A method for detecting mRNA-specific immune activation and its application

Abstract

The present invention discloses a method for detecting mRNA-specific immune activation, which comprises the following steps: preparing mRNA, including preparing a linearized template, in vitro transcription, capping, removing the linearized template, and purifying mRNA; preparing iDC cells, transfecting the iDC cells, and loading the mRNA onto the DC cells by electroporation or chemical transfection; inducing mDC from iDC, culturing, and plating for the Elispot assay; performing Elispot staining and detection. The present invention mainly designs a method for directly detecting mRNA-specific immune activation by Elispot. The method of the present invention can directly detect mRNA-specific immune activation, and has the advantages and characteristics of short detection time, high convenience, less consumption of experimental cells, and low detection cost, and can be well applied to detecting the effect of mRNA vaccine-specific immune activation.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology research, and specifically relates to a method for detecting mRNA-specific immune activation and its application. Background Art

[0002] mRNA vaccines introduce mRNA containing the encoded antigen protein into the human body, directly translate it to form the corresponding antigen protein, thereby inducing a specific immune response in the body and achieving the effect of preventive immunity. Due to the impact of the novel coronavirus, mRNA vaccines have received more extensive attention. Since mRNA was first successfully extracted in 1960, its value has played an important role in the fields of infectious disease vaccines and cancer treatment 60 years later. And the detection of mRNA-specific immune activation is an important aspect of testing the safety and effectiveness of vaccines. The specific immune activation analysis can specifically correspond to the detection and analysis of the immune mechanism of cellular immunity.

[0003] The mechanism of action of cellular immunity mainly includes two aspects, the direct killing effect of sensitized T cells and the synergistic killing effect of the cytokines they release. Therefore, the commonly used cellular immune analysis indicators for mRNA vaccines can also be roughly divided into two categories: T cell-related phenotypic indicators and related cytokine analysis. The immunological method for directly detecting the content of cytokines is the most commonly used detection method for evaluating the cellular immune response induced by vaccines.

[0004] Enzyme-linked Immunospot Assay (ELISPOT) combines cell culture technology and ELISA technology, and is a cellular immunological detection technique that can in-situ detect antibody-secreting cells or cytokine-secreting cells. It is most commonly used for the cellular immune evaluation of vaccines.

[0005] In many studies, the Elispot technique has been used to detect the number of antigen-specific cytotoxic T cells (CTLs) in the peripheral blood lymphocytes of immunized animals. Recently, the Elispot technique has been used to analyze and evaluate the detection of peptide-specific T lymphocytes from the PBMCs (peripheral blood mononuclear cells) of infectious disease patients and vaccine trials for inducing tumor-specific T cells in cancer patients. Regarding the detection of the immunogenicity of neoantigens in cancer patients, the Elispot detection method generally used in the field of T cell immune function detection reported in the literature is to synthesize tumor-specific full-sequence short peptides in vitro as a peptide library, load them onto antigen-presenting cells, dendritic cells (DCs), mix the peptide-stimulated dendritic cells with T lymphocytes and incubate them, and detect whether the antigen peptide is specifically immunologically activated. This method detects the specific immunological activation effect of the peptide, and there is a lack of a method for directly detecting the specific immunological activation effect of mRNA vaccines. Summary of the Invention

[0006] The object of the present invention is to provide a new method for detecting the specific immunological activation of mRNA, which can directly detect the specific immunological activation effect of mRNA vaccines.

[0007] The first aspect of the present invention provides a method for detecting the specific immunological activation of mRNA.

[0008] A method for detecting the specific immunological activation of mRNA includes the following steps:

[0009] S100. Prepare purified mRNA;

[0010] S200. Collect iDC cells;

[0011] S300. iDC transfection: Transfect the purified mRNA into iDC cells to obtain iDC cells loaded with and expressing the antigen mRNA;

[0012] S400: Induce and differentiate iDCs into mDCs for culture to obtain mDC cells loaded with and expressing the antigen mRNA;

[0013] S500. Seed the Elispot experiment: Mix the mDC cells loaded with and expressing the antigen mRNA with lymphocytes to obtain a cell suspension;

[0014] Add the Elispot special serum-free culture medium to the wells of the pre-coated Elispot plate for activation;

[0015] Add the cell suspension to the wells of the activated pre-coated plate.

[0016] S600: Perform staining and detection.

[0017] In some embodiments, preparing the mRNA comprises the following steps:

[0018] S101: Prepare linearized template;

[0019] S102: In vitro transcription;

[0020] S103: Capping;

[0021] S104: Remove linearized template;

[0022] S105: Purify mRNA.

[0023] In some embodiments, in the transfection of iDCs in step S300, the ratio of mRNA to iDC cells is: when the number of iDC cells is about 5×10 4 cells, the added amount of mRNA is 0.5 μg - 5 μg, preferably 0.8 μg - 4 μg, more preferably 1 μg - 2 μg.

[0024] In some embodiments, the iDC cells can be isolated and induced from human PBMCs, can be isolated from animals such as mice, and in one embodiment, are preferably isolated and induced from human PBMCs.

[0025] In some embodiments, the transfection of iDC cells is to load mRNA onto DC cells by electroporation or chemical transfection.

[0026] In some embodiments, by transient stable electroporation, the transfection conditions are: pulse voltage 200 - 1500 V, pulse duration 5 - 50 ms, and number of pulses 1 - 3 times.

[0027] In some embodiments, in the obtained iDC cells loaded with antigen-expressing mRNA, the mRNA is pseudouridine-modified.

[0028] The second object of the present invention is to provide the application of the above detection method in the detection of the specific immune activation effect of mRNA vaccines.

[0029] The present invention mainly designs a method for directly detecting mRNA-specific immune activation by using Elispot. The detection method and platform of the present invention load mRNA into antigen-presenting cells dendritic cells (DC cells), especially first transfecting iDC cells with mRNA, and then inducing the culture of mDC by iDC cells loaded with mRNA expressing antigens, translating to form antigens in the cytoplasm, so as to directly verify the specific immune activation effect of the tumor antigens expressed by mRNA. Further, the present invention can well activate specific T cells and obtain detection results related to antigenicity by optimizing the dosage ratio of mRNA transfected into iDC cells.

[0030] The method of the present invention can directly detect the specific immune activation effect of mRNA vaccines, and has the advantages and characteristics of short detection time, high convenience, less consumption of experimental cells, and low detection cost. Brief Description of the Drawings

[0031] Figure 1 It is an experimental diagram of the spots in the Elispot plate wells in Example 1 and Example 2 of the present invention.

[0032] Figure 2 is Figure 1 the statistical chart of the spot data.

[0033] Figure 3 It is an experimental diagram of the spots in the wells of the Elispot plate in Example 3 of the present invention.

[0034] Figure 4 It is an experimental diagram of the spots in the wells of the Elispot plate in Example 4 and Example 5 of the present invention.

[0035] Figure 5 It is the HCMV antigen expression plasmid constructed by the present invention.

[0036] Figure 6 It is the OVA antigen expression plasmid constructed by the present invention. Detailed Description of the Invention

[0037] For the experimental methods without specific conditions in the following examples of the present invention, they are usually in accordance with conventional conditions or in accordance with the conditions recommended by the manufacturer. All kinds of commonly used chemical reagents used in the examples are commercially available products.

[0038] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not used to limit the present invention.

[0039] The terms "comprise" and "have" and any variations thereof in the present invention are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product or device that includes a series of steps is not limited to the listed steps or components, but may optionally further include steps not listed, or may optionally further include other steps or components inherent to these processes, methods, products or devices.

[0040] The "plurality" mentioned in the present invention means two or more. "And / or" describes the relationship between associated objects and indicates that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. The character " / " generally indicates that the associated objects before and after are in an "or" relationship.

[0041] The antigen-encoding mRNA in the present invention is synthesized by in vitro transcription using a kit. First, a plasmid vector that can conveniently and rapidly load and stably express antigenic epitopes is established. Cytomegalovirus is a herpesvirus group DNA virus belonging to the β-herpesvirus subfamily, including human, murine, bovine, porcine, feline cytomegaloviruses, etc. Among them, the one causing human diseases is human cytomegalovirus (HCMV). HCMV is very widely infected in the population, CMV-positive samples are easily obtained, and the CMV peptide pool is also relatively mature in research. Therefore, the present invention uses CMV antigen as the specific antigen. In addition, OVA protein is also a commonly used simulated antigen in the laboratory. In the present invention, OVA antigen is used as an irrelevant antigen in the control immune evaluation for detailed illustration. In the mRNA antigen synthesis of the present invention, the antigen human cytomegalovirus protein (HCMV) and ovalbumin (OVA) are selected as the encoding antigens for experiments. They are respectively named CMV mRNA and OVA mRNA. According to conventional methods, different sites of mRNA can be modified, including capping modification at the 5' end, adding more than 100 poly-A at the 3' end, and using linker sequences and signal peptides to connect antigenic epitopes, so as to enhance the stability of the in vitro transcribed mRNA. The designed antigenic epitope sequence can be replaced with epitopes of different tumor antigens as needed, so it is applicable to the detection of the specific immune activation effect of different tumor antigen vaccines. In addition, studies have shown that modified nucleotides, such as using pseudo-UTP to replace conventional nucleotides in mRNA, can improve the stability and stress response of mRNA.

[0042] The antigen-encoding sequence of cytomegalovirus (HCMV) in the examples of the present invention is as follows:

[0043]

[0044] Among them, the italic part is 5’UTR&3’UTR, the underlined part is SP (signal peptide), the bold part is Linker, the italic and underlined part is the Antigen expression sequence, and the shaded part is MITD (MHC class I trafficking signal).

[0045] The antigen-encoding sequence corresponding to the OVA polypeptide in the examples of the present invention is as follows:

[0046]

[0047]

[0048] Among them, the italic part is 5’UTR&3’UTR, the underlined part is SP (signal peptide), the bold part is Linker, the italic and underlined part is the Antigen expression sequence, and the shaded part is MITD (MHC class I trafficking signal).

[0049] For the constructed plasmids, please refer to Figure 5 and Figure 6 .

[0050] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosed content of the present invention more thorough and comprehensive.

[0051]

[0052]

[0053]

[0054] Example 1

[0055] The method for detecting the specific immune activation effect of mRNA in this example includes the following steps:

[0056] S100: mRNA preparation

[0057] 1 Preparation of linearized template

[0058] 1.1 Plasmid linearization

[0059] Thaw and mix 10x Reaction buffer (cut smart) at room temperature, and place the mixed EcoRI-HF on an ice box.

[0060] Configure the enzyme digestion system, mix well, and react in a metal bath at 37°C for 1 hour.

[0061] Plasmid linearization enzyme digestion system:

[0062]

[0063]

[0064] 1.2 Linearized plasmid purification

[0065] 1.2.1 Add 200ul of the mixed AMPureXP beads to 200ul of the above enzyme digestion system, Vortex to mix, and incubate at room temperature for 10 minutes.

[0066] 1.2.2 Place the reaction tube on a magnetic rack, absorb the magnet for 5 minutes, and discard the supernatant.

[0067] 1.2.3 Add 1 ml of 70% ethanol to wash the beads, resuspend the beads using vortex and let stand for 20 seconds.

[0068] 1.2.4 Place the reaction tube on a magnetic rack, absorb for 5 minutes, discard the supernatant, and repeat the washing step once.

[0069] 1.2.5 Aspirate the 70% ethanol and dry the magnetic beads at room temperature for 10 minutes.

[0070] 1.2.6 Add 100ul RNase-free water for elution, incubate for 5 minutes, and repeat the elution once.

[0071] 1.2.7 After measuring the concentration by Nanodrop, the linearization efficiency was determined by agarose gel electrophoresis.

[0072] The purified linearized plasmid was used as the DNA template for in vitro transcription in the next step.

[0073] 2 In vitro transcription

[0074] Thaw NTPs and 10x Reaction buffer at room temperature and mix well. Place IVT Enzyme mix on ice.

[0075] Prepare the transcription system according to the table below, mix thoroughly, and react in a metal bath at 37°C for 3 hours.

[0076] Transcription system:

[0077]

[0078]

[0079] The reaction product after transcription is used as RNA solution for the capping step.

[0080] 3' Capping

[0081] 3.1 Thaw and mix the 10x Capping buffer and GTP at room temperature. Thaw and mix the SAM in the Custom RNA Kit on ice. Place the Capping Enzyme and 2'-O-Methyltransferase on an ice box.

[0082] 3.2 Prepare the RNA solution: Add 101 ul of RNase-free water to 99 ul of the transcription completion system.

[0083] Prepare the capping system according to the following table, mix well, and react in a metal bath at 37 °C for 1 hour.

[0084] Capping system:

[0085]

[0086]

[0087] 4 Removal of linearized template

[0088] Place DNase I on an ice box.

[0089] Prepare the plasmid template removal system according to the following table. After mixing well, react in a metal bath at 37 °C for 0.5 hour.

[0090] Plasmid template removal system:

[0091] Name Quantity Capped RNA 400ul DNase I, RNase-free 5ul

[0092] 5 mRNA purification

[0093] 1.5.1 Vortex the MyOne COOH Dynabeads (concentration 10 mg / ml). Prepare 100 ul of magnetic beads for every 1 mg of RNA. For the above in vitro transcription (IVT) reaction, 0.6 mg of Dynabeads can be prepared, i.e., 60 ul, and pipette it into a centrifuge tube.

[0094] 1.5.2 Place the centrifuge tube on a magnetic stand, magnetize for 30 seconds - 1 minute, and discard the supernatant.

[0095] 1.5.3 Mix the RNA to be purified with the magnetic beads, then add 2 volumes of RNA Binding Buffer, pipette to mix evenly, and place it on a metal bath at 1000 rpm and mix at room temperature for 10 minutes.

[0096] 1.5.4 Place the centrifuge tube on the magnetic stand, magnetize for 2 - 3 minutes, discard the supernatant. Add 1 ml of 70% ethanol, and resuspend the magnetic beads by vortexing. Place it on the magnetic stand, magnetize for 30 seconds - 1 minute, discard the supernatant, and repeat the washing once.

[0097] 1.5.5 Remove the residual ethanol, dry at room temperature on the magnetic stand for 5 - 10 minutes, avoiding the magnetic beads from being too dry.

[0098] 1.5.6 Remove the centrifuge tube from the magnetic stand, add 100 ul of RNase-free water, resuspend the magnetic beads by vortexing for 20 seconds, and incubate at 65 °C at 1000 rpm for 5 minutes.

[0099] 1.5.7 Place the centrifuge tube on the magnetic stand, magnetize for 30 seconds - 1 minute.

[0100] 1.5.8 Collect the supernatant into a new centrifuge tube. Measure the purity and concentration using Nanodrop, then detect the concentration using the Qubit4 kit, calculate the total yield of purified RNA, and identify it by gel electrophoresis (Urea-TBE gel).

[0101] S200: DC Separation Induction

[0102] 2.1 On the first day: Resuscitate the CMV-positive PBMCs purchased from Miaoshun Biology, seed them at 1 - 2x10 7 cells / well in a 6-well cell culture plate. Resuspend the cell culture medium: AIM-V; volume: 2 mL / well. Place it in a 37 °C cell culture incubator for 2 h;

[0103] 2.2 After 2 h, collect the suspended lymphocytes in the culture plate into a 15 mL centrifuge tube, wash the cells twice with 1 mL of 1x PBS, and collect the washing solution into the above centrifuge tube. Centrifuge at 320 g at room temperature for 7 min to collect the suspended lymphocytes and store them frozen for later use;

[0104] 2.3 The adherent cells after 2 h: Supplement one well in the 6-well culture plate with 2 mL of iDC culture medium. iDC culture medium: AIM-V containing 500 U / mL of IL-4 and 800 U / mL of GM-CSF, place it in a 37 °C cell culture incubator for culture. Culture until the 7th day, and change half of the iDc Medium (culture medium) during this period.

[0105] 2.4 On the seventh day: Harvest the iDC cells (immature dendritic cells), collect the culture supernatant in the 6-well cell culture plate into a 15 mL centrifuge tube, wash the cells twice with 1X PBS, collect the washing solution into the above centrifuge tube, and then use 1 mL Use the Cell Detachment Solution to digest the cells for 2 - 3 minutes. Pipette the digested cells and observe under an inverted fluorescence microscope to confirm that the cells are completely detached. Collect the cell mixture into the above centrifuge tube, and wash the cells twice with the same 1X PBS. Collect the washing solution into the above centrifuge tube. Centrifuge at 320g for 7 minutes at room temperature to collect the iDC cells.

[0106] S300: iDC Transfection

[0107] 3.1 Cell culture medium: iDC culture medium (AIM-V containing 500 U / mL IL-4 and 800 U / mL GM-CSF); Volume: 300 uL / well. Prepare in advance and preheat in a 37°C cell culture incubator.

[0108] 3.2 The collected iDC cells are washed with PBS (without Ca 2+ and Mg 2+ ), and centrifuged at 400g for 5 minutes at room temperature.

[0109] 3.3 According to the experimental groups (mainly including blank control; experimental group), resuspend the iDC cells with 10 ul of R Buffer. The obtained DC cell suspension (the number of cells is 5×10 4 - 5×10 5 cells) is mixed with the grouped mRNA (mass is 0 - 5 μg). In the present invention, preferably, for 5×10 4 - 5×10 5 DC cells, the mass of mRNA added is 0.5 μg - 5 μg (preferably 0.5 μg - 2 μg). In this example, the number of cells in the DC cell suspension is approximately 5×10 4 cells, and the added amount of mRNA is 1 μg.

[0110] 3.4 Apply the Neon transfection system for transient stable electroporation (pulse voltage 200 - 1500 V (preferably 800 - 1200 V in this example), pulse duration 5 - 50 ms (preferably 5 - 10 ms in this example), pulse number 1 - 3 times). All operations are carried out under sterile conditions; after transient stable electroporation, seed the electroporated cells into a preheated cell plate. Incubate in a carbon dioxide incubator at 37°C and 5% CO 2 for 24 h to obtain iDC cells loaded with and expressing antigen - encoding mRNA.

[0111] S400: iDC Induction of mDC (Mature DC Cells)

[0112] After culturing the iDCs transfected by instantaneous stable electrotransfection for 24 h, GM-CSF at a concentration of 1600 U / ml, IL-4 at 1000 U / ml, TNF-α at 10 ng / ml, IL-1β at 10 ng / ml, IL-6 at 320 ng / ml, and PGE-2 at 2 μg / ml were added to the cell culture medium, and the cells were cultured in a carbon dioxide incubator at 37 °C and 5% CO 2 for 24 h to obtain mDC cells loaded with mRNA expressing the antigen.

[0113] S500: Seeding the plate for the Elispot assay

[0114] 5.1 Thaw the suspended lymphocytes in advance: Thaw the suspended lymphocytes cryopreserved in step 2.2 18 - 24 h in advance, culture them in a low-adhesion 24-well culture plate, resuspend the cell culture medium: AIM-V + 10% hAB, volume: 2 mL / well, and place them in a 37 °C cell culture incubator overnight.

[0115] 5.2 Prepare the cell suspension: Mix the mDc cells loaded with mRNA expressing different antigens and the suspended lymphocytes thawed in advance with a micropipette, aspirate 20 μl of the cell mixture, dilute it 2-fold with trypan blue for cell counting and record the cell viability.

[0116] 5.3 According to the experimental design groups (mainly including experimental groups, positive controls, and blank controls), set three replicates for each group. According to E:T = 10:1; E = suspended lymphocytes, T = mDc cells loaded with antigen = 2X10 4 . Calculate the required number of cells and the corresponding volume of cell suspension. Aspirate the required amount of cells into 1.5 ml centrifuge tubes with different labels.

[0117]

[0118]

[0119] 5.4 Place the centrifuge tubes in a centrifuge and centrifuge at 320 g at room temperature for 7 min to collect the cells. Resuspend the cells with the serum-free medium specific for Elispot according to a final volume of 200 μl / well to prepare the cell suspension.

[0120] 5.5 Activation of the pre-coated plate: Add the serum-free medium specific for Elispot to the wells of the pre-coated plate of the Elispot kit to be activated, 200 μl per well; after standing at room temperature for 10 min, pour out the medium.

[0121] 5.6 Add the cell suspension: According to a final volume of 200 μl / well, add the cell suspension prepared in step 5.1.4 to the corresponding wells of the activated pre-coated plate.

[0122] 5.7 Addition of stimulants: Specific positive control group, add CMV polypeptide at a concentration of 1 μg / mL to the corresponding wells of the pre-coated plate after activation. Positive control group: add PHA at a concentration of 2.5 μg / mL to the corresponding wells of the pre-coated plate after activation. Incubate in a carbon dioxide incubator at 37°C and 5% CO 2 for 24 h.

[0123] S600: Elispot staining

[0124] 6.1 Cell lysis: Pour out the cells and culture medium in the wells, add ice-cold deionized water, 200 μL / well, and incubate at 4°C for 10 min;

[0125] 6.2 Plate washing: Pour out the liquid in the wells, add 1x Washing buffer, 200 μL / well, wash 5 - 7 times, and let it stand for 30 - 60 seconds each time. For the last time, blot dry on absorbent paper;

[0126] 6.3 Incubation with detection antibody: Dilute the biotinylated antibody with Dilution buffer R (1×) (1:100) to prepare the working solution, and then add it to each experimental well, 100 μL / well. Incubate at 37°C for 1 hr;

[0127] 6.4 Plate washing: Pour out the liquid in the wells, add 1x Washing buffer, 200 μL / well, wash 5 times, and let it stand for 30 - 60 seconds each time. For the last time, blot dry on absorbent paper;

[0128] 6.5 Incubation with streptavidin-HRP: Dilute streptavidin-HRP with Dilution buffer R (1×) (1:100) to prepare the working solution, and then add it to each experimental well, 100 μL / well. Incubate at 37°C for 1 hr;

[0129] 6.6 Plate washing: Pour out the liquid in the wells, add 1x Washing buffer, 200 μL / well, wash 5 times, and let it stand for 30 - 60 seconds each time. For the last time, blot dry on absorbent paper;

[0130] 6.7 Color development: Add freshly prepared AEC color development solution to each experimental well, 100 μL / well. Incubate at room temperature in the dark for 15 - 45 min (it is more appropriate to develop color at 37°C for 30 min);

[0131]

[0132] 6.8 Terminate color development: Pour out the liquid in the well, uncover the base of the plate, wash the front, back, and the base 3 - 5 times with deionized water / tap water to terminate color development. Place the plate in a cool place at room temperature and wait for it to dry naturally, then close the base; subsequently, perform spot counting on the ELISPOT plate.

[0133] Example 2:

[0134] The operation steps are the same as those in Example 1, except that the mRNA is pseudouridine - modified CMV mRNA. The pseudouridine modification in this example replaces the uridine in the CDS (coding region) with pseudouridine.

[0135] Blank control example

[0136] The purpose of this example is to exclude the influence of blank mDC and suspended lymphocytes.

[0137] Among them, a total of 3 blank control examples are designed for the centrifuge tubes in step 5.1.3, which are: adding only suspended lymphocytes (T - cells), adding only blank mDC without loaded mRNA, and adding only suspended lymphocytes + blank mDC without loaded mRNA.

[0138] Specific positive control example 1

[0139] Other steps in this example are the same as those in blank control example 1, except that the substances added to the centrifuge tube in step 5.3 are blank mDc + suspended lymphocytes + 1 μg / mL (CMV polypeptide). The purpose is to prove the detection efficacy of the detection method of the present invention.

[0140] Non - specific positive control example 1

[0141] Other steps in this example are the same as those in blank control example 1, except that the substances added to the centrifuge tube in step 5.3 are blank mDc + suspended lymphocytes + 2.5 μg / mL PHA. The purpose is to prove the reliability of the whole system.

[0142] Non - specific positive control example 2

[0143] Other steps in this example are the same as those in Example 1, except that the antigen expressed by the mRNA loaded on iDC is OVA. The purpose is to prove that the T cells activated by the mRNA of the present invention are specific rather than non - specific.

[0144] Non - specific positive control example 3

[0145] Other steps in this example are the same as those in Example 1, except that the antigen expressed by the mRNA loaded on iDC is OVA and the mRNA is pseudouridine - modified. The purpose is to prove that the T cells activated by the mRNA of the present invention are specific rather than non - specific.

[0146] As can be seen from Figure 1 , there are three replicas in each group. The first 3 columns are blank control examples. There are almost no spots in the Elispot plates of the first 3 columns, indicating that suspended lymphocytes and mDC cells without loaded mRNA hardly activate specific T cells. The 7th column is the spot pattern of the first specific positive control example, and its additive is blank mDc + suspended lymphocytes + 1 μg / mL (CMV peptide). As can be seen from the spot pattern of the 7th column, the number of specific T cells activated by CMV polypeptide is very large. The 8th column is the spot pattern of the first non-specific positive control example. As can be seen from the spots in the 8th column, the experimental system designed in the present invention is reliable. The 4th column is the spot pattern of the first embodiment of the present invention, and its additive is suspended lymphocytes and mDC loaded with CMV mRNA. As can be seen from the spot pattern of the 4th column, the mDC cells loaded with CMV-mRNA in the present invention can activate specific T cells. The 5th column is the spot pattern of the second embodiment, further verifying that the present invention can detect the specific immune activation effect of mRNA. The 6th column is the spot pattern of the first non-specific control example. The spots in the 6th column are very few and almost equivalent to those of the blank control example, indicating that the mRNA in the present invention activates specific T cells.

[0147] The transfection of both the first and second embodiments was carried out on iDCs.

[0148] Figure 2 The spot statistical chart also further illustrates the above conclusion.

[0149] Example 3

[0150] Other steps are the same as those in the first embodiment, except that in S300: before transfecting iDc with mRNA, according to the steps of S400: inducing iDC to mDC, iDC is induced into mDC and then transfected with mRNA.

[0151] Figure 3 is the Elispot map of Example 3. Figure 3 The first and second columns of Figure 3 are blank control examples; the 3rd column is the OVA non-specific control example; the 4th column is the spot pattern of Example 3; the 5th column is the CMV specific positive control example, and the 6th column is the PHA non-specific positive control example. Through Figure 1 it can be seen that the 4th column is almost the same as the blank control example and there are almost no spots. There is a very large difference from the spot pattern results of

[0152] Example 4

[0153] Other steps are the same as those in Example 1, except that in S300: the ratio between the number of iDC cells and the mass of mRNA is different in the iDc transfection step. In this example, and in step 3.3, the number of cells in the DC cell suspension is approximately 2x10 5 cells, and the amount of mRNA added is 21 μg).

[0154] Example 5

[0155] Other steps are the same as those in Example 4, except that in this example, in step 3.3, the number of cells in the DC cell suspension is approximately 2x10 5 cells, and the amount of mRNA added is 0.1 μg.

[0156] For the experimental results of Example 4 and Example 5, please refer to Figure 4 . Figure 4 Columns 1 to 3 in are blank control examples; column 4 is the dot blot of Example 4, column 5 is the dot blot of Example 5, column 6 is the dot blot of the OVA non-specific control example (mRNA dosage is 10 μg); column 7 is the CMV-specific positive control example, and column 8 is the PHA non-specific positive control example. It can be seen from Figure 4 that Figure 4 there are almost no dots in columns 4 to 5 of, indicating that specific T cells are not activated. Further comparison with Figure 1 shows that when the number of cells in the DC cell suspension is approximately 2x10 5 cells, it is not advisable to add 21 μg or 0.1 μg of mRNA.

[0157] Example 6

[0158] Other steps are the same as those in Example 4, except that in this example, in step 3.3, the number of cells in the DC cell suspension is approximately 5x10 5 cells, and the amount of mRNA added is 4.5 μg. It is found through experiments that there are almost no dots on the dot blot of Example 6. It can be seen that the amount of mRNA added is very crucial and directly affects the detection results.

[0159] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art can also make various equivalent variations or substitutions without departing from the spirit of the present invention, and these equivalent variations or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A method for detecting mRNA-specific immune activation, characterized in that, it comprises the following steps: S100. Obtain purified mRNA; S200. Collect iDC cells; S300. iDC transfection: Transfect the purified mRNA into iDC cells to obtain iDC cells loaded with mRNA expressing the antigen; the ratio of the mRNA to the iDC cells is: 1 μg : 5×10 4 ; S400. Induce and differentiate iDC into mDC for culture to obtain mDC cells loaded with mRNA expressing an antigen; S500. Seed the Elispot experiment: Mix the mDC cells loaded with mRNA expressing an antigen with lymphocytes to obtain a cell suspension; Add serum-free medium to the wells of the pre-coated plate and activate; Add the cell suspension to the wells of the activated pre-coated plate; S600: Perform staining and detection.

2. The method according to claim 1, characterized in that, the obtaining of the purified mRNA comprises the following steps: S101: Preparation of a linearized template; S102: In vitro transcription; S103: Capping; S104: Removal of the linearized template; S105: Purification of mRNA.

3. The method according to any one of claims 1-2, characterized in that, the iDC cells are isolated and induced from human PBMC.

4. The method according to any one of claims 1-2, characterized in that, the iDC cells are transfected by electroporation or chemical transfection to load mRNA onto the iDC cells.

5. The method according to claim 4, characterized in that, by transient stable electroporation, the transfection conditions are: pulse voltage 200-1500V, pulse duration 5-50ms, and pulse number 1-3 times.

6. The method according to any one of claims 1-2, characterized in that, in the obtained iDC cells loaded with mRNA expressing an antigen, the mRNA is pseudouridine-modified.

7. Use of the method for detecting mRNA-specific immune activation according to any one of claims 1-6 in detecting the specific immune activation effect of an mRNA vaccine.

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