Multi-antigen expressed circRNA vaccine and preparation method thereof

Abstract

The invention provides a circRNA vaccine expressed by multiple antigens and a preparation method of the circRNA vaccine, and belongs to the technical field of biological medicines, and the circRNA vaccine comprises a circular RNA (circRNA) sequence capable of simultaneously expressing two or more antigen sequences. The circRNA vaccine provided by the invention can be used for simultaneously expressing any two or three of HBV antigens HBsAg and HBcAg and therapeutic polypeptide, and also can be used for expressing a corresponding RSV (Respiratory Syndrome Virus) trimer F antigen. The circRNA vaccine for mammal injection is obtained through in-vitro gene synthesis, in-vitro transcription reaction, linear RNA (Ribonucleic Acid) cyclization, hydrogel / amino acid nanoparticle preparation and the like. HBVamp can be reduced for healthy people; the traditional Chinese medicine composition can be used for relieving and eliminating the symptoms, stabilizing the illness state, reducing the virus transmission, reducing the dosage, shortening the treatment course, delaying / reversing the disease course and improving the prognosis and life quality aiming at infected persons, and can be used for relieving and eliminating the symptoms, stabilizing the illness state, reducing the virus transmission, reducing the dosage, shortening the treatment course and improving the prognosis and life quality.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a circRNA vaccine expressing multiple antigens and a preparation method thereof. Background Art

[0002] Injectable vaccines are considered the most effective way to control viral infections. Vaccines currently in use are mainly divided into two categories based on their ingredients: vaccines containing complete viruses and vaccines containing partial viral components. The former are mainly inactivated vaccines and attenuated vaccines, while the latter are mainly recombinant protein vaccines, vector vaccines and nucleic acid vaccines. Inactivated vaccines and attenuated vaccines need to handle the infectious viruses produced during the production process, and the finished vaccines need to undergo extensive safety testing before being put into use. Recombinant protein vaccines are highly dependent on host cells during the production process, and their production capacity is easily limited. In addition, inactivated vaccines, attenuated vaccines, recombinant protein vaccines and DNA vaccines also face problems such as allergic reactions caused by residual host DNA or the DNA sequence itself used as a vaccine.

[0003] Pneumonia caused by SARS-CoV-2 coronavirus infection has caused a serious global public health emergency. In the fight against the coronavirus, RNA nucleic acid vaccines have played an unimaginable role. Nucleic acids developed based on mRNA technology provide a potential path for the development of immunogenic vaccines. There is no need to deal with infectious viruses during the production process, the production cycle is short, and there is no host DNA residue. RNA molecules have a shorter half-life than DNA and do not have the ability to integrate into the host cell genome. They only need to enter the cytoplasm of the host cell to exert their effects, and they will be quickly cleared in the host body, so they are considered to have higher safety. However, its short half-life brings safety as well as the defect of a short duration of action.

[0004] circRNA is a type of RNA molecule whose head and tail are covalently linked to form a circular structure. Due to the lack of a 3' end, it is resistant to nucleases and has a long half-life. It effectively solves the defects of the current linear RNA vaccine, such as short shelf life and high storage and transportation costs. It is considered to be the most promising next-generation nucleic acid vaccine technology route. The existing mainstream circRNA synthesis technology route is more difficult to synthesize circRNAs longer than 4000nt, with low circularization efficiency and great difficulty in purification. Therefore, the main circRNA vaccines currently under development are all single antigen / peptide expression. The combined expression of multiple antigens / peptides requires the simultaneous use of multiple circRNA sequences, which greatly increases the probability of adverse reactions such as allergies in the receptor. Summary of the invention

[0005] In view of the defects and shortcomings of existing circRNA vaccines that can only express a single antigen or polypeptide, the present invention provides a method for preparing a circRNA vaccine that can use a single circRNA sequence to express two or more antigens and polypeptides.

[0006] The present invention is implemented by the following technical scheme: a circRNA vaccine expressing multiple antigens, characterized in that all sequences involved in the present invention include:

[0007] IRES sequence SEQ NO.1

[0008] ACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATA

[0009] TTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTTCTTGACGAGCATTCCTAGGG

[0010] GTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGA

[0011] AGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGA

[0012] CAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTG

[0013] CCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGG

[0014] CTGAAGGATGCCCAGAAGGTACCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTA

[0015] CATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATA;

[0016] Self-catalytic intron upstream sequence SEQ NO.2

[0017] TACCTAGATATTTTCATTCCAGCGGTTTTTTTCCTAGTCTCACCTACACAGGTTCCAGTGAGCTCAGG

[0018] AGTTTACCTTCATGTCCGTGGCACAGCCCATAATTACCCTTCTCTGTCCAGGGATGTATATTTGCATC

[0019] TTTCCTTCAACTTATGAAGTGAATGGAGGGGAAATGTACAGTTTTAATTGAGTTTGAAGTAGCTTTC

[0020] ATCTGACCAGCCCTGTGCTGGAGCGTGCCTCTTCTTCTGTGAGCAGAGGGGCTACTGCGTGGGAAATGGCTAAACCGCCTGCTTTGTCTTTCACTGCAG;

[0021] Self-catalytic intron downstream sequence SEQ NO.3 GTAAGGGCAGCGGCGAGGTCCCTCATGTGGGCGACCCTCCTGCGGGAAGGGAGCTTGCACAGGTGTTGCCCTTGGTCCCTAACCTGTCAAGGTCAGTGGCCCTCTGCTGACCTTGTTGCCCTTACAGTATTAGTGGGAATGGGAGTTGTCAAGTTGCTGTTTACCAAGTTCAGAAGTAATCTCTATCTCCTTTGAAATAAATTGACAATGTACGAGATTCATTGAGATGACATTTGCCACACCTTTCATTCAAAAGATGTTTATTGAACATGTACCAGGTACTAGATTCTGGGGGA;

[0022] HBV HBsAg sequence SEQ NO.4

[0023] ATGGAGAACATCACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGATCACCCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTCCTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTATTGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACCTGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTTGCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCATCCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGCTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAGCATCGTGAGTCCCTTTATACCGCTGTTACCAATTTTCTTTTGTCTCTGGGTATACATTTAA;

[0024] HBcAg sequence SEQ NO.5

[0025] W R T S H Q D S / D P C S C Y R R G F S C / Q E S S Q YR R V / T R G G L LS I F / G D H P C V L A K I R S P Q PP I T H Q P P V L Q F V L V I A G C V C GV L S Y S S S S C C Y A S S S Y W F F W I I K V C C P F V L / F Q D QQ Q P V RD H A K P A R L L L K A T L C F P H V A V Q N L R M E I A P V F P S H R P G LS Q N T Y G S G P Q S V S L G S V Y / C H L F S G S / G F P P L F G F Q L Y G / CGI G G Q V C T A S / V P L Y R C Y Q F S F V S G Y T F;

[0026] SEQ NO.6

[0027] <h2 style=";text-align:left;direction:ltr">;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0028] <h2 style=";text-align:left;direction:ltr"> SEQ NO.7<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0029] <h2 style=";text-align:left;direction:ltr"> WTSIHTRNLELLWSCSASCHR IFSLPFETFSTPP LP CIVRPWSRRNTARHTIRHSGR LSCAGVSS / TWPPGSVA TW KTPPRGSL / SVTSMLTWA / R SD NSCGSTFPVLRSE ER PFL ST SFPSACGFALHQH ID HLTRQSCLHFPKPLL SD GEADRL DG EHPRLAVEDPSRRVADVLNLG SLSA;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0030] RSV antigen is the 3-mer Fusion protein nucleic acid sequence SEQ NO.8;

[0031] RSV antigen is the amino acid sequence of the 3-mer Fusion protein SEQ NO.9;

[0032] Therapeutic polypeptide nucleic acid sequence SEQ NO.10

[0033] ;

[0034] Therapeutic polypeptide protein sequence SEQ NO.11

[0035] MALTFALLVALLVLSCKSSCSVGCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQF QKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILA VRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSSFSLSTNLQESLRSKE;

[0036] Nucleic acid sequence of human Tissue plasminogen activator (tPA) signal peptide SEQ NO.12

[0037] GATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCAGCCAGGAAATCCATGCCCGATTCAGAAGA;

[0038] Amino acid sequence of human Tissue plasminogen activator (tPA) signal peptide SEQ NO.13

[0039] DAMKRGLCCVLLLCGAVFVSPSQEIHARFRR;

[0040] Upstream sequence of QRE SEQ NO.14

[0041] TCTAGGTAGCCATGTGTGACTAGTGGCCACCATAATGGGCTTCACGGTACTAGGTGACTTACTTGACTTCTTTGTGTTCCAGTTTTCCCATCTGTACAGATGAGATCATATAAGTTACCTTTTGTGGTTACTATATGAATTAAATAAAATAATATAGTTAAGAAGGAGTTTAACACTATGCTGGAGCAGAATATTTCCATGAGAAATGGTAGTTAATTATAATGACATACATTTAATAAACAAGTTATTTTGCTTTTGAGCATTAATTTAAACCTGTTTATTTTTGTTCTTCCCTTCCAG;

[0042] Downstream sequence of QRE SEQ NO.15

[0043] GTAATTTTATTTTTGTAAAATTCTATTTTAAAAAATAAGCCATTTTGTATTATGGTTTATGATTTATAGGAGTTGTAACTTTGTGTACAGTATGTTTGAATGTGTATACACAGTGTAGGTATCTTCCTTACTTAGATGTGAAAACACAAAGTAGTAGTAATCTTTTTTT CATTCCGAGTCCTTACCTCAACCTACAGTAGCTCTGGAAACTAACTGTTCTCTGTTAGTCATTGTCTGTACTTAAAGTCAAGCGCTGGTGGGCAGGAAACTCAGGAGTAATATTTAATCTATTTCAGATAGAGTGTTCTGTCTTCTGGCATATAAAGTTTGATGGTTA.

[0044] 2. A method for preparing a circRNA vaccine expressing multiple antigens, characterized in that: a process for preparing an in vitro circRNA vaccine in a laboratory environment, the process mainly comprising the following steps:

[0045] Step 1: Preparation of linear DNA template: construct a DNA template containing a linear RNA sequence for subsequent in vitro transcription. Use PCR to clone the required elements (such as IRES sequence, m6A sequence, tPA sequence, protein-encoding ORF region, etc.) from different templates, and use overlap PCR to connect them to form a complete linear RNA DNA template;

[0046] Step 2 Linearization of IVT (in vitro transcription) template: Prepare linearized DNA template for in vitro transcription, add T7 promoter sequence to the 5' end of the upstream primer, and obtain IVT template by PCR amplification; clone the IVT template DNA sequence into the plasmid vector containing T7 promoter, amplify the plasmid by E. coli, and select appropriate restriction sites for linearization; transform the plasmid into JM 109 competent cells, obtain high-concentration plasmid DNA by shaking and plasmid extraction; use EcoR V and other endonucleases for enzyme digestion, confirm linearization by agarose gel electrophoresis and purify;

[0047] Step 3: Synthesis and purification of linear RNA: Synthesize linear RNA by in vitro transcription and purify it. Use T7 RNA Polymerase for in vitro transcription to generate linear RNA. Add Recombinant DNase I to digest the DNA template. Use Monarch RNA Cleanup Kit for purification.

[0048] Step 4: Catalytic ligation and purification of circRNA: Linear RNA is catalytically ligated into circRNA and purified, and linear RNA is circularized into circRNA using RNA ligase (such as T4 RNA ligase 1 or T4 RNA ligase 2), or the 5' and 3' catalytic intron fragments in linear RNA are ligated into circRNA using the self-catalytic properties of group I or group II introns; RNase R treatment is used to remove unligated linear RNA and other linear RNA contaminants; in order to enhance the interaction between host proteins and circRNA, a QRE sequence is introduced into circRNA, and Monarch RNA Cleanup Kit is used for purification again; quality assessment: the quality and yield of circRNA are evaluated by agarose gel electrophoresis; subsequent detection and analysis are performed on the circularization effect, purification effect, safety and efficacy of the vaccine, such as: the circRNA vaccine prepared by S1 and verified by S2 is co-incubated with positively charged nano-polylysine to obtain a circRNA vaccine that can be used for mouse injection.

[0049] Furthermore, the circRNA vaccine can simultaneously express any two or three of the HBV antigens HBsAg, HBcAg and a therapeutic polypeptide, and the therapeutic polypeptide is human α-interferon.

[0050] Furthermore, the circRNA vaccine can express the corresponding RSV 3-mer F antigen and is used to treat hepatitis caused by HBV infection.

[0051] Furthermore, the polypeptide of the RSV trimer F antigen comprises a multimerization domain, and the multimerization domain is the C-terminal Foldon domain of T4 fiber protein that mediates the trimerization of T4 fiber protein.

[0052] Furthermore, a human tissue plasminogen activator (tPA) signal peptide is added to the front section of the ORF.

[0053] Furthermore, QRE sequences were used to enhance the interaction between host proteins and circRNAs.

[0054] Furthermore, the carrier of the circRNA vaccine is a nano hydrogel carrier and a nano polylysine carrier, and the dosage form is selected from a prefilled injection solution, an injection solution or a lyophilized powder injection.

[0055] The beneficial effects of the present invention are:

[0056] 1. Preventive vaccination of healthy people can eliminate or reduce the risk of HBV and RSV infection, alleviate (delay) clinical symptoms after possible infection, and reduce the probability of severe clinical symptoms and complications after possible infection.

[0057] 2. Therapeutic vaccination of infected people can reduce or eliminate one or more symptoms caused by viral infection, stabilize the condition of infected people, reduce the probability of virus transmission, reduce the use of existing drugs, shorten the clinical cure cycle, reverse or delay disease progression, improve prognosis and improve the quality of life of infected people. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 FIG. 1 is a schematic diagram of the structure of HBV circRNA encoding multiple antigens or polypeptides according to an embodiment of the present invention.

[0059] Figure 2 This is a schematic diagram of the structure of the RSV circRNA vaccine according to one embodiment of the present invention.

[0060] Figure 3 The figure is the agarose gel electrophoresis result of circRNA and its precursor linear RNA (Linear RNA) according to one embodiment of the present invention.

[0061] Figure 4 Dynamic light scattering (DLS) according to one embodiment of the present invention detects the particle size of the circRNA vaccine described in the present invention.

[0062] Figure 5 This is the humoral immune response of the circRNA vaccine of one embodiment of the present invention.

[0063] Figure 6 This is the cellular immune response of the circRNA vaccine of one embodiment of the present invention.

[0064] Figure 7 This is a safety verification of the circRNA vaccine of one embodiment of the present invention.

[0065] Figure 8 The mice injected with circRNAHBsAg-HBcAg according to one embodiment of the present invention effectively resist HBV pseudovirus infection. (Refer to therapeutic hepatitis B vaccine Figure 2 )

[0066] Fig. 9 The circRNA HBsAg-HBcAg-interferon of one embodiment of the present invention achieves functional cure of HBV. (Refer to therapeutic hepatitis B vaccine Figure 3 ) DETAILED DESCRIPTION

[0067] Example 1

[0068] S1: Construction of circRNA IVT plasmid template and in vitro preparation of circRNA

[0069] This example illustrates the preparation of an in vitro circRNA vaccine on a laboratory scale using the circRNA design and synthesis technology provided by the present invention. First, a linear DNA chain is synthesized, which contains a template sequence that can be used to produce linear RNA in vitro. The structure of linear RNA is as follows: Figure 1 and Figure 2 As shown. The linear RNA sequence includes: IRES sequence and m6A sequence used as translation start sequence; tPA sequence used as polypeptide exocytosis guide peptide; ORF region encoding HBV HBsAg, HBcAg and RSV 3-mer Fusion protein; RBS sequence and m6A sequence between two ORF regions as translation start signal; 2A peptide used as connecting peptide between two ORF regions; and Foldon sequence to promote protein folding. Among them, if the circRNA does not use the Foldon sequence, if the 2A peptide is used as the connecting sequence of the two ORFs, a stop codon (TAA or TAG or TGA) is added to the end of the last ORF; if the circRNA uses the Foldon sequence, a stop codon is added to the end of the Foldon sequence.

[0070] The linear DNA template of linear RNA can be prepared by cloning the required originals from different templates using the traditional PCR method, and then connecting them through overlap PCR to obtain the complete DNA template sequence of linear RNA. This template can also be obtained from commercial companies through gene synthesis. The IVT template can be prepared by PCR or by cloning the template into any plasmid vector and amplifying it through Escherichia coli. When using the PCR method to amplify the IVT template, it is necessary to add the T7 promoter sequence to the 5' end of the upstream primer; when using plasmids for IVT preparation, the template DNA sequence can be cloned into a plasmid vector containing the T7 promoter, and the multiple cloning site that does not exist in the template can be selected for connection. If the vector does not contain restriction sites such as EcoR V that can produce blunt ends, they can be added inside the selected multiple cloning site to facilitate the preparation of linearized IVT templates. After the plasmid is prepared, it is transformed into JM 109 competent cells (TaKaRa). The operation process is as follows: 1) After thawing JM109 competent cells on ice, gently mix the competent cells and plasmid DNA at a ratio of 10 ng plasmid DNA per 100 μL cells; 2) After ice bathing for 30 minutes, place at 42°C for 45 seconds, and then ice bath again for 2 minutes; 3) Add SOC or LB medium at a ratio of 1:9, and shake the bacteria at 37°C and 180 rpm for 60 minutes; 4) Take an appropriate amount of bacterial solution and spread it on the plate culture medium added with the corresponding antibiotics, and culture it at 37°C overnight.

[0071] Take a 12mL sterile shake tube (Biosharp), add 5mL of LB medium with the corresponding antibiotics added, then pick a single clone from the overnight culture plate, inoculate it in the shake tube, and culture it at 37°C and 180rpm for 16-24 hours. Then use MiniBEST Plasmid Purification Kit (TaKaRa) to extract the plasmid according to the steps described in the instructions. After Nanodrop quantification, use EcoR V and other endonucleases that can produce flat ends for enzyme digestion to achieve linearization of the IVT template. After enzyme digestion, perform agarose gel electrophoresis on the product to confirm linearization and purify it through agarose gel recovery.

[0072] Linear RNA was prepared using T7 RNA Polymerase (TaKaRa). After the reaction, Recombinant DNase I (RNase-free) (TaKaRa) was added and incubated at 37°C for 30 minutes to digest the DNA template. The DNA was then purified using Monarch RNA Cleanup Kit (NEB). A secondary purification was performed if necessary. If the prepared circRNA uses group I or group II introns for catalytic ligation, it is necessary to provide any of the above linear RNAs under conditions that activate the 5' catalytic group I or group II intron fragment and the 3' catalytic group I or group II intron fragment to provide a circRNA product, wherein the linear RNA comprises: a 3' catalytic group I or group II intron fragment located at the 5' end flank of the 3' exon sequence that can be recognized by group I or group II intron, and a 5' catalytic group I intron or group II fragment located at the 3' end flank of the 5' exon sequence that can be recognized by group I or group II intron, thereby obtaining a circRNA vaccine through autocatalysis, and using RNaseR treatment and then using Monarch RNA Cleanup Kit (NEB) to separate the prepared circRNA. The quality of the circRNA was evaluated by agarose gel electrophoresis.

[0073] S2: Gel electrophoresis and RNase R resistance identification of circRNA

[0074] The main purpose of this step is to detect the circularization effect, purification effect and resistance to linear RNA enzyme RNase R of the prepared circRNA. The three ORF regions of the circRNA used in this step encode 3×His (18 amino acids), 2×FLAG (16 amino acids) and 2×HA (18 amino acids) tags, respectively, to verify the feasibility of the circRNA preparation method of the present invention.

[0075] The circRNA prepared and purified as described in S1 was analyzed by agarose gel electrophoresis for circRNA and linear RNA. Figure 3 As shown in the figure, the T7 IVT product is single and has poor resistance to RNase R; the electrophoresis speed of the circularized circRNA is accelerated and has strong resistance to RNase R. Since the circRNA is head-to-tail connected and does not have a typical 5' or 3' end, the circRNA is resistant to RNase R. Therefore, the above agarose nucleic acid electrophoresis results confirm the circularization of the circRNA.

[0076] S3: Humoral immune response of mice to circRNA vaccine

[0077] The circRNA vaccine prepared by S1 and verified by S2 was co-incubated with positively charged nano-polylysine to obtain a circRNA vaccine for mouse injection. The specific operation method is as follows: dissolve nano-polylysine and circRNA in RNase-free water at a molar ratio of 1:2, mix well and react in the dark for 30 minutes, centrifuge at 8000g for 15 minutes at room temperature, discard the supernatant and wash twice with RNase-free water, detect by chromatography until there is no free circRNA, freeze-dry, and obtain a circRNA vaccine for injection. Dynamic light scattering determined that the particle size range of the prepared nucleic acid drug was 15-100nm, with an average particle size of 55nm ( Figure 4 ).

[0078] S3.1: Humoral immune response of mice after circRNA vaccine injection

[0079] 8-week-old C57BL / 6J mice were randomly divided into 6 groups and injected with placebo, 0.01mg / kg, 0.1mg / kg, 1mg / kg, 10mg / kg, and 20mg / kg of circRNA vaccine through the thigh muscle, with a total injection volume of 50μL. The second booster injection was injected on the 14th day after the injection, and blood was collected from the inferior vena cava on the 14th day after the second injection to separate the serum. Half of the whole blood obtained from the inferior vena cava was added to a centrifuge tube containing EDTA anticoagulant for blood cell analysis; the other half was not added with anticoagulant, left at room temperature for 30 minutes, and centrifuged at 4000rpm for 10 minutes at room temperature to obtain serum samples. First, the specific antibody IgG titers of HBV and RSV in the serum were detected by ELISA. As the injection dose increased, the titer of IgG in the serum increased significantly. The HBsAg and HBcAg specific IgG titers in the 20 mg / kg group exceeded 60,000 and 30,000, respectively, and the RSV fusion protein specific antibody IgG titer exceeded 80,000, while no specific IgG production was detected in the placebo group. The same serum was subjected to an in vitro pseudovirus neutralization test, and the results showed that the neutralization titers of the three circRNA vaccines exceeded 5000, of which the neutralization titer of IgG induced by circfusion exceeded 8000 ( Figure 5 B).

[0080] S3.2: Cellular immune response in mice after circRNA vaccine injection

[0081] Figure 6The specific T cell immune response of mice immunized with circRNA-HBsAg-HBcAg and circRNA-fusion vaccines is shown. In this step, flow cytometry (FACS) was used to analyze the percentage of positive cells for the corresponding cytokines in CD8+ and CD4+ T cells. The spleen of the experimental mice after blood was drawn from the inferior vena cava in S3.1 was separated, the spleen was crushed with tweezers, collagenase type III was added, 5 mL (400 U / ml) per spleen, digested at 37 degrees for 20 minutes, and filtered through a cell sieve to obtain a single cell suspension.

[0082] Take a sterile 15mL centrifuge tube, add lymphocyte separation solution, draw the single cell suspension just prepared and slowly add it to the centrifuge tube. The volume ratio of separation solution to cell suspension is 1:2. Be careful not to break the interface between lymphocyte separation solution and cell suspension. Centrifuge at 1500rpm for 20 minutes at room temperature. Discard the supernatant, draw the white blood cell layer, add CD4+ (Novozyme, CS102-01) or CD8+ (Novozyme, CS103-01) T cell purification magnetic beads according to the instructions, and separate CD4+ and CD8+ T cells by negative sorting. Then, IFN-γ and IL-2 positive cells in CD4+ and CD8+ T cells were sorted and counted by FACS, and IL-10 positive cells in both T cells were sorted and counted. Figure 6 AB shows the results of FACS sorting and counting of IFN-γ and IL-2 positive cells in CD4+T cells; Figure 6 CD shows the results of FACS sorting and counting of IFN-γ and IL-2 positive cells in CD8+T cells; Figure 6 E shows the results of sorting and counting IL-10 positive cells in two types of T cells. The data are shown as mean ± SEM, with three experimental mice in each group. The circRNA vaccine of the present invention can induce a significant increase in the count of cytokine IFN-γ and IL-2 positive cells after injection, but there is no significant effect on the induction of cytokine IL-10 positive cells, which proves its effective induction of corresponding memory T cells.

[0083] S4: Safety verification of circRNA vaccines

[0084] When blood was collected from the mice in S3.1, half of the whole blood collected from the inferior vena cava was added to a centrifuge tube containing EDTA anticoagulant for blood cell analysis; the other half was not added with anticoagulant, left at room temperature for 30 minutes, and centrifuged at 4000 rpm for 10 minutes at room temperature to obtain serum samples. The obtained blood was cleared for IgG detection as described in S3.1 and also for corresponding serum biochemical analysis. Figure 7A shows the results of blood cell analysis, where RBC is red blood cell count, HGB is hemoglobin, WBC is total white blood cell count, NEU is neutrophil count, LYM is lymphocyte count, MONO is monocyte count, EOS is eosinophil count, BASO is basophil count, and PLT is platelet count. It can be seen that the circRNA vaccine described in the present invention can effectively induce the generation of lymphocytes after vaccination. Combined with the test results described in S3.1 and S3.2, the increased lymphocytes are mainly effector T cells and effector B cells. In addition, although the total white blood cell count level of the vaccination group increased, it did not exceed 4-9×10 9 The normal reference range of white blood cells is 100 / mL, so the total white blood cell count can be considered to be at a normal level. Figure 7 B shows the results of serum biochemical analysis, where ALT is alanine aminotransferase, AST is aspartate aminotransferase, ALP is alkaline phosphatase, GLU is glucose, CREA is serum creatinine, TBIL is total bilirubin, DBIL is direct bilirubin, IBIL is indirect bilirubin, ALB is albumin, GLB is globulin, TP is total protein, BUN is blood urea nitrogen, HBDH is α-hydroxybutyrate dehydrogenase, and CK is creatine kinase. It can be seen that the various blood biochemical indicators after the circRNA vaccine of the present invention were not significantly increased compared with the placebo group, indicating that the injection of the circRNA vaccine had no substantial damage to the main organs of the whole body. The results of blood cell analysis and blood biochemical analysis jointly proved the safety of the circRNA vaccine of the present invention.

[0085] S5: Mice injected with the circRNAHBsAg-HBcAg vaccine of the present invention effectively resist HBV pseudovirus infection

[0086] Eight-week-old C57BL / 6J mice were randomly selected and divided into three groups. The WT group was not injected, and the other two groups were injected with a placebo and 20 mg / kg of circRNAHBsAg-HBcAg vaccine through the thigh muscle, respectively, with a total injection volume of 50 μL. The second booster injection was injected on the 14th day after the injection, and on the 28th day after the first injection, the mice were injected with 100 μL of 1×1010 vg / mL AAV-HBV 1.3 virus using PBS as a solvent through the tail vein. After 28 days of normal feeding after injection, blood was collected from the inferior vena cava for serum HBV DNA, HBsAg and HBcAg detection. The HBV DNA test was performed using the careHBV (Qiagen) kit, and all operations were performed according to the instructions. HBsAg and HBeAg were tested using the commercially available HBsAg and HBeAg ELISA kits from Shanghai Kehua Company. The results are as shown in Figure 8As shown in the figure, the levels of HBV DNA (A), antigen HBsAG (B) and antigen HBeAg (C) in the serum of mice injected with circRNAHBsAg-HBcAg vaccine were significantly decreased. circRNAHBsAg-HBcAg better protected the experimental mice exposed to HBV.

[0087] S6: Injection of the circRNAHBsAg-HBcAg-interferon of the present invention achieves functional cure of HBV in HBV model mice

[0088] 8-week-old C57BL / 6J mice were randomly divided into 3 groups, of which the WT group was used as a blank control group, the Placebo group and the Treatment group were injected with 1×1010vg / mL of AAV-HBV 1.3 virus, and 28 days later, the Placebo group was injected with a placebo through the thigh muscle, and the Treatment group was injected with 20mg / kg of circRNAHBsAg-HBcAg vaccine. After the first injection, the vaccine was injected again every 14 days according to the same operation, for a total of four injections. 3 months and 6 months after the last injection, the HBV DNA level, HBsAg, HBsAg IgG, ALT and AST levels in the serum were detected, and the liver tissue was taken to detect the level of HBV virus in stem cells. The detection methods of HBV DNA, HBsAg, HBsAg IgG, ALT and AST were the same as S4 and S5. The stem cell cccDNA was detected by real-time fluorescence quantitative PCR (RT-qPCR). After total DNA was extracted from mouse hepatocytes, the DNA was treated with Xma I (NEB) and Sac I endonucleases and purified for RT-qPCR detection. The upstream and downstream primer sequences were 5′-TGCACTTCGCTTCACCT-3′ and 5′-AGGGGCATTTGGTGGTC-3′, respectively.

[0089] like Fig. 9 As shown in the figure, after 4 injections of the vaccine, the C57 / BL 6J cells carrying HBV virus can continuously reduce the level of HBV DNA in the serum for up to 6 months (A); the serum antigen HBsAg is at a low level, and no serum HBV DNA is detected in one mouse in the treatment group (B); the level of specific antibody IgG to the antigen HBsAg is high, and no serum HBsAg is detected in one mouse in the treatment group (C); liver injury indicators ALT and AST continue to improve (DE); cccDNA levels in hepatocytes are at extremely low levels (F). In general, it has the ability to achieve functional cure for HBV.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and prove its effectiveness and safety, rather than to limit it. Although the above embodiments describe the present invention in detail, professionals in the field should understand that it can still be modified according to the technical solutions described in the above embodiments, or some or all of the technical features therein can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A circRNA vaccine expressing multiple antigens, characterized in that: All the sequences involved in the circRNA vaccine are as follows: IRES sequence SEQNO.1 ACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATA; Self-catalytic intron upstream sequence SEQNO.2 TACCTAGATATTTTCATTCCAGCGGTTTTTTTCCTAGTCTCACCTACACAGGTTCCAGTGAGCTCAGGAGTTTACCTTCATGTCCGTGGCACAGCCCATAATTACCCTTCTCTGTCCAGGGATGTATATTTGCATCTTTCCTTCAACTTATGAAGTGAATGGAGGGGAAATGTACAGTTTTAATTGAGTTTGAAGTAGCTTTCATCTGACCAGCCCTGTGCTGGAGCGTGCCTCTTCTTCTGTGAGCAGAGGGGCTACTGCGTGGGAAATGGCTAAACCGCCTGCTTTGTCTTTCACTGCAG; Self-catalytic intron downstream sequence SEQNO.3 GTAAGGGCAGCGGCGAGGTCCCTCATGTGGGCGACCCTCCTGCGGGAAGGGAGCTTGCACAGGTGTTGCCCTTGGTCCCTAACCTGTCAAGGTCAGTGGCCCTCTGCTGACCTTGTTGCCCTTACAGTATTAGTGGGAATGGGAGTTGTCAAGTTGCTGTTTACCAAGTTCAGAAGTAATCTCTATCTCCTTTGAAATAAATTGACAATGTACGAGATTCATTGAGATGACATTTGCCACACCTTTCATTCAAAAGATGTTTATTGAACATGTACCAGGTACTAGATTCTGGGGGA; HBV HBsAg sequence SEQNO.4 ATGGAGAACATCACATCAGGATTCCTAGGACCCCTGCTCGTGTTACAGGCGGGGTTTTTCTTGTTGACAAGAATCCTCACAATACCGCAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGGATCACCCGTGTGTCTTGGCCAAAATTCGCAGTCCCCAACCTCCAATCACTCACCAACCTCCTGTCCTCCAATTTGTCCTGGTTATCGCTGGATGTGTCTGCGGCGTTTTATCATATTCCTCTTCATCCTGCTGCTATGCCTCATCTTCTTATTGGTTCTTCTGGATTATCAAGGTATGTTGCCCGTTTGTCCTCTAATTCCAGGATCAACAACAACCAGTACGGGACCATGCAAAACCTGCACGACTCCTGCTCAAGGCAACTCTATGTTTCCCTCATGTTGCTGTACAAAACCTACGGATGGAAATTGCACCTGTATTCCCATCCCATCGTCCTGGGCTTTCGCAAAATACCTATGGGAGTGGGCCTCAGTCCGTTTCTCTTGGCTCAGTTTACTAGTGCCATTTGTTCAGTGGTTCGTAGGGCTTTCCCCCACTGTTTGGCTTTCAGCTATATGGATGATGTGGTATTGGGGGCCAAGTCTGTACAGCATCGTGAGTCCCTTTATACCGCTGTTACCAATTTTCTTTTGTCTCTGGGTATACATTTAA; HBcAg sequence SEQ NO.5 WRTSHQDS / DPCSCYRRGFSC / QESSQYRRV / TRGGLLSIF / GDHPCVLAKIRSPQPPITHQPPVLQFVLVIAGCVCGVLSYSSSSCCYASSSYWFFWIIKVCCPFVL / FQDQQQPVRDHAKPARLLLKATLCFPHVAVQNLRMEIAPVFPSHRPGLSQNTYGSGPQSVSLGSVY / CHLFSGS / GFPPLFGFQLYG / CGIGGQVCTAS / VPLYRCYQFSFVSGYTF; SEQ NO.6 ATGGACATCGATCCATACAAGGAATTTGGAGCTTCTGTGGAGCTGCTCAGCTTCCTGCCATCGGATTTTTTCCCTTCCATTCGAGACCTTCTCGACACCGCCTCTGCCCTGTATCGTGAGGCCCTGGAGTCGCCGGAACACTGCTCGCCACACCATACGGCACTCAGGCAGGCTATCCTGTGCTGGGGTGAGCTCATGAACCTGGCCACCTGGGTCGGTAGCAACTTGGAAGACCCCGCCTCGCGGGAGCTTGTAGTCAGTTACGTCAATGTTAACATGGGCCTAAAGATCAGACAACTCTTGTGGTTCCACATTTCCTGTCTTACGTTCGGAAGAGAGACCGTTCTTGAGTACCTCGTTTCCTTCGGCGTGTGGATTCGCACTCCACCAGCATATCGACCACCTAACGCGCCAATCCTGTCTACACTTCCCGAAACCACTGTTGTCAGACGGAGAGGCAGATCGCCTAGACGGAGAACACCCTCGCCTCGCCGTCGAAGATCCCAGTCGCCGCGTCGCAGACGTTCTCAATCTCGGGAGTCTCAGTGCTAA; SEQ NO.7 WTSIHTRNLELLWSCSASCHRIFSLPFETFSTPPLPCIVRPWSRRNTARHTIRHSGRLSCAGVSS / TWPPGSVATWKTPPRGSL / SVTSMLTWA / RSDNSCGSTFPVLRSEERPFLSTSFPSACGFALHQHIDHLTRQSCLHFPKPLLSDGEADRLDGEHPRLAVEDPSRRVADVLNLGSLSA; RSV antigen is the 3-mer Fusion protein nucleic acid sequence SEQ NO.8; RSV antigen is the amino acid sequence of the 3-mer Fusion protein SEQ NO.9; Therapeutic polypeptide nucleic acid sequence SEQNO.10 ; Therapeutic polypeptide protein sequence SEQNO.11 MALTFALLVALLVLSCKSSCSVGCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSSFSLSTNLQESLRSKE; Human Tissueplasminogenactivator (tPA) signal peptide nucleic acid sequence SEQ NO.12 GATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCAGCCAGGAAATCCATGCCCGATTCAGAAGA; Human Tissueplasminogenactivator (tPA) signal peptide amino acid sequence SEQ NO.13 DAMKRGLCCVLLLCGAVFVSPSQEIHARFRR; QRE upstream sequence SEQNO.14 TCTAGGTAGCCATGTGTGACTAGTGGCCACCATAATGGGCTTCACGGTACTAGGTGACTTACTTGACTTCTTTGTGTTCCAGTTTTCCCATCTGTACAGATGAGATCATATAAGTTACCTTTTGTGGTTACTATATGAATTAAATAAAT AATATAGTTAAGAAGGAGTTTAACACTATGCTGGAGCAGAATATTTCCATGAGAAATGGTAGTTAATTATAATGACATACATTTAATAAACAAGTTATTTTGCTTTTGAGCATTAATTTAAACCTGTTTTATTTTTGTTTCTTCCCTTCCAG; QRE downstream sequence SEQNO.15 GTAATTTTATTTTTGTAAAATTCTATTTTAAAAAATAAGCCATTTTGTATTATGGTTTATGATTTATAGGAGTTGTAACTTTGTGTACAGTATGTTTGAATGTGTATACACAGTGTAGGTATCTTCCTTACTTAGATGTGAAAACACAAAGTAGTAGTAATCTTTTTTT CATTCCGAGTCCTTACCTCAACCTACAGTAGCTCTGGAAACTAACTGTTCTCTGTTAGTCATTGTCTGTACTTAAAGTCAAGCGCTGGTGGGCAGGAAACTCAGGAGTAATATTTAATCTATTTCAGATAGAGTGTTCTGTCTTCTGGCATATAAAGTTTGATGGTTA.

2. A method for preparing a circRNA vaccine expressing multiple antigens, characterized in that: The following steps are involved: Step 1: Preparation of linear DNA template: construct a DNA template containing a linear RNA sequence for subsequent in vitro transcription, clone the required elements (such as IRES sequence, m6A sequence, tPA sequence, protein-encoding ORF region, etc.) from different templates by PCR, and connect them using overlap PCR to form a complete linear RNA DNA template; Step 2 Linearization of IVT (in vitro transcription) template: Prepare linearized DNA template for in vitro transcription, add T7 promoter sequence to the 5' end of the upstream primer, and obtain IVT template by PCR amplification; clone the IVT template DNA sequence into a plasmid vector containing T7 promoter, amplify the plasmid by E. coli, and select appropriate restriction sites for linearization; transform the plasmid into JM 109 competent cells, obtain high-concentration plasmid DNA by shaking and plasmid extraction; use EcoR V and other endonucleases for restriction digestion, confirm linearization by agarose gel electrophoresis and purify; Step 3: Synthesis and purification of linear RNA: Synthesize linear RNA by in vitro transcription and purify it. Use T7 RNA Polymerase for in vitro transcription to generate linear RNA. Add Recombinant DNase I to digest the DNA template. Use Monarch RNA Cleanup Kit for purification and secondary purification. Step 4: Catalytic ligation and purification of circRNA: Linear RNA is catalytically ligated into circRNA and purified, and linear RNA is circularized into circRNA using RNA ligase (such as T4 RNA ligase 1 or T4 RNA ligase 2), or the 5' and 3' catalytic intron fragments in linear RNA are ligated into circRNA using the autocatalytic properties of group I or group II introns; RNase R treatment is used to remove unligated linear RNA and other linear RNA contaminants; in order to enhance the interaction between host proteins and circRNA, a QRE sequence is introduced into circRNA, and Monarch RNA Cleanup Kit is used for purification again; quality assessment: the quality and yield of circRNA are evaluated by agarose gel electrophoresis; subsequent detection and analysis are performed on the circularization effect, purification effect, safety and efficacy of the vaccine, such as: the circRNA vaccine prepared by S1 and verified by S2 is co-incubated with positively charged nano-polylysine to obtain a circRNA vaccine that can be used for mouse injection.

3. The method for preparing a circRNA vaccine expressing multiple antigens according to claim 2, characterized in that: The circRNA vaccine can simultaneously express any two or three of the HBV antigens HBsAg, HBcAg and a therapeutic polypeptide, wherein the therapeutic polypeptide is human α-interferon.

4. The method for preparing a circRNA vaccine expressing multiple antigens according to claim 2, characterized in that: The circRNA vaccine can express the corresponding RSV 3-mer F antigen and is used to treat hepatitis caused by HBV infection.

5. The method for preparing a circRNA vaccine expressing multiple antigens according to claim 2, characterized in that: The polypeptide of the RSV trimer F antigen comprises a multimerization domain, and the multimerization domain is the C-terminal Foldon domain of the T4 fiber protein that mediates the trimerization of the T4 fiber protein.

6. The method for preparing a circRNA vaccine expressing multiple antigens according to claim 2, characterized in that: A human tissue plasminogen activator (tPA) signal peptide is added to the front section of the ORF.

7. The method for preparing a circRNA vaccine expressing multiple antigens according to claim 2, characterized in that: QRE sequences were used to enhance the interaction between host proteins and circRNAs.

8. The method for preparing a circRNA vaccine expressing multiple antigens according to claim 2, characterized in that: The carriers of the circRNA vaccine are nano hydrogel carriers and nano polylysine carriers, and the dosage form is selected from prefilled injection solution, injection solution or lyophilized powder injection.

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