Dengue virus circular mRNA vaccine

Abstract

This invention discloses a circular mRNA vaccine for dengue virus, belonging to the field of nucleic acid vaccine technology. The circular mRNA molecule expressed by this invention, which expresses the tetravalent dengue virus EDIII tandem antigen, uniformly expresses the EDIII domains of four serotypes (DENV-1 to DENV-4). The uniform expression of the four serotype EDIII domains is achieved primarily through multiple combinations of the four serotype EDIII domains, the T2A / P2A self-cleaving peptide, the Furin restriction site, and the flexible linker peptide (GGGS) arranged in a 4-way configuration. This design further ensures a balanced and efficient immune response in the tetravalent dengue virus mRNA vaccine containing this circular mRNA molecule, reduces ADE epitope immune responses, effectively avoids the risk of ADE effects, and the process can be scaled up industrially.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid vaccine technology, specifically relating to a dengue virus circular mRNA vaccine. Background Technology

[0002] Dengue fever is an acute vector-borne infectious disease caused by the dengue virus (DENV) and transmitted by Aedes mosquitoes. Typical symptoms include high fever (up to 40°C), severe headache, eye pain, muscle and joint pain (often called "breakbone fever"), rash, and bleeding tendency. Most patients have a good prognosis, but some cases can progress to dengue hemorrhagic fever or dengue shock syndrome, leading to severe complications such as plasma leakage and multiple organ failure, with a high mortality rate, especially posing a significant threat to children and adolescents. According to the World Health Organization, up to 400 million people are infected with dengue virus globally each year, of whom 96 million show clinical symptoms, and approximately 10,000 die. About 3.9 billion people in tropical and subtropical regions are at risk of infection. In 2024, a total of 14.4 million confirmed cases were reported globally, with the Americas, Southeast Asia, and the Western Pacific being high-incidence areas, placing a heavy economic burden on global public health systems.

[0003] The primary vectors of dengue virus are Aedes aegypti and Aedes albopictus mosquitoes. After a mosquito bites an infected person, the virus replicates and multiplies within the mosquito, then spreads through bites of healthy individuals. Peak transmission occurs two hours after sunrise and two hours before sunset. The virus can thrive in damp environments such as stagnant water containers and tree holes. Influenced by factors such as global climate change, rapid urbanization, and frequent cross-border movement of people, its spread continues to expand, and it has been reported to be prevalent in more than 100 countries and regions. Currently, there are no specific antiviral drugs for dengue fever; clinical treatment primarily focuses on symptomatic and supportive care. Vaccination remains the most effective means of preventing infection and reducing the disease burden.

[0004] Dengue fever vaccine development began in the 20th century. After decades of exploration, only a few vaccines have been approved for marketing or entered the late stage of clinical trials worldwide. This has led to a research and development pattern in which multiple technical routes are being pursued in parallel. However, all existing vaccines have significant limitations.

[0005] (1) CYD-TDV (Dengvaxia®) uses yellow fever vaccine 17D as a backbone to chimeric express prM and E proteins of the four serotypes of DENV. This vaccine has a good protective effect against those who have been previously infected with DENV, but its protective efficacy against DENV-1 and DENV-2 is low (50.3% and 42.3%, respectively). Furthermore, individuals who have not been vaccinated against DENV may have an increased risk of severe dengue fever after vaccination, so its use is restricted to people with a history of infection.

[0006] (2) TAK-003 (Qdenga®) is a quadrivalent vaccine based on DENV-2 PDK-53 with replacement of prM and E proteins. This vaccine can induce seroconversion in all four serotypes, but the neutralizing antibody response against DENV-4 is weak, and its long-term protective efficacy still needs to be observed; (3) TV003 / TV005, a live attenuated vaccine developed by the National Institute of Allergy and Infectious Diseases in the United States, has entered Phase III clinical trials. A single dose can induce a high seroconversion rate, but the DENV-2 component requires an increased dose to achieve balanced immunity. A common drawback of live attenuated vaccines is the risk of virulence reversion, insufficient safety for immunocompromised individuals, pregnant women, and the elderly, and the potential to induce antibody-dependent enhancement (ADE), leading to more severe illness when subsequently infected with other serotypes.

[0007] Dengue virus belongs to the genus Flaviviridae in the family Flaviviridae. It is a single-stranded positive-sense RNA virus with a genome length of approximately 11,000 bases, encoding three structural proteins (C, prM, and E) and seven non-structural proteins (NS1-NS5). The E protein is crucial for viral invasion of host cells, mediating viral attachment and membrane fusion. Its extracellular domains (EDⅠ-EDⅢ) are the main targets of neutralizing antibodies. The virus has four serotypes (DENV1-DENV4), with approximately 65%-70% amino acid sequence homology among them. Infection with one serotype only provides lifelong immunity to that serotype, offering no cross-protection against other serotypes. In fact, antibody-dependent enhancement (ADE) may increase the risk of severe reinfection—non-neutralizing cross-antibodies bind to heterologous viruses, promoting viral entry into host cells via the Fcγ receptor and exacerbating infection. The viral genome is prone to antigenic drift, and the conserved epitopes in the E protein fusion loop region easily induce ADE-related antibodies, posing a significant challenge to vaccine development. Inactivated vaccines have good safety profiles but weak immunogenicity, requiring multiple doses. Recombinant protein subunit vaccines primarily target the E protein or EDIII domain, offering good safety but limited immunogenicity. They rely on adjuvants and are difficult to induce balanced protection against the four serotypes. Virus-like particle (VLP) vaccines can mimic the natural structure of viruses, but their production processes are complex and costly, and early versions suffered from insufficient immunogenicity.

[0008] In recent years, mRNA vaccines have emerged as a new direction in dengue fever vaccine development due to their unique advantages. They involve synthesizing mRNA sequences encoding the target antigen through in vitro transcription, encapsulating them in lipid nanoparticles (LNPs), and then introducing them into the human body. The antigens are directly translated and expressed in the cytoplasm, inducing a dual humoral and cellular immune response. Compared to traditional vaccines, mRNA vaccines offer advantages such as not requiring live virus culture, shorter development cycles, no risk of infection or insertional mutations, and the ability to precisely control antigen expression levels and duration through optimization of the untranslated region (UTR) sequence and delivery system. Moderna's mRNA-1893 Zika virus vaccine has completed Phase I clinical trials, inducing a significant neutralizing antibody response at a 100 μg dose, and demonstrating good immunogenicity in both previously exposed and unexposed flavivirus individuals.

[0009] Based on mRNA technology, circular RNA (circRNA) vaccines, as a new generation of nucleic acid vaccine platforms, have shown greater application potential and can compensate for the low conversion rate of mRNA vaccines. Circular RNA eliminates the 5' cap and 3' poly(A) tail through covalently closed circularization, which resists degradation by nucleases in vivo. Its stability at 37°C is more than 3 times higher than that of traditional linear mRNA, significantly prolonging antigen expression time. This addresses the shortcomings of existing vaccines, such as short antibody duration and the need for multiple booster immunizations, and also overcomes the problem of unstable in vivo expression of traditional mRNA vaccines. Summary of the Invention

[0010] The purpose of this invention is to address the shortcomings of existing technologies in quadrivalent dengue virus vaccines, such as inconsistent antigen expression, uneven serotype immune responses, and the weak and easily suppressed immunogenicity of DENV-2 type. This invention provides a circular mRNA vaccine for dengue virus; specifically, it relates to a quadrivalent dengue virus EDIII tandem antigen, a circular mRNA molecule encoding this antigen, the vaccine, and its preparation method. The circular mRNA molecule expressing the quadrivalent dengue virus EDIII tandem antigen provided by this invention uniformly expresses the EDIII domains of the four serotypes (DENV-1 to DENV-4). It achieves uniform expression of the four serotype EDIII domains primarily through multiple combinations of the four serotype EDIII domains, the use of T2A / P2A self-cleaving peptides, Furin restriction sites, and the flexible linker peptide (GGGS) in a 4-way arrangement design. This further results in a balanced and efficient immune response in the quadrivalent dengue virus mRNA vaccine containing this circular mRNA molecule, reducing ADE epitope immune responses and effectively avoiding the risk of ADE effects. The process can be scaled up industrially.

[0011] The objective of this invention is achieved through the following technical solution: <First Aspect> This invention relates to a tetravalent dengue virus EDⅢ tandem antigen, the structure of which is tpA signal peptide-D2EDⅢ-(GGGS)4-D2EDⅢ-Furin restriction site-D3EDⅢ-T2A self-cleaving peptide-D4EDⅢ-P2A self-cleaving peptide-D1EDⅢ, wherein D1EDⅢ is mutated by the F108A ADE epitope.

[0012] Preferably, the amino acid sequence of the dengue virus tetravalent EDIII tandem antigen is shown in SEQ ID NO.1.

[0013] Preferably, the nucleotide sequence of the dengue virus tetravalent EDIII tandem antigen is shown in SEQ ID NO.25.

[0014] <Second aspect> This invention relates to a circular mRNA sequence expressing the above-mentioned tetravalent dengue virus EDIII tandem antigen, comprising exon E1 / E2 sequence, IRES sequence, tpA signal peptide sequence, nucleotide sequence of the above-mentioned tetravalent dengue virus EDIII tandem antigen, and stop codon sequence.

[0015] Preferably, the circular mRNA sequence expressing the above-mentioned dengue virus tetravalent EDⅢ tandem antigen is as shown in SEQ ID NO.2, or is a circular mRNA sequence with more than 90% homology.

[0016] <Third aspect> This invention relates to a nucleotide sequence comprising an EcoRI restriction site, a T7 promoter sequence, intron fragment I, an IRES sequence, a tpA signal peptide sequence, the nucleotide sequence of the aforementioned dengue virus tetravalent EDIII tandem antigen, a stop codon sequence, intron fragment II, and an XbaI restriction site, which can be used to prepare a circular mRNA sequence expressing the aforementioned dengue virus tetravalent EDIII tandem antigen in vitro.

[0017] Preferably, the nucleotide sequence is as shown in SEQ ID NO.3, or a nucleotide sequence with more than 90% homology to SEQ ID NO.3.

[0018] <Fourth Aspect> This invention relates to a plasmid vector comprising the above-described nucleotide sequence. Preferably, the plasmid vector is obtained by inserting the above-described nucleotide sequence into the pUC57 vector via double digestion with EcoRI and XbaI.

[0019] <Fifth Aspect> This invention relates to a dengue virus circular mRNA vaccine comprising the aforementioned circular mRNA molecule expressing the tetravalent dengue virus EDIII tandem antigen and lipid nanoparticles.

[0020] <Sixth Aspect> This invention relates to a recombinant *Escherichia coli* strain, which is obtained by transforming *E. coli* with the aforementioned plasmid vector. This recombinant *E. coli* strain can be used for the mass production of the aforementioned plasmid vector, which contains the aforementioned nucleotide SEQ ID NO.3, and is subsequently used for the in vitro preparation of dengue virus circular mRNA and its vaccine.

[0021] <Seventh Aspect> This invention relates to a strain of Escherichia coli PC004, with accession number CCTCC M2026349.

[0022] The Escherichia coli PD004 of this invention was deposited with the China Center for Type Culture Collection on February 5, 2026, at Wuhan University, Wuhan, China, with accession number CCTCC M2026349.

[0023] Compared with the prior art, the present invention has the following beneficial effects: This invention primarily utilizes multiple combinations of four serotype EDⅢ domains, a T2A / P2A self-cleaving peptide, a Furin restriction site, and a flexible linker peptide (GGGS) to design a tetravalent dengue virus EDⅢ tandem antigen, a circular mRNA molecule encoding this antigen, a vaccine, and a method for its preparation. This ensures consistent in vitro expression levels of the four serotype EDⅢ domain antigens, resulting in a highly efficient and balanced immune response to the E protein of the four serotypes. This overcomes the shortcomings of existing technologies where the immunogenicity of DENV-2 is weak and easily suppressed, effectively avoiding the risk of ADE (antidepressant effect), and the process can be scaled up industrially. Attached Figure Description

[0024] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 The results of Western blot analysis of circular mRNA expression of the D1EDⅢ domain; Figure 2 The results of Western blot analysis of circular mRNA expression of the D2EDⅢ domain; Figure 3 The results of Western blot analysis of circular mRNA expression of the D3EDⅢ domain; Figure 4 The results of Western blot analysis of circular mRNA expression of the D4EDⅢ domain; Figure 5 The results of D1-E antibody titer assay in mice immunized with circular mRNA vaccine; Figure 6 The results show the titer of D2-E antibodies in mice immunized with a circular mRNA vaccine. Figure 7 The results of D3-E antibody titer assay in mice immunized with circular mRNA vaccine; Figure 8 The results show the titer of D4-E antibodies in mice immunized with a circular mRNA vaccine. Detailed Implementation

[0025] The present invention will be described in detail below with reference to embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0026] Example 1: Construction of gene template plasmid; To express different tandem combinations of the EDIII domain from four different dengue virus serotypes, this embodiment selected a circular mRNA technology platform for expression differential verification. The template plasmid construction method for the circular mRNA technology platform was as follows: EcoRI restriction site, T7 promoter, intron fragment I, IRES, different tandem combinations of the EDIII domain from the four different serotypes, intron fragment II, and XbaI restriction site were sequentially linked, followed by double digestion with EcoRI and XbaI and insertion into the pUC57 vector. The sequences used to construct the vector plasmid are shown in Table 1. To facilitate the detection of EDIII expression from the four different serotypes using different constructed circular mRNAs, different tags were added to the C-terminus of the EDIII amino acid sequences of the four different serotypes. Specifically, a 6*His tag was added to the C-terminus of D1EDIII, a Flag tag was added to the C-terminus of D2EDIII, an HA tag was added to the C-terminus of D3EDIII, and a Myc tag was added to the C-terminus of D4EDIII. The tandem combinations of the EDⅢ domains of the four different serotypes could be directly linked sequentially or randomly linked using a combination of T2A / P2A self-cleaving peptide, Furin restriction site, and flexible linker peptide (GGGS). The EDⅢ domains, tags, and linker peptide sequences of the four different serotypes are shown in Table 2. Gene fragment synthesis and vector plasmid cloning and construction were outsourced to Suzhou GenScript Biotech Co., Ltd.

[0027] Table 1. Sequences used for vector plasmid construction

[0028] Table 2. Sequences used in the construction of antigen tandem combinations

[0029] .

[0030] Example 2: Preparation of circular mRNA; Each antigen combination vector plasmid synthesized and constructed by Genscript Biotech was digested with BsaI to convert the plasmid DNA into linear cDNA. The enzyme digestion reaction system is as follows: 1 mg of plasmid DNA 2 ml of 10× buffer solution (100mM Tris-HCl pH 7.0, 100mM MgCl2, 1mg / ml BSA), 1 ml of BsaI enzyme Add enzyme-free water to bring the total to 20ml.

[0031] The enzyme was digested overnight at 37°C. The digested products were recovered using a universal DNA recovery kit (Tiangen Biotech Co., Ltd.).

[0032] The recovered linear cDNA template was subjected to in vitro transcription. The in vitro transcription reaction system is as follows: 1 mg of linear cDNA 2 ml of 10× transcription buffer (400mM Tris-HCl pH 8.0, 200mM MgCl2, 50mM DTT, 10mM spermidine) 1 ml of NTP (20 mM each), T7 RNA polymerase 200 KU, RNase inhibitor 2KU, Add enzyme-free water to a final volume of 20ml.

[0033] Incubate at 37°C and 220 rpm with shaking for 3-5 hours, then add 1 KU DNase I and react at 37°C for 15-30 min. The crude transcript is then purified using a centrifugal column RNA purification kit (NEB).

[0034] The purified linear RNA was cyclized in the following system: 1 mg of linear RNA 1 ml of 10× cyclization buffer (500mM Tris-HCl pH 7.0, 100mM MgCl2, 20mM DTT), GTP 0.5ml, Add enzyme-free water to bring the volume to 10ml.

[0035] Incubate at 55°C for 15-30 min, then add 5 ml of 7.5M lithium chloride solution and incubate overnight at -20°C. Centrifuge at 5000 rpm for 20 min at 4°C using a refrigerated centrifuge, collect the precipitate, wash the precipitate twice with ice-cold anhydrous ethanol, dissolve the precipitate in 1 ml of enzyme-free water, and determine the mRNA concentration using Nanodrop.

[0036] Example 3: Expression and detection of circular mRNA; The circular mRNAs of each antigen combination prepared in Example 2 were transfected into 24-well plates containing monolayers of 293T cells using the TransIT®-mRNATransfection Kit at a rate of 500 ng / well. The transfection conditions were as follows: Circular mRNA 500 ng / well TransIT 2μl / well Boost 2μl / well 293T cells 1×10 5 / hole, 1 ml of DMEM complete culture medium per well; Cells were incubated in 24-well plates at 37°C and 5% CO2 for 48 hours, and then collected. Since the four different serotypes had different tags attached to the EDIII terminus, antibodies corresponding to the tags were used for detection. The levels of these tags corresponded to the EDIII expression levels of each serotype. Western blotting was performed on cells transfected with circular mRNA of each antigen combination using their respective tag antibodies. The primary antibody used was the tag antibody, and the secondary antibody was HRP-labeled goat anti-rabbit antibody (Thermo). The 6×His tag antibody, Flag tag antibody, HA tag antibody, and Myc tag antibody were all from Yisheng Biotechnology (Shanghai) Co., Ltd. Results are shown below. Figures 1-4 A total of 13 antigen combinations were tested. Among them, the expression levels of EDⅢ in the four serotypes of antigen combination DENV-PD004 (structure: tpA signal peptide-D2EDⅢ-(GGGS)4-D2EDⅢ-Furin-D3EDⅢ-T2A-D4EDⅢ-P2A-D1EDⅢ) were relatively uniform and high. Therefore, it was selected for further study.

[0037] Example 4: E. coli transformation and preparation of recombinant plasmids The plasmid corresponding to the antigen combination DENV-PD004 obtained in Example 3 was transformed into *E. coli* DH5α competent cells using the calcium chloride method and plated on LB agar plates containing the corresponding antibiotic (100 μg / ml ampicillin). The plates were incubated at 37°C. When colonies were clearly visible, plump single colonies were picked and placed in 10 ml of liquid LB agar containing the corresponding antibiotic (100 μg / ml ampicillin). The culture was incubated at 37°C until the OD600 reached 1.0-3.0, yielding the seed culture. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) A 3% inoculum was used to inoculate the seed culture into a 5L fermenter for fermentation. When the OD600 value of the cells reached approximately 80-100, the cells were collected by centrifugation. Excess seed culture was added to a final concentration of 8% glycerol and stored at -80℃. The resulting culture was named *Escherichia coli* PD004. After sequencing verification, it was deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC M 2026349.

[0038] Take 200g of harvested E. coli fermentation cells and add 1.6L of 50mM Tris-1 solution (stored at 4℃) at a cell mass to solution volume ratio of 1:8. HL, 10mM EDTA, pH 8.0), resuspend the bacterial cells by stirring. Then add 1.6L of Solution II (0.2M NaOH, 1.0% SDS), stir slowly to mix, and let stand for 3 minutes. After 10 min, add 1.6 L of pre-cooled Solution III (3M KAC, pH 5.5) at 4℃, stir slowly to mix, and let stand for 30 min. Finally, add 1.6 L of 2M CaCl2 solution, stir slowly to mix, and let stand for 1 h. The lysis solution separates into two layers: a solid upper layer and a liquid lower layer. The liquid is pumped out using a peristaltic pump and filtered through a 1.0 μm pore size filter to remove impurities. The lysis solution is then filtered through a Quickstand ultrafiltration system using a 300 KD hollow fiber ultrafiltration membrane and concentrated for 5 minutes. The plasmid was diluted 10-fold to obtain a crude pure solution. This crude solution was then purified using an AKTA purifier system, employing both a DEAE (GE Healthcare Life Science, USA) and a Capto™ Core 700 (GE Healthcare Life Science, USA) column according to the supplier's recommended buffer system. Finally, the solution was concentrated and percolated using a Quickstand ultrafiltration system with a 100KD hollow fiber ultrafiltration membrane (GE Healthcare Life Science, USA, membrane area 500 cm², membrane fiber tube diameter 1 mm). The membrane negative pressure (TMP) was 1 psi, and the concentration factor was 5. 10 times the volume, with a percolation ratio of 4. The template plasmid DNA corresponding to the antigen combination DENV-PD004 was prepared by multiplying the antigen by 12.

[0039] Example 5: Preparation and Analysis of Circular mRNA Vaccine To further investigate the immunogenicity of the antigen combination DENV-PD004 obtained in Example 3 against four serotypes in vivo, the plasmid DNA prepared in Example 4 was first used to prepare circular mRNA according to the method in Example 2. The prepared circular mRNA was diluted to 200 μg / mL with 10 mM citrate / sodium citrate buffer at pH 4.0 to prepare solution I. Cationic lipid SM102, cholesterol (5-cholesterol-3β-ol), DSPC (distearate phosphatidylcholine), and PEG-DMG (polyethylene glycol dimyristate) were dissolved in ethanol at a molar ratio of 50:38.5:10:1.5 to obtain solution II. Solution I and solution II were mixed using a microfluidic device at an N / P ratio of 7:1 at a flow rate of 20 ml / min. The mixture was immediately diluted with Tris-HCl solution (pH 7.4, containing 8% sucrose (w / v)), and the ethanol in the solution was removed by tangential flow filtration and concentrated to a circular mRNA concentration of 50 μg / mL to obtain the dengue virus circular mRNA vaccine.

[0040] The particle size, polydispersity index (PDI), and surface potential of LNP particles were measured using dynamic light scattering on a Malvern Zetasizer Nano-ZEN 3600 (Malvern) potential-laser particle size analyzer. The encapsulation efficiency was determined using the Quant-iT™ RiboGreen™ RNA Assay Kit (Invitrogen™ R11490). All four physical parameters of the vaccine sample were within the theoretical range and met the requirements for vaccine immunization (results are shown in Table 3).

[0041] Table 3 Characterization of dengue virus circular mRNA vaccine samples

[0042] Example 6: Animal immunization and detection; To investigate the immunogenicity of the dengue virus circular mRNA vaccine against four serotypes, Balb / c mice were immunized with the aforementioned dengue virus circular mRNA vaccine at doses of 2 μg, 5 μg, 10 μg, and 20 μg, with five mice immunized at each dose. A booster immunization with the same dose was administered three weeks after the initial immunization. Mice injected with saline served as negative controls. Blood was collected on day 14 post-boost immunization to separate serum. Antibody titers in the separated serum were detected using an indirect ELISA method based on the E protein of the four serotypes. Recombinant E proteins for all four serotypes were purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd. The main steps of the indirect ELISA detection method are as follows: (1) Dilute the recombinant E protein to 2 μg / ml with 0.1M bicarbonate buffer (pH 9.6), add 100 μl / well to a 96-well microplate (Costar), and incubate overnight at 4°C. After washing 3 times, add 5% BSA and block at 37°C for 2 hours. Pat dry, dry at 37°C, and store at 4°C for later use.

[0043] (2) The collected mouse serum was diluted 100 times with PBS and then serially diluted. Each dilution of the sample was added to the 96-well plate coated in the previous step and reacted at 37 °C for 1 hour. After washing 3 times, 100 μl of HRP-labeled rabbit anti-mouse IgG (Sigma) was added to each well and reacted at 37 °C for 1 hour. After washing 5 times, TMB substrate (Beyotime) was added and the color was developed for 10 min. The OD value at 450 nm was measured.

[0044] (3) Antibody titer calculation: The antibody titer is the highest serum dilution factor whose OD value is greater than twice the OD value of the PBS control well.

[0045] The detection of recombinant E protein in four serotypes and the antibody titer in the serum of mice immunized with dengue virus circular mRNA vaccine are as follows: Figures 5-8 The results showed that the serum of mice immunized with the circular mRNA vaccine produced high levels of antibodies against the E protein of all four serotypes, and the antibody titers remained consistent at both low and high doses. This indicates that the vaccine can be stably and uniformly expressed in vivo and provides balanced immune responses to each serotype, effectively avoiding the risk of ADE effects.

[0046] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A tetravalent dengue virus EDIII tandem antigen, the structure of which is tpA signal peptide - D2EDIII-(GGGS)4-D2EDIII-Furin restriction site - D3EDIII-T2A self-cleaving peptide - D4EDIII-P2A self-cleaving peptide - D1EDIII; wherein, The amino acid sequence of D1EDⅢ is shown in SEQ ID NO.10, the amino acid sequence of D2EDⅢ is shown in SEQ ID NO.12, the amino acid sequence of D3EDⅢ is shown in SEQ ID NO.14, and the amino acid sequence of D4EDⅢ is shown in SEQ ID NO.

16.

2. The dengue virus tetravalent EDIII tandem antigen according to claim 1, characterized in that, The amino acid sequence of the tetravalent dengue virus EDIII tandem antigen is shown in SEQ ID NO.1; preferably, the nucleotide sequence of the tetravalent dengue virus EDIII tandem antigen is shown in SEQ ID NO.

25.

3. A circular mRNA sequence expressing the tetravalent dengue virus EDIII tandem antigen as described in claim 1, characterized in that, The circular mRNA sequence includes exon E1 / E2 sequence, IRES sequence, tpA signal peptide sequence, nucleotide sequence of the dengue virus tetravalent EDIII tandem antigen, and stop codon sequence.

4. The circular mRNA sequence according to claim 3, characterized in that, The circular mRNA sequence is shown in SEQ ID NO.2, or is an RNA sequence with more than 90% homology to SEQ ID NO.

2.

5. A nucleotide sequence, characterized in that, The nucleotide sequence includes an EcoRI restriction site, a T7 promoter sequence, intron fragment I, an IRES sequence, a tpA signal peptide sequence, the nucleotide sequence of the dengue virus tetravalent EDIII tandem antigen as described in claim 1, a stop codon sequence, intron fragment II, and an XbaI restriction site.

6. The nucleotide sequence according to claim 5, characterized in that, The nucleotide sequence is as shown in SEQ ID NO.3, or is a nucleotide sequence with more than 90% homology to SEQ ID NO.

3.

7. A plasmid vector, characterized in that, The plasmid vector contains the nucleotide sequence as described in claim 5; preferably, the plasmid vector is constructed by inserting the nucleotide sequence into the pUC57 vector via double digestion with EcoRI and XbaI.

8. A dengue virus circular mRNA vaccine, characterized in that, The circular mRNA vaccine comprises a circular mRNA sequence expressing the tetravalent dengue virus EDIII tandem antigen as described in claim 3 and lipid nanoparticles.

9. A recombinant Escherichia coli, characterized in that, The recombinant Escherichia coli is obtained by transforming the plasmid vector as described in claim 7 into Escherichia coli.

10. An Escherichia coli PC004 strain, with accession number CCTCC NO: M 2026349.

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