Cyclic RNA vaccine against PRRS virus and construction method and application thereof

Abstract

The application discloses an anti-proliferation and respiratory syndrome (PRRS) virus circular RNA vaccine and a construction method and application thereof. The circular RNA vaccine comprises circular RNA and a pharmaceutically acceptable carrier and the like. Coding elements in the circular RNA can encode GP4, GP2, GP5 and M proteins of a porcine reproductive and respiratory syndrome virus. The circular RNA vaccine has high stability and good safety, and after immunizing pigs, no adverse reactions are generated, high levels of neutralizing antibodies are induced in the pigs after immunization, and high levels of cellular immune responses are activated, and the immunization protection rate can reach 100%. Meanwhile, the preparation process does not need to be carried out on virus isolation, greatly shortens the research and development cycle, and the production process is not restricted by biological factors such as virus titers, is easy to realize standardized and large-scale production, and can effectively reduce production costs.

Description

Technical Field

[0001] This application relates to a circular RNA (circRNA) vaccine, and more particularly to a circular RNA vaccine against porcine reproductive and respiratory syndrome (PRRS) virus, its construction method and application, its preparation method and application, belonging to the field of animal immunotherapy technology. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a serious epidemic disease that severely impacts the growth of pigs. Its main symptoms manifest as reproductive and respiratory problems at different growth stages, including abortion in sows, fetal death or premature birth in piglets, as well as fever, hemorrhage, and respiratory syndromes. The most prominent characteristic is the bluish-purple discoloration of the ears in infected pigs, commonly known as "blue ear disease." PRRS typically establishes long-term infection through multiple complex mechanisms, which is one of the biggest obstacles to controlling the disease. Furthermore, due to the high mutation and recombination rate and immunosuppressive properties of the porcine reproductive and respiratory syndrome virus (PRRSV), existing vaccines cannot provide broad-spectrum cross-protection, posing a significant challenge to disease control.

[0003] PRRSV is a single-stranded positive-sense RNA virus belonging to the genus β-arteritisvirus. The nucleocapsid of PRRSV has smooth icosahedral symmetry; its diameter is approximately 30 nanometers, and it is enveloped by a single membrane. The PRRSV genome is approximately 15.4 kb, containing at least 10 open reading frames (ORFs) encoding at least 14 non-structural proteins (NSPs) and 8 structural proteins (GP2a, GP2b, GP3, GP4, GP5, GP5a, M, and N). Currently, there are two main genotypes of porcine reproductive and respiratory syndrome virus (PRRSV): PRRSV-1 and PRRSV-2.

[0004] Currently, vaccines against PRRSV are mainly inactivated or attenuated vaccines prepared based on virus isolation and culture. These vaccines have inherent limitations, such as long development cycles and high production costs. Furthermore, the frequent mutations of PRRSV lead to changes in the antigenicity of circulating strains, further limiting the effectiveness of these vaccines and making complete control of PRRS difficult. Although researchers have been continuously committed to technological innovation in PRRS vaccines in recent years and have achieved some results in the fields of viral vector vaccines and DNA vaccines, effectively controlling and eradicating PRRS through these vaccines still faces significant challenges. Summary of the Invention

[0005] The main objective of this application is to provide a circular RNA vaccine against PRRS virus, its construction method, and its application, in order to overcome the shortcomings of the prior art.

[0006] To achieve the aforementioned objectives, this application employs the following technical solution.

[0007] According to a first aspect of this application, a circular RNA is provided comprising coding elements, said coding elements comprising codon-optimized and deleted signal peptides and / or transmembrane regions of porcine reproductive and respiratory syndrome virus GP2, GP4, GP5, M protein coding sequences.

[0008] In one embodiment, the encoding element comprises: The first nucleotide sequence is used to encode the GP2 protein; The second nucleotide sequence is used to encode the GP4 protein; The third nucleotide sequence is used to encode the GP5 protein; The fourth nucleotide sequence is used to encode the M protein.

[0009] Furthermore, the first nucleotide sequence may be the sequence shown in SEQ ID NO:1 or an extended or truncated sequence thereof, particularly a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO:1. More preferably, the first nucleotide sequence is as shown in SEQ ID NO:1.

[0010] Furthermore, the second nucleotide sequence may be the sequence shown in SEQ ID NO:2 or an extended or truncated sequence thereof, particularly a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO:2. More preferably, the second nucleotide sequence is as shown in SEQ ID NO:2.

[0011] Furthermore, the third nucleotide sequence can be the sequence shown in SEQ ID NO:3 or an extended or truncated sequence thereof, particularly a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO:3. More preferably, the third nucleotide sequence is as shown in SEQ ID NO:3.

[0012] Furthermore, the fourth nucleotide sequence can be the sequence shown in SEQ ID NO:4 or an extended or truncated sequence thereof, particularly a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO:4. More preferably, the fourth nucleotide sequence is as shown in SEQ ID NO:4.

[0013] In this application, the nucleotide sequences of the structural proteins GP2, GP4, GP5, and M, which can induce high levels of neutralizing antibodies by PRRSV, are optimized to reduce their secondary structures, making them more suitable for in vitro transcription and circularization of circular RNA. At the same time, by deleting the signal peptide and transmembrane region of the target protein, it is made more suitable for expression in porcine cells.

[0014] In one embodiment, the first, second, third, and fourth nucleotide sequences are tandemly linked, with adjacent nucleotide sequences connected by a fifth nucleotide sequence. The fifth nucleotide sequence encodes a P2A peptide, a self-splicing peptide that enables the independent expression of multiple proteins in the same mRNA transcript during translation, thereby achieving multi-gene co-expression. By using the P2A peptide to tandemly link four optimized nucleotide sequences in the order GP2, GP4, GP5, and M, the four structural proteins GP2, GP4, GP5, and M can be independently expressed during translation, reducing the complexity of subsequent purification processes and significantly lowering production costs.

[0015] Furthermore, the fifth nucleotide sequence can be the sequence shown in SEQ ID NO:5 or an extended or truncated sequence thereof, particularly a sequence that is more than 95% identical to the full-length sequence of SEQ ID NO:5. More preferably, the fifth nucleotide sequence is as shown in SEQ ID NO:5.

[0016] In one embodiment, the coding element is formed by sequentially connecting a first nucleotide sequence, a fifth nucleotide sequence, a second nucleotide sequence, a fifth nucleotide sequence, a third nucleotide sequence, a fifth nucleotide sequence, and a fourth nucleotide sequence.

[0017] In one embodiment, the circular RNA is generated based on type II intron self-cleavage circularization, and its vector comprises the following elements connected in sequence: intron fragment I, IRES (internal ribosome entry site), T7 promoter, coding element, intron fragment II, and T7 terminator. This design eliminates dependence on exon sequences, producing "scar-free" circular RNA without scar sequences, thereby eliminating the potential influence of scar sequences.

[0018] According to a second aspect of this application, a recombinant vector is provided, comprising a recombinant nucleic acid molecule, said recombinant nucleic acid molecule comprising a second nucleotide sequence, a first nucleotide sequence, a third nucleotide sequence and a fourth nucleotide sequence in sequence connected in series, wherein adjacent nucleotide sequences in the second nucleotide sequence, the first nucleotide sequence, the third nucleotide sequence and the fourth nucleotide sequence are linked by a fifth nucleotide sequence.

[0019] In one embodiment, the recombinant vector comprises intron fragment I, IRES, T7 promoter, coding element, intron fragment II, and T7 terminator connected in sequence; wherein the coding element comprises the recombinant nucleic acid molecule.

[0020] According to a third aspect of this application, a method for preparing circular RNA is provided, comprising using the recombinant vector to transcribe precursor RNA and further preparing it into circular RNA.

[0021] In one embodiment, the preparation method may include the following steps: (1) Design, optimize and synthesize nucleotide sequences encoding PRRSV GP2, GP4, GP5 and M proteins, and synthesize plasmid DNA of circular RNA; (2) The target nucleotide sequence is ligated to a plasmid to obtain a recombinant vector, and the vector is linearized using a restriction endonuclease; (3) Using T7 RNA polymerase as a template, T7 RNA transcription was performed and self-cleaving circularization was completed; (4) The circular RNA was precipitated and purified by lithium chloride method and other methods, and further purified by high performance liquid chromatography (HPLC) and other methods to obtain the target circular RNA.

[0022] This application involves deleting the signal peptide and transmembrane region from the four protective antigen sequences of PRRSV (GP2, GP4, GP5, and M) that can induce neutralizing antibodies, and then tandem them with the P2A peptide. The four tandemly transcribed proteins can be transcribed into individually expressed proteins in vivo. The prepared circular RNA enhances the protective efficacy of the vaccine while reducing the complex process of purifying the four circular RNAs separately, greatly saving costs and improving efficiency.

[0023] Furthermore, this application applies type II intron self-splicing technology of circular RNA to the field of PRRSV vaccine development, eliminating scar sequences that may affect the body, making it safer and more reliable. In addition, the circular RNA prepared in this application undergoes self-circularization during in vitro transcription, eliminating the need for additional in vitro circularization operations, simplifying the process and overcoming the bottlenecks of complex, costly, and limited immunization effects in existing protein vaccine production processes. This provides a new strategy and technical approach for the prevention and control of porcine reproductive and respiratory syndrome (PRRS).

[0024] Furthermore, the purified target circular RNA can be encapsulated using a commercially available lipid nanoparticle transfection kit. For example, the circular RNA can preferably be encapsulated using the LNP transfection kit from HanzBio to improve the transfection efficiency and stability of the circular RNA, thereby further enhancing immunogenicity and protective efficacy.

[0025] According to a fourth aspect of this application, a cell is provided comprising the recombinant vector. The cell may include, but is not limited to, *Escherichia coli* DH5α competent strains.

[0026] According to a fifth aspect of this application, a host cell is provided, including, but not limited to, porcine kidney cells transfected with the circular RNA, such as PK-15.

[0027] According to a sixth aspect of this application, a complex is provided comprising lipid nanoparticles encapsulating the circular RNA.

[0028] In one embodiment, the lipid nanoparticles encapsulate circular RNA at a rate greater than 90%.

[0029] In one embodiment, the surface potential of the composite is electronegative.

[0030] According to a seventh aspect of this application, an immune composition is provided, comprising: The circular RNA or the complex described herein; And, pharmaceutically acceptable carriers.

[0031] According to the eighth aspect of this application, the use of the circular RNA, the recombinant vector, the complex, or the immune composition in the production of an agent for inducing an immune response against porcine reproductive and respiratory syndrome virus infection in test animals or for preventing and / or treating animals infected with porcine reproductive and respiratory syndrome virus is provided.

[0032] Furthermore, the animals mentioned include pigs.

[0033] According to the ninth aspect of this application, the use of the circular RNA, the recombinant vector, the complex, or the immune composition in the preparation of a porcine reproductive and respiratory syndrome virus vaccine is provided.

[0034] This application utilizes a circular RNA method to prepare a PRRSV vaccine. Because circular RNA lacks free ends and forms a covalently closed continuous loop, it significantly resists degradation by intracellular and extracellular nucleases, prolonging the vaccine's protective effect in vivo and inducing a more potent and durable immune response. Circular RNA vaccines do not contain infectious components, degrade into nucleotides in vivo, and do not integrate into the host genome, demonstrating good safety for use in pigs.

[0035] According to the tenth aspect of this application, a porcine reproductive and respiratory syndrome virus circular RNA vaccine is provided, comprising: The circular RNA or the complex; And, pharmaceutically acceptable carriers.

[0036] In some cases, the vaccine may also include more than one pharmaceutically acceptable excipient, adjuvant, or carrier. For example, the vaccine may also contain other adjuvants. The term "adjuvant" means any molecule added to the vaccine described herein to enhance the immunogenicity of the antigen encoded by the gene.

[0037] Furthermore, when applying the vaccine, only an effective amount needs to be administered to the animals. The "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, the desired effect.

[0038] According to the eleventh aspect of this application, a method is provided for inducing an immune response against porcine reproductive and respiratory syndrome virus (PRRSV) infection or for protecting a test animal from PRRSV infection, the method comprising administering the vaccine to the test animal. Further, the animal includes a pig.

[0039] Compared to existing technologies, the circular RNA vaccine provided in this application exhibits high stability and good safety, with no adverse reactions observed after immunization of pigs. Following immunization, it induces high levels of neutralizing antibodies in pigs and activates a high level of cellular immune response, providing nearly 100% immune protection, exceeding the level of existing vaccines. Furthermore, its preparation method eliminates the need for virus isolation, significantly shortening the research and development cycle. The production process is not constrained by biological factors such as virus titer, facilitating standardization and large-scale production, and effectively reducing production costs. Attached Figure Description

[0040] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0041] Figure 1 This is a graph showing the detection of neutralizing antibody titers in mice during Example 1. Figure 2 This is a schematic diagram of the circRNA expression cassette structure and self-circularization in Example 2; Figure 3 The images shown are agarose gel electrophoresis images of circRNA before and after purification in Example 2. Figure 4 This is a Western blot image showing the expression of circRNA in PK-15 cells in Example 2; Figure 5 This is a graph showing the body temperature changes of piglets after challenge in Example 3; Figure 6 This is a statistical curve of the mortality rate of piglets after challenge in Example 3; Figure 7 This is a graph showing the results of virus content determination in the serum of animals after challenge in Example 3; Figure 8 It is CD4 in Example 3 +A statistical chart showing the percentage of IFN-γ positive cells in T cell subsets; Figure 9 It is CD8 in Example 3 + A statistical chart showing the percentage of IFN-γ positive cells in T cell subsets. Detailed Implementation

[0042] The present application is further illustrated below by way of examples. All reagents and raw materials used in the following examples are commercially available, and experimental methods without specific conditions are generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers. Furthermore, unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this application employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques have been well described in existing literature.

[0043] In the following embodiments, this application improves the expression efficiency of porcine reproductive and respiratory syndrome virus (PRRSV) GP2, GP4, GP5, and M proteins through codon optimization, while also improving the self-circularization efficiency of circular RNA. The optimized nucleotide sequence of GP2 (with signal peptide and transmembrane region deletions) is shown in SEQ ID NO.1. The optimized nucleotide sequence of GP4 (with signal peptide and transmembrane region deletions) is shown in SEQ ID NO.2. The optimized nucleotide sequence of GP5 (with signal peptide and transmembrane region deletions) is shown in SEQ ID NO.3. The optimized nucleotide sequence of M (with transmembrane region deletions) is shown in SEQ ID NO.4. The optimized nucleotide sequences of GP2, GP4, GP5, and M are tandemly linked with the nucleotide sequence of the P2A peptide (SEQ ID NO.5) to achieve separate expression. Key considerations during codon optimization include: codon bias of the target expression species, adaptation to the restriction endonuclease sites of the vector used, the overall GC content of the sequence, and other sequence features that may affect expression or stability.

[0044] The following embodiments of this application synthesize a recombinant vector containing the above-mentioned nucleotide sequence, linearize the vector by enzyme digestion, transcribe it in vitro, purify it to obtain circular RNA, and then encapsulate it with lipid nanoparticles to form the final circular RNA vaccine.

[0045] Example 1: Identification of PRRSV circular RNA vaccine antigen 1.1 Antigen Preparation Referring to *Molecular Cloning: A Laboratory Manual (4th Edition)* (J. Sambrook & MR. Green, Science Press, March 2017, Volume 1, Chapter 3), the gene sequences of PRRSV GP2, GP4, GP5, and M proteins were cloned into the pET28a(+) prokaryotic expression plasmid, respectively. Expression was performed using an *E. coli* expression system, and GP2, GP4, GP5, and M were all successfully expressed. The expressed proteins were purified by affinity chromatography using Ni-IMAC packing material.

[0046] 1.2 Mouse Immunization Mice were immunized with purified PRRSV GP2, GP4, GP5, and M proteins, and the immunization conditions for each group are shown in Table 1. Mice were boosted immunized 21 days after the initial immunization. Serum samples were collected on day 7 after the booster immunization for neutralizing antibody detection.

[0047] Table 1. Grouping details of experimental animals

[0048] *The immunization dose is a mixture of equal amounts of several proteins, with a total of 50 μg per animal.

[0049] 1.3 Neutralizing antibody detection Blood was collected from the orbital rim of mice on day 28 post-immunization. The separated serum was inactivated at 56 °C. Marc-145 cells were used for neutralization experiments, with PRRSV CH / SCHY / 2018 strain as the neutralizing antigen. The level of PRRSV neutralizing antibodies in the serum was measured, and the highest dilution that completely protected the cells from damage was taken as the neutralizing titer of the serum sample. The results of the neutralization experiment are as follows: Figure 1 As shown, the neutralizing antibody level in the GP2, GP4, GP5, and M mixed immunization group was higher than that in the GP4, GP5, and M mixed immunization group, the GP2, GP5, and M mixed immunization group, the GP2, GP4, and M mixed immunization group, the GP2, GP4, and GP5 mixed immunization group, and the blank control group. Therefore, GP2, GP4, GP5, and M proteins were selected as immunogens to construct a circular RNA vaccine.

[0050] Example 2: Construction of PRRSV circular RNA 2.1 Construction of Recombinant Plasmids The recombinant DNA template plasmid (hereinafter defined as the experimental plasmid) was synthesized by Genewiz Biotechnology Co., Ltd. This recombinant DNA template plasmid is formed by sequentially linking the T7 promoter, intron fragment I, IRES, the nucleotide sequence of the PRRSV protein, intron fragment II, and the T7 terminator. The nucleotide sequence of the PRRSV protein is formed by tandemly linking the nucleotide sequences of PRRSV protein GP2, GP4, GP5, and M (with the signal peptide and transmembrane region deleted) via P2A.

[0051] As a control, the following recombinant DNA template plasmid was synthesized by Genewiz Biotechnology Co., Ltd.: Control plasmid a: The difference between this plasmid and the recombinant DNA template plasmid is that the nucleotide sequence of the PRRSV protein is formed by tandemly connecting the nucleotide sequences of PRRSV protein GP5, GP2, GP4, and M (with the signal peptide and transmembrane region deleted) via P2A.

[0052] Control plasmid b: The difference between this plasmid and the recombinant DNA template plasmid is that the nucleotide sequence of the PRRSV protein is formed by tandemly connecting the nucleotide sequences of PRRSV protein M, GP2, GP4, and GP5 (with the signal peptide and transmembrane region deleted) via P2A.

[0053] Control plasmid c: The difference between this plasmid and the recombinant DNA template plasmid is that the nucleotide sequence of the PRRSV protein is formed by tandemly connecting the nucleotide sequences of PRRSV protein GP4, GP2, GP5, and M (with the signal peptide and transmembrane region deleted) via P2A.

[0054] Each recombinant DNA template plasmid was transformed into Escherichia coli DH5α competent strain. Single clones were randomly selected and inoculated into 100 mL of LB liquid medium containing penicillin resistance. The culture was carried out in a shaker at 37 °C. Recombinant DNA plasmids for preparing circular RNA were extracted according to the instructions of the Omega endotoxin-free plasmid DNA extraction kit. After determining the concentration of recombinant DNA plasmids, they were stored at -20 °C.

[0055] 2.2 Linearization of Recombinant Plasmid Enzyme Digestion The recombinant DNA plasmids extracted in the previous step were linearized by restriction endonuclease Xba I (Thermo Scientific), prepared according to the restriction digestion system shown in Table 2, and incubated in a 37°C water bath for 30 min.

[0056] Table 2 Enzyme digestion system

[0057] The linearized plasmid after enzyme digestion was extracted with phenol-chloroform and then precipitated with ethanol to obtain the purified linearized plasmid. Finally, the concentration was measured with an ultra-micro UV spectrophotometer and stored at -20℃.

[0058] 2.3 In vitro transcription of linearized plasmids and RNA self-circulation The linearized plasmids prepared using the above process were transcribed in vitro according to the Promega RiboMAX™ Large Scale RNA Production Systems T7 kit instructions at 37°C for 4 hours. The transcribed linear RNA underwent self-circularization through intron splicing to form circular RNA. (See [link to documentation]). Figure 2 The transcription system is shown in Table 3 below.

[0059] Table 3 In vitro transcription system

[0060] After transcription, RQ1 RNase-Free DNase was added and the mixture was treated at 37°C for 30 min to remove the linearized RNA template.

[0061] 2.4 Purification of Circular RNA 1) Each product from step 2.1 was initially purified by lithium chloride precipitation. RNA was precipitated using 5M lithium chloride to remove residual enzymes and other impurities from the product.

[0062] 2) Circular RNA was purified by HPLC to remove uncircularized linear RNA and cleaved introns. An Aglient 1260 Infinity II high-performance liquid chromatograph was used with a Sepax SRT SEC-1000 size-exclusion column. Elution was performed with buffer (10 mM Tris, 0.5 mM EDTA, DEPC water) at a flow rate of 1 min / ml. The target circular RNA fraction was collected based on the UV detector signal. The purification process was observed by RNA electrophoresis. The fraction containing the target circular RNA was concentrated using ultrafiltration to obtain the purified circular RNA. The purification results are shown below. Figure 3 As shown, the purified circular RNA was stored at -80℃.

[0063] 2.5 Validation of Circular RNA Expression in Cells Purified circular RNAs were transfected into 70% confluence adherent porcine kidney cells (PK-15) using Lipofectamine MessengerMAX (Invitrogen). After 48 hours of transfection, the cell supernatant was removed, and the cells were gently washed with PBS followed by lysis with RIPA lysis buffer. The lysed cells were collected and centrifuged at 8000 rpm for 10 minutes at 4°C. The supernatant was then mixed with 5× Loading Buffer and boiled for Western blot analysis. Monoclonal antibodies against GP2, GP4, GP5, and M proteins were used as primary antibodies to verify the expression of circular RNA in porcine cells. Figure 4 It can be seen that, for circular RNA based on the above experimental plasmids, PRRSV GP2, GP4, GP5, and M proteins tandemly linked on the same circular RNA can be independently expressed in PK-15.

[0064] For circular RNAs constructed based on control plasmids a, b, and c, the GP2, GP4, GP5, and M proteins tandemly linked to each circular RNA can also be expressed independently in PK-15, but the expression levels are significantly lower than those of circular RNAs constructed based on experimental plasmids.

[0065] Example 3: Immunization Experiment with Circular RNA Vaccine Against Porcine Reproductive and Respiratory Syndrome 3.1 LNP Preparation of circular RNA After the purified circular RNA (based on the experimental plasmid) was determined to be of a certain concentration, it was used to prepare a lipid nanoparticle / RNA composition using commercial LNP (HanzBio). The composition met the following requirements: the particle size was 102.3 nm, the polydispersity index was 0.22, indicating that the LNP particles carrying the circular RNA were of moderate size and uniformly distributed; the encapsulation efficiency reached 92.42%, indicating good encapsulation effect; the surface potential was negative, avoiding cytotoxicity caused by positive charges on the nanoparticle surface, and the biocompatibility was good; the encapsulation concentration was ≥0.113 mg / mL, which can be directly used as a PRRSV circular RNA vaccine for animal immunization.

[0066] 3.2 Immunoassay 3.2.1 Animal Immunization Twenty healthy 4-week-old piglets, free from infection with porcine reproductive and respiratory syndrome virus (PRRSV), African swine fever virus (ASFV), classical swine fever virus (CSFV), and porcine circovirus type 2 (PCV2), were purchased from a pig farm in Jiangsu Province, China. The piglets were randomly divided into four groups and housed in separate rooms. The vaccine group received an intramuscular injection of 50 μg of anti-PRRSV circular RNA vaccine via the neck, or a commercially available inactivated PRRSV vaccine (JXA1-R) as a positive control. The control group and the PRRSV challenge group received phosphate-buffered saline (PBS). A booster immunization was administered 21 days after the initial immunization. Serum samples were collected on day 7 post-boost immunization for neutralizing antibody detection. The immunization conditions for each group are shown in Table 4.

[0067] Table 4. Grouping details of experimental animals

[0068] 3.2.2 Virus Attack Animals were challenged 28 days after immunization. Basal body temperature was measured 3 days prior to challenge. On the day of challenge, the animals were weighed and their body temperature was measured. All piglets were inoculated with PRRSV via intramuscular and intranasal administration. CH / SCHY / 2018 is a highly virulent poison; the challenge dose is 2 × 10⁻⁶. 5 TCID50 / head, 1.5 mL intramuscular injection, 1.5 mL nasal drop injection.

[0069] 3.2.3 Neutralizing antibody detection Blood samples were collected from the ear vein on day 14 and day 28 post-immunization. The separated serum was inactivated at 56 °C. Marc-145 cells were used for neutralization experiments, with PRRSV CH / SCHY / 2018 strain as the neutralizing antigen. The level of PRRSV neutralizing antibodies in the serum was measured, and the highest dilution that completely protected the cells from damage was taken as the neutralizing titer of the serum sample. The results of the neutralization experiments are shown in Table 5.

[0070] Table 5 Neutralization Experiment Results

[0071] 3.2.4 Clinical symptom monitoring Piglets were isolated after being challenged with the virus. Their rectal temperature was measured and recorded daily, and their clinical signs were observed. In the challenge control group, fever (≥40.5℃), dyspnea, lethargy, and anorexia began to appear on day 2 post-challenge. As the disease progressed, lameness, tremors, unsteady gait, and obvious cyanosis appeared on the abdomen, ear tips, and abdominal skin. In the circular RNA vaccine group, the animals' body temperature fluctuated around 39.5℃ after challenge, remaining normal, and no obvious clinical symptoms such as coughing, dyspnea, anorexia, diarrhea, or fever were observed. In the inactivated vaccine group, two piglets developed fever, dyspnea, lethargy, and anorexia, while the other three piglets had normal body temperatures and no obvious clinical symptoms were observed. According to the PRRSV vaccine quality standards, in the challenge control group, all 5 out of 5 pigs should develop the disease, and at least 2 out of 5 should die. In the immunized pigs, at least 4 out of 5 should survive. Statistical results show that in the PRRSV inactivated vaccine group, 4 out of 5 pigs survived (80% survival rate); in the PRRSV circular RNA vaccine group, 5 out of 5 pigs survived (100% survival rate); and in the control group, 4 out of 5 experimental pigs died, and 5 out of 5 developed the disease. The body temperature change curves and survival rates of piglets after challenge are shown below. Figure 5 , 6 As shown.

[0072] 3.2.5 Detection of viral load in serum and lung tissue PRRSV quantitative PCR was used to detect the viral load in the blood of all pigs after immunization and challenge, and to evaluate the duration of viremia after challenge. Figure 7 As shown, compared with the challenge control group, the serum viral load in the circular RNA vaccine immunization group was significantly reduced at 7 and 14 days post-infection, and no virus was detected in the serum at 21 days post-infection. The blood viral load in the inactivated vaccine immunization group also decreased to some extent.

[0073] 3.2.6 Flow cytometry analysis Blood was collected from piglets on day 28 post-immunization, and peripheral blood mononuclear cells (PBMCs) were isolated to assess cellular immune responses. The proportion of IFN-γ-producing CD4+ and CD8+ cells was analyzed using intracellular immunofluorescence. The isolated PBMCs were restimulated in vitro with purified PRRSV viral particles (10 µg / mL), with a Golgi apparatus inhibitor (BDBiosciences) added at the later stage of stimulation to induce cytokine accumulation within the cells. Cell surface antigens were labeled with anti-CD3, CD4, and CD8a fluorescent antibodies (BioLegend) to differentiate T cell subsets. Cells were then fixed and ruptured, and stained with IFN-γ (BioLegend) fluorescent antibody. Finally, data were collected and analyzed using flow cytometry and FlowJo software to calculate the percentage of IFN-γ-positive cells in each T cell subset. It was observed that on day 28 post-immunization, the circular RNA vaccine significantly increased the proportion of IFN-γ-producing CD4+ and CD8+ T cells compared to the inactivated vaccine and the blank control group. Figure 8 , 9 As shown, the results indicate that the circular RNA vaccine can significantly induce cellular immune responses in piglets.

[0074] In summary, the embodiments of this application optimize and tandem the nucleic acid sequences of PRRSV structural proteins GP2, GP4, GP5, and M, thereby optimizing expression, enhancing immunogenicity, simplifying the tedious steps of purifying each protein separately, enhancing efficacy, and reducing production costs. Immunoprotection experimental data fully validate that the PRRSV circular RNA vaccine obtained in this application has good immunogenicity; the survival rate of piglets vaccinated with the PRRSV circular RNA vaccine is higher than that of the inactivated vaccine group and the PBS control group.

[0075] It should be understood that the embodiments described above are only some, not all, of the embodiments of this application. The detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

Claims

1. A circular RNA, characterized in that, The element comprises a coding element consisting of a first nucleotide sequence, a second nucleotide sequence, a third nucleotide sequence, and a fourth nucleotide sequence connected in series, wherein adjacent nucleotide sequences are linked by a fifth nucleotide sequence. The first nucleotide sequence, as shown in SEQ ID NO.1, encodes the GP2 protein; The second nucleotide sequence, as shown in SEQ ID NO.2, encodes the GP4 protein; The third nucleotide sequence, as shown in SEQ ID NO.3, is used to encode the GP5 protein; The fourth nucleotide sequence, as shown in SEQ ID NO.4, is used to encode the M protein; The fifth nucleotide sequence, as shown in SEQ ID NO.5, is used to encode the P2A peptide.

2. A recombinant vector for preparing the circular RNA of claim 1, characterized in that: The recombinant vector comprises, in sequence, intron fragment I, IRES, T7 promoter, coding element as described in claim 1, intron fragment II, and T7 terminator.

3. A method for preparing circular RNA, characterized in that, This includes using the recombinant vector described in claim 2 to transcribe precursor RNA, which is then further prepared into circular RNA.

4. A complex, characterized in that, The invention comprises lipid nanoparticles carrying circular RNA, wherein the circular RNA comprises the circular RNA of claim 1.

5. The complex according to claim 4, characterized in that: The lipid nanoparticles encapsulate circular RNA at a rate greater than 90%.

6. The complex according to claim 4, characterized in that: The surface potential of the composite is electronegative.

7. An immune composition, characterized in that, include: The circular RNA of claim 1 or the complex of any one of claims 4-6; And, pharmaceutically acceptable carriers.

8. Use of the circular RNA of claim 1, the recombinant vector of claim 2, the complex of any one of claims 4-6, or the immune composition of claim 7 in the production of an agent for preventing infection of animals with porcine reproductive and respiratory syndrome virus, wherein the animal is a pig.

9. The use of the circular RNA of claim 1, the recombinant vector of claim 2, the complex of any one of claims 4-6, or the immune composition of claim 7 in the preparation of a porcine reproductive and respiratory syndrome virus vaccine.

10. A circular RNA vaccine against porcine reproductive and respiratory syndrome virus, characterized in that, include: The circular RNA of claim 1 or the complex of any one of claims 4-6; And, pharmaceutically acceptable carriers.

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