African swine fever virus p30 protein mRNA vaccine and preparation method and application

Abstract

An mRNA, comprising RNA transcribed from each of a 5'-UTR sequence, a 3'-UTR sequence, a signal peptide sequence and an antigen protein coding gene sequence. When the antigen protein coding gene sequence is an ASFV virulence protein, such as a p30 protein, the mRNA prepared is injected into a pig, and a high level of antibody can be produced one week after the second immunization. Accordingly, the mRNA provided by the application can be used as an active substance for the preparation of a vaccine, such as the application in the preparation of an African swine fever virus vaccine.

Description

TECHNICAL FIELD

[0001] The present application relates to a vaccine, in particular to a nucleic acid vaccine, and its application in African swine fever virus vaccine. BACKGROUND

[0002] African swine fever (ASF) is a highly contagious disease caused by African swine fever virus (ASFV). ASFV mainly infects domestic pigs and wild boars, and the mortality rate can reach 100%. In 1921, ASF was first reported in Kenya, and quickly spread to eastern and southern countries on the African continent. In 2007, Georgia reported the first ASF in Eastern Europe, forming a new ASF epidemic area in Eastern Europe. In 2018, ASF was first reported in China, and quickly spread in many regions of the country. The outbreak and prevalence of ASF have caused huge economic losses to China's pig industry.

[0003] ASFV is a linear double-stranded DNA virus with a capsid structure, belonging to the Asfivirus genus of the Asfarviridae family. The ASFV genome is large, encoding more than 150 proteins. Among them, the p30 protein is a phosphorylated protein encoded by the CP204L gene of ASFV, which is expressed in the early stage of ASFV infection and is one of the most immunogenic proteins. It is often used as a target for ASFV serological detection. In addition, the p30 protein is often considered a protective antigen of ASFV. Studies have shown that the p30 protein can induce neutralizing antibodies against the virus. A recent study showed that p30, CD2V and K205R immunization can delay the onset of viremia in pigs and provide partial protection against ASFV challenge with the virulent strain HLJ / 18.

[0004] Currently, the prevention and control of many animal diseases mainly rely on traditional vaccines, such as inactivated vaccines, attenuated vaccines, vector vaccines and subunit vaccines, etc. Although scholars have conducted a lot of research on ASF vaccines, there is still no safe and effective vaccine strategy.

[0005] mRNA vaccines involve introducing mRNA encoding an antigenic protein into the body, where it is expressed, inducing a specific immune response and thus providing immune protection. mRNA vaccines offer advantages such as rapid response to pathogen mutations, simple production processes, high production efficiency, and ease of scalability. Unlike traditional vaccines that rely on antigens or attenuated viruses to stimulate the immune system, mRNA vaccines do not contain antigens themselves, but instead primarily consist of mRNA encoding the antigen. While these antigen-encoding mRNAs can be translated into antigenic proteins within cells, the key challenge remains whether they are immunogenic and can stimulate an immune response in the body to produce antibodies. mRNA vaccines have shown great potential, however, no mRNA vaccines for animal use are currently commercially available. In light of this, the present invention was proposed. Summary of the Invention

[0006] One object of the present invention is to provide an mRNA artificially produced by molecular biology technology, which can be expressed in vivo.

[0007] Another object of the present invention is to provide an mRNA that can be expressed in an organism and induce the organism to produce a specific immune response and antibodies.

[0008] Another object of the present invention is to provide an mRNA that can express the p30 protein of ASFV in an organism and induce the organism to produce a specific immune response and antibodies.

[0009] Another object of the present invention is to provide an mRNA that can express the p30 protein of ASFV in pigs, induce the pigs to produce a specific immune response, produce antibodies, and thus form an immune barrier against ASFV.

[0010] The fifth object of the present invention is to provide a method for preparing a vaccine, which is conducive to the preparation of an mRNA vaccine against ASFV.

[0011] The sixth object of the present invention is to provide an application of mRNA in the preparation of ASFV vaccine.

[0012] An mRNA comprises RNA transcribed from each of the following sequences:

[0013] 5'-UTR sequence, encoding the 5' untranslated region of porcine TUBA1A and its homologous sequence;

[0014] 3'-UTR sequence, encoding the 3' untranslated region of porcine TUBA1A and its homologous sequence;

[0015] A signal peptide sequence encoding a signal polypeptide of porcine IFNB1 and a homologous sequence thereof; and

[0016] Antigen protein coding gene sequence.

[0017] The mRNA of the present application also comprises a cap (Cap) structure, which is arranged at the 5' end of the mRNA and is denoted as: 5' cap structure. Common cap structures are, for example, Cap0, Cap1 and Cap2. Cap0 refers to the ribose of the terminal nucleotide being unmethylated, Cap1 refers to the ribose of one terminal nucleotide being methylated, and Cap2 refers to the ribose of two terminal nucleotides being methylated. Specifically, for example, m7G5'ppp5'Np, m7G5'ppp5'NmpNp and m7G5'ppp5'NmpNmpNp, etc.

[0018] The mRNA of the present application also comprises a poly A sequence.

[0019] One 5'-UTR embodiment of the mRNA of the present application has a sequence comprising the nucleotide sequence shown in SEQ ID NO. 1, and a sequence having more than 70% homology thereto.

[0020] Another 5'-UTR embodiment of the mRNA of the present application has a sequence comprising the nucleotide sequence shown in SEQ ID NO. 1, and a sequence having more than 75% homology thereto.

[0021] Another 5'-UTR embodiment of the mRNA of the present application has a sequence comprising the nucleotide sequence shown in SEQ ID NO. 1, and a sequence having more than 80% homology thereto.

[0022] Another 5'-UTR embodiment of the mRNA of the present application has a sequence comprising the nucleotide sequence shown in SEQ ID NO. 1, and a sequence having more than 85% homology thereto.

[0023] Another 5'-UTR embodiment of the mRNA of the present application has a sequence comprising the nucleotide sequence shown in SEQ ID NO. 1, and a sequence having more than 90% homology thereto.

[0024] Another 5'-UTR embodiment of the mRNA of the present application has a sequence comprising the nucleotide sequence shown in SEQ ID NO. 1, and a sequence having more than 95% homology thereto.

[0025] One 3'-UTR embodiment of the mRNA of the present application has a sequence comprising the nucleotide sequence shown in SEQ ID NO. 2, and a sequence having more than 70% homology thereto.

[0026] Another 3'-UTR embodiment of the mRNA of the present application has a sequence comprising the nucleotide sequence shown in SEQ ID NO. 2, and a sequence having more than 75% homology thereto.

[0027] Another embodiment of the 3'-UTR of the mRNA of the present application, the sequence comprises the nucleotide sequence as shown in SEQ ID NO. 2, and a sequence with more than 80% homology thereof.

[0028] Another embodiment of the 3'-UTR of the mRNA of the present application, the sequence comprises the nucleotide sequence as shown in SEQ ID NO. 2, and a sequence with more than 85% homology thereof.

[0029] Another embodiment of the 3'-UTR of the mRNA of the present application, the sequence comprises the nucleotide sequence as shown in SEQ ID NO. 2, and a sequence with more than 90% homology thereof.

[0030] Another embodiment of the 3'-UTR of the mRNA of the present application, the sequence comprises the nucleotide sequence as shown in SEQ ID NO. 2, and a sequence with more than 95% homology thereof.

[0031] Another embodiment of the 3'-UTR of the mRNA of the present application, the sequence comprises the nucleotide sequence as shown in SEQ ID NO. 2, and a sequence with more than 95% homology thereof.

[0032] Another embodiment of the signal peptide sequence of the mRNA of the present application, the sequence comprises the nucleotide sequence as shown in SEQ ID NO. 3, and a sequence with more than 70% homology thereof.

[0033] Another embodiment of the signal peptide sequence of the mRNA of the present application, the sequence comprises the nucleotide sequence as shown in SEQ ID NO. 3, and a sequence with more than 75% homology thereof.

[0034] Another embodiment of the signal peptide sequence of the mRNA of the present application, the sequence comprises the nucleotide sequence as shown in SEQ ID NO. 3, and a sequence with more than 80% homology thereof.

[0035] Another embodiment of the signal peptide sequence of the mRNA of the present application, the sequence comprises the nucleotide sequence as shown in SEQ ID NO. 3, and a sequence with more than 85% homology thereof.

[0036] Another embodiment of the signal peptide sequence of the mRNA of the present application, the sequence comprises the nucleotide sequence as shown in SEQ ID NO. 3, and a sequence with more than 90% homology thereof.

[0037] Another embodiment of the signal peptide sequence of the mRNA of the present application, the sequence comprises the nucleotide sequence as shown in SEQ ID NO. 3, and a sequence with more than 95% homology thereof.

[0038] The mRNA vaccine of the present application, the antigen protein coding gene sequence adopts the nucleic acid sequence corresponding to the ASFV virulence protein, such as: p30 protein and its homologous protein, such as: the sequence shown in SEQ ID NO. 4 and its homologous sequence.

[0039] Another specific embodiment of the mRNA antigen protein coding gene sequence of the present application, the sequence comprises the nucleotide sequence shown in SEQ ID NO. 4, and the sequence with more than 70% homology thereof.

[0040] Another specific embodiment of the mRNA antigen protein coding gene sequence of the present application, the sequence comprises the nucleotide sequence shown in SEQ ID NO. 4, and the sequence with more than 75% homology thereof.

[0041] Another specific embodiment of the mRNA antigen protein coding gene sequence of the present application, the sequence comprises the nucleotide sequence shown in SEQ ID NO. 4, and the sequence with more than 80% homology thereof.

[0042] Another specific embodiment of the mRNA antigen protein coding gene sequence of the present application, the sequence comprises the nucleotide sequence shown in SEQ ID NO. 4, and the sequence with more than 85% homology thereof.

[0043] Another specific embodiment of the mRNA antigen protein coding gene sequence of the present application, the sequence comprises the nucleotide sequence shown in SEQ ID NO. 4, and the sequence with more than 90% homology thereof.

[0044] Another specific embodiment of the mRNA antigen protein coding gene sequence of the present application, the sequence comprises the nucleotide sequence shown in SEQ ID NO. 4, and the sequence with more than 95% homology thereof.

[0045] Another mRNA, which is transcribed from an artificial plasmid, the plasmid comprising:

[0046] a promoter (such as: T7 promoter);

[0047] a 5'-UTR sequence, which is the 5' untranslated region encoding pig TUBA1A and its homologous sequence;

[0048] a 3'-UTR sequence, which is the 3' untranslated region encoding pig TUBA1A and its homologous sequence;

[0049] a signal peptide sequence, which is the signal polypeptide encoding pig IFNB1 and its homologous sequence; and

[0050] an antigen protein coding gene sequence.

[0051] Another mRNA, which is transcribed from a pUC57 plasmid, the plasmid comprising:

[0052] a promoter (such as: T7 promoter);

[0053] a 5'-UTR sequence, which is a 5' untranslated region of porcine TUBA1A and a homologous sequence thereof;

[0054] a 3'-UTR sequence, which is a 3' untranslated region of porcine TUBA1A and a homologous sequence thereof;

[0055] a signal peptide sequence, which is a signal polypeptide of porcine IFNB1 and a homologous sequence thereof; and

[0056] an antigen protein coding gene sequence.

[0057] The mRNA provided by the present application can be prepared by the following method:

[0058] First, a 5'-UTR sequence (such as, but not limited to, a 5' untranslated region of porcine TUBA1A and a homologous sequence thereof), a 3'-UTR sequence (such as, but not limited to, a 3' untranslated region of porcine TUBA1A and a homologous sequence thereof), a signal peptide sequence (such as, but not limited to, a signal polypeptide of porcine IFNB1 and a homologous sequence thereof), and an antigen protein coding gene sequence (such as, but not limited to, a p30 protein of ASFV) are cloned into a plasmid (such as, but not limited to, pUC57);

[0059] Then, the above plasmid is used as a template, a nucleotide sequence containing a promoter (such as: T7) is used as an upstream primer (such as: SEQ ID NO. 5), and a sequence containing ployT and part of the 3'-UTR sequence is used as a downstream primer (such as: SEQ ID NO. 6) to perform PCR reaction to prepare an in vitro transcription template containing ployA;

[0060] Next, a transcription enzyme (such as: T7 transcription enzyme) and a capping enzyme perform in vitro transcription, and a 5' cap structure is added to the 5' end of the mRNA, and the mRNA is obtained.

[0061] The mRNA of the present application is used as a drug (vaccine) when it is used to act on in vitro cells or in vivo cells to achieve the purpose of antibody production by cells.

[0062] The mRNA of the present application is mixed with other excipients to prepare a drug (vaccine) for anti-virus (such as: ASFV).

[0063] These pharmaceutical excipients can be various conventional excipients used in various formulations, such as, but not limited to, isotonic agents, buffers, flavoring agents, excipients, fillers, binders, disintegrants, and lubricants, etc. They can also be selected for compatibility with the substance, such as emulsifiers, solubilizers, bacteriostatic agents, analgesics, and antioxidants, etc. Such excipients can effectively improve the stability and solubility of the compounds contained in the composition or modify the release rate and absorption rate of the compounds, thereby improving the metabolism of various compounds in the body and enhancing the administration effect of the composition.

[0064] In aqueous injection, the excipients generally include isotonic agents and buffers, and necessary emulsifiers (such as Tweeen-80, Pluronic, and Poloxamer, etc.), solubilizers, and bacteriostatic agents, etc. In addition, it also includes other pharmaceutically acceptable pharmaceutical excipients, such as antioxidants, pH adjusters, and analgesics, etc.

[0065] The excipients used for preparing oral liquid formulations generally include solvents, and necessary flavoring agents, bacteriostatic agents, emulsifiers, and coloring agents, etc.

[0066] The excipients used for preparing tablets generally include fillers (such as starch, sugar powder, dextrin, lactose, compressible starch, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, and mannitol, etc.), binders (such as ethanol, starch paste, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, gelatin solution, sucrose solution, and aqueous or alcoholic solution of polyvinylpyrrolidone, etc.), disintegrants (such as dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, and cross-linked sodium carboxymethyl cellulose), and lubricants (such as magnesium stearate, microcrystalline silica, talc, hydrogenated vegetable oil, polyethylene glycol 4,000, polyethylene glycol 6,000, and magnesium lauryl sulfate, etc.), etc.

[0067] The excipients used for preparing emulsions are generally water, oil (such as fatty acids), emulsifiers, and necessary preservatives and flavoring agents, etc.

[0068] Various excipients are combined with the mRNA to form a dosage form that is beneficial for drug delivery, such as, but not limited to, aqueous injection, powder injection, pill, powder, tablet, patch, suppository, emulsion, cream, gel, granule, capsule, aerosol, spray, powder spray, sustained-release preparation, and controlled-release preparation. In addition, excipients such as, but not limited to, gelatin, albumin, chitosan, polyether, and polyester macromolecular materials such as, but not limited to, polyethylene glycol, polyurethane, polycarbonate, and copolymers thereof can be used to achieve specific drug delivery purposes or methods, such as sustained-release drug delivery, controlled-release drug delivery, and pulse drug delivery. The so-called "beneficial for drug delivery" mainly refers to, but is not limited to, improving therapeutic effect, improving bioavailability, reducing toxic side effects, and improving patient compliance, etc.

[0069] The mRNA of the present application is combined with other excipients, such as liposome wrapping, to further improve the efficacy of the mRNA.

[0070] The mRNA of the present application is also loaded or coated on a stent material as an active ingredient, such as a biocompatible degradable material mixed to form a microneedle and a microneedle array thereof, or loaded in a metal microneedle to form a microneedle chip. When the microneedle pierces the skin, the mRNA is released in the dermis layer, improving the vaccine efficacy of the mRNA.

[0071] It has been verified that the prepared mRNA is injected into pigs, and a high level of antibody, such as an antibody against the p30 protein of ASFV, can be produced one week after two immunizations, which can be used as an active substance for the preparation of a vaccine, such as the preparation of an African swine fever virus vaccine.

[0072] The p30 protein mRNA provided by the present application is composed of a selected 5'UTR sequence, a signal peptide sequence, a 3'UTR sequence, and a p30 coding gene sequence, and is constructed by transcription. After immunizing pigs, the mRNA can produce a high level of antibody. Compared with other several mRNA vaccines tried, the present application not only realizes the preparation of mRNA against the p30 protein of African swine fever virus, but also realizes the production of specific antibody against the p30 protein of African swine fever virus in pigs. The provided mRNA can be used as an active substance for the preparation of an African swine fever virus vaccine.

[0073] The p30 protein mRNA described in the present application has no risk of introducing toxic substances compared with traditional vaccines, has high safety, and has a short production cycle. BRIEF DESCRIPTION OF DRAWINGS

[0074] Figure 1 A schematic diagram of an artificial plasmid used to prepare the mRNA vaccine of the present application;

[0075] Figure 2The agarose gel electrophoresis chart of mRNA vaccine prepared by the present application;

[0076] Figure 3 The mRNA gel electrophoresis chart after in vitro transcription of mRNA vaccine prepared by the present application;

[0077] Figure 4 The IFA chart of PK15 cells transfected with p30 mRNA;

[0078] Figure 5 The ELISA result chart after p30 mRNA immunization of pigs. DETAILED DESCRIPTION

[0079] The technical solutions of the present application are described in detail below in combination with the drawings. The embodiments of the present application are only used to illustrate the technical solutions of the present application and not to limit the same. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the spirit and scope of the technical solutions of the present application, and all of them should be covered in the scope of the claims of the present application.

[0080] Example 1 p30 mRNA sequence and preparation method thereof

[0081] In this embodiment, the UTR sequence, signal peptide sequence, p30 gene coding sequence and synthetic gene sequence of the present application are provided. The specific sequences are shown in Table 1 below.

[0082] Table 1

[0083]

[0084]

[0085] Preparation of p30 mRNA:

[0086] The mRNA preparation process of the African swine fever virus p30 protein provided in this embodiment is as follows:

[0087] (1) DNA sequence synthesis: design the DNA sequence, and the sequence of the target fragment is 5' UTR sequence, signal peptide sequence, p30 gene coding sequence, and 3' UTR sequence in sequence. The DNA sequence is synthesized by Shengong Bioengineering (Shanghai) Co., Ltd. and cloned into pUC57 plasmid to obtain recombinant plasmid pUC57-p30 plasmid, see Figure 1 .

[0088] The 5'UTR sequence and the 3'UTR sequence described in this embodiment are both non-coding regions of porcine TUBA1A. The signal peptide sequence described in this embodiment is a signal peptide sequence of porcine IFNB1. The p30 gene coding sequence described in this embodiment is an optimized sequence.

[0089] (2) PCR template preparation: prepare a 50 μl PCR reaction system, including: 50 ng of plasmid containing the target DNA, 1 μM of upstream primer and downstream primer, 25 μl of DreamTaq PCR master mix (2x), and enzyme-free water to make up 50 μl. The PCR reaction conditions are as follows: 95°C pre-denaturation for 3 min, 95°C denaturation for 30 s, 56°C pre-denaturation for 30 s, 72°C extension for 1 min, 35 cycles, and 72°C final extension for 10 min. The PCR template containing poly A nucleic acid is obtained.

[0090] In this embodiment, the PCR product is purified by using a PCR product purification kit, and the purified PCR product is subjected to nucleic acid gel electrophoresis, and the electrophoresis result is shown in Figure 2 The primer set sequence used in this embodiment is shown in Table 2.

[0091] Table 2

[0092]

[0093] (3) In vitro transcription and capping and purification: use an in vitro transcription and capping kit to perform in vitro transcription on the PCR product template sequence containing a T7 promoter. In a 20 μl system, add: 10 μl of 2x NTP / CAP, 2 μl of 10x Reaction buffer, 0.2 μg of PCR amplified template, 2 μl of Enzyme mix, and enzyme-free water to make up 20 μl. After incubation at 37°C for 1 h, add 1 μl of TURBO DNase to the reaction system, mix well, and incubate at 37°C for 15 min. After the reaction is completed, use an mRNA purification kit to purify the obtained mRNA. Perform agarose gel electrophoresis analysis on the purified mRNA, and the electrophoresis result is shown in Figure 3 , where the arrow is the mRNA band.

[0094] Example 2: p30 mRNA expression identification

[0095] In this embodiment, the expression analysis of the p30 mRNA in PK15 cells is as follows:

[0096] (1) Prepare the p30 mRNA according to Example 1.

[0097] (2) Seed the PK15 cells in a 12-well plate, and when the cells grow to 80-90% confluence, perform p30 mRNA transfection.

[0098] (3) mRNA transfection: According to the lip2000 transfection reagent instruction, 2 pg p30 mRNA was mixed with 4 pl liposome lip2000 uniformly, and was placed at room temperature for 30 min. The mRNA-liposome mixture was inoculated into PK15 cells.

[0099] (4) Indirect immunofluorescence detection: After transfection for 24 h, the cells were fixed, and the monoclonal antibody of mouse anti-p30 protein prepared in the laboratory was incubated. After the incubation of the primary antibody, the secondary antibody of rabbit anti-mouse was incubated, and the expression of p30 was detected under a fluorescence microscope. The detection results are shown in Figure 4 , which show that the p30 protein is successfully expressed in the PK15 cells.

[0100] Example 3 p30 mRNA immunization of pigs

[0101] In this example, the pigs were immunized with the aforementioned p30 mRNA, and the specific antibody level of the pigs after immunization was detected.

[0102] The p30 mRNA was wrapped with a lipid nanoparticle (LNP), and the LNP wrapping was completed by Shanghai Lan'ou Biomedicine Co., Ltd. The 30-day-old commercial pigs were randomly divided into 2 groups, with 2 pigs in each group. The pigs in the experimental group were injected with 50 pg mRNA (100 pl) intramuscularly, and the pigs in the control group were injected with the same amount of LNP intramuscularly. The pigs were boosted once 21 days after the initial immunization. After the initial immunization, blood was collected on days 21, 28, 35, and 42, and serum was collected. The p30 antibody level was determined by using the indirect ELISA method constructed in the laboratory (Chinese Journal of Animal Infectious Diseases [J], 2022, 30(03), 158-165).

[0103] The ELISA detection results are shown in Figure 5 , and the OD450 determination results show the antibody levels of the p30 mRNA immunization group and the LNP control group. After the second immunization, the p30 mRNA can rapidly stimulate the pigs to produce specific antibodies against the p30 protein of African swine fever virus (OD450 value of about 3.8) within one week, and high levels of antibodies can be detected on day 21 after the second immunization. After the immunization of the pigs in the LNP control group, the antibody level did not change significantly.

[0104] Accordingly, the technical solutions provided in this example not only realize the preparation of mRNA against the p30 protein of African swine fever virus, but also realize the production of specific antibodies against the p30 protein of African swine fever virus in pigs by mRNA. The provided mRNA can be used as an active substance for the preparation of vaccines against African swine fever virus.

Claims

1. A nucleic acid molecule, characterized in that As shown in SEQ ID NO.

5.

2. A method for preparing mRNA of a p30 protein of African swine fever virus, comprising: cloning a 5'-UTR sequence, a 3'-UTR sequence, a signal peptide sequence and an antigen protein coding gene sequence into a plasmid first; then, using the plasmid as a template, using a nucleotide containing a promoter as an upstream primer and a nucleotide containing ployT and part of the 3'-UTR as a downstream primer to perform PCR reaction to prepare an in vitro transcription template containing ployA; then, performing in vitro transcription by a transcription enzyme and a capping enzyme and adding a 5' cap structure to the 5' end of the mRNA, thereby obtaining the mRNA; the 5'-UTR sequence is a nucleotide sequence as shown in SEQ ID NO. 1; the 3'-UTR sequence is a nucleotide sequence as shown in SEQ ID NO. 2; the signal peptide sequence is a nucleotide sequence as shown in SEQ ID NO. 3; the antigen protein coding gene sequence is a nucleotide sequence as shown in SEQ ID NO. 4.

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