Porcine epidemic diarrhea mRNA vaccine and preparation method thereof

Abstract

The application relates to the technical field of bioengineering, and discloses a porcine epidemic diarrhea mRNA vaccine and a preparation method thereof, which comprises an mRNA sequence encoding the amino acid sequence of S protein of GIIa, GIIb and GIIc type porcine epidemic diarrhea virus with mutations, the mutated mRNA sequence has higher expression and better immunostimulatory effect, the expression is increased by 10 times, and excellent immunization effect is achieved in mice and piglets; the trivalent combined vaccine has broad-spectrum immunization effect and can reduce diarrhea of piglets, and the immunization effect is far superior to that of commercial vaccines tested in experiments. The mutated mRNA vaccine has outstanding progress and is suitable for popularization and application.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bioengineering, and particularly relates to a porcine epidemic diarrhea mRNA vaccine and a preparation method thereof. BACKGROUND

[0002] The statements in this section merely provide background information related to the present disclosure and can not constitute the prior art.

[0003] Porcine epidemic diarrhea (PED) is an acute infectious disease that poses a serious threat to the health of pigs, caused by porcine epidemic diarrhea virus (PEDV) in the Coronaviridae family. The virus is highly contagious and can affect pigs of all ages, especially newborn piglets, with clinical symptoms including severe vomiting, diarrhea, listlessness, and decreased appetite. In severe cases, it can lead to dehydration and rapid weight loss, and even death, with a mortality rate of newborn piglets as high as 80% to 100%.

[0004] Since 2010, the PEDV G2 group of variant strains has spread widely around the world, with significantly improved pathogenicity and transmission speed compared to the original strain, further increasing the challenge of effectively preventing and controlling porcine epidemic diarrhea.

[0005] Currently, there is no specific drug available for treating PEDV-infected pigs, and vaccination is the main means of preventing and controlling the spread of PED. The main vaccines used on the market are PED inactivated vaccines and attenuated live vaccines. Although these two vaccines have played a certain role in preventing and controlling PED, the immunogenicity of inactivated vaccines is relatively weak, and multiple vaccinations are usually required to enhance immune effectiveness. Although attenuated live vaccines have strong immunogenicity, they have the potential risk of virulence returning to strength and producing new recombinant strains. In addition, the continuous variation of PEDV makes the antigens of existing vaccines not completely matched with the epidemic strains, resulting in a decrease in immune protection. There is a coexistence of multiple PEDV subgroups in China, which may be one of the reasons for the poor immune effect of traditional vaccines. Moreover, the development and production cycle of traditional vaccines is relatively long, making it difficult to quickly respond to the emergence of new strains.

[0006] Compared with traditional vaccines, mRNA vaccines have shown significant advantages. First, mRNA vaccines can stimulate strong immune responses, activating both humoral and cellular immunity, providing comprehensive protection. Second, mRNA vaccines are highly safe, as they do not involve live viruses in the production process, eliminating the risk of reverse transcription and virulence return. Finally, mRNA vaccines are extremely fast to develop and produce, with high programmability, allowing rapid adjustment of vaccine sequences to address new pathogen variants, which is particularly critical in the face of highly mutated pathogens, emerging and emerging epidemics.

[0007] In addition, due to the high mutability of PEDV virus, although the majority of the current epidemic strains are GII genotype, different subgroups are prevalent in different regions, and cross protection between different subgroups is poor. Currently available PEDV vaccines are only for one genotype subtype, so the vaccine prevention and control effect is generally poor, and there is an urgent need for a PEDV vaccine with broad-spectrum protection.

[0008] Prior art: CN113274491A and CN118147173A disclose an RNA vaccine for porcine epidemic diarrhea and a construction method thereof. Both cases use the coding sequence of S protein to prepare mRNA to prepare a vaccine against a single genotype. The PEDV S protein designed without sequence mutation has the problems of low expression efficiency and poor stability, which need to be solved urgently. SUMMARY

[0009] The purpose of the present application is to address the problems of low expression efficiency and poor stability of current mRNA vaccines, and to provide a porcine epidemic diarrhea mRNA vaccine and a preparation method thereof, which improves the expression efficiency and immune level of the mRNA vaccine through mutation.

[0010] The technical solution of the present application is as follows:

[0011] A porcine epidemic diarrhea mRNA vaccine includes the following mRNA fragment, which encodes the amino acid sequence of S protein, S1 protein or S protein receptor binding region of GIIa, GIIb or GIIc type porcine epidemic diarrhea virus with one or more mutations as follows:

[0012] F896P: F phenylalanine at position 896 is mutated to P proline;

[0013] A970P: A alanine at position 970 is mutated to P proline;

[0014] S975P: S serine at position 975 is mutated to P proline;

[0015] A1032P: A alanine at position 1032 is mutated to P proline;

[0016] G964C: G glycine at position 964 mutated to C cysteine;

[0017] A971C: A alanine at position 971 mutated to C cysteine;

[0018] L861C: L leucine at position 861 mutated to C cysteine;

[0019] G959C: G glycine at position 959 mutated to C cysteine;

[0020] M1028F: M methionine at position 1028 mutated to F phenylalanine;

[0021] T1050D: T threonine at position 959 mutated to D aspartic acid;

[0022] The gene sequence of wild type S protein of GIIa, GIIb and GIIc type is shown in SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6;

[0023] The amino acid sequence of S protein of GIIa, GIIb and GIIc type containing the above 10 mutations is shown in SEQ ID NO. 11, SEQ ID NO. 12 and SEQ ID NO. 13 respectively, and the nucleic acid sequence of S protein of GIIa, GIIb and GIIc type containing the above 10 mutations is shown in SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO. 16.

[0024] According to a preferred embodiment, it further comprises a nanoliposome particle, the mRNA fragment is wrapped in the nanoliposome particle, and the nanoliposome particle is DMG-PEG2000, SM-102, cholesterol, Dlin-MC3-DMA, ALC-1059 or DSPC; the particle size of the nanoliposome particle after wrapping the mRNA fragment is 50-200 nm.

[0025] According to a preferred embodiment, the nitrogen to phosphorus ratio of the encapsulation system of the nanoliposome particle is 3:1-15:1. More preferably, the nitrogen to phosphorus ratio is 3:1, 8:1, 10:1 or 15:1.

[0026] According to a preferred embodiment, it further comprises one or more of a buffer, a protective agent and / or an adjuvant, and the buffer is PBS or Tris buffer, and the pH range is 6.0-8.0.

[0027] According to a preferred embodiment, the vaccine preparation form is a nasal spray, an oral agent or an injection.

[0028] Another aspect of the present application provides a method for preparing an mRNA vaccine as described above, comprising the following steps:

[0029] Step (1): connecting the vector and the target gene fragment to construct an in vitro expression vector;

[0030] Step (2): transferring the in vitro expression vector constructed in step (1) into a competent cell for amplification, lysing the cell, and purifying to obtain a supercoiled plasmid;

[0031] Step (3): linearizing the supercoiled plasmid obtained in step (2) to obtain mRNA, and purifying the obtained mRNA to obtain an mRNA stock solution.

[0032] Step (4): encapsulating the stock mRNA with a lipid nanoparticle, and performing ultrafiltration and preparation to obtain a vaccine finished product.

[0033] According to a preferred embodiment, the competent cell is an E. coli competent cell. Preferably, it can be DH5-alpha, TOP10, stbl2, stbl3, JM109 or other E. coli cells with the same plasmid amplification function.

[0034] Preferably, the vector comprises a promoter, a 5'UTR, a Kozak sequence, a 3'UTR and a polyA tail.

[0035] Preferably, the vector further comprises a high-copy plasmid replication initiation site, a kanamycin resistance gene, and a resistance gene promoter.

[0036] The promoter is a T7 promoter; the Kozak sequence is "GCCACC"; the 5'UTR and 3'UTR are UTR regions of high expression abundance protein sequences such as alpha globin, beta globin, heat shock protein, and albumin.

[0037] The polyA tail is 60 adenine nucleotides (A) + GGG + 60 adenine nucleotides (A). Preferably, the polyA tail can be 60-200 continuous or discontinuous adenine nucleotides.

[0038] The "target gene fragment" is a target gene sequence encoding the mRNA fragment as described above; preferably, it can also include some known functional modified fragments.

[0039] In one possible implementation, the mRMA termination codon encoding the PEDV virus is one or more of TGA, TAA or TAG.

[0040] According to a preferred embodiment, the way of purifying the plasmid in step (2) is clarified filtration, chromatography, ultrafiltration. The chromatography filler is anion exchange filler, hydrophobic chromatography filler, molecular sieve. Or other chromatography fillers with the same effect.

[0041] According to a preferred embodiment, the linearization process in step (3) specifically includes the following sub-steps:

[0042] Step (3.1): supercoiled plasmid enzyme linearization;

[0043] Step (3.2): in vitro transcription reaction: transcribe the linearized sequence obtained in step (3.1) while capping, add T7 transcriptase, ATP, CTP, GTP, Pseudo-UTP and cap1 cap analog.

[0044] According to a preferred embodiment, step (3.2) can further include the following sub-steps:

[0045] Step (3.2.1): in vitro transcription reaction: transcribe the linearized sequence obtained in step (3.1), add T7 transcriptase, ATP, CTP and Pseudo-UTP;

[0046] Step (3.2.2): configure the capping system for the product of step (3.2.1), add GTP, SAM, capping enzyme and methyltransferase.

[0047] According to a preferred embodiment, the way of purifying the mRNA in step (3) is dT affinity chromatography, Core400 chromatography filler or other chromatography fillers with the same effect.

[0048] According to a preferred embodiment, the lipid nanoparticles in step (4) are selected from DMG-PEG2000, SM-102, cholesterol, Dlin-MC3-DMA, ALC-1059 or DSPC. The encapsulated liposome particle diameter is in the range of 50-200nm.

[0049] According to a preferred embodiment, the encapsulation process in step (4) is T-mix, microfluidic or IJPM technology.

[0050] According to a preferred embodiment, the nitrogen to phosphorus ratio of the encapsulation system in step (4) is 3:1-15:1. More preferably, the nitrogen to phosphorus ratio is 3:1, 8:1, 10:1 or 15:1.

[0051] According to a preferred embodiment, the formula of the vaccine preparation in step (4) comprises mRNA-encapsulated liposome particles, a buffer, a protective agent and an adjuvant; the buffer is PBS or Tris buffer, with a pH range of 6.0-8.0; the protective agent component is sucrose. The adjuvant is chitosan.

[0052] According to a preferred embodiment, the PEDV virus vaccine is in the form of a nasal spray, an oral agent or an injection.

[0053] According to a preferred embodiment, the PEDV virus vaccine is used for twice or multiple immunization.

[0054] Compared with the prior art, the beneficial effects of the present application are:

[0055] 1. A porcine epidemic diarrhea mRNA vaccine and a preparation method thereof, the monovalent PEDV mRNA vaccine provided by the present application induces an increase of more than 10 times in the expression amount of a target protein in vivo, and GIIa-S, GIIb-S and GIIc-S induce a higher level of humoral immunity in mice than the commercial vaccine of the prior art.

[0056] 2. The trivalent PEDV mRNA vaccine GII-triValent provided by the present application induces a high level of neutralizing antibody against three GII epidemic strains in mice, and the antibody level is significantly higher than that of the commercial vaccine of the prior art.

[0057] 3. The monovalent and trivalent mRNA vaccines provided by the present application induce a significantly higher level of humoral immunity in piglets than the commercial vaccine of the prior art, and are higher than the optimal immunization strategy (attenuation + inactivation), have a better broad-spectrum protection effect, and can effectively solve the practical problem that the prior art has poor prevention and control effect in use.

[0058] 4. The trivalent vaccine provided by the present application shows complete protection against mixed infection of three GII genotype PEDV viruses in piglet challenge experiments, and the protection is reflected in high neutralizing antibody level, low virus shedding and pathology. The data in all aspects show that the protection effect of the trivalent PEDV mRNA vaccine GII-triValent is significantly better than the optimal immunization strategy of the commercial vaccine of the prior art.

[0059] 5. The process provided by the present application is different from the laboratory process, and is a scalable process for industrial production, and the prepared plasmid and RNA meet the commercial production standards. BRIEF DESCRIPTION OF DRAWINGS

[0060] Figure 1 It is the pcDNA3.1 plasmid map in Example 1.

[0061] Figure 2pIVT-D1-Kan-BsaI plasmid map in Example 1;

[0062] Figure 3 pIVT-PEDV-S plasmid map in Example 1;

[0063] Figure 4 Detection concentration of the spiral plasmid in Example 1;

[0064] Figure 5 Protein expression and antibody level of 10 mutants in Example 2;

[0065] Figure 6 Neutralizing antibody level of random combination screening of 10 mutants in Example 2;

[0066] Figure 7 Protein expression data of the target protein before and after mutation of different genotypes of 10 mutants in Example 2;

[0067] Figure 8 Protein band of the target protein after mutation of different genotypes of 10 mutants in Example 2;

[0068] Figure 9 IgG detection results of different groups of mice in Example 3;

[0069] Figure 10 Neutralization titers of the antibodies to be tested of each group of mice in Example 3; wherein (A) is the GIIa strain neutralization titer; (B) is the GIIb strain neutralization titer; (C) is the GIIc strain neutralization titer;

[0070] Figure 11 Body weight changes of each group of mice in Example 3;

[0071] Figure 12 Body temperature and body weight changes of piglets after challenge in Example 4; wherein (A) is the body temperature monitoring (B) is the body weight change of experimental animals 14 days after challenge;

[0072] Figure 13 Immunogenicity data of the mRNA vaccine of the application in piglets in Example 4;

[0073] Figure 14 Diarrhea data of each group of piglets in Example 4;

[0074] Figure 15 qPCR detection data of anal swab PEDV virus content of each group of piglets in Example 4;

[0075] Figure 16 Dissection of small intestinal tissue of each group of piglets in Example 4 and preparation of typical pathological sections. DETAILED DESCRIPTION

[0076] The specific examples listed in the present application are only as examples of the present application, and the present application is not limited to the specific examples described below. Any equivalent modifications and substitutions of the examples described below made by those skilled in the art are also within the scope of the present application. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present application should be encompassed within the scope of the present application. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. All reagents or instruments not mentioned by the manufacturer are conventional products that can be purchased on the market. In order to better illustrate the present application, numerous specific details are given in the following detailed description of the embodiments. Those skilled in the art should understand that the present application can also be implemented without some specific details. In some embodiments, methods, means, apparatuses and steps that are well known to those skilled in the art are not described in detail in order to highlight the main idea of the present application.

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise indicated, all measurements used in the description herein are made in international units, and the numerical values and numerical ranges appearing in the present application should be understood to encompass the systematic errors that are inevitable in industrial production.

[0078] The features and properties of the present application are further described in detail below in conjunction with the examples.

[0079] Example 1 Preparation method of a porcine epidemic diarrhea mRNA vaccine

[0080] (1) Synthesis of mRNA vaccine coding sequence (target gene fragment)

[0081] The spike protein (Spike protein, S protein) coding sequence in the mRNA vaccine is based on the S gene sequence SEQ ID NO. 1 (GenBank: KM287429.1) of the GIIa type PEDV epidemic strain, the S gene sequence SEQ ID NO. 2 (GenBank: JX188454.1) of the GIIb type PEDV epidemic strain, and the S gene sequence SEQ ID NO. 3 (GenBank: KU847996.1) of the GIIc type PEDV epidemic strain. According to SEQ ID NO. 11~13, the corresponding amino acid sequences are mutated.

[0082] The wild type and mutant S protein gene coding sequences are codon optimized and further designed to replace the rare codons of pigs in the sequence; under the premise of avoiding specific enzyme cutting sites such as XbaI, SalI, Bsa I and unfavorable motifs, the sequence is modified to appropriately improve the translation efficiency of the antigen protein in the host cell. The present application compares SEQ ID NO. 4 PEDV-GIIa-wt, SEQ ID NO. 5 PEDV-GIIb-wt, SEQ ID NO. 6 PEDV-GIIc-wt three S protein coding sequences and SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13 three mutant protein sequences and their coding sequences SEQ ID NO. 14 PEDV-GIIa-S, SEQ ID NO. 15 PEDV-GIIb-S, and SEQ ID NO. 16 PEDV-GIIc-S as S antigen protein coding sequences in PEDV mRNA vaccine, which are synthesized by a gene synthesis company and cloned into pcDNA3.1 vector (plasmid map Figure 1 ).

[0083] (2) mRNA vaccine transcription vector construction

[0084] The in vitro transcription vector pIVT-D1-Kan-BsaI sequence of the porcine epidemic diarrhea mRNA vaccine contains the following elements: T7 promoter SEQ ID NO. 7, 5' untranslated region (5' UTR) SEQ ID NO. 8, S antigen protein coding gene, 3' untranslated region (3' UTR) SEQ ID NO. 9 and polyadenylate (Poly A) SEQ ID NO. 10, and the downstream of the Poly A tail structure is connected with the enzyme cutting site (Bsa I) for linearization of the plasmid (plasmid map Figure 2 ).

[0085] The synthesized DNA template sequence and pIVT-D1-Kan-BsaI vector are double enzyme cut with XbalI and SalI and connected with T4 ligase overnight to generate the pIVT-PEDV-S recombinant vector (plasmid map Figure 3 ), which is transformed into stbl3 competent cells and single colonies are selected for preservation.

[0086] (3) mRNA in vitro transcription plasmid preparation

[0087] A. The transformant strain of the pIVT plasmid is fermented and cultured, lysed with 0.2% NaOH, 1% SDS solution, neutralized with 2M acetic acid potassium acetate solution, and centrifuged at 8000 rpm for 30 minutes to harvest the supernatant.

[0088] B. 6FF molecular sieve chromatography (purchased from Cytiva): mainly remove E. coli RNA; 6FF chromatography eluent, collect the first elution peak sample, UV 260 rises to 50 mAU and above to start collecting, and falls to 500 mAU and below to stop collecting. The linear flow rate of the chromatography process is not higher than 800 cm / h.

[0089] C. PS hydrophobic chromatography (Plasmid Select (PS) purchased from Cytiva): column packing name: Plasmid Select; mainly bind supercoiled plasmid DNA to remove open-loop plasmid DNA; PS chromatography liquid A balances the Plasmid Select (PS) chromatography column;

[0090] Load the 6FF chromatography collected liquid, and the loading capacity is not higher than 1 mg plasmid / mL packing.

[0091] After the loading is completed, rinse with not less than 1 column volume of PS chromatography liquid A. Then elute with not less than 1 column volume of 30% PS chromatography liquid B, and finally elute with PS chromatography liquid B, UV 260 rises to 500 mAU and above to start collecting, and falls to 1000 mAU and below to stop collecting. The linear flow rate of the chromatography process is not higher than 200 cm / h.

[0092] D. Ultrafiltration concentration. Concentrate the PS chromatography collected liquid to 10±5 times using an ultrafiltration system with a molecular weight cutoff of 300kD, the inlet pressure of the ultrafiltration process is not higher than 1 bar, the backflow end pressure is not higher than 1 bar, and the permeation end pressure is not higher than 1 bar. Dilute the ultrafiltration concentrated collected liquid 6±2 times with 30Q diluent (100mmol / L Tris-HCl, 10mmol / L EDTA), which is the 30Q chromatography loading liquid.

[0093] E. 30Q ion exchange chromatography: packing name: 30Q (purchased from Cytiva); mainly remove endotoxin and E. coli protein impurities, and finally obtain high-purity supercoiled plasmid DNA.

[0094] 30Q chromatography liquid A is used for balancing, 50% 30Q chromatography liquid B is used for elution, UV 260 rises to 200 mAU and above to start collecting, and falls to 300 mAU and below to stop collecting. The linear flow rate of the chromatography process is not higher than 200 cm / h.

[0095] F. Ultrafiltration concentration. Concentrate the 30Q chromatography collected liquid to 10±5 times using an ultrafiltration system with a molecular weight cutoff of 300kD, the inlet pressure of the ultrafiltration process is not higher than 1 bar, the backflow end pressure is not higher than 1 bar, and the permeation end pressure is not higher than 1 bar.

[0096] The above process can obtain high-purity transcription template plasmid with superhelix rate of 80% or more. For example Figure 4 .

[0097] (4) Linearization

[0098] The plasmid obtained in step (3) is digested with Bsa I (purchased from Nanjing Novozyme) restriction endonuclease, and the reaction time is 2 h. The reaction system is shown in Table 1:

[0099] Table 1 Linearization enzyme digestion reaction system

[0100]

[0101] The harvested linearized product is purified according to the C-F steps in step (3), which is not repeated. The plasmid digestion effect is identified by 1% agarose nucleic acid gel electrophoresis at 150 V for 20 min. The correct target fragment is obtained.

[0102] (5) In vitro transcription to prepare mRNA

[0103] The following raw materials are purchased from Nanjing Shenji. After preparing the reaction system, the reaction is carried out at 37°C and 10 rpm for 4 hours. After the reaction is completed, 4 ml of 0.5M EDTA is added to the reaction system to terminate the reaction.

[0104]

[0105] (6) mRNA purification

[0106] Oligo dT (purchased from Thermofisher) affinity column chromatography (mRNA chromatography 1) can selectively purify target mRNA containing polyA tail, and can remove template DNA, incomplete mRNA, proteins and NTPs in the reaction system to the maximum extent. The main chromatography parameters are as follows:

[0107] RNA chromatography solution A: 20 mmol / L Tris-HCl, pH 7.5, 2 mmol / L EDTA, 500 mmol / L NaCl

[0108] RNA chromatography solution B: 20 mmol / L Tris-HCl, pH 7.5

[0109] Add 1 / 9 volume of transcription product to 4 mol / L NaCl. dT affinity chromatography: (filler name: Oligo dT) Equilibrate the Oligo dT chromatography column with not less than 1 column volume of dT chromatography liquid A, and load the dT chromatography sample with a volume not higher than 0.5 column volumes and a linear flow rate not higher than 150 cm / h. After loading, elute with not less than 2 column volumes of dT chromatography liquid A at a linear flow rate not higher than 150 cm / h. Then elute with not less than 2 column volumes of 50% dT chromatography liquid B at a linear flow rate not higher than 300 cm / h. Finally, elute with dT chromatography liquid B, and start collecting when the UV 260 rises to 50 mAU or above, and stop collecting when the UV 260 drops to 50 mAU or below.

[0110] Add 20KU DNaseI enzyme to the harvest liquid to remove DNA templates.

[0111] Second step chromatography Core400 chromatography:

[0112] Equilibrate the Core 400 chromatography column (filler name: Capto Core400; purchased from Cytiva) with not less than 1 column volume of dT chromatography liquid B. Load the DNase I digestion liquid with a volume not higher than 3 column volumes, and start collecting when the UV 260 rises to 50 mAU or above. After loading, elute with not less than 2 column volumes of dT chromatography liquid B, and collect the material during the elution. Stop collecting when the UV 260 drops to 100 mAU or below, which is the Core chromatography collection liquid. The linear flow rate during chromatography is not higher than 160 cm / h.

[0113] Use a 100kD tangential flow system to concentrate the collected chromatography flow by 5±1 times, then perform 10 times liquid exchange with 2.5mmol / L sodium citrate (pH 6.4), and finally add 1~4 volumes of 2.5 mmol / L sodium citrate (pH 6.4). After filtering and sterilizing, measure the mRNA concentration with a NanoDrop spectrophotometer. Analyze the purity of mRNA by capillary electrophoresis.

[0114] Through this process, the purity of mRNA can reach more than 95%, and the integrity of mRNA is good.

[0115] (7) Lipid nanoparticle encapsulation

[0116] Liposome raw materials:

[0117]

[0118] Prepare the lipid alcohol solution according to the following table

[0119]

[0120] Take the frozen mRNA and place it in the ice bath along with the container; remove when all the mRNA has thawed; dilute with 25 mM sodium citrate buffer to a final concentration of 167 pg / mL; turn on the microfluidic instrument; open the settings interface and input the desired parameters (MyaGen R-SDM chip, flow rate 20 mL / min; aqueous: organic = 3: 1; pre-waste 0.5 mL, post-waste 0 mL; PNI regular chip, flow rate 18 mL / min; aqueous: organic = 3: 1; pre-waste 0.5 mL); draw up the appropriate lipid / alcohol solution and mRNA buffer using a syringe according to the set conditions and install them on the instrument; begin preparing the LNP stock solution (draw up 1.5 mL of mRNA solution and 0.5 mL of lipid / alcohol solution and prepare approximately 1.5 mL of LNP stock solution); dilute the collected LNP stock solution 5-fold with PBS buffer (or Tris-HCl buffer) and concentrate back to the original volume using an ultrafiltration tube (centrifugation parameters: 2000g, 10 min, 25°C, repeat dilution and ultrafiltration 2 times); sucrose buffer exchange: replace the ultrafiltrated LNP solution with a sucrose solution to a final sucrose concentration of 5%; filter the product using a 0.22 pm membrane to obtain the final LNP solution; place the LNP solution in an enzyme-free sterile container; obtain the final LNP solution encapsulating the mRNA fragment and store it in a refrigerator at 2-8°C, -15 to -25°C, or -65 to -85°C.

[0121] Example 2 Screening of mutant protein sequences

[0122] After preparing mRNA-LNP according to the above process steps using mRNA encoding the sequences of 42 mutant proteins, and detecting the expression levels of the target proteins and the ability to induce neutralizing antibodies in mice, it was found that 10 mutants were significantly better than the wild-type Spike sequence in terms of protein expression and antibody levels. As shown in Table 1. Figure 5

[0123] The ten candidate mutants are:

[0124] 1: F896P: F phenylalanine at position 896 is mutated to P proline;

[0125] 2: A970P: A alanine at position 970 is mutated to P proline;

[0126] 3: S975P: S serine at position 975 is mutated to P proline;

[0127] 4: A1032P: A alanine at position 1032 is mutated to P proline;

[0128] ​5: G964C: G glycine at position 964 mutated to C cysteine;

[0129] 6: A971C: A alanine at position 971 mutated to C cysteine;

[0130] 7: L861C: L leucine at position 861 mutated to C cysteine;

[0131] 8: G959C: G glycine at position 959 mutated to C cysteine;

[0132] 9: M1028F: M methionine at position 1028 mutated to F phenylalanine;

[0133] 10: T1050D: T threonine at position 959 mutated to D aspartic acid.

[0134] Random combination screening was performed on the above 10 candidate mutations, 10 different combinations were tested, and it was found that when the ten mutation points existed at the same time, the expression amount of the target protein increased by more than 12 times, and the neutralizing antibody level increased significantly (such as Figure 6 ).

[0135] Candidate vaccine preparation and detection

[0136] The mRNA vaccine containing the target mutation (ten mutations existing at the same time) of the GIIa / b / c genotype was prepared according to the steps of Example 1 above, and the particle size of the lipid nanoparticles was detected by dynamic light scattering technology. The particle size of the lipid nanoparticles was normally distributed, and the particle size of the lipid nanoparticles was uniformly distributed between 80-120 nm. The ribogreen kit (purchased from invitrogen) was used to detect the encapsulation rate according to the kit instructions, and the encapsulation rate reached more than 95%. The data before and after the mutation of each subtype are as follows:

[0137]

[0138] 2 μg of mRNA-LNP was used to infect 80% confluent HEK293 cells in a 6-well plate, and the sample was collected after 24 hours. The lysate was harvested, and the S protein content was detected by Elisa, and the protein expression was detected by WB.

[0139] First, sample for the difference of the target protein expression test, compared the mutant sequence GIIa-S, GIIb-S, GIIc-S and wild type sequence WTa, WTb, WTc protein expression. Take the primary antibody coated overnight ELISA plate, discard the coating solution, add 300 μl washing solution per well, wash 3 times, add blocking solution 200 μl / well, cover the sealing plate film, placed in 37℃ enzyme-labeled plate constant temperature oscillator incubate 1h. Take out the blocked ELISA plate, discard the blocking solution, add 300 μl washing solution per well, wash 5 times, each interval inverted on the blotting paper to dry the residual liquid in the hole. Prepare the standard and test sample, test sample for 3 biological repeats, cover the sealing plate film, placed in 37℃ enzyme-labeled plate constant temperature oscillator incubate 2h. Take out the ELISA plate, discard the sample, add 300 μl washing solution per well, wash 5 times, add 100 μl HRP secondary antibody working solution per well, cover the sealing plate film, placed in 37℃ enzyme-labeled plate constant temperature oscillator incubate 1h. Take out the ELISA plate, discard the detection antibody, add 300 μl washing solution per well, wash 5 times, each interval inverted on the blotting paper to dry the residual liquid in the hole. Add 100 μl TMB color developing solution to the ELISA plate, cover the sealing plate film, placed in 37℃ enzyme-labeled plate constant temperature oscillator develop color for 3-7 minutes. Color developing solution needs to be taken out from 2-8℃ refrigerator for 10ml light protection to room temperature. Stop: add 100 μl / well prepared stop solution to the ELISA plate, gently mix, put into the enzyme-labeled instrument to read the plate within 30 minutes. According to the standard curve to calculate the target protein expression.

[0140] The results further show that the expression of the S protein designed by mutation is significantly improved, with an increase of more than 10 times, and is suitable for different genotypes of PEDV Spike, such as Figure 7 as shown.

[0141] Then take the sample and use 10% SDS-PAGE gel for SDS-PAGE electrophoresis of protein sample, the conditions are constant voltage 80 V electrophoresis for 30 min, then constant voltage 120 V electrophoresis for 60 min. After electrophoresis, transfer the protein to PVDF membrane, the conditions are constant current 250 mA for 90 min. After transfer, block the PVDF membrane with 5% skim milk powder at 4℃ overnight, then wash the membrane with PBS buffer for 3 times, 5 min each time. Add anti-PEDV S protein monoclonal antibody (purchased from Zonogen company) diluted 1000 times with PBS buffer, incubate at room temperature for 1 h. Then wash the membrane with PBST buffer for 3 times, 10 min each time. Add 20000 times diluted horseradish peroxidase labeled goat anti-mouse IgG (purchased from abcam company), incubate at room temperature for 1 h. Then wash the membrane with PBST buffer for 3 times, 10 min each time. Finally, wash with PBS once, and use enhanced ECL luminescent liquid (purchased from Biyun Tian company) to perform ECL luminescence operation according to the instructions. The results are as followsFigure 8 As shown, a clear band of about 200 kDa appeared in all the test groups, which was consistent with the expectation.

[0142] Example 3 Safety and efficacy test of candidate vaccine in mice

[0143] Test grouping and immunization:

[0144]

[0145] To verify the immune effect of the PEDV mRNA vaccine in the application, SPF Balb / c mouse animal tests were carried out. 120 6-8 week old Balb / c female mice were randomly divided into 8 groups. The mice in group 1 were inoculated with PBS as a blank control group, the mice in group 2 were inoculated with 1 μg GIIa-S vaccine (mRNA-LNP), the mice in group 3 were inoculated with 1 μg GIIb-S vaccine, the mice in group 4 were inoculated with 1 μg GIIc-S vaccine, the mice in group 5 were inoculated with 1 μg GII-triValent trivalent vaccine, the mice in group 6 were inoculated with 3 μg GII-triValent trivalent vaccine, the mice in group 7 were inoculated with 100 μl (1 / 10 dose) of the approved for use PEDV inactivated vaccine of Keweicun Company, and the mice in group 8 were inoculated with 100 μl (1 / 10 dose) of the approved for use PEDV attenuated live vaccine of Keweicun Company, and all groups were boosted once at the same dose 21 days after the first immunization. The PEDV Spike (classic strain CV777) specific antibody IgG and GIIa, GIIb, GIIc strain neutralization titers were detected 14 days and 35 days after the first immunization. The mice were weighed every other day during the test period, and the abnormal reactions at the inoculation site and the animal mental state were observed.

[0146] a) Detection of S protein specific IgG antibodies

[0147] On the 14th day and the 35th day after the first immunization, blood was collected from the retro-orbital plexus of the mice and serum was prepared. In an Elisa plate, 5 sera from each group were diluted 200 times to detect Spike specific IgG at a concentration of 2 μg / ml of S protein coated overnight (Spike protein purchased from Novozyme). The serum was added to the ELISA plate and incubated at 37°C for 1 hour, washed 5 times with 0.05% PBST, added 1:2000 diluted HRP labeled anti-mouse IgG secondary antibody (purchased from Abeam company), incubated at 37°C for 30 minutes, washed 5 times with 0.05% PBST, added TMB substrate (purchased from Biyun Tian company), and the absorbance at 650 nm was read using a microplate reader after the color development was stopped.

[0148] The IgG detection results are as follows: Figure 9As shown, the mRNA vaccine induced high level of humoral immune response in mice, the OD 650nm value of serum S protein specific IgG antibody of mRNA vaccine mice was significantly higher than that of two commercial vaccines, the IgG antibody level of the same dose group was close, and the specific IgG antibody level of the GII-trivalent vaccine showed a dose-dependent manner. It is proved that the GII-trivalent PEDV vaccine has stronger immunogenicity than the existing commercial vaccine.

[0149] b) True virus neutralizing antibody titer detection

[0150] The true virus neutralizing antibody titer detection can verify whether the antibodies produced in the animal body have neutralizing effect on the virus, so as to reflect the level of protective antibodies. In order to prove whether the multivalent PEDV vaccine of the application has a broader spectrum of protection, the neutralizing titer of mouse serum against three GII genotype PEDV viruses 14 days after the second immunization (day 35) was detected. The GIIa (SC1607-03 strain), GIIb (SC1803-12 strain) and GIIc type (SC1602-01 strain) PEDV strains were cultured in the laboratory and prepared into virus culture solution. After the virus solution was neutralized with an equal volume of mouse serum of different dilutions at 37°C for 1 h, it was added to the monolayer culture of Vero cells in a 96-well plate, 5 replicate wells were set for each dilution of each sample, and the Vero cell pathological changes were observed daily until stable, and the data was observed for 3-5 days and the number of wells with pathological changes was counted. The neutralizing titer of the antibodies of each group of mice was calculated using the Reed-Muench formula, and the results are as follows Figure 10 As shown in A, B and C, the S protein antigens of GIIa, GIIb and GIIc genotypes can induce high levels of neutralizing antibodies against homologous strains, and the neutralizing antibody levels induced by heterologous strains are relatively low, which also proves to some extent that the phenomenon that the single PEDV vaccine has poor prevention and control effect in reality. At the same time, the neutralizing antibody titer induced by the mRNA vaccine is significantly higher than that of the commercial inactivated vaccine and attenuated live vaccine, which proves that its immune effect is superior to the existing vaccines. In addition, the GII-trivalent PEDV vaccine of the application induces neutralizing antibody levels much higher than those of commercial vaccines against three laboratory strains, which proves that the vaccine has potential protection against different epidemic strains and has a broad spectrum that the existing vaccines do not have.

[0151] The above test results show that the mRNA vaccine expressing PEDV-S protein prepared by the application can effectively induce humoral immune response after immunizing mice, and the high level of neutralizing antibodies induced have neutralizing effect on PEDV GII genotype 3 subgroups, and the immune effect is much better than that of the commercial vaccine tested in the experiment.

[0152] c) Safety of the mRNA vaccine

[0153] During the observation period, the mice in the 8th group showed a certain degree of mental fatigue after immunization, and the mice in the other groups were normal, and there was no obvious swelling and induration at the injection site. The body weight change is shown in Figure 11 , and overall, there is little difference between the vaccine immunization group and the negative control group, showing fluctuation and rising. It proves that the mRNA vaccine has good safety.

[0154] Example 4 In vivo effectiveness test of candidate vaccine piglets

[0155] To verify the effectiveness of the vaccine in vivo, the effectiveness verification in piglet back was carried out. PEDV specific IgG antibody negative sows were selected, and 42 piglets were divided into 7 groups after delivery. Different groups were immunized according to the table at 7 days and 21 days with an interval of 14 days. The positive control group is attenuated live vaccine from Kefa company, and inactivated vaccine is used for booster immunization. The reason for choosing this vaccination strategy is that the current breeding base generally believes that this sequential immunization program is the best immunization method for the existing vaccine.

[0156] Test grouping and immunization:

[0157]

[0158] Blood was collected 14 days and 28 days after the first immunization to detect serum specific IgG antibody and neutralizing antibody level against GII genotype three subtypes. 14 days after the second immunization, piglets were orally infected with live virus for challenge test. To verify the effectiveness of the vaccine for different strains, the oral virus is a mixture of three laboratory isolated strains, and the three strains are mixed at a ratio of 1:1:1, and the total virus content is 3x10 7 TCID 50 . After challenge, the body temperature, diarrhea, body weight gain and anal swab of piglets were detected.

[0159] a) The body temperature and body weight change after challenge are shown in Figure 12 A and B. Figure 12 A shows that the PBS group, GIIa-S (mRNA-LNP) group, GIIb-S group, GIIc-S group and commercial vaccine group all have an upward trend 3-4 days after challenge, while the two groups of trivalent vaccine GII-triValent show normal fluctuation. Figure 12B shows the percentage change in body weight of piglets 14 days post challenge, where the PBS group had an average weight gain of 32% post challenge, the GIIa-S group had a 64% gain, the GIIb-S group had a 70% gain, the GIIc-S group had a 51% gain, the commercial vaccine group had a 47% gain, the GII-triValent 10 pg group had a 93% gain, the GII-triValent 30 pg group had a 97% gain, and the unchallenged control animals had a 89% gain. The results show that the mixed virus challenge had a significant impact on the normal growth of the animals, and that the commercial vaccine group did not significantly improve compared to the PBS control group, and that the monovalent mRNA vaccine groups only reduced the impact to a certain extent, while the trivalent mRNA vaccine of the present application had no impact on the growth performance of the piglets. This proves that the trivalent PEDV vaccine of the present application has its actual innovative value and significant effect superior to the prior art.

[0160] b) Immunogenicity in piglets as Figure 13 A, B, C, D. The detection method is as described in step b) of Example 3. The results show that the mRNA vaccine can induce high levels of S protein specific antibodies and neutralizing antibodies 14 days after one dose. Overall, the immunogenicity of the mRNA vaccine group is significantly superior to the commercial vaccine produced by the prior art. The heterologous neutralizing antibodies induced by the S protein antigen are lower than the homologous strain, which proves the necessity of the multivalent PEDV vaccine, and the multivalent PEDV vaccine of the present application induces high levels of neutralizing antibodies against the three GII epidemic strains, which is consistent with the mouse data, proving its broad-spectrum immunogenicity.

[0161] c) The diarrhea of piglets after challenge was observed, and according to the shape of the excrement, it was divided into severe diarrhea (liquid excrement), mild diarrhea (semi-solid excrement), and healthy (solid excrement), and recorded. The results are shown in Figure 14 , and the data show that in each group of 6 test animals, the PBS group ( Figure 14 A) all had severe diarrhea and 3 / 6 animals still had mild diarrhea until the 13th day. The monovalent mRNA vaccine groups also had diarrhea symptoms, of which the GIIa-S and GIIb-S groups were mild diarrhea ( Figure 14 B, 14C), and the diarrhea symptoms disappeared at the end of the observation period. The GIIc-S group had 2 animals ( Figure 14 D) with severe diarrhea, and maintained mild diarrhea until the end of the observation period, and the remaining 4 had mild diarrhea, and the diarrhea disappeared at the end of the observation period. The commercial vaccine group ( Figure 14 E) had 5 animals with severe diarrhea, but improved compared to the PBS group, and all animals turned to mild diarrhea by the end of the observation period. It is worth noting that the two dose groups of the trivalent PEDV vaccine of the present application (10 pg group and 30 pg group) did not have diarrhea symptoms ( Figure 14 F, Figure 14G), which had 100% protection effect on mixed virus infection. The above data show that although the monovalent mRNA vaccine has improved effect compared with the commercial vaccine, the protection of mixed infection is still limited for the genotype diverse PEDV, while the trivalent PEDV vaccine GII-triValent in the present application has a significant advantage in preventing multiple genotype virus infection, and achieves 100% protection effect in animal experiments.

[0162] d) The PEDV virus content of the animal anal swab was detected according to the “SN / T 1699-2017 Quarantine Technical Specification for Porcine Epidemic Diarrhea” (detection of PEDV virus content in animal anal swab) Figure 15 ), and the results showed that the trivalent PEDV vaccine GII-triValent could clear the anal virus shedding (CT>40) at 5 days after challenge, while the monovalent mRNA vaccine group still had a small amount of shedding (35<CT<40) until 14 days, and the commercial vaccine group had a decrease in virus load compared with the PBS control group, but failed to significantly inhibit the shedding.

[0163] e) The small intestine tissue of the animal was dissected and typical pathological sections were prepared, as shown in Figure 16 , the small intestine pathological sections of the PBS group and the commercial vaccine group showed obvious small intestine villus shedding, the monovalent mRNA vaccine group was significantly improved compared with the PBS group but still had thinning of the intestinal wall and shortening of the villi. The small intestine sections of the two dose groups of the trivalent mRNA vaccine showed no obvious pathology. It is proved that the commercial vaccine of the prior art has poor protection effect on mixed virus infection, and the trivalent mRNA vaccine GII-triValent has ideal protection effect on mixed virus infection.

[0164] The above examples only express the specific embodiments of the present application, which are described in detail and in detail, but cannot be understood as limiting the protection scope of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the technical scheme concept of the present application, a number of modifications and improvements can be made, which are within the protection scope of the present application.

Claims

1. A porcine epidemic diarrhea mRNA vaccine, characterized in that, The mRNA fragment is transcribed from a DNA sequence as shown in SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO.

16.

2. The porcine epidemic diarrhea mRNA vaccine according to claim 1, characterized in that The mRNA fragment is further wrapped in a nanoliposome particle, and the nanoliposome particle is DMG-PEG2000, SM-102, cholesterol, Dlin-MC3-DMA, ALC-1059 or DSPC; the particle size of the nanoliposome particle after wrapping the mRNA fragment is 50-200 nm.

3. The porcine epidemic diarrhea mRNA vaccine according to claim 2, characterized in that The nitrogen-phosphorus ratio of the encapsulation system of the nanoliposome particle is 3:1-15:

1.

4. The porcine epidemic diarrhea mRNA vaccine of claim 1, wherein the porcine epidemic diarrhea mRNA vaccine is a vaccine for preventing the porcine epidemic diarrhea virus infection in a pig. The vaccine preparation further comprises one or more of a buffer, a protective agent and / or an adjuvant, wherein the buffer is PBS or Tris buffer, and the pH range is 6.0-8.

0.

5. The porcine epidemic diarrhea mRNA vaccine according to claim 4, characterized in that The vaccine preparation is in the form of a nasal spray, an oral agent or an injection.

6. The method of claim 1-5, wherein the porcine epidemic diarrhea mRNA vaccine is prepared by, The method comprises the following steps: Step (1): connecting a vector and a target gene sequence encoding the mRNA fragment as claimed in claim 1 to construct an in vitro expression vector; Step (2): amplifying the in vitro expression vector constructed in step (1) by transforming into a competent cell, lysing the cell and purifying to obtain a supercoiled plasmid; Step (3): linearizing the supercoiled plasmid obtained in step (2) to obtain mRNA, and purifying the obtained mRNA to obtain an mRNA stock solution; Step (4): encapsulating the mRNA stock solution in a lipid nanoparticle, and performing ultrafiltration and preparation to obtain a vaccine finished product; The linearization in step (3) comprises the following sub-steps: Step (3.1): enzyme digestion linearization of the supercoiled plasmid; Step (3.2): in vitro transcription reaction: transcribing the linearized sequence obtained in step (3.1) and simultaneously capping, adding T7 transcription enzyme, ATP, CTP, GTP, Pseudo-UTP and cap1 cap analog.

7. The method of claim 6, wherein the porcine epidemic diarrhea mRNA vaccine is prepared by, The vector in step (1) comprises a promoter, a 5'UTR, a Kozak sequence, a 3'UTR and a polyA tail.

8. The method of claim 6, wherein the porcine epidemic diarrhea mRNA vaccine is prepared by, The competent cell is an E. coli competent cell.

9. The method of claim 8, wherein the porcine epidemic diarrhea mRNA vaccine is prepared by, The E. coli competent cell is DH5-alpha, TOP10, stbl2, stbl3 or JM109.

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