Rabies virus circular RNA vaccine as well as preparation and application thereof

Abstract

The invention relates to a rabies virus vaccine, in particular to a rabies virus circular RNA (Ribonucleic Acid) vaccine as well as preparation and application thereof. The rabies virus CircRNA vaccine comprises CircRNA and a vaccine vector, wherein the CircRNA is used for expressing optimized rabies virus immunogen. The rabies virus CircRNA vaccine is prepared, and the stability and the protein expression efficiency of the optimized sequence are remarkably improved at the same time. The vaccine can rapidly induce high-level anti-rabies virus immune response, and has no obvious toxic or side effect. According to the rabies inactivated vaccine, the immunizing frequency of the existing rabies inactivated vaccine can be reduced, the problems of virus pollution and the like are not involved, and the rabies inactivated vaccine is more economical, safer and more efficient.

Description

Technical Field

[0001] The present invention relates to rabies virus vaccine, in particular to rabies virus circular RNA vaccine and its preparation and application Background Art

[0002] Rabies is a highly lethal zoonotic infectious disease caused by the rabies virus (RABV), which has been found in more than 150 countries and regions around the world. RABV is neurotropic. When humans are scratched or bitten by infected animals, the virus gradually invades the nervous system, causing severe nervous system inflammation such as encephalitis and myelitis, with a mortality rate of nearly 100%. Wild animals are the main hosts of RABV, and the infection of humans and domestic animals is mainly caused by contact with wild animals carrying rabies virus.

[0003] There is currently no specific drug to treat rabies, and it can only be prevented and controlled with the help of vaccines. Vaccinating dogs with rabies vaccines is the most effective measure to control rabies. The veterinary rabies vaccines currently approved for marketing are mainly inactivated vaccines, which still have the following shortcomings: First, the preparation process of inactivated vaccines is complicated, involving a large number of cell amplification and virus amplification, and there are certain safety risks; second, the immune efficacy of such vaccines still needs to be improved. The amplification of rabies virus has problems such as low virus titer and low effective antigen amount, which makes the inactivated vaccine have the risk of immune failure. The current pet rabies vaccine immunization process in my country is: puppies are first immunized after 3 months of age, and then they need to be boosted once a year. However, even if pets are vaccinated with rabies vaccines on time, animals still have the risk of unqualified immunization and carrying rabies virus. The veterinary rabies vaccines currently approved for marketing are mainly inactivated vaccines. However, there are many technical difficulties and safety risks in the production process of inactivated vaccines, including complex preparation processes, low virus titers, and low effective antigen amounts. More importantly, the immunogenicity of inactivated vaccines is relatively low, and there is a risk of immune failure, requiring animals to be vaccinated once a year. This complicated immunization procedure has invisibly restricted the promotion of veterinary rabies vaccines. Therefore, a new economical, safe, and efficient veterinary rabies vaccine is urgently needed to be developed.

[0004] The rabies virus G protein is its main antigen, which can effectively stimulate the proliferation of specific helper T cells (Th) and cytotoxic T cells (CTL), and induce the body to produce specific antibodies. The G protein of Rabies lyssavirus is a type I glycoprotein composed of 505 amino acids. Its full-length amino acid sequence can be found in (genbank: ACR39382.1. Monoclonal antibodies and vaccines targeting G protein can provide effective protection for the host and are widely used in vaccine research and development.

[0005] mRNA vaccines are an emerging type of vaccine that are superior to inactivated vaccines in inducing long-term neutralizing antibodies and cellular immunity. Companies including Curevac, SMIC and Livanda have laid out rabies mRNA vaccine pipelines. However, mRNA vaccines still have problems such as poor stability and excessive immunogenicity. Replacing normal uracil with pseudouracil during transcription can reduce the activation of TLR7 / TLR8, reduce intracellular inflammatory response, and improve the stability and immune effect of mRNA vaccines. However, the use of modified nucleotides increases the cost of vaccines. The latest study in Nature shows that the incorporation of N1-methylpseudouracil significantly increases the +1 ribosomal frameshift during translation of mRNA, and this frameshift mainly occurs between two characteristic sliding sequences. The mutant protein produced by the +1 ribosomal frameshift can induce unnecessary T cell immune responses in mice and humans. If the off-target unintended protein has biological activity, mRNA vaccines containing modified nucleotides may pose a potential threat to the health of subjects. The above-mentioned defects of linear mRNA are further magnified in the field of veterinary vaccines. Therefore, how to avoid ribosomal frameshifting and off-target unexpected protein production caused by modified nucleotides while ensuring vaccine stability and immunogenicity is a challenge that needs to be urgently addressed in the field of veterinary rabies vaccines. Summary of the invention

[0006] Purpose of the Invention

[0007] In order to overcome the deficiencies of the prior art, the present invention provides a stable and safe veterinary rabies virus CircRNA vaccine that does not require modified nucleotides and a preparation method thereof, so as to achieve the purpose of lifelong protection for companion animals including dogs with a single immunization.

[0008] Technical Solution

[0009] To achieve the above objectives, the present invention discloses a rabies virus circRNA vaccine, characterized in that the vaccine comprises: (1) a circRNA for expressing an optimized rabies virus immunogen, wherein the immunogen is a G protein; and (2) a vaccine vector, wherein the vaccine vector is a lipid nanoparticle (LNP) that can be utilized by the circRNA.

[0010] In some embodiments, the G protein is the entire protein, or the extramembrane portion of the G protein, or a G protein multimer;

[0011] In some embodiments, the rabies virus is selected from the following group: PV strain, Flury strain, SAD strain, aG strain, CTN strain, and their derivatives or multiple combinations thereof.

[0012] In some embodiments, the rabies virus is the CTN-1 strain.

[0013] In some embodiments, the optimized nucleotide sequence is such as SEQ ID NO: 1 or 2.

[0014] Preferably, the circRNA sequence has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence shown in SEQ ID NO: 1 or 2, or at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutated mRNA sequence; the mutation is selected from insertion, deletion and / or substitution, and the substitution is preferably a codon substitution of a synonymous amino acid.

[0015] In some embodiments, such as Figure 1 As shown, the circRNA precursor nucleotide sequence has the following structure: A1-A2-A3-A4-A5-A6: A1 is a homology arm; A2 is a 3'intron element; A3 is an E2 sequence, preferably a human immunoglobulin κ light chain variable region signal peptide coding sequence; A4 is a spacer element; A5 is an IRES; A6 is a rabies virus antigen coding sequence, wherein the rabies virus antigen selects G protein, the extramembrane part of G protein, or the entire G protein; A7 is a spacer element; A8 is an E2 sequence; A9 is a 5'intron element; and A10 is a homology arm.

[0016] After the circRNA precursor sequence is sheared (A7-A10) and cyclized, the final circRNA structure is A1-A6.

[0017] The circRNA nucleotide sequence is SEQ ID NO: 5 or 6.

[0018] In some embodiments, the lipid nanoparticles include polyethylene glycol lipids, neutral lipids, cationic lipids, and cholesterol or cholesterol analogs.

[0019] In some embodiments, the cationic lipids include but are not limited to Dlin-MC3-DMA, DODMA, DODAP, ALC-0315 or SM102.

[0020] In some embodiments, the average particle size of the lipid nanoparticles is in the range of 10-500 nm. Preferably, the average particle size of the lipid nanoparticles is in the range of 100-150 nm.

[0021] In some embodiments, the mass ratio of lipid to optimized mRNA is 10-30: 1. Preferably, the mass ratio of lipid to optimized mRNA is 20:1.

[0022] The second aspect of the present invention provides a plurality of isolated nucleic acid fragments encoding any one of the above-mentioned G proteins, or encoding any one of the above-mentioned proteins.

[0023] The third aspect of the present invention provides a variety of vectors, which contain the above-mentioned nucleic acid fragments, preferably, lipid nanoparticles that can be used by CircRNA.

[0024] The fourth aspect of the present invention provides a method for preparing the above-mentioned rabies virus CircRNA vaccine, which comprises introducing the nucleic acid fragment into the body of the administration subject.

[0025] The fifth aspect of the present invention provides a medicine comprising any one of the above-mentioned CircRNA vaccines.

[0026] The sixth aspect of the present invention provides any one of the above-mentioned CircRNA vaccines, which can induce a specific immune response against rabies virus in the body of a subject to which it is administered.

[0027] Application of the rabies virus circRNA vaccine in the preparation of drugs for preventing or treating rabies

[0028] The seventh aspect of the present invention provides any of the above-mentioned circRNA vaccines, the product obtained by the method, or the use of the drug for preparing a drug for preventing rabies virus infection in an individual in need thereof; optionally, the virus is rabies virus.

[0029] The eighth aspect of the present invention provides a method for preventing viral infection in an individual in need thereof, comprising administering to the individual any of the above-mentioned CircRNA vaccines, or the product obtained by the method; optionally, the virus is selected as rabies virus.

[0030] Key points of the present invention

[0031] 1. Expressing rabies virus optimized immunogen CircRNA, wherein the immunogen is G protein;

[0032] 2. Vaccine vector, which is a lipid nanoparticle that can be utilized by CircRNA.

[0033] Beneficial Effects

[0034] Circular RNA (circRNA) is a type of circular closed single-stranded RNA molecule. When it has an internal ribosome binding site (IRES), it can translate proteins in a cap-independent manner. Compared with traditional linear mRNA, the circular structure of circRNA brings unique advantages: first, the complete circular structure of circRNA can protect it from degradation by nuclease exonucleases, significantly improve its stability, and achieve room temperature storage and repeated freeze-thaw storage. Secondly, the cytotoxicity and side effects of linear mRNA are partly attributed to its high immunogenicity. Although the incorporation of modified nucleotides can reduce the activation of intracellular TLRs and thus reduce immunogenicity, the ribosomal frameshift caused by modified nucleotides brings more uncertain risks. CircRNA still shows low immunogenicity in the absence of nucleotide modification, and its safety is higher. Finally, circRNA can mediate higher and more persistent antigen expression, prolong the antigen presentation process and germinal center reaction, thereby enhancing the acquired immune response and producing more neutralizing antibodies. In summary, circRNA has better safety, stability, and manufacturing simplicity, and has become a new direction that has attracted much attention in the field of vaccines.

[0035] The development of circRNA vaccines includes multiple steps, such as antigen selection, antigen sequence design and optimization, and delivery system selection. How to optimize the circRNA sequence and achieve in vivo delivery are two major technical barriers. Among them, the ideal circRNA sequence optimization scheme must achieve the following two goals at the same time: (1) improve the stability of the circRNA secondary structure to delay immune failure caused by circRNA degradation; (2) improve the translation efficiency of circRNA so that it can produce a large amount of target antigen protein.

[0036] The present invention designs an optimized circRNA vaccine expressing rabies G protein. Under the same conditions, the preferred sequence circRNA of the present invention can mediate the expression of G protein in vitro, and its duration is significantly longer than that of linear mRNA. In a mouse model, the preferred circRNA vaccine of the present invention can quickly induce a high level of immune response against rabies virus G protein in mice, and the antibody level is significantly higher than that of the inactivated vaccine and the unoptimized circRNA vaccine that have been marketed. Compared with the linear mRNA vaccine with the same sequence, the preferred circRNA vaccine can induce a stronger specific T cell response in mice without obvious weight changes. The attack protection test shows that a single immunization of 2μg of the preferred circRNA vaccine can completely protect mice against 100LD50 rabies virus attack infection, and its protective effect is comparable to that of 5μg of mRNA vaccine containing modified nucleotides, and is significantly better than the protective effect of immunization with commercially available inactivated vaccines.

[0037] The present invention prepares a rabies virus CircRNA vaccine, and the stability and protein expression efficiency of the optimized sequence are significantly improved at the same time. The vaccine can quickly induce a high level of anti-rabies virus immune response without obvious toxic side effects. The present invention can reduce the number of immunization doses of existing rabies inactivated vaccines, and does not involve problems such as virus contamination, and is a more economical, safe and efficient rabies vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 , schematic diagram of the structure of circRNA precursor; A1-A2-A3-A4-A5-A6: A1 is the homology arm; A2 is the 3'intron element; A3 is the E2 sequence, preferably the signal peptide coding sequence of the variable region of human immunoglobulin κ light chain; A4 is the spacer element; A5 is IRES; A6 is the rabies virus antigen coding sequence, wherein the rabies virus antigen selects the G protein, the extramembrane part of the G protein, or the entire G protein; A7 is the spacer element; A8 is the E2 sequence; A9 is the 5'intron element; A10 is the homology arm.

[0039] Figure 2 , RNA-mediated in vitro expression levels, i.e., FCM detection results of HEK293T cells transfected with circRNA or mRNA for 24 and 72 hours, respectively, where (A) is the expression of G protein in 293T cells 24 hours after transfection, and (B) is the expression of G protein 72 hours after transfection;

[0040] Figure 3 , Rabies G protein-specific antibody response after immunization of mice with circRNA vaccines of different sequences. Compared with the PBS group, *: P < 0.05, **: P < 0.01, ***: P < 0.001. Compared with the inactivated vaccine, #: P < 0.05, ##: P < 0.01, ###: P < 0.001. There was no significant difference in antibody titer at each time point between the circRNA-9 group and the inactivated vaccine, which is not marked;

[0041] Figure 4 、Rabies G protein-specific T cell response after circRNA-10 immunization of mice. (A) Representative ELIspot detection graph; (B) Statistical graph;

[0042] Figure 5 , the challenge and protection survival rate of mice immunized with rabies circRNA; 14 days after the mice were immunized, 100×IMLD50 RABV street virus strain HuNPB3 was injected intramuscularly, and the mortality rate of mice within 14 days of the challenge was observed and recorded. DETAILED DESCRIPTION

[0043] The present invention will be further described below in conjunction with specific examples, and the advantages and features of the present invention will become clearer as the description proceeds. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0044] The embodiments of the present invention are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0045] Example 1 Synthesis of mRNA

[0046] The present invention provides an mRNA designed based on the amino acid sequence optimization of the G protein of lyssavirus, and the specific process is as follows:

[0047] 1.1G protein nucleotide sequence optimization

[0048] The amino acid sequence of Lyssavirus G protein (see GenBank: ACR39382.1), the nucleotide sequence of G protein (see GenBank: FJ959397.1).

[0049] The present invention adopts conventional molecular biological methods in the art to construct relevant plasmids. Plasmids with corresponding G protein encoding nucleotide sequences (SEQ ID NO: 1-2) were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. The corresponding nucleotide sequences are shown in Table 1, and r1-r2 are the insertion sequence numbers.

[0050] Table 1. Codon-optimized nucleotide sequence of antigen G protein

[0051]

[0052]

[0053]

[0054] 1.2 CircRNA preparation and protein expression identification

[0055] The nucleotide fragment encoding the G protein in the recombinant plasmid was amplified by polymerase chain reaction (PCR). First, the linearized DNA template encoding the G protein was amplified using PCR reagent (ThermoScientific, Cat#F531L) and primers. Transcription was performed in vitro according to the instructions of the HiScribe T7 High Yield RNA Synthesis Kit (NEB, Cat#E2040S). After in vitro transcription, DNA enzyme I (Vazyme, Cat#EN401-01-AA) was used to remove the DNA template to obtain a high-purity linear RNA precursor. The linear RNA was circularized using the intron-exon replacement system (Permuted introns and exons, PIE). The uncircularized linear RNA was removed using the exonuclease activity of RNase R (Novoprotein, Cat#E224). According to the instructions of Monarch RNA Purification Kit (NEB, Cat# T2050L), impurities such as proteins and salts in RNA were removed, and finally antigen CircRNA containing basic elements that can be stably translated in vivo was obtained. Its structure is as follows Figure 1 As shown, the DNA template sequence is shown in Table 2, the corresponding circular RNA nucleotide sequence is shown in Table 3, SEQ ID NO.5 is the CircRNA obtained after cyclization and purification, namely CircRNA-9, SEQ ID NO.6 is the CircRNA obtained after cyclization and purification, namely CircRNA-10, wherein the U in the mRNA number indicates that UTP is regular UTP, and Ψ indicates that UTP is replaced by modified nucleotides.

[0056] Table 2. Nucleotide sequences of DNA templates

[0057]

[0058]

[0059]

[0060]

[0061]

[0062] Table 3. Nucleotide sequences of circular RNA

[0063]

[0064]

[0065]

[0066]

[0067] Example 2 Preparation and detection of Lyssavirus circRNA vaccine

[0068] LNP-CircRNA particles were prepared using a microfluidic system. The CircRNA solution was replaced with a citric acid buffer solution to adjust the final concentration of CircRNA to 0.05 mg / mL. According to the molar ratio of cationic lipid / auxiliary phospholipid / cholesterol / polyethylene glycol lipid = 50 / 10 / 38.5 / 1.5, appropriate amounts of lipids were taken and dissolved in anhydrous ethanol to prepare a mixed solution with a final concentration of 2.85 mg / mL. 2 mL of CircRNA citric acid buffer and 0.67 mL of lipid mixture were drawn with a syringe and injected into the microfluidic chip. According to the operating instructions of the microfluidic instrument (Suzhou Wenhao Microfluidic Technology Co., Ltd.), the LNP-CircRNA mixture solution was prepared and collected. The obtained LNP-CircRNA mixture solution was ultrafiltered and concentrated, and the concentrated sample was collected to obtain the Lyssa rabies virus CircRNA vaccine.

[0069] Flow cytometry (FCM) was used to identify the G protein expression ability of LNP-circRNA. HEK293T cells with good morphology (purchased from the National Model and Specialty Experimental Cell Resource Bank, Shanghai, China) were taken and cultured at 2x10 5Cells / well were inoculated in a 12-well plate and cultured in a constant temperature incubator at 37°C and 5% CO2. When the cells covered 70%-80% of the surface of the bottom plate, 1 μg of CircRNA or mRNA / well was added to the plate wells and cultured for 24 or 72 hours. On the day of the FCM experiment, the cells were rinsed with PBS buffer (VivaCell, Cat#C3580-0500), 500 μL of trypsin (New Cyme, Cat#C100C1) was added for digestion, and 1 mL of DMEM complete medium supplemented with 10% fetal bovine serum (DMEM, VivaCell, Cat#C3110-0500; fetal bovine serum, VivaCell, Cat#C04001-500) was used to terminate the reaction. The cell turbidity was transferred to a 1.5 mL EP tube by blowing and centrifuging at 300×g, 4°C for 5 minutes, and the supernatant was discarded and washed twice with PBS buffer. Add 100 μL of 1:200 diluted anti-rabies virus monoclonal antibody (Santa Cruz, Cat# sc-57995) to the washed cell sample, resuspend and incubate on ice for 30 minutes. After incubation, wash the cells twice with 1xPBS buffer by centrifugation, and the centrifugation parameters are set to 300×g, 4°C, and 5 minutes. Add 100 μL of 1:200 diluted AlexaFlour633 goat anti-mouse IgG antibody (FisherScientific, A21052) to the washed cell sample, resuspend and incubate on ice in the dark for 30 minutes. After incubation, wash the cell sample twice with 1 mL PBS buffer by centrifugation, and the centrifugation parameters are set to 300×g, 4°C, and 5 minutes. Resuspend the cell sample, filter and use a flow cytometer (Thermofisher, model AttuneNxT) for protein expression detection. The detection results are shown in Figure 2 .

[0070] like Figure 2 As shown in the figure, both circRNA and mRNA can achieve the level expression of rabies G protein in vitro. 24 hours after transfection, the expression levels of rabies G protein mediated by circRNA-9, circRNA-10 and the corresponding linear mRNA-9 and mRNA-10 were significantly equivalent. 72 hours after transfection, the expression level of G protein in each transfection group decreased significantly, among which the decrease in the circRNA-9 and circRNA-10 transfection groups was significantly lower than that in the linear mRNA group, and there was no significant difference between the circRNA-9 and circRNA-10 groups. The above results indicate that the stability of circular RNA increases and the expression time of exogenous protein is prolonged.

[0071] Example 3 In vivo experiments in mice demonstrate the ability of circRNA vaccines to induce antibody production

[0072] In order to evaluate the ability of the above-mentioned different CircRNA vaccines to induce antibodies against G protein, the in vivo efficacy experiment in mice was used for identification. BALB / c female mice aged 6 to 8 weeks were grouped, with 8 mice in each group. The specific grouping scheme is shown in Table 4. At the same time, a PBS group and an inactivated vaccine (Rebelan) group were set up, with 8 mice in each group. On day 0, each CircRNA vaccine group was intramuscularly injected with 2μg LNP-CircRNA, and blood was collected on days 14, 28, 42, and 56 after immunization. The collected blood samples were allowed to stand at room temperature for 2h, and then centrifuged at 3000rpm and 4℃ for 10min to separate the serum and carry out the detection of rabies G protein-specific binding antibodies and neutralizing antibodies. The weight of mice was monitored regularly during the immunization period.

[0073] Table 4. Mouse immunization schedule

[0074]

[0075] Enzyme-linked immunosorbent assay (ELISA) was used to measure the antibody levels targeting G protein in each group of serum samples. 10x ELISA coating solution (New Saimei, Cat#E30500) was diluted to 1x coating working solution, and the antigen protein CTN-1-G (purchased from Nanjing Detai Bioengineering Co., Ltd.) was diluted to a final concentration of 1 μg / mL with the coating working solution to obtain a coating antigen solution. The coating antigen solution was added to a 96-well EIA / RIA transparent flat-bottom polystyrene microplate (Corning, Cat#3590), 100 μL per well, and placed at 4°C overnight. The next day, discard the coating solution, wash the plate 4 times with 200 μL ELISA cleaning solution (New Saimei, Cat#E21000), pat dry and add 100 μL of antigen blocking solution containing 3% bovine serum albumin (Solebo, Cat#A8020), and place it at 37°C for blocking for 2 hours. Discard the blocking solution, wash 3 times with 200 μL ELISA cleaning solution and pat dry, dilute the serum sample 1:100 or 1:10000 with PBS buffer containing 1% bovine serum albumin (i.e., antibody diluent), take 100 μL and add it to the above plate. Then move the plate into a 37°C incubator and incubate for 2 hours. Discard the serum sample, wash the plate 5 times with ELISA cleaning solution, pat dry, add 100 μL of 1:10000 diluted Goat Anti-Mouse IgG (H+L)-HRP (Southern Biotech, Cat#1036-05), and incubate at 37°C for 1 hour. Discard the secondary antibody, wash the plate 5 times with 200 μL ELISA cleaning solution, pat dry, add 100 μL TMB, react at room temperature in the dark for 15 minutes, terminate with 50 μL ELISA stop solution (New Saimei, Cat#E40500), and then read the absorbance value at 450 nm using a microplate reader (TECAN, model MultiskanMk3). The results are shown in Figure 3 .

[0076] like Figure 3 As shown in the figure, antibodies targeting G protein were detected in the serum of each experimental group, and the IgG antibody levels induced by each circRNA vaccine group were significantly higher than those of the PBS group 2 weeks after immunization. The binding antibody levels of mice in the circRNA-10 immunization group were significantly better than those of the inactivated vaccine at 14, 28, 42, and 56 days after immunization (P<0.001), while the antibody levels of the circRNA-9 immunization group and the inactivated vaccine group were comparable, with no significant difference. In summary, circRNA vaccines can mediate rabies-specific antibody responses in mice, among which the preferred sequence circRNA-10 can induce a stable and high level of humoral immune response, which is significantly better than the inactivated vaccine that has been marketed.

[0077] Example 4 In vivo experiments in mice demonstrate the ability of circRNA vaccines to induce the production of specific T cells

[0078] In order to evaluate the ability of the above-mentioned preferred CircRNA vaccine to induce specific T cells, it was identified by in vivo efficacy experiments in mice and compared with the corresponding linear mRNA vaccines. BALB / c female mice aged 6 to 8 weeks were grouped, with 6 mice in each group. The specific grouping scheme is shown in Table 5. At the same time, a PBS group and an inactivated vaccine (Rebelan) group were set up, with 6 mice in each group. On day 0, immunization was performed according to the dose shown in Table 5. 14 days after immunization, the mice were euthanized, the spleens were collected, and ELISpot detection was performed. Mouse spleen immune cells were isolated, and the cell concentration was adjusted to 2×10 6 / mL. According to the instructions of the reagent manufacturer, add 200μL PBS to each well of the pre-coated 96-well plate of the IFN-γ ELISpot kit, wash the plate 4 times, then discard the PBS, add 200μL immune cell culture medium to each well, and block at 37℃ for 30min~1h. Discard the blocking solution, add 200μL diluted spleen immune cell suspension to each well, add 1μg CTN-1-G protein to the antigen stimulation well to make the final concentration 5μg / mL, and set non-stimulation wells and PMA / Ionomycin mixture stimulation wells for each sample as negative and positive controls respectively. Then place the above ELISpot 96-well plate in a 37℃ 5% CO2 cell culture incubator for 20h. Take out the ELISpot plate, discard the liquid in the plate, wash each well with 200μL PBS 5 times, and pat dry the liquid. Add 100μL diluted R4-6A2-biotin primary antibody working solution to each well and incubate in a 37℃ biochemical incubator for 2h. Take out the ELISpot plate, discard the liquid in the plate, wash each well with 200μL PBS 5 times, pat dry the liquid, add 100μL diluted Streptavidin-ALP working solution to each well, and incubate at 37℃ for 1h. After the incubation, discard the liquid in the plate, wash each well with 200μL PBS 5 times, and pat dry the liquid. Add 100μL of substrate filtered through a 0.45μm filter membrane to each well, incubate at room temperature away from light, and observe the degree of color development every 3 to 5 minutes. After the color development is completed, pour out the liquid in the well, uncover the base of the plate, rinse the front and back of the 96-well plate and the base with pure water 3 to 5 times to stop the color development. Place the 96-well plate in a cool place at room temperature, wait for it to dry naturally, then close the base, and observe and count using an enzyme-linked immunosorbent spot analyzer. See the results. Figure 4 .

[0079] Table 5. Immunization scheme for mice in each group

[0080]

[0081] like Figure 4As shown in the figure, 14 days after immunization, specific T cells secreting IFN-γ were detected in the spleens of mice in each experimental group after stimulation with rabies G protein, indicating that the vaccine immunization mediated a rabies G protein-specific T cell response in mice. The number of T cells produced in the spleens of mice immunized with CircRNA-10 was higher than that in the mRNA-10 and inactivated vaccine groups, indicating that the preferred sequence CircRNA-10 can induce a high level of cellular immune response, which is significantly better than the inactivated vaccine that has been marketed.

[0082] Example 5 Mouse challenge protection test

[0083] To evaluate the protective effect of circRNA vaccines and compare them with linear mRNA containing modified nucleotides, a rabies virus attack infection experiment was conducted. 6-8 week old BALB / c female mice were grouped, with 5 mice in each group. The specific grouping scheme is shown in Table 6. At the same time, a PBS group and an inactivated vaccine (Rebelan) group were set up, with 5 mice in each group. On day 0, immunization was performed according to the dose shown in Table 5. 14 days after immunization, mice were attacked and infected in an ABSL-3 animal biosafety laboratory with intramuscular injection of 100×IMLD50 RABV street virus strain HuNPB3. The mortality rate of mice within 14 days of the attack was observed and recorded. The results are shown in Figure 5 .

[0084] Table 6 Immunization scheme for mice in each group

[0085]

[0086] like Figure 5 As shown, a single immunization of 2 μg of the preferred sequence CircRNA-10 can completely protect mice from the lethal attack of rabies virus (5 / 5, 100%), which is equivalent to the protective effect of the immunization group of 5 μg of linear mRNA containing modified nucleotides (5 / 5, 100%), while the survival rates of the inactivated vaccine group and the PBS group were 40% and 20%, respectively. In summary, a single immunization of the rabies CircRNA vaccine prepared by the present invention can induce mice to produce an effective antiviral immune response, which is sufficient to protect mice from the lethal attack of rabies virus.

[0087] In summary, the Lyssavirus circRNA vaccine prepared by the present invention has stability and immunogenicity, can mediate the continuous expression of rabies G protein in vitro, and has significantly better stability than linear mRNA vaccines; in vivo, circRNA immunization can induce mice to produce high levels of cellular and humoral immune responses, and protect mice against lethal doses of virus attack infection, among which 2μg circRNA-10 has a protective effect equivalent to 5μg mRNA containing modified nucleotides, which is significantly better than existing inactivated vaccines.

Claims

1. A rabies virus circRNA vaccine, characterized in that: The invention comprises CircRNA for expressing optimized rabies virus immunogen and a vaccine vector, wherein the immunogen is G protein; and the vaccine vector is a lipid nanoparticle.

2. The rabies virus circRNA vaccine according to claim 1, characterized in that: The rabies virus is selected from PV strain, Flury strain, SAD strain, aG strain, CTN strain and their derivative strains or multiple combinations thereof.

3. The rabies virus circRNA vaccine according to claim 1, characterized in that: The rabies virus is CTN-1 strain.

4. The rabies virus circRNA vaccine according to claim 3, characterized in that: The G protein is the whole G protein, or the extramembrane part of the G protein, or the G protein polymer.

5. The rabies virus circRNA vaccine according to claim 4, characterized in that: The optimized nucleotide sequence of the G protein is shown in SEQ ID NO: 1 or 2.

6. The rabies virus circRNA vaccine according to claim 5, characterized in that: The circRNA nucleotide sequence is shown in SEQ ID NO: 5 or 6.

7. The rabies virus circRNA vaccine according to claim 1, characterized in that: The lipid nanoparticles include polyethylene glycol lipids, neutral lipids, cationic lipids and cholesterol or cholesterol analogs.

8. The rabies virus circRNA vaccine according to claim 7, characterized in that: The cationic lipids include but are not limited to Dlin-MC3-DMA, DODMA, DODAP, ALC-0315 or SM102.

9. The rabies virus circRNA vaccine according to claim 8, characterized in that: The mass ratio of the cationic lipid to the optimized nucleotide sequence is 10-30:

1.

10. Use of the rabies virus circRNA vaccine according to any one of claims 1 to 9 in the preparation of a drug for preventing or treating rabies.

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