An mRNA vaccine for preventing rift valley fever and a preparation method thereof

Abstract

The application discloses an mRNA vaccine for preventing rift valley fever and a preparation method thereof. The vaccine comprises liposome-wrapped mRNA-GnGc, and the mRNA-GnGc comprises a secretory signal peptide and a gene of a rift valley fever virus glycoprotein Gn+Gc sequence; the application can express the antigen of the rift valley fever virus by using one mRNA, has strong immunogenicity, and has good protection effect. The application has important significance for the research of the rift valley fever vaccine.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of vaccines, and particularly relates to an mRNA vaccine for preventing Rift Valley fever and a preparation method thereof. BACKGROUND

[0002] Rift Valley fever (RVF) is a zoonosis caused by Rift Valley fever virus (RVFV) that is transmitted by mosquitoes or contact, and poses a great threat to animal husbandry production and human health. The World Organization for Animal Health lists it as a notifiable disease. However, so far, there is no approved human vaccine for preventing RVF. Therefore, it is of great significance to research a safe and effective Rift Valley fever vaccine for responding to Rift Valley fever outbreaks.

[0003] RVFV has a wide host range and can infect a variety of animals, but mainly causes abortion, birth of malformed fetuses in ruminants, especially sheep, and death of newborn calves. After human infection, most people show self-limiting fever, but some infected people can develop severe symptoms such as hemorrhagic fever, meningoencephalitis, fulminant hepatitis, retinitis, and even blindness.

[0004] RVFV belongs to the family of Phenuroviridae and the genus of Phlebovirus, and is a segmented negative-strand RNA virus with a capsid. Its genome contains three segments, L (Large), M (Medium), and S (Small). The glycoproteins Gn and Gc encoded by the M segment form a heterodimer on the surface of the virion and play an important role in virus adsorption and invasion. They are the main regions where neutralizing epitopes exist and are the main target proteins for vaccine development.

[0005] Vaccination is the most economical and effective method for the prevention and control of RVF epidemic, and the current research on RVF vaccine mainly includes traditional RVFV inactivated vaccine, attenuated live vaccine, subunit vaccine, recombinant vector vaccine and the like, each of which has its own advantages and disadvantages. The earliest developed RVF vaccine is formalin inactivated vaccine, such as NDBR103 vaccine, TSI GSD 200 and the like, but most of them need to be immunized for multiple times to obtain durable immunity. Among attenuated live vaccines, the widely used ones are Smithburn vaccine, MP-12 and Clone 13 vaccine, although the attenuated vaccine has strong immunogenicity and can induce durable immunity of livestock after single immunization, but it has a certain risk of causing abortion and producing deformed fetus of pregnant livestock. In addition, some attenuated live vaccines also have the possibility of reverse virulence and genetic reassortment with wild strains. The subunit vaccine has high safety, but its immune effect still needs to be further explored. In order to overcome the problems existing in the above vaccines, many scholars turn their attention to viral live vector vaccine, and at present, a variety of viral vectors have been used for the research of RVF vaccine, including Newcastle disease virus, chimpanzee adenovirus (ChAdOx1), vaccinia virus ankara strain, human adenovirus type 5 and the like, but the pre-existing immunity of animals to the vector virus may affect the protective efficacy of the recombinant vector vaccine.

[0006] Among the above-mentioned RVF vaccines, only three vaccines for human use have entered clinical trials, which are TSI-GSD-200, MP-12 and ChAdOx1 RVF vaccine, and the three vaccines are relatively safe in human body, but TSI-GSD-200 needs to be immunized for 4 times to obtain durable immunity; MP-12 still has a certain risk of teratogenicity to animals; ChAdOx1 RVF has a low pre-existing immunity rate to the vector, and is safe in animal body, but its ability to induce immune response is significantly lower than that of Ad5 RVF vaccine.

[0007] The purpose of the present application is to provide a safe and efficient RVF vaccine to realize effective prevention and control of epidemic and effective response to RVF epidemic. SUMMARY

[0008] To achieve the above-mentioned object, the concept of the present application is to provide an mRNA expressing optimized immunogens of Rift Valley fever virus as a vaccine. Based on the above-mentioned concept, the present application compares and analyzes the ability of different antigen fragment mRNA vaccines to induce immune responses in mice, and screens out an immunogen combination with better immune effect, i.e., the antigen combination of Gn protein and Gc protein. Therefore, the present application first provides a polynucleotide molecule encoding glycoprotein Gn+Gc of Rift Valley fever virus, the sequence of which is shown in SEQ ID NO. 1. The polynucleotide sequence encoding glycoprotein Gn+Gc of Rift Valley fever virus is a sequence based on the wild sequence of G protein of Rift Valley fever virus vaccine strain MP12 (GenBank sequence number: DQ380208.1) Gn+Gc and codon optimized for mammalian cell expression.

[0009] Secondly, to obtain an mRNA vaccine (mRNA-GnGc) based on the antigen combination of Gn protein and Gc protein as an immunogen, the present application constructs a transcription template of the mRNA vaccine, i.e., a polynucleotide molecule containing the above-mentioned polynucleotide molecule encoding glycoprotein Gn+Gc of Rift Valley fever virus, further containing 5'UTR encoding DNA and signal peptide encoding DNA at the 5' end of the polynucleotide molecule, and further containing 3'UTR encoding DNA and poly(A) tail at the 3' end of the polynucleotide molecule.

[0010] In a preferred embodiment, the sequence of the 5'UTR encoding DNA is shown in SEQ ID NO. 9, the sequence of the signal peptide encoding DNA is shown in SEQ ID NO. 7, the sequence of the 3'UTR encoding DNA is shown in SEQ ID NO. 10, and the sequence of the poly(A) tail is shown in SEQ ID NO. 11.

[0011] Thirdly, the present application provides an mRNA obtained by transcription with the above-mentioned polynucleotide molecule as a template.

[0012] In a preferred embodiment, the mRNA further contains a cap structure at the 5' end.

[0013] More preferably, the mRNA has the following structure: A1-A2-A3-A4-A5-A6:

[0014] A1 is a 5' cap structure;

[0015] A2 is a 5' UTR element;

[0016] A3 is a signal peptide encoding sequence, preferably a tPA signal peptide encoding sequence;

[0017] A4 is a Rift Valley fever virus antigen coding sequence, wherein the antigen is selected from the group consisting of Gn and Gc proteins;

[0018] A5 is a 3' UTR element;

[0019] A6 is a poly(A) tail structure.

[0020] In one embodiment of the present application, the cap structure is Cap1.

[0021] More preferably, the mRNA contains modified nucleotides and / or unmodified nucleotides, wherein the modified nucleotides are mainly pseudo-UTP replacing all UTP in the sequence.

[0022] Fourthly, the present application provides a liposome nanoparticle encapsulating the above mRNA.

[0023] Finally, the present application provides a preparation method of the above liposome nanoparticle, which comprises the following steps:

[0024] (1) forming a lipid mixture of heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate (SM-102), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and mPEG-DMG-2K, and preparing a solution containing the above mRNA;

[0025] (2) mixing the lipid mixture obtained in step (1) with the solution containing the above mRNA.

[0026] In a preferred embodiment, heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate (SM-102), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and mPEG-DMG-2K are combined in a ratio of 50:10:38.5:1.5 in step (1).

[0027] In a preferred embodiment, the mass ratio of the lipid mixture to the mRNA in step (2) is 10:1~1:1.

[0028] The mRNA can be delivered into the body to produce a strong immune response, and has the characteristics of high safety, good immunogenicity and easy production.

[0029] This invention provides an mRNA-GnGc vaccine for the prevention of Rift Valley fever virus infection. Immunization of mice with this vaccine induces strong humoral and cellular immune responses. Immunization of rhesus monkeys with this vaccine also induces strong humoral and cellular immune responses. Infection of mice with a lethal dose of the virus, and observation of weight changes and survival, showed that vaccine-immunized mice did not experience significant weight changes and all survived, indicating that the vaccine provides good protection for mice. This invention is of significant importance for the research of vaccines against Rift Valley fever virus infection. Attached Figure Description

[0030] Figure 1 A schematic diagram for constructing an mRNA vaccine;

[0031] Figure 2 This diagram illustrates the results of linearized restriction enzyme digestion of the mRNA transcription template. Lane 1: Linearized mRNA-Gn (4878bp); Lane 2: mRNA-Gn plasmid (4878bp); Lane 3: Linearized mRNA-GnGc (6399bp); Lane 4: mRNA-GnGc plasmid (6399bp); Lane 5: Linearized mRNA-Gc (4788bp); Lane 6: mRNA-Gc plasmid (4788bp).

[0032] Figure 3 A graph showing the expression levels of binding antibodies induced by mRNA vaccines with different antigen fragments in mice;

[0033] Figure 4 A graph showing the expression levels of neutralizing antibodies induced in mice by mRNA vaccines with different antigen fragments;

[0034] Figure 5 To induce the secretion of IFN-γ, TNF-α, and IL-2 by mRNA vaccines with different antigen fragments in mice, CD8+ cells were tested. + A graph showing the number of T cells;

[0035] Figure 6 Figure showing the expression level of neutralizing antibodies induced by mRNA-GnGc in rhesus monkeys;

[0036] Figure 7 A graph showing the number of T cells that secrete IFN-γ, IL-2, and IL-4 induced by mRNA-GnGc in rhesus monkeys;

[0037] Figure 8 The graph shows the weight change curve of mice immunized with mRNA-GnGc after infection with RVFV.

[0038] Figure 9The survival curve of mice immunized with mRNA-GnGc after infection with RVFV is shown in the figure. DETAILED DESCRIPTION

[0039] In order to make the above-mentioned purposes, technical solutions and advantages of the present application more clear and obvious, the present application will be further described in detail below with reference to the drawings and specific embodiments.

[0040] Example 1. Construction of recombinant plasmid

[0041] Referring to the G protein sequence of the Rift Valley fever virus vaccine strain MP12 (GenBank sequence number: DQ380208.1), the Gn+Gc (SEQ:2) Gn (SEQ:4) and Gc (SEQ:6) protein sequences of RVFV were obtained, and the codon-optimized Gn+Gc nucleotide sequence (SEQ ID NO:1) (the GC content of the optimized sequence was increased from 46.2% to 59.1%, and the codon adaptability index was increased from 0.42 to 0.85) adapted to mammalian cell expression, the Gn nucleotide sequence (SEQ ID NO:3) and the Gc (SEQ ID NO:5) nucleotide sequence were obtained using biology software (General Biosystems Co., Ltd.). In order to obtain secretory expression, the tPA signal peptide sequence (amino acid sequence: SEQ ID NO:8, nucleotide sequence: SEQ ID NO:7) was added at the N-terminus of the mature protein. In order to construct the mRNA vaccine, the 5'UTR (nucleotide sequence: SEQ ID NO:9) was added at the N-terminus of the target antigen, and the 3'UTR (nucleotide sequence: SEQ ID NO:10) and poly A (nucleotide sequence: SEQ ID NO:11) were added at the C-terminus, and the EcoR I restriction enzyme cutting site (GAATTC) (see Figure 1 ). The designed sequence was synthesized by a gene synthesis company (General Biosystems Co., Ltd.) using conventional technical means in the art, and the target fragment was constructed into the pUC57 vector through enzyme digestion, ligation, transformation of competent cells, cloning and sequencing identification, and then the positive strain identified correctly was amplified and cultured, and the plasmid was extracted. The plasmid was linearized by using the restriction enzyme EcoR I at 37°C for 2h, and the template linearization was confirmed by nucleic acid gel electrophoresis (see Figure 2 ).

[0042] Example 2. Preparation of mRNA-Gn, mRNA-Gc and mRNA-GnGc

[0043] In vitro transcription was performed using T7 High Yield RNA Transcription Kit (N¹ -Me-Pseudo UTP) (Cat No: DD4202-01) reagents from Novagen.

[0044] In vitro transcription reaction system (20 μl): 1 μg linearized plasmid, 2 μl (100 mM) ATP, 2 μl (100 mM) GTP, 2 μl (100 mM) CTP, 2 μl (100 mM) N1-Methylpseudo-UTP, 2 μl 10 × Transcrption Buffer, 2 μl T7 RNA Polymerase Mix, add RNase-free ddH2O to make up the total reaction system to 20 μl.

[0045] In vitro transcription procedure: vortex mixing, centrifuge briefly to collect, incubate at 37°C in PCR instrument for 2 h, after the reaction is completed, add 1 μl (1 U / μl) DNase I to the system, incubate at 37°C for 15 min, for DNA template digestion.

[0046] Purification: use VAHTS RNA Clean Beads (N412-01) of Qiagen to purify, obtain purified RNA, concentration is 1500 ng / μl, total mass is 150 μg.

[0047] mRNA capping reaction uses Vaccinia Capping Enzyme (10 U / μl) (DD4109-01) and mRNA Cap-2'-O-Methyltransferase (DD4110-01) reagents of Qiagen.

[0048] mRNA capping procedure: take 10 μg RNA, dilute to 14 μl with RNase-free ddH2O, heat at 65°C for 5 min, then place on ice for 5 min; add 2 μl 10 × Capping Reaction buffer, 1 μl (10 mM) GTP, 1 μl (32 mM) SAM, 1 μl (10 U / μl) Vaccinia Capping Enzyme, 1 μl (50 U / μl) 2'-O-Methyltransferase, react at 37°C for 1 h. The Vaccinia Capping Enzyme can add the cap structure m7Gppp to the 5' end of the RNA to obtain Cap0 structure, and the 2'-O-Methyltransferase uses SAM as a methyl donor to add a methyl group to the 2'-O site of the first nucleotide adjacent to the cap structure (Cap0) at the 5' end of the RNA, forming mRNA with Cap1 structure.

[0049] Recovery: use VAHTS RNA Clean Beads of Qiagen for purification, obtain purified RNA, the purified sample band is single, concentration is 1000 ng / μl.

[0050] Example 3. Liposome nanoparticle encapsulated mRNA

[0051] SM-102 (Heptadecan-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl) amino)octanoate), Xiamen SinoBiologics Biotech Co., Ltd., Cat# 2089251-47-6), DSPC (1,2- Distearoyl-sn-glycero-3-phosphocholine, Avanti Polar Lipids, Cat# 816-94-4), Cholesterol (Avanti Polar Lipids, Cat# 57-88-5) and mPEG-DTA-2K (Methoxy polyethylene glycol ditetradecylacetamide, Xiamen SinoBiologics Biotech Co., Ltd., Cat# 1849616-42-7) were dispersed in absolute ethanol with the ratio of 50:10:38.5:1.5, and then shaken to mix well and stored at 4°C. Subsequently, the lipid mixture and mRNA dissolved in sodium acetate buffer were added to two inlets of the INano™ L nanoparticle preparation platform (Miannan (Shanghai) Instrument Technology Co., Ltd.) respectively, and the preparation of liposome nanoparticles was carried out at a fixed flow rate (0.1-60 ml / min) and ratio (10:1-1:1), in which the optimal ratio of aqueous phase to lipid phase was 3:1. The LNP-mRNA product was collected, and after ultrafiltration with a 30 kD ultrafiltration tube, it was dispersed in PBS and stored at 4°C for use.

[0052] Example 4. LNP-mRNA particle size and encapsulation rate detection

[0053] 4.1 The product was diluted 1:10 with PBS, and the nanoparticle size was characterized by Malvern nanoparticle size potential instrument. The average particle size was 76.86 nm, and the polydispersity index (PI) was 0.02.

[0054] 4.2 The encapsulated mRNA was detected by Quant-it RiboGreen RNA assay kit. The samples were diluted with TE and TE buffer containing 0.5% Triton X-100 respectively, and incubated at 37°C for 10 min.

[0055] 4.3 The above samples were added to a 96-well plate, and a known concentration of standard was added at the same time, followed by the addition of TE solution containing RiboGreen dye, and then reacted at room temperature for 3 min. Subsequently, the plate was placed in a multifunctional enzyme labeler, and the fluorescence value (excitation wavelength 480 nm, emission wavelength 520 nm) was selected for encapsulation rate detection. The encapsulation rate of the sample was about 92%.

[0056] Example 5. Evaluation of the efficacy of different antigen fragment vaccines in mice

[0057] 5.1 Immunization scheme design

[0058] Twenty-four 6-8 week old BALB / c female mice were randomly divided into 4 groups (6 mice per group), namely mRNA-Gn immunization group, mRNA-Gc immunization group, mRNA-GnGc immunization group and LNP negative control group. The method of intramuscular injection was used, and the immunization dose was 5 μg per mouse. The first immunization was performed on the first day of the experiment, and the booster immunization was performed on the 14th day of the experiment. Blood was collected at 2, 3 and 4 weeks after immunization, and serum was separated for binding antibody and neutralizing antibody determination. The mice were euthanized at 4 weeks after immunization, and the spleen was taken for cellular immune determination.

[0059] 5.2 Antigen-specific binding antibody titer determination

[0060] 5.2.1 The RVFV Gn and Gc proteins were diluted to 2 μg / ml with ELISA coating solution, and 100 μl was added to each well of a 96-well plate, and incubated at 4°C overnight.

[0061] 5.2.2 After washing 3 times with PBST, 100 μl of 2% BSA in PBS buffer was added to each well and incubated at 37°C for 1 h, and then washed 3 times with PBST.

[0062] 5.2.3 The serum samples were gradient diluted with diluent, 100 μl per well, and incubated at 37°C for 1 h, and then washed 3 times with PBST.

[0063] 5.2.4 100 μl of HRP-labeled goat anti-human IgG secondary antibody (1:10000 dilution) was added to each well, and incubated at 37°C for 1 h, and then washed 3 times with PBST.

[0064] 5.2.5 100 μl of TMB single-component color developing solution was added to each well, and color developed at room temperature for 5 min, then 50 μl of stop solution was added, and finally the OD450nm-OD630nm value was read with a microplate reader.

[0065] 5.2.6 ELISA results: Gn and Gc specific binding antibody detection results are shown in Figure 3 The upper graph is the Gn specific binding antibody detection result. The mRNA-Gn immunization group and the mRNA-GnGc immunization group can produce binding antibodies against Gn after 2 immunizations, and the binding antibody titer (10 4 or 10 2(Left and right). The following figure shows the detection results of Gc-specific binding antibodies. Both the mRNA-Gc immunization group and the mRNA-GnGc immunization group produced high levels of Gc-binding antibodies after two immunizations, and the titer of binding antibodies in the mRNA-Gc immunization group was significantly higher than that in the mRNA-GnGc immunization group, approximately 2.3-3 times higher. Results Summary: Mice immunized with mRNA-GnGc can produce specific IgG antibodies against both Gn and Gc, and the titer of binding antibodies against Gc (10... 4 The levels (around 10) are higher than those of antibodies against Gn (10). 2 The levels were around 100%, but all were lower than the binding antibody levels induced by mRNA vaccines containing a single antigen fragment. No antibodies were produced in the negative control group.

[0066] 5.3 Neutralizing antibody titer determination

[0067] 5.3.1 Serially diluted mouse serum from mice 4 weeks after inactivation was added to 100 TCID45. 50 The RVFV MP-12 virus (rMP-12-eGFP) rescued using reverse genetics was mixed and incubated at 37°C for 1 h, then added to Vero E6 cells and cultured at 37°C for 48 h.

[0068] 5.3.2 Cells were fixed with 4% paraformaldehyde and incubated at room temperature for 2 hours. They were then washed three times with PBS, stained with DAPI, and the levels of infected cells (eGFP) and total cells (DAPI) were quantified using a Celigo (Nexcelcom) imaging system. Infectivity was measured by assessing the accumulation of eGFP in Vero E6 cells. FRNT 50 Defined as the reciprocal of the serum dilution, which shows 50% inhibition of viral replication compared to the level of infection in the virus wells alone.

[0069] 5.3.3 Results are as follows Figure 4 The results showed that no neutralizing antibodies were detected in mice immunized with mRNA-Gn, while mice immunized with mRNA-Gc and mRNA-GnGc produced high titers of neutralizing antibodies, with titers reaching 6806 and 6904, respectively. No neutralizing antibodies were detected in the control group.

[0070] 5.4 Intracellular cytokine staining

[0071] To prepare a mouse spleen cell suspension, 2.4 × 10⁻⁶ cells were added to each well of a 24-well plate. 6The cells were then stimulated with Gn and Gc polypeptide library (2 pg / ml final concentration of each polypeptide), and GolgiStop was added to each well as a cytokine secretion blocker. A PMA stimulation group was set as a positive control, and a group without any stimulant was set as a negative control. The cells were incubated in a 37°C cell incubator for 6 h. Then the cells were collected by centrifugation, subjected to antibody staining, and then detected on a FACSCanton flow cytometer.

[0072] The results show that the three vaccine strains can induce a cellular immune response in mice, but mRNA-GnGc induces CD8 + T cells were significantly higher than those in the other groups, which were 2.03, 2.32, and 2.18 times those in the Gn group, and 1.97, 3.77, and 3.8 times those in the Gc group, respectively Figure 5 .

[0073] In summary, mRNA-GnGc can induce appropriate humoral immune response and significant cellular immune response. The mRNA-GnGc with better immune effect was selected as a candidate vaccine strain, and its immunogenicity was further verified in rhesus monkeys.

[0074] Example 6. Evaluation of the ability of mRNA-GnGc vaccine to induce immune response in rhesus monkeys

[0075] 6.1 Design of immunization scheme

[0076] Five 5-6-year-old rhesus monkeys were randomly divided into two groups, three of which were immunized with mRNA-GnGc vaccine, and two of which were negative controls. The mRNA-GnGc immunization dose was 100 pg per monkey, and the first immunization was performed on the first day of the test, and the booster immunization was performed on the 14th day of the test. Blood was collected before immunization and at 2, 3, and 4 weeks after immunization to isolate serum for neutralizing antibody determination. Anticoagulated blood was collected at 3 and 4 weeks after immunization to isolate peripheral blood mononuclear cells (PBMCs), and ELISPOT was used to detect the production of cytokines by cells.

[0077] 6.2 Determination of neutralizing antibody titer

[0078] 6.2.1 The inactivated rhesus monkey serum was gradiently diluted and mixed with 100 TCID 50 of RVFV MP-12 virus (rMP-12-eGFP) rescued by reverse genetic manipulation technology with a GFP tag, incubated at 37°C for 1 h, and then added to VeroE6 cells, which were incubated at 37°C for 48 h.

[0079] 6.2.2 Cells were fixed with 4% paraformaldehyde for 2h at room temperature, followed by 3 washes with PBS, and stained with DAPI. Infected cells (eGFP) and total cells (DAPI) were quantified using a Celigo (Nexcelcom) imager. Infectivity was measured by assessing the accumulation of eGFP in Vero E6 cells. FRNT50 was defined as the reciprocal of the serum dilution that showed 50% inhibition of viral replication compared to the level of infection in virus-only wells.

[0080] 6.2.3 Results are shown in Figure 6.2.3 Figure 6 It was shown that mRNA-GnGc boosted Rhesus monkeys to produce high titer of neutralizing antibodies, and the titer reached 10898 one week after boosting, while the control group did not produce neutralizing antibodies.

[0081] 6.3 Enzyme-linked immunospot assay (ELISPOT)

[0082] 6.3.1 Pre-coated 96-well plates (MabTech) with anti-monkey IFN-γ, anti-monkey IL-2 and anti-human IL-4 were washed 4 times with sterile PBS, 200 μl per well.

[0083] 6.3.2 200 μl of RPMI 1640 medium containing 10% FBS and double antibodies were added to each well, and incubated at room temperature for 2h.

[0084] 6.3.3 Peripheral blood mononuclear cells (PBMCs) were isolated.

[0085] 6.3.4 4 x 105 PBMCs were added to each well, and the full peptide library of RVFV Gn+Gc was added, with a final concentration of 2 μg / ml per polypeptide. Anti-CD3 monoclonal antibody was added as a positive control, and no stimulant was added as a negative control. The cells were incubated in a cell incubator for 18h. 5

[0086] 6.3.5 The cell suspension was discarded, and washed 5 times with PBS.

[0087] 6.3.6 The detection antibody was diluted to 1 μg / mL with PBS containing 0.5% FBS, and 100 μl was added to each well, and incubated at room temperature for 2h.

[0088] 6.3.7 The supernatant was discarded, and washed 5 times with PBS, 200 μl per well.

[0089] 6.3.8 Streptavidin-HRP was diluted with PBS containing 0.5% FBS, and 100 μl was added to each well, and incubated at room temperature for 1h.

[0090] 6.3.9 The supernatant was discarded, and washed 5 times with PBS, 200 μl per well.​

[0091] 6.3.10 Add 100 μΐ of substrate per well and monitor spot formation (about 10 min).

[0092] 6.3.11 Wash with deionized water to stop the reaction and let the plate dry at room temperature until completely dry

[0093] 6.3.12 Image using enzyme-linked spot imager and analyze.

[0094] 6.3.13 Results are shown in Figure 7 It is shown that mRNA-GnGc vaccine can significantly stimulate T cells to secrete IFN-γ, IL-2 and IL-4 compared with the control group, indicating that mRNA-GnGc can induce significant T cell response in rhesus monkeys in vivo.

[0095] The immunogenicity studies in mice and rhesus monkeys showed that mRNA-GnGc can induce good humoral and cellular immunity, and the experimental animals are safe. Further, a pharmacodynamic study was carried out on a lethal model of interferon receptor knockout mice (Shanghai South Model Organism Technology Co., Ltd.) to evaluate the protective efficacy.

[0096] Example 7. Analysis of the protective ability of mRNA-GnGc vaccine on mice

[0097] Ten 6-8 week old type I interferon receptor knockout mice were randomly divided into two groups (5 mice in each group), namely mRNA-GnGc immunization group and LNP negative control group. The method of intramuscular injection was used, the immunization dose of mRNA-GnGc was 5 μg per mouse, the first immunization was carried out on the first day of the experiment, and the booster immunization was carried out on the 14th day of the experiment. Fourteen days after the booster immunization, 2 x 105.0 TCID 4 TCID 50 of RVFV rMP-12 strain were injected intraperitoneally, and then the body weight changes and survival of the mice were observed daily for 14 days.

[0098] Results: The body weight changes are shown in Figure 8 , the survival is shown in Figure 9 , the body weight of the mice in the LNP negative control group decreased significantly after challenge, by about 17%-22%, and all died 3-4 days after challenge, while the body weight of the mice in the mRNA-GnGc vaccine immunization group did not change significantly and all survived during the entire observation period (14 days), with a survival rate of 100%.

[0099] From the above results, it can be seen that mRNA-GnGc immunized mice have a preventive protective effect on Rift Valley fever virus infection.

[0100] The above has carried out the detailed introduction to the embodiment of the application, the principle and implementation mode of the application have been described in this paper by applying specific examples, but the application is not limited to the specific details in the above implementation mode, within the technical concept range of the application, the technical scheme of the application can be variously simply modified, and these simple modifications all belong to the protection range of the application.

Claims

1. A nucleic acid molecule containing a polynucleotide molecule encoding the glycoprotein Gn+Gc of Rift Valley fever virus, characterized in that, The sequence of the polynucleotide molecule is shown in SEQ ID NO.

1. The 5' end of the polynucleotide molecule also contains a 5'UTR encoding DNA and a signal peptide encoding DNA, and the 3' end of the polynucleotide molecule also contains a 3'UTR encoding DNA and a poly(A) tail. The sequence of the 5'UTR encoding DNA is shown in SEQ ID NO.9, the sequence of the signal peptide encoding DNA is shown in SEQ ID NO.7, the sequence of the 3'UTR encoding DNA is shown in SEQ ID NO.10, and the sequence of the poly(A) tail is shown in SEQ ID NO.

11.

2. mRNA obtained by transcription using the nucleic acid molecule of claim 1 as a template.

3. The mRNA according to claim 2, characterized in that, The 5' end of the mRNA also contains a cap structure.

4. The mRNA according to claim 3, characterized in that, The hat structure is Cap1.

5. A liposome nanoparticle encapsulating the mRNA of any one of claims 2-4.

6. The method for preparing liposome nanoparticles according to claim 5, characterized in that, The method includes the following steps: (1) A lipid mixture is formed by heptadecano-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate, 1,2-distearate-sn-glycerol-3-phosphate choline, cholesterol and methoxy polyethylene glycol bistetradecyl acetamide, and a solution containing the mRNA of any one of claims 2-4 is prepared; (2) Mix the lipid mixture obtained in step (1) with a solution containing the mRNA of any one of claims 2-4.

7. The method according to claim 6, characterized in that, In step (1), heptadecano-9-yl-8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)amino)octanoate, 1,2-distearate-sn-glycerol-3-phosphocholine, cholesterol and methoxy polyethylene glycol bistetradecyl acetamide are combined in a ratio of 50:10:38.5:1.

5.

8. The method according to claim 7, characterized in that, In step (2), the mass ratio of lipid mixture to mRNA is 10:1 to 1:1.

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