Creation and application of PEDV and TGEV circular RNA combined vaccine

Abstract

The invention discloses creation and application of a PEDV (porcine epidemic diarrhea virus) and TGEV (transmissible gastroenteritis virus) circular RNA (ribonucleic acid) combined vaccine. The vaccine composition provided by the invention is composed of a PEDV circular RNA vaccine and a TGEV circular RNA vaccine, can simultaneously prevent and treat two infectious diseases, namely porcine epidemic diarrhea virus and transmissible gastroenteritis virus, and compared with a single vaccine, the bivalent vaccine composition does not generate an immune interference phenomenon, the immune effect of the vaccine is not reduced, and the vaccine composition can be used for preventing and treating the porcine epidemic diarrhea virus and the transmissible gastroenteritis virus. And adverse reactions caused by multiple inoculation immunization can be avoided, and the method has wide application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically, relates to the creation and application of a circular RNA bivalent vaccine for porcine epidemic diarrhea virus and porcine transmissible gastroenteritis virus. Background Art

[0002] China is one of the countries with the richest livestock resources and a long history of animal husbandry in the world. According to statistics from the Food and Agriculture Organization of the United Nations, China's animal husbandry has developed rapidly, and its pork, mutton and egg production ranks first in the world. In particular, the pig industry occupies a very important position. According to data from the National Bureau of Statistics, China's pork production in 2021 was 52.9593 million tons, making it the world's largest pork producer. Faced with the current situation of such a large pig population, many problems have also arisen, especially the occurrence of animal diseases, coupled with the increase in zoonotic diseases, which has led to a surge in public health pressure in various countries, seriously affecting human health and the development of the pig industry.

[0003] Both PEDV and TGEV belong to the genus Alphacoronavirus, which is a single-stranded positive-sense RNA virus. Both viruses have been identified as the causative agent of pandemic swine diarrhea, causing significant losses to the pig industry. TGEV was first documented in the United States in 1946 and subsequently confirmed in Europe, Asia, Africa, and South America. The first discovery of PEDV dates back to the early 1970s. Since 2010, highly pathogenic strains of PEDV have gradually spread in pig populations and have continued to be prevalent in many countries in Asia, North America, and Europe, especially in newborn piglets less than 10 days old, with a mortality rate of 100% after PEDV infection. The widespread spread of PEDV and TGEV around the world has brought serious economic consequences to the global pig industry. In the face of complex and variable diarrhea epidemics, vaccination has become the most effective preventive measure. Due to the serious co-infection of PEDV and TGEV, a single vaccination cannot provide protection against both virus infections at the same time. Therefore, bivalent inactivated vaccines or live attenuated vaccines against PEDV and TGEV have been mainly used for piglet protection. However, the effectiveness and safety of current vaccines are not enough. Therefore, there is an urgent need to develop new bivalent vaccines that are both safe and effective against PEDV and TGEV.

[0004] The surface glycoprotein (S) of PEDV and TGEV is located in the outermost layer of the virus particle. The S protein plays a vital role in the host cell binding process, so its full-length or truncated form has become an important target for vaccine development. Studies have shown that the conserved epitope (COE) region of PEDVS1 acts as a neutralizing epitope, while the N-terminal domain (NTD) region acts as a potential co-receptor binding region. The S1 domain of TGEV contains four antigenic sites (or two antigenic sites), namely C, B, D and A (or NTD and RBD), arranged in sequence from the N-terminus to the C-terminus. Among these sites, both A and D are able to induce the production of neutralizing antibodies against TGEV in the host body. Therefore, in the immunogenicity studies related to PEDV or TGEV vaccines, the full-length S protein or specific domains, such as S1 or COE regions, are widely used.

[0005] Recent studies have shown that mRNA vaccines have significant advantages over traditional vaccines, including the ability to flexibly target emerging or mutated pathogens and the ability to quickly design, generate, and induce strong immune responses. Studies on PEDV mRNA vaccines have also been reported and have produced promising immune effects. However, the main limitation of mRNA vaccines is that they are easily degraded by RNases and need to be stored and transported at low temperatures. This poses a major challenge to the development of veterinary vaccines.

[0006] It has been established that circRNAs can be translated into proteins through a cap-independent mechanism, which makes them potentially powerful alternatives to mRNA. Unlike linear mRNA, circRNA has excellent stability due to its covalently closed circular structure. In addition, circRNA exhibits reduced immunogenicity and maintains sustained and efficient protein expression capabilities even in the absence of nucleotide modifications. In addition, the absence of capping and tailing reactions in circRNA synthesis further reduces production costs. These advantages make circRNA an attractive candidate for vaccine development, especially in the field of veterinary vaccines. Based on the above advantages, we developed circRNA vaccines against PEDV and TGEV. Summary of the invention

[0007] In order to make up for the shortcomings of the prior art, the main purpose of the present invention is to use the circular RNA vaccine platform to develop a multivalent and multi-linked vaccine, in order to produce a protective effect in response to infections from different pathogens, especially by optimizing the dose of various antigen components to immunize the body to achieve a similar level of protection against infections from different viruses.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A first aspect of the present invention provides a circular RNA.

[0010] Further, the circular RNA includes any one of the following:

[0011] 1) The circular RNA comprises a coding element, and the coding element encodes a polypeptide comprising the S1 region of the PEDV S protein and the Fc region of the porcine immunoglobulin;

[0012] 2) the circular RNA comprises a coding element, and the coding element encodes a polypeptide comprising the S1 region of the TGEV S protein;

[0013] Preferably, the PEDV S protein S1 region has an amino acid sequence as shown in SEQ ID NO.5, or an amino acid sequence having at least 90% identity with SEQ ID NO.5.

[0014] Preferably, the porcine immunoglobulin Fc region includes the Fc region of porcine IgG, IgA, IgD, IgE or IgM.

[0015] Preferably, the porcine immunoglobulin Fc region is the Fc region of porcine IgG.

[0016] More preferably, the amino acid sequence of the Fc region of the porcine IgG has the amino acid sequence shown in SEQ ID NO.6, or an amino acid sequence having at least 90% identity with SEQ ID NO.6.

[0017] Preferably, the amino acid sequence of the S1 region of the truncated fragment of the TGEV S protein has the amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence that has at least 90% identity with SEQ ID NO.7.

[0018] Furthermore, the nucleotide sequence encoding the S1 region of the PEDV S protein comprises the sequence shown in SEQ ID NO.2, or a sequence having at least 90% identity with the sequence shown in SEQ ID NO.2.

[0019] Furthermore, the nucleotide sequence encoding the Fc region of porcine IgG comprises the sequence shown in SEQ ID NO.3, or a sequence having at least 90% identity with the sequence shown in SEQ ID NO.3.

[0020] Furthermore, the nucleotide sequence encoding the S1 region of the truncated fragment of the TGEV S protein comprises the sequence shown in SEQ ID NO.4, or a sequence having at least 90% identity with the sequence shown in SEQ ID NO.4.

[0021] In the present invention, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein and are amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component).

[0022] In the present invention, the term "circular RNA" refers to an RNA molecule in a closed circular shape. In the present invention, the circular RNA used has protein translation activity and can also be called "circular RNA".

[0023] In the present invention, the truncated fragment S1 region of the PEDV S protein has an amino acid sequence shown in SEQ ID NO.5, or an amino acid sequence with a homology of more than 90% to SEQ ID NO.5; similarly, the truncated fragment S1 region of the TGEV S protein has an amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence with a homology of more than 90% to SEQ ID NO.7.

[0024] In the present invention, the terms "sequence identity" and "percentage of identity" refer to the percentage of nucleotides or amino acids that are identical (i.e., identical) between two or more polynucleotides or polypeptides. The sequence identity between two or more polynucleotides or polypeptides can be determined by the following method: the nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned and the number of positions containing the same nucleotide or amino acid residue in the aligned polynucleotides or polypeptides is scored, and compared with the number of positions containing different nucleotides or amino acid residues in the aligned polynucleotides or polypeptides. The polynucleotides may differ at one position, for example, by containing different nucleotides (i.e., substitutions or mutations) or missing nucleotides (i.e., nucleotide insertions or nucleotide deletions in one or two polynucleotides). The polypeptides may differ at one position, for example, by containing different amino acids (i.e., substitutions or mutations) or missing amino acids (i.e., amino acid insertions or amino acid deletions in one or two polypeptides). The sequence identity can be calculated by dividing the number of positions containing the same nucleotide or amino acid residue by the total number of amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing the identical nucleotide or amino acid residue by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide and multiplying by 100.

[0025] In the present invention, the amino acid sequence of the truncated fragment S1 region of the PEDV S protein obtained by modification also belongs to the protection scope of the present invention. The term "modification" refers to any chemical modification of the amino acid sequence, such as substitution, deletion, insertion and / or addition of amino acids. The term "substitution" refers to the replacement of one or more amino acids by different amino acids. "Deletion" refers to the reduction of one or more amino acids in the amino acid sequence. "Insertion" or "addition" refers to the change in the amino acid sequence resulting in the increase of one or more amino acids compared to the naturally occurring molecule.

[0026] In this article, "nucleotide" includes deoxyribonucleotides and ribonucleotides and their derivatives. As used herein, "ribonucleotide" is a constituent substance of ribonucleic acid (RNA), consisting of one molecule of base, one molecule of pentose, and one molecule of phosphoric acid, which refers to a nucleotide with a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. "Deoxyribonucleotide" is a constituent substance of deoxyribonucleic acid (DNA), also consisting of one molecule of base, one molecule of pentose, and one molecule of phosphoric acid, which refers to a nucleotide in which the hydroxyl group at the 2' position of the β-D-ribofuranosyl group is replaced by hydrogen, and is the main chemical component of chromosomes. "Nucleotide" is usually referred to by a single letter representing the base: "A (a)" refers to deoxyadenosine or adenylic acid containing adenine, "C (c)" refers to deoxycytidine or cytidine containing cytosine, "G (g)" refers to deoxyguanosine or guanylate containing guanine, "U (u)" refers to uridylate containing uracil, and "T (t)" refers to deoxythymidylate containing thymine.

[0027] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to a polymer of deoxyribonucleotides (deoxyribonucleic acid, DNA) or a polymer of ribonucleotides (ribonucleic acid, RNA). "Polynucleotide sequence", "nucleic acid sequence" and "nucleotide sequence" are used interchangeably to refer to the order of nucleotides in a polynucleotide. It should be understood by those skilled in the art that a DNA coding strand (sense strand) and the RNA it encodes can be considered to have the same nucleotide sequence, and the deoxythymidylic acid in the DNA coding strand sequence corresponds to the uridine acid in the RNA sequence it encodes.

[0028] The second aspect of the present invention provides a linear RNA or a recombinant nucleic acid molecule.

[0029] Furthermore, the linear RNA undergoes a self-cleavage reaction and then cyclizes to form the circular RNA as described in the first aspect of the present invention.

[0030] Furthermore, the recombinant nucleic acid molecule forms the circular RNA described in the first aspect of the present invention after transcription.

[0031] In the present invention, the term "linear RNA" refers to an RNA precursor that can be circularized to form a circular RNA, which is generally transcribed from a linear DNA molecule and can also be called "linear RNA".

[0032] In the present invention, the term "linear RNA" refers to RNA with translation function including a 5' cap structure (5' Cap), a 3' polyadenosine tail (PolyAtail), a 5' untranslational region (5' UTR), a 3' untranslational region (3' UTR) and an open reading frame (Open reading frame, ORF) and other structures, which can also be called "linear RNA".

[0033] The third aspect of the present invention provides an expression vector.

[0034] Furthermore, the vector comprises the linear RNA described in the second aspect of the present invention.

[0035] As used herein, the term "expression" includes transcription and / or translation of a nucleotide sequence. Thus, expression may involve the production of transcripts and / or polypeptides. The term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA (transcript). The term "in vitro transcription" refers to the in vitro synthesis of RNA, particularly mRNA, in a cell-free system (e.g., in an appropriate cell extract) (see, e.g., Pardi et al., 1996).

[0036] N., Muramatsu H., Weissman D., Karikó K. (2013). In: Rabinovich P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation. Methods in Molecular Biology (Methods and Protocols), vol 969. Humana Press, Totowa, NJ.). A vector that can be used to produce a transcript is also called a "transcription vector", which contains a regulatory sequence required for transcription. The term "transcription" covers "in vitro transcription".

[0037] As used herein, "encoding" refers to the inherent properties of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA can be used as a template to synthesize polymers and macromolecules in other biological processes, as long as there is a clear nucleotide sequence or a clear amino acid sequence. Therefore, a gene encodes a protein when the gene's mRNA produces a protein in a cell or other biological system through transcription and translation.

[0038] A fourth aspect of the present invention provides a porcine epidemic diarrhea virus circular RNA vaccine.

[0039] Furthermore, the vaccine comprises the circular RNA described in 1) of the first aspect of the present invention.

[0040] Preferably, the vaccine comprises a lipid encapsulating the circular RNA.

[0041] Preferably, the lipid comprises a cationic lipid, a phospholipid, a steroid and a polyethylene glycol-modified lipid.

[0042] Preferably, the vaccine comprises lipid nanoparticles or lipid polymer complexes.

[0043] In the present invention, the term "vaccine" means that the vaccine is an autoimmune preparation for preventing infectious diseases made by artificially attenuating, inactivating or using transgenic methods for pathogenic microorganisms (such as bacteria, rickettsia, viruses, etc.) and their metabolites. In some embodiments, the vaccine in the present disclosure is a PEDV circular RNA vaccine and a TGEV circular RNA vaccine. In some embodiments, the vaccine in the present disclosure is a nucleic acid vaccine, which comprises a recombinant nucleic acid molecule, a recombinant expression vector, a linear RNA or a circular RNA that expresses an antigenic polypeptide. In some more specific embodiments, the vaccine in the present disclosure is a nucleic acid vaccine, which comprises a circular RNA that expresses an antigenic polypeptide.

[0044] As used herein, the term "lipid" refers to an organic compound comprising a hydrophobic portion and optionally also a hydrophilic portion. Lipids are generally insoluble in water but soluble in many organic solvents. Typically, amphipathic lipids comprising a hydrophobic portion and a hydrophilic portion can be organized into a lipid bilayer structure in an aqueous environment, for example, in the form of vesicles. Lipids may include, but are not limited to, fatty acids, glycerides, phospholipids, sphingolipids, glycolipids, steroids, and cholesterol esters, etc.

[0045] As used herein, "lipid nanoparticle" or "LNP" refers to a lipid vesicle having a uniform lipid core, which is a particle formed by lipids, the lipid components undergoing intermolecular interactions to form a nanostructured entity. Nucleic acids are encapsulated in the lipids.

[0046] The fifth aspect of the present invention provides a porcine transmissible gastroenteritis virus circular RNA vaccine.

[0047] Furthermore, the vaccine comprises the circular RNA described in 2) in the first aspect of the present invention.

[0048] Furthermore, the vaccine comprises lipids that encapsulate the circular RNA.

[0049] Preferably, the lipid comprises a cationic lipid, a phospholipid, a steroid and a polyethylene glycol-modified lipid.

[0050] Preferably, the vaccine comprises lipid nanoparticles or lipid polymer complexes.

[0051] A sixth aspect of the present invention provides a composition, which is any one of the following:

[0052] 1) A vaccine composition, comprising the porcine epidemic diarrhea virus circular RNA vaccine described in the fourth aspect and the porcine transmissible gastroenteritis virus circular RNA vaccine described in the fifth aspect;

[0053] 2) A circular RNA composition, comprising the circular RNA described in 1) and the circular RNA described in 2) in the first aspect of the present invention.

[0054] Preferably, the mixing ratio of the porcine epidemic diarrhea virus circular RNA vaccine and the porcine transmissible gastroenteritis virus circular RNA vaccine is 4:1 to 8:1.

[0055] Preferably, the mixing ratio of the circular RNA described in 1) and the circular RNA described in 2) in the first aspect is 4:1 to 8:1.

[0056] The seventh aspect of the present invention provides a vaccine preparation.

[0057] Furthermore, the vaccine preparation comprises the vaccine composition described in the sixth aspect of the present invention and an inactivated diarrhea virus bivalent vaccine.

[0058] Preferably, the vaccine preparation contains two dosage units, the first dosage unit contains the inactivated diarrhea virus bivalent vaccine, and the second dosage unit contains the vaccine composition described in the sixth aspect of the present invention.

[0059] Preferably, the first dosage unit is used in the initial immunization, and the second dosage unit is used in the secondary immunization.

[0060] Preferably, the inactivated diarrhea virus bivalent vaccine is Coxiel.

[0061] An eighth aspect of the present invention provides a method for immunizing pigs.

[0062] Furthermore, the method comprises administering to pigs the porcine epidemic diarrhea virus circular RNA vaccine described in the fourth aspect, the porcine transmissible gastroenteritis virus circular RNA vaccine described in the fifth aspect of the present invention, the composition described in the sixth aspect of the present invention, or the vaccine preparation described in the seventh aspect of the present invention.

[0063] The ninth aspect of the present invention provides any one of the following applications.

[0064] Furthermore, the application includes:

[0065] 1) Use of the circular RNA described in the first aspect of the present invention, the linear RNA described in the second aspect of the present invention, or the expression vector described in the third aspect of the present invention in the preparation of a circular RNA vaccine for porcine epidemic diarrhea virus or a circular RNA vaccine for porcine transmissible gastroenteritis virus;

[0066] 2) Use of the porcine epidemic diarrhea virus circular RNA vaccine described in the fourth aspect of the present invention, the porcine transmissible gastroenteritis virus circular RNA vaccine described in the fifth aspect of the present invention, the composition described in the sixth aspect of the present invention, or the vaccine preparation described in the seventh aspect of the present invention in the preparation of a drug against porcine epidemic diarrhea virus and / or porcine transmissible gastroenteritis virus;

[0067] 3) Use of the combination of the composition described in the sixth aspect of the present invention and an inactivated diarrhea virus bivalent vaccine in the preparation of a drug against porcine epidemic diarrhea virus and / or porcine transmissible gastroenteritis virus.

[0068] In the present invention, "treatment" means: after suffering from a disease, the subject is exposed to (e.g., administered) the vaccine composition of the present invention, thereby alleviating the symptoms of the disease compared to when the subject is not exposed, and does not necessarily mean that the symptoms of the disease are completely suppressed. Suffering from a disease means: the body has symptoms of the disease.

[0069] In the present invention, "prevention" means that before contracting a disease, by exposing a subject to the circular vaccine composition of the present invention, the probability of contracting a disease and / or the symptoms after contracting a disease are reduced compared to when the subject has not been exposed, and it does not necessarily mean that the disease must be completely suppressed.

[0070] The present invention also provides a method for preventing and / or treating porcine epidemic diarrhea virus and / or porcine transmissible gastroenteritis virus, which comprises administering to a subject an effective amount of the above-mentioned vaccine composition or the vaccine preparation described in the seventh aspect of the present invention; preferably, the administration is performed by injection.

[0071] In the present invention, the term "individual", "patient" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0072] In the present invention, the terms "comprising", "having", "including" or "containing" may mean inclusive or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising", "having", "including" or "containing" may also mean closed, excluding additional, uncited elements or method steps.

[0073] In the present invention, the word "may" includes both performing a certain process and not performing a certain process.

[0074] Advantages and benefits of the present invention:

[0075] Compared with a single vaccine, the vaccine combination provided by the present invention does not produce the immune interference phenomenon between vaccines that is usually seen in the prior art of injecting two or more vaccines at the same time, and the vaccine immune effect is not reduced. The antibodies produced are equivalent to the immune effect of a single vaccine. While reducing the cost of immunization, it saves immunization procedures and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 circRNA PS1 CircRNA TS1 Characterization diagram, 1A-1E are PEDVS and TGEVS antigen pattern diagrams respectively ( Figure 1 A) circRNA PS1 CircRNA TS1 Cyclization Figure 1 B) Schematic diagram of ring sequencing ( Figure 1 C) and circRNA vaccine model diagram ( Figure 1 E), in addition to circRNA PS1 CircRNA TS1 In vitro transfection expression verification diagram ( Figure 1 D)

[0077] Figure 2 circRNA PS1 and circRNA TS1 In vivo immunogenicity assessment of vaccines, Figure 2 A is the mouse immunization flow chart; Figure 2 B is different doses of circRNA PS1 - Analysis of PS1-specific IgG antibody titers induced by LNP vaccine; Figure 2 C represents different doses of circRNA TS1 - Analysis of TS1-specific IgG antibody titers induced by LNP vaccine; Figure 3 circRNA PS1F CircRNA TS1F In vivo and in vitro characterization diagram. Figure 3 A is circRNA PS1F CircRNA TS1F In vitro transfection expression verification diagram, Figure 3 B is the mouse immunization flow chart; Figure 3 C is 10 μg of circRNA PS1-LNP and circRNA PS1F - Analysis of PS1-specific IgG antibody titers induced by LNP vaccine;

[0078] Figure 3 D is 10 μg of circRNA TS1 -LNP and circRNA TS1F - Analysis of TS1-specific IgG antibody titers induced by LNP vaccine;

[0079] Figure 4 For equal doses of circRNA PS1F -LNP and circRNA TS1 -LNP can inhibit the production of PS1 antibodies;

[0080] Figure 4 A is the mouse immunization flow chart; Figure 4 B is the analysis of circRNA inoculated simultaneously PS1F -LNP and circRNA TS1 -LNP-induced PS1-specific IgG antibody titers, as measured by circRNA PS1F -LNP or LNP individuals as control; Figure 4 C is for analysis of circRNA inoculated simultaneously PS1F -LNP and circRNA TS1 -LNP-induced TS1-specific IgG antibody titers, with single LNP or circRNA TS1 -LNP as control; Figure 4 D was treated with the same dose (2 μg) of circRNA PS1F and circRNA TS1 After 48 hours of transfection of HEK293F cells, the expression of PSIF in the supernatant was analyzed. As a control, circRNA was transfected alone. PS1F Or with unrelated circRNA GFP Co-transfection;

[0081] Figure 5 To optimize circRNA PS1F -LNP and circRNA TS1 -LNP dosage to enhance the efficacy of the bivalent vaccine against PEDV and TGEV; Figure 5 A is a schematic diagram of the mouse immunization process; Figure 5 B Single LNP was used as control, and the concentrations of 10 μg and 20 μg circRNA were detected. PS1F Comparative analysis of vaccine-induced PS1-specific IgG antibody titers; Figure 5 C is a single LNP or circRNA PS1F-LNP (20 μg) was used as control, and cells were inoculated with different doses (2.5, 5.0 or 10 μg) of circRNA TS1 circRNA PS1F -LNP (20 μg) and circRNA TS1 -Analysis of PS1-specific IgG antibody titers induced by LNP; Figure 5 D is a single LNP or circRNA TS1 -LNP (2.5, 5.0 or 10 μg) was used as control and circRNA was inoculated at the same time PS1F -LNP (20 μg) and containing different doses of circRNA TS1 (2.5, 5.0 or 10 μg) of circRNA TS1 -LNP induced TS1-specific IgG antibody titers; Figure 5 E is the detection of specific T cell response induced by circular RNA vaccine by flow cytometry, with 20 μg circRNA PS1F and 5 μg circRNA TS1 After immunization with the bivalent circRNA vaccine, CD8 + T cells secreting IFNγ accounted for CD8 + Percentage of T cells;

[0082] Figure 6 The inactivated PEDV and TGEV dual vaccine and the optimized circRNA containing 20 μg PS1F and 5 μg circRNA TS1 Comparison of different immunization schedules of bivalent circRNA vaccines; Figure 6 A-6B is the analysis of serum PS1 and TS1 specific IgG antibody titers after primary and booster immunization of mice with different immunization strategies; Figure 6 C-6D is the analysis of PS1- and TS1-specific IgG1 and IgG2a antibody titers in mouse sera after enhanced immunization with different immunization strategies; Figure 6 E-6F was calculated by calculating the ratio of IgG2a / IgG1 in PS1- and TS1-specific antibodies; Figure 6 G-6H is a test for neutralization of PGEV and TGEV real viruses using the serum of mice after immunization with different enhanced immunization strategies. DETAILED DESCRIPTION

[0083] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments. Unless otherwise specified, the equipment and reagents used in each embodiment and test example can be obtained from commercial sources. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Unless otherwise specified, all technical means in the examples of the present invention are conventional means well known to those skilled in the art, such as enzyme digestion, transformation, bacterial culture, in vitro transcription, etc.

[0084] Based on the information contained in this application, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined process, properties or components, because these embodiments and other descriptions are only for illustrating specific aspects of the present invention. In fact, various changes that the technicians in this field or related fields can obviously make to the embodiments of the present invention are all covered within the scope of the appended claims.

[0085] Example 1 Preparation and characterization of circular RNA vaccines

[0086] Press Figure 1 Design a circular RNA plasmid vector, digest the plasmid with enzymes, perform in vitro transcription using the NEB in vitro transcription kit, add additional GTP to improve the circularization efficiency of the circular RNA, and purify the circular RNA by HPLC. The specific steps are as follows:

[0087] (1) First, the empty vector containing ePIE-CVB3 was cut open with EcoRV, and the genes PEDV S1 (PS1), TGEV S1 (TS1), PEDV S1 pFc (PS1F), and TGEV S1pFc (TS1F) were constructed on the plasmid using the homologous recombination method and extracted according to the instructions of the Tiangen Plasmid Extraction Kit.

[0088] The circular RNA vector sequence is abbreviated as: ePIE-CVB3, and the nucleotide sequence is as follows:

[0089] CGTACGTCGAGCATCGTAATACGACTCACTATAGGGCGAATTGGGAGACCCTCGACCGT

[0090] CGATTGTCCACTGGTCAACAATAGATGACTTACAACTAATCGGAAGGTGCAGAGACTCGACG

[0091] GGAGCTACCCTAACGTCAAGACGAGGGTAAAGAGAGAGTCCAATTCTCAAAGCCAATAGGCA

[0092] GTAGCGAAAGCTGCAAGAGAATGAAAATCCGTTGACCTTAAACGGTCGTGTGGGTTCAAGTC

[0093] CCTCCACCCCCACGCCGGAAACGCAATAGCCGAAAAACAAAAAACAAAAAAAACAAAAAAA

[0094] AAACCAAAAAAACAAAACACATTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTG

[0095] GGCGCTAGCACTCTGGTATCACGGTACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGT

[0096] AACTTAGAAGTAACACACACCGATCAACAGTCAGCGTGGCACACCAGCCACGTTTTGATCAA

[0097] GCACTTCTGTTACCCCGGACTGAGTATCAATAGACTGCTCACGCGGTTGAAGGAGAAAGCGT

[0098] TCGTTATCCGGCCAACTACTTCGAAAAACCTAGTAACACCGTGGAAGTTGCAGAGTGTTTCGC

[0099] TCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCATTCCCCACGGGCGACCGTGG

[0100] CGGTGGCTGCGTTGGCGGCCTGCCCATGGGGAAACCCATGGGACGCTCTAATACAGACATGG

[0101] TGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTG

[0102] CGGAGCACACACCCTCAAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTGCAGCGGAAC

[0103] CGACTACTTTGGGTGTCCGTGTTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAATTGAGA

[0104] GATCGTTACCATATAGCTATTGGATTGGCCATCCGGTGACTAATAGAGCTATTATATATCCCTTTG

[0105] TTGGGTTTATACCACTTAGCTTGAAAGAGGTTAAAACATTACAATTCATTGTTAAGTTGAATAC

[0106] AGCAAAGATATCGATATCAAAAAACAAAAAACAAAACGGCTATTATGCGTTACCGGCGAGAC

[0107] GCTACGGACTTAAATAATTGAGCCTTAAAGAAGAAATTCTTTAAGTGGATGCTCTCAAACTCA

[0108] GGGAAACCTAAATCTAGTTATAGACAAGGCAATCCTGAGCCAAGCCGAAGTAGTAATTAGTAA

[0109] GACCAGTGGACAATCGACGGATAACAGCATATCTAGGCGGCCGCGAATTC(SEQ ID NO.1).

[0110] The target fragment PEDV S1 (abbreviated as PS1), whose nucleotide sequence is:

[0111]

[0112] The target fragment pFc (abbreviated as F), whose nucleotide sequence is:

[0113] GTGGCTGGCCCTAGCGTGTTCATCTTCCCACCTAAGCCTAAGGACACCCTGATGATCAGCCAGACACCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCAAAGAACACGCCGAGGTGCAGTTCTCTTGGTACGTGGACGGCGTGGAAGTGCACACCGCCGAGACAAGACCCAAAGAGGAACAGTTCAACAGCACCTACAGAGTGGTGTCCGTGCTGCCCATCCAGCACCAGGATTGGCTGAAGGGCAAAGAGTTCAAGTGCAAAGTGAACAACGTGGACCTGCCTGCTCCTATCACCAGAACCATCAGCAAGGCCATCGGCCAGAGCAGAGAACCCCAGGTGTACACACTTCCTCCACCAGCCGAGGAACTGAGCCGGTCTAAAGTGACCGTGACCTGCCTCGTGATCGGCTTCTACCCTCCTGACATTCACGTGGAATGGAAGTCCAACGGCCAGCCTGAGCCTGAGGGCAACTACAGAACAACCCCTCCACAGCAGGATGTGGACGGCACATTCTTTCTGTACAGCAAGCTGGCCGTGGATAAGGCCAGATGGGATCACGGCGAGACATTCGAGTGTGCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCTATCAGCAAGACCCAGGGCAAG (SEQ ID NO.3).

[0114] The target fragment TGEV S1 (abbreviated as TS1), whose nucleotide sequence is:

[0115]

[0116] The amino acid sequence of the PEDV S1 (PS1) region is

[0117] ATMETDTLLLWVLLLWVPGSTGDLPQDVTRCSANTNFRRFFSKFNVQAPAVV

[0118] VLGGYLPIGENQGVNSTWYCAGQHPTASGVHGIFLSHIRGGHGFEIGISQEPFD

[0119] PSGYQLYLHKATNGNTNATARLRICQFPSIKTLGPTANNDVTTGRNCLFNKAIP

[0120] AHMSEHSVVGITWDNDRVTVFSDKIYYFYFKNDWSRVATKCYNSGGCAMQY

[0121] VYEPTYYMLNVTSAGEDGISYQPCTANCIGYAANVFATEPNGHIPEGFSFNNW

[0122] FLLSNDSTLVHGKVASNQPLLVNCLLAIPKIYGLGQFFSFNKTIDGVCNGAAVQ

[0123] RAPEALRFNINDTSVILAEGSIVLHTALGTNFSFVCGNSSDPHLATFAIPLGATQ

[0124] VPYYCFLKVDTYNSTVYKFLAVLPPTVREIVITKYGDVYVNGFGYLHLGLLD

[0125] AVTINFTGHGTDDDVSGFWTIASTNFVDALIEVQGTAIQRILYCDDPVSQLKCS

[0126] QVAFDLDDGFYPISSRNLLSHEQPTSFVTLPSFNDHSFVNITVSASFGGHSGAN

[0127] LIASDTTINGFSSFCVDTRQFTISLFYNVTNSYGYVSNSQDSNCPFTLQSVNDY

[0128] LSFSKFCVSTSLLASACTIDLFGYPEFGSGVKFTSLYFQFTKGELITGTPKPLEG

[0129] VTDVSFMTLDVCTKYTIYGFKGEGIITLTNSSFLAGVYYTSDSGQLLAFKNVTSGAVYSVTPCSFSEQAAYVDDDIVGVISSLSNSTF (SEQ ID NO.5); wherein, amino acids 1-2 (AT) are amino acids corresponding to the Kozak sequence, and amino acids 3-23 (METDTLLLWVLLLWVPGSTGD) are amino acids corresponding to the fragment Igκ of the antibody light chain.

[0130] The amino acid sequence of the Fc region of porcine IgG is

[0131] VAGPSVFIFPPKPKDTLMISQTPEVTCVVVDVSKEHAEVQFSWYVDGVEVHT

[0132] AETRPKEEQFNSTYRVVSVLPIQHQDWLKGKEFKCKVNNVDLPAPITRTISKAI

[0133] GQSREPQVYTLPPPAEELSRSKVTVTCLVIGFYPPDIHVEWKSNGQPEPEGNYR

[0134] TTPPQQDVDGTFFLYSKLAVDKARWDHGETFECAVMHEALHNHYTQKSISKTQGK(SEQ ID NO.6);

[0135] The amino acid sequence of the TGEV S1 (TS1) region is

[0136] ATMETDTLLLWVLLLWVPGSTGDIGNHWNLIETFLLNYSSRLPPNSDVVLGD

[0137] YFPTVQPWFNCIRNDSNDLYVTLENLKALYWDYATENITWNHRQRLNVVVN

[0138] GYPYSITVTTTRNFNSAEGAIICICKGSPPTTTTESSLTCNWGSECRLNHKFPICP

[0139] SNSEANCGNMLYGLQWFADEVVAYLHGASYRISFENQWSGTVTLGDMRATT

[0140] LEVAGTLVDLWWFNPVYDVSYYRVNNKNGTTVVSNCTDQCASYVANVFTTQ

[0141] PGGFIPSDFSFNNWFLLTNSSTLVSGKLVTKQPLLVNCLWPVPSFEEAASTFCFE

[0142] GAGFDQCNGAVLNNTVDVIRFNLNFTTNVQSGKGATVFSLNTTGGVTLEISCY

[0143] YDTVSDSSFFSYGEIPFGVTDGPRYCYVLYNGTALKYLGTLPPSVKEIAISKWG

[0144] HFYINGYNFFSTFPIDCISFNLTTGDSDVFWTIAYTSYTEALVQVENTAITKVTY

[0145] CNSHVNNIKCSQLTANLNNGFYPVSSSEVGLVNKSVVLLPSFYTHTIVNITIDL

[0146] GMKRSGYGQPIASTLSNITLPMQDNNTDVYCIRSDQFSVYVHSTCKSSLWDNI

[0147] FKRNCTDVLDATAVIKTGTCPFSFDKLNNYLTFNKFCLSLSPVGANCKFDVAA

[0148] RTRTNEQVVRSLYVIYEEGDNIVGVPSDNSGLHDLSVLHLDSCTDYNIYGRTG

[0149] VGIIRQTNRTLLSGLYYTSLSGDLLGFKNVSDGVIYSVTPCDVSAQAAVIDGTI

[0150] VGAITSINSELLGLTHWTTTPNFYYYSIYNYTNDRTRGTAIDSNDVDCEPVITYSNIGVCKNGAFVFINVTHSDGDVQPIST (SEQ ID NO.7); wherein, amino acids 1-2 (AT) are amino acids corresponding to the Kozak sequence, and amino acids 3-23 (METDTLLLWVLLLWVPGSTGD) are amino acids corresponding to the fragment Igκ of the antibody light chain.

[0151] (2) Plasmid linearization can be performed using a single restriction enzyme site previously reserved in the plasmid DNA. The linearization can be performed by referring to the instructions for the Takara restriction endonuclease.

[0152] (3) Linearized plasmid purification:

[0153] 1) Add water to 500 μL of the digested product, add an equal volume of organic solvent (phenol / chloroform / isoamyl alcohol 25:24:1), mix well, and centrifuge at 4°C, 12,000 rpm for 15 min;

[0154] 2) After centrifugation, transfer the DNA in the upper aqueous phase to a new EP tube, aspirate about 400 μL; add an equal volume of chloroform, a total volume of 800 μL, vortex to mix; centrifuge at 4°C, 12000 rpm for 15 min;

[0155] 3) Take 300 μL of the supernatant and put it into a new centrifuge tube, add 30 μL of 3M sodium acetate; then add twice the volume (660 μL) of pre-cooled anhydrous ethanol; mix thoroughly and precipitate at -20℃ for at least 30 minutes;

[0156] 4) Centrifuge at 14000 rpm for 15 min at 4°C to collect DNA precipitate;

[0157] 5) After removing the supernatant, slowly add 750 μL of ice-cold 70% ethanol to the EP tube; shake gently, centrifuge at 4°C, 14,000 rpm for 5 min, and collect the DNA precipitate;

[0158] 6) Repeat step 5);

[0159] 7) Discard the supernatant, invert the centrifuge tube and let it dry; add 20 μL of RNase-free water to dissolve.

[0160] (4) In vitro transcription:

[0161] 1) Use NEB's in vitro transcription kit to prepare the system in an EP tube according to Table 1:

[0162] Table 1 In vitro transcription system

[0163] Element Quantity (μL) 10XReactionbuffer 2 ATP 2 GTP 2 UTP 2 CTP 2 T7RNApolymeraseMix 2 Template DNA (1 μg / μL) 1 RNase-free water 7 Total 20

[0164] 2) Gently flick to mix and centrifuge to the bottom of the tube. Place the reactants in a PCR instrument and incubate at 37°C for 2 hours.

[0165] 3) DNase I (Takara) was added to degrade the DNA template, and the reaction system was prepared according to Table 2. After mixing, the mixture was placed in a 37°C metal bath for 20-30 min.

[0166] Table 2 DNA degradation system

[0167]

[0168]

[0169] 4) After the incubation, add 2.5 μL of 0.5 M EDTA to the reaction mixture, mix well, centrifuge, place in an 80°C water bath for 2 min to inactivate DNase I, and then perform RNA column purification to obtain RNA.

[0170] Circularization: Add cyclization buffer (50mM Tris-HCl, 10mMMgCl2, 1mM DTT, pH7.5) and GTP to the final concentration of 2mM / L to the RNA component obtained by in vitro transcription, incubate at 55℃ for 15min, and then purify the RNA column to obtain the reaction product containing circular RNA. The splicing site sequencing results showed that the circular RNA splicing reaction occurred at the expected position. The expression was verified by transfecting 293F cells ( Figure 1 D and Figure 3 A).

[0171] Example 2 Determination of PS1 and TS1-specific IgG in mouse serum (circRNA at different doses) PS1 -LNP and circRNA TS1 -LNP)( Figure 2 )

[0172] 45 female Balb / c mice aged 6-8 weeks were divided into 9 groups, including CircRNA PS1 -LNP (1, 2.5, 5, 10 μg) and circRNA TS1 -LNP (1, 2.5, 5, 10 μg) and LNP group. Vaccination was given by intramuscular injection, once every 2 weeks, for a total of two immunizations. Blood was collected 14 days after the first and second immunizations, and ELISA was performed to detect the antibody level in the serum. The results are shown in Figure 2 .

[0173] The results of antibody level detection showed that the antibody levels against TS1 and PS1 in serum increased in a dose-dependent manner. TS1 When the LNP vaccination doses were 2.5 μg, 5 μg, and 10 μg, the specific antibodies against TGEV S1 in the serum increased steadily, and the antibody endpoint titer of TGEV S1 in the 10 μg vaccine group was close to 10 6 The circRNA PS1When the LNP vaccination doses were 2.5μg, 5μg, and 10μg, the specific antibodies against PEDV S1 in the serum increased significantly, especially in the group vaccinated with 10μg of vaccine, the average increase in antibody titer was more than ten times compared with the group vaccinated with 5μg of vaccine ( Figure 2 B-2C).

[0174] Example 3 PS1 (circRNA) in mouse serum PS1F (10μg)-LNP and circRNA PS1 (10 μg)-LNP) and TS1-specific IgG (circRNA TS1F (10μg)-LNP and circRNA TS1 (10μg)-LNP) determination ( Figure 3 )

[0175] We will circRNA PS1F (10μg)-LNP, circRNA PS1 (10 μg)-LNP and circRNA TS1F (10μg)-LNP and circRNA TS1 The (10 μg)-LNP vaccine (10 μg) was injected intramuscularly into 6-8 week old Balb / c female mice. Serum samples were collected 14 days after the first immunization and booster immunization, and the PS1 and TS1 serum specific antibodies were quantitatively detected by enzyme-linked immunosorbent assay (ELISA). Figure 3 B). The results showed that in the evaluation of PS1-specific antibodies, the circRNAPS1 vaccine group containing the pFc tag elicited significantly higher antibody levels than the group without the pFc tag. In contrast, when evaluating the level of specific antibodies against TS1, fusion with the pFc tag did not enhance its immunogenicity ( Figure 3 C-3D). In summary, our results suggest that for some antigens, fusion of an Fc tag can significantly enhance the immunogenicity of the antigen.

[0176] Example 4 Determination of PS1 and TS1-specific IgG in mouse serum (circRNA PS1F -LNP&

[0177] CircRNA TS1 -LNP (10μg+10μg)

[0178] Next, we will circRNA PS1F (10μg)-LNP and circRNA TS1 (10 μg)-LNP equal amounts were mixed and injected intramuscularly into 6-8 week old Balb / c female mice, and circRNA was setPS1F (10μg)-LNP vaccine group and circRNA TS1 (10μg)-LNP vaccine group was used as control ( Figure 4 A). Serum samples were collected 14 days after the first immunization and booster immunization, and enzyme-linked immunosorbent assay (ELISA) was used to quantitatively detect serum-specific antibodies against PEDV S1 and TGEV S1. The results showed that circRNA PS1F (10μg)-LNP and circRNA TS1 (10μg)-LNP equal amount mixed vaccine group and circRNA PS1F After booster immunization, the specific antibodies against PS1 in the (10μg)-LNP vaccine group and the equivalent dose group of the bivalent vaccine were significantly lower than those in the circRNA PS1F (10μg)-LNP vaccine group ( Figure 4 B). After booster immunization, there was no significant difference in the level of TS1-specific antibodies induced between the bivalent vaccine equal-dose group and the LNP-circRNATS1 (10 μg) vaccine group ( Figure 4 C). At the same time, in vitro transfection experiments proved that this phenomenon was not caused by expression differences ( Figure 4 D) These experimental results suggest that there may be antigen competition when two circRNA vaccines are administered at equal doses, further suppressing the level of specific antibodies induced in vivo by antigens with poor immunogenicity.

[0179] Example 5 PS1-specific IgG determination in mouse serum (circRNA PS1F (10μg)-LNP and circRNA PS1F (20μg)-LNP)

[0180] To address the phenomenon of weakened PS1-specific antibodies in the medium-dose vaccination group of circular RNA bivalent vaccine, we first tried to increase the expression of circRNA PS1F -LNP inoculation dose to improve the immunogenicity of PS1. 10μg and 20μg of circRNA were respectively PS1F -LNP vaccine was injected intramuscularly into 6-8 week old Balb / c female mice. After two vaccinations 14 days apart, the results of ELISA showed that 20 μg of circRNA PS1F -LNP vaccine group induced PS1-specific antibodies significantly higher than the 10μg vaccine group ( Figure 5 A-5B).

[0181] Example 6 Determination of PS1 and TS1-specific IgG in mouse serum (circRNA PS1F -LNP&

[0182] circRNA TS1 -LNP(20μg+10μg, 20μg+5μg, 20μg+2.5μg)( Figure 5 )

[0183] Next, we will circRNA PS1F (20μg)-LNP&circRNA TS1 (10μg)-LNP mixed, after the first immunization and booster immunization, the results of enzyme-linked immunosorbent assay showed that circRNA PS1F (20μg)-LNP&circRNA TS1 The level of specific antibodies against PS1 induced in the serum of the (10μg)-LNP vaccine group was still significantly lower than that of circRNA vaccination alone. PS1F The antibody level of the (20μg)-LNP vaccine group was significantly different from that of the TS1 group. Figure 2 ) It can be seen that circRNA TS1 When the inoculation doses of -LNP were 2.5μg, 5μg, and 10μg, the specific antibody against TS1 in the serum increased steadily, and the specific antibody levels against TGEV S1 induced by each dose group were slightly different. We speculate that reducing circRNA TS1 -LNP vaccination doses have the potential to reduce circRNA TS1 The immunogenicity of circRNA was improved while suppressing the production of PS1-specific antibodies. PS1F (20μg)-LNP&circRNA TS1 (5μg)-LNP vaccine group and circRNA PS1F (20μg)-LNP&circRNA TS1 The (2.5μg)-LNP vaccine group was injected intramuscularly into 6-8 week old Balb / c female mice. After two immunizations with an interval of 14 days, the results of enzyme-linked immunosorbent assay showed that circRNA PS1F (20μg)-LNP&circRNA TS1 (5μg)-LNP vaccine group and circRNA PS1F (20μg)-LNP&circRNA TS1 The ability of (2.5μg)-LNP vaccine group to induce PS1-specific antibodies was significantly higher than that of circRNA PS1F (20μg)-LNP&circRNA TS1 (10μg)-LNP vaccine group, and circRNA PS1F(20μg)-LNP&circRNA TS1 (5μg)-LNP vaccine group and circRNA PS1F (20μg)-LNP&circRNA TS1 The ability of (2.5μg)-LNP vaccine group to induce PS1-specific antibodies and circRNA PS1F -LNP (20μg) vaccine group was similar ( Figure 5 C) The specific antibodies against TS1 induced by the above two-combination vaccine groups were compared with those induced by circRNA alone. TS1 -LNP vaccine doses of 2.5 μg, 5 μg, and 10 μg induced similar levels of specific antibodies against TS1 ( Figure 5 D) In ​​general, by increasing circRNA PS1F -LNP vaccine dose and appropriate reduction of circRNA TS1 -LNP vaccine doses can detect high levels of specific antibodies against PS1 and TS1 in the serum. In view of this, we chose circRNA PS1F (20μg)-LNP&circRNA TS1 The (5μg)-LNP vaccine group was used for subsequent experiments. Next, we obtained single cell suspensions from the spleens of immunized mice in the vaccine group and the control group. After corresponding protein stimulation, flow cytometry detection revealed that the vaccine group could induce specific T cell responses ( Figure 5 E).

[0184] Example 7 Comparison of different immunization programs, vaccines including inactivated diarrhea virus bivalent vaccine (IAV) and circRNA PS1F&TS1 -LNP(20μg+5μg)(CircRNA vaccine)( Figure 6 )

[0185] Recent studies have shown that heterologous vaccination can induce stronger immune responses against pathogens compared to homologous vaccination. Therefore, we decided to investigate whether the combination of circRNA vaccines with commercial inactivated vaccine (IAV, purchased from Wuhan Keqian Biological Co., Ltd., trade name Kexianning) could induce stronger immune responses and provide enhanced protection against PEDV and TGEV infection. Within the framework of prime-boost immunization, four different immunization regimens were established according to the vaccine type: IAV-IAV, circRNA-circRNA, IAV-circRNA, and circRNA-IAV. Figure 5After the immunization schedule shown, ELISA was used to detect the specific antibodies induced against PS1 and TS1 in serum samples. The ELISA results showed that the antibody titers against PS1 in the IAV-circRNA and circRNA-circRNA vaccine groups were significantly higher than those in the other two groups. However, the antibody titers against TS1 were similar in all four groups ( Figure 6 A-6B).

[0186] In addition, we further investigated the specific IgG1 and IgG2a elicited by various immune pathways. When measuring the levels of anti-PS1-specific IgG1 and IgG2a antibodies, both the IAV-IAV and circRNA-IAV groups showed a Th2-biased antibody response (IgG2a / IgG1<1). On the other hand, the IAV-circRNA group and the circRNA-circRNA group showed a tendency towards a Th1 response. When measuring the levels of specific IgG1 and IgG2a antibodies against TS1, only the IAV-IAV immunization group showed a Th2-biased antibody response (IgG2a / IgG1<1), while the other three groups all showed a stronger preference for Th1 response compared with the IAV-IAV group ( Figure 6 C-6F). These results indicate that our circRNA vaccine has significant advantages in inducing Th1 immune response.

[0187] In addition, we performed real virus neutralization experiments with PEDV and TGEV ( Figure 6 G-6H), the results showed that the neutralization titer of the IAV-circRNA group against TGEV was significantly higher than that of the other three groups. However, all four groups had similar neutralization effects against PEDV. Overall, the IAV-circRNA vaccination program is a superior choice for preventing PEDV and TGEV infections in pig production.

[0188] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A circular RNA, characterized in that The circular RNA includes any of the following: 1) The circular RNA comprises a coding element, and the coding element encodes a polypeptide comprising the S1 region of the PEDV S protein and the Fc region of a porcine immunoglobulin; 2) the circular RNA comprises a coding element, and the coding element encodes a polypeptide comprising the S1 region of the TGEV S protein; Preferably, the PEDV S protein S1 region has an amino acid sequence as shown in SEQ ID NO.5, or an amino acid sequence having at least 90% identity with SEQ ID NO.5; Preferably, the porcine immunoglobulin Fc region comprises the Fc region of porcine IgG, IgA, IgD, IgE or IgM; Preferably, the porcine immunoglobulin Fc region is the Fc region of porcine IgG; More preferably, the amino acid sequence of the Fc region of the porcine IgG has the amino acid sequence shown in SEQ ID NO.6, or an amino acid sequence having at least 90% identity with SEQ ID NO.6; Preferably, the amino acid sequence of the S1 region of the truncated fragment of the TGEV S protein has the amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence that has at least 90% identity with SEQ ID NO.

7.

2. The circular RNA according to claim 1, characterized in that The nucleotide sequence encoding the S1 region of the PEDV S protein comprises the sequence shown in SEQ ID NO.2, or a sequence having at least 90% identity with the sequence shown in SEQ ID NO.2; The nucleotide sequence encoding the Fc region of porcine IgG comprises the sequence shown in SEQ ID NO.3, or a sequence having at least 90% identity with the sequence shown in SEQ ID NO.3; The nucleotide sequence encoding the S1 region of the truncated fragment of the TGEV S protein comprises the sequence shown in SEQ ID NO.4, or a sequence having at least 90% identity with the sequence shown in SEQ ID NO.

4.

3. A linear RNA or recombinant nucleic acid molecule, characterized in that: The linear RNA undergoes self-cleavage reaction and then cyclizes to form the circular RNA as described in any one of claims 1-2; the recombinant nucleic acid molecule is transcribed to form the circular RNA as described in any one of claims 1-2.

4. An expression vector, characterized in that: The vector comprises the linear RNA according to claim 3.

5. A porcine epidemic diarrhea virus circular RNA vaccine, characterized in that: The vaccine comprises the circular RNA described in claim 1) ; Preferably, the vaccine comprises a lipid encapsulating the circular RNA; Preferably, the lipids comprise cationic lipids, phospholipids, steroids and polyethylene glycol-modified lipids; Preferably, the vaccine comprises lipid nanoparticles or lipid polymer complexes.

6. A porcine transmissible gastroenteritis virus circular RNA vaccine, characterized in that: The vaccine comprises the circular RNA described in claim 1 (2); Preferably, the vaccine comprises a lipid encapsulating the circular RNA; Preferably, the lipid comprises a cationic lipid, a phospholipid, a steroid and a polyethylene glycol-modified lipid; Preferably, the vaccine comprises lipid nanoparticles or lipid polymer complexes.

7. A composition, characterized in that The composition is any of the following: 1) A vaccine composition, characterized in that the vaccine composition comprises the porcine epidemic diarrhea virus circular RNA vaccine according to claim 5 and the porcine transmissible gastroenteritis virus circular RNA vaccine according to claim 6; 2) A circular RNA composition, characterized in that the circular RNA composition comprises the circular RNA described in 1) and the circular RNA described in 2) in claim 1; Preferably, the mixing ratio of the porcine epidemic diarrhea virus circular RNA vaccine and the porcine transmissible gastroenteritis virus circular RNA vaccine is 4:1 to 8:1; Preferably, the mixing ratio of the circular RNA described in 1) and the circular RNA described in 2) in claim 1 is 4:1 to 8:

1.

8. A vaccine preparation, characterized in that The vaccine preparation comprises the vaccine composition of claim 7 and an inactivated diarrhea virus bivalent vaccine; Preferably, the vaccine preparation contains two dosage units, the first dosage unit contains the inactivated diarrhea virus bivalent vaccine, and the second dosage unit contains the vaccine composition according to claim 7; Preferably, the first dosage unit is used in the initial immunization, and the second dosage unit is used in the secondary immunization; Preferably, the inactivated diarrhea virus bivalent vaccine is Coxiel.

9. A method for immunizing pigs, characterized in that: The method comprises administering the vaccine of claim 5, the vaccine of claim 6, the vaccine composition of claim 7, or the vaccine formulation of claim 8 to the pig.

10. Any of the following applications, characterized in that: The applications include: 1) Use of the circular RNA according to any one of claims 1 to 2, the linear RNA according to claim 3, or the expression vector according to claim 4 in the preparation of a circular RNA vaccine for porcine epidemic diarrhea virus or a circular RNA vaccine for porcine transmissible gastroenteritis virus; 2) Use of the porcine epidemic diarrhea virus circular RNA vaccine of claim 5, the porcine transmissible gastroenteritis virus circular RNA vaccine of claim 6, the composition of claim 7 or the vaccine preparation of claim 8 in the preparation of drugs against porcine epidemic diarrhea virus and / or porcine transmissible gastroenteritis virus; 3) Use of the composition of claim 7 in combination with an inactivated diarrhea virus bivalent vaccine in the preparation of a drug against porcine epidemic diarrhea virus and / or porcine transmissible gastroenteritis virus.

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