An mRNA vaccine of pseudorabies virus and a preparation method thereof

Abstract

The application discloses an mRNA vaccine mechanism preparation method of pseudorabies virus and belongs to the technical field of biological immunity. The technical scheme is as follows: an mRNA vaccine of pseudorabies virus, wherein the antigen coding sequence of the vaccine is obtained according to the gE protein sequence of the pseudorabies virus. The mRNA vaccine is obtained by taking the gE protein of the pseudorabies virus, which is usually neglected in the preparation of vaccines in the prior art, as the design basis of the antigen. The mRNA vaccine obtained by referring to the design is tested, and the test result shows that the mRNA vaccine is relatively other pseudorabies.

Description

Technical Field

[0001] This application belongs to the field of bioimmunotechnology, and more specifically, relates to an mRNA vaccine for pseudorabies virus and a method for preparing the same. Background Technology

[0002] Pseudorabies is a highly contagious, septicemic, and deadly infectious disease caused by pseudorabies virus (PRV). Pigs of all ages are susceptible. In pregnant sows, PRV infection can lead to abortion, stillbirth, mummified fetuses, or weak piglets. In piglets, PRV infection can cause high fever, neurological symptoms, and respiratory distress; newborn piglets infected with PRV often exhibit neurological symptoms. In breeding pigs, PRV infection can lead to infertility, anestrus in sows causing breeding difficulties, and testicular swelling and atrophy in boars, resulting in loss of breeding ability.

[0003] Pseudorabies virus (PRV) is a double-stranded DNA virus with a genome length of approximately 150 kb and an average G+C content as high as 74%. PRV consists of a unique long segment (UL), a unique short segment (US), flanking terminal repeats (TR), and internal repeats (IR). Current research has found that gE is the major virulence gene of PRV, but it is not essential for viral replication.

[0004] Therefore, silencing the virulence gene gE can reduce the virulence of the virus without affecting immunogenicity. The resulting attenuated strain can be used as a candidate strain for vaccine to control and eradicate pseudorabies, which is also the design direction of the attenuated strain vaccines currently on the market.

[0005] Meanwhile, since gE is the main virulence gene of wild-type strains, and with the use of a large number of attenuated vaccines that silence the gE virulence gene, the gE virulence gene is considered an important marker for determining whether pigs are infected with wild-type strains. As a result, a variety of detection kits for the gE virulence gene have emerged.

[0006] mRNA vaccines are a third-generation vaccine technology that uses a delivery system to introduce mRNA encoding an antigen target into the body, where it is translated into antigen proteins to elicit a specific immune response. They are mainly divided into three types: non-replicating, self-amplifying, and trans-amplifying. They offer advantages such as short development cycles, simple production processes, and a dual immunization mechanism. The principle behind mRNA vaccines is to utilize viral gene sequences, rather than the virus itself, to generate antigen proteins through host cells, inducing humoral and T-cell immune responses. Furthermore, mRNA vaccines do not carry viral components and pose no risk of infection.

[0007] Chinese patent application 202311227274.X discloses the preparation and application of a pseudorabies virus circular RNA molecule, wherein the circular RNA contains coding elements encoding any one of the pseudorabies virus gD protein, gB protein, gC protein, and gK protein;

[0008] Further observation of the instructions for the above scheme reveals that: "The five glycoproteins (gB, gC, gD, gH, and gI proteins) that have been identified on the PRV viral particle envelope are usually selected as targets for subunit vaccine research." It is evident that the mainstream view in the existing technology does not include the design of using gE protein as a target.

[0009] Furthermore, there are precedents in the prior art for antigen design of gE protein of other viruses in the herpesvirus genus. For example, Chinese patent application 202411739924.3 discloses a varicella-zoster virus mRNA vaccine and its preparation method and application, the antigen of which is designed based on gD2, gE1 or gC1.

[0010] However, as mentioned earlier in this application, although PRV belongs to the herpesvirus genus, in the process of PRV virus prevention and control, gE, as the main virulence gene of PRV virus, appears more often as a silenced virulence gene in attenuated vaccines and as a marker of wild-type strain infection in detection kits, rather than as a precedent for using it to prepare mRNA vaccines.

[0011] Therefore, the technical problem to be solved in this application is: how to provide an mRNA vaccine that uses gE protein as the basis for antigen design, which is different from the existing technology. Summary of the Invention

[0012] The main objective of this application is to provide an mRNA vaccine that uses gE protein as the basis for antigen design, which differs from existing technologies.

[0013] To achieve the above objectives, this application provides an mRNA vaccine for pseudorabies virus, wherein the antigen coding sequence of the vaccine is designed based on the gE protein sequence of pseudorabies virus.

[0014] Furthermore, in the actual experimental process, this application also compared the neutralizing antibody level of mRNA vaccines prepared using antigens designed with gC and the viral load in brain tissue after challenge. According to the experimental results, the mRNA vaccines designed using gE protein as the antigen design basis have better neutralizing antibody titers and also have a good inhibitory effect on viral load after viral infection.

[0015] Preferably, the nucleotide sequence of the antigen encoding sequence of the vaccine is as shown in SEQ ID NO: 1.

[0016] Preferably, the mRNA vaccine is obtained by cloning the antigen coding sequence into a self-replicating vector or a non-replicating vector to prepare a synthetic plasmid, followed by in vitro transcription, capping, and LNP coating.

[0017] The self-replicating vector is pSFV;

[0018] The non-replicating vector is pVAX1.

[0019] Preferably, the self-replicating vector pSFV includes a T7 promoter sequence, a 5'UTR region, the coding regions of SFV non-structural proteins 1-4, a P2A region, a 3'UTR region, and a 3' terminal Poly A tail;

[0020] The non-replicating vector pVAX1 includes a T7 promoter sequence, a 5' UTR region, a 3' UTR region, and a 3' terminal Poly A tail.

[0021] Preferably, the LNP is obtained by mixing SM-102, DSPC, cholesterol and DMG-PEG2000 in anhydrous ethanol;

[0022] Furthermore, the molar ratio of SM-102, DSPC, cholesterol, and DMG-PEG2000 is 45–65:8–12:35–40:1–2.

[0023] In addition, this application also discloses a method for preparing the above-mentioned mRNA vaccine, comprising the following steps:

[0024] Step 1: Design the antigen coding sequence of the vaccine based on the gE protein sequence of pseudorabies virus;

[0025] Step 2: After cloning the antigen coding sequence into a self-replicating vector or a non-replicating vector to obtain a synthetic plasmid, it is then transcribed and capped in vitro to obtain mRNA;

[0026] Step 3: Coat the mRNA obtained in Step 2 with LNP to obtain the mRNA vaccine.

[0027] One of the above-mentioned technical solutions in this application has at least one of the following advantages or beneficial effects: This application selects the gE protein, the main virulence gene of pseudorabies virus, which is often overlooked in the prior art, as the basis for designing the mRNA antigen. It compares the neutralizing antibody level and viral load in brain tissue after challenge with the mRNA vaccine prepared using the antigen designed with gC. According to the experimental results, the mRNA vaccine obtained by using gE protein as the design basis for the antigen has a better neutralizing antibody titer and also has a good inhibitory effect on viral load after viral infection. Attached Figure Description

[0028] Figure 1 The image shows the agarose gel electrophoresis results of PCR gene amplification during the mRNA synthesis plasmids for expressing antigen proteins in pseudorabies virus self-replicating and non-replicating mRNA-gE vaccines.

[0029] Figure 2 Figure 1 shows the agarose gel electrophoresis results of the self-replicating and non-replicating mRNAs of pseudorabies virus antigen protein after in vitro transcription and capping.

[0030] Figure 3 The image shows the results of Western blotting of pseudorabies virus gE protein expressed by self-replicating and non-replicating mRNA-gE transfected with the antigen protein in cells.

[0031] Figure 4 The agarose gel electrophoresis verification results of PCR gene amplification during the mRNA synthesis plasmid for expressing antigen protein in non-replicating mRNA-gC vaccine.

[0032] Figure 5 The image shows the results of Western blotting of non-replicating mRNA-gC expressing pseudorabies virus gc protein.

[0033] Figure 6 A comparison of neutralizing antibody levels for gE-mRNA and gC-mRNA;

[0034] Figure 7 A comparison of the protective capabilities of gE-mRNA and gC-mRNA;

[0035] Figure 8 The level of pseudorabies virus gE protein-specific antibodies in serum samples;

[0036] Figure 9 The level of pseudorabies virus-specific neutralizing antibodies in serum samples;

[0037] Figure 10 Results of spleen lymphocyte typing after immunization;

[0038] Figure 11 Comparison of spleen lymphocyte typing in the PBS group, LNP-SFV-gE-mRNA group, and LNP-pVAX1-gE-mRNA group;

[0039] Figure 12 This describes the survival status of mice in different groups after pseudorabies virus infection. Detailed Implementation

[0040] The present application will be clearly and completely described below with reference to its embodiments. It should be noted that, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0041] Example 1: Construction of self-replicating and non-replicating mRNA synthesis plasmids expressing antigen proteins

[0042] The antigen-coding sequence of the mRNA vaccine in this application was designed based on the pseudorabies virus gE protein sequence (Gene ID: 80532578) in the NCBI protein database (https: / / www.ncbi.nlm.nih.gov / protein / ), as shown in SEQ ID NO: 1; in addition, the encoded antigen gene sequence is shown in SEQ ID NO: 2.

[0043] The antigen gene was synthesized and cloned into the pSFV self-replicating vector and the pVAX1 non-replicating vector. The pSFV self-replicating vector includes the T7 promoter sequence, 5'UTR region, coding regions of SFV non-structural proteins 1-4, P2A region, 3'UTR region, and 3' poly A tail. The pVAX1 non-replicating vector includes the T7 promoter sequence, 5'UTR region, 3'UTR region, and 3' poly A tail. Two Spe I restriction sites are present simultaneously before the T7 promoter and after the 3'UTR, allowing for effective removal of non-transcribed fragments using Spe I. The obtained pSFV-gE and pVAX1-gE plasmids were stored in *E. coli* DH5α competent cells. Before use, the strains were inoculated into LB liquid medium containing kanamycin resistance and cultured at 37°C in a shaker. The mRNA expressing the cloned antigen protein was extracted into the synthetic plasmid according to the plasmid DNA kit instructions, and the concentration was determined before storage and use.

[0044] like Figure 1 The image shown is an agarose gel electrophoresis image after amplification of the pseudorabies virus gE protein gene. A single band was obtained after PCR amplification, and the band size was as expected.

[0045] Example 2: Linearization of self-replicating and non-replicating mRNA synthesis plasmids expressing antigen proteins

[0046] The pseudorabies virus gE protein self-replicating and non-replicating mRNA synthesis plasmids pSFV-gE and pVAX1-gE have two Spe I restriction sites both before the T7 promoter and after the 3'UTR. The vector plasmids were linearized by restriction endonuclease Spe I. The linearization digestion reaction was performed according to the following steps:

[0047] The enzyme digestion reaction system was prepared according to the system shown in Table 1. After thoroughly mixing the plasmid pSFV-gE and pVAX1-gE reaction systems, the enzyme digestion reaction was carried out at 37℃ for 1 h. After the reaction was completed, 5 μL of the reaction solution from each group was taken for agarose gel electrophoresis to detect the enzyme digestion results.

[0048] Table 1. Single enzyme digestion system of plasmid pSFV-gE or pVAX1-gE

[0049] Reactive components Dosage plasmid pSFV-gE or pVAX1-gE 20 μg SpeI 15 μL 10×CutSmart 20 μL <![CDATA[RNase-free ddH2O]]> to 200 μL

[0050] Dilute the 200 μL digestion system from the previous step with an equal volume of RNase-free ddH2O. Add an equal volume of DNA extraction phenol reagent and chloroform in a 1:1 ratio to the digestion system. Mix well and centrifuge at 12000 rpm for 10 min. Transfer the supernatant to a new RNase-free centrifuge tube, add an equal volume of chloroform, invert to mix, and centrifuge at 12000 rpm for 3 min. Transfer the supernatant to a new centrifuge tube. Add 20 µL of 3 mol / L NaAc to the solution and mix thoroughly. Add 3 volumes of anhydrous ethanol, mix well, and incubate at -80℃ for 60 min to precipitate. After precipitation, centrifuge at 12000 rpm at 4℃ for 15 min and remove the supernatant. Add 1 mL of 75% ethanol (RNase-free), vortex, and centrifuge at 12000 rpm for 5 min. Discard the supernatant. Repeat the steps once. Centrifuge at 12000 rpm for 2 min to remove all ethanol from the centrifuge tube. Add 20 μL of RNase-free ddH2O to dissolve the precipitate at the bottom of the tube, and measure the DNA concentration using a NanoDrop 2000c for subsequent experiments.

[0051] Example 3: In vitro transcription capping to obtain self-replicating and non-replicating mRNAs expressing antigen proteins

[0052] The T7 promoter upstream of the target gene in the mRNA-synthesized plasmid was used to prepare and express the self-replicating and non-replicating antigen protein mRNAs of pseudorabies virus via in vitro transcription.

[0053] First, take 1-2 μg of linearized self-replicating or non-replicating mRNA plasmid and synthesize mRNA using T7 RNA polymerase at 37℃. The in vitro transcription reaction system is shown in Table 2.

[0054] Table 2 In vitro transcription reaction system

[0055] Reactive components Dosage Transcription Buffer 4 μL ATP (100mM) 4 μL UTP (100mM) 4 μL GTP (100mM) 4 μL CTP (100mM) 4 μL DNA transcription template 2 μg T7 RNA Polymerase Mix 4μL m7G 2μL <![CDATA[RNase-free ddH2O]]> to 40 μL

[0056] After mixing the system and briefly centrifuging, the mixture was placed at 37°C and reacted for 3 h. Subsequently, 1 μL LDNase I was added to the reaction system, and the mixture was digested at 37°C for 15 min to remove the DNA template.

[0057] To obtain fully transcribed mRNA, oligo(dT) fiber columns were used to purify the fully transcribed mRNA:

[0058] (1) Pretreatment of oligo(dT) fiber column: oligo(dT) cellulose was treated with DEPC and 0.1M NaOH in sequence, and then the column was washed with 1× loading buffer (20 mM Tris-HCl, 0.5 M NaCl, 1 mM EDTA and 0.1% SLS) until pH < 8.0.

[0059] (2) Sample loading: After dissolving the mRNA, place it in a 65℃ incubator for 5 min and then immediately cool it to room temperature. Add an equal volume of 2× loading buffer, mix well, and then add the mixture to an oligo(dT) fiber column for loading. Collect the eluent and determine the OD. 260 Close to 0.

[0060] (3) Elution: Elute the column with 2-3 column volumes of eluent (10 mM Tris-HCl, 1 mM EDTA and 0.05% SDS), collect the eluent and determine the OD. 260 The mRNA obtained by electrophoresis is the target mRNA.

[0061] like Figure 2 The image shows the agarose gel electrophoresis results of the self-replicating and non-replicating mRNA of the pseudorabies virus antigen protein after in vitro transcription and capping. After the in vitro transcription and capping reaction, a single band of mRNA was obtained, and the band size was as expected.

[0062] Example 4: Detection of antigen protein expression by self-replicating mRNA expressing antigen protein

[0063] 1) Resuscitation of HEK-293T cells

[0064] Take one vial of frozen HEK-293T cell line and place it in a 37°C water bath, gently shaking to thaw. Transfer the cell suspension to a centrifuge tube and slowly add 2 mL of 10% FBS High glu-DMEM medium, then centrifuge at 1000 rpm for 5 min at room temperature. Discard the supernatant and resuspend the cell pellet in 1 mL of complete culture medium. Transfer the cell suspension to a prepared T25 adherent culture flask, add 4 mL of complete culture medium, and then incubate at 37°C in a 5% CO2 cell culture incubator.

[0065] 2) Cell passage

[0066] Once the cell density reaches 90%, passage can begin. Discard the culture medium, add 1 mL of DPBS to the bottom of the flask, and gently shake to wash away any floating dead cells. Repeat this step once, then remove the DPBS. Add 1 mL of trypsin to the flask and incubate at 37°C for 1-2 minutes. After removing from the incubator, observe under a microscope. Once the cells become rounded, immediately add 1 mL of culture medium containing 10% fetal bovine serum and mix thoroughly to stop digestion. Transfer the mixed cell suspension to a 1.5 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes at room temperature. After centrifugation, discard the supernatant and resuspend the cells in 1 mL of complete cell culture medium. Add the cell suspension to the culture flask, then add 4 mL of complete cell culture medium. Shake the flask to mix thoroughly and incubate at 37°C in a 5% CO2 cell culture incubator.

[0067] 3) Plasmid transfection into HEK-293T cells

[0068] Resuspend the cells in 2 mL of 10% complete culture medium, add an appropriate amount of cell resuspension to each well of a 24-well plate, and then add 450 μL of complete cell culture medium to bring the cell density to approximately 4.5 × 10⁻⁶ cells per well. 4 Gently shake the culture plate to distribute the cells evenly. Incubate overnight at 37°C with 5% CO2. Observe the cells the next day. When the cell density reaches approximately 70-80%, transfect the cells using Lipofectamine 3000 transfection reagent and incubate at 37°C. Six hours after transfection, discard the culture medium and replace it with complete medium containing 3% FBS.

[0069] 4) Western blot detection of antigen protein secretion expression.

[0070] Forty-eight hours after cell transfection, cells and culture supernatant were collected and lysed using cell lysis buffer. After centrifugation, the supernatant was collected and loaded with sample buffer, followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, protein samples from the protein gel were electroblotted onto a PVDF membrane. The PVDF membrane was then blocked with 5% skim milk for at least 3 hours. Subsequently, the expression of the antigen protein was detected using a specific mouse polyclonal antibody against gE protein.

[0071] like Figure 3 The figure shown is a Western blotting result of the self-replicating and non-replicating mRNA expression of pseudorabies virus gE protein in cells transfected with antigen protein.

[0072] Example 5: Preparation of self-replicating and non-replicating mRNA vaccines coated with lipid nanoparticles

[0073] mRNA is a negatively charged biological macromolecule, making it difficult to cross negatively charged cell membranes via passive transport. Lipid nanoparticles (LNPs) can be used to deliver RNA, making them an effective drug delivery method for mRNA vaccines. The preparation of the self-replicating mRNA vaccine LNP-SFV-gE-mRNA and the non-replicating mRNA vaccine LNP-pVAX1-gE-mRNA using lipid nanoparticles was carried out according to the following steps:

[0074] SM-102, DSPC, cholesterol, and DMG-PEG2000 were dissolved in 100 μL of anhydrous ethanol at a molar ratio of 50:10:38.5:1.5, with a total mass of 480 μg. This ethanol solution was then mixed with 300 μL of 20 mM sodium acetate buffer containing 10 μg of the mRNA prepared in Example 3 using microfluidics to prepare nanoparticles. The prepared LNP-mRNA mixture was diluted to 15 mL with DPBS and transferred to a 30 kDa ultrafiltration tube. Ultrafiltration was performed at 4°C and 1000 g until the mRNA concentration in the mixture reached 1 μg / μL, yielding the final product: self-replicating LNP-SFV-gE-mRNA and non-replicating LNP-pVAX1-gE-mRNA.

[0075] Example 6: Preparation of gC-mRNA vaccine and comparison of the effects of gE-mRNA and gC-mRNA

[0076] The preparation of non-replicating gC-mRNA, wherein the preparation method of the synthetic plasmid (pVAX1-gC) is the same as in Example 1, except that the gE protein coding sequence is replaced by the comparative antigen coding sequence designed using the pseudorabies virus gC protein sequence (Gene ID: 80532556) and cloned into the pVAX1 non-replicating vector. The specific comparative antigen coding sequence is shown in SEQ ID NO: 3, and the gene sequence of the antigen it encodes is shown in SEQ ID NO: 4.

[0077] The subsequent preparation process of the mRNA vaccine follows the procedures outlined in Examples 2-5 of this application, yielding the gC-mRNA vaccine. The PCR amplification verification results of the synthesized plasmid and the WB test results of the vaccine are referenced in the preparation process. Figure 4-5 ;

[0078] Twenty 6-week-old female BALB / c mice were then randomly divided into four groups: a negative control group, a positive challenge control group, a non-replicating gE-mRNA vaccine group (pVAX1-gE group), and a non-replicating gC-mRNA vaccine group (pVAX1-gC group). BALB / c mice were injected with LNP-pVAX1-gE-mRNA or LNP-pVAX1-gC-mRNA vaccine intramuscularly or subcutaneously on days 0 and 14. On day 14 after the second immunization, blood was collected via the orbital route to collect serum samples. Subsequently, a challenge experiment was conducted, with 100*LD doses administered subcutaneously in the neck. 50 The pseudorabies virus was detected. Clinical symptoms and mortality in mice were observed and recorded daily after inoculation with the virus. The level of specific neutralizing antibodies against the pseudorabies virus in serum was determined by virus neutralization assay after immunization with the mRNA vaccine.

[0079] The results of the comparison of neutralizing antibodies can be referenced. Figure 6 It is evident that mRNA vaccines prepared using gE protein produce higher levels of neutralizing antibodies compared to mRNA vaccines prepared using traditional gC protein. Further observation is needed. Figure 7 It is evident that mice in the experimental group injected with gE-mRNA vaccine (non-replicating gE-mRNA vaccine group) had a higher survival rate;

[0080] In summary, the mRNA vaccine prepared by this application using the gE protein of pseudorabies virus, which is neglected by traditional techniques, has a higher immunoprotective effect than the mRNA vaccine prepared using the gC protein commonly used by traditional techniques. This result is beyond our expectations.

[0081] Example 7 Immunization experiment of pseudorabies virus self-replicating and non-replicating mRNA vaccines

[0082] 1) Proliferation of pseudorabies virus and TCID 50 Measurement

[0083] Resuscitate Vero cells and infect them with porcine pseudorabies virus after 2-3 passages when they are in good condition. Incubate at 37°C for about 24 hours until the cells become fully cytopathic. Collect the cells and freeze-thaw them three times. Centrifuge at 8000 rpm at 4°C and collect the supernatant to obtain the virus solution.

[0084] Healthy Vero cells were seeded into 96-well plates. When the cell density reached approximately 90%, the cells were inoculated with serially diluted 10-fold virus solution. After incubation for 1 hour, the virus solution was discarded, and the cells were washed 2-3 times with DPBS. Culture medium containing 2% serum was then added, and the cells were cultured further. The number of wells showing cytopathic effects (CPE) was observed and recorded daily, and TCID was calculated using the Reed-Muench method. 50 .

[0085] 2) Median lethal dose (LD50) of the virus 50 Measurement

[0086] The obtained viral fluid was serially diluted 10-fold. Thirty 10-week-old female BALB / c mice were randomly divided into 6 groups. Five groups were infected with different dilutions of pseudorabies virus via subcutaneous injection in the neck, while the remaining group served as a negative control. Clinical symptoms and mortality were observed and recorded daily after inoculation for 15 days. The LD50 was calculated using the Reed-Muench method. 50 .

[0087] 3) The immunogenicity assessment of self-replicating and non-replicating mRNA vaccines shall be conducted according to the following steps:

[0088] Twenty 6-week-old female BALB / c mice were randomly divided into four groups: negative control group, positive challenge control group, self-replicating mRNA vaccine group (SFV-gE), and non-replicating mRNA vaccine group (pVAX1-gE). BALB / c mice were injected intramuscularly or subcutaneously with LNP-SFV-gE-mRNA or LNP-pVAX1-gE-mRNA vaccines on days 0 and 14. On day 14 after the second immunization, serum samples were collected via the orbital route. Subsequently, challenge experiments were conducted by subcutaneous inoculation of 100*LD via the neck. 50 The pseudorabies virus was detected. Clinical symptoms and mortality in mice were observed and recorded daily after inoculation.

[0089] The level of specific antibodies against pseudorabies virus gE protein in serum after immunization with mRNA vaccine was determined by enzyme-linked immunosorbent assay (ELISA): Serum sample and HRP-labeled detection antibody were sequentially added to microwells pre-coated with pseudorabies virus gE protein, followed by incubation and thorough washing. The sample was then developed with TMB substrate, and the color was converted to a final yellow color by 1M sulfuric acid. The absorbance (OD value) was measured at 450 nm using an ELISA reader.

[0090] The level of specific neutralizing antibodies against pseudorabies virus in serum after immunization with mRNA vaccine was detected by virus neutralization: Healthy Vero cells were seeded into 96-well plates until the cell density reached approximately 90%. Immunized mouse serum was inactivated at 56°C for 30 min. The pseudorabies virus solution was diluted to 200*TCID. 50Inactivated mouse serum was serially diluted 2-fold and mixed 1:1 with diluted pseudorabies virus solution, then inoculated into cells. After 1 hour of incubation, the virus solution was discarded, cells were washed 2-3 times with DPBS, and cultured again with medium containing 2% serum. The number of cytopathic wells was observed and recorded daily, and the results were calculated using the Reed-Muench method.

[0091] like Figure 8 As shown, the level of specific antibodies against the pseudorabies virus gE protein in serum samples was determined. Animals immunized with both the self-replicating mRNA vaccine (LNP-SFV-gE-mRNA) and the non-replicating mRNA vaccine (LNP-pVAX1-gE-mRNA) produced different levels of specific antibodies. The neutralizing antibody level in the SFV-gE group was significantly higher than that in the pVAX1-gE group. The data indicate that both the self-replicating and non-replicating pseudorabies virus mRNA vaccines can effectively activate humoral immunity to produce specific antibodies against the gE protein.

[0092] like Figure 9 As shown, the levels of pseudorabies virus-specific neutralizing antibodies in serum samples were determined. Animals immunized with both the self-replicating mRNA vaccine (LNP-SFV-gE-mRNA) and the non-replicating mRNA vaccine (LNP-pVAX1-gE-mRNA) produced different levels of specific neutralizing antibodies. The neutralizing antibody levels in the SFV-gE group were significantly higher than those in the pVAX1-gE group. These data indicate that both self-replicating and non-replicating pseudorabies virus mRNA vaccines can effectively activate humoral immunity to produce specific neutralizing antibodies against porcine pseudorabies virus.

[0093] like Figure 10-11 As shown, the T lymphocyte typing of mouse spleen was determined. Immunization with both the self-replicating mRNA vaccine (LNP-SFV-gE-mRNA) and the non-replicating mRNA vaccine (LNP-pVAX1-gE-mRNA) effectively stimulated cellular immune responses, with the cellular immune response level of LNP-SFV-gE-mRNA being significantly higher than that of LNP-pVAX1-gE-mRNA.

[0094] like Figure 12 The figure shows the survival of mice in each group after pseudorabies virus infection. All mice in the negative control group survived healthily, while all mice in the positive control group died within 6 days. The survival rate of mice immunized with the self-replicating mRNA vaccine (LNP-SFV-gE-mRNA) was 80%, and the survival rate of mice immunized with the non-replicating mRNA vaccine (LNP-pVAX1-gE-mRNA) was 60%.

Claims

1. An mRNA vaccine for pseudorabies virus, characterized in that, The antigen coding sequence of the vaccine was designed based on the gE protein sequence of pseudorabies virus.

2. The pseudorabies virus mRNA vaccine according to claim 1, characterized in that, The nucleotide sequence of the antigen encoding sequence of the vaccine is shown in SEQ ID NO:

1.

3. The pseudorabies virus mRNA vaccine according to claim 1, characterized in that, The mRNA vaccine is obtained by cloning the antigen coding sequence into a self-replicating vector or a non-replicating vector to prepare a synthetic plasmid, followed by in vitro transcription, capping, and LNP coating. The self-replicating vector is pSFV; The non-replicating vector is pVAX1.

4. The pseudorabies virus mRNA vaccine according to claim 3, characterized in that, The self-replicating vector pSFV includes a T7 promoter sequence, a 5'UTR region, the coding regions of SFV non-structural proteins 1-4, a P2A region, a 3'UTR region, and a 3' terminal PolyA tail; The non-replicating vector pVAX1 includes a T7 promoter sequence, a 5' UTR region, a 3' UTR region, and a 3' terminal Poly A tail.

5. The pseudorabies virus mRNA vaccine according to claim 1, characterized in that, The LNP was obtained by mixing SM-102, DSPC, cholesterol, and DMG-PEG2000 in anhydrous ethanol; Furthermore, the molar ratio of SM-102, DSPC, cholesterol, and DMG-PEG2000 is 45–65:8–12:35–40:1–2.

6. A method for preparing the mRNA vaccine according to any one of claims 1-5, characterized in that, Includes the following steps: Step 1: Design the antigen coding sequence of the vaccine based on the gE protein sequence of pseudorabies virus; Step 2: After cloning the antigen coding sequence into a self-replicating vector or a non-replicating vector to obtain a synthetic plasmid, it is then transcribed and capped in vitro to obtain mRNA; Step 3: Coat the mRNA obtained in Step 2 with LNP to obtain an mRNA vaccine.

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