Poxvirus recombinant chimeric antigen as well as circular RNA vaccine and application thereof

Abstract

The invention provides a poxvirus recombinant chimeric antigen as well as a circular RNA (Ribonucleic Acid) vaccine and application thereof. The invention designs a recombinant chimeric antigen aiming at pox virus, the recombinant chimeric antigen comprises antigens B6 and M1, circRNA comprising the recombinant chimeric antigen is constructed by using an optimized inverted intron-exon (PIE) system, the constructed circRNA vaccine encodes tandem fusion protein in different virion forms of monkey pox, the chimeric antigen is expressed in vivo, and the recombinant chimeric antigen is obtained. The chimeric immunogen can effectively stimulate the specific immunoreaction of an organism aiming at the chimeric immunogen, meanwhile, the production efficiency can be improved, and the production cost can be reduced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a poxvirus recombinant chimeric antigen, a circular RNA vaccine thereof and applications thereof. Background Art

[0002] The genus Orthopoxvirus includes monkeypox virus (MPXV), variola virus (VARV), cowpox virus (CPXV), vaccinia virus (VACV), and ectromelia virus (ECTV). It is an important class of biohazard pathogens that pose a serious threat to human health. Especially since 2022, there has been a surge in monkeypox virus outbreaks in many countries, bringing a crisis to global public health. However, there is currently no protective vaccine directly targeting monkeypox virus for epidemic prevention and control.

[0003] Since orthopoxviruses have high homology at the genome level and are closely related to each other in terms of immunogens, they have cross-protective activity. Previously, the FDA approved two attenuated vaccines that were used to prevent smallpox for pre-exposure vaccination to prevent and control orthopoxviruses including monkeypox. They are ACAM2000® and JYNNEOSTM vaccines. Among them, ACAM2000® is a replicating vaccine that has the ability to replicate in the human body. After vaccination, there is a risk of encephalitis, myocarditis, progressive vaccinia infection, etc., and it is not suitable for vaccination of young children, pregnant women, and people with low or impaired immune function; JYNNEOSTM is a non-replicating vaccine. Since it cannot replicate in the human body, its safety has been improved compared to replicating vaccines, but the immune effect has also been greatly reduced. Therefore, its ability to curb the spread of monkeypox virus and eradicate the virus remains to be determined. Therefore, in view of the severe form of the epidemic and the various drawbacks of existing vaccines, it is urgent to use new technologies to develop a new generation of vaccines with clear immunogenic components, clear mechanisms of action, safety and effectiveness to help disease prevention and control.

[0004] During the COVID-19 pandemic, the rapid development of mRNA technology is changing vaccine development and has attracted widespread attention. Subsequently, multiple mRNA vaccine candidates are also being developed in the field of emerging infectious diseases against other important pathogens. Regarding the recent monkeypox epidemic, some studies have shown that mRNA-based vaccines can effectively prevent MPXV in mice and non-human primates. However, due to their inherent instability, unnecessary immunogenicity and limited half-life, current mRNA vaccines still have certain limitations in terms of widespread application. circRNA is a covalently closed circular structure. Because it does not have a 5ʼ-cap and 3ʼ-poly (A) tail structure, it can be protected from nuclease degradation compared with linear mRNA, and has high stability and a longer half-life. In addition, even without nucleotide modification, circRNA has low immunogenicity. These advantages make circRNA more suitable for vaccine development. Summary of the invention

[0005] To make up for the deficiencies of the prior art, the present invention provides a poxvirus recombinant chimeric antigen, a circular RNA vaccine thereof and applications thereof.

[0006] To achieve the above purpose, the present invention adopts the following technical solution.

[0007] The first aspect of the present invention provides a poxvirus recombinant chimeric antigen, wherein the recombinant chimeric antigen comprises any two or more of B6, M1, A35 and A29.

[0008] Furthermore, the recombinant chimeric antigen is selected from any two of B6, M1 and A29.

[0009] Furthermore, the recombinant chimeric antigen is selected from B6 and M1.

[0010] Further, B6 and M1 are connected sequentially.

[0011] Furthermore, the amino acid sequence of B6 is as shown in SEQ ID NO: 2, or one or more amino acids are substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO: 2.

[0012] Furthermore, the amino acid sequence of M1 is as shown in SEQ ID NO: 3, or one or more amino acids are substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO: 3.

[0013] The second aspect of the present invention provides a polynucleotide encoding the recombinant chimeric antigen according to the first aspect of the present invention.

[0014] Furthermore, the polynucleotide encoding B6 has at least 80% sequence identity with the sequence shown in SEQ ID NO:4.

[0015] Furthermore, the polynucleotide encoding M1 has at least 80% sequence identity with the sequence shown in SEQ ID NO:5.

[0016] Furthermore, the sequence of the polynucleotide encoding B6 is shown in SEQ ID NO:4.

[0017] Furthermore, the sequence of the polynucleotide encoding M1 is shown in SEQ ID NO:5.

[0018] The third aspect of the present invention provides a circRNA, wherein the circRNA comprises the polynucleotide described in the second aspect of the present invention.

[0019] Furthermore, the circRNA also includes a signal peptide.

[0020] Furthermore, the circRNA also includes an IRES sequence.

[0021] Furthermore, the IRES sequence is selected from CVB3 virus.

[0022] Furthermore, the circRNA also includes other regulatory elements.

[0023] The fourth aspect of the present invention provides a nucleic acid construct, which comprises the polynucleotide described in the second aspect of the present invention or the circRNA described in the third aspect of the present invention.

[0024] The fifth aspect of the present invention provides a vector, which comprises the polynucleotide described in the second aspect of the present invention, the circRNA described in the third aspect of the present invention, or the nucleic acid construct described in the fourth aspect of the present invention.

[0025] The sixth aspect of the present invention provides a host cell, which comprises the polynucleotide described in the second aspect of the present invention, the circRNA described in the third aspect of the present invention, the nucleic acid construct described in the fourth aspect of the present invention, or the vector described in the fifth aspect of the present invention.

[0026] The seventh aspect of the present invention provides a vaccine, which comprises the recombinant chimeric antigen described in the first aspect of the present invention, the polynucleotide described in the second aspect of the present invention, the circRNA described in the third aspect of the present invention, the nucleic acid construct described in the fourth aspect of the present invention, the vector described in the fifth aspect of the present invention, or the host cell described in the sixth aspect of the present invention.

[0027] Furthermore, the vaccine is selected from nucleic acid vaccines.

[0028] Furthermore, the nucleic acid vaccine is selected from RNA vaccine.

[0029] Furthermore, the RNA vaccine is selected from circRNA vaccine.

[0030] Furthermore, the circRNA vaccine also includes an IRES sequence.

[0031] Furthermore, the IRES sequence is selected from CVB3 virus.

[0032] The eighth aspect of the present invention provides a pharmaceutical composition, which comprises the recombinant chimeric antigen described in the first aspect of the present invention, the polynucleotide described in the second aspect of the present invention, the circRNA described in the third aspect of the present invention, the nucleic acid construct described in the fourth aspect of the present invention, the vector described in the fifth aspect of the present invention, the host cell described in the sixth aspect of the present invention, or the vaccine described in the seventh aspect of the present invention.

[0033] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0034] The ninth aspect of the present invention provides a method for constructing the circRNA described in the third aspect of the present invention, the method comprising constructing a plasmid of nucleotides expressing the recombinant chimeric antigen described in the first aspect of the present invention.

[0035] Furthermore, the plasmid also includes an IRES sequence.

[0036] Furthermore, the IRES sequence is selected from CVB3 virus.

[0037] Furthermore, the plasmid also includes introns, homology arms, and spacer regions.

[0038] Furthermore, the intron is selected from group I introns.

[0039] Furthermore, the method further comprises circularizing the RNA.

[0040] Furthermore, using GTP and Mg + Circularize RNA.

[0041] Furthermore, the method also includes purifying circRNA.

[0042] The tenth aspect of the present invention provides the use of the recombinant chimeric antigen described in the first aspect of the present invention, the polynucleotide described in the second aspect of the present invention, the circRNA described in the third aspect of the present invention, the nucleic acid construct described in the fourth aspect of the present invention, the vector described in the fifth aspect of the present invention, the host cell described in the sixth aspect of the present invention or the vaccine described in the seventh aspect of the present invention in preventing poxvirus infection or in preparing a pharmaceutical composition for treating poxvirus infection.

[0043] Furthermore, the poxvirus is selected from monkeypox virus.

[0044] Advantages and beneficial effects of the present invention: The present application designs a recombinant chimeric antigen against poxvirus, which includes antigens B6 and M1. A circRNA including the above-mentioned recombinant chimeric antigen is constructed using an optimized inverted intron-exon (PIE) system. The constructed circRNA vaccine encodes a tandem fusion protein in the form of different monkeypox virus particles. The chimeric immunogen is expressed in vivo, which can effectively stimulate the body's specific immune response to the chimeric immunogen, while also improving production efficiency and reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a graph evaluating the immunogenicity and protective efficacy of the monovalent circRNA vaccine, wherein 1A is a graph of the immunization program of the monovalent circRNA vaccine, 1B is a graph of the specific antibody immunization against B6, 1C is a graph of the specific antibody immunization against M1, 1D is a graph of the specific antibody immunization against A35, 1E is a graph of the specific antibody immunization against A29, 1F is a graph of the weight statistics of mice immunized with the monovalent circRNA vaccine after being infected with the virus, and 1G is a graph of the survival rate statistics of mice immunized with the monovalent circRNA vaccine after being infected with the virus; Figure 2 It is a diagram of the construction and analysis of the chimeric antigen circRNA vaccine, wherein 2A is a flowchart of the construction of the chimeric antigen circRNA vaccine, and 2B-2D are diagrams of the expression of the chimeric antigen circRNA vaccine; Figure 3 It is a graph of immunogenicity evaluation of chimeric antigen circRNA vaccine, wherein 3A is a graph of immunization program of chimeric antigen circRNA vaccine, 3B is a graph of specific antibody level of B6 antigen, 3C is a graph of specific antibody level of M1 antigen, 3D is a graph of specific antibody level of A35 antigen, and 3E is a graph of specific antibody level of A29 antigen; Figure 4 4A is a graph showing the body weight of mice immunized with chimeric antigen circRNA vaccine after in vivo challenge, and 4B is a graph showing the survival rate of mice immunized with chimeric antigen circRNA vaccine after in vivo challenge; Figure 5 It is the circRNA after high-dose challenge B6A29 and circRNA B6M1 Vaccine in vivo protection effect diagram, among which 5A is circRNA B6A29 and circRNA B6M1 Body weight of immunized mice after high-dose challenge. 5B is a circRNA B6A29and circRNA B6M1 Survival rate of immunized mice after high-dose challenge. DETAILED DESCRIPTION

[0046] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0047] The present invention provides a poxvirus recombinant chimeric antigen, wherein the recombinant chimeric antigen comprises any two or more of B6, M1, A35 and A29.

[0048] In some embodiments, the recombinant chimeric antigen may include any two, three, or four of B6, M1, A35, and A29, for example, B6 and M1, B6 and A35, B6 and A29, B6, M1 and A29, B6, M1, A35, and A29, etc.

[0049] In a preferred embodiment, the recombinant chimeric antigen is selected from any one of the following groups: B6 and M1, B6 and A29.

[0050] In a more preferred embodiment, the recombinant chimeric antigen is selected from B6 and M1.

[0051] In a specific embodiment, B6 and M1 are connected sequentially.

[0052] The amino acid sequence of B6 is as shown in SEQ ID NO: 2, or one or more amino acids are substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO: 2.

[0053] In some embodiments, the substitution, deletion or insertion of one or more amino acids may be, for example, a substitution, deletion or insertion of one, two or three amino acids, which does not affect the function of the amino acids.

[0054] Recombinant chimeric antigens can be used in the detection of poxviruses.

[0055] In some embodiments, the recombinant chimeric antigen can be labeled with a detectable marker, and any marker and labeling method known to those skilled in the art can be used. Commonly used markers may include enzymes (such as HRP, β-galactosidase, alkaline phosphatase, etc.), radioactive isotopes (such as 32 P or 125I), biotin, digoxin, colloidal metals (such as colloidal gold, etc.), fluorescent dyes (such as fluorescein, rhodamine, Texas Red, etc.), chemiluminescent compounds or bioluminescent compounds (such as dioxetane, luminol or acridinium, etc.). Any labeling steps well known in the art can be used, such as covalent coupling of enzymes or biotin groups, iodination, phosphorylation, biotinylation, etc.

[0056] The present invention provides a polynucleotide encoding the above-mentioned recombinant chimeric antigen.

[0057] In some embodiments, the polynucleotide sequence can be codon optimized. A codon optimized sequence can be a sequence in which codons in a polynucleotide encoding a polypeptide have been substituted to increase the expression, stability and / or activity of the polypeptide. Factors that affect codon optimization include, but are not limited to, one or more of the following: (i) variation in codon bias between two or more organisms or genes or synthetically constructed bias tables, (ii) variation in the degree of codon bias within an organism, gene, or group of genes, (iii) systematic variation in codons including their context, (iv) variation in codons according to their decoding tRNA, (v) variation in codons according to GC% overall or at a position in a triplet, (vi) variation in the degree of similarity to a reference sequence (e.g., a naturally occurring sequence), (vii) variation in the codon frequency cutoff, (viii) structural properties of mRNA transcribed from a DNA sequence, (ix) prior knowledge about the function of a DNA sequence on which a codon substitution set is designed, and / or (x) systematic variation in codon sets for each amino acid.

[0058] The present invention provides a circRNA, which comprises the above-mentioned polynucleotide.

[0059] In some embodiments, circRNA is also interchangeably referred to as circular RNA, microcircRNA, microcircRNA, or small circRNA.

[0060] In some embodiments, the circRNA further comprises an IRES sequence, IRES (internal ribosome entry site) refers to an RNA element that allows translation to be initiated in a cap-independent manner as part of a larger process of protein synthesis. IRES can be derived from DNA or RNA of organisms, including but not limited to viruses, mammals, and insects (e.g., fruit flies). In a preferred embodiment, the IRES is derived from a virus, such as ABPV_IGRpred, AEV, ALPV_IGRpred, BQCV_IGRpred, BVDV1_1-385, BVDV1_29-391, CrPV_SNCR, CrPV_IGR, crTMV_IREScp, crTMV-IRESmp75, crTM_VIRESmp228, crTMV_IREScp, crTMV_IREScp, CSFV, CVB3, DCVIGR, EMCV-R, EoPVSNTR, ERAV_245-961, ERBV_162-920, EV71_1-748, FeLV-Notch2, FMDV_type_C, GBV-A, GBV-B, GBV-C, gypsy_env, gypsyD5, gypsyD2, HAV_ HM175, HCV_type_1a, HiPV_IGRpred, HIV-1, HoCV1_IGRpred, HRV-2, IAPV_IGRpred, idefix, KBV _IGRpred, LINE-1_ORF1_-101_to_-1, LINE-1_ORF1_-302_to_-202, LINE-1_-ORF2_-138to_-86 , LINE-1_ORF1_-44to_-1, PSIV_IGR, PV_type1_Mahoney, PV_type3_Leon, REV-A, RhPV_5NCR, Rh PV_IGR, SINV1_IGRpred, SV40_661-830, TMEV, TMV_UI_IRESmp228, TRV_5NTR, TrV_IGR or TSV_IGR.

[0061] In a specific embodiment, the IRES sequence is selected from the CVB3 virus.

[0062] The circRNA also includes other regulatory elements.

[0063] In some embodiments, the other regulatory elements refer to nucleotide sequences in the non-coding region that regulate the translation of the coding region, including but not limited to start codons, stop codons, Kozak sequences, and any other regulatory elements.

[0064] The present invention provides a nucleic acid construct, which comprises the above-mentioned polynucleotide or the above-mentioned circRNA.

[0065] In some embodiments, a nucleic acid construct refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a native gene or modified in a manner that does not otherwise exist in nature or is synthesized to contain a nucleic acid segment.

[0066] The present invention provides a vector, which comprises the above-mentioned polynucleotide, the above-mentioned circRNA or the above-mentioned nucleic acid construct.

[0067] In some embodiments, a vector is a replicon that can be attached to another DNA segment, i.e., an insert, so that the attached segment can be replicated in a cell, such as a plasmid, a bacmid, a phage, a virus, a virion, or a viscous body. A vector can be a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. A vector encompasses any genetic element that can replicate when combined with appropriate regulatory elements and can transfer a gene sequence to a cell. According to some embodiments, a vector can be an expression vector or a recombinant vector.

[0068] The present invention provides a host cell, which comprises the above-mentioned polynucleotide, the above-mentioned circRNA, the above-mentioned nucleic acid construct or the above-mentioned vector.

[0069] In some embodiments, host cells include any cell type that is susceptible to transformation, transfection, transduction, etc. of the nucleic acid construct or vector of the present application. As a non-limiting example, the host cell can be any of isolated primary cells, pluripotent stem cells, CD34+ cells, induced pluripotent stem cells, or many immortalized cell lines (e.g., HepG2 cells). Alternatively, the host cell can be an in situ or in vivo cell in a tissue, organ, or organism.

[0070] The present invention provides a vaccine, which comprises the above-mentioned recombinant chimeric antigen, the above-mentioned polynucleotide, the above-mentioned circRNA, the above-mentioned nucleic acid construct, the above-mentioned vector or the above-mentioned host cell.

[0071] In some embodiments, the types of vaccines include inactivated vaccines, live attenuated vaccines, recombinant protein vaccines, vector vaccines and nucleic acid vaccines.

[0072] In a preferred embodiment, the type of vaccine is selected from nucleic acid vaccines, including DNA vaccines and RNA vaccines.

[0073] In a more preferred embodiment, the nucleic acid vaccine is selected from RNA vaccines, including mRNA vaccines and circRNA vaccines.

[0074] In a specific embodiment, the RNA vaccine is selected from a circRNA vaccine.

[0075] In some embodiments, the circRNA vaccine also includes a delivery vector.

[0076] In a specific embodiment, the delivery vehicle is selected from LNP (lipid nanoparticles), which may include any lipid capable of forming particles, and the one or more microcircRNAs are attached to the particles, or the one or more microcircRNAs are encapsulated in the particles. Lipids refer to a group of organic compounds that are derivatives of fatty acids (such as esters) and are generally characterized by being insoluble in water but soluble in a variety of organic solvents. Lipids are generally divided into at least three categories: (1) simple lipids, including fats, oils, and waxes; (2) complex lipids, including phospholipids and glycolipids; and (3) derived lipids, such as steroids.

[0077] The present invention provides a pharmaceutical composition, which comprises the above-mentioned recombinant chimeric antigen, the above-mentioned polynucleotide, the above-mentioned circRNA, the above-mentioned nucleic acid construct, the above-mentioned vector, the above-mentioned host cell or the above-mentioned vaccine.

[0078] The pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0079] In some embodiments, pharmaceutically acceptable excipients refer to clinically useful solvents, dispersion media, coatings, isotonic and absorption delaying agents, buffers and excipients, such as phosphate buffered saline solutions (PBS), aqueous solutions and emulsions of glucose or mannitol, such as oil-in-water or water-in-oil emulsions, and various types of wetting agents, immunostimulants and / or adjuvants. The pharmaceutical excipients used in the composition depend on the intended mode of administration of the active agent. Some examples of materials that can be used as pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as 80, phosphates, glycine, sorbic acid or potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride or zinc salts), colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, methylcellulose, hydroxypropyl methylcellulose, sheep Gross fat, sugars (such as lactose, glucose and sucrose); starches (such as corn starch and potato starch); cellulose and its derivatives (such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate); powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol and phosphate buffered solution. In addition, other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, mold release agents, coating agents, sweeteners, flavoring agents and fragrances, preservatives and antioxidants may also be present in the composition according to the judgment of the formulator.

[0080] The present invention provides use of the above-mentioned recombinant chimeric antigen, the above-mentioned polynucleotide, the above-mentioned circRNA, the above-mentioned nucleic acid construct, the above-mentioned vector, the above-mentioned host cell or the above-mentioned vaccine in preventing poxvirus infection or in preparing a pharmaceutical composition for treating poxvirus infection.

[0081] In some embodiments, the poxvirus comprises monkeypox virus, smallpox virus, cowpox virus and / or vaccinia virus.

[0082] In a specific embodiment, the poxvirus is selected from monkeypox virus.

[0083] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0084] Example

[0085] 1. Experimental methods (1) circRNA synthesis The universal sequence of the PIE system containing the type I self-splicing intron, CVB3-IRES fragment, spacer and homology arms was cloned into the linearized pUC57 plasmid containing the T7 RNA polymerase promoter to construct the circular RNA linearization template. Subsequently, the fragments encoding different antigens were amplified by PCR and cloned into the vector backbone digested with EcoRV. These plasmids were linearized by EcoRI digestion, and the digestion products were then separated and extracted by agarose gel electrophoresis. The concentration of the target fragment was increased to more than 500-1000 ng / μL (in RNase-free water) to obtain the DNA template for in vitro transcription, which was then stored at -20°C.

[0086] The linearized plasmid DNA template was used to synthesize the circRNA precursor by in vitro transcription using the HiScribe® T7 High Yield RNA Synthesis Kit. After the in vitro transcription reaction, it was treated with DNase I (RNase-free), and then GTP was added to a final concentration of 2 mM, and a buffer containing magnesium was added to promote the cyclization of the precursor. Finally, the RNA was column purified using the Monarch® RNA Purification Kit.

[0087] (2) circRNA purification, quality control and expression analysis High-quality circRNA was isolated and purified from the RNA cyclization mixture by HPLC using an Elite EClassical 3200 series HPLC system. RNA was detected and collected based on UV absorption measurements at 260 nm. The resulting circRNA was desalted by passing it through a desalting column, precipitated with 5 M ammonium acetate, and then resuspended in RNase-free water.

[0088] For splice site sequencing, reverse transcription was performed using TransScript® II One-Step gDNA Removal and cDNA Synthesis Supermix provided by TransGen Biotech. PCR products for sequencing were generated using gene-specific primers spanning the splice sites. All PCR products were verified by Sanger sequencing.

[0089] In each well of a 6-well plate, the purified circRNA was transfected into HEK293 cells using Invitrogen's Lipofectamine® MessengerMAXTM reagent according to the manufacturer's protocol. 48 hours after transfection, the cell culture supernatant was collected and the expression of the antigen was verified by ELISA.

[0090] (3) LNP encapsulation of circRNA LNP-circRNAs were prepared by mixing an aqueous phase containing circRNA with an ethanol phase containing lipid and cholesterol components using a microfluidic mixing device. The device uses chaotic mixing properties to induce fluid folding in a laminar state, thereby promoting the repeated formation of uniform LNPs. The aqueous and ethanol phases were mixed in a microfluidic device at a ratio of 1:3 (volume ratio). After preparation, LNP-circRNAs were dialyzed for 12 hours using a membrane with a molecular weight cutoff of 3.5 kDa to remove salt. The dialyzed LNP-circRNAs were diluted 100 times with deionized water, and 10 microliters of the sample was then dropped onto a carbon-coated copper grid and dried under vacuum to remove residual moisture.

[0091] (4) Mouse experiments For immunization of circRNA vaccine candidates, LNP-circRNAs dry powder was dissolved in RNase-free water at a concentration of 100 μg / mL. Each BALB / c mouse was inoculated with 100 μL by intramuscular injection, with an interval of 14 days between two inoculations. Serum was collected 13 days after each inoculation for ELISA testing.

[0092] To evaluate the protective effect of candidate vaccines in vivo, VACV was used for challenge test. Mice were anesthetized with isoflurane and inhaled 50 μL of low dose (2×10 5 PFUs) or high dose (1×10 6 The mice were weighed every day for 14 days, during which time they were euthanized when their body weight decreased by 20%.

[0093] (5) ELISA 96-well ELISA plates were coated with 2 μg / mL protein or 1 μg / mL antibody overnight and then blocked with 10% skim milk in PBST. Serum samples or cell supernatants were diluted and added to each well for incubation, and then 1:4000 diluted goat anti-mouse IgG-horseradish peroxidase antibody or anti-His-tag mAb-HRP-Direct was added to the ELISA plate for incubation and color development with color development solution. Finally, the reaction was stopped with 2 M hydrochloric acid, and the absorbance was measured at 450 nm using a PerkinElmer microplate reader.

[0094] 2. Experimental results Four circRNAs targeting major MPXV antigens (B6, M1, A35, and A29) were designed using the optimized inverted intron-exon (PIE) system.

[0095] Then, according to Figure 1 The immunization schedule shown in A evaluated the monovalent circRNA vaccine (circRNA B6 、circRNA M1 、circRNA A35 and circRNA A29 ) to investigate the immunogenicity and protective efficacy of the vaccine. Randomly grouped BALB / c mice were inoculated intramuscularly with 100 μL of a vaccine containing 8 μg of monovalent circular RNA, and 100 μL of LNP as a negative control. Each vaccine was administered twice, with an interval of 14 days between each dose. Serum samples were collected on the 13th day after each immunization, and specific antibodies against the corresponding antigens were detected by enzyme-linked immunosorbent assay (ELISA). It can be observed that specific antibodies against B6 and M1 were significantly stimulated after the first immunization, which was significantly different from the LNP group, and further increased after booster immunization; antibodies against A29 were not significantly stimulated after the first immunization, but were significantly increased after booster immunization, and the titer was slightly lower than the antibody level stimulated by B6 and M1; however, antibodies against A35 were still not observed to be significantly produced after booster immunization ( Figure 1 To further evaluate their protective efficacy, 15 days after booster immunization, immunized mice were intranasally inoculated with 1×10 6 PFUs VACV-WR, and initial weight loss was observed in all vaccine groups. A35 、circRNA A29 The LNP group provided almost no protection, and the weight loss reached the euthanasia threshold (20%) on the 4th day, and all died ( Figure 1 F and 1G). It is worth noting that circRNA B6 and circRNA M1 The 1:1 ratio and 2:1 ratio, respectively, experienced initial weight loss but ultimately provided 80% and 50% protection against lethal viral challenge ( Figure 1 F and 1G). These findings suggest that B6 and M1 are promising antigen candidates for mixed immunogen design, or their combination will provide better immune protection effects.

[0096] Based on the above experimental results, the combination of A35 and A29 antigens was excluded, and the coding sequences of B6A29, A29B6, B6M1, M1B6, A35M1 and M1A35 were integrated into the circRNA template vector to prepare circRNA B6A29 、circRNA A29B6 、circRNA B6M1 、circRNA M1B6 、circRNAA35M1 and circRNA M1A35 ( Figure 2 A). The purified circRNA B6A29 、circRNA A29B6 、circRNA B6M1 、circRNA M1B6 、circRNA A35M1 and circRNA M1A35 Transfected into HEK293 cells to verify their expression, given the importance of antigen expression levels in circRNA vaccine development, circRNAs were screened by ELISA analysis B6A29 、circRNA B6M1 and circRNA A35M1 ( Figure 2 B-2D) and encapsulated them into LNPs for further study.

[0097] Then according to Figure 3 The immunization protocol shown in A evaluated circRNA B6A29 、circRNA B6M1 and circRNA A35M1 Immunogenicity of the vaccine. 8 μg of circRNA B6A29 , 8 μg of circRNA B6M1 and 8 μg of circRNA A35M1 BALB / c mice were immunized by intramuscular injection. LNP was used as a negative control to detect specific antibodies in serum samples. The results showed that circRNA B6A29 A certain level of antibodies was induced only after the second immunization, while circRNA B6M1 There is a good stimulation effect after the first immunization, and it further increases after the booster immunization. The level of induced specific antibodies increases steadily, which is significantly higher than that of circRNA B6A29 and LNP group ( Figure 3 B). In terms of the level of specific antibodies against M1 antigen, circRNA A35M1 It was still after the second immunization that a certain level of antibodies was induced, and the differences within the group were large, while circRNA B6M1 Based on the antibody level stimulated by the first immunization, it was further increased after the booster immunization, and the induced specific antibody level was more balanced, with small differences within the group, and was significantly higher than circRNA A35M1 and LNP group ( Figure 3 C). As for A35 and A29 antigens, circRNA A35M1 and circRNA B6A29No significant antibody levels were stimulated in the first immunization, and although a certain antibody level was induced after the second immunization, there was no statistically significant difference compared with the LNP group ( Figure 3 D and 3E).

[0098] To further explore circRNA B6A29 、circRNA B6M1 and circRNA A35M1 In vivo challenge protection effect, mice vaccinated according to the above immunization schedule were intranasally inoculated with 2×10 5 The mice were challenged with 100 PFUs VACV-WR and their body weights were monitored daily for 2 weeks. Figure 3 A). The mice inoculated with LNP continued to lose weight after the virus challenge, and all died after reaching the euthanasia threshold on the 5th day after infection. A35M1 The mice also continued to lose weight after infection, and some mice began to die on the 4th day after infection, ultimately providing 40% protection against the virus ( Figure 4 A and 4B). B6A29 and circRNA B6M1 The immunized mice did not lose significant weight throughout the experiment, and both provided complete protection ( Figure 4 A and 4B).

[0099] To further evaluate circRNA B6A29 and circRNA B6M1 To evaluate the protective effect, we then inoculated circRNA again under the aforementioned immunization program. B6A29 、circRNA B6M1 and LNP, inoculated as before, 1×10 6 PFUs VACV-WR was used to attack the virus ( Figure 3 A). After a high lethal dose of virus, receiving circRNA B6A29 The mice immunized with LNP showed severe weight loss. Compared with the previous group, all mice in the LNP group reached the euthanasia threshold on the 4th day after infection. B6A29 The mice in the group began to die on the third day after infection, and ultimately only about 14% of the mice were protected from the virus ( Figure 5 A and 5B). In contrast, circRNA B6M1 The mice showed a slight initial weight loss, which then recovered rapidly, and the mice were completely protected ( Figure 5A and 5B). These results together indicate that circRNA B6M1 Vaccines are promising candidates for MPXV vaccine development.

[0100] Sequences involved in this application: ePIE base sequence:

[0101] B6 amino acid (T20-H279, C140S): TCTVPTMNNAKLTSTETSFNDKQKVTFTCDSGYHSLDPNAVCETDKWKYENPCKKMCTVSDYVSELYDKPLYEVNSTMTLSCNGETKYFRCEEKNGNTSWNDTVTCPNAECQPLQLEHGSSQPVKEKYSFGEYMTINCDVGYEVIGVSYISCTANSWNVIPSCQQKCDIPSLSNGLISGSTFSIGGVIHLSCKSGFTLTGSPSSTCIDGKWNPILPTCVRSNEEFDPVDDGPDDETDLSKLSKDVVQYEQEIESLEATYH (SEQ ID NO:2).

[0102] M1 amino acid (A3-G181): AAASIQTTVNTLSERISSKLEQEANASAQTKCDIEIGNFYIRQNHGCNITVKNMCSADADAQLDAVLSAATETYSGLTPEQKAYVPAMFTAALNIQTSVNTVVRDFENYVKQTCNSSAVVDNKLKIQNVIIDECYGAPGSPTNLEFINTGSSKGNCAIKALMQLTTKATTQIAPRQVAG (SEQ ID NO:3).

[0103] B6 nucleotide: ACTTGCACCGTGCCCACCATGAACAACGCCAAGCTGACCAGCACCGAGACCAGCTTCAACGACAAGCAGAAGGTCACCTTCACTTGCGACAGCGGATACCACTCTCTGGACCCTAACGCTGTCTGCGAGACCGACAAGTGGAAGTACGAGAACCCTTGCAAGAAGATGTGCACCGTGTCCGACTACGTGTCCGAGCTGTACGACAAGCCCCTGTACGAGGTCAACAGCACCATGACCCTCTCTTGCAACGGCGAGACCAAGTACTTCCGCTGCGAGGAGAAGAATGGAAACACCTCTTGGAACGACACCGTGACTTGCCCTAACGCCGAGTGTCAGCCTCTGCAGCTGGAACACGGAAGCTCTCAGCCCGTGAAGGAGAAGTACAGCTTCGGCGAGTACATGACCATCAATTGCGACGTGGGCTACGAGGTCATCGGCGTGTCCTACATCTCTTGCACCGCCAACTCTTGGAACGTGATCCCCTCTTGCCAGCAGAAGTGCGACATCCCTAGCCTGTCTAACGGCCTGATCAGCGGAAGCACCTTCAGCATCGGCGGAGTGATCCACCTGTCTTGCAAGAGCGGCTTCACCCTGACAGGCAGCCCTAGCAGCACCTGTATCGACGGCAAGTGGAACCCTATTCTGCCTACCTGCGTGCGGAGCAACGAGGAGTTCGACCCAGTGGACGATGGACCAGACGACGAGACCGATCTGAGCAAGCTGAGCAAGGACGTGGTGCAGTACGAGCAGGAGATCGAGAGCCTGGAAGCCACCTACCAC (SEQ ID NO:4).

[0104] M1 nucleotide: GCCGCCGCCTCTATCCAGACCACAGTGAACACCCTGAGCGAGCGGATCAGCTCCAAGCTGGAGCAGGAGGCAAACGCAAGCGCCCAGACAAAGTGCGACATCGAGATCGGCAATTTCTACATCAGACAGAACCACGGCTGCAATATCACCGTGAAGAATATGTGCTCCGCCGACGCAGATGCACAGCTGGATGCCGTGCTGTCCGCCGCAACCGAGACATACTCTGGCCTGACCCCTGAGCAGAAGGCCTATGTGCCAGCCATGTTCACAGCCGCCCTGAACATCCAGACCAGCGTGAATACAGTGGTGCGGGACTTTGAGAACTACGTGAAGCAGACCTGCAATTCTAGCGCCGTGGTGGACAACAAGCTGAAGATCCAGAATGTGATCATCGATGAGTGTTATGGAGCACCAGGATCCCCTACCAACCTGGAGTTCATCAATACAGGCTCCTCTAAGGGCAACTGCGCCATCAAGGCCCTGATGCAGCTGACCACAAAGGCCACCACACAGATCGCACCTAGGCAGGTGGCAGGA (SEQ ID NO:5).

[0105] A 29 amino acids (M1 - E110, C71 / 72S): MDGTLFPGDDDLAIPATEFFSTKAAKNPETKREAIVKAYGDDNEETLKQRLTNLEKKITNITTKFEQIEKSSKRNDEVLFRLENHAETLRAAMISLAKKIDVQTGRHPYE (SEQ ID NO:6).

[0106] A 35 amino acids (S90 - T181): STTQYDHKESCNGLYYQGSCYILHSDYKSFEDAKANCAAESSTLPNKSDVLTTWLIDYVEDTWGSDGNPITKTTSDYQDSDVSQEVRKYFCT (SEQ ID NO:7).

[0107] A 29 nucleotides: ATGGATGGTACCCTGTTCCCCGGCGATGACGACTTGGCCATCCCCGCGACCGAGTTTTTCTCCACTAAGGCGGCGAAGAACCCGGAGACAAAGCGGGAGGCCATCGTCAAGGCGTACGGGGACGACAATGAGGAGACGCTGAAGCAGCGTCTCACGAACTTGGAGAA GAAAATTACAAACATTACGACCAAGTTCGAGCAGATTGAGAAGAGCAGCAAGAGAAACGATGAAGTCCTCTTCCGGCTGGAGAATCACGCAGAGACACTAAGGGCTGCGATGATTTCTCTAGCCAAGAAGATAGACGTCCAGACGGGGCGCCATCCATACGAG (SEQ ID NO:8).

[0108] A35 Nucleotide: AGCACCACCCAGTACGACCACAAAGAGAGCTGCAACGGCCTGTACTACCAAGGCTCTTGTTACATCCTGCACAGCGACTATAAGAGCTTCGAGGATGCTAAGGCCAATTGCGCCGCCGAGAGCAGCACACTGCCTAACAA GTCCGACGTGCTGACCACCTGGCTGATCGACTACGTGGAAGATACATGGGGAAGCGACGGCAACCCCATCACAAAAACCACAAGCGATTATCAGGACAGCGACGTGTCTCAGGAGGTTAGAAAGTACTTTTGCACC (SEQ ID NO:9).

[0109] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A poxvirus recombinant chimeric antigen, characterized in that: The recombinant chimeric antigen includes any two or more of B6, M1, A35 and A29; Preferably, the recombinant chimeric antigen is selected from any two of B6, M1 and A29; Preferably, the recombinant chimeric antigen is selected from B6 and M1; Preferably, B6 and M1 are connected sequentially; Preferably, the amino acid sequence of B6 is as shown in SEQ ID NO: 2, or one or more amino acids are substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO: 2; Preferably, the amino acid sequence of M1 is as shown in SEQ ID NO: 3, or one or more amino acids are substituted, deleted or inserted in the amino acid sequence shown in SEQ ID NO:

3.

2. A polynucleotide, characterized in that The polynucleotide encodes the recombinant chimeric antigen according to claim 1; Preferably, the polynucleotide encoding B6 has at least 80% sequence identity with the sequence shown in SEQ ID NO:4; Preferably, the polynucleotide encoding M1 has at least 80% sequence identity with the sequence shown in SEQ ID NO:5; Preferably, the sequence of the polynucleotide encoding B6 is shown in SEQ ID NO: 4; Preferably, the sequence of the polynucleotide encoding M1 is shown as SEQ ID NO:

5.

3. A circRNA, characterized in that: The circRNA comprises the polynucleotide of claim 2; Preferably, the circRNA further comprises a signal peptide; Preferably, the circRNA further comprises an IRES sequence; Preferably, the IRES sequence is selected from CVB3 virus; Preferably, the circRNA also includes other regulatory elements.

4. A nucleic acid construct, characterized in that The nucleic acid construct comprises the polynucleotide according to claim 2 or the circRNA according to claim 3.

5. A carrier, characterized in that The vector comprises the polynucleotide according to claim 2, the circRNA according to claim 3 or the nucleic acid construct according to claim 4.

6. A host cell, characterized in that The host cell comprises the polynucleotide of claim 2, the circRNA of claim 3, the nucleic acid construct of claim 4 or the vector of claim 5.

7. A vaccine, characterized in that The vaccine comprises the recombinant chimeric antigen of claim 1, the polynucleotide of claim 2, the circRNA of claim 3, the nucleic acid construct of claim 4, the vector of claim 5 or the host cell of claim 6; Preferably, the vaccine is selected from nucleic acid vaccines; Preferably, the nucleic acid vaccine is selected from RNA vaccines; Preferably, the RNA vaccine is selected from circRNA vaccines; Preferably, the circRNA vaccine further comprises an IRES sequence; Preferably, the IRES sequence is selected from CVB3 virus.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the recombinant chimeric antigen of claim 1, the polynucleotide of claim 2, the circRNA of claim 3, the nucleic acid construct of claim 4, the vector of claim 5, the host cell of claim 6 or the vaccine of claim 7; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

9. The method for constructing circRNA according to claim 3, characterized in that: The method comprises constructing a plasmid for expressing the nucleotide sequence of the recombinant chimeric antigen of claim 1; Preferably, the plasmid further comprises an IRES sequence; Preferably, the IRES sequence is selected from CVB3 virus; Preferably, the plasmid further comprises introns, homology arms, and spacer regions; Preferably, the intron is selected from group I introns; Preferably, the method further comprises circularizing the RNA; Preferably, GTP and Mg-containing + Circularized RNA; Preferably, the method further comprises purifying circRNA.

10. Use of the recombinant chimeric antigen according to claim 1, the polynucleotide according to claim 2, the circRNA according to claim 3, the nucleic acid construct according to claim 4, the vector according to claim 5, the host cell according to claim 6 or the vaccine according to claim 7 in preventing poxvirus infection or in preparing a pharmaceutical composition for treating poxvirus infection; Preferably, the poxvirus is selected from monkeypox virus.

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