Pseudorabies immunogen composition and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a pseudorabies immunogen composition and application thereof. The invention provides a pseudorabies immunogen composition. The pseudorabies immunogen composition 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 pseudorabies vaccine is prepared by adopting a self-replicating RNA technology, and the pseudorabies virus infection can be protected, so that the defect of low pseudorabies virus protection efficiency of the conventional pseudorabies vaccine is effectively overcome.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a pseudorabies immunogen composition and its use. Background Art

[0002] Porcine pseudorabies is an acute infectious disease caused by Pseudorabies virus (PRV). Currently, pigs are the only known natural hosts of PRV. Its infection can cause a large number of neonatal piglets to die, and the morbidity and mortality rate can reach 100%. The morbidity rate of weaned piglets is 20% - 40%, and the mortality rate is 10% - 20%. The main manifestations are neurological symptoms, diarrhea, vomiting, etc. The main manifestations of fattening pigs' respiration are difficulty, growth stagnation, etc.; adult pigs are generally latently infected with mild symptoms; pregnant sows infected may have abortions and stillbirths; boars are often infertile after infection. This disease breaks out in pig farms and is one of the major infectious diseases endangering the global pig industry.

[0003] PRV, also known as Suid herpesvirus 1 (SuHV-1) or Aujeszky's disease virus (ADV), belongs to the Alphaherpesvirinae subfamily of the Herpesviridae family. PRV is a double-stranded DNA virus with a viral genome of approximately 145 kb, encoding 70 - 100 proteins, most of which are capsid proteins, envelope proteins, tegument 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) have been identified on the envelope of the virion. During the infection process, gC, gB, gD, gH, and gL are involved in virus entry and are the main antigens that stimulate the host innate immune response. Among them, gD is the main glycoprotein of PRV and can stimulate the body to produce neutralizing antibodies against PRV.

[0004] Vaccine immunization is the main means to prevent and control porcine pseudorabies. More and more evidence shows that gD has less mutation characteristics and is a key protein to activate humoral and cellular immune responses, making it a promising target for the development of new vaccines. And the expression level of the protein directly affects the immunization dose, immune effect, and vaccine cost.

[0005] The traditional vaccines used in the current pig industry have many disadvantages. Inactivated vaccines or subunit vaccines have deficiencies such as low immune efficacy and large immune side reactions; live attenuated vaccines have the risk of reversion to virulence, and other components of the virus are prone to cause adverse reactions or the DNA of the virus undergoes recombination, etc., resulting in risks such as some genetic mutations. At the same time, traditional vaccines also have problems such as a long production cycle, high production cost, and relatively lagging characteristics in application for emergency situations.

[0006] In view of the above-mentioned many problems of the current pseudorabies vaccine, the market demand for vaccines that can effectively provide protection, have a short production cycle, and low cost has become increasingly urgent. In recent years, with the application of mRNA vaccines in COVID-19 prevention, it has been confirmed that mRNA vaccines are an effective means to quickly respond to epidemic mutant strains.

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

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

[0009] 1. Safety: Since mRNA is a non-infectious and non-integrating platform, there is no potential risk of infection or insertional mutation. In addition, its in vivo half-life can be regulated by using various modification and delivery methods. And research shows that nucleic acid modification can also downregulate the innate immunogenicity of mRNA to further improve safety and reduce side effects.

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

[0011] 3. High production efficiency: mRNA vaccines have the potential for rapid, inexpensive, and scalable manufacturing. The reduction in the dosage used can greatly save production costs, reduce the selling price of drugs, and benefit a wider range of farmers. At the same time, mRNA vaccines have the advantages of a simple and rapid production process, good safety, and easy development of multi-valent and multi-component vaccines, which are significantly superior to subunit vaccines, inactivated vaccines, and live attenuated vaccines.

[0012] mRNA vaccines have been applied in the research of various infectious diseases and tumor treatments, including conventional mRNA vaccine technology, self-replicating RNA vaccine technology, trans-amplifying RNA vaccine technology, and circular RNA vaccine technology. Among them, the self-replicating RNA vaccine is a better alternative. The difference from ordinary mRNA is that in addition to carrying the coding sequence (open reading frame, ORF) of the antigen, the RNA also carries the 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 mRNA vaccines, but also has the advantages of low dosage, low toxicity and side effects, and lower production cost. However, at present, no self-replicating RNA vaccine for preventing porcine pseudorabies has been developed. Summary of the Invention

[0013] Therefore, the technical problem to be solved by the present invention is to provide a pseudorabies immunogen composition and its use.

[0014] For this purpose, the present invention provides the following technical solutions:

[0015] The embodiments disclosed in the present invention relate to a pseudorabies immunogen composition, comprising at least one antigen saRNA molecule; the antigen saRNA molecule comprises at least 1 open reading frame ORF, and the open reading frame ORF encodes a polypeptide comprising the gD antigen from pseudorabies virus.

[0016] In some embodiments, the pseudorabies virus can be selected from the dominant epidemic strains of Chinese pseudorabies virus since 2010; preferably, the PRV AH02LA variant strain is selected. This strain is a PRV variant 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 show that intranasal PRV AH02LA infection results in a morbidity or mortality rate of 100% 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 classical strains, including high fever, respiratory distress, depression, anorexia, systemic neurological symptoms, and diarrhea.

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

[0018] In some embodiments, the polypeptide sequence of the gD antigen of the PRV AH02LA variant is an amino acid sequence having a homology of 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 more than 99.99% with the amino acid sequence shown in SEQ ID NO.1.

[0019] In some embodiments, the DNA sequence of the polypeptide of the gD antigen of the PRV AH02LA variant is codon-optimized and is the nucleotide sequence shown in SEQ ID NO.2 or a nucleotide sequence having a homology of more than 80% therewith.

[0020] In some embodiments, the DNA sequence of the polypeptide of the gD antigen of the PRV AH02LA variant is a nucleotide sequence having a homology of 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 more than 99.99% with the nucleotide sequence shown in SEQ ID NO.2.

[0021] In some embodiments, the DNA sequence of the polypeptide of the gD antigen of the PRV AH02LA variant is codon-optimized and is the nucleotide sequence shown in SEQ ID NO.3 or a nucleotide sequence having a homology of more than 80% therewith.

[0022] In some embodiments, the DNA sequence of the polypeptide of the gD antigen of the PRV AH02LA variant is a nucleotide sequence having a homology of 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 more than 99.99% with the nucleotide sequence shown in SEQ ID NO.3.

[0023] In some embodiments, the antigen saRNA molecule sequentially includes the following elements in the 5'-3' direction: 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 a 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 level of the gD antigen, thereby affecting the immune effect. The signal peptide coding gene sequence is contained at the N-terminus of the gD antigen coding gene sequence, or the signal peptide coding gene sequence can be inserted between two sequence fragments of the sgP sequence and the gD antigen coding gene sequence.

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

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

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

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

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

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

[0031] In a preferred embodiment, a signal peptide is fused to the N-terminus of the gD antigen polypeptide, and the gD antigen polypeptide is expressed under the guidance of a signal peptide with high specific expression. For example, the porcine interleukin-2 signal peptide sequence is as shown in EQ ID NO.4, or an amino acid sequence having more than 80% homology therewith. The porcine interleukin-2 signal peptide sequence has been codon-optimized, such as the nucleotide sequence shown in SEQ ID NO.5 or a nucleotide sequence having more than 80% homology therewith.

[0032] In a preferred embodiment, the signal peptide sequence is an amino acid sequence having a homology of 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 more than 99.99% with the amino acid sequence shown in SEQ ID NO.4.

[0033] In a preferred embodiment, the N-terminus of the gD antigen polypeptide is fused with a signal peptide, and the gD antigen polypeptide is expressed under the guidance of a specifically highly expressed signal peptide. The signal peptide sequence is as shown in SEQ ID NO.6, or an amino acid sequence having a homology of more than 80% therewith.

[0034] In a preferred embodiment, the N-terminus of the gD antigen polypeptide is fused with a signal peptide, and the gD antigen polypeptide is expressed under the guidance of a specifically highly expressed signal peptide. The signal peptide sequence is as shown in EQ ID NO.7, or an amino acid sequence having a homology of more than 80% therewith.

[0035] In an optimal embodiment, a signal peptide is provided upstream of the gD antigen, and the gD antigen polypeptide is expressed under the guidance of a specifically highly expressed signal peptide, which can increase the expression level of the antigen polypeptide and thus trigger a stronger immune effect.

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

[0037] The embodiments disclosed in the present invention relate to a biological material, including any one of the following:

[0038] A1. A recombinant vector plasmid containing the saRNA molecular sequence described above;

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

[0040] The embodiments disclosed in the present invention 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 PEG lipid after structural modification. The composition ratios of the lipid nanoparticles are as follows: ionizable cationic lipid (20 - 60 wt%), non-cationic lipid (20 - 55 wt%), cholesterol (19.5 - 55 wt%), and PEG lipid after structural modification (0.5 - 5 wt%). Among them, 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 PEG lipid after structural modification can be DMG-PEG 2000. After these lipid substances are mixed in a certain proportion, they are processed with saRNA in proportion to form saRNA lipid nanoparticles. The processing of the nanoparticles can be carried out using a microfluidic nanometer or other methods. The size of the nanoparticle diameter, the particle size distribution (PDI), and the charge carried on its surface (zeta potential) are crucial for the in vivo absorption of LNP and can determine the efficiency of LNP entering cells. The saRNA lipid nanoparticles can induce the body to produce neutralizing antibodies to protect the body from killing pseudorabies virus when entering the body. After being absorbed by the body cells, the saRNA lipid nanoparticles enter the body cells. In the body cells, the lipid vesicles rupture, releasing the encapsulated saRNA, which triggers protein synthesis by means of the cell's own protein translation system.

[0042] The uses of the pseudorabies immunogen composition, the biological material, or the pharmaceutical composition disclosed in the present invention include:

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

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

[0045] (3) Use in the preparation of a product for a pseudorabies-related detection reagent. The product includes a reagent or a kit.

[0046] 1. A pseudorabies immunogen composition provided by the present invention includes at least one antigen saRNA molecule; the antigen saRNA molecule contains at least 1 open reading frame (ORF), and the open reading frame (ORF) encodes a polypeptide containing the gD antigen from a pseudorabies virus strain; the present invention uses the self-replicating RNA technology to produce a pseudorabies vaccine and can protect against pseudorabies virus infection, effectively solving the drawback of the low protection efficiency of current pseudorabies vaccines against pseudorabies virus.

[0047] 2. A pseudorabies immunogen composition provided by the present invention, in which a replicase gene is introduced into the antigen saRNA molecule, and the RNA targeting the antigen can be replicated in large quantities, so that the antigen protein can be expressed in large quantities under the condition of a low vaccination dose and the expression time of the antigen protein is very long. Therefore, the body can continuously produce an immune response, and thus the immune time of the body against pseudorabies virus is correspondingly prolonged. Compared with traditional inactivated vaccines, the most direct advantage is the low vaccine dosage and small side effects; furthermore, the reduction of the dosage used can save a large amount of production costs.

[0048] 3. A pseudorabies immunogen composition provided by the present invention, the antigen saRNA molecule further includes an sg promoter (sgP), a signal peptide coding gene sequence and / or a sequence encoding a replicase. The signal peptide coding gene sequence is contained at the N-terminus of the gD antigen coding gene sequence, or the signal peptide coding gene sequence can also be inserted between two sequence fragments of the sgP sequence and the gD antigen coding gene sequence; by setting a signal peptide upstream of the gF antigen polypeptide, the gD antigen polypeptide is guided by a highly specific signal peptide for expression, which can increase the expression amount of the antigen polypeptide and thus trigger a stronger immune effect. In addition, compared with the original signal peptide of the virus, the signal peptide screened in the present invention can specifically increase the expression amount of the gD protein in a porcine cell line. Therefore, in practical applications, the fusion antigen obtained by combining the signal peptide screened in the present invention with gD can reduce the immune dose while maintaining a strong immune effect, reducing the costs of pig farms and farmers. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0050] Figure 1 It is a schematic structural diagram of the saRNA molecule containing gD in Example 1 of the present invention;

[0051] Figure 2It is a representative electrophoresis diagram of the linearized plasmid and the recombinant plasmid before enzyme digestion in Example 2 of the present invention; in the figure, from left to right, lane 1 is the DNA molecular weight standard; lane 2: undigested JF-AV02 plasmid; lane 3: digested JF-AV02 plasmid; lane 4 is the DNA molecular weight standard; lane 5: undigested JF-AV03 plasmid; lane 6: digested JF-AV03 plasmid;

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

[0053] Figure 4 It is a detection diagram of the expression levels of two codon-optimized saRNAs of gD after transfection of cells in step (3) of Example 4 of the present invention; in the figure, the base of logx is 10;

[0054] A: Detection of the expression level of the reporter protein after transfection of the porcine ST cell line with gD-1 / 2HiBiT; the antigen ORF nucleic acid sequence of gD1 / HiBiT is selected from the antigen ORF nucleic acid sequence consisting of SEQ ID NO.9, SEQ ID NO.2, and HiBiT in sequence from the 5' to 3' direction; the antigen ORF of gD2 / HiBiT is the antigen ORF nucleic acid sequence consisting of SEQ ID NO.9, SEQ ID NO.3, and HiBiT in sequence from the 5' to 3' direction;

[0055] B: Detection of the expression level of the reporter protein after transfection of the porcine PK-15 cell line with gD-1 / 2HiBiT, the antigen ORF nucleic acid sequence of gD1 / HiBiT consists of SEQ ID NO.9, SEQ ID NO.2, and HiBiT in sequence from the 5' to 3' direction; gD2 / HiBiT is the antigen ORF nucleic acid sequence consisting of SEQ ID NO.9, SEQ ID NO.3, and HiBiT in sequence from the 5' to 3' direction;

[0056] Figure 5 It is the detection result of the expression levels of each liposome nanoparticle in the porcine PK15 cell line and the porcine ST cell line and the immune efficacy in animals in Example 5 of the present invention, wherein:

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

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

[0059] C is the result of the relative titer diagram of neutralizing antibodies generated by the immune efficacy of each liposome nanoparticle in animals;

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

[0061] Figure 6 It is the result of the green fluorescent protein expression level of differently modified nano-liposome particles in Example 6 of the present invention; in the figure: A: unmodified, B: modified with 50% m5CTP alone, C: modified with 50% ψTP alone, D: modified with 50% m5CTP + 50% ψTP;

[0062] Figure 7 It is the statistical result of the green fluorescent protein expression level of nano-liposome particles with different ratios in Example 6 of the present invention;

[0063] Figure 8 It is the experiment of JF-Luc RNA LNPs and mRNA-Lucferase LNP in animals in Example 7 of the present invention; in the first row, for the four-mouse group, from left to right are mRNA-Lucferase LNP 5 μg, JF-Luc RNA LNPs 5 μg, mRNA-Lucferase LNP 2.5 μg, JF-Luc RNA LNPs 2.5 μg; for the two-mouse group, the non-detectable mRNA-Lucferase mouse is removed, and from left to right, JF-Luc RNA LNPs 5 μg and JF-Luc RNA LNPs 2.5 μg are left;

[0064] Figure 9 It is the detection chart of the serum neutralizing antibody titer after immunizing piglets with vaccines such as JF-AV01-RNA LNPs in Example 8 of the present invention; in the figure, compared with the control group, * indicates p < 0.05, **** indicates p < 0.0001;

[0065] Figure 10 It is the change chart of cytokines in the body after immunizing piglets with vaccines such as JF-AV01-RNA LNPs in Example 8 of the present invention; in the figure, A is the average level of IFN-γ in each group; B is the average level of IL-4 in each group; compared with the control group, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001;

[0066] Figure 11It is the graph of the body temperature change of piglets after challenge with vaccines such as JF-AV01-RNA LNPs in Example 8 of the present invention;

[0067] Figure 12 It is the plasmid map of the recombinant plasmid JF-AV01 in Example 1 of the present invention. Detailed implementation mode

[0068] The following embodiments are provided to better further understand the present invention, which is not limited to the described optimal implementation mode and can be easily understood and implemented by researchers in this industry. It does not limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with those of other existing technologies falls within the protection scope of the present invention.

[0069] For those not specifying specific experimental steps or conditions in the embodiments, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments not specifying the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0070] For ease of understanding, the present invention provides the following scientific background information and definitions. Any technical feature disclosed herein can be part of each embodiment of the present invention. Other technical features may 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 the response of various cytokines to antigens.

[0072] Humoral immunity / humoral immune response: Humoral immunity generally refers to antibody production and its accompanying auxiliary processes. The typical characteristics of the humoral immune response may 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, with untranslated regions on both its upstream 5' side and downstream, namely 5' UTR and 3' UTR. Eukaryotic mRNA molecules also have 5' cap and 3' tail structures at both ends. The mRNA of prokaryotic cells generally does not have a tail, but the mRNA of viruses infecting eukaryotic cells generally has a poly-A tail.

[0074] Open reading frame, i.e., 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] The cap structure, referring to the cap structure at the 5' end of mRNA, is of great significance for stabilizing mRNA and its translation.

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

[0077] The 3' untranslated region (3' UTR) is the transcribed sequence after the stop codon, containing a polyadenylation signal. Mature mRNA generally has a poly(A) tail with a length of 20 - 200 bases added to its 3' end, which can prevent degradation by exonucleases, is also related to nuclear pore transport to the cytoplasm, and can also regulate the translation process.

[0078] The signal peptide is located at the N-terminus of the secreted protein. It is generally composed of 15 - 30 amino acids and includes three regions: a positively charged N-terminus, called the basic amino terminus; and an intermediate hydrophobic sequence. When the signal peptide sequence is synthesized, it is recognized by the signal recognition particle (SRP), protein synthesis pauses or slows down, and the signal recognition particle carries the ribosome to the endoplasmic reticulum, where protein synthesis resumes. Under the guidance of the signal peptide, the newly synthesized protein enters the lumen of the endoplasmic reticulum. The signal peptide sequence is then excised by signal peptidase. If a stop-transfer sequence exists at the C-terminus of the nascent peptide chain, it may not be excised by signal peptidase. The differences in the efficiency and functional characteristics of the signal peptide directly affect the yield of the subsequent protein induced to be synthesized. Therefore, optimizing the signal peptide is a feasible way to improve protein yield.

[0079] Codon optimization. A codon is a triplet ribonucleotide sequence used to encode an amino acid. Some different codons can encode the same amino acid, which are called synonymous codons. There is codon bias in different organisms, that is, uneven codon usage frequencies. The secondary structure of the coding region and codon selection can both affect translation efficiency. Excessive secondary structure and rare codons will both reduce the translation speed. Therefore, through codon optimization, the expression level of the protein can be improved.

[0080] The DNA sequences in the following examples were all synthesized by Nanjing Genscript Biotech Co., Ltd.

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

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

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

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

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

[0086] The attenuated LA - 2017 vaccine was provided by the 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 tag HiBiT used in the following examples is a commercial tag developed by Promega Corporation. As described by Marie K. Schwinn et al., fusing it to the C - terminus of the target polypeptide can directly reflect the expression level of the target protein.

[0092] Example 1 Construction of Recombinant Plasmid

[0093] (1) Selection of Sequences

[0094] In this example, the vector sequence used refers to the published invention patent: CN 117205309 A. Replace the nucleotide sequence (from the 7622bp to the 8340bp) in the original vector JF - FV01 with the series of sequences described in this invention; the coding gene of the signal peptide and the polypeptide of the gD antigen of the pseudorabies virus strain is the coding gene of the amino acid sequence shown in SEQ ID NO.1.

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

[0096] (2) Construction of Recombinant Plasmid

[0097] Prepare the vector JF-AV01 containing each component of saRNA, and the plasmid map is as Figure 12 shown. The T7 promoter (T7promotor), sg promoter (sgP), and chloramphenicol resistance sequence (chloramphenicol) are all common sequences. The template vector sequence used in the present invention is selected from CN 117205309 A, in which the nucleotide sequence (bp 7622 - bp 8340) in the original vector JF-FV01 is replaced with the series of sequences described in the present invention. The main molecular cloning elements are as Figure 1 shown, specifically as follows:

[0098] 1) Select ApaI restriction endonuclease (Nanjing Novoprotein Co., Ltd.) to linearize 1 μg of the JF-FV01 template plasmid. The reaction system is: 2 μL of reaction buffer, 1 μL of ApaI restriction endonuclease, 1 μL of template plasmid, and add enzyme-free water to 20 μL. React at 37 °C for 15 minutes.

[0099] 2) Use a DNA recovery kit (TaKaRa) to recover the linearized template according to the instructions.

[0100] 3) Select 15 - 25 bp homologous recombination arms at 1 - 200 nucleotides upstream and 1 - 200 nucleotides downstream of the cloning site of the template plasmid, design the homologous arms upstream and downstream of the signal peptide - gD polypeptide nucleotide respectively, and hand them over to Nanjing GenScript Co., Ltd. for synthesis. Carry out homologous recombination ligation according to the instructions of the homologous recombination kit (Nanjing Novoprotein Co., Ltd.). React at 37 °C for 30 minutes.

[0101] 4) Thaw the cloning competent cells (Beijing Solarbio Science & Technology Co., Ltd.) on ice. Take 10 μL of the recombinant product and add it to 100 μL of the competent cells. Gently flick the tube wall to mix (do not shake). Let it stand on ice for 30 minutes; after heat shock in a 42 °C water bath for 45 seconds, immediately place it on ice to cool for 2 minutes. Add 900 μL of LB medium without antibiotics, and shake the bacteria at 37 °C for 1 hour (rotation speed 200 - 250 rpm). Preheat the LB solid medium plate with the corresponding resistance in a 37 °C incubator. Centrifuge at 5000 rpm for 5 minutes, discard 900 μL of the supernatant. Resuspend the bacterial cells with the remaining medium, and gently spread them evenly on the plate containing chloramphenicol resistance with a sterile spreading rod. Incubate inverted in a 37 °C incubator for 12 hours.

[0102] 5) Pick well - growing monoclonal colonies and inoculate them into LB medium containing chloramphenicol resistance, and expand the culture at 37 °C and 200 rpm.

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

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

[0105] The nucleic acid sequence of gD antigen is SEQ ID NO.3, the signal peptide sequence is SEQ ID NO.4, and the constructed recombinant plasmid is named JF-AV01. The plasmid map is shown in Figure 12 As shown. The nucleic acid sequence of the gD antigen is SEQ ID NO.2, the signal peptide nucleic acid sequence is SEQ ID NO.7, and a 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 SEQ ID NO.7, and a HiBiT tag is added to the 3' end of the gD antigen nucleic acid sequence. The constructed recombinant plasmid is named JF-AV03. Similarly, the polypeptide sequence of the gD antigen is replaced with the sequence of firefly luciferase, and the signal peptide encoding gene sequence is not added. The constructed recombinant plasmid is named JF-Luc. The immune antigen comparison table is shown in the following table:

[0106] Table 2: Immunogen and sequence comparison table

[0107]

[0108] Example 2 Preparation of saRNA Plasmid

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

[0110] (1) Enzyme digestion

[0111] The recombinant plasmid JF-AV01-5 or JF-Luc control in Example 1 was used as a template plasmid, and restriction endonuclease BspQI was used for digestion. The digestion system is shown in the following table (taking 200 μl reaction system as an example, Table 3): digestion conditions: 50°C, reaction time 15-60 minutes. Then, the linearized plasmid was recovered using the Qiagen plasmid purification kit. The representative electrophoresis of the linearized plasmid and the recombinant plasmid before digestion is shown in Figure 2 Any commercially available endonuclease can be used.

[0112] Table 3. DNA template linearization reaction system

[0113] Component Dosage / μl Final Concentration Template Plasmid 20 μg 200 ng / μl BspQI 10 μl 1 U / μl BspQI Buffer 20 μl 1× Enzyme-Free Water Make up to 200 μl -

[0114] (2) In vitro transcription and synthesis of saRNA

[0115] The raw materials for in vitro synthesis of saRNA are the above-mentioned linearized DNA template, dNTP mixture, 10× transcription buffer, T7 RNA polymerase, inorganic pyrophosphatase, RNase inhibitor, and enzyme-free water. The reaction conditions are a water bath at 37°C for 2 - 4 hours, and the reaction system is shown in Table 4. All the above raw materials are commercially available. 5-Methylcytosine is not incorporated into the CTP solution; pseudouridine is not incorporated into the UTP solution.

[0116] Table 4. Reagent components and dosages required for a 20 μl in vitro transcription system.

[0117] Component Dosage Final Concentration 10× Transcription Buffer 2 μl 1× T7 RNA Polymerase 2 μl 20 U / μl Inorganic Pyrophosphatase 2 μl 5 mU / μl RNase Inhibitor 1 μl 2 U / μl ATP Solution 2 μl 5 mM / μl CTP Solution 2 μl 5 mM / μl GTP Solution 2 μl 5 mM / μl UTP Solution 2 μl 5 mM / μl Template DNA 1 μl 25 ng / μl Enzyme-Free Water Make up to 20 ul -

[0118] (3) Digestion of linear DNA template

[0119] Add DNase I to the saRNA obtained in step (2). Add 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] Precipitate and purify the saRNA from the digested reaction solution in step (3) using sodium acetate to obtain purified saRNA. The specific steps are as follows: Transfer the nucleic acid-containing solution to a new Eppendorf centrifuge tube using a pipette, and at the same time measure the volume of the nucleic acid solution. Add 1 / 10 volume of 3 mol / L sodium acetate buffer. Make the final concentration of sodium acetate 0.3 mo1 / L; after inverting and mixing evenly, accurately add 2 volumes of pre-cooled absolute ethanol equal to the volume of the nucleic acid solution, invert and mix evenly, place in an ice bath for 15 - 30 min or 20 - 30 min, and centrifuge at 16000 g for 30 minutes.

[0122] (5) Capping

[0123] Incubate the purified saRNA in step (4) at 65°C for 1 - 30 min. Then perform a capping reaction using a commercially available RNA capping kit. Here, the cap compound can have different structures. In this example, the 5’ cap structure selects 7mG(5')ppp N. Taking a 20 μl reaction system as an example (see Table 5). The reaction conditions are a water bath at 37°C for 1 - 4 h.

[0124] Table 5. Reagent components and dosages required for a 20 μl RNA capping reaction system.

[0125] Component Dosage Final Concentration 10× Capping Buffer 2 μl 1× Vaccinia Virus Capping Enzyme 1 μl 0.5 U / μl GTP Solution 1 μl 0.5 mM S-Adenosylmethionine 1 μl 0.1 mM saRNA 10 μg 500 ng / μl Enzyme-Free Water Make up to 20 μl -

[0126] (6) Purification

[0127] Precipitate and purify the reaction product obtained in step (5) using sodium acetate (the same method as in step (4)) to obtain purified saRNA.

[0128] (7) Analysis

[0129] Perform capillary electrophoresis analysis on the saRNA containing the gD antigen sequence prepared in step (6) (named JF-AV01~5 saRNA). Sampling, electrophoresis, and analysis are all carried out according to the operation steps of the merchant (Agilent RNA-FA kit). The exemplary capillary electrophoresis analysis result of JF-AV02 is as Figure 3 shown. The RNA integrity of the gD antigen is 90.1%, which is greater than 80%. Similarly, similar results with RNA integrity greater than 80% are also obtained for the gD antigens of JF-AV01 saRNA, JF-AV03 - 5 saRNA. The above indicates that all saRNAs are successfully constructed.

[0130] Example 3 Preparation of Lipid Nanoparticles (Lipid nanoparticle, LNP)

[0131] This example provides a method for preparing lipid nanoparticles, which can be used as an active ingredient of a drug. The method includes the following steps:

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

[0133] Take SM-102, DSPC, cholesterol, and DMG-PEG 2000, dissolve them in absolute ethanol so that the concentration of SM-102 is 50 mM, the concentration of DSPC is 20 mM, the concentration of cholesterol is 50 mM, and the concentration of DMG-PEG 2000 is 20 mM to obtain Solution B;

[0134] Simultaneously load Solutions A and B into a microfluidic mixer (Mai'anna). The volume ratio of Solution A to Solution B is 8:1, and the total flow rate is 3 ml / min. Collect the effluent. Transfer the effluent to a dialysis tube (30K specification), centrifuge and concentrate it until the saRNA concentration is 0.5 mg / ml, and then filter it through a 0.22 μm filter membrane. Collect the filtrate to prepare saRNA lipid nanoparticles, that is, obtain the JF-AV01 saRNA LNPs solution correspondingly.

[0135] The solutions of JF-AV01, JF-AV02, JF-AV03, JF-AV04, JF-AV05 and JF-Luc RNA saRNA LNPs (which can be abbreviated as JF-Luc RNA LNPs, and so on) were used to measure the particle size, PDI (particle distribution index) and zeta (Zeta potential) potential of LNPs with a Malvern instrument (Malvern, England). The encapsulation efficiency of 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) Entrapment Efficiency (%) JF-Luc LNPs 75.84 0.141 -2.62 100％ JF-AV01LNPs 74.94 0.136 -2.06 100％ JF-AV02LNPs 74.56 0.130 -1.88 100％ JF-AV03LNPs 75.43 0.127 -2.01 100％ JF-AV04LNPs 75.05 0.125 -2.34 100％ JF-AV05LNPs 74.87 0.135 -1.91 100％

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

[0139] (1) Take a 96-well plate, inoculate ST or PK-15 cells (1×10 4 cells / well), and culture them in DMEM medium (Thermo Fisher Scientific) containing 10% serum and 1% double antibody for 16 - 18 hours.

[0140] (2) After completing step (1), add two kinds 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), and culture for 24 hours. In order to detect antigen expression, the recombinant plasmid in Example 1 used when preparing the liposome nanoparticles was added with a HiBit tag after the two gD sequences respectively. Among them, the DNA sequence corresponding to gD1 includes SEQ ID NO.9, SEQ ID NO.2, HiBit in sequence from 5'-3' direction, and the DNA sequence corresponding to gD2 includes SEQ ID NO.9, SEQ ID NO.3, HiBit in sequence from 5'-3' direction.

[0141] (3) After completing step (2), add HiBit reagent (Promega) according to the supplier's instructions to detect chemiluminescence. As Figure 4 shown in A, the expression levels of the two sequences are similar in the porcine cell line ST. As Figure 4 shown in B, the expression level of gD2 is slightly higher than that of gD-1 in the porcine cell line PK-15. For the gD antigen, the nucleic acid sequence SEQ ID NO.3 was selected for subsequent signal peptide screening and animal immunization challenge experiments.

[0142] Example 5 Screening of High-Expression Signal Peptides

[0143] I. Expression of Antigens Guided by Different Signal Peptides in Cells

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

[0145] II. Detection of the Immune Efficacy of Antigens Guided by Different Signal Peptides in Vivo

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

[0147] (1) Immunize 4-6-week-old Balb / c mice with JF-AV03 LNPs (containing the original virus-derived signal peptide of gD, sp3), JF-AV04 LNPs (containing the signal peptide of porcine interleukin-2, sp1), JF-AV05 LNPs (the signal peptide of porcine interleukin-4, sp2), and JF-Luc LNPs, 5 mice in each group, and perform intramuscular injection in the leg. Select a dose of 1 μg (1 μg refers to the RNA content encapsulated in the liposome nanoparticles) for 1 immunization. After 21 days of immunization, collect orbital blood and separate serum.

[0148] (2) ELISA assay: Coat the ELISA plate with pseudorabies gD protein (Wuhan Huamei Company) overnight at 4°C; Gradient dilute the serum in (1) above with a dilution factor of 3, and incubate it with the gD protein coated on 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 one hour. After completing the above steps, wash the plate, use the ELISA two-component chromogenic solution (Beijing Solarbio Company) to develop color at room temperature. After the color development is completed, add the termination reagent, and use a microplate reader (FlexStation3) to detect the absorbance value OD450nm.

[0149] The results showed that all three signal peptides successfully directed the expression of gD, as Figure 5 shown in C below, and produced antibodies against the gD antigen. Among them, the group of mice with gD guided by sp1 showed higher serum neutralizing antibody levels compared to sp2 and sp3, indicating that the expression of gD antigen guided by sp1 can generate strong immune response and protective efficacy in the body. Therefore, the porcine interleukin-2 signal peptide sequence (sp1) SEQ ID NO.4 was preferably used as the signal peptide to direct the expression of gD antigen. Referring to Example 1, the C-terminal HiBiT tag of the gD antigen in JF-AV04 was deleted to form the JF-AV01 vector to prepare liposome nanoparticles for subsequent immune challenge experiments.

[0150] Example 6 investigated the effect of different nucleotide modifications on the expression level of saRNA

[0151] This example investigated the effect of the incorporation of 5-methylcytidine m5CTP and pseudouridine ψTP into the CTP solution and / or UTP solution in the in vitro transcription synthesis of saRNA in Example 2 on the expression efficiency of saRNA.

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

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

[0154] (2) Single 50% m5CTP modification group: 50% of the CTP in the CTP solution in terms of molar ratio was replaced by m5CTP.

[0155] (3) Single 50% ψTP modification group: 50% of the UTP in the UTP solution in terms of molar ratio was replaced by pseudouridine ψTP.

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

[0157] Then the remaining steps are carried out according to Example 1, Example 2 and Example 3, and 4 groups of JF-EGFP-saRNA NPs solutions are obtained correspondingly (in this experiment, the gD antigen in Example 1 is replaced with the coding gene of EGFP), corresponding to unmodified, 50% m5CTP modification alone, 50% ψTP modification alone, and 50% m5CTP + 50% ψTP modification.

[0158] II. Detection of expression efficiency of different modifications

[0159] Transfect HEK293 cells with 750 ng of unmodified, 50% m5CTP-modified, 50% ψTP-modified, and 50% m5CTP + 50% ψTP-modified saRNA-EGFP nanoliposome particles for 48 hours. The transfection method is as follows: Seed 100,000 HEK293T cells per well in a 12-well plate and culture overnight. After the cells adhere, transfer the nanoliposome particles containing 750 ng of saRNA (referring to the RNA content encapsulated in the liposome nanoparticles) to the cell culture wells and detect after 24 hours of transfection.

[0160] Detect the expression level of green fluorescent protein in cells by flow cytometry experiment using a flow cytometer (Thermo Fisher, Attune NxT). The results are as Figure 6 shown. In this experiment, the saRNA-EGFP with 50% m5CTP modification has the highest expression efficiency, followed by the unmodified saRNA-EGFP, then the saRNA-EGFP with 50% ψTP modification, and the expression level of 50% m5CTP modification + 50% ψTP modification is the lowest.

[0161] III. Detection of expression efficiency of different ratios of the same modification

[0162] Transfection method: Transfect HEK293 cells with 50% ψTP-modified, 50% m5CTP-modified, and 100% m5CTP-modified (replace CTP with m5CTP at a molar ratio of 100% in the CTP solution in the 50% m5CTP modification group alone in (2)) saRNA-EGFP nanoliposome particles at two doses of 375 ng and 750 ng (referring to the RNA content encapsulated in the liposome nanoparticles) for 48 hours (the method is the same as that in step II. Detection of expression efficiency of different modifications).

[0163] Detect the expression level of green fluorescent protein in cells by flow cytometry experiment using a flow cytometer (Thermo Fisher, Attune NxT). The results are as Figure 7As shown, the transfection efficiency increases with the increase of RNA dose. The expression efficiency of m5CTP-modified saRNA-EGFP is better than that of ψTP-modified saRNA-EGFP, and with the increase of the 50% m5CTP incorporation ratio, the expression level increases accordingly. Referring to the transfection positive rate of 50% ψTP modification alone, the transfection positive rate of 50% m5CTP modification alone is higher than that of 50% ψTP modification alone, and the transfection positive rate of 100% m5CTP modification alone is the highest among groups at the same dose.

[0164] Example 7

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

[0166] Among them, mRNA-Lucferase is an mRNA molecule: the preparation method is basically the same as that of JF-Luc RNA saRNA, except that the coding replicase elements NSP1, 2, 3, 4 and sgP elements are not added. The obtained mRNA-Lucferase was used to prepare mRNA-Lucferase LNPs according to Example 3.

[0167] I. Experimental method

[0168] 4-6-week-old Balb / c mice were injected intramuscularly in the leg with JF-Luc RNA LNPs and mRNA-Lucferase LNPs. Two doses (5 μg and 2.5 μg, referring to the RNA content encapsulated in the liposome nanoparticles) were selected for a single injection, and the Lucferase luciferase signal was detected by a multifunctional microplate reader (FlexStation3) on days 9, 11, 21, 30, 41, and 50.

[0169] The results are as Figure 8 shown. The expression level of the optimized JF-Luc RNA LNPs is better than that of mRNA-Lucferase LNP. mRNA-Lucferase LNP was undetectable on day 11, but JF-Luc RNA LNPs still had strong expression signals, and the luciferase expression duration of JF-Luc RNALNPs was extended to day 50.

[0170] Example 8 RNA vaccine evaluation

[0171] I. Design of piglet immunization plan

[0172] For PRV-negative piglets aged 28 to 35 days, the immunization method on Day 0 (the 0th day) was intramuscular injection of liposomal nanoparticles, with an inoculation volume of 0.5 mL. A booster immunization was carried out on Day 28 (the 28th day) (3 groups). The specific immunization plan is shown in Table 7 below;

[0173] Observe for 14 days after inoculation, observing clinical symptoms such as spirit and appetite every day, and weighing on the 7th and 14th days; Collect serum on days 7, 14, 21, 28, 35, and 42 to measure neutralizing antibodies (fixed virus-diluted serum method) and cytokine secretion levels (Elabscience);

[0174] On Day 42, 3 pigs were selected from each group for virus challenge; Observe for 14 days after virus challenge, observing clinical symptoms, body temperature response, etc. every day, collecting nasal swabs every day, detecting the virus excretion situation by PCR and virus isolation methods, and detecting the virus titer (fixed virus-diluted serum method). On the 14th day after virus challenge, perform necropsy, take tissues such as brain, liver, spleen, lung, lymph, and tonsil, observe the lesion situation and make pathological sections.

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

[0176] Table 7. Immunization plan

[0177]

[0178]

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

[0180] II. Detection of neutralizing antibodies

[0181] For PRV-negative piglets aged 28 to 35 days, blood was collected from the anterior vena cava on days 14, 21, 28, 35, and 42 after the first immunization, serum was separated, and the PRV neutralizing antibodies in the serum were detected. The data results are as follows:

[0182] Table 8. Average neutralizing antibody titers of each group on days 14, 21, 28, 35, and 42 after the first immunization

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

[0184] The results are as Figure 9As shown in the above table, on days 14 and 21 after the first immunization, the average antibody levels in the JF-AV01 RNA LNPs immunization groups were comparable to those of the live vaccine and inactivated vaccine, with no significant difference; the JF-AV01 RNA LNPs 50 μg immunization group was higher than the JF-AV01 RNA LNPs 5 μg immunization group. On day 28 after the first immunization, the single immunization group of the live vaccine and the second immunization group of the inactivated vaccine were higher than the three JF-AV01 RNA LNPs immunization groups. Some groups were boost-immunized on day 28. On day 7 after the boost immunization, the neutralizing antibody level in the JF-AV01 RNA LNPs 5 μg second immunization group increased significantly, and the neutralization titer could reach 2 -7.65 (1:200), which was significantly higher than that of the live vaccine 2 -4.34 (1:20) and the inactivated vaccine 2 -4.78 (1:27) immunization groups (P<0.0001); after the boost immunization, the neutralizing antibody in the JF-AV01 RNA LNPs 5 μg second immunization group maintained a high level. On day 14 after the boost immunization, the neutralization titer could reach 2 -8.08 . Single immunization with JF-AV01 RNA LNPs showed a protection efficiency comparable to that of traditional vaccines. After the second immunization, the protection efficiency of the JF-AV01 RNA LNPs vaccine far exceeded that of the attenuated vaccine and inactivated vaccine. It was proved that the saRNA vaccine of the present invention was far superior to traditional vaccines in long-term immunity.

[0185] III. Cytokine detection

[0186] Using the double antibody sandwich ELISA method, the JF-AV01 saRNA LNPs 5 μg second immunization group, LA2017 live vaccine single immunization group, LA-A inactivated vaccine second immunization group, and JF-Luc control group were selected to detect the levels of IL-4 and IFN-γ cytokines in the serum samples of piglets on day 21 and day 35 (day 7 after the boost immunization) after the first immunization ( Figure 10 A and B in).

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

[0188]

[0189]

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

[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] On day 21 after the first immunization, the IFN-γ level in the secondary immunization group with 5 μg of JF-AV01 RNA LNPs was significantly higher than that in the control group (P = 0.0018). There was no significant difference between the live vaccine LA2017 primary immunization group and the control group, and the IFN-γ level in the secondary immunization group with LA-A inactivated vaccine was significantly higher than that in the control group (P = 0.0196). On day 7 after the booster immunization, the IFN-γ level in the secondary immunization group with 5 μg of JF-AV01 RNA LNPs was significantly higher than that in the control group (P < 0.0001), the live vaccine LA2017 group (P = 0.0003), and the secondary immunization group with LA-A inactivated vaccine (P = 0.0010). On day 21 after the first immunization, there was no significant difference in the IL-4 cytokine level among all groups. On day 7 after the booster immunization, the IL-4 level in the secondary immunization group with 5 μg of JF-AV01 RNA LNPs was significantly higher than that in the control group (P < 0.01) and the live vaccine LA2017 group (P < 0.01). This experiment proved that the saRNA vaccine of the present invention could induce stronger immune responses in the body, thereby enhancing the body's protective ability against PRV.

[0193] III. Viral challenge protection experiment

[0194] The body temperature changes of piglets after viral challenge are shown in the following table, as shown in the following table and Figure 11 shown.

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

[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 of each group after viral challenge (°C)

[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 day 3 after viral challenge, the body temperatures of piglets in the viral challenge control group all reached above 41 °C, and typical symptoms such as reduced appetite, listlessness, abdominal breathing, and purulent nasal discharge began to appear. One piglet in the control group died on day 7 after viral challenge, and all the remaining piglets in the control group were diseased. Symptoms such as anorexia, ataxia, unconsciousness, and dyspnea appeared before necropsy at the end of the viral challenge observation.

[0200] In the piglets immunized once with 50 μg of JF-AV01 RNA LNPs, one piglet had a body temperature exceeding 41°C for 4 days. Another piglet still had symptoms such as loss of appetite, listlessness, and purulent nasal discharge before necropsy at the end of the observation period. The clinical symptoms of the remaining piglets disappeared on the 9th day. In the piglets immunized once with 5 μg of JF-AV01 RNA LNPs, one piglet had a body temperature exceeding 41°C for 3 days during the observation period, and the clinical symptoms (body temperature exceeding 41°C) of all piglets in this group disappeared on the 10th day. The piglets in the JF-AV01 RNA LNPs 5 μg twice immunized group only showed mild symptoms after challenge, manifested as reduced appetite and listlessness, and the symptoms disappeared on the 8th day. In the piglets in the LA-A inactivated vaccine twice immunized group, one piglet had a body temperature exceeding 41°C and lasted for 3 days after challenge. On the 6th day, all piglets had loss of appetite and listlessness, and the clinical symptoms disappeared on the 8th day. The piglets in the LA2017 live vaccine once immunized group only showed mild symptoms after challenge, manifested as reduced appetite and listlessness, and the symptoms disappeared on the 6th day, and individual piglets lasted until the 9th day. The results of the challenge protection efficiency are shown in Table 13. This experimental case proves that the low-dose booster immunization of the saRNA of the present invention can completely protect piglets from death caused by PRV challenge.

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

[0202] Vaccine Group Number of Protected Animals Protection Rate JF-AV01 RNA LNPs-50 μg 1 / 3 33.33％ JF-AV01RNA LNPs-5 μg 2 / 3 66.66％ JF-AV01RNA LNPs-5 μg-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 embodiments are only examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims

1. A pseudorabies immunogen composition, characterized in that: 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 pseudorabies virus.

2. The pseudorabies immunogen composition according to claim 1, characterized in that The pseudorabies virus is selected from the PRV AH02LA variant.

3. The pseudorabies immunogen composition according to claim 2, characterized in that: The polypeptide of the gD antigen of the PRV AH02LA variant is an amino acid sequence as shown in SEQ ID NO.1 or an amino acid sequence having more than 80% homology thereto; and / or, the DNA sequence of the polypeptide of the gD antigen of the PRV AH02LA variant is codon-optimized and is a nucleotide sequence as shown in SEQ ID NO.2 or a nucleotide sequence having more than 80% homology thereto; And / or, the DNA sequence of the polypeptide of the gD antigen of the PRV AH02LA variant is codon-optimized and is a nucleotide sequence as shown in SEQ ID NO.3 or a nucleotide sequence having more than 80% homology thereto.

4. The pseudorabies immunogen composition according to claim 3, characterized in that The antigen saRNA molecule includes the following elements in the 5'-3' direction: 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; And / or, the 5'UTR sequence is an optimized 5'UTR sequence of human β-globin; And / or, the 3'UTR sequence is a series of 2-3 optimized 3'UTR sequences of human β-globin or 3'UTR sequences of African clawed frog β-globin; And / or, the poly(A) tail has a length of 30-200 nucleotides.

5. The pseudorabies immunogen composition according to claim 4, characterized in that The antigen saRNA molecule also includes an sg promoter, a signal peptide encoding gene sequence, and a sequence encoding a replicase.

6. The pseudorabies immunogen composition according to claim 5, characterized in that The antigen saRNA molecule includes the following elements in the 5'-3' direction: a 5' cap structure, a 5'UTR sequence, a gene sequence encoding a replicase, an sgP promoter, a signal peptide encoding gene sequence, a gD antigen encoding gene sequence, a poly (A) tail and a 3'UTR sequence; And / or, the signal peptide is selected from porcine interleukin 2 signal peptide, porcine interleukin 4 signal peptide or PRV gD protein original signal peptide.

7. The pseudorabies immunogen composition according to claim 6, characterized in that The sequence encoding the replicase is the coding gene sequence of NSP 1, 2, 3, 4; And / or, the porcine interleukin-2 signal peptide sequence is an amino acid sequence as shown in SEQ ID NO.4 or an amino acid sequence having more than 80% homology thereto; And / or, the porcine interleukin-2 signal peptide sequence is codon-optimized, such as the nucleotide sequence shown in SEQ ID NO.5 or a nucleotide sequence having more than 80% homology thereto; And / or, the porcine interleukin-4 signal peptide sequence is the amino acid sequence shown in SEQ ID NO.6 or an amino acid sequence having more than 80% homology thereto; And / or, the original signal peptide sequence of the PRV gD protein is the amino acid sequence shown in SEQ ID NO.7 or an amino acid sequence having more than 80% homology thereto.

8. The pseudorabies immunogen composition according to claim 7, characterized in that Synthesized by in vitro transcription.

9. The pseudorabies immunogen composition according to claim 8, characterized in that 5-methylcytosine is doped into the CTP solution in the in vitro transcription system, and / or pseudouridine is doped into the UTP solution, and the molar ratio of 5-methylcytosine and / or pseudouridine is 10-100%.

10. A biomaterial, characterized in that: Includes any of the following: A1. A recombinant vector plasmid containing the antigen saRNA molecule described in any one of claims 1 to 9; A2. A host cell containing the recombinant vector described in A1.

11. A pharmaceutical composition, characterized in that The invention comprises the pseudorabies immunogen composition according to any one of claims 1 to 9, and a pharmaceutically acceptable carrier.

12. The use of the pseudorabies immunogen composition according to any one of claims 1 to 9, the biological material according to claim 10 or the pharmaceutical composition according to claim 11 comprises: (1) Use in the preparation of vaccines for the prevention of pseudorabies in pigs; (2) Use in the preparation of products for inhibiting pseudorabies virus; (3) Use in the preparation of pseudorabies related detection reagent products.

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