Method for enhancing immune response of mammals by using electroporation device
The application of electrical pulses to mammals through electroporation devices solves the problem of low antigen expression levels in traditional vaccine injection methods, achieving a more efficient immune response and smaller tissue damage.
Patent Information
- Application Number
- CN202411869766.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-18
- Publication Date
- 2025-04-11
AI Technical Summary
Traditional vaccine injection methods lead to a low expression level of antigen in the body, making it difficult to effectively stimulate the immune response of mammals.
Electroporation devices are used to apply electrical pulses to the subcutaneous tissue of mammals to enhance the efficiency of vaccine entry into cells, and to reduce tissue damage and bleeding through multiple injections and adjustment of electroporation device parameters.
It improves the efficiency of vaccine entry into cells, enhances the immune response, and provides a vaccine immunity method that is less harmful to the subject.
Smart Images
Figure CN119791889A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of electroporation device-assisted mammalian vaccine injection, and in particular to a method for enhancing the immune response of mammals by using electroporation device-assisted vaccination and booster immunization. Background Art
[0002] Mammalian skin tissue has different cell layers, which are divided into the epidermis, dermis and subcutaneous layer. The skin can not only act as a physical barrier to resist pathogens, but also play a protective role through immune function. The epidermis of the skin contains a large number of Langerhans cells, which are important antigen-presenting cells. The muscle-developed areas have rich blood circulation, which can enable the injected vaccine components to quickly enter the blood circulation and then be taken up by immune cells to initiate the body's immune response process. The method of vaccination is a key factor affecting the immune effect, especially nucleic acid vaccines. When nucleic acid vaccines are injected using traditional methods, the expression level of antigens in the body has always been low. Therefore, how to choose a suitable vaccine injection method is an important means to improve the immune effect. Summary of the invention
[0003] In view of the above technical problems in the related art, the present invention provides a method for enhancing the immune response of a mammal using an electroporation device, which can solve the above problems.
[0004] Electroporation instrument is a professional instrument and equipment based on electroporation technology to achieve the introduction of exogenous substances (such as DNA, RNA, protein, drugs, etc.) into cells. Electroporation technology applies a short high-intensity electric pulse to the target tissue to change the phospholipid bilayer structure of the cell and produce instantaneous, reversible micropores. Extracellular substances diffuse into the cell through this pore. At the same time, under the action of the external electric field, the charged molecules or ions produce electrophoresis and electroosmosis, thereby increasing the cell's uptake of exogenous substances. When the electric shock ends, the cell biomembrane will repair itself and restore its original barrier function. This process may cause slight tissue damage. However, this damage is not completely without benefit. It can induce cytokines and lymphocyte infiltration, induce antigen-presenting cells to the injection site, and thus enhance the immune response to a certain extent.
[0005] Electroporation can be used to deliver a variety of molecules: dyes, tracers, chemicals, antibodies, and nucleic acids. Electroporation has been used in different tissues and animal models to improve the efficiency of vaccine entry into cells and the immune response. Improving the electroporator or adjusting the parameters can increase the efficiency of vaccine transfer and reduce the amount of vaccine used while ensuring the production of target levels of antigen. However, there are still several problems with injecting vaccines using electroporators, including differential heat generation and cell death caused by the failure of the electroporator to reclose in time. In order to mitigate these side effects, we need to understand how electroporation works and why heat is generated. When a voltage (V) is applied to the electrodes implanted in the tissue, it generates an electric field ("E"), which is the applied voltage ("V") divided by the distance between the electrodes ("d"). E = V / d. The flow of ions opens the electroporator and allows the movement of molecules into the subject's cells during electroporation. The flow of charge in a medium between two points with a potential difference in a conductor is called current (I). The heat (W) generated by electroporation is related to the resistance (R) and the current and the action time (t), W = I2Rt. Electroporation does not occur below the threshold, while cells will be destroyed if it exceeds the upper limit. There are many parameters that affect its delivery effect, including: 1) the interval between injection and electroporation; 2) electroporation pulse voltage; 3) pulse time; 4) number of pulses; 5) pulse interval, etc. There are large differences between different tissues, such as skin thickness, fat content, number of blood vessels, etc. Therefore, it is necessary to adjust the parameters of the electroporator according to the species and transfer location, so as to obtain the best immune effect while avoiding skin and tissue damage and bleeding as much as possible.
[0006] To achieve the above technical objectives, the technical solution of the present invention is implemented as follows: A method for enhancing the immune response of a mammal using an electroporation device comprises administering a first push injection of a vaccine composition to a first tissue site of the mammal at a first time; then administering a second push injection of the vaccine composition to a second tissue site of the mammal at a second time; and then administering a third push injection of the vaccine composition to a third tissue site of the mammal at a third time.
[0007] Furthermore, an electroporation pulse is administered before, simultaneously with, or after the first bolus injection, the second bolus injection, and the third bolus injection.
[0008] Furthermore, the conditions of the electroporation pulses given before, simultaneously with or after the first bolus injection, the second bolus injection and the third bolus injection are: voltage 50V, pulse frequency 1Hz, pulse interval 50ms.
[0009] Furthermore, the second bolus injection is administered 7 days after the first bolus injection, and the third bolus injection is administered 7 days after the second bolus injection.
[0010] Furthermore, the mammal is an animal.
[0011] Furthermore, the vaccine composition comprises a polypeptide antigen.
[0012] Furthermore, the vaccine composition comprises a nucleic acid encoding an antigen, and the antigen is expressed by the nucleic acid in the mammal.
[0013] Furthermore, the first tissue includes subcutaneous tissue and muscle tissue of the limbs, shoulders and buttocks of a mammal.
[0014] Furthermore, the second tissue site includes subcutaneous tissue and muscle tissue of the limbs, shoulders and buttocks of the mammal excluding the first injection site.
[0015] Furthermore, the third site tissue includes subcutaneous tissue and muscle tissue of the limbs, shoulders, and buttocks of the mammal excluding the second injection site.
[0016] Beneficial effects of the invention: The present application provides a more efficient and stronger method for inducing an immune response, and is also a vaccine immunization method that causes less damage to the subject. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0018] The present invention is further described in detail below with reference to the accompanying drawings.
[0019] Figure 1 Schematic diagram of the proportion of CD8+T cells in the peripheral blood of mice described in the embodiment of the present invention; Figure 2 is a schematic diagram of the proportion of NKT cells in the peripheral blood of mice according to an embodiment of the present invention; Figure 3 It is a schematic diagram of the tumor growth curve of B16OVA cell line tumor-bearing mice treated with the nucleic acid vaccine described in the embodiment of the present invention (preventive model, vaccine was injected on days -21, -14, and -7, and tumor cells were inoculated on day 0). DETAILED DESCRIPTION
[0020] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present invention.
[0021] like Figure 1-3 As shown, according to the present invention, a method for enhancing the immune response of a mammal using an electroporation device is disclosed, comprising administering a first push injection of a vaccine composition to a first tissue site of the mammal at a first time; then administering a second push injection of the vaccine composition to a second tissue site of the mammal at a second time; and then administering a third push injection of the vaccine composition to a third tissue site of the mammal at a third time.
[0022] The present invention relates to a method for delivering a vaccine assisted by an electroporation device, wherein the vaccine can be directed against bacteria, viruses, cancer and other disease states, and can enhance the body's immunity and treat tumors by adjusting the working parameters of the electroporator and injecting the vaccine multiple times. On the one hand, it includes administering a vaccine composition to the skin and / or muscle. In the embodiment, the administration scheme of the vaccine composition is an initial push injection to a first tissue, and then a booster injection is injected every 7 days at a tissue different from the previous injection site, and two booster injections are injected continuously. The initial injection site can be muscle tissue, and the subsequent booster injections can be pushed into the skin tissue. On the other hand, it includes an auxiliary method of an electroporation device, which can adjust the parameters of the electroporation device to obtain the most suitable use conditions, so as to achieve the highest immune effect under the condition of minimal damage. The pulse voltage is increased to achieve the best immune effect under the condition of low pulse frequency and less damage. Another aspect includes electroporation-assisted administration of vaccines, wherein the vaccine can be attenuated or inactivated bacteria / viruses; it can be composed of recombinant proteins, polypeptides or peptides; it can also be a nucleic acid vaccine encoding one or more antigens.
[0023] Embodiment 1: Electroporation device assisted immunity and enhanced the ability of vaccines to promote the infiltration of specific CD8+T cells and innate immune cells into the tumor microenvironment. The specific method was as follows: 8-week-old C57BL / 6N female mice were adapted to the new environment for one week; after one week of adaptation, different doses of nucleic acid vaccine were injected into the left anterior tibia of the mice; electroporation was given or not after vaccine injection; the low-dose nucleic acid vaccine and no electroporation group were recorded as C1; the high-dose nucleic acid vaccine and no electroporation group were recorded as C2; the low-dose nucleic acid vaccine was electroporated, and the electroporation parameters were: 25 V voltage, 10 Hz frequency, and 50 ms interval, and the group was recorded as L1; the high-dose nucleic acid vaccine was electroporated, and the electroporation parameters were: 25 V voltage, 10 Hz frequency, and 50 ms interval, and the group was recorded as L2; the low-dose nucleic acid vaccine was electroporated, and the electroporation parameters were: 50 V voltage, 1 Hz frequency, and 50 ms interval, and the group was recorded as L2 ms, group H1; high dose of nucleic acid vaccine, electroporation, electroporation parameters: 50 V voltage, 1 Hz frequency, interval 50 ms, group H2; peripheral blood of mice was collected, red blood cell lysis buffer was added to lyse red blood cells; the prepared peripheral blood mononuclear cells were stained and analyzed by flow cytometry to detect CD8+T cells, NKT cells (such as Figure 1-2 as shown).
[0024] Then proceed as follows: digest the cells with trypsin to prepare a single-cell suspension and adjust the cell concentration to 2 × 10 5 / 100µl; the cell suspension was subcutaneously inoculated into mice, which was recorded as day 0; the nucleic acid vaccine was injected into the left anterior muscle, right anterior muscle and left posterior muscle of the tibia of mice on days -21, -14 and -7, respectively; electroporation was given or not after vaccine injection; the low-dose nucleic acid vaccine group without electroporation was recorded as C1; the high-dose nucleic acid vaccine group without electroporation was recorded as C2; the low-dose nucleic acid vaccine group with electroporation, the electroporation parameters were: 25 V voltage, 10 Hz frequency, 50 ms interval, the group was recorded as L1; the high-dose nucleic acid vaccine group with electroporation, the electroporation parameters were: 25 V voltage, 10 Hz frequency, 50 ms interval, the group was recorded as L2; the low-dose nucleic acid vaccine group with electroporation, the electroporation parameters were: 50 V voltage, 1 Hz frequency, 50 ms interval, the group was recorded as H1; the high-dose nucleic acid vaccine group with electroporation, the electroporation parameters were: 50 V voltage, 1 Hz frequency, 50 ms, group H2; record the subcutaneous tumor volume of mice (such as Figure 3 ).
[0025] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A method for enhancing the immune response of a mammal using an electroporation device, characterized in that: The method comprises administering a first push injection of the vaccine composition to a first tissue site of the mammal at a first time; then administering a second push injection of the vaccine composition to a second tissue site of the mammal at a second time; and then administering a third push injection of the vaccine composition to a third tissue site of the mammal at a third time.
2. A method for enhancing the immune response of a mammal using an electroporation device according to claim 1, characterized in that: The electroporation pulse is administered before, simultaneously with, or after the first bolus, the second bolus, and the third bolus.
3. A method for enhancing the immune response of a mammal using an electroporation device according to claim 2, characterized in that: The conditions of the electroporation pulses given before, simultaneously with or after the first bolus injection, the second bolus injection and the third bolus injection are: voltage 50V, pulse frequency 1Hz, pulse interval 50ms.
4. A method for enhancing the immune response of a mammal using an electroporation device according to claim 1, characterized in that: The second bolus injection is given 7 days after the first bolus injection, and the third bolus injection is given 7 days after the second bolus injection.
5. A method for enhancing the immune response of a mammal using an electroporation device according to claim 1, characterized in that: The mammal is an animal.
6. A method for enhancing the immune response of a mammal using an electroporation device according to claim 1, characterized in that: The vaccine composition comprises a polypeptide antigen.
7. A method for enhancing the immune response of a mammal using an electroporation device according to claim 1, characterized in that: The vaccine composition comprises a nucleic acid encoding an antigen, and the antigen is expressed by the nucleic acid in the mammal.
8. The method of claim 1, wherein the method comprises: The first tissue site includes subcutaneous tissue and muscle tissue of the limbs, shoulders and buttocks of a mammal.
9. A method for enhancing the immune response of a mammal using an electroporation device according to claim 1, characterized in that: The second site tissue includes subcutaneous tissue and muscle tissue of the limbs, shoulders and buttocks of the mammal excluding the first injection site.
10. The method of claim 1, wherein: The third site tissue includes subcutaneous tissue and muscle tissue of the limbs, shoulders and buttocks of the mammal excluding the second injection site.