as chicken, Ostriches, peafowl.
Can adopt conventional method to carry out the separation of mononuclearcell, for example: venous blood collection, with concentration expressed in percentage by volume is that the heparin of 2.5-5% is received or the sodium citrate solution anticoagulation of 2.5-5%, separates obtaining mononuclearcell again from anticoagulation by density with lymphocyte separation medium.Described lymphocyte separation medium can adopt commercially available getting final product.
For obtaining purer mononuclearcell, can carry out purification to mononuclearcell, purification process can be: mononuclearcell is carried out In vitro culture, treat cell attachment after, remove suspension cell, obtain the mononuclearcell of purification.
The condition of in vitro culture of described mononuclearcell can be: cultivate 1-7d with complete culture solutions such as RPMI1640 or TCM199 down at 36-38 ℃.
Described pathogen can be pathogenic microorganism (comprising virus), parasite, or the microorganism of deactivation or parasite; The part component of described source of disease body can be protein, RNA of pathogen etc.
Described culture medium altogether adopts the conventional animal cell culture medium that adds concentration expressed in percentage by volume 8-12% serum to get final product.Described serum can be hyclone etc., and Zooblast culture medium can be complete mediums such as RPMI1640 or TCM199.
Vaccine of the present invention can be fed back to it subcutaneous, lymph node, muscle, blood, mucosa etc. and can cause immunoreactive region of interest in use, reaches effect to disease prevention and/or treatment by bringing out immunoreation in the body.
The invention provides a kind of cell mediated immunity vaccine and preparation method thereof.This vaccine is with the single or multiple pathogen of Monocytes or dendritic cell and purification or its part component, or pathological material of disease is cultivated altogether, under conditions in vitro, realized antigen presentation, can bring out immunoreation after the feedback body is interior, reached effect disease prevention and/or treatment.Vaccine of the present invention has the following advantages: 1) immunity height, this vaccine can cause specific antibody consumingly and produce, and antibody titer can reach 1: 10-100000; 2) safe, detected external attack pathogen digestion situation in Monocytes after 24 hours, the result does not find the pathogen of surviving; 3) broad spectrum activity height, the specific immunity of animal body are all realized by Monocytes or dendritic cell, so this vaccine is applicable to all pathogenic organisms except the pathogen of attacking immunocyte; 4) Monocytes or dendritic cell have the ability of automatic processing and process antigen, epitope than artificial screening is more accurate, speed is faster, can be described as automatization's handling machine of antigen processing, therefore handle unknown pathogen with this vaccine, can be used as a kind of preventive means at illness outbreak initial stage; 4) evident in efficacy, white diarrhea disease, pig erysipelas disease etc. is all had better therapeutic effect.In addition, the separation of Monocytes, dendritic cell, purification and extracorporeal culturing method are simple, can obtain in a large number, thereby the preparation method of this vaccine are simple, with low cost, have the feasibility of suitability for industrialized production.Vaccine of the present invention and preparation method thereof will play a significant role at the prevention and the immunization therapy aspect of medical science, animal production field, particularly various autoimmune diseases, has a extensive future.
Below in conjunction with specific embodiment the present invention is described in further details.
Figure of description
Fig. 1 is with the acridine orange of purification of individual nucleus (AO) dyeing, the dyeing of Ji's nurse Sa and CD14 indirect IF staining qualification result
The specific embodiment
Method therefor is conventional method if no special instructions among the following embodiment.
The preparation of embodiment 1, white diarrhea disease vaccine and detection thereof
One, preparation vaccine
1, mononuclearcell obtains and purification
1) separation of mononuclearcell
From chicken, separate mononuclearcell, concrete grammar is: with venous blood collection position sterilization under the chicken wing, venous blood collection, the adding concentration expressed in percentage by volume is 5% heparin sodium aqua anticoagulation, obtains mononuclearcell with lymphocyte separation medium (available from Cedarlane company) by the density separation again from anticoagulation.
2) purification of mononuclearcell
Use the RPMI1640 complete culture solution at 37 ℃, 5%CO mononuclearcell
2Cultivate 8-24h in guarantor and the humidity incubator, after treating cell attachment, remove suspension cell, attached cell is carried out acridine orange (AO) dyeing, the dyeing of Ji's nurse Sa and CD14 indirect IF staining identifies, (a-1 is that new isolating mononuclearcell (also having some other cell) carries out the painted result of acridine orange to the result as shown in Figure 1, the cell of three kinds of arrow logos is respectively mononuclearcell, granulocyte, lymphocyte from top to bottom, and a-2 is the observed result under the fluorescence microscope.B-1 carries out the painted result of Ji's nurse Sa for the mononuclearcell of cultivating 7d, and b-2 is that part is amplified.C-1 is the result that the mononuclearcell of cultivation 7d carries out the indirect immunostaining of CD14, and c-2 is the observed result (scale is 30 μ m) under the fluorescence microscope), show to have obtained the higher mononuclearcell of purity.
3) acquisition of Monocytes
The mononuclearcell of purification was cultivated 5 days down at 37 ℃ with the RPMI1640 complete culture solution, obtained Monocytes.
3, the preparation of white diarrhea disease vaccine
Get Pullorum Disease Salmonella and Monocytes by 1000: 1 mixed, with the CO of the RPMI1640 complete medium that adds concentration expressed in percentage by volume 10% hyclone at 37 ℃
2Cultivate 1h in the incubator altogether, the pathogen that flush away is not digested is divided two groups, continues respectively to cultivate 1h (A group), 24h (B group) hour, has been engulfed the immunocyte of pathogenic bacteria, can be used as the white diarrhea disease vaccine.
Two, titration
The B group immunocyte that step 1 is obtained feeds back to the subcutaneous location of chicken by 1000/dosage only, to bring out immunoreation.Injection back 14d detects the antibody titer of immune chicken with the ELISA method, and the result reaches 1: 10-100000 shows that the white diarrhea disease vaccine for preparing with the inventive method can bring out the immunoreation of body.
Three, safety detects
Two groups of immunocytes of A, B that step 1 is obtained wash 3 times with PBS, add 1mL 1%Triton-X100 cell lysis film then and discharge bacterium in the born of the same parents, dilute 100 times then, get 200 μ l and coat on the nutrient broth agar flat board (purchasing), count clump count after 24 hours in Beijing extensive and profound in meaning star biotechnology responsibility company limited.Bacterium colony appears in A as a result, and the B group is not found bacterium colony, illustrates that pathogen has been digested by immunocyte, does not have corresponding pathogen to produce, and proves that the vaccine with the inventive method preparation has higher biological safety.
Four, immune effect detects
The B group immunocyte that step 1 is obtained feeds back to the healthy chicken individuality of not contacted Pullorum Disease Salmonella by 1000/dosage only, makes it obtain immunological memory.Be 10 with concentration then
7The Salmonella counteracting toxic substances of individual/mL, the result obtains 100% protection, shows that the Pullorum Disease salmonella vaccine with the inventive method preparation has effect preferably to white diarrhea disease.
The preparation of embodiment 2, swine erysipelas vaccine and detection thereof
One, preparation vaccine
1, mononuclearcell obtains and purification
1) separation of mononuclearcell
From pig, separate mononuclearcell, concrete grammar is: pig vena cava anterior blood sampling site is sterilized, venous blood collection, adding concentration expressed in percentage by volume are 5% sodium citrate solution anticoagulation, obtain mononuclearcell with lymphocyte separation medium by the density separation again from anticoagulation.
2) purification of mononuclearcell
Mononuclearcell is cultivated 24-48h with the RPMI1640 complete culture solution in 37 ℃ of incubators, after treating cell attachment, remove suspension cell, attached cell is carried out acridine orange (AO) dyeing, the dyeing of Ji's nurse Sa and CD14 indirect IF staining identify, qualification result shows and has obtained the higher mononuclearcell of purity.
3) acquisition of Monocytes
The mononuclearcell of purification was cultivated 7 days down at 37 ℃ with the RPMI1640 complete culture solution, obtained Monocytes.
2, the preparation of swine erysipelas vaccine
Get bacillus rhusiopathiae suis and Monocytes by 10000: 1 mixed, with the CO of the RPMI1640 complete medium that adds concentration expressed in percentage by volume 10% hyclone at 37 ℃
2Cultivate 1h in the incubator altogether, the pathogen that flush away is not digested continues to cultivate 24h, has been engulfed the immunocyte of pathogenic bacteria, can be used as swine erysipelas vaccine.
Two, titration
The immunocyte that step 1 is obtained feeds back to the lymph node of pig by 1000/dosage only, to bring out immunoreation.Injection back 14d, with the antibody titer of ELISA method detection immune swine, the result reaches 1: 10-100000 shows that the swine erysipelas vaccine with the inventive method preparation can bring out the immunoreation of body.
Three, safety detects
The immunocyte that step 1 is obtained washs 3 times with PBS, adds 1mL 1%Triton-X 100 cell lysis films then and discharges bacterium in the born of the same parents, dilutes 100 times then, gets 200 μ l and coats on the nutrient broth agar flat board, counts clump count behind the 24h.The result does not find bacterium colony, illustrates that pathogen is digested by immunocyte, does not have corresponding pathogen to produce, and proves that the vaccine with the inventive method preparation has higher biological safety.
Four, immune effect detects
The immunocyte that step 1 is obtained feeds back to the lymph node that does not infect the bacillus rhusiopathiae suis pig by 1000/dosage only, makes it obtain immunological memory.Use 10 then
7The bacillus rhusiopathiae suis counteracting toxic substances of individual/mL is found to obtain protection, shows that the swine erysipelas vaccine with the inventive method preparation has effect preferably to the pig erysipelas disease.
The preparation of embodiment 3, bovine tuberculosis vaccine and detection thereof
One, preparation vaccine
1, mononuclearcell obtains and purification
1) separation of mononuclearcell
From cattle, separate mononuclearcell, concrete grammar is: sterilized in people's venous blood collection position, venous blood collection, adding volumetric concentration are 5% sodium citrate solution anticoagulation, obtain mononuclearcell with lymphocyte separation medium by the density separation again from anticoagulation.
2) purification of mononuclearcell
Mononuclearcell is cultivated 24-72h with the TCM199 complete culture solution in 37 ℃ of incubators, after treating cell attachment, remove suspension cell, attached cell is carried out acridine orange (AO) dyeing, the dyeing of Ji's nurse Sa and CD14 indirect IF staining identify, qualification result shows and has obtained the higher mononuclearcell of purity.
3) acquisition of dendritic cell
The mononuclearcell of purification was cultivated 3 days down at 37 ℃ with the TCM199 complete culture solution of 50 μ g/mL GM-CSF, obtained dendritic cell.
2, the preparation of bovine tuberculosis vaccine
Get mycobacterium tuberculosis var bovis and dendritic cell by 100: 1 mixed, with the CO of the TCM199 complete medium that adds concentration expressed in percentage by volume 10% hyclone at 37 ℃
2Cultivate 1h in the incubator altogether, the pathogen that flush away is not digested continues to cultivate 48h hour, has been engulfed the immunocyte of pathogenic bacteria, can be used as the bovine tuberculosis vaccine.
Two, titration
The immunocyte that step 1 is obtained feeds back to the lymph node of cattle by 1000/dosage only, to bring out immunoreation.Injection back 14d, with the antibody titer of ELISA method detection immune cattle, the result reaches 1: 10-1000000 shows that the bovine tuberculosis vaccine with the inventive method preparation can bring out the immunoreation of body.
Three, safety detects
The immunocyte that step 1 is obtained washs 3 times with PBS, adds 1mL 1%Triton-X 100 cell lysis films then and discharges bacterium in the born of the same parents, dilutes 100 times then, gets 200 μ l and coats on the nutrient broth agar flat board, counts clump count after 24 hours.The result does not find bacterium colony, illustrates that pathogen is digested by immunocyte, does not have corresponding pathogen to produce, and proves that the vaccine with the inventive method preparation has higher biological safety.
Four, immune effect detects
The immunocyte that step 1 is obtained feeds back to the not healthy cattle cattle lymph node of infected cattle mycobacterium tuberculosis by 1000/dosage only, makes it obtain immunological memory.Be 10 with concentration then
7The mycobacterium tuberculosis var bovis counteracting toxic substances of individual/mL finds that cattle obtains immunoprotection, shows that the bovine tuberculosis vaccine with the inventive method preparation has effect preferably to bovine tuberculosis.
Claims (10)
1, a kind of cell mediated immunity vaccine is with Monocytes or dendritic cell and purified single or multiple pathogen or its part component, or the immunocyte that obtains after cultivating altogether of not purified pathological material of disease.
2, vaccine according to claim 1 is characterized in that: described pathogen is pathogenic microorganism, parasite, or the pathogenic microorganism of deactivation or parasite; The part component of described source of disease body is the protein or the RNA of pathogen.
3, vaccine according to claim 1 and 2 is characterized in that: described pathological material of disease is a tumor cell.
4, a kind of method for preparing the described vaccine of claim 1 is with Monocytes or dendritic cell and single or multiple pathogen or its part component, or pathological material of disease cultivates 24-72h altogether under 36-38 ℃, cleans cell, obtains vaccine; The ratio of described Monocytes or dendritic cell and pathogen is 1: 1-10000, the ratio of described Monocytes or dendritic cell and pathological material of disease is 1: 1-10000.
5, method according to claim 4 is characterized in that: described Monocytes or dendritic cell derive from peripheral blood, bone marrow, the tissue of animal, or obtain through external evoked differentiation.
6, method according to claim 5, it is characterized in that: the external evoked differentiation method of described Monocytes or dendritic cell is: mononuclearcell is cultivated 1-10d with being added with RPMI1640 or the TCM199 complete culture solution that concentration is 10-100 μ g/mL cytokine down at 36-38 ℃, obtain Monocytes or dendritic cell; Described cytokine is one or more among GM-CSF, M-CSF, IL-4 and the IL-6.
7, method according to claim 6 is characterized in that: described mononuclearcell derives from mammal or birds; Described mammal behaviour, cattle, pig, dog, cat or deer, described birds are chicken, Ostriches or peafowl.
8, method according to claim 7, it is characterized in that: the method for mononuclearcell being carried out purification is: mononuclearcell is cultivated 5-7d with RPMI1640 or TCM199 complete culture solution down at 36-38 ℃, after treating cell attachment, remove suspension cell, obtain the mononuclearcell of purification.
9, according to each described method of claim 4-8, it is characterized in that: described pathogen is pathogenic microorganism, parasite, or the microorganism of deactivation or parasite; The part component of described source of disease body is the protein or the RNA of pathogen.
10, according to each described method of claim 4-8, it is characterized in that: described culture medium altogether is for adding the RPMI1640 or the TCM199 complete medium of concentration expressed in percentage by volume 8-12% serum.
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| CNA2006101136080A CN1951499A (en) | 2006-10-09 | 2006-10-09 | A cell mediated immunity vaccine and preparation method thereof |
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| CNA2006101136080A CN1951499A (en) | 2006-10-09 | 2006-10-09 | A cell mediated immunity vaccine and preparation method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107630000A (en) * | 2017-11-06 | 2018-01-26 | 中国农业大学 | A kind of kit of domestic animal derived from peripheral blood macrophage separation and culture |
| US20190381158A1 (en) * | 2016-02-04 | 2019-12-19 | Duke University | Cell-based vaccine compositions and methods of use |
| US12252545B2 (en) | 2019-12-11 | 2025-03-18 | Myeloid Therapeutics, Inc. | Therapeutic cell compositions and methods of manufacturing and use thereof |
| US12319925B2 (en) | 2021-05-11 | 2025-06-03 | Myeloid Therapeutics, Inc. | Methods and compositions for genomic integration |
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2006
- 2006-10-09 CN CNA2006101136080A patent/CN1951499A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20190381158A1 (en) * | 2016-02-04 | 2019-12-19 | Duke University | Cell-based vaccine compositions and methods of use |
| US12220451B2 (en) * | 2016-02-04 | 2025-02-11 | Duke University | Cell-based vaccine compositions and methods of use |
| CN107630000A (en) * | 2017-11-06 | 2018-01-26 | 中国农业大学 | A kind of kit of domestic animal derived from peripheral blood macrophage separation and culture |
| CN107630000B (en) * | 2017-11-06 | 2021-07-13 | 中国农业大学 | A kit for isolating and culturing macrophages derived from peripheral blood of livestock |
| US12252545B2 (en) | 2019-12-11 | 2025-03-18 | Myeloid Therapeutics, Inc. | Therapeutic cell compositions and methods of manufacturing and use thereof |
| US12319925B2 (en) | 2021-05-11 | 2025-06-03 | Myeloid Therapeutics, Inc. | Methods and compositions for genomic integration |
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