CN114432261B - Preparation method of targeted delivery vaccine and erythrocyte vaccine - Google Patents
Preparation method of targeted delivery vaccine and erythrocyte vaccine Download PDFInfo
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- CN114432261B CN114432261B CN202210177998.7A CN202210177998A CN114432261B CN 114432261 B CN114432261 B CN 114432261B CN 202210177998 A CN202210177998 A CN 202210177998A CN 114432261 B CN114432261 B CN 114432261B
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- A61K9/0012—Galenical forms characterised by the site of application
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Abstract
The invention provides a preparation method of a targeted delivery vaccine and a red blood cell vaccine, which can realize the timeliness of the vaccine taken by antigen presenting cells, and has the advantages of more rapid and efficient effect, wherein the antigen presenting cells are widely distributed in peripheral blood and various tissues, and main target organs are peripheral immune organs such as lymph nodes and spleen.
Description
Technical Field
The invention relates to the technical field of vaccines, in particular to a preparation method of a targeted delivery vaccine and a red blood cell vaccine.
Background
The current vaccination mode is subcutaneous injection, and has the main advantages that the skin is the subcutaneous tissue of the maximum immune organ of the human body and has rich antigen presenting cells, and the vaccination mode is safe, simple and easy to get up; however, antigen uptake is dependent on the tendency of antigen presenting cells to vaccinate, and subsequent antigen homing to lymph nodes or spleen further activates T cells to function, with limitations in timeliness and vaccine uptake efficiency.
Accordingly, the prior art has drawbacks and needs improvement.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of a targeted delivery vaccine and a red blood cell vaccine, and the antigen uptake efficiency and timeliness are greatly improved.
The technical scheme of the invention is as follows: a method of preparing a targeted delivery vaccine is provided, comprising the following steps.
S1: peripheral blood was obtained and stored in anticoagulation blood collection tubes.
S2: the erythrocytes of the peripheral blood are separated from lymphocytes, and the erythrocytes are collected.
S3: the collected erythrocytes were washed with 0.01M PBS or 0.9% physiological saline-centrifuged several times to obtain erythrocytes after primary centrifugation.
S4: adding 55.6% -83.3% 1xPBS or 0.9% physiological saline into the centrifuged red blood cells for dilution, then adding vaccine components, and preserving at low temperature for 0.1-8 hours to obtain a mixed red blood cell and vaccine mixture.
S5: adjusting PBS concentration in the mixture of red blood cells and vaccine to 0.01M, and preserving at low temperature for 0.1-8 hours; or adjusting NaCl concentration in the mixture of erythrocyte and vaccine to 0.9%, and preserving at low temperature for 0.1-8 hr.
S6: erythrocytes were collected by centrifugation and subsequently incubated with CD47 antibody or CpG for 0.1-8 hours.
S7: washing with 0.01M PBS or 0.9% physiological saline, centrifuging for several times to obtain red blood cells after centrifuging again, and obtaining the red blood cell vaccine, namely the targeted delivery vaccine.
The red blood cells are cells which are rich in the peripheral blood of the human body and are easy to obtain, and mature cells are mainly used as oxygen transport carriers and have the following characteristics: 1) No cell nucleus, less organelles in the cell and clear components; 2) Immune inert cells, long blood circulation time and short time can circulate all tissues and organs of the organism; 3) The biconcave disc shape has a relatively high surface area; 4) Better film stability and deformability. In view of the characteristics of the erythrocytes, the erythrocytes are modified to load the vaccine and target antigen presenting cells, so that the antigen uptake efficiency and timeliness can be greatly improved.
In step S2, the method for separating erythrocytes from lymphocytes may be any of the following methods: lymphocyte separation liquid separation method, hemodialysis separation method, and density gradient centrifugation method.
In step S3, the surface of the red blood cell contains neither an antigen A nor an antigen B, and is used for preparing a universal red blood cell vaccine and preparing and vaccinating autologous and allogeneic red blood cell vaccines; the blood transfusion principle is suitable for the application principle of the erythrocyte vaccine.
In step S4, further comprising: the centrifuged red blood cells are subjected to electric shock in advance, the working voltage of the electric shock is 150V, and the electric shock is 15ms. The electric shock allows more efficient entry of vaccine components into erythrocytes. Preferably, the electric shock is once.
In step S5, the vaccine component is a component having immunomodulation.
The immunomodulating component comprises an antigen polypeptide sequence and/or an antigen mRNA sequence and/or an antigen DNA sequence and/or an inactivated pathogenic microorganism and/or an attenuated pathogenic microorganism and/or a TLR receptor agonist.
In steps S3 and S7, the number of times of washing-centrifuging is two.
The erythrocyte vaccine is used for preparing medicines for preventing and treating immune related diseases; the immune-related diseases include: cancer, pathogenic infectious diseases, autoimmune deficiency diseases; the cancer comprises: b-cell lymphoma, T-cell lymphoma, mycosis fungoides, hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, cancer of the nervous system, head and neck cancer, squamous cell carcinoma of the head and neck, lung cancer, small cell lung cancer, non-small cell lung cancer, neuroblastoma, glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, melanoma, basal cell carcinoma, squamous cell carcinoma, liver cancer, squamous cell carcinoma of the mouth, throat and lung, cervical cancer, breast cancer, renal cancer, genitourinary system cancer, lung cancer, esophageal cancer, head and neck cancer, large intestine cancer, hematopoietic system cancer; testicular cancer; colorectal cancer, prostate cancer, AIDS-related lymphoma or AIDS-related sarcoma; pathogenic infectious diseases include: local tissue and systemic inflammatory response caused by invasion of any one pathogen of bacteria, viruses, fungi and parasites into human body; the autoimmune deficiency diseases include: chronic lymphothyroiditis, hyperthyroidism, type 1 diabetes, myasthenia gravis, chronic ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pneumonecropathy syndrome (goodpasture syndrome), pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple cerebral sclerosis, acute idiopathic polyneuritis, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, polyarteritis nodosa, wegener granulomatosis.
The erythrocyte vaccine is injected by intravenous injection.
The peripheral blood is autologous peripheral blood or allogeneic O-type blood.
The invention also provides a red blood cell vaccine, and the preparation method of the targeted delivery vaccine is prepared.
The term "antigen" is a substance that induces an immune response.
The term "vaccine" is understood to mean a composition for generating immunity for the prevention and/or treatment of diseases. Accordingly, vaccines are drugs containing antigens and are intended for use in humans or animals by vaccination to produce specific defenses and protective substances.
By adopting the scheme, the invention provides the preparation method of the targeted delivery vaccine and the erythrocyte vaccine, which can realize the timeliness of the vaccine taken by antigen presenting cells, and has quicker and more efficient effect, and the main target organs are peripheral immune organs such as lymph nodes and spleen; the erythrocyte vaccine can be phagocytized specifically after contacting the antigen presenting cells, and then activate organism specific immunity in lymphoid organs to generate specific immunity aiming at vaccine components, thereby helping the organism to regulate and remove tumor cells or pathogenic microorganisms through autoimmune functions, regulating autoimmune diseases and reducing inflammatory reactions. Can be applied to tumor immunotherapy, tumor recurrence prevention, preparation of therapeutic or prophylactic pathogenic microorganism vaccine and treatment of autoimmune diseases.
Drawings
FIG. 1 is a flow chart of the present invention;
FIG. 2 is a graph showing the comparison of the results of centrifugation by adding PBS in different concentration ranges to the centrifuged red blood cells;
FIG. 3 is a graph showing the results of hemoglobin measurement of the supernatant of the sample of FIG. 2;
FIG. 4 is a comparison of 25ug/ml of 62.5% PBS+Cy7-labeled polypeptide vaccine+centrifuged erythrocytes, pure water+centrifuged erythrocytes, 100% PBS+Cy7-labeled polypeptide vaccine 25ug/ml for lysis-centrifugation;
FIG. 5 is a graph showing the results of detecting Cy7 from the supernatant of the sample of FIG. 4;
FIG. 6 shows that after intravenous injection of erythrocyte vaccines, the erythrocyte vaccines can be rapidly targeted to and enriched in spleen organs;
FIG. 7 shows that antigen presenting cells of spleen, peripheral blood, liver can specifically phagocytize erythrocyte vaccines and take on an activated state.
Detailed Description
The invention will be described in detail below with reference to the drawings and the specific embodiments.
Referring to fig. 1, the present invention provides a method for preparing a targeted delivery vaccine, which can achieve efficient uptake of the prepared erythrocyte vaccine by antigen presenting cells, and comprises the following steps.
S1: obtaining autologous peripheral blood of an experimental mouse and storing the autologous peripheral blood in an anticoagulation blood collection tube; the peripheral blood adopts autologous peripheral blood, and the erythrocyte vaccine prepared from the autologous peripheral blood can reduce immune response.
S2: separating erythrocytes and lymphocytes in the peripheral blood by using lymphocyte separation liquid, and collecting the erythrocytes;
s3: the collected erythrocytes were then washed with 0.01M PBS or 0.9% physiological saline-centrifuged 2 times;
s4: adding 1xPBS (diluted by pure water) with the concentration range of 33.3% -100.0% into the centrifuged red blood cells, preserving for 1-2 hours at low temperature, and obtaining the result as shown in figure 2, wherein the red blood cells with the concentration higher than 62.5% are complete, and the concentration lower than 55.6% are hemolyzed; the results of hemoglobin detection of the supernatant are shown in FIG. 3, and are consistent with those of FIG. 2;
s5: selecting red blood cells after centrifugation, wherein the concentration of 62.5% PBS is dilution concentration plus 25ug/ml of Cy 7-labeled polypeptide vaccine, and the control is pure water to dissolve red blood cells and Cy 7-labeled polypeptide vaccine dissolved in 100% PBS, preserving for 1-2 hours at low temperature, and centrifuging to obtain the result shown in figure 4; the results of Cy7 detection of the supernatant are shown in FIG. 5; calculating the loading rate of the red blood cells to be 37% according to the detection result of Cy 7; regulating the concentration of the mixture of the red blood cells and the vaccine to be 0.01M PBS concentration or 0.9 percent physiological saline, and preserving for 1-2 hours at low temperature;
s6: red blood cells were collected by centrifugation and then incubated with CD47 antibody or CpG for 1 hour;
s7: washing with 0.01M PBS or 0.9% physiological saline-centrifuging for 2 times, collecting erythrocyte to obtain erythrocyte vaccine;
s8: the red cell vaccine prepared by intravenous injection at the tail of the mouse is anesthetized in half an hour after injection, and the enrichment condition of the red cell vaccine in the mouse is detected by in vivo imaging of the small animal, and the result is shown in fig. 6, wherein the red cell vaccine is mainly enriched in spleen organs;
s9: taking spleen, liver and peripheral blood of a healthy mouse, separating erythrocytes and lymphocytes by using lymphocyte separating liquid, and collecting lymphocytes;
s10: lymphocyte concentration was adjusted to 5 x 10 with mouse lymphocyte culture broth 6 Per ml, respectively using100 ul/well was seeded in 96-well plates; adding 0.01M PBS into a blank group, adding the erythrocyte vaccine prepared in the step S7 into a test group, and adding the semi-finished erythrocyte vaccine prepared in the step S5 into a control group; incubation at 37 ℃ for 48 hours, followed by collection of cells for detection of antigen presenting cell surface markers using flow cytometry; specifically, cells were suspended in FACS buffer (1×pbs, containing 2 mM EDTA and 1% fbs) and then stained with the appropriate antibody for 20 minutes in the dark at 4 ℃. Flow cytometer data were obtained on a Facsariiii flow cytometer (BD Biosciences) and analyzed using FlowJo v10 software (TreeStar) and FCS Express 7 software. As shown in fig. 7, the semi-finished erythrocyte vaccine without CD47 antibody is not phagocytized by antigen presenting cells and has no antigen presenting cell activating effect, but the erythrocyte vaccine described in the invention can be specifically taken in by antigen presenting cells and is in an activated state, and the surface erythrocyte vaccine can precisely target and activate the antigen presenting cells of peripheral blood, liver, spleen and other organs.
In summary, erythrocytes are abundant and accessible cells in the peripheral blood of mammals, and mature cells are mainly used as oxygen transport carriers and have the following characteristics: 1) No cell nucleus, less organelles in the cell and clear components; 2) Immune inert cells, long blood circulation time and short time can circulate all tissues and organs of the organism; 3) The biconcave disc shape has a relatively high surface area; 4) Better film stability and deformability. In view of the characteristics of the erythrocytes, the erythrocytes are modified to load the vaccine and target antigen presenting cells, so that the antigen uptake efficiency and timeliness can be greatly improved. The invention provides an intravenous injection erythrocyte carrier vaccine of a targeted spleen antigen presenting cell, which can realize rapid enrichment of the vaccine in the spleen and is accurately taken in by the antigen presenting cell.
In step S2, the method for separating erythrocytes from lymphocytes may be any of the following methods: lymphocyte separation liquid separation method, hemodialysis separation method, and density gradient centrifugation method.
In step S3, the surface of the red blood cell contains neither an antigen A nor an antigen B, and is used for preparing a universal red blood cell vaccine and preparing and vaccinating autologous and allogeneic red blood cell vaccines; the blood transfusion principle is suitable for the application principle of the erythrocyte vaccine.
In step S4, further comprising: the centrifuged red blood cells are subjected to electric shock in advance, the working voltage of the electric shock is 150V, and the electric shock is 15ms. The electric shock allows more efficient entry of vaccine components into erythrocytes. Preferably, the electric shock is once.
In step S5, the vaccine component is a component having immunomodulation. The immunomodulating component comprises an antigen polypeptide sequence and/or an antigen mRNA sequence and/or an antigen DNA sequence and/or an inactivated pathogenic microorganism and/or an attenuated pathogenic microorganism and/or a TLR receptor agonist.
The erythrocyte vaccine is used for preparing medicines for preventing and treating immune related diseases; the immune-related diseases include: cancer, pathogenic infectious diseases, autoimmune deficiency diseases; the cancer comprises: b-cell lymphoma, T-cell lymphoma, mycosis fungoides, hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, cancer of the nervous system, head and neck cancer, squamous cell carcinoma of the head and neck, lung cancer, small cell lung cancer, non-small cell lung cancer, neuroblastoma, glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, melanoma, basal cell carcinoma, squamous cell carcinoma, liver cancer, squamous cell carcinoma of the mouth, throat and lung, cervical cancer, breast cancer, renal cancer, genitourinary system cancer, lung cancer, esophageal cancer, head and neck cancer, large intestine cancer, hematopoietic system cancer; testicular cancer; colorectal cancer, prostate cancer, AIDS-related lymphoma or AIDS-related sarcoma; pathogenic infectious diseases include: local tissue and systemic inflammatory response caused by invasion of any one pathogen of bacteria, viruses, fungi and parasites into human body; the autoimmune deficiency diseases include: chronic lymphothyroiditis, hyperthyroidism, type 1 diabetes, myasthenia gravis, chronic ulcerative colitis, pernicious anemia with chronic atrophic gastritis, pneumonecropathy syndrome (goodpasture syndrome), pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple cerebral sclerosis, acute idiopathic polyneuritis, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, polyarteritis nodosa, wegener granulomatosis.
The erythrocyte vaccine is injected by intravenous injection.
In summary, the invention provides a preparation method of a targeted delivery vaccine and a erythrocyte vaccine, which can realize the timeliness of the vaccine taken by antigen presenting cells, and has quicker and more efficient effect, and the main target organs are peripheral immune organs such as lymph nodes and spleen; antigen presenting cells are widely distributed in peripheral blood and various tissues. Can realize the rapid enrichment of vaccine in spleen and can be accurately taken up by antigen presenting cells.
The foregoing description of the preferred embodiment of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (7)
1. A method of preparing a targeted delivery vaccine comprising the steps of:
s1: peripheral blood is obtained and stored in an anticoagulation blood collection tube;
s2: separating erythrocytes from lymphocytes of the peripheral blood, and collecting the erythrocytes;
s3: washing the collected red blood cells with 0.01M PBS or 0.9% physiological saline, centrifuging for several times to obtain red blood cells after primary centrifugation;
s4: adding 55.6% -83.3% 1xPBS or 0.9% physiological saline into the centrifuged red blood cells for dilution, then adding vaccine components, and preserving at low temperature for 0.1-8 hours to obtain a mixed red blood cell and vaccine mixture;
s5: adjusting PBS concentration in the mixture of red blood cells and vaccine to 0.01M, and preserving at low temperature for 0.1-8 hours; or adjusting NaCl concentration in the mixture of erythrocyte and vaccine to 0.9%, and preserving at low temperature for 0.1-8 hr;
s6: centrifuging to collect red blood cells, and then adding CD47 antibody or CpG to incubate for 0.1-8 hours;
s7: washing with 0.01M PBS or 0.9% physiological saline and centrifuging for several times to obtain red blood cells after centrifuging again, wherein the red blood cells are red blood cell vaccines, namely targeted delivery vaccines;
the vaccine component is a component with immunoregulation function; the immunomodulating component comprises an antigen polypeptide sequence and/or an antigen mRNA sequence and/or an inactivated pathogenic microorganism and/or an attenuated pathogenic microorganism and/or a TLR receptor agonist.
2. The method of claim 1, wherein in step S2, the method of separating erythrocytes from lymphocytes is any one of the following: lymphocyte separation liquid separation method, hemodialysis separation method, and density gradient centrifugation method.
3. The method of claim 1, wherein in step S4, further comprising: the centrifuged red blood cells are subjected to electric shock in advance, the working voltage of the electric shock is 150V, and the electric shock is 15ms.
4. The method of claim 1, wherein the washing-centrifuging is performed twice in steps S3 and S7.
5. The method for preparing a targeted delivery vaccine according to claim 1, wherein the erythrocyte vaccine is injected by intravenous injection.
6. The method for preparing a targeted delivery vaccine according to claim 1, wherein the peripheral blood is autologous peripheral blood or allogeneic O-type blood.
7. An erythrocyte vaccine prepared by the method of preparing the targeted delivery vaccine of any one of claims 1-6.
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