A pseudorabies immunogen composition and uses thereof

Abstract

The present application relates to the technical field of biotechnology, in particular to a pseudorabies immunogen composition and application thereof. The present application provides a pseudorabies immunogen composition, which comprises at least one antigen saRNA (self-replicating RNA) molecule; the antigen saRNA molecule comprises at least one open reading frame (ORF), and the open reading frame ORF encodes a polypeptide comprising a gD antigen from a pseudorabies virus. The present application uses the self-replicating RNA technology to prepare a pseudorabies vaccine, and can protect the infection of the pseudorabies virus, which effectively solves the shortcoming of low protection efficiency of the previous pseudorabies vaccine on the pseudorabies virus.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a pseudorabies immunogen composition and use thereof. BACKGROUND

[0002] Pseudorabies of pigs is an acute infectious disease caused by Pseudorabies virus (PRV). At present, pigs are the only known natural host of PRV, and its infection can cause a large number of deaths in newborn piglets, with a morbidity of 100%. The morbidity of weaned piglets is 20% to 40%, and the mortality is 10% to 20%, mainly showing symptoms such as neurological symptoms, diarrhea, vomiting, etc. Respiratory symptoms of fattening pigs mainly include difficulty, growth arrest, etc.; adult pigs are generally subclinical infection, with mild symptoms; pregnant sows infected can have abortion, stillbirth; boars infected are often infertile. The disease is epidemic in pig farms, and is one of the major infectious diseases that endanger the global pig industry.

[0003] PRV, also known as SuHV-1 or Aujeszky's disease virus (ADV), belongs to the herpesvirus subfamily of the herpesvirus family. PRV is a double-stranded DNA virus, with a viral genome of about 145 kb, encoding 70 to 100 proteins, most of which are capsid proteins, envelope proteins, epidermal proteins and enzymes. Eleven glycoproteins (gB, gC, gD, gE, gG, gH, gI, gK, gL, gM and gN) and four transmembrane proteins (UL20, UL43, US9 and UL24) are identified on the envelope of the virion. During the infection process, gC, gB, gD, gH and gL are involved in the invasion of the virus and are the main antigens that stimulate the host's innate immune response. Among them, gD is the main glycoprotein of PRV, which can stimulate the body to produce neutralizing antibodies against PRV.

[0004] Vaccine immunization is the main means of preventing and controlling pseudorabies of pigs. More and more evidence shows that gD has less mutation characteristics and is a key protein that activates the humoral and cellular immune response, making it a promising target for developing new vaccines. The expression amount of the protein directly affects the immune dose, the immune effect and the cost of the vaccine.

[0005] The traditional vaccines currently used in the pig industry have many shortcomings. Inactivated vaccines or subunit vaccines have low immune efficacy, large immune side effects, etc.; attenuated live vaccines have the risk of reversion, and other components of the virus can easily cause adverse reactions or DNA recombination of the virus, etc. leading to some genetic mutation risks. At the same time, traditional vaccines also have the characteristics of long production cycle, high production cost, and relatively lagging application in emergency situations.

[0006] In view of the above problems of the current pseudorabies vaccine, the market demand for a vaccine that can effectively provide effective protection, has a short production cycle and low cost has become increasingly urgent. In recent years, with the application of mRNA vaccine in the prevention of new crown, it is confirmed that mRNA vaccine is an effective and rapid prevention and control means for epidemic mutant strains.

[0007] mRNA vaccine is a new type of vaccine that clones the encoded antigen gene of interest into the corresponding plasmid vector, produces a large amount of mRNA through in vitro transcription, and encapsulates the mRNA in a nanocarrier, and then delivers it into the host body. After vaccination and uptake by antigen-presenting cells, mRNA is transported to the cytoplasm, and after antigen processing, it triggers an MHC presentation cascade. Therefore, antigen-presenting cells can activate CD8 + and CD4 + T cells after presenting tumor-associated antigens on MHC class I and MHC class II. In addition, CD4 + T cells can co-activate antigen-specific B cells and induce a humoral immune response. As antigen-presenting cells, B cells can in turn activate CD4 + T cells after internalizing extracellular proteins and presenting them to MHC class II on B cells.

[0008] Compared with subunit vaccines, inactivated virus vaccines and attenuated live virus vaccines, and DNA-based vaccines, mRNA vaccines have several significant advantages:

[0009] 1. Safety: Since mRNA is a non-infectious, non-integrating platform, there is no potential risk of infection or insertion mutation. In addition, its in vivo half-life can be adjusted by using various modifications and delivery methods. Studies have shown that nucleic acid modification can also down-regulate the inherent immunogenicity of mRNA to further improve safety and reduce side effects.

[0010] 2. Effectiveness: Various modifications make mRNA more stable and highly translatable, and by constructing mRNA onto a carrier molecule, it can be quickly taken up and expressed in the cytoplasm, enabling effective in vivo delivery. mRNA is the smallest genetic carrier, so it avoids anti-carrier immune responses and can be administered repeatedly.

[0011] 3. High production efficiency: mRNA vaccines have the potential for rapid, inexpensive and scalable manufacturing, with reduced dosage, which can greatly save production costs and reduce the sales price of drugs, benefiting a wider range of farmers. At the same time, mRNA vaccines have the advantages of simple and fast production process, good safety, and ease of development of multi-link and multi-valent vaccines, which are significantly superior to subunit vaccines or inactivated vaccines and attenuated vaccines.

[0012] mRNA vaccines have been used in a variety of infectious diseases and tumor treatment research. Including conventional mRNA vaccine technology, self-replicating RNA vaccine technology, trans-amplifying RNA vaccine technology and circular RNA (Circular RNA) vaccine technology. Among them, self-replicating RNA vaccine is a better alternative. In addition to carrying the coding sequence (open reading frame, ORF) of the antigen, the RNA also carries a sequence of an RNA replicase (nsp1, 2, 3, 4). After this RNA replicase is produced, it can use the coding sequence of the antigen as a template to produce more copies of the antigen saRNA molecule. Therefore, the self-replicating RNA vaccine not only has all the advantages of the mRNA vaccine, but also has the advantages of low dosage, small side effects and lower production cost. However, there is currently no self-replicating RNA vaccine for preventing porcine pseudorabies. SUMMARY

[0013] Therefore, the technical problem to be solved by the present application is to provide a pseudorabies immunogen composition and uses thereof.

[0014] To this end, the present application provides the following technical solutions:

[0015] The embodiments disclosed in the present application relate to a pseudorabies immunogen composition, comprising at least one antigen saRNA molecule; the antigen saRNA molecule comprises at least one open reading frame ORF, and the open reading frame ORF encodes a polypeptide comprising a gD antigen from a pseudorabies virus.

[0016] In some embodiments, the pseudorabies virus can be selected from the Chinese pseudorabies virus dominant epidemic strain since 2010; preferably, the PRV AH02LA variant strain is selected. This strain is a PRV variant strain isolated from Anhui, China in 2012. Sequence analysis shows that AH02LA belongs to the same branch as other new PRV variants isolated in China after 2011. Research data shows that intranasal PRV AH02LA infection causes 100% morbidity or mortality in 28-day-old and 70-day-old pigs, and the main clinical symptoms of weaned piglets are similar to those of other virulent strains and classic strains, including high fever, respiratory distress, depression, anorexia, general nervous system symptoms and diarrhea.

[0017] In some embodiments, the polypeptide of the gD antigen of the PRV AH02LA variant strain is an amino acid sequence as shown in SEQ ID NO. 1 or an amino acid sequence having 80% or more homology thereto.

[0018] In some embodiments, the polypeptide sequence of the gD antigen of the PRV AH02LA variant strain is an amino acid sequence having 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.1%, 99.2%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 99.99% or more homology to the amino acid sequence as set forth in SEQ ID NO. 1.

[0019] In some embodiments, the DNA sequence of the polypeptide of the gD antigen of the PRV AH02LA variant strain is codon-optimized to be a nucleotide sequence as set forth in SEQ ID NO. 2 or a nucleotide sequence having 80% or more homology thereto.

[0020] In some embodiments, the DNA sequence of the polypeptide of the gD antigen of the PRV AH02LA variant strain is a nucleotide sequence having 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.1%, 99.2%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 99.99% or more homology to the nucleotide sequence as set forth in SEQ ID NO. 2.

[0021] In some embodiments, the DNA sequence of the polypeptide of the gD antigen of the PRV AH02LA variant strain is codon-optimized to be a nucleotide sequence as set forth in SEQ ID NO. 3 or a nucleotide sequence having 80% or more homology thereto.

[0022] In some embodiments, the DNA sequence of the polypeptide of the gD antigen of the PRV AH02LA variant strain is a nucleotide sequence having 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.1%, 99.2%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 99.99% or more homology to the nucleotide sequence as set forth in SEQ ID NO. 3.

[0023] In some embodiments, the antigen saRNA molecule comprises, in the 5’-3’ direction, the following elements in sequence: a 5’ cap structure, a 5’ UTR sequence, a coding gene sequence of the gD antigen, a poly(A) tail, and a 3’ UTR sequence.

[0024] As a preferred embodiment, the antigen saRNA molecule further comprises an sg promoter (sgP), a signal peptide coding gene sequence and / or a sequence encoding replicase. The sgP is located upstream of the gD antigen coding gene and downstream of the 5'UTR sequence. The signal peptide coding gene sequence can optimize the expression amount of the gD antigen, thereby affecting the immune effect. The signal peptide coding gene sequence is inserted at the N-terminus of the gD antigen coding gene sequence, or the signal peptide coding gene sequence is inserted between the sgP sequence and the two sequence fragments of the gD antigen coding gene sequence.

[0025] In some embodiments, the 5'UTR sequence is an optimized 5'UTR sequence of human beta-globin.

[0026] In some embodiments, the 3'UTR sequence is a tandem of 2-3 optimized 3'UTR sequences of human beta-globin or a 3'UTR sequence of Xenopus beta-globin.

[0027] In some embodiments, the poly(A) tail has a length of 30-200 nucleotides.

[0028] In some embodiments, the RNA molecule comprises, in the 5'-3' direction, the following elements in order: a 5' cap structure, a 5'UTR sequence, a coding gene sequence encoding replicase, an sgP promoter, a signal peptide coding gene sequence, a gD antigen coding gene sequence, a poly(A) tail and a 3'UTR sequence.

[0029] In some embodiments, the sequence encoding replicase is a coding gene sequence of NSP 1, 2, 3, 4.

[0030] In a preferred embodiment, the signal peptide is selected from a pig interleukin 2 signal peptide, a pig interleukin 4 signal peptide or a PRV gD protein original signal peptide.

[0031] In a preferred embodiment, the gD antigen polypeptide is fused with a signal peptide at the N-terminus, and the gD antigen polypeptide is guided to express by a specific high expression signal peptide, such as a pig interleukin 2 signal peptide sequence as shown in SEQ ID NO. 4, or an amino acid sequence having 80% or more homology thereto. The pig interleukin 2 signal peptide sequence is codon-optimized, such as the nucleotide sequence as shown in SEQ ID NO. 5 or a nucleotide sequence having 80% or more homology thereto.

[0032] In preferred embodiments, the signal peptide sequence is an amino acid sequence having 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, 99.1%, 99.2%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 99.99% or more homology with the amino acid sequence as set forth in SEQ ID NO. 4.

[0033] In preferred embodiments, the gD antigen polypeptide is N-terminally fused with a signal peptide, and the gD antigen polypeptide is specifically guided to express by the signal peptide, and the signal peptide sequence is as set forth in SEQ ID NO. 6, or an amino acid sequence having 80% or more homology therewith.

[0034] In preferred embodiments, the gD antigen polypeptide is N-terminally fused with a signal peptide, and the gD antigen polypeptide is specifically guided to express by the signal peptide, and the signal peptide sequence is as set forth in SEQ ID NO. 7, or an amino acid sequence having 80% or more homology therewith.

[0035] In preferred embodiments, the gD antigen polypeptide is N-terminally fused with a signal peptide, and the gD antigen polypeptide is specifically guided to express by the signal peptide, and the signal peptide sequence is as set forth in SEQ ID NO. 7, or an amino acid sequence having 80% or more homology therewith.

[0036] In some embodiments, during in vitro transcription, 5-methylcytosine is incorporated into the CTP solution in the in vitro transcription system, and / or pseudouridine is incorporated into the UTP solution in the in vitro transcription system, and the molar ratio of 5-methylcytosine and / or pseudouridine incorporated is 10-100%. Further preferably, the molar ratio of 5-methylcytosine and / or pseudouridine incorporated can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a range between any two of the above values. Preferably, during in vitro transcription, the molar ratio of pseudouridine (ψTP) incorporated into the UTP solution in the in vitro transcription system is 50%. Further preferably, during in vitro transcription, the molar ratio of 5-methylcytosine (m5CTP) incorporated into the CTP solution in the in vitro transcription system is 50% or 100%.

[0037] Embodiments disclosed in the present application relate to a biomaterial, comprising any one of the following:

[0038] A1. A recombinant vector plasmid containing the sequence of the saRNA molecule;

[0039] A2. A host cell containing the recombinant vector described in A1.

[0040] The embodiments disclosed in the present application relate to a pharmaceutical composition comprising the pseudorabies immunogen composition and a pharmaceutically acceptable carrier.

[0041] In some embodiments, the carrier comprises lipid nanoparticles (LNP). The lipid nanoparticles are composed of an ionizable cationic lipid, a non-cationic lipid, a sterol, and a structurally modified PEG lipid. The composition of the lipid nanoparticles is ionizable cationic lipid (20-60 wt%), non-cationic lipid (20-55 wt%), cholesterol (19.5-55 wt%), and structurally modified PEG lipid (0.5-5 wt%). The ionizable cationic lipid can be DLin-MC3-DMA or SM-102, etc. The non-cationic lipid (neutral lipid) can be DSPC. The sterol is cholesterol. The structurally modified PEG lipid can be DMG-PEG 2000. These lipids are mixed in a certain proportion, and then processed into saRNA liposome nanoparticles together with saRNA in proportion. The nanoparticles can be processed using microfluidic nanometers or other methods. The size of the nanoparticles, the particle size distribution (PDI), and the charge (zeta potential) on the surface of the nanoparticles are crucial for the in vivo absorption of LNP and can determine the efficiency of LNP entering cells. The saRNA liposome nanoparticles can induce the body to produce neutralizing antibodies to protect the body from killing the pseudorabies virus. After the saRNA liposome nanoparticles are absorbed by cells, they enter the body cells. In the body cells, the liposomes rupture and release the encapsulated saRNA, which triggers protein synthesis with the help of the cell's own protein translation system.

[0042] The embodiments disclosed in the present application relate to the use of the pseudorabies immunogen composition, the biomaterial, or the pharmaceutical composition, which includes:

[0043] (1) the use in the preparation of a vaccine for preventing porcine pseudorabies;

[0044] (2) the use in the preparation of a product for inhibiting the pseudorabies virus;

[0045] (3) the use in the preparation of a product for detecting pseudorabies. The product includes a reagent or a kit.

[0046] 1. The pseudorabies immunogen composition provided by the application comprises at least one antigen saRNA molecule; the antigen saRNA molecule comprises at least one open reading frame ORF, and the open reading frame ORF encodes a polypeptide comprising a gD antigen from a pseudorabies virus strain; the application uses the technology of self-replicating RNA to produce a pseudorabies vaccine and can protect against infection of a pseudorabies virus, which effectively solves the shortcoming of low protection efficiency of the current pseudorabies vaccine against the pseudorabies virus.

[0047] 2. The pseudorabies immunogen composition provided by the application, the replication enzyme gene is introduced into the antigen saRNA molecule, so that the RNA of the targeted antigen can be replicated in large quantities, the antigen protein can be expressed in large quantities under the condition of low inoculation dose, the antigen protein expression time is very long, the body can continuously produce an immune response, and the immune time of the body to the pseudorabies virus is correspondingly prolonged, and compared with the traditional split vaccine, the most direct advantage is that the vaccine has a low use dose and small side effects; in addition, the reduction of the use dose can greatly save the production cost.

[0048] 3. The pseudorabies immunogen composition provided by the application, the antigen saRNA molecule further comprises an sg promoter (sgP), a signal peptide coding gene sequence and / or a sequence encoding a replication enzyme. The signal peptide coding gene sequence is contained in the N terminal of the gD antigen coding gene sequence, or the signal peptide coding gene sequence is inserted between the sgP sequence and the two sequence fragments of the gD antigen coding gene sequence; by providing the signal peptide upstream of the gF antigen polypeptide, the gD antigen polypeptide is guided to express by the specific high expression signal peptide, the expression amount of the antigen polypeptide can be increased, and a stronger immune effect can be triggered. In addition, the signal peptide screened in the application can specifically increase the expression amount of the gD protein in a pig cell line compared with the original signal peptide of the virus. Therefore, in actual application, the fusion antigen obtained by using the signal peptide screened in the application in combination with gD can maintain a strong immune effect while reducing the immune dose and reducing the cost of pig farms and breeders. BRIEF DESCRIPTION OF DRAWINGS

[0049] In order to more clearly illustrate the specific embodiments of the application or the technical solutions in the prior art, the drawings needed in the specific embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0050] Figure 1 is a structural schematic diagram of the saRNA molecule comprising gD in Example 1 of the application;

[0051] Figure 2Figure 1 is a representative electrophoretogram of the recombinant plasmid before linearization and enzyme digestion in Example 2 of the present application; in the figure, from left to right, lane 1 is a DNA molecular weight marker; lane 2: un-digested JF-AV02 plasmid; lane 3: JF-AV02 plasmid after enzyme digestion; lane 4 is a DNA molecular weight marker; lane 5: un-digested JF-AV03 plasmid; lane 6: JF-AV03 plasmid after enzyme digestion;

[0052] Figure 3 Figure 2 is a representative result of capillary electrophoresis analysis of JF-AV02 RNA in the (7) analysis step in Example 2 of the present application, the gD saRNA fragment integrity is more than 90%;

[0053] Figure 4 Figure 3 is a graph of the expression amount detection of gD after transfection of two codon-optimized saRNAs in Example 4 step (3); the base of logx is 10;

[0054] A: gD-1 / 2HiBiT transfection of pig ST cell line after the expression amount detection of reporter protein; gD1 / HiBiT antigen ORF nucleic acid sequence is composed of SEQ ID NO. 9, SEQ ID NO. 2, HiBiT in the order from 5' to 3' direction; gD2 / HiBiT antigen ORF is composed of SEQ ID NO. 9, SEQ ID NO. 3, HiBiT in the order from 5' to 3' direction;

[0055] B: gD-1 / 2HiBiT transfection of pig PK-15 cell line after the expression amount detection of reporter protein, gD1 / HiBiT antigen ORF nucleic acid sequence is composed of SEQ ID NO. 9, SEQ ID NO. 2, HiBiT in the order from 5' to 3' direction; gD2 / HiBiT is composed of SEQ ID NO. 9, SEQ ID NO. 3, HiBiT in the order from 5' to 3' direction;

[0056] Figure 5 Figure 4 is the expression amount of each liposome nanoparticle in pig PK15 cell line, pig ST cell line and the immune efficacy detection results in animals in Example 5 of the present application, wherein:

[0057] A is the gD protein expression amount of each liposome nanoparticle in pig PK15 cell line;

[0058] B is the gD protein expression amount of each liposome nanoparticle in pig ST cell line;

[0059] C is the relative titer graph result of neutralizing antibodies produced by each liposome nanoparticle in animals to trigger immune efficacy;

[0060] In the figure, sp1 is a liposome nanoparticle with a signal peptide sequence of the N-terminal signal peptide of porcine interleukin 2 (IL-2); sp2 is a liposome nanoparticle with a signal peptide sequence of the N-terminal signal peptide of porcine interleukin 4 (IL-4); sp3 is a liposome nanoparticle with a signal peptide sequence of the N-terminal natural signal peptide of PRV virus gD protein; Luc represents JF-Luc LNP; and compared with SP3, ** represents p<0.01, and *** represents p<0.001;

[0061] Figure 6 is the green fluorescent protein expression result of the nanoliposome particles with different modifications in embodiment 6 of the present application; in the figure: A: unmodified, B: 50% m5CTP modification alone, C: 50% ψTP modification alone, and D: 50% m5CTP+50% ψTP modification;

[0062] Figure 7 is the green fluorescent protein expression statistical result of the nanoliposome particles with different proportions in embodiment 6 of the present application;

[0063] Figure 8 is the experiment of JF-Luc RNA LNP and mRNA-Lucferase LNP in animals in embodiment 7 of the present application; the first row is four mice groups from left to right in order of mRNA-Lucferase LNP 5 μg, JF-Luc RNA LNP 5 μg, mRNA-Lucferase LNP 2.5 μg, and JF-Luc RNA LNP 2.5 μg; the two mice groups are the mice groups of mRNA-Lucferase that are not detectable, and from left to right, the remaining JF-Luc RNA LNP 5 μg and JF-Luc RNA LNP 2.5 μg;

[0064] Figure 9 is the detection graph of serum neutralizing antibody titer after immunizing piglets with JF-AV01-RNA LNP and other vaccines in embodiment 8 of the present application; in the figure, compared with the control group, * represents p<0.05, and **** represents p<0.0001;

[0065] Figure 10 is the graph of changes in cytokines in vivo after immunizing piglets with JF-AV01-RNA LNP and other vaccines in embodiment 8 of the present application; in the figure, A is the average level of IFN-γ of each group; B is the average level of IL-4 of each group; compared with the control group, * represents p<0.05, ** represents p<0.01, *** represents p<0.001, and **** represents p<0.0001;

[0066] Figure 11Figure 8 is a graph showing the body temperature changes of piglets after immunization with JF-AV01-RNA LNPs and challenge in Example 8 of the present application;

[0067] Figure 12 Figure 1 is a plasmid map of the recombinant plasmid JF-AV01 in Example 1 of the present application. DETAILED DESCRIPTION

[0068] The following examples are provided to better enable those skilled in the art to further understand and practice the application. They are not intended to limit the scope of the application in any way. The contents and scope of the present application are not limited to the contents of the examples, and any person skilled in the art can easily understand and implement them. Any product that is the same or similar to the present application obtained by the disclosure of the present application or by combining the present application with other prior art features falls within the scope of the present application.

[0069] The specific experimental steps or conditions are not indicated in the examples, and can be performed according to the conventional experimental steps described in the literature in the art or the operation or conditions. The reagents or instruments used are not indicated by the manufacturer, and are conventional reagent products that can be obtained by purchase.

[0070] In order to facilitate understanding, the present application provides the following scientific background information and definitions. Any technical feature disclosed herein can be part of each embodiment of the present application. Other technical features can also be provided in the context of the specification.

[0071] Cellular immunity / cellular immune response: Cellular immunity is generally associated with the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T lymphocytes, and various cytokine responses to antigens.

[0072] Humoral immunity / humoral immune response: Humoral immunity generally refers to antibody production and its accompanying helper processes. Typical features of a humoral immune response can be Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation, and memory cell production.

[0073] The main sequence of mature mRNA is the coding region open reading frame, and there are non-coding regions upstream 5' side and downstream, i.e. 5' UTR and 3' UTR. There are also 5' cap and 3' tail structures at both ends of eukaryotic mRNA molecules. The mRNA of prokaryotic cells generally has no tail, but the viral mRNA that infects eukaryotic cells generally has a poly A tail.

[0074] Open reading frame, ORF, is a continuous DNA or RNA sequence that starts with a start codon (ATG or AUG) and ends with a stop codon (TAA, TAG, TGA or UAA, UAG, UGA). An ORF usually encodes a protein or a fusion protein.

[0075] Cap, refers to the cap structure at the 5' end of mRNA, which is important for the stability of mRNA and its translation.

[0076] 5' untranslated region (5'UTR) is a short sequence between the cap structure and the start codon of the coding region, which is a highly sensitive region for translation initiation. The length, secondary structure and the number of start codons of 5'UTR can affect the efficiency of translation initiation. The length of 5'UTR is generally 100-200 nucleotides.

[0077] 3' untranslated region (3'UTR) is the transcription sequence after the stop codon, which contains the tailing signal. The mature mRNA generally has a poly A tail of 20-200 bases at its 3' end, which can prevent exonuclease degradation, is related to nuclear pore transport to the cytoplasm, and can also regulate the translation process.

[0078] Signal peptide is located at the N-terminus of secreted proteins. It is generally composed of 15-30 amino acids. It includes three regions: a positively charged N-terminus, called basic amino-terminal; a middle hydrophobic sequence. After the synthesis of the signal peptide sequence, it is recognized by the signal recognition particle (SRP), and protein synthesis is paused or slowed down. The signal recognition particle carries the ribosome to the endoplasmic reticulum, and protein synthesis starts again. Under the guidance of the signal peptide, the newly synthesized protein enters the endoplasmic reticulum lumen. The signal peptide sequence is then removed by signal peptidase. The termination transport sequence exists at the C-terminus of the nascent peptide chain. It can also not be removed by signal peptidase. The efficiency and functional characteristics of the signal peptide directly affect the subsequent protein expression yield induced by the signal peptide. Therefore, optimization of the signal peptide is a feasible path to improve protein yield.

[0079] Codon optimization. Codon is a triplet of ribonucleotide sequence used to encode amino acids. Some different codons can encode the same amino acid, which is called synonymous codon. There is a preference for codons in different organisms, i.e. uneven codon usage frequency. The secondary structure of the coding region and codon selection can affect the translation efficiency, and excessive secondary structure and rare codons can reduce the translation speed. Therefore, through codon optimization, the expression level of protein can be improved.

[0080] The DNA sequences in the following examples were synthesized by Nanjing Kingsriver Biotechnology Co., Ltd.

[0081] Liposome SM-102: MedChemExpresss; CAS No.: 2089251-47-6.

[0082] DMG-PEG 2000: Merk Company, CAS No.: 1397695-86-1.

[0083] DSPC (Distearyphosphatidylcholine): Merk, CAS#: 63-89-8.

[0084] Cholesterol: Merk, CAS#: 57-88-5.

[0085] LA-A inactivated vaccine was provided by Jiangsu Academy of Agricultural Sciences.

[0086] LA-2017 attenuated vaccine was provided by Jiangsu Academy of Agricultural Sciences.

[0087] The sequences involved in the following examples are shown in the following table:

[0088] Table 1, Sequences

[0089]

[0090]

[0091] The label HiBiT used in the following examples is a commercial label developed by Promega, see Marie K. Schwinn et al. for description, which is fused at the C-terminus of the polypeptide of interest, and can directly feedback the expression amount of the protein of interest.

[0092] Example 1 Construction of recombinant plasmid

[0093] (1) Selection of sequences

[0094] In this embodiment, the vector sequence used is referred to the published patent application CN 117205309 A, in which the nucleotide sequence (from 7622bp to 8340bp) in the original vector JF-FV01 is replaced by the sequence described in the present application; the coding gene of the signal peptide and the polypeptide of the gD antigen of the pseudorabies virus strain are the coding gene of the amino acid sequence shown in SEQ ID NO. 1.

[0095] Through sequence analysis and codon optimization, the above-mentioned nucleotide sequence of the polypeptide of the gD antigen from the pseudorabies virus strain is shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively. The signal peptide sequence is selected from the original signal peptide of PRV gD protein, and can also be selected from the signal peptide of porcine interleukin 2 and the signal peptide of porcine interleukin 4, as shown in SEQ ID NO. 4, SEQ ID NO. 6, and SEQ ID NO. 7, and the coding gene is shown in SEQ ID NO. 5, SEQ ID NO. 8, and SEQ ID NO. 9. The label at the C-terminus of the coding gene of the gD antigen polypeptide is the HiBiT label.

[0096] (2) Construction of recombinant plasmid

[0097] The vector JF-AV01 containing each component of the saRNA was prepared, and the plasmid map is shown in Figure 12 The T7 promoter, sg promoter, and chloramphenicol resistance sequence are common sequences. The template vector sequence used in the application is selected from CN 117205309 A, in which the nucleotide sequence (7622bp-8340bp) in the original vector JF-FV01 is replaced with the series of sequences described in the application, and the main molecular cloning elements are shown in Figure 1 as follows:

[0098] 1) 1 μg of JF-FV01 template plasmid was linearized by selecting ApaI restriction endonuclease (Nanjing Novozyme), and the reaction system was: reaction buffer 2 μL, ApaI restriction endonuclease 1 μL, template plasmid 1 μL, supplemented with enzyme-free water to 20 μL, and reacted at 37°C for 15 minutes.

[0099] 2) The linearized template was recovered according to the steps of the DNA recovery kit (TaKaRa).

[0100] 3) At 1-200 nucleotides upstream and downstream of the template plasmid cloning site, 15-25 bp homologous recombination arms were selected, respectively, and the homologous arms were designed upstream and downstream of the signal peptide-gD polypeptide nucleotide, and were synthesized by Nanjing Kingsway. According to the instructions of the homologous recombination kit (Nanjing Novozyme), homologous recombination was performed. Reaction at 37°C for 30 minutes.

[0101] 4) Thaw the cloning competent cells (Beijing Solabio) on ice, take 10 μL of the recombination product and add it to 100 μL of the competent cells, gently shake the tube wall to mix (do not shake to mix), and stand on ice for 30 minutes; after 42°C water bath heat shock for 45 seconds, immediately place on ice for cooling for 2 minutes. Add 900 μL of LB medium without antibiotics, and shake at 37°C for 1 hour (rotation speed 200-250 rpm). The corresponding LB solid medium containing antibiotics was preheated in a 37°C incubator. Centrifuge at 5000 rpm for 5 minutes, discard 900 μL of supernatant. Resuspend the bacterial cells with the remaining medium, and gently spread them on the plate containing chloramphenicol-resistant plates with a sterile spreader. Incubate in a 37°C incubator for 12 hours.

[0102] 5) Pick well-growing single colonies and inoculate them in LB medium containing chloramphenicol, and incubate at 37°C, 200 rpm.

[0103] 6) Extract the target plasmid according to the instructions of the plasmid extraction kit (Beijing Tiangen).

[0104] 7) The plasmid construction in the present application is carried out according to the above method.

[0105] The nucleic acid sequence of the gD antigen is SEQ ID NO. 3, the signal peptide sequence is selected as SEQ ID NO. 4, the constructed recombinant plasmid is named JF-AV01, and the plasmid map is as shown in Figure 12 The nucleic acid sequence of the gD antigen is SEQ ID NO. 2, the signal peptide nucleic acid sequence is selected as SEQ ID NO. 7, and the HiBiT tag is added to the 3' end of the gD antigen nucleic acid sequence. The constructed recombinant plasmid is named JF-AV02. The nucleic acid sequence of the gD antigen is SEQ ID NO. 3, the signal peptide nucleic acid sequence is selected as SEQ ID NO. 7, and the HiBiT tag is added to the 3' end of the gD antigen nucleic acid sequence. The constructed recombinant plasmid is named JF-AV03. In this way, the polypeptide sequence of the gD antigen is replaced by the sequence of firefly luciferase, and no signal peptide coding gene sequence is added. The constructed recombinant plasmid is named JF-Luc. The immunogen control table is shown in the following table:

[0106] Table 2: Immunogen and sequence control table

[0107]

[0108] Example 2 Preparation of saRNA plasmid

[0109] The present embodiment provides a method for preparing a saRNA plasmid, comprising the following steps:

[0110] (1) Enzymatic digestion

[0111] Take the recombinant plasmid JF-AV01-5 or JF-Luc control in Example 1 as the template plasmid, and use restriction endonuclease BspQI for enzymatic digestion, respectively. The enzyme digestion system is shown in the following table (take 200 μl reaction system as an example, Table 3): The enzyme digestion conditions are: 50°C, and the reaction time is 15-60 minutes. Then use Qiagen plasmid purification kit to recover the linearized plasmid. The representative electrophoretogram of the linearized plasmid and the recombinant plasmid before enzyme digestion is shown in Figure 2 Any commercialized endonuclease can be used.

[0112] Table 3. DNA template linearization reaction system

[0113] Component Amount / μl Final concentration Template plasmid 20μg 200ng / μl BspQI 10μl 1U / μl BspQI buffer 20μl 1× Enzyme-free water Supplemented to 200μl -

[0114] (2) In vitro transcription to synthesize saRNA

[0115] The in vitro synthesis of saRNA raw materials are linear DNA template, dNTP mixture, 10x transcription buffer, T7 RNA polymerase, inorganic pyrophosphatase, RNAase inhibitor, and enzyme-free water. The reaction conditions are 37°C water bath, 2-4 hours, and the reaction system is shown in Table 4. The raw materials can be commercially available. No 5-methylcytosine is added to the CTP solution; no pseudouridine is added to the UTP solution.

[0116] Table 4. The required components and amounts of each reagent for a 20 μl in vitro transcription system.

[0117] Component Amount Final concentration 10× transcription buffer 2μl 1× T7 RNA polymerase 2μl 20U / μl Inorganic pyrophosphatase 2μl 5mU / μl RNase inhibitor 1μl 2U / μl ATP solution 2μl 5mM / μl CTP solution 2μl 5mM / μl GTP solution 2μl 5mM / μl UTP solution 2μl 5mM / μl Template DNA 1μl 25ng / μl Enzyme-free water Supplemented to 20ul -

[0118] (3) Digestion of linear DNA template

[0119] To the saRNA obtained in step (2), add DNase I, 5 U of DNase I per 1 μg of saRNA, and incubate at 37°C for 15-60 minutes.

[0120] (4) Purification of saRNA

[0121] The reaction solution after digestion in step (3) is precipitated and purified with sodium acetate to obtain purified saRNA. The specific steps are as follows: the nucleic acid-containing solution is transferred to a new Eppendorf centrifuge tube with a pipette, and the volume of the nucleic acid solution is measured. Add 1 / 10 volume of 3 mol / L sodium acetate buffer. The final concentration of sodium acetate is 0.3 mol / L; mix well, accurately add 2 volumes of pre-cooled anhydrous ethanol, mix well, and place in ice bath for 15-30 min or 20-30 min, centrifuge at 16000g for 30 min.

[0122] (5) Capping

[0123] The purified saRNA in step (4) is incubated at 65°C for 1-30 min. Then a commercially available RNA capping kit is used for capping reaction. The cap compound here can be of different structures. In this example, the 5' cap structure is selected as 7mG(5')ppp N. Take a 20 μl reaction system as an example (see Table 5). Reaction conditions: 37°C water bath, 1-4h.

[0124] Table 5. The required components and amounts of each reagent for a 20 μl RNA capping reaction system.

[0125] Component Amount Final concentration 10× capping buffer 2μl 1× Vaccinia virus capping enzyme 1μl 0.5U / μl GTP solution 1μl 0.5mM S-adenosyl methionine 1μl 0.1mM saRNA 10μg 500ng / μl Enzyme-free water Supplemented to 20μl -

[0126] (6) Purification

[0127] The reaction product obtained in step (5) was precipitated and purified with sodium acetate (the method is the same as step (4)), to obtain the purified saRNA.

[0128] (7) Analysis

[0129] The saRNA containing gD antigen sequence prepared in step (6) (named JF-AV01-5 saRNA) was subjected to capillary electrophoresis analysis (Agilent Fragment Analyzer 5400) for sample loading, electrophoresis, and analysis, which were all performed according to the manufacturer's operation (Agilent RNA-FA kit) steps. The exemplary capillary electrophoresis analysis result of JF-AV02 is shown in FIG. 5, and the RNA integrity of gD antigen is 90.1%, which is greater than 80%. Similarly, the RNA integrity of gD antigen of JF-AV01 saRNA and JF-AV03-5 saRNA also achieved similar results greater than 80%. The above shows that the saRNA is successfully constructed. Figure 3

[0130] Example 3 Preparation of Lipid Nanoparticle (LNP)

[0131] The present embodiment provides a preparation method of a lipid nanoparticle, which can be used as an effective component of a drug. The method comprises the following steps:

[0132] Any one of the saRNAs prepared in Example 2 (JF-AV02 saRNA as an example) was diluted to a concentration of 5 μg / ml with a sodium acetate buffer solution with pH = 4 and 50 mM to obtain solution A;

[0133] SM-102, DSPC, cholesterol, and DMG-PEG 2000 were dissolved in pure ethanol to obtain solution B, in which the concentration of SM-102 was 50 mM, the concentration of DSPC was 20 mM, the concentration of cholesterol was 50 mM, and the concentration of DMG-PEG 2000 was 20 mM;

[0134] Each solution A and solution B were simultaneously added to a microfluidic mixer (Microlit), and the volume ratio of solution A to solution B was 8:1, and the total flow rate was 3 ml / min. The effluent was collected. The effluent was transferred to a dialysis tube (30K specification), and centrifuged to concentrate the saRNA to a concentration of 0.5 mg / ml, and then filtered with a 0.22 μm filter membrane, and the filtrate was collected, to prepare the saRNA lipid nanoparticle, i.e., to obtain the JF-AV01 saRNA LNP solution.

[0135] ​The particle size, PDI (particle distribution index), and zeta (Zeta potential) potential of JF-AV01, JF-AV02, JF-AV03, JF-AV04, JF-AV05, and JF-Luc RNA saRNA LNP (which can be abbreviated as JF-Luc RNA LNPs, and so on) solutions were measured using a Malvern instrument (Malvern, England). The encapsulation efficiency of the LNPs was calculated using an RNA Assay Kit (Thermo Fisher Scientific). The results are shown in Table 6 below.

[0136] Table 6: LNP characterization data

[0137] Solution Particle size (nm) Polydispersity index (PDI) Zeta potential (Mv) Encapsulation efficiency (%) JF-Luc LNPs 75.84 0.141 -2.62 100％ JF-AV01 LNPs 74.94 0.136 -2.06 100％ JF-AV02 LNPs 74.56 0.130 -1.88 100％ JF-AV03 LNPs 75.43 0.127 -2.01 100％ JF-AV04 LNPs 75.05 0.125 -2.34 100％ JF-AV05 LNPs 74.87 0.135 -1.91 100％

[0138] Example 4 In vitro expression of saRNA liposome nanoparticles

[0139] (1) A 96-well plate was taken, and ST or PK-15 cells (1 x 10 4 cells / well) were seeded and cultured in DMEM medium (Thermo Fisher Scientific) containing 10% serum and 1% double antibody for 16-18 hours.

[0140] (2) After step (1) was completed, two types of gD (JF-AV02 LNPs, JF-AV03 LNPs) prepared according to Example 3 (50 ng, 25 ng, 12.5 ng, 6.25 ng, 3.13 ng, 1.56 ng, referring to the RNA content encapsulated in the liposome nanoparticles) were added and cultured for 24 hours. In order to detect antigen expression, the recombinant plasmid used in Example 1 was added with a HiBit tag after the two gD sequences, respectively, wherein the gD1 corresponds to a DNA sequence including SEQ ID NO. 9, SEQ ID NO. 2, HiBit in turn from the 5'-3' direction, and the gD2 corresponds to a DNA sequence including SEQ ID NO. 9, SEQ ID NO. 3, HiBit in turn from the 5'-3' direction.

[0141] (3) After step (2) was completed, HiBit reagent (Promega) was added according to the supplier's instructions, and chemiluminescence was detected, as shown in Figure 4 A, the expression amounts of the two sequences in the pig cell line ST were similar, as shown in Figure 4 B, the expression amount of gD2 in the pig cell line PK-15 was slightly higher than that of gD-1. For the gD antigen, the nucleic acid sequence SEQ ID NO. 3 was selected for subsequent further signal peptide screening and animal immunization and challenge experiments.

[0142] Example 5 High expression signal peptide screening

[0143] I. Different signal peptides guide the expression of antigens in cells

[0144] The original N-terminal signal peptide sequence of JF-AV03 was replaced, and the replacement steps were the same as in Example 1. The original signal peptide sequence (sp3) of SEQ ID NO. 7 in the original JF-AV03 vector was replaced with the porcine interleukin 2 signal peptide sequence (sp1) SEQ ID NO. 4 to form JF-AV04, and the original signal peptide sequence (sp3) of SEQ ID NO. 7 in the original JF-AV03 vector was replaced with the porcine interleukin 4 signal peptide sequence (sp2) SEQ ID NO. 6 to form JF-AV05. Compared with the original signal peptide sequence (sp3) of SEQ ID NO. 7, the expression amount was compared, and the 96-well cell culture plate was prepared for inoculation of porcine PK-15 cell line and porcine ST cell line according to step (1) in Example 4. Add three kinds of gD saRNA liposome nanoparticles containing different signal peptides prepared according to Example 3 (300 ng, 150 ng, 75 ng, 37.5 ng, 18.75 ng, referring to the RNA content encapsulated in the liposome nanoparticles), and then add HiBit reagent (Promega) according to the supplier's instructions after 24 hours of culture. The chemiluminescence was detected according to the supplier's instructions (Promega) (A, B), and it can be seen that the chemiluminescence corresponding to the porcine interleukin 2 signal peptide is significantly higher than the other two groups, indicating that the porcine interleukin 2 signal peptide guided gD protein in vitro expression is superior to the porcine interleukin 4 and the original virus source signal peptide of gD, suggesting that the porcine interleukin 2 signal peptide guided gD antigen has in vivo expression ability. Figure 5

[0145] II. Detection of immune efficacy of antigens guided by different signal peptides in vivo.

[0146] Detection of immune efficacy of gD antigens guided by porcine interleukin 2 signal peptide (sp1), porcine interleukin 4 signal peptide (sp2), and gD original virus source signal peptide (sp3) in vivo. The experimental method is as follows:

[0147] (1) JF-AV03 LNPs (containing gD original virus source signal peptide, sp3), JF-AV04 LNPs (containing porcine interleukin 2 signal peptide, sp1), JF-AV05 LNPs (porcine interleukin 4 signal peptide, sp2), and JF-Luc LNPs were used to immunize 4-6 week old Balb / c mice, 5 mice in each group, and the leg muscle was injected with 1 μg (1 μg refers to the RNA content encapsulated in the liposome nanoparticles) dose of immunization once. After 21 days of immunization, the orbital blood was collected, and the serum was separated.

[0148] ​(2) ELISA detection: use pseudorabies gD protein (Wuhan Huamei Company) to coat ELISA plate at 4°C overnight; the serum in (1) above is gradiently diluted, the dilution factor is 3, and incubated with the coated gD protein in the ELISA plate at room temperature for 1 hour. After washing the plate, add HRP-conjugated anti-mouse IgG antibody and incubate at room temperature for 1 hour. After completing the above steps, wash the plate, use ELISA double-component color developing liquid (Beijing Solabio Company) to develop color at room temperature, after the color development is completed, add the termination reagent, and use an enzyme label instrument (FlexStation3) to detect the absorbance value OD450nm.

[0149] The results show that: three signal peptides successfully guide gD expression, as shown in Figure 5 C, and produce antibodies against gD antigen. Among them, the gD group mice guided by sp1 show higher serum neutralizing antibody levels than sp2 and sp3, indicating that sp1 guided gD antigen expression can produce strong body immunity and protection efficacy. Therefore, the porcine interleukin 2 signal peptide sequence (sp1) SEQ ID NO. 4 is preferred as the signal peptide for guiding gD antigen expression, and the HiBiT tag at the carbon terminal of gD antigen in JF-AV04 is deleted to form JF-AV01 carrier to prepare liposome nanoparticles for subsequent immunization and challenge experiments.

[0150] Example 6: Investigation of the effect of different nucleotide modifications on the expression amount of saRNA

[0151] This example investigates the effect of the incorporation of 5-methylcytosine m5CTP and pseudouridine ψTP in the CTP solution and / or UTP solution on the expression efficiency of saRNA synthesized by in vitro transcription.

[0152] I. The experimental groups are as follows:

[0153] (1) Unmodified group: neither the CTP solution nor the UTP solution is doped with m5CTP and pseudouridine ψTP.

[0154] (2) 50% m5CTP modification group alone: 50% of the CTP in the CTP solution is replaced by m5CTP.

[0155] (3) 50% ψTP modification group alone: 50% of the UTP in the UTP solution is replaced by pseudouridine ψTP.

[0156] (4) 50% m5CTP + 50% ψTP modification group: 50% of the CTP in the CTP solution is replaced by m5CTP, and at the same time, 50% of the UTP in the UTP solution is replaced by pseudouridine ψTP.

[0157] Then the remaining steps are implemented according to Example 1, Example 2 and Example 3, to obtain four groups of JF-EGFP-saRNALNPs solutions (in this experiment, the gD antigen in Example 1 is replaced by the coding gene of EGFP), corresponding to unmodified, 50% m5CTP modification alone, 50% ψTP modification alone, and 50% m5CTP+50% ψTP modification.

[0158] II. Expression efficiency detection of different modifications

[0159] 750 ng of unmodified, 50% m5CTP modified, 50% ψTP modified, and 50% m5CTP+50% ψTP modified saRNA-EGFP nanoliposome particles were used to transfect HEK293 cells for 48 hours. The transfection method was as follows: 100,000 HEK293T cells per well were plated in a 12-well plate and cultured overnight. After the cells adhered, nanoliposome particles containing 750 ng of saRNA (referring to the RNA content encapsulated in the liposome nanoparticles) were transferred to the cell culture well. After 24 hours of transfection, the expression of green fluorescent protein in the cells was detected.

[0160] The expression of green fluorescent protein in the cells was detected by flow cytometry (Thermo Fisher, Attune NxT). The results are shown in Figure 6 In this experiment, the expression efficiency of 50% m5CTP modified saRNA-EGFP was the highest, followed by unmodified saRNA-EGFP, then 50% ψTP modified saRNA-EGFP, and the expression of 50% m5CTP modification+50% ψTP modification was the lowest.

[0161] III. Expression efficiency detection of different proportions of the same modification

[0162] The transfection method was as follows: 50% ψTP modified, 50% m5CTP modified, and 100% m5CTP modified (the 100% molar proportion of CTP in the CTP solution in (2) 50% m5CTP modification alone was replaced by m5CTP) saRNA-EGFP nanoliposome particles were used to transfect HEK293 cells for 48 hours at two doses of 375 ng and 750 ng (referring to the RNA content encapsulated in the liposome nanoparticles).

[0163] The expression of green fluorescent protein in the cells was detected by flow cytometry (Thermo Fisher, Attune NxT). The results are shown in Figure 7As shown, transfection efficiency increased with increasing RNA dose. m5CTP-modified saRNA-EGFP had better expression efficiency than ψTP-modified saRNA-EGFP, and the expression level increased with increasing 50% m5CTP incorporation ratio, and the transfection positive rate of 50% m5CTP modification alone was higher than that of 50% ψTP modification alone, and the transfection positive rate of 100% m5CTP modification alone was the highest among the groups at the same dose.

[0164] Example 7

[0165] This example investigates the expression of JF-Luc RNA LNPs and mRNA-Lucferase LNPs prepared in Example 3 in animals in vivo.

[0166] wherein mRNA-Lucferase is an mRNA molecule: the preparation method is basically the same as that of JF-Luc RNA saRNA, except that no replicase element NSP1, 2, 3, 4 and sgP element are added. The obtained mRNA-Lucferase is used to prepare mRNA-Lucferase LNPs according to Example 3.

[0167] I. Experimental methods

[0168] JF-Luc RNA LNPs and mRNA-Lucferase LNPs are used for leg muscle injection of 4-6 week old Balb / c mice, two doses (5 μg and 2.5 μg, referring to the RNA content encapsulated in the liposome nanoparticles) are selected for injection once, and the Lucferase luciferase signal is detected by a multifunctional enzyme label instrument (FlexStation3) on days 9, 11, 21, 30, 41 and 50.

[0169] As shown in Figure 8 the results, the expression of the optimized JF-Luc RNA LNPs is better than that of mRNA-Lucferase LNP. The mRNA-Lucferase LNP is not detectable on day 11, but the JF-Luc RNA LNP still has a strong expression signal, and the luciferase expression of the JF-Luc RNA LNP is prolonged to day 50.

[0170] Example 8 RNA vaccine evaluation

[0171] I. Design of piglet immunization scheme

[0172] 28-35 day-old PRV-negative piglets were immunized with Day 0 (day 0) liposome nanoparticles by intramuscular injection, with a vaccination volume of 0.5 mL, and a booster immunization on Day 28 (day 28) (group 3). The specific immunization scheme is shown in Table 7 below.

[0173] After vaccination, 14 days of observation, daily observation of mental and appetite and other clinical symptoms, and weighing on the 7th day and the 14th day; serum was collected on 7, 14, 21, 28, 35, 42 d for neutralizing antibody (fixed virus-diluted serum method) and cytokine secretion level (Elabscience) detection;

[0174] On Day 42, 3 pigs from each group were subjected to challenge; after challenge, 14 days of observation, daily observation of clinical symptoms, body temperature response, etc., daily collection of nasal swabs, detection of virus shedding by PCR and virus isolation method, and detection of virus titer (fixed virus-diluted serum method), necropsy on the 14th day after challenge, collection of brain, liver, spleen, lung, lymph and tonsil tissues, observation of pathological changes and pathological section.

[0175] The negative control JF-Luc LNPs, the low-dose group injected with JF-AV01 RNA LNPs prepared according to Example 3, the immunization dose of 50 μg for the high-dose group and 5 μg for the low-dose group. The saRNA plasmid used in the preparation of the JF-AV01 RNA LNPs according to Example 3 was replaced with m5CTP having a molar ratio of 100% in the in vitro transcription system (Table 4) during the preparation process according to Example 2.

[0176] Table 7, immunization scheme

[0177]

[0178]

[0179] The immunization dose of the liposome nanoparticles refers to the RNA dose encapsulated in the liposome nanoparticles.

[0180] Two, neutralizing antibody detection

[0181] 28-35 day-old PRV-negative piglets were immunized with Day 0 (day 0) liposome nanoparticles by intramuscular injection, with a vaccination volume of 0.5 mL, and a booster immunization on Day 28 (day 28) (group 3). The specific immunization scheme is shown in Table 7 below.

[0182] Table 8, average neutralizing antibody titer of each group after the first immunization on 14, 21, 28, 35, 42 d

[0183] Group 14d 21d 28d 35d 42d 1 2 -1.99 ]] 2 -2.16 ]] 2 -2.24 ]] 2 -1.88 ]] 2 -1.99 ]] 2 2 -1.07 ]] 2 -1.66 ]] 2 -1.88 ]] 2 -1.84 ]] 2 -1.70 ]]> 3 2 -1.27 ]] 2 -1.57 ]] 2 -2.33 ]] 2 -7.65 ]] 2 -8.08 ]] 4 2 -2.82 ]] 2 -3.10 ]] 2 -3.68 ]] 2 -4.34 ]] 2 -2.58 ]] 5 2 -3.07 ]] 2 -2.60 ]] 2 -4.88 ]] 2 -4.78 ]] 2 -3.85 ]] 6 0 0 0 0 0

[0184] The results are as follows Figure 9As shown in the above table, the average antibody of the JF-AV01 RNA LNP immunization group was equivalent to that of the live vaccine and inactivated vaccine at 14 and 21 days after the first immunization, and there was no significant difference; the JF-AV01 RNA LNP 50 μg immunization group was higher than the JF-AV01 RNA LNP 5 μg immunization group. The live vaccine one-time immunization group and the inactivated vaccine two-time immunization group were higher than the three JF-AV01 RNA LNP immunization groups at 28 days after the first immunization. Some groups were boosted at 28 days, and at 7 days after the boosting, the neutralizing antibody level of the JF-AV01 RNA LNP 5 μg two-time immunization group was significantly improved, and the neutralizing titer could reach 2 -7.65 (1:200) was significantly higher than that of the live vaccine 2 -4.34 (1:20) and the inactivated vaccine 2 -4.78 (1:27) immunization group (P<0.0001); the neutralizing antibody of the JF-AV01 RNA LNP 5 μg two-time immunization group maintained a high level after the boosting, and at 14 days after the boosting, the neutralizing titer could reach 2 -8.08 The single-time immunization JF-AV01 RNA LNPs showed a protection efficiency equivalent to that of the traditional vaccine, and after the two-time immunization, the JF-AV01 RNA LNP vaccine protection efficiency was far higher than that of the attenuated vaccine and the inactivated vaccine. It was proved that the saRNA vaccine of the application had a long-term immunity and was far superior to the ability of the traditional vaccine.

[0185] III. Cytokine detection

[0186] The IL-4 and IFN-γ cytokine levels of the serum samples of the JF-AV01 saRNA LNP 5 μg two-time immunization group, the LA2017 live vaccine one-time immunization group, the LA-A inactivated vaccine two-time immunization group, and the JF-Luc control group at 21 days and 35 days (7 days after the boosting) after the first immunization were detected by the double antibody sandwich ELISA method. Figure 10 A and B).

[0187] Table 9 Average level of IFN-γ (pg / ml) of each group at 21 and 35 days after the first immunization

[0188]

[0189]

[0190] Table 10 Average level of IL-4 (pg / ml) of each group at 21 and 35 days after the first immunization

[0191] Group 21d 35d 3 70130.00 50726.67 4 31103.33 12343.33 5 42156.67 17003.33 6 11043.33 4580.00

[0192] The IFN-γ of the JF-AV01 RNA LNP 5 μg secondary immunization group was significantly higher than that of the control group (P=0.0018) 21 days after the first immunization, the LA2017 live vaccine primary immunization group had no significant difference with the control group, and the LA-A inactivated vaccine secondary immunization group was significantly higher than the control group (P=0.0196). The IFN-γ of the JF-AV01 RNA LNP 5 μg secondary immunization group was significantly higher than that of the control group (P<0.0001), the LA2017 live vaccine group (P=0.0003) and the LA-A inactivated vaccine secondary immunization group (P=0.0010) 7 days after the booster immunization. There was no significant difference in the IL-4 cytokine level between each group 21 days after the first immunization. The IL-4 cytokine level of the JF-AV01 RNA LNP 5 μg secondary immunization group was significantly higher than that of the control group (P<0.01) and the LA2017 live vaccine group (P<0.01) 7 days after the booster immunization. The experiment proves that the saRNA vaccine of the application can induce stronger immunity of the body and further enhance the protection ability of the body to PRV.

[0193] III. Challenge protection experiment

[0194] The body temperature changes of the piglets after challenge are shown in the following table, and the average temperature (°C) of each group after challenge is shown in the following table and Figure 11

[0195] Table 11 Average temperature (°C) of each group after challenge

[0196] Group 1d 2d 3d 4d 5d 1 39.77 40.93 40.50 40.67 41.17 2 39.80 40.37 40.50 40.33 40.43 3 40.07 40.00 40.63 40.60 40.67 4 39.67 40.40 40.67 40.37 39.90 5 39.67 40.27 40.70 40.60 40.07 6 40.07 40.70 41.17 40.77 40.30

[0197] Table 12 Average temperature (°C) of each group after challenge

[0198] Group 6d 7d 8d 9d 10d 1 40.43 39.70 40.03 39.90 39.60 2 40.87 40.37 39.83 39.87 39.07 3 40.60 40.03 39.97 39.33 39.43 4 39.00 39.40 39.73 39.67 39.37 5 39.60 39.90 39.60 39.67 39.73 6 39.27 39.00 38.65 38.85 39.70

[0199] On the 3rd day after challenge, the body temperature of the piglets in the challenge control group reached above 41 °C, and the typical symptoms such as decreased appetite, depression, abdominal breathing and purulent nasal discharge began to appear. On the 7th day after challenge, one piglet in the control group died, and all the piglets in the control group were sick, and all the piglets in the control group appeared symptoms such as loss of appetite, ataxia, unconsciousness and dyspnea before autopsy at the end of the challenge observation.

[0200] ​One pig in the JF-AV01 RNA LNP 50 μg single immunization group had a body temperature over 41°C for 4 days, and another pig in the group still had symptoms such as anorexia, listlessness, and purulent nasal discharge before autopsy at the end of the observation, and the rest of the piglets disappeared on the 9th day; one pig in the JF-AV01 RNA LNP 5 μg single immunization group had a body temperature over 41°C for 3 days during the observation, and all piglets in the group disappeared on the 10th day; only one pig in the JF-AV01 RNA LNP 5 μg double immunization group had mild symptoms after challenge, showing anorexia and listlessness, and the symptoms disappeared on the 8th day; one pig in the LA-A inactivated vaccine double immunization group had a body temperature over 41°C for 3 days after challenge, and all piglets had anorexia and listlessness on the 6th day, and the clinical symptoms disappeared on the 8th day; only one pig in the LA2017 live vaccine single immunization group had mild symptoms after challenge, showing anorexia and listlessness, and the symptoms disappeared on the 6th day, and some piglets continued until the 9th day. The results of the protection efficiency after challenge are shown in Table 13. The case of the present experiment proves that the saRNA low-dose booster immunization of the present application can completely protect piglets from PRV challenge and death.

[0201] Table 13. Protection efficiency of piglets after challenge

[0202] Vaccine group Protected head Protection rate JF-AV01 RNA LNPs-50μg 1 / 3 33.33％ JF-AV01 RNA LNPs-5μg 2 / 3 66.66％ JF-AV01 RNA LNPs-5μg-Twice 3 / 3 100％ PRV LA 2017 3 / 3 100％ PRV LA-A Twice 2 / 3 66.66％ JF-Luc 0 / 3 0

[0203] Obviously, the above examples are only examples for clearly illustrating but not limiting the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. All the embodiments do not need to be exhausted here, and the obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A pseudorabies immunogen composition, characterized in that, It includes an antigen saRNA molecule; the antigen saRNA molecule contains an open reading frame (ORF) that encodes a polypeptide of the gD antigen of pseudorabies virus; The pseudorabies virus mentioned is a PRV AH02LA variant; The polypeptide of the gD antigen of the PRV AH02LA mutant strain has the amino acid sequence shown in SEQ ID NO.1; The DNA sequence of the polypeptide of the gD antigen of the PRV AH02LA mutant strain has been codon-optimized and is the nucleotide sequence shown in SEQ ID NO.

2. Alternatively, the DNA sequence of the polypeptide of the gD antigen of the PRV AH02LA mutant strain has been codon-optimized to be the nucleotide sequence shown in SEQ ID NO.3; The antigen saRNA molecule comprises the following elements in sequence along the 5'-3' direction: a 5' cap structure, a 5' UTR sequence, a gene sequence encoding a replicase, an sgP promoter, a gene sequence encoding a signal peptide, a gene sequence encoding a gD antigen, a poly(A) tail, and a 3' UTR sequence. The 5'UTR sequence is an optimized 5'UTR sequence of human β-globin; The 3'UTR sequence is a tandem optimized 3'UTR sequence of 2-3 human β-globin or the 3'UTR sequence of Xenopus β-globin; The poly(A) tail is 30-200 nucleotides in length; The signal peptide is selected from porcine interleukin-2 signal peptide, porcine interleukin-4 signal peptide, or PRV gD protein original signal peptide.

2. The pseudorabies immunogen composition of claim 1, wherein, The sequences encoding the replicase are the gene sequences encoding NSP1, NSP2, NSP3, and NSP4. The porcine interleukin-2 signal peptide sequence is the amino acid sequence shown in SEQ ID NO.

4. Alternatively, the porcine interleukin-2 signal peptide sequence has been codon-optimized to be the nucleotide sequence shown in SEQ ID NO.5; The porcine interleukin-4 signal peptide sequence is the amino acid sequence shown in SEQ ID NO. 6; The original signal peptide sequence of the PRV gD protein is the amino acid sequence shown in SEQ ID NO.

7.

3. The pseudorabies immunogen composition according to claim 2, characterized in that, The antigen saRNA molecule is synthesized via in vitro transcription.

4. The pseudorabies immunogen composition according to claim 3, characterized in that, In the in vitro transcription system, 5-methylcytosine is incorporated into the CTP solution, and / or pseudouridine is incorporated into the UTP solution, with the molar ratio of 5-methylcytosine and / or pseudouridine being 10-100% respectively.

5. A biomaterial, characterized in that, Including any of the following: A1. A recombinant vector plasmid containing the antigen saRNA molecule as described in any one of claims 1-4; A2. Host cells containing the recombinant vector described in A1.

6. A pharmaceutical composition, characterized in that, It comprises the pseudorabies immunogen composition according to any one of claims 1-4, and a pharmaceutically acceptable carrier.

7. The uses of the pseudorabies immunogen composition according to any one of claims 1-4, the biological material according to claim 5, or the pharmaceutical composition according to claim 6 are as follows: (1) Use in the preparation of a vaccine to prevent pseudorabies in pigs; (2) Use in the preparation of products that inhibit pseudorabies virus.

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