MRNA vaccine composition for combined treatment of porcine circovirus and application of mRNA vaccine composition

Abstract

The invention discloses an mRNA vaccine composition for combined treatment of porcine circovirus and application of the mRNA vaccine composition. The combination comprises a nucleotide combination, the nucleotide combination comprises a sequence for coding the Cap protein of the porcine circovirus and a CpG adjuvant, and the sequence for coding the Cap protein of the porcine circovirus and the CpG adjuvant are respectively assembled into a porcine circovirus mRNA vaccine and a CpG adjuvant mRNA vaccine. In order to solve the problem that the existing mRNA vaccine generally depends on inherent immune stimulation of mRNA and LNP, and generally, an exogenous adjuvant is not additionally added, so that the immune response intensity or persistence is insufficient possibly, the CpG adjuvant and the PCV2 Cap mRNA-LNP vaccine are creatively combined for use; experiments show that the combination can significantly improve the neutralizing antibody level and enhance the immune response of Th1 type cells compared with mRNA independent immunization, has good safety, and can be used for preventing and / or treating PCV2 infection.

Description

Technical Field

[0001] This invention relates to the field of viral vaccine technology, to an mRNA vaccine and its application, and more particularly to an mRNA vaccine combination for the combined treatment of porcine circovirus and its application. Background Technology

[0002] Porcine circovirus (PCV) is a major pathogen causing diseases in pigs. PCV exists in two forms: PCV1 and PCV2. PCV2 is the primary pathogen causing porcine circovirus disease (PCVD), while PCV1 typically does not. The Cap and Rep proteins are two important protein components of PCV2. The Cap protein is a structural protein of the virus, forming the outer shell of the viral particle and playing a role in protecting the genome and aiding in host infection. The Rep protein, on the other hand, is a replication protein of PCV2. As a non-structural protein, it is mainly involved in viral replication and proliferation. The Rep protein plays a crucial role in recognizing the replication initiation site and initiating replication in the PCV2 genome, and is essential for infectivity and viral load control. The Cap protein is the main antigenic protein of PCV2, capable of inducing the host to produce specific antibodies and enhancing the immune system's response to PCV2. Current vaccine development for PCV2 primarily focuses on the Cap protein.

[0003] mRNA vaccines are highly customizable, allowing for the design of specific mRNA sequences based on different viral variants, thereby enhancing vaccine adaptability and efficacy. Studies indicate that mRNA vaccines are low-risk, highly effective, rapidly developable, and inexpensive to produce. They can also initiate the synthesis of target proteins within the host, eliciting a durable immune response. This means that the development of PCV2-based mRNA vaccines could potentially provide long-lasting immune protection for pig herds, reducing the risk of reinfection and virus transmission.

[0004] Despite the advantages of short development cycles and rapid customization, the immunogenicity of mRNA vaccines is still affected by factors such as antigen expression levels, delivery efficiency, and the activation mode of the host's innate immune system. From the current technological development path, most mRNA vaccine platforms (especially those using lipid nanoparticles (LNPs) for delivery) typically do not include traditional exogenous adjuvants in their formulation design. This is because mRNA and LNPs themselves can induce certain innate immune stimulation and exhibit an "adjuvant-like effect." However, in immune protection against viral infections such as PCV2, relying solely on the mRNA-LNP system may still result in insufficient strength or persistence of the immune response, necessitating further enhancement of immunogenicity. Adjuvants can enhance vaccine immunogenicity by activating innate immunity and promoting antigen presentation. Among them, CpG oligodeoxynucleotides (CpG ODNs), as TLR9 (Toll-like receptor 9) agonists, can promote antibody production and preferentially induce Th1-type immune responses, demonstrating potential for antiviral immune enhancement. In human medicine, CpG adjuvants (CpG ODN adjuvants) have been successfully used as key components in products such as hepatitis B vaccines, demonstrating their safety and efficacy. In veterinary medicine, CpG adjuvants have also been extensively studied, showing the potential to enhance the immunogenicity of various vaccines. However, there is a lack of systematic public reports on combining CpG adjuvants with mRNA-LNP vaccines encoding the PCV2 Cap protein to achieve synergistic immune enhancement. Therefore, the research and development of an mRNA vaccine that can induce stronger and more comprehensive immune protection through the combined application of CpG adjuvants has become a current research direction in the vaccine field. Summary of the Invention

[0005] To achieve the above objectives, the present invention aims to provide a combination of mRNA vaccines for the combined treatment of porcine circovirus and its application, thereby generating stronger and more comprehensive immune protection and better treatment and / or prevention of porcine circovirus.

[0006] One aspect of the present invention provides a nucleotide combination comprising a sequence encoding a Cap protein of porcine circovirus and a CpG adjuvant, wherein the amino acid sequence of the Cap protein of porcine circovirus is shown in SEQ ID NO: 1, and the nucleotide sequence of the CpG adjuvant is shown in SEQ ID NO: 2.

[0007] In a preferred embodiment of the present invention, the sequence of the Cap protein encoding porcine circovirus is shown in SEQ ID NO:3 or SEQ ID NO:4.

[0008] In a preferred embodiment of the present invention, the 5' end of the sequence encoding the Cap protein of porcine circovirus is connected to a 5' UTR sequence, and the 3' end of the sequence encoding the Cap protein of porcine circovirus is also connected to a 3' UTR sequence.

[0009] On the other hand, the present invention provides an mRNA vaccine combination comprising the above-mentioned nucleotide combination, wherein the sequence encoding the Cap protein of porcine circovirus and the CpG adjuvant are respectively assembled into a porcine circovirus mRNA vaccine and a CpG adjuvant mRNA vaccine.

[0010] In a preferred embodiment of the present invention, the weight ratio of the porcine circovirus mRNA vaccine to the CpG adjuvant mRNA vaccine is (1~10):(1~3).

[0011] In a preferred embodiment of the present invention, the weight ratio of the porcine circovirus mRNA vaccine to the CpG adjuvant mRNA vaccine is 10:3.

[0012] In a preferred embodiment of the present invention, the dosage of the porcine circovirus mRNA vaccine is 1-10 μg / mL, and the dosage of the CpG adjuvant mRNA vaccine is 1-3 μg / mL.

[0013] Preferably, the dosage of the porcine circovirus mRNA vaccine is 10 μg / mL, and the dosage of the CpG adjuvant mRNA vaccine is 3 μg / mL.

[0014] In a preferred embodiment of the present invention, the mRNA in the mRNA vaccine is encapsulated in liposome nanoparticles, and the particle size of the liposome nanoparticles is preferably 50~200nm.

[0015] In a preferred embodiment of the present invention, the liposome nanoparticles are selected from at least one of cationic lipids, distearate phosphatidylcholine (DSPC), cholesterol, and DMG-2000;

[0016] More preferably, the liposome nanoparticles are composed of the following components in parts by weight: 30-80 parts of cationic lipids, 5-15 parts of distearate phosphatidylcholine (DSPC), 24-52 parts of cholesterol, and 1-2 parts of DMG-2000.

[0017] More preferably, the liposome nanoparticles are composed of the following components in parts by weight: 50 parts cationic lipids, 10 parts distearate phosphatidylcholine (DSPC), 38.5 parts cholesterol, and 1.5 parts DMG-2000.

[0018] In another aspect, the present invention also provides a kit comprising a first formulation and a second formulation, wherein the first formulation comprises a porcine circovirus mRNA vaccine and pharmaceutically acceptable excipients, and the second formulation comprises a CpG adjuvant-packaged CpG adjuvant-packaged CpG adjuvant-packaged mRNA vaccine and pharmaceutically acceptable excipients; the nucleotide sequence of the porcine circovirus mRNA vaccine comprises a sequence encoding the Cap protein of porcine circovirus, the amino acid sequence of the Cap protein of porcine circovirus is shown in SEQ ID NO: 1, and the nucleotide sequence of the CpG adjuvant is shown in SEQ ID NO: 2.

[0019] In a preferred embodiment of the present invention, the weight ratio of the porcine circovirus mRNA vaccine to the CpG adjuvant mRNA vaccine is (1~10):(1~3).

[0020] Preferably, the weight ratio of the porcine circovirus mRNA vaccine to the CpG adjuvant mRNA vaccine is 10:3.

[0021] In a preferred embodiment of the present invention, the dosage of the porcine circovirus mRNA vaccine is 1-10 μg / mL, and the dosage of the CpG adjuvant mRNA vaccine is 1-3 μg / mL.

[0022] Preferably, the dosage of the porcine circovirus mRNA vaccine is 10 μg / mL, and the dosage of the CpG adjuvant mRNA vaccine is 3 μg / mL.

[0023] In a preferred embodiment of the present invention, the sequence of the Cap protein encoding porcine circovirus is shown in SEQ ID NO:3;

[0024] Preferably, the 5' end of the sequence encoding the Cap protein of porcine circovirus is connected to a 5' UTR sequence, and the 3' end of the sequence encoding the Cap protein of porcine circovirus is also connected to a 3' UTR sequence.

[0025] In another aspect, the present invention also provides a pharmaceutical composition comprising the above-described mRNA vaccine composition, and further comprising pharmaceutically acceptable components.

[0026] In another aspect, the present invention also provides the use of the above-described nucleotide combination or the above-described mRNA vaccine combination, or the above-described pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of porcine circovirus.

[0027] In another aspect, the present invention also provides an appropriate amount of the above-mentioned combination of nucleotides or the above-mentioned appropriate amount of mRNA vaccine combination, or the above-mentioned appropriate amount of pharmaceutical composition, applied to a desired receptor.

[0028] The beneficial effects of this invention are:

[0029] 1. Adjuvant synergistic effect

[0030] Existing technologies generally consider that mRNA vaccines (especially those delivered using lipid nanoparticles (LNPs)) typically acquire immunogenicity by relying on the innate immune stimulation induced by the mRNA itself and the LNP carrier. Therefore, their formulations often employ a basic "mRNA + LNP" structure, usually without the addition of exogenous adjuvants. However, the inventors, in their first combination of CpG adjuvant and PCV2 Cap mRNA-LNP vaccine, unexpectedly discovered that only CpG1 significantly increased neutralizing antibody levels, while adjuvants CpG2 and CpG3 did not significantly increase neutralizing antibody levels. Further experiments with CpG1 showed that its addition induced a stronger Th1-type cellular immune response. Experiments demonstrated that the PCV2 mRNA + CpG group, compared to the mRNA-only group, induced a higher proportion of antigen-specific CD8⁺ T cells and a more active germinal center B cell response, exhibiting a synergistic effect and producing a more efficient and longer-lasting immune protection than a single mRNA vaccine. Meanwhile, the addition of CpG adjuvant did not cause significant safety issues, and the vaccine composition demonstrated good safety in mouse models. 2. Vaccine Design

[0031] This study used the porcine circovirus Cap protein gene sequence as a basis and optimized the codons to improve translation efficiency. A signal peptide was added before the RNA sequence encoding the Cap protein to increase protein secretion. Furthermore, a T7 promoter, a 5'UTR, and a "Cap 101" structure were added to the front of the signal peptide sequence. A 3'UTR and a poly A tail were added to the rear of the RNA sequence encoding the Cap protein to improve the translation efficiency and stability of the mRNA vaccine, thus constructing a candidate mRNA vaccine expressing the Cap antigen. A candidate mRNA vaccine with high secretion levels was also constructed by expressing the porcine circovirus Cap protein.

[0032] The expression and extracellular secretion of the constructed candidate mRNA vaccine in HEK-293T cells were verified by in vitro cell transfection and Western blotting experiments.

[0033] 3. Immune effect

[0034] The plasmid expressing the expression was used to prepare mRNA via in vitro transcription. Quality analysis, including lipid nanoparticle packaging and electron microscopy, was then performed to prepare a porcine circotropic mRNA vaccine. Immunization of mice with the constructed candidate mRNA vaccine induced high levels of IgG antibodies against PCV2. Attached Figure Description

[0035] The accompanying drawings, which form part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0036] The invention will be more clearly understood with reference to the accompanying drawings and the following detailed description, wherein:

[0037] Figure 1 The image shows the agarose gel electrophoresis results of in vitro transcribed mRNA in Example 2 of the present invention, where PCV2 mRNA represents the mRNA of the Cap antigen of PCV2;

[0038] Figure 2 This is the Western Blot detection result of the in vitro transcription mRNA protein expression verification experiment in Example 3 of the present invention, where M represents protein marker, 1 is blank control supernatant; 2 is Cap mRNA transfection cell supernatant; 3 is blank control cell lysate; 4 is Cap mRNA transfection cell lysate.

[0039] Figure 3 This is an electron microscopy analysis result of LNP after packaging in Embodiment 4 of the present invention;

[0040] Figure 4 This is a graph showing the particle size analysis results of LNP after packaging in Example 4 of the present invention;

[0041] Figure 5 These are flowcharts of animal experiments in Examples 5 and 6 of the present invention;

[0042] Figure 6 The figures in Example 5 of this invention are the Cap protein-specific IgG antibody titer and the neutralizing antibody titer after immunizing mice, where A is the specific IgG antibody titer and B is the neutralizing antibody titer.

[0043] Figure 7 This is the change in the content of specific CD4+ T cells and CD8+ T cells in spleen lymphocytes after immunizing mice in Example 6 of the present invention. Among them, A is a representative flow cytometry result, B is a statistical graph of CD4+ T cells, and C is a statistical graph of CD8+ T cells.

[0044] Figure 8 This is the change in GC B cell content in the inguinal lymph nodes of mice after immunization in Example 6 of the present invention. A is a representative flow cytometry result graph, and B is a cell number statistics graph.

[0045] Figure 9 This is the change in the content of Tfh cells in the inguinal lymph nodes of mice after immunization in Example 6 of the present invention. A is a representative flow cytometry result graph, and B is a cell number statistics graph.

[0046] Figure 10The results of the detection of IFN-γ specific cellular immune response of spleen lymphocytes in Example 6 of the present invention are shown in the left figure, which is the ELISpot image and the right figure is the spot statistical results.

[0047] Figure 11 This is the detection result of IL-4 specific cellular immune response of spleen lymphocytes in Example 6 of the present invention, wherein the left figure is the ELISpot image and the right figure is the spot statistical result;

[0048] Figure 12A These are photographs of different organs and tissues taken after a single overdose immunization with the vaccine in Example 7 of this invention.

[0049] Figure 12B This is an image showing the HE staining results of different organs after a single overdose immunization with the vaccine in Example 6 of this invention;

[0050] In this context, Cap mRNA represents the experimental group of porcine circovirus mRNA vaccine, and Cap mRNA+CpG1, CpG2, or CpG3 represent the experimental groups of porcine circovirus mRNA vaccine combined with CpG1, CpG2, or CpG3, respectively. Detailed Implementation

[0051] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0052] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use.

[0053] Techniques, methods, and equipment known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques and methods should be considered part of the specification.

[0054] Unless otherwise specified, all reagents and materials mentioned in this article are available from conventional sources.

[0055] Unless otherwise specified, all mice used in this article were purchased from Spiford Biotechnology Co., Ltd.

[0056] Unless otherwise specified, "Cap mRNA-LNP", "Cap mRNA vaccine" or "Cap mRNA LNP" in this article all refer to "porcine circovirus mRNA vaccine".

[0057] Unless otherwise specified, there are three CpG adjuvants in this article, referred to as CpG1, CpG2 and CpG3.

[0058] Unless otherwise specified, "CpG-LNP", "CpG LNP", "CpG1-LNP" or "CpG1 LNP" in this article refer to "CpG adjuvanted mRNA vaccine".

[0059] Unless otherwise specified, "Porcine circovirus Cap protein" in this article refers to "Porcine circovirus PCV2 Cap protein".

[0060] Example 1. Construction of an antigen expression vector for a porcine circovirus mRNA vaccine.

[0061] I. The construction method of antigen expression vector plasmid is as follows:

[0062] (1) The following gene sequences were synthesized by Shanghai Sangon Biotech Co., Ltd.: from 5' to 3' end, they are T7 promoter (SEQ ID NO:8), 5'UTR (SEQ ID NO:5), kozak sequence (GCCACC), tPA signal peptide sequence (SEQ ID NO:9), Cap sequence (SEQ ID NO:4), 3'UTR (SEQ ID NO:6), and polyA (SEQ ID NO:7).

[0063] (2) Primers were synthesized at Beijing Qingke Biotechnology Co., Ltd.: Primer F: GCCTCAGGCCACCATGACCTACCCCCGCCG (SEQ ID NO:12); Primer R: GCGGTACCTTAGGGGTTCAGGGTGGGGG (SEQ ID NO:1:3).

[0064] The Cap gene sequence was obtained by PCR amplification using the synthesized plasmid as a template and the PrimeSTAR Max DNA Polymerase enzyme from TAKARA.

[0065]

[0066] (3) Double digestion was performed using Xho I and Kpn I restriction endonucleases from TransGen, and the DNA fragment containing the Cap gene and the pUC57 vector fragment were recovered by agarose gel electrophoresis.

[0067] (4) Perform PCR products on 1% agarose gel electrophoresis and recover PCR fragment DNA using an Axygen gel recovery kit.

[0068] (5) Homologous recombination was performed using Takara T4 DNA ligase. The pUC57 vector fragment recovered in (3) and the Cap fragment recovered in (4) were ligated. After reacting at 25°C for 15 min, the mixture was added to competent DH5α cells from Qingke Company. The cells were then placed on ice for 30 min, heat-shocked at 42°C for 90 s, placed on ice for 2 min, and 1 mL of LB medium was added. The cells were shaken at 200 rpm for 30 min at 37°C, centrifuged at 2000 rpm for 10 min, and then resuspended in 100 μL of LB medium. The cells were then spread on a Cannabis-resistant solid agar plate and colonies were picked the next day.

[0069] (6) Plasmids were extracted from 2 mL of bacterial culture using TransGen's plasmid DNA mini kit. The plasmids were then digested with Xho I and Kpn I restriction endonucleases for identification. The recombinant plasmid sequence was confirmed to be correct by sequencing by Qingke Company.

[0070] (7) The CpG sequence was synthesized by Qingke Biotechnology, as shown in SEQ ID NO:2, and all nucleotides were thiolated.

[0071] Example 2. mRNA transcription validation assay

[0072] The plasmid obtained in Example 1 was linearized by digestion with Bsa I restriction endonuclease from Yisheng Biotechnology Co., Ltd. The plasmid was then recovered using a DNA recovery kit from TransGen Co., Ltd.

[0073] In vitro transcription was performed using the T7 Transcription Kit from Yisheng Biotechnology Co., Ltd. The PCR reaction was carried out at 37°C for 2 hours. 1.5 volumes of lithium chloride were added to precipitate the mRNA, which was then resuspended in DEPC water. RNA concentration was measured using a Nanodrop instrument, and the mRNA was stored at -80°C.

[0074] A 1% agarose gel was prepared, and the quality of in vitro transcribed RNA was verified by agarose gel electrophoresis. The results are as follows: Figure 1 As shown, from Figure 1 As can be seen, the in vitro transcribed mRNA bands are clear and uniform. The mRNA sequence of the PCV2 Cap antigen is shown in SEQ ID NO:3. Other sequences are as follows:

[0075] The DNA sequence of the 5' untranslated region (5'UTR) is shown in SEQ ID NO:5:

[0076] GAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCGCTAGCCTCGAG

[0077] The DNA sequence of the 3' untranslated region (3'UTR) is shown in SEQ ID NO:6:

[0078] GATATCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTG

[0079] Polyadenylate (poly A) has the sequence shown in SEQ ID NO:7 and contains 104 bases A:

[0080] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0081] The DNA sequence of the T7 promoter is shown in SEQ ID NO:8:

[0082] TAATACGACTCACTATAGG

[0083] The DNA sequence of the signal peptide tPA is shown in SEQ ID NO:9:

[0084] ATGGACGCCATGAAGAGGGGGCTGTGCTGCGTGCTGCTGCTGTGCGGAGCCGTGTTCGTGAGCGCCTCC.

[0085] The Cap antigen DNA sequence of PCV2 is shown in SEQ ID NO:4:

[0086] ATGACCTACCCCCGCCGCCGCTACCGCCGCCGCCGCCACCGCCCCCGGAGCCACCTGGGCCAGATCCTGCGCCGCCGGCCCTGGCTGGTGCACCCCCGCCACCGCTACCGCTGGCGCCGGAAGAACGGCATCTTCAACACCCGCCTGTCCCGCACCTTCGGCTACACCATCAAGCGCACCACCGTCAAGACCCCCAGCTGGGCCGTGGACATGATGCGCTTCAACATCAACGACTTCCTGCCCCCCGGCGGCGGCAGCAACCCCCGCTCCGTGCCCTTCGAGTACTACCGCATCCGCAAGGTGAAGGTGGAGTTCTGGCCCTGCAGCCCCATCACCCAGGGCGACCGCGGCGTGGGCAGCAGCGCCGTGATCCTGGACGACAACTTCGTGACCAAGGCCACCGCCCTGACCTACGACCCCTACGTGAACTACAGCAGCCGCCACACCATCACCCAGCCCTTCAGCTACCACAGCCGCTACTTCACCCCCAAGCCCGTGCTGGACAGCACCATCGACTACCTGCAGCCCAACAACAAGCGCAACCAGCTGTGGCTGCGCCTGCAGACCGCCGGGAACGTGGACCACGTGGGCCTGGGCACCGCCTTCGAGAACAGCATCTACGACCAGGAGTACAACATCCGGGTCACCATGTACGTGCAGTTCCGCGAGTTCAACCTGAAGGCCCCCACCCTGAACCCC

[0087] The amino acid sequence of the Cap antigen of PCV2 is shown in SEQ ID NO: 1:

[0088] MHLDCKPEFSYAIAKDERIGQLGAEGLTTTWKEYSPGMKLEDTMVIAWCEDGKLMYLQRCTRETRYLAILHTRALPTSVVFKKLFDGRKQEDVVEMNDNFEFGLCPCDAKPIVRGKFNTTLLNGPAFQMVCPIGWTGTVSCTSFNMDTLATTVVRTYRRSKPFPHRQGCITQKNLGEDLHNCILGGNWTCVPGDQLLYKGGSIESCKWCGYQFKESEGLPHYPIGKCKLENETGYRLVDSTSCNREGVAIVPQGTLKCKIGKTTVQVIAMDTKLGPMPCRPYEIISSEGPVEKTACTFNYTKTLKNKYFEPRDSYFQQYMLKGEYQYWFDLEVTDHHRDYFAESILVVVVALLGGRYVLWLLVTYMVLSEQKALG。

[0089] The Cap antigen mRNA sequence of PCV2 is shown in SEQ ID NO:3:

[0090]

[0091] The CpG1 sequence is shown in SEQ ID NO:2:

[0092] TCGTCGTTGTCGTTCGGGCGGCG (all are thio-modified).

[0093] The CpG2 sequence is shown in SEQ ID NO:10:

[0094] GGGGGACGATCGTCGGGGGG (All are thio-modified)

[0095] The CpG3 sequence is shown in SEQ ID NO:11:

[0096] TCGTCGTTGTCGTTTTGTCGTT (all are thio-modified).

[0097] Example 3. Cap protein expression verification experiment

[0098] The purified in vitro transcribed Cap mRNA from Example 2 was transfected into HEK-293T cells using TransGen's PEI reagent for expression verification. 293T cells were evenly seeded in 24-well plates, and RNA was transfected using PEI, with 0.8 μg of mRNA per well. After 48 h of transfection, the cell supernatant was collected, loading buffer was added for sample processing, and after SDS-PAGE electrophoresis, the cells were incubated with anti-Cap protein antibody from Kemiao Biotechnology and HRP-labeled goat anti-rabbit IgG antibody from TransGen. Western blotting was performed using ECL to obtain the results; the results are shown below. Figure 2 As shown in the figure, compared with the control group, the experimental group transfected with Cap mRNA showed protein bands of the expected size, indicating that Cap mRNA can be expressed and secreted in cells.

[0099] Example 4. Preparation of mRNA vaccine

[0100] The plasmid successfully constructed in Example 1 was linearized by single-enzyme digestion with Bsa I restriction endonuclease. The DNA was then recovered using a DNA fragment recovery kit.

[0101] The linearized DNA molecules were transcribed in vitro using the T7 High Yield RNA Transcription Kit (N1-Me-Pseudo UTP) from Yisheng Biotechnology Co., Ltd. This reaction yielded a large amount of mRNA modified with N1-Me-Pseudo UTP. Cap101 from Cangzhou Weikexin Biotechnology Co., Ltd. was then added to cap the transcribed mRNA. The capped mRNA molecules were extracted using lithium chloride and packaged to prepare an LNP-mRNA vaccine.

[0102] The specific experimental steps are as follows: First, the alcohol phase was prepared by dissolving the lipids in anhydrous ethanol at a mass ratio of cationic lipid (SM102):distearate phosphatidylcholine (DSPC):cholesterol:DMG-2000 = 50:10:38.5:1.5. Next, the aqueous phase was prepared using 50 mM citrate buffer (pH 4.0) as the solute for dissolving the mRNA. The mRNA was then packaged using a microfluidic device with an alcohol phase:aqueous phase ratio of 1:3 (volume ratio). Next, the mRNA was diluted with RNase-free PBS buffer and concentrated using a 30 kDa ultrafiltration tube. An equal volume of 20% sucrose PBS solution was added to adjust the mRNA concentration to 60 μg / mL and the sucrose concentration to 10%. Finally, the mRNA was filtered through a 0.22 μm filter to obtain the prepared Cap LNP-mRNA vaccine, which was then aliquoted and stored at -20°C.

[0103] The liposome nanoparticles contain at least one of cationic lipids, distearate phosphatidylcholine (DSPC), cholesterol, and DMG-2000.

[0104] The process of encapsulating CpG adjuvants into mRNA vaccines is the same as above.

[0105] The results were observed using a transmission electron microscope as follows: Figure 3 As shown, from Figure 3 It can be seen that the prepared Cap mRNA-LNP and CpG1-LNP vaccine particles are uniform and consistent (images of other CpG adjuvant vaccines are not shown).

[0106] Particle size distribution was detected by dynamic light, and the results are as follows: Figure 4 As shown, from Figure 4 It can be seen that the prepared Cap mRNA-LNP and CpG-LNP1 vaccines have uniform particle size distribution (images of other CpG adjuvant vaccines are not shown).

[0107] Example 5. Immunological evaluation experiment in mice with different CpG adjuvants

[0108] The immunization effect of the mRNA vaccine prepared in Example 4 on BALB / c mice was evaluated. Twenty-five 8-week-old SPF BALB / c mice purchased from Liaoning Changsheng Biotechnology Co., Ltd. were randomly divided into 5 groups (n=5 per group). The mice were immunized intramuscularly with 10 μg Cap mRNA vaccine, 10 μg Cap mRNA vaccine, and 3 μg CpG1 mRNA, 10 μg Cap mRNA vaccine, 3 μg CpG2 mRNA, and 10 μg Cap mRNA vaccine, respectively. One hour later, 3 μg CpG3 mRNA was injected intramuscularly at the same location. A blank control group was also included. Two weeks after immunization, a booster immunization was performed using the same dose and volume of vaccine as the initial immunization. The results were evaluated two weeks later, following the procedure described below. Figure 5 As shown.

[0109] The titer of specific anti-Cap antibodies in immune serum was analyzed using an indirect ELISA method based on Cap protein as the coating antigen. Purified Cap protein (200 ng / well) was coated onto the ELISA plate. Serum samples separated at different time points after booster immunization were serially diluted and added to the corresponding ELISA plates. HRP-labeled goat anti-mouse IgG antibody and TMB were added sequentially. After colorimetric termination, the OD value was measured using A450 to determine the antibody titer. Results are as follows: Figure 6 As shown in A, the results indicate that the serum-specific anti-Cap antibody titer increased in mice immunized with the Cap mRNA vaccine. Among the three different CpGs added, only CpG1 significantly enhanced the level of specific anti-Cap IgG antibodies induced by the Cap mRNA vaccine, indicating that the addition of CpG1 can effectively induce humoral immune response, while CpG2 and CpG3 had no significant effect.

[0110] The level of neutralizing antibodies in mouse serum was then measured. Mouse serum (inactivated at 56°C for 30 min) was serially diluted twofold with serum-free maintenance medium. Each dilution was replaced with an equal volume of standardized PCV2 virus suspension (containing 200 TCID50 per 50 μL). 50 Mix to achieve a final attack dose of 100 TCID per well. 50Serum-virus mixtures were incubated at 37°C and 5% CO2 for 60 minutes to allow antibody-virus interaction. After incubation, 100 μL of each mixture was transferred to monolayers of PK-15 cells cultured in 96-well tissue culture plates and incubated at 37°C for 1 hour. The inoculum was then discarded and replaced with fresh maintenance medium supplemented with 2% fetal bovine serum, and the cells were cultured for another 72 hours to allow viral replication. After incubation, cells were fixed with 80% acetone, and viral infection was detected using an immunoperoxidase monolayer assay (IPMA): PCV2-specific monoclonal primary antibody was used, followed by HRP-labeled secondary antibody, with 3,3'-diaminobenzidine as the chromogenic substrate. Neutralization titer (NT) was measured. 50 The reciprocal of the highest serum dilution that reduces the number of viral foci by ≥50% compared to the viral control group is defined and calculated using the Reed-Muench method. Results are as follows: Figure 6 As shown in B, the Cap mRNA vaccine significantly enhanced the level of neutralizing antibodies in the serum after immunization. Among the three different CpGs, only CpG1 could significantly increase the level of neutralizing antibodies again, while CpG2 and CpG3 had no significant effect.

[0111] Example 6. Mouse Immunological Evaluation Experiment of CpG1

[0112] The immunization effect of the mRNA vaccine prepared in Example 4 on BALB / c mice was evaluated. Fifteen 8-week-old SPF BALB / c mice purchased from Liaoning Changsheng Biotechnology Co., Ltd. were randomly divided into three groups of five mice each. The mice were immunized intramuscularly with 10 μg of Cap mRNA vaccine, 10 μg of Cap mRNA vaccine, and 100 μL of PBS (as a blank control group) at the same site one hour later. Two weeks after immunization, a booster immunization was performed using the same dose and volume of vaccine as the initial immunization. The results were evaluated two weeks later, following the procedure described below. Figure 5 As shown.

[0113] Splenic lymphocytes from mice were isolated and added to 96-well plates. The cells were stimulated with a synthetic Cap protein peptide library (QPFSYHSRY, NPRSVFEY, HTITQPFSY, ATALTYDPY, KRNQLWLRL, VHPRHRYRW, RYFTPKPVL, SYHSRYFTP, RPRSHLGQI, mixed in equal proportions) at a concentration of 18 μg / mL / well. The cells were cultured at 37°C for 6 hours, followed by flow cytometry analysis to detect CD4+ in the spleen. + T cells and CD8 + Changes in T cell variables. Results as follows: Figure 7As shown, immunization with Cap mRNALNP can significantly increase CD4 levels. + T cells and CD8 + The level of T cells was further increased after the addition of CpG, which boosted CD4 count. + T cells and CD8 + The level of T cells.

[0114] Inguinal lymph nodes of mice were isolated, and changes in the content of GC B cells and Tfh cells were detected by flow cytometry. Results are as follows: Figure 8 and Figure 9 As shown, the results indicate that CpG can significantly enhance the content of GC B cells and Tfh cells, and the effect is better than that of the Cap mRNA-LNP group alone.

[0115] The isolated spleen cells were added to 96-well plates and stimulated with a synthetic Cap protein polypeptide library (as above) at a concentration of 4.5 μg / mL / well. The plates were incubated at 37°C for 48 hours. ELIS pot was used to detect IFN-γ and IL-4-specific cellular immune responses. Results are as follows: Figure 10 and Figure 11 As shown, the results indicate that CpG can significantly enhance the specific cellular immune response to IFN-γ and IL-4, and the effect is better than that of the Cap mRNA-LNP group alone.

[0116] Example 7 Safety Evaluation

[0117] To investigate the safety of the vaccine and CpG in mice, Cap mRNA-LNP or Cap mRNA-LNP combined with CpG1-LNP were injected intraperitoneally into mice at a single dose of 5 times the normal dose, with 5 mice in each group. A blank control group was also included. All mice were 8 weeks old Balb / c and purchased from Liaoning Changsheng Biotechnology Co., Ltd. Two weeks after intervention, the mice were dissected for histopathological examination and HE staining. The results are as follows: Figure 12A-12B As shown, no abnormal lesions were observed in the major organs (heart, liver, spleen, lungs, and kidneys) of each vaccine group, confirming that the vaccine composition has good safety.

[0118] In summary, this invention mainly provides a combined composition of PCV2 Cap mRNA-LNP vaccine and CpG ODN adjuvant and its application. By introducing CpG adjuvant into the optimized Cap mRNA-LNP delivery system, it solves the problem that existing mRNA-LNP vaccines usually do not add adjuvants, resulting in insufficient immune response strength and Th1 cellular immunity, and the need to improve the duration of immunity. Thus, under the premise of good safety, it achieves synergistic enhancement of humoral and cellular immunity, and obtains higher and more lasting protective effects.

[0119] The description of this invention is given for illustrative and descriptive purposes only and is not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of the invention and to enable those skilled in the art to understand the invention and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A nucleotide combination comprising a sequence encoding a Cap protein of porcine circovirus and a CpG adjuvant, wherein, The amino acid sequence of the Cap protein of the porcine circovirus is shown in SEQ ID NO:1, and the nucleotide sequence of the CpG adjuvant is shown in SEQ ID NO:

2.

2. The nucleotide combination according to claim 1, characterized in that, The sequence of the Cap protein encoding porcine circovirus is shown in SEQ ID NO:3 or SEQ ID NO:4; Preferably, the 5' end of the sequence encoding the Cap protein of porcine circovirus is connected to a 5' UTR sequence, and the 3' end of the sequence encoding the Cap protein of porcine circovirus is also connected to a 3' UTR sequence.

3. An mRNA vaccine combination comprising the nucleotide combination of claim 1 or 2, wherein the sequence encoding the Cap protein of porcine circovirus and the CpG adjuvant are respectively assembled into a porcine circovirus mRNA vaccine and a CpG adjuvant mRNA vaccine; Preferably, the weight ratio of the porcine circovirus mRNA vaccine to the CpG adjuvant mRNA vaccine is (1~10):(1~3). More preferably, the weight ratio of the porcine circovirus mRNA vaccine to the CpG adjuvant mRNA vaccine is 10:

3.

4. The mRNA vaccine combination according to claim 3, characterized in that, The dosage of the porcine circovirus mRNA vaccine is 1-10 μg / dose, and the dosage of the CpG adjuvanted mRNA vaccine is 1-3 μg / dose. Preferably, the dosage of the porcine circovirus mRNA vaccine is 10 μg / dose, and the dosage of the CpG adjuvanted mRNA vaccine is 3 μg / dose.

5. The mRNA vaccine combination according to claim 3 or 4, characterized in that, The mRNA in the mRNA vaccine is encapsulated in liposome nanoparticles, and the particle size of the liposome nanoparticles is preferably 50~200nm. Preferably, the liposome nanoparticles are selected from at least one of cationic lipids, distearate phosphatidylcholine (DSPC), cholesterol, and DMG-2000; More preferably, the liposome nanoparticles are composed of the following components in parts by weight: 30-80 parts of cationic lipids, 5-15 parts of distearate phosphatidylcholine (DSPC), 24-52 parts of cholesterol, and 1-2 parts of DMG-2000. More preferably, the liposome nanoparticles are composed of the following components in parts by weight: 50 parts cationic lipids, 10 parts distearate phosphatidylcholine (DSPC), 38.5 parts cholesterol, and 1.5 parts DMG-2000.

6. A kit comprising a first formulation and a second formulation, the first formulation comprising a porcine circovirus mRNA vaccine and pharmaceutically acceptable excipients, the second formulation comprising a CpG adjuvant-packaged CpG adjuvant-packaged CpG adjuvant-packaged mRNA vaccine and pharmaceutically acceptable excipients; the nucleotide sequence of the porcine circovirus mRNA vaccine comprising a sequence encoding the Cap protein of porcine circovirus, the amino acid sequence of the Cap protein of porcine circovirus being as shown in SEQ ID NO: 1, and the nucleotide sequence of the CpG adjuvant being as shown in SEQ ID NO:

2.

7. The reagent kit according to claim 6, characterized in that, The weight ratio of the porcine circovirus mRNA vaccine to CpG adjuvant is (1~10):(1~3). Preferably, the weight ratio of the porcine circovirus mRNA vaccine to the CpG adjuvant is 10:

3.

8. The reagent kit according to claim 7, characterized in that, The dosage of the porcine circovirus mRNA vaccine is 1-10 μg / dose, and the dosage of the CpG adjuvant is 1-3 μg / dose. Preferably, the dosage of the porcine circovirus mRNA vaccine is 10 μg / dose, and the dosage of the CpG adjuvant is 3 μg / dose.

9. The reagent kit according to any one of claims 6 to 8, characterized in that, The sequence encoding the porcine circovirus Cap protein is shown in SEQ ID NO:3; Preferably, the 5' end of the sequence encoding the Cap protein of porcine circovirus is connected to a 5' UTR sequence, and the 3' end of the sequence encoding the Cap protein of porcine circovirus is also connected to a 3' UTR sequence.

10. A pharmaceutical composition comprising the mRNA vaccine combination of any one of claims 3 to 5, further comprising pharmaceutically acceptable excipients.

11. The use of the nucleotide combination according to claim 1 or 2, or the mRNA vaccine combination according to any one of claims 3 to 5, or the pharmaceutical composition according to claim 10 in the preparation of a medicament for the prevention and / or treatment of porcine circovirus.

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