Modified mrna vaccine for dengue virus

Abstract

The present invention provides a modified mRNA vaccine for dengue virus. Specifically, the present invention provides an mRNA vaccine composition for dengue virus, comprising: (a) mRNA for expressing an immunogen of dengue virus, the immunogen of dengue virus being selected from the group consisting of E80 protein (i.e. the extra-membrane portion of envelope protein E), prME protein, NS1, or a combination thereof; and (b) a pharmaceutically acceptable carrier. Experiments show that the mRNA vaccine of the present invention can induce a super-high level of neutralizing antibodies, which is 10-50 times higher than the antibody level induced by other vaccines in mice, and can induce a strong T cell response against dengue virus, thereby providing complete protection for the immunized animals.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and more specifically relates to a modified mRNA vaccine for dengue virus. Background Technology

[0002] Dengue virus belongs to the Flaviviridae family and the Flavivirus genus. It is transmitted by mosquitoes such as Aedes aegypti and Aedes albopictus, and can cause severe symptoms such as dengue fever and dengue hemorrhagic fever. It is widely distributed in tropical and subtropical regions worldwide, infecting approximately 390 million people annually. Most dengue virus infections are asymptomatic, while those who do develop symptoms experience headache, fever, muscle pain, and arthritis; severe cases can be life-threatening. If a dengue patient is reinfected with a different serotype of dengue virus, the likelihood of developing severe dengue fever increases.

[0003] Dengue virus is an enveloped, single-stranded RNA virus containing four serotypes (DENV-1, DENV-2, DENV-3, and DENV-4), encoding three structural proteins (C, prM, and E) and seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). The envelope protein E is a transmembrane protein, with its extramembrane portion comprising 80% of the total protein (E80), and consists of three domains: EDI, EDII, and EDIII.

[0004] Currently, recombinant subunit vaccines for dengue virus mainly include subunit protein vaccines, adenovirus vector vaccines, and DNA vaccines.

[0005] An effective vaccine should be able to prevent all four serotypes of dengue virus simultaneously. Currently, the only commercially available dengue vaccine is the quadrivalent chimeric attenuated vaccine (Dengvaxia) developed by Sanofi Pasteur in France. However, in early 2018, 17 children who received this vaccine died from dengue virus infection, and some others developed rashes. Sanofi states that the vaccine is protective for those who have already been exposed to mosquito-borne virus infection, but may cause infection to worsen in those who have not been previously exposed.

[0006] Therefore, there is an urgent need in this field to develop new dengue virus vaccines that can more effectively induce neutralizing antibodies against dengue virus and provide more effective protection. Summary of the Invention

[0007] The purpose of this invention is to provide a new dengue virus vaccine that more effectively induces neutralizing antibodies against dengue virus and provides more effective protection.

[0008] In a first aspect of the invention, a dengue virus mRNA vaccine composition is provided, the vaccine composition comprising:

[0009] (a) mRNA expressing a dengue virus immunogen, wherein the dengue virus immunogen is selected from the group consisting of: E80 protein (i.e., the extracellular portion of envelope protein E), prME protein, NS1, or combinations thereof; and

[0010] (b) Pharmaceutically acceptable carriers.

[0011] In another preferred embodiment, the dengue virus is selected from the group consisting of serotypes DENV-1, DENV-2, DENV-3, DENV-4, or combinations thereof.

[0012] In another preferred embodiment, the dengue virus is serotype DENV-2.

[0013] In another preferred embodiment, the mRNA has the structure of Formula I:

[0014] Z1-Z2-Z3-Z4-Z5-Z6 (I)

[0015] In the formula,

[0016] Z1 is an uncapped or 5' capped component;

[0017] Z2 is a 5'-UTR element;

[0018] Z3 is a signal peptide coding sequence, preferably Z3 is an IgE signal peptide coding sequence;

[0019] Z4 is the coding sequence for the dengue virus immunogen, which is selected from the following groups: E80 protein, prME protein, and NS1;

[0020] Z5 is a 3-UTR component;

[0021] Z6 is a polyA tail component.

[0022] In another preferred embodiment, the 5' capping element is selected from the group consisting of: cap0 (m7G5'ppp5'Np), cap1 (m7G5'ppp5'NmpNp) and cap2 (m7G5'ppp5'NmpNmpNp); preferably cap1.

[0023] In another preferred embodiment, the mRNA is encapsulated in liposomes to form liposome nanoparticles.

[0024] In another preferred embodiment, the average particle size of the liposome nanoparticles is 10-500 nm, more preferably 20-250 nm, and even more preferably 50-100 nm.

[0025] In another preferred embodiment, the liposome nanoparticles comprise an outer liposome layer and an inner mRNA.

[0026] In another preferred embodiment, the components of the liposome layer are selected from the group consisting of PEG-modified lipids (such as DMG-PEG2000), neutral lipids (such as DSPC), cationic lipids (such as D-Lin-MC3-DMA), cholesterol, or combinations thereof.

[0027] In another preferred embodiment, the liposome layer of the nanoparticles comprises PEG-modified lipids (DMG-PEG 2000), neutral lipids (DSPC), cationic lipids (D-Lin-MC3-DMA), and cholesterol.

[0028] In another preferred embodiment, the PEG-modified lipid is selected from the group consisting of DMG-PEG 2000, C14-PEG2000, C16-PEG2000, or combinations thereof.

[0029] In another preferred embodiment, the cationic lipid is selected from the group consisting of DODAP, D-Lin-MC3-DMA, DODMA, or combinations thereof.

[0030] In another preferred embodiment, the vaccine composition includes mRNA for expressing the E80 protein and mRNA for expressing the NS1 protein.

[0031] In another preferred embodiment, the molar ratio of the mRNA used to express the E80 protein to the mRNA used to express the NS1 protein is 1:99 to 99:1, more preferably 1:10 to 10:1.

[0032] In another preferred embodiment, the dengue virus immunogen includes E80, prME, and NS1 of dengue virus type II.

[0033] In another preferred embodiment, the mRNA itself in the vaccine composition may also act as an adjuvant.

[0034] In a second aspect of the invention, an mRNA is provided having the structure of Formula I:

[0035] Z1-Z2-Z3-Z4-Z5-Z6 (I)

[0036] In the formula,

[0037] The definitions of Z1, Z2, Z3, Z4, Z5, and Z6 are as described above.

[0038] In another preferred embodiment, the mRNA is present in the form of liposome nanoparticles.

[0039] In another preferred embodiment, the liposome nanoparticles comprise an outer liposome layer and an inner mRNA.

[0040] In a third aspect of the invention, there is provided the use of the dengue virus mRNA vaccine composition of the first aspect of the invention or the mRNA of the second aspect of the invention, which is used to prepare a medicament for (a) preventing dengue virus; (b) inducing the production of dengue virus-specific antibodies in mammals; and / or (c) inducing a dengue virus-T cell response in mammals.

[0041] In a fourth aspect of the invention, a method for preparing liposome nanoparticles encapsulated with mRNA is provided, comprising the steps of:

[0042] (S1) Provide mRNA having the structure of Formula I:

[0043] Z1-Z2-Z3-Z4-Z5-Z6 (I)

[0044] In the formula,

[0045] The definitions of Z1, Z2, Z3, Z4, Z5, and Z6 are as described above; and

[0046] (S2) The mRNA is mixed with liposome nanoparticles and incubated to form liposome nanoparticles encapsulating mRNA.

[0047] In another preferred embodiment, in step (S2), mixing and incubation are carried out at 35°C–42°C.

[0048] In another preferred embodiment, the incubation time is 0.1-3 hours, more preferably 0.5-1.5 hours.

[0049] In another preferred embodiment, the mRNA is prepared using the following steps:

[0050] (i) Obtain a linearized product by PCR, the linearized product containing the coding sequence of the dengue virus immunogen;

[0051] (ii) Using the linearized product as a template, the initial mRNA sequence is obtained by in vitro transcription;

[0052] (iii) Add 5'cap1 (N7mGpppGm) to the 5' end of the mRNA sequence and add a poly(A) tail to the 3' end to obtain the mRNA with translational function.

[0053] In another preferred embodiment, the mRNA is mixed with empty liposome nanoparticles.

[0054] In another preferred embodiment, the empty-loaded liposome nanoparticles are prepared using the following steps.

[0055] (W1) Four lipids were dissolved in ethanol. These four lipids were PEG-modified lipids (DMG-PEG 2000) and neutral lipids (DSPC) from Avanti Polar Lipids, cationic lipids (D-Lin-MC3-DMA) from Medchemexpress, and cholesterol from Sigma.

[0056] (W2) Four lipids are mixed in a predetermined molar ratio and homogenized by a liposome extruder to obtain empty liposome nanoparticles.

[0057] In another preferred embodiment, the molar ratio is DLin-MC3-DMA (MC3): DSPC: cholesterol: DMG-PEG 2000 = (50±10): (10±2): (38.5±7.7): (1.5±0.3), more preferably 50:10:38.5:1.5.

[0058] In a fifth aspect of the invention, a method for preventing dengue virus infection is provided, comprising the steps of administering to a desired subject the dengue virus mRNA vaccine composition of the first aspect of the invention, or the mRNA of the second aspect, or liposome nanoparticles containing said mRNA.

[0059] In another preferred embodiment, the objects include humans and non-human mammals (such as rodents, for example, mice and rats).

[0060] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0061] Figure 1The expression levels of DENV2 E80, prME, and NS1 mRNAs in HEK-293T cells and the morphological observation of liposome nanoparticles (LNPs) are shown. (a) Schematic diagram of the mRNA vaccine structure used in this invention. 5'cap1, 5'UTR, signal peptide, antigens (DENV2 E80, prME, and NS1), 3'UTR, and poly(A) tail. (b) mRNA transfection into HEK-293T cells to express the target proteins, analyzed by flow cytometry using antibodies against dengue virus E protein and NS1 protein, respectively. (c) Schematic diagram of LNP structure. (d) Cryo-electron microscopy observation of the morphology and size of the prepared LNPs.

[0062] Figure 2 The immunogenicity of the DENV2 E80-mRNA vaccine in mice is shown. (a) Schematic diagram of Balb / c mice immunized with the mRNA vaccine. A total of three immunizations were administered, each two weeks apart. Spleen and blood samples were collected at week six for immunogenicity testing. (b, c, d) Antibody reaction detection in immune serum. DENV2-specific antibody responses were detected using ELISA assays with DENV2 (16681), E80, and NS1 proteins derived from Drosophila cells (C6 / 36) as coating antigens. Serum from 6 weeks post-immunization was serially diluted for testing. (e) Neutralizing antibody titer detection in immune serum, demonstrating the neutralizing capacity of the immune serum against the DENV2 16681 strain. (f) Antigen-specific T cell response was detected using the Elispot assay. Splenic lymphocytes from mice 6 weeks post-immunization were used for specific stimulation and negative controls to analyze IFN-γ production. Four peptide libraries covering the entire E80 protein (P1, P2, P3, P4) and the NS1 single peptide P... 265-273 Specific stimulation of splenic lymphocytes was performed using PBS and PBS, respectively, with PBS serving as a negative control. Figure 2 In numbers b through 2e, each symbol represents a mouse. The horizontal line represents the mean of that group.

[0063] Figure 3The results of dose investigation of DENV2 E80-mRNA and NS1-mRNA are shown. Mice were immunized three times with LNP-mRNA vaccines containing 20 μg E80, 10 μg E80, 5 μg E80, 20 μg NS1, 10 μg NS1, and 5 μg NS1, respectively. (a, b, c) ELISA experiments were performed using DENV2 (16681), E80, and NS1 proteins derived from Drosophila cells (C6 / 36) as coating antigens to detect DENV2-specific antibody responses. Serum was serially diluted 6 weeks after immunization. (d) Neutralizing antibody titers of the immune serum were detected, demonstrating the neutralizing capacity of the immune serum against the DENV2 16681 strain. (e) Antigen-specific T-cell responses were detected using the Elispot assay. Splenic lymphocytes from mice 6 weeks after immunization were used for specific stimulation and negative controls to analyze IFN-γ production. Using two peptide libraries (P2 and P4) of E80, and NS1 single peptide P 265-273 Specific stimulation of splenic lymphocytes was performed using PBS and PBS, respectively, with PBS serving as a negative control. Figure 3 In numbers a through 3d, each symbol represents a mouse. The horizontal line represents the mean of that group.

[0064] Figure 4 The protective effect of the DENV2 vaccine on mice was demonstrated. Mice were immunized three times with 10 μg E80, 10 μg NS1, 10 μg E80+NS1 LNP mRNA vaccines, and Empty-LNP mRNA vaccine, respectively. Two weeks after the three immunizations, mice were challenged, and blood samples were collected on days 2, 3, and 4 post-challenge, as well as a spleen sample on day 4, to detect viral load. (a, b, c) DENV2-specific antibody responses were detected using ELISA assays with DENV2 (16681), E80, and NS1 proteins derived from Drosophila cells (C6 / 36) as coating antigens. Serum from mice 6 weeks post-immunization was serially diluted. (d) Neutralizing antibody titers in the immune serum were measured, demonstrating the neutralizing capacity of the immune serum against DENV2 16681 and DENV2 LP strains. (e) Antigen-specific T-cell responses were detected using the Elispot assay. Splenic lymphocytes from mice 6 weeks post-immunization were used for specific stimulation and negative control experiments to analyze IFN-γ production. Using two peptide libraries (P2 and P4) of E80, and NS1 single peptide P 265-273 Specific stimulation of splenic lymphocytes was performed using PBS, with PBS serving as a negative control. (f, g) Viral load in mouse blood and in mouse spleen on day 4 post-challenge were detected using qRT-PCR. Figure 4In the symbols a, b, c, d, f, and g, each symbol represents a mouse. The horizontal line represents the mean of the group.

[0065] Figure 5 The antibody-dependent enhancement response (ADE) of DENV2 was demonstrated. (a) Serum from patients immunized with 10 μg of the E80, E80+NS1, and Empty-LNP mRNA vaccines was used to detect the ADE of DENV2. The serum was serially diluted and incubated with the virus at 37°C for 1 h, and then used to infect K562 cells. After 48 h, these cells were stained with antibodies and analyzed by flow cytometry. (b) Antibody typing experiments were performed on serum from patients immunized with 10 μg of the E80, E80+NS1, and Empty-LNP mRNA vaccines. Detailed Implementation

[0066] Through extensive and in-depth research, the inventors have developed, for the first time, a novel mRNA vaccine encapsulated in liposome nanoparticles—a modified mRNA vaccine targeting dengue virus type II. Unexpectedly, this mRNA vaccine not only enhances specific T-cell responses but also induces extremely high levels of neutralizing antibodies, 10-50 times higher than the antibody levels induced by other vaccines in mice. This invention was completed based on these findings.

[0067] Specifically, the inventors successfully prepared three mRNA vaccines against dengue virus type II using liposome nanoparticles to encapsulate mRNA vaccines, targeting the viral prME protein, the extracellular region of the envelope protein E gene (E80), and the NS1 protein, respectively. Experimental results showed that all three mRNAs prepared in vitro could express specific proteins intracellularly; the prepared LNPs had a stable structure and morphology, and high efficiency in encapsulating mRNA; the LNP-encapsulated mRNA could effectively express proteins in mice; all three mRNA vaccines (E80-mRNA, prME-mRNA, and NS1-mRNA) could induce specific antibody and T-cell responses; and the E80-mRNA and E80+NS1-mRNA vaccines provided complete protection in mice.

[0068] the term

[0069] As used herein, the terms “mRNA of the present invention”, “mRNA of the present invention for the prevention of dengue virus”, “mRNA molecule of the present invention”, and “mRNA sequence of the present invention” are used interchangeably to refer to mRNA oligonucleotides having the structure of Formula I.

[0070] dengue virus

[0071] Dengue virus is an enveloped single-stranded RNA virus, classified into four serotypes (DENV-1, DENV-2, DENV-3, and DENV-4), which encode three viral structural proteins (C, prM, and E) and seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). 1 The E80 protein is the extracellular domain of the envelope protein (accounting for 80% of the N-terminus of the E protein), while the NS1 protein is a non-structural protein.

[0072] Dengue virus infection causes flu-like symptoms, and infection with the four types of dengue virus can worsen the symptoms, increasing the difficulty of prevention.

[0073] mRNA vaccine

[0074] This invention also provides an mRNA vaccine for dengue virus prevention and a method for preparing the same. The dengue virus mRNA vaccine of this invention has advantages such as relatively long expression time, strong immunogenicity, and good protective effect.

[0075] Typically, the method of the present invention includes: firstly obtaining the expression gene (or coding sequence) of the antigen protein by PCR, then obtaining the corresponding mRNA by in vitro transcription technology, and finally encapsulating the mRNA with liposome nanoparticles to obtain a highly efficient mRNA vaccine.

[0076] mRNA vaccines are in vitro prepared mRNA vaccines with expressive activity. Their main structure includes 5' and 3' UTRs and an open reading frame containing the expressed antigen. Compared to DNA vaccines, they do not require any nuclear localization signals and pose no risk of integration into the genome. mRNA vaccines encapsulated in liposomes do not require any other adjuvants; the liposomes and RNA themselves act as adjuvants. Furthermore, they can simultaneously activate both MHCII and MHCII presentation pathways, thereby significantly improving the immunogenicity of the vaccine.

[0077] To enhance the expression level of mRNA vaccines, this invention selects a series of nucleic acid elements that promote mRNA expression. These elements include (but are not limited to): a 5' UTR, a signal peptide, an antigen sequence, and a 3' UTR.

[0078] In this invention, the preferred dengue virus proteins are E80 and NS1 proteins. In the mRNA vaccine of this invention, the mRNAs encoding these dengue virus proteins are respectively...

[0079] In this invention, SEQ ID No: 1 is the dengue virus type II prME DNA sequence; SEQ ID No: 3 is the dengue virus type II E80 DNA sequence; and SEQ ID No: 5 is the dengue virus type II NS1 DNA sequence. The mRNA sequence corresponds to SEQ ID No.: 1, 3, or 5, respectively.

[0080] In addition, SEQ ID Nos. 2, 4 and 6 provide the amino acid sequences of dengue virus type II prME, E80 and NS1, respectively.

[0081] In a preferred embodiment, the mRNA vaccine of the present invention comprises mRNA targeting dengue virus E80 and NS1 in 1 to 4 of the four serotypes (DENV-1, DENV-2, DENV-3 and DENV-4).

[0082] Liposome nanoparticles

[0083] The present invention also provides a liposome nanoparticle, which is used to encapsulate an in vitro prepared mRNA vaccine, thereby assisting the mRNA vaccine to enter the animal body and express proteins.

[0084] In another preferred embodiment, the liposome nanoparticles comprise four components: DLin-MC3-DMA (MC3), DSPC, cholesterol, and DMG-PEG 2000. These four lipids can form stable liposome nanoparticles in vitro, protecting mRNA from degradation by external nucleases and facilitating mRNA entry into the animal body to exert its function.

[0085] In another preferred embodiment, the liposome nanoparticles are relatively uniform in size, approximately 80 nm, and can be effectively transported within an animal body.

[0086] Composition and method of application

[0087] The present invention also provides a composition comprising the liposome nanoparticles of the present invention and the mRNA vaccine of the present invention.

[0088] The compositions of the present invention include pharmaceutical compositions and vaccine compositions.

[0089] The compositions of the present invention can be monovalent (for one dengue virus serotype) or polyvalent (for four dengue virus serotypes); at the same time, they can contain only one nucleotide (E80 or NS1) or multiple nucleotides (E80 plus NS1).

[0090] The pharmaceutical or vaccine compositions of the present invention can be prepared into various dosage forms, including (but not limited to): injections, suspensions, sprays, etc.

[0091] The main advantages of this invention include:

[0092] (1) A modified dengue virus mRNA vaccine encapsulated by liposome nanoparticles was developed for the first time;

[0093] (2) The E80-mRNA vaccine of the present invention can produce high titers of neutralizing antibodies in mice;

[0094] (3) The E80-mRNA and NS1-mRNA vaccines of the present invention can generate specific T cell responses in mice;

[0095] (4) The E80-mRNA vaccine of the present invention can improve complete protection in mice and clear viremia.

[0096] (5) The NS1-mRNA of the present invention can basically clear viremia.

[0097] (6) Combined immunization with NS1-mRNA and E80-mRNA vaccines showed a synergistic effect, specifically: a) both provided 100% complete protection against viral infection in mice ( Figure 4 f, g); b) Enhance the intensity of E protein-specific T cell responses ( Figure 4 f); c) Reduced ADE side effects of DENV2 ( Figure 5 a) and d) altered the distribution of IgG subclasses. Figure 5 b) Increase Th1 T cell response and decrease Th2 T cell response.

[0098] (7) The method for preparing the mRNA vaccine of the present invention is convenient and quick, and the speed is better than other existing vaccine technologies.

[0099] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in *Molecular Cloning: A Laboratory Manual* by Sambrook J. et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0100] Unless otherwise specified, all experimental materials and reagents used in the following examples are available from commercially available sources.

[0101] Sequence information

[0102] SEQ ID No: 1 and 2 are the prME DNA sequence and amino acid sequence of dengue virus type II;

[0103] SEQ ID Nos. 3 and 4 are the DNA and amino acid sequences of dengue virus type II E80;

[0104] SEQ ID Nos. 5 and 6 are the DNA and amino acid sequences of dengue virus type II NS1.

[0105] SEQ ID No: 7 is a 5'UTR element;

[0106] SEQ ID No: 8 is the signal peptide coding sequence;

[0107] SEQ ID No: 9 is a 3'UTR element.

[0108] Materials and methods

[0109] 1. Cells, viruses

[0110] C6 / 36 cells and Vero cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were cultured in DMEM medium containing 10% bovine serum. The DENV2 strain used in this invention was strain 16681.

[0111] 2. Antibodies

[0112] Anti-dengue virus antibody (purchased from Genetex); Anti-dengue virus NS1 protein antibody (purchased from Sigma)

[0113] 3. mRNA preparation

[0114] Three plasmids (5'UTR-IgE-E80-3'UTR-puc57; 5'UTR-IgE-prME-3'UTR-puc57; 5'UTR-IgE-NS1-3'UTR-puc57) were synthesized from Jereh Biotechnology. The target fragments were amplified from these three plasmids using PCR technology, and the purified PCR products were used as templates for in vitro transcription. The corresponding mRNAs were then prepared using NEB's mRNA Cap 2´-O-Methyltransferase kit and E. coli Poly(A) Polymerase kit. Finally, the integrity and size of the mRNAs were observed by agarose gel electrophoresis.

[0115] 4. Transfect HEK-293T cells to express proteins

[0116] The prepared mRNA was transfected into HEK-293T cells using the commercially available Lipsome2000 transfection kit. The cells were cultured in 10% FBSDMEM medium. Cells were harvested 36 hours after transfection, and the target protein was then detected. Untransfected cells served as a negative control (Mock group).

[0117] 5. FACS detection of protein expression

[0118] The collected cells were fixed and perforated using 4% PFA and perforation solution. They were stained with a specific antibody against DENV2 (4G2) and a FITC-labeled anti-mouse antibody. Finally, the expression level of the target protein was analyzed by flow cytometry.

[0119] 6. Liposome nanoparticles encapsulate mRNA

[0120] The four lipids (D-Lin-MC3-DMA, DSPC, cholesterol, and DMG-PEG 2000) were dissolved in ethanol and mixed in a certain molar ratio (D-Lin-MC3-DMA: DSPC: cholesterol: DMG-PEG2000 = 50:10:38.5:1.5). The liposome mixture was added to a liposome extruder (Avestin LF1) and passed through an 80 nm filter membrane. The prepared liposome nanoparticles and mRNA were then incubated for a certain period of time to finally obtain a liposome-encapsulated mRNA vaccine.

[0121] 7. Mouse Immunization

[0122] The animal experiments were approved by the Animal Committee of the Shanghai Pasteur Institute. All Balb / c mice used in this experiment were purchased from Vital River Pharmaceuticals.

[0123] Mice were immunized with Empty-LNP, E80-mRNA, prME-mRNA, and NS1-mRNA, respectively. Empty-LNP (LNP without mRNA encapsulation) served as a negative control. Immunization was performed three times, at weeks 0, 2, and 4, by intramuscular injection of 40 μl per mouse. At week 6, mice were sacrificed, and spleens and blood were collected for Elispot assays, ELISA assays, and neutralization titer determination.

[0124] 8. Specific T cell detection assay

[0125] The spleen of the mouse was removed, and 1 ml of 1640 medium was added to a sterile culture dish. The cells were then ground in a 40 μm cell sieve. The collected immune cells from the spleen were lysed using erythrocyte lysis buffer, and then counted. 1.65 × 10⁶ cells were added to each well of an Elispot 96-well plate coated with anti-IFN-γ. 6 Spleen cells were treated with different stimulants: DENV2's E804 peptide library (P1, P2, P3, P4), and DENV2's NS1 single peptide P. 265-273 PBS was used as a negative control. After 48 hours, the cells were washed off, stained with IFN-γ specific antibody, then incubated with streptavidin-HRP at room temperature for 1 hour, and developed with BCP / NCIP substrate. Finally, the spots were counted using a CTL instrument.

[0126] 9. Antibody titer detection experiment

[0127] ELISA assay: ELISA plates were coated with DENV2 virus from C6 / 36 cells and viral culture supernatant from Vero cells (containing a large amount of secretory NS1 protein), respectively. After blocking with 5% milk, 100 μl of serum at 2-fold serial dilutions was added to each well and incubated at 37°C for 1 h. Then, IgG-HRP secondary antibody was added, and finally, TMB chromogenic solution was used for color development. The reaction was terminated with 1M HCl, and the OD450 was read on a microplate reader.

[0128] PRNT assay: Serum samples were incubated at 56°C for 30 min to inactivate complement. One day in advance, 70,000 Vero cells were seeded into 48-well plates. The next day, serum was serially diluted, with 50 μl of serum and 100 PFU of dengue virus 16681 per well incubated at 37°C for 1 hour. The cells were then added to the seeded 48-well plates for infection. Finally, the virus and serum complexes were removed, and the cells were cultured for four days with 700 μl of 1.5% CMC. On the fourth day, the cell plates were harvested, and virus-infected cells were fixed with 4% PFA. After staining with anti-dengue virus antibody for 3 h, secondary antibody (mouse antibody) was added and incubated at 37°C for 2 h. Finally, color development was performed using a BCP / NCIP kit, and the cells were observed and counted. The neutralizing titer in the serum sample was EC50, indicating the serum dilution that inhibited 50% of virus-infected cells.

[0129] 10. Statistics

[0130] All statistical analyses were performed using GraghPadPrism5. Specific IgG antibody responses and neutralizing titers for DENV2 and NS1 were analyzed using a two-tailed Student's t-test.

[0131] Example 1 II Dengue virus mRNA vaccine

[0132] 1.1 mRNA vaccine

[0133] In this embodiment, the constructed mRNA vaccine includes prME, E80, and NS1, as shown in the schematic diagram below. Figure 1 As shown in a. mRNA-prME, mRNA-E80, and mRNA-NS1 express prME, E80, and NS1 proteins, respectively.

[0134] HEK-293T cells were transfected with the three mRNAs mentioned above, and untransfected cells were used as a negative control (Mock group). The expressed proteins were identified using flow cytometry. Cells were collected 36 hours after transfection and stained with dengue virus E protein (4G2) and NS1 protein-specific antibodies, followed by FITC-labeled secondary antibody staining. Finally, the expression of specific proteins was detected by flow cytometry.

[0135] like Figure 1 As shown in b, the results indicate that all three mRNAs can express the corresponding proteins intracellularly, namely prME protein, E80 protein, and NS1 protein.

[0136] 1.2 Liposome Nanoparticles (LNP)

[0137] The inventors constructed liposome nanoparticles using four lipid components. The lipids were dissolved in anhydrous ethanol and mixed in a specific ratio. The nanoparticles were then homogenized using a liposome extruder equipped with an 80nm filter membrane. A schematic diagram of the structure is shown below. Figure 1 As shown in c.

[0138] To verify whether the liposome nanoparticles (LNPs) were successfully prepared, the LNPs were observed using cryo-electron microscopy.

[0139] The results show that, Figure 1 As shown in d, most LNPs have a particle size of around 80 nm.

[0140] Example 2: DENV2 E80-mRNA, prME-mRNA, and NS1-mRNA vaccines induced T cell and antibody responses.

[0141] In this embodiment, the immunogenicity of modified nucleotide (1mψ) E80-mRNA, prME-mRNA and NS1-mRNA vaccines was evaluated by antibody reaction and T cell reaction.

[0142] 2.1 Antibody reaction

[0143] BALB / cA mice (n=4 per group) were immunized with 20 μg of E80-mRNA, prME-mRNAP, NS1-mRNA, and Empty-LNP mRNA vaccines at weeks 0, 2, and 4, respectively, with Empty-LNP serving as a negative control. Serum and spleen were collected at week 6. ELISA experiments were performed using DENV2(16681) virus, E80 protein, and NS1 protein derived from insect cells as coating antigens to detect specific antibody responses.

[0144] like Figure 2 As shown in b, c, and d, the E80 mRNA vaccine produced strong specific antibodies against both DENV2 and E80 proteins; the prME-mRNA vaccine produced specific antibodies against DENV2, but no significant antibody response against E80. The NS1-mRNA vaccine produced strong specific antibodies against the NS1 protein.

[0145] like Figure 2 As shown in e, the E80-mRNA vaccine immune serum has a strong neutralizing effect on the virus.

[0146] 2.2 T cell response

[0147] For the ELISPOT experiment, four peptide libraries from DENV2 E80 and NS1 single peptide P were used. 265-273 PBS and splenic lymphocytes were specifically stimulated, with PBS serving as a negative control, to detect DENV2-specific T cell responses.

[0148] like Figure 2 As shown in f, the results indicate that all three vaccine groups—E80-mRNA, prME-mRNAP, and NS1-mRNA—produced strong T-cell responses.

[0149] Example 3: Different doses of E80 and NS1 mRNA vaccines can all produce strong T-cell responses and specific antibody levels.

[0150] In this embodiment, the immunization effects of different doses of E80-mRNA-LNP and NS1-mRNA-LNP vaccines were evaluated.

[0151] 3.1 Specific antibody response

[0152] Seven groups of BALB / cA mice (n=3 per group) were immunized at weeks 0, 2, and 4 with E80-mRNA (20 μg), E80-mRNA (10 μg), E80-mRNA (5 μg), NS1-mRNA (20 μg), NS1-mRNA (10 μg), NS1-mRNA (5 μg), and empty-LNP (empty-LNP), respectively, with Empty-LNP serving as a negative control. Serum and spleen were collected at week 6. ELISA experiments were performed using insect cell-derived DENV2 (16681), E80 protein, and NS1 protein as coating antigens to detect DENV2-specific antibody responses.

[0153] like Figure 3 As shown in a, 3b, and 3c, the results indicate that both the E80-mRNA and NS1-mRNA vaccines produced significant antibody responses, with no significant differences between different doses.

[0154] In addition, such as Figure 3 As shown in d, the three groups of E80-mRNA vaccine immune sera produced a strong neutralizing effect on the standard strain DENV2 (16681), and there was no significant difference in neutralizing titers among the three groups.

[0155] 3.1 T cell response

[0156] For the ELISPOT experiment, two peptide libraries, P2 and P4, from DENV2 E80, and the NS1 single peptide P were used. 265-273 PBS and splenic lymphocytes were specifically stimulated, with PBS serving as a negative control, to detect DENV2-specific T cell responses.

[0157] like Figure 3 As shown in e, the results indicate that all six groups of vaccines, including E80-mRNA and NS1-mRNA, produced strong T-cell responses.

[0158] The above results indicate that different doses of the E80 and NS1 mRNA vaccines of the present invention can produce strong T-cell responses and specific antibody levels.

[0159] In subsequent experiments, representative 10 μg E80 and 10 μg NS1 mRNA vaccines were selected for immune protection experiments.

[0160] Example 4: DENV2 mRNA vaccine can protect mice against DENV2 virus.

[0161] In this embodiment, the protective efficacy of the mRNA vaccine was further evaluated. The method is as follows: Four immunization groups were set up. BALB / cA mice (n=10 per group) were immunized with 10 μg of E80-mRNA, NS1-mRNA, E80+NS1 mRNA, and unencapsulated LNP (Empty-LNP) at weeks 0, 2, and 4, respectively, with Empty-LNP serving as a negative control. At week 6, anti-IFNRα / β antibodies were injected intraperitoneally, and subcutaneous challenge with DNEV2-LP was initiated the following day. Blood samples were collected on days 2, 3, and 4 post-challenge, and the spleen was collected on day 4 for viral load detection.

[0162] result

[0163] like Figure 4 As shown in a, 4b, and 4c, all three groups of immune sera containing E80-mRNA, NS1-mRNA, and E80+NS1-mRNA produced specific antibody responses.

[0164] Figure 4 The results show that the above-mentioned E80-mRNA and E80+NS1-mRNA vaccines have a strong neutralizing effect on both the standard strain DENV2 (16681) and the DENV2-LP used for challenge in our laboratory.

[0165] Figure 4 e indicates that all three groups of immune sera produced a strong T-cell response.

[0166] like Figure 4 As shown in f and 4g, on day 4, the viral load in the blood of mice in the Empty-LNP group was significantly different from that in the E80-mRNA, NS1-mRNA, and E80+NS1 mRNA groups, and the viral load in the blood of the E80-mRNA, NS1-mRNA, and E80+NS1 mRNA groups was completely cleared on day 4. On day 4, a small amount of virus was detected in the spleen of the NS1-mRNA immunized group, while no virus was detected in the E80-mRNA and E80+NS1-mRNA immunized groups.

[0167] The above results indicate that the mRNA-E80 and mRNA-E80-NS1 vaccines can completely protect BALB / cA mice against DENV2 virus, and the mRNA-NS1 vaccine can clear viremia, but a small amount remains in the spleen.

[0168] Example 5: Antibody-dependent enhancement response and antibody typing of DENV2

[0169] 5.1 Detection of Antibody-Dependent Enhancement (ADE)

[0170] Serum samples from patients immunized with E80-mRNA, E80+NS1-mRNA, and Empty-LNP mRNA vaccines were used to detect the antibody-dependent enhancement (ADE) response of DENV2 (16681).

[0171] The results are as follows Figure 5 As shown in Figure a, the viral infection rate reached its maximum (approximately 10%) in the serum of the E80-mRNA immunization group at a dilution of around 25,600. The viral infection rate in the serum of the E80+NS1-mRNA immunization group reached its maximum (approximately 7%) at dilutions between 25,600 and 102,400. This indicates that when E80 and NS1 are used in combination, the NS1 antibody can reduce the ADE response of the E80-mRNA vaccine, playing a synergistic protective role. This is beneficial for improving the protective effect of the vaccine and reducing or minimizing side effects.

[0172] 5.2 Antibody typing

[0173] Antibody typing experiments were performed on serum samples from the three vaccines administered above.

[0174] The results are as follows Figure 5 As shown in b, the proportion of IgG1 in the E80-NS1-mRNA-immunized serum is slightly higher than that in the E80-mRNA-immunized serum, indicating a stronger Th2 cell response; while the proportion of IgG2a in the E80-mRNA-immunized serum is slightly higher than that in the E80-NS1-mRNA-immunized serum, indicating a stronger Th1 cell response.

[0175] discuss

[0176] Currently, there is no specific treatment for dengue virus, making the development of a vaccine with good preventive effects extremely urgent.

[0177] Several aspects of dengue virus warrant attention, primarily antibody-dependent enhancement (ADE) and vascular leakage caused by the NS1 protein. 2 The ADE reaction mainly refers to the development of lifelong immunity to a specific type of dengue virus after initial infection. When the body is reinfected with a different type of virus, some weakly neutralizing or non-neutralizing antibodies bind to the virus, forming antigen-antibody complexes. These antibodies, relying on their Fcγ-terminus, bind to the Fcγ receptors on macrophages, promoting viral aggregation and infecting macrophages, thus exacerbating disease progression. 3-5 The NS1 protein is part of the viral replication complex. NS1 hexamers released into the extracytoplasm may disrupt endothelial glycogen through inflammation-dependent or inflammation-independent pathways, thereby causing antigenemia and pro-inflammatory responses, increasing the risk of vascular leakage. 6, 7In dengue virus vaccine research, the E protein has become the main target antigen due to its multiple neutralizing sites, while the NS1 protein, which lacks neutralizing sites, is often overlooked.

[0178] Compared to traditional subunit vaccines, mRNA vaccines offer advantages such as simpler administration, shorter treatment time, and higher potency. While both mRNA and DNA vaccines belong to the category of nucleic acid vaccines, mRNA vaccines do not require any nuclear localization signals and have no possibility of integration into the genome.

[0179] Currently, this technology has not been applied to the field of dengue virus. This invention is the first to prepare a dengue virus mRNA vaccine encapsulated by liposome nanoparticles, aiming to reduce the cost of vaccine preparation and achieve balanced protection against four serotypes of the virus.

[0180] The experimental results of this invention demonstrate that the prepared liposome nanoparticles (LNPs) can effectively encapsulate mRNA vaccines, forming highly immunogenic mRNA vaccines without the need for other adjuvants. In in vitro cell experiments, all three mRNA vaccines, prME, E80, and NS1, expressed specific proteins intracellularly.

[0181] In in vivo animal immunization experiments, all three mRNA vaccines produced strong T-cell responses and antibody levels, with E80 inducing the highest titer of neutralizing antibodies. Subsequent in vivo protective experiments also confirmed that the E80-mRNA vaccine, NS1-mRNA, and E80+NS1-mRNA all provided good protective effects.

[0182] Surprisingly, the E80-mRNA vaccine can induce extremely high levels of neutralizing antibodies. Figure 4 e) It induces antibody levels 10-50 times higher in mice than other vaccines; it also provides complete protection (100% complete protection) in mice, meaning that dengue virus is undetectable in the blood and spleen. Figure 4 f,g).

[0183] In addition, immunization with NS1-mRNA alone can provide partial protection in mice from viremia. Figure 4 g, Day 4).

[0184] Furthermore, synergistic effects were observed when NS1-mRNA and E80-mRNA vaccines were administered in combination. Specifically, this manifested as: a) both vaccines providing 100% complete protection against viral infection in mice (…). Figure 4 f, g); b) Enhance the intensity of E protein-specific T cell responses ( Figure 4 f); c) Reduced ADE side effects of DENV2 ( Figure 5 a) and ) altered the distribution of IgG subclasses ( Figure 5b) This indicates that Th1 T cell responses were increased and Th2 T cell responses were decreased.

[0185] Since the E80-mRNA vaccine already possesses excellent protective efficacy (100% complete protection), the protective efficacy of the NS1-mRNA+E80-mRNA vaccine in combined immunization remains 100%. However, the increased intensity of the E protein-specific T-cell response, the reduced ADE side effects of DENV2, and the altered distribution of IgG subclasses all suggest a synergistic effect. For example, the reduced ADE side effects of DENV2 are more conducive to providing protection and may further reduce the risk of infection and severe illness in animals after infection.

[0186] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0187] References

[0188] 1. Simmons, CP, Farrar, JJ, Nguyen v, V, and Wills, B (2012). Dengue. The New England journal of medicine366: 1423-1432.

[0189] 2. Guzman, MG, Halstead, SB, Artsob, H, Buchy, P, Farrar, J, Gubler, DJ, et al. (2010). Dengue: a continuing global threat. Nature reviewsMicrobiology8: S7-16.

[0190] 3.Halstead, SB, Nimmannitya, S, and Cohen, SN (1970). Observationsrelated to pathogenesis of dengue hemorrhagic fever. IV. Relation of diseaseseverity to antibody response and virus recovered. The Yale journal ofbiology and medicine42: 311-328.

[0191] 4.Beatty, PR, Puerta-Guardo, H, Killingbeck, SS, Glasner, DR,Hopkins, K, and Harris, E (2015). Dengue virus NS1 triggers endothelialpermeability and vascular leak that is prevented by NS1 vaccination. Sciencetranslational medicine7: 304ra141.

[0192] 5.Wilder-Smith A, OE, Horstick O, Wills B. (2019). Dengue. Lancet393: 350.

[0193] 6.Mackenzie, JM, Jones, MK, and Young, PR (1996). Immunolocalizationof the dengue virus nonstructural glycoprotein NS1 suggests a role in viralRNA replication. Virology220: 232-240.

[0194] 7. Hafirassou, ML, Meertens, L, Umana-Diaz, C, Labeau, A, Dejarnac, O, Bonnet-Madin, L, et al. (2018). A Global Interactome Map of the Dengue VirusNS1 Identifies Virus Restriction and Dependency Host Factors. Cell reports22:1364. sequence list <110> Shanghai Public Health Clinical Center <120> Modified mRNA vaccine for dengue virus <130> P2020-0391 <160> 9 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1950 <212> DNA <213> Dengue virus <400> 1 atgattgtgt ctaggcagga gaagggaaag tccctcctgt tcaagacaga ggacggagtg 60 aacatgtgca cactcatggc tatggacctc ggcgagctat gcgaggacac aattacatac 120 aagtgcccac tgctgcggca gaacgagccc gaggacatcg actgctggtg caactctaca 180 tctacctggg tgacatacgg aacatgcaca actatggggg agcacagacg cgagaagcgg 240 tccgtggctc tcgtgcccca cgtgggaatg ggcctggaga cccggacaga gacctggatg 300 tcttctgagg gcgcttggaa gcacgtgcag cgcattgaga cctggattct ccgccaccct 360 ggcttcacca tgatggccgc tattctcgct tacacaattg gcacaaccca cttccagcgc 420 gctctcattt tcattctgct caccgccgtg accccatcta tgaccatgag atgcatcggc 480 atgtccaacc gcgacttcgt ggagggcgtg tccggcggat cttgggtgga cattgtgctt 540 gagcacgggt cttgcgtgac cacaatggct aagaacaagc ctacactcga cttcgagctg 600 attaagaccg aggctaagca gcccgctaca ctgcgcaagt actgcattga ggctaagctc 660 acaaacacca caacagagtc ccggtgccct acccagggcg agccatctct caacgaggag 720 caggacaagc ggttcgtgtg caagcactct atggtggacc gcggatgggg aaacggatgc 780 gggctgttcg gaaagggcgg aattgtgaca tgcgctatgt tcagatgcaa gaagaacatg 840 gagggaaagg tggtgcagcc tgagaacctt gaatacacca ttgtgattac ccctcactcc 900 ggggaggagc acgccgtggg aaacgacaca ggcaagcacg gaaaggagat taagattacc 960 cctcagtcta gcatcacaga ggccgaattg accggatacg gcacagtgac aatggagtgc 1020 tctcctagaa caggcctcga cttcaacgag atggtgctgc tccagatgga gaacaaggct 1080 tggctcgtgc accgccagtg gttcctcgac ctgccactgc cttggctgcc gggagccgac 1140 acccagggat ctaactggat acagaaggag acactcgtga ccttcaagaa cccccacgct 1200 aagaagcagg acgtggtggt gctgggttca caggagggcg ccatgcacac cgctctgaca 1260 ggcgctacag agattcagat gtctagcggc aacctcctgt tcaccggcca cctgaagtgc 1320 cgcctccgca tggacaagct ccagctgaag ggaatgtcct actctatgtg caccggaaag 1380 ttcaaggtgg tgaaggagat cgctgagacc cagcacggaa ccattgtgat tagagtgcag 1440 tacgagggcg acggcagccc ttgcaagatc cctttcgaga tcatggacct cgaaaagcgc 1500 cacgtgctgg gacgcctgat cacagtgaac ccaattgtga ccgagaagga ctccccagtg 1560 aacattgagg ctgagcctcc tttcggcgac tcttacatca ttatcggagt ggagccgggt 1620 cagctgaagc tcaactggtt caagaagggg tcttccattg gccagatgtt cgagacaaca 1680 atgcgcggcg ccaagcgcat ggctattctc ggcgacacag cttgggactt cgggtccctg 1740 ggcggcgtgt tcaccagcat cggaaaggcc ctccaccagg tgttcggggc tatttacggg 1800 gccgctttca gcggcgtgtc ttggacaatg aagattctga tcggggtgat tattacctgg 1860 atcggaatga actcccggtc taccagcctg tctgtgacac tggtgctcgt gggaattgtg 1920 accctgtacc tgggagtgat ggtgcaggcc 1950 <210> 2 <211> 650 <212> PRT <213> Dengue virus <400> 2 Met Ile Val Ser Arg Gln Glu Lys Gly Lys Ser Leu Leu Phe Lys Thr 1 5 10 15 Glu Asp Gly Val Asn Met Cys Thr Leu Met Ala Met Asp Leu Gly Glu 20 25 30 Leu Cys Glu Asp Thr Ile Thr Tyr Lys Cys Pro Leu Leu Arg Gln Asn 35 40 45 Glu Pro Glu Asp Ile Asp Cys Trp Cys Asn Ser Thr Ser Thr Trp Val 50 55 60 Thr Tyr Gly Thr Cys Thr Thr Met Gly Glu His Arg Arg Glu Lys Arg 65 70 75 80 Ser Val Ala Leu Val Pro His Val Gly Met Gly Leu Glu Thr Arg Thr 85 90 95 Glu Thr Trp Met Ser Ser Glu Gly Ala Trp Lys His Val Gln Arg Ile 100 105 110 Glu Thr Trp Ile Leu Arg His Pro Gly Phe Thr Met Met Ala Ala Ile 115 120 125 Leu Ala Tyr Thr Ile Gly Thr Thr His Phe Gln Arg Ala Leu Ile Phe 130 135 140 Ile Leu Leu Thr Ala Val Thr Pro Ser Met Thr Met Arg Cys Ile Gly 145 150 155 160 Met Ser Asn Arg Asp Phe Val Glu Gly Val Ser Gly Gly Ser Trp Val 165 170 175 Asp Ile Val Leu Glu His Gly Ser Cys Val Thr Thr Met Ala Lys Asn 180 185 190 Lys Pro Thr Leu Asp Phe Glu Leu Ile Lys Thr Glu Ala Lys Gln Pro 195 200 205 Ala Thr Leu Arg Lys Tyr Cys Ile Glu Ala Lys Leu Thr Asn Thr Thr 210 215 220 Thr Glu Ser Arg Cys Pro Thr Gln Gly Glu Pro Ser Leu Asn Glu Glu 225 230 235 240 Gln Asp Lys Arg Phe Val Cys Lys His Ser Met Val Asp Arg Gly Trp 245 250 255 Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Gly Ile Val Thr Cys Ala 260 265 270 Met Phe Arg Cys Lys Lys Asn Met Glu Gly Lys Val Val Gln Pro Glu 275 280 285 Asn Leu Glu Tyr Thr Ile Val Ile Thr Pro His Ser Gly Glu Glu His 290 295 300 Ala Val Gly Asn Asp Thr Gly Lys His Gly Lys Glu Ile Lys Ile Thr 305 310 315 320 Pro Gln Ser Ser Ile Thr Glu Ala Glu Leu Thr Gly Tyr Gly Thr Val 325 330 335 Thr Met Glu Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Val 340 345 350 Leu Leu Gln Met Glu Asn Lys Ala Trp Leu Val His Arg Gln Trp Phe 355 360 365 Leu Asp Leu Pro Leu Pro Trp Leu Pro Gly Ala Asp Thr Gln Gly Ser 370 375 380 Asn Trp Ile Gln Lys Glu Thr Leu Val Thr Phe Lys Asn Pro His Ala 385 390 395 400 Lys Lys Gln Asp Val Val Val Leu Gly Ser Gln Glu Gly Ala Met His 405 410 415 Thr Ala Leu Thr Gly Ala Thr Glu Ile Gln Met Ser Ser Gly Asn Leu 420 425 430 Leu Phe Thr Gly His Leu Lys Cys Arg Leu Arg Met Asp Lys Leu Gln 435 440 445 Leu Lys Gly Met Ser Tyr Ser Met Cys Thr Gly Lys Phe Lys Val Val 450 455 460 Lys Glu Ile Ala Glu Thr Gln His Gly Thr Ile Val Ile Arg Val Gln 465 470 475 480 Tyr Glu Gly Asp Gly Ser Pro Cys Lys Ile Pro Phe Glu Ile Met Asp 485 490 495 Leu Glu Lys Arg His Val Leu Gly Arg Leu Ile Thr Val Asn Pro Ile 500 505 510 Val Thr Glu Lys Asp Ser Pro Val Asn Ile Glu Ala Glu Pro Pro Phe 515 520 525 Gly Asp Ser Tyr Ile Ile Ile Gly Val Glu Pro Gly Gln Leu Lys Leu 530 535 540 Asn Trp Phe Lys Lys Gly Ser Ser Ile Gly Gln Met Phe Glu Thr Thr 545 550 555 560 Met Arg Gly Ala Lys Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp 565 570 575 Phe Gly Ser Leu Gly Gly Val Phe Thr Ser Ile Gly Lys Ala Leu His 580 585 590 Gln Val Phe Gly Ala Ile Tyr Gly Ala Ala Phe Ser Gly Val Ser Trp 595 600 605 Thr Met Lys Ile Leu Ile Gly Val Ile Ile Thr Trp Ile Gly Met Asn 610 615 620 Ser Arg Ser Thr Ser Leu Ser Val Thr Leu Val Leu Val Gly Ile Val 625 630 635 640 Thr Leu Tyr Leu Gly Val Met Val Gln Ala 645 650 <210> 3 <211> 1182 <212> DNA <213> Dengue virus <400> 3 atgcgctgca tcggaatgtc caaccgcgac ttcgtggagg gagtgtccgg cgggtcttgg 60 gtggacattg tgttggagca cgggtcttgc gtgaccacaa tggctaagaa caagcctacc 120 ctcgacttcg agctgattaa gacagaggct aagcagcccg ctacactccg caagtactgc 180 attgaggcta agctcacaaa cacaaccaca gagtcgaggt gccctaccca gggcgagcca tctctcaacg aggagcagga caagcgcttc gtgtgcaagc actctatggt ggaccgcgga tggggaacg gatgcggcct gttcggaag ggcggaattg tgacatgcgc tatgttcaga 360 tgcaagaaga acatggaggg aaaggtggtg cagcccgaga acctaggta cacaattgtg attaccccac actctggcga ggagcacgcc gtgggaacg acaccggaa gcacggaag richness ttacccctca gtcttctatt richness agctgaccgg attack 540 gtgactatgg agtgctctcc tagaacaggc ctcgacttca acgagatggt gctgctccag atggagaaca aggcttggct cgtgcaccgg cagtggttcc tcgacctgcc actgccttgg cttcctggag ccgacaccca gggatctaac tggattcaga aggagacact ggtgacattc 720 aagaaccctc acgctaagaa gcaggacgtg gtggtgctgg ggtcccagga gggcgccatg 780 840. cacaccgctc tgaccggggc cagagatc cagatgtcta gcggcaacct gctgttcaca ggccacctga agtgccgcct gcgcatggac aagctccagc tgaagggcat gtcttactct 900 atgtgcaccg gaaagttcaa ggtggtgaag gagatcgccg agacccagca cggcaccatc 960 gtgattagag tgcagtacga gggcgacggc agcccttgca agattccttt cgagatcatg 1020 gacttagaaa agcgccacgt gctgggacgc ctcatcacag tgaacccaat tgtgacagag 1080 aaggactccc ctgtgaacat tgaggctgag ccacctttcg gcgactccta catcattatc 1140 ggcgtggagc caggacagct gaagctcaac tggttcaaga ag 1182 <210> 4 <211> 394 <212> PRT <213> Dengue virus <400> 4 Met Arg Cys Ile Gly Met Ser Asn Arg Asp Phe Val Glu Gly Val Ser 1 5 10 15 Gly Gly Ser Trp Val Asp Ile Val Leu Glu His Gly Ser Cys Val Thr 20 25 30 Thr Met Ala Lys Asn Lys Pro Thr Leu Asp Phe Glu Leu Ile Lys Thr 35 40 45 Glu Ala Lys Gln Pro Ala Thr Leu Arg Lys Tyr Cys Ile Glu Ala Lys 50 55 60 Leu Thr Asn Thr Thr Thr Glu Ser Arg Cys Pro Thr Gln Gly Glu Pro 65 70 75 80 Ser Leu Asn Glu Glu Gln Asp Lys Arg Phe Val Cys Lys His Ser Met 85 90 95 Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Gly 100 105 110 Ile Val Thr Cys Ala Met Phe Arg Cys Lys Lys Asn Met Glu Gly Lys 115 120 125 Val Val Gln Pro Glu Asn Leu Glu Tyr Thr Ile Val Ile Thr Pro His 130 135 140 Ser Gly Glu Glu His Ala Val Gly Asn Asp Thr Gly Lys His Gly Lys 145 150 155 160 Glu Ile Lys Ile Thr Pro Gln Ser Ser Ile Thr Glu Ala Glu Leu Thr 165 170 175 Gly Tyr Gly Thr Val Thr Met Glu Cys Ser Pro Arg Thr Gly Leu Asp 180 185 190 Phe Asn Glu Met Val Leu Leu Gln Met Glu Asn Lys Ala Trp Leu Val 195 200 205 His Arg Gln Trp Phe Leu Asp Leu Pro Leu Pro Trp Leu Pro Gly Ala 210 215 220 Asp Thr Gln Gly Ser Asn Trp Ile Gln Lys Glu Thr Leu Val Thr Phe 225 230 235 240 Lys Asn Pro His Ala Lys Lys Gln Asp Val Val Val Leu Gly Ser Gln 245 250 255 Glu Gly Ala Met His Thr Ala Leu Thr Gly Ala Thr Glu Ile Gln Met 260 265 270 Ser Ser Gly Asn Leu Leu Phe Thr Gly His Leu Lys Cys Arg Leu Arg 275 280 285 Met Asp Lys Leu Gln Leu Lys Gly Met Ser Tyr Ser Met Cys Thr Gly 290 295 300 Lys Phe Lys Val Val Lys Glu Ile Ala Glu Thr Gln His Gly Thr Ile 305 310 315 320 Val Ile Arg Val Gln Tyr Glu Gly Asp Gly Ser Pro Cys Lys Ile Pro 325 330 335 Phe Glu Ile Met Asp Leu Glu Lys Arg His Val Leu Gly Arg Leu Ile 340 345 350 Thr Val Asn Pro Ile Val Thr Glu Lys Asp Ser Pro Val Asn Ile Glu 355 360 365 Ala Glu Pro Pro Phe Gly Asp Ser Tyr Ile Ile Ile Gly Val Glu Pro 370 375 380 Gly Gln Leu Lys Leu Asn Trp Phe Lys Lys 385 390 <210> 5 <211> 1068 <212> DNA <213> Dengue virus <400> 5 gactccggat gcgtggtgtc ttggaagaac aaggagttga agtgcgggtc cggaattttc 60 attaccgaca acgtgcacac ctggacagag cagtacaagt tccagccaga gtcccctagc 120 aagctcgcat cggctattca gaaggctcac gaggagggaa tttgcgggat tcggtctgtg 180 acccgcttag agaacctgat gtggaagcag attacccctg agctgaacca catcctgtct 240 gagaacgagg tgaagctgac cattatgaca ggcgacatta agggcatcat gcaggccgga 300 aagcgcagcc tcaggcccca gcctacagaa ctaaagtact cttggaaaac atggggaaag 360 gctaagatgc tgtctaccga gtctcacaac cagaccttcc tgatcgacgg gccagagacc 420 gccgagtgcc caaacacaaa ccgcgcttgg aactccttag aagtggagga ctacgggttc 480 ggcgtgttca ccaccaacat ttggctcaag ctgaaggaga agcaggacgt gttctgcgac 540 tctaagctga tgagcgccgc tattaaggac aacagggctg tgcacgccga catgggatac 600 tggattgagt ccgccctcaa cgacacatgg aagattgaga aggcttcttt cattgaggtg 660 aagaactgcc actggcctaa gtcccacacc ctgtggtcta acggcgtgtt ggaatctgag 720 atgattattc caaagaacct cgctggcccc gtgtcccagc acaactacag acccggatac 780 cacacccaga tcaccgggcc ttggcacctg ggcaagttgg agatggactt cgacttctgc 840 gacggcacaa cagtggtggt gaccgaggac tgcggaaaca gggggccatc tctgcgcaca 900 accaccgcct cgggcaagct cattacagag tggtgctgta ggtcttgcac actgcctcct 960 ctccgctacc gcggcgagga cggatgctgg tacggcatgg agattcgccc actcaaggag 1020 aaggaggaga acctggtgaa ctctctcgtg acagccggcc acggatga 1068 <210> 6 <211> 355 <212> PRT <213> Dengue virus <400> 6 Asp Ser Gly Cys Val Val Ser Trp Lys Asn Lys Glu Leu Lys Cys Gly 1 5 10 15 Ser Gly Ile Phe Ile Thr Asp Asn Val His Thr Trp Thr Glu Gln Tyr 20 25 30 Lys Phe Gln Pro Glu Ser Pro Ser Lys Leu Ala Ser Ala Ile Gln Lys 35 40 45 Ala His Glu Glu Gly Ile Cys Gly Ile Arg Ser Val Thr Arg Leu Glu 50 55 60 Asn Leu Met Trp Lys Gln Ile Thr Pro Glu Leu Asn His Ile Leu Ser 65 70 75 80 Glu Asn Glu Val Lys Leu Thr Ile Met Thr Gly Asp Ile Lys Gly Ile 85 90 95 Met Gln Ala Gly Lys Arg Ser Leu Arg Pro Gln Pro Thr Glu Leu Lys 100 105 110 Tyr Ser Trp Lys Thr Trp Gly Lys Ala Lys Met Leu Ser Thr Glu Ser 115 120 125 His Asn Gln Thr Phe Leu Ile Asp Gly Pro Glu Thr Ala Glu Cys Pro 130 135 140 Asn Thr Asn Arg Ala Trp Asn Ser Leu Glu Val Glu Asp Tyr Gly Phe 145 150 155 160 Gly Val Phe Thr Thr Asn Ile Trp Leu Lys Leu Lys Glu Lys Gln Asp 165 170 175 Val Phe Cys Asp Ser Lys Leu Met Ser Ala Ala Ile Lys Asp Asn Arg 180 185 190 Ala Val His Ala Asp Met Gly Tyr Trp Ile Glu Ser Ala Leu Asn Asp 195 200 205 Thr Trp Lys Ile Glu Lys Ala Ser Phe Ile Glu Val Lys Asn Cys His 210 215 220 Trp Pro Lys Ser His Thr Leu Trp Ser Asn Gly Val Leu Glu Ser Glu 225 230 235 240 Met Ile Ile Pro Lys Asn Leu Ala Gly Pro Val Ser Gln His Asn Tyr 245 250 255 Arg Pro Gly Tyr His Thr Gln Ile Thr Gly Pro Trp His Leu Gly Lys 260 265 270 Leu Glu Met Asp Phe Asp Phe Cys Asp Gly Thr Thr Val Val Val Thr 275 280 285 Glu Asp Cys Gly Asn Arg Gly Pro Ser Leu Arg Thr Thr Thr Ala Ser 290 295 300 Gly Lys Leu Ile Thr Glu Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro 305 310 315 320 Leu Arg Tyr Arg Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu Ile Arg 325 330 335 Pro Leu Lys Glu Lys Glu Glu Asn Leu Val Asn Ser Leu Val Thr Ala 340 345 350 Gly His Gly 355 <210> 7 <211> 44 <212> DNA <213> Artificial Sequence <400> 7 aaataagaga gaaaagaaga gtaagaagaa atataagagc cacc 44 <210> 8 <211> 54 <212> DNA <213> Artificial Sequence <400> 8 atggactgga cctggattct gttcctcgtg gccgccgcta ctcgtgtgca ctct 54 <210> 9 <211> 110[[ID=三十二]] <212> DNA <213> Artificial Sequence <400> 9 tgataatagg ctggagcctc ggtggccatg cttcttgccc cttgggcctc cccccagccc 60 ctcctcccct tcctgcaccc gtacccccgt ggtctttgaa taaagtctga 110

Claims

1. A dengue virus mRNA vaccine composition, characterized in that, The vaccine composition contains: (a) mRNA expressing the dengue virus immunogen, wherein the dengue virus immunogen is the E80 protein, the extracellular portion of envelope protein E; and (b) A pharmaceutically acceptable carrier; The vaccine composition further contains mRNA for expressing the dengue virus immunogen NS1, and the molar ratio of mRNA for expressing the E80 protein to mRNA for expressing the NS1 protein is 1:10 to 10:

1. The mRNA described has the structure of Formula I: Z1-Z2-Z3-Z4-Z5-Z6 (I) In the formula, Z1 is an uncapped or 5' capped component; Z2 is a 5'-UTR element; Z3 is the signal peptide coding sequence; Z4 is the coding sequence for the dengue virus immunogen, in which the dengue virus immunogen is either the E80 protein or the NS1 protein; Z5 is a 3'-UTR element; Z6 is a polyA tail component; The amino acid sequence of the E80 protein is shown in SEQ ID No: 4, and the amino acid sequence of the NS1 protein is shown in SEQ ID No:

6. The mRNA is encapsulated in liposomes, thereby forming liposome nanoparticles; The dengue virus mentioned is serotype DENV-2.

2. The vaccine composition according to claim 1, characterized in that, The weight ratio of mRNA for expressing E80 protein to mRNA for expressing NS1 protein in the vaccine composition is 5 μg: 5 μg.

3. The vaccine composition according to claim 2, characterized in that, The mRNA described has the structure of Formula I: Z1-Z2-Z3-Z4-Z5-Z6 (I) In the formula, Z1 is a 5' capped component; Z2 is a 5'-UTR element; Z3 is the signal peptide coding sequence; Z4 is the coding sequence for the dengue virus immunogen, in which the dengue virus immunogen is either the E80 protein or NS1; Z5 is a 3-UTR component; Z6 is a polyA tail component.

4. The vaccine composition according to claim 3, characterized in that, Z3 is the coding sequence for the IgE signal peptide.

5. The vaccine composition according to claim 1, characterized in that, The average particle size of the liposome nanoparticles is 10-500 nm.

6. The vaccine composition according to claim 1, characterized in that, The liposome nanoparticles comprise an outer liposome layer and an inner mRNA layer.

7. The vaccine composition according to claim 6, characterized in that, The components of the liposome layer are selected from the group consisting of PEG-modified lipids, neutral lipids, cationic lipids, cholesterol, or combinations thereof.

8. The vaccine composition according to claim 7, characterized in that, The liposome layer of the nanoparticles includes DMG-PEG2000, DSPC, D-Lin-MC3-DMA, and cholesterol.

9. The vaccine composition according to claim 8, characterized in that, The molar ratio was DLin-MC3-DMA:DSPC:cholesterol:DMG-PEG2000 = (50±10):(10±2):(38.5±7.7):(1.5±0.3).

10. The vaccine composition according to claim 7, characterized in that, The PEG-modified lipids are selected from the group consisting of DMG-PEG 2000, C14-PEG2000, C16-PEG2000, or combinations thereof.

11. An mRNA combination, characterized in that, It consists of mRNA for expressing E80 protein and mRNA for expressing NS1 protein, and the molar ratio of mRNA for expressing E80 protein to mRNA for expressing NS1 protein is 1:10 to 10:

1. The mRNA described has the structure of Formula I: Z1-Z2-Z3-Z4-Z5-Z6 (I) In the formula, Z1 is an uncapped or 5' capped component; Z2 is a 5'-UTR element; Z3 is the signal peptide coding sequence; Z4 is the coding sequence for the dengue virus immunogen, in which the dengue virus immunogen is either the E80 protein or the NS1 protein; Z5 is a 3'-UTR element; Z6 is a polyA tail component; The amino acid sequence of the E80 protein is shown in SEQ ID No: 4, and the amino acid sequence of the NS1 protein is shown in SEQ ID No:

6.

12. The mRNA combination as described in claim 11, characterized in that, Z3 is the coding sequence for the IgE signal peptide.

13. Use of a dengue virus mRNA vaccine composition as claimed in claim 1 or the mRNA composition as claimed in claim 11, characterized in that, The preparation is used to develop a drug for (a) preventing dengue virus; (b) inducing specific antibodies against dengue virus in mammals; and / or (c) inducing T-cell responses against dengue virus in mammals.

14. A method for preparing liposome nanoparticles encapsulated with mRNA, characterized in that, Including steps: (S1) Provides an mRNA combination consisting of mRNA for expressing E80 protein and mRNA for expressing NS1 protein, wherein the molar ratio of mRNA for expressing E80 protein to mRNA for expressing NS1 protein is 1:10 to 10:

1. The mRNA described has the structure of Formula I: Z1-Z2-Z3-Z4-Z5-Z6 (I) In the formula, Z1 is an uncapped or 5' capped component; Z2 is a 5'-UTR element; Z3 is the signal peptide coding sequence; Z4 is the coding sequence for the dengue virus immunogen, in which the dengue virus immunogen is either the E80 protein or the NS1 protein; Z5 is a 3-UTR component; Z6 is a polyA tail component; The amino acid sequence of the E80 protein is shown in SEQ ID No: 4, and the amino acid sequence of the NS1 protein is shown in SEQ ID No: 6; and (S2) The mRNA is mixed with liposome nanoparticles and incubated to form liposome nanoparticles encapsulating mRNA.

15. The method as described in claim 14, characterized in that, Z3 is the coding sequence for the IgE signal peptide.

16. The method as described in claim 14, characterized in that, In step (S2), mixing and incubation are carried out at 35°C–42°C.

17. The method as described in claim 14, characterized in that, The incubation time is 0.1-3 hours.

18. The method as described in claim 14, characterized in that, The mRNA was prepared using the following steps: (i) Obtain a linearized product by PCR, the linearized product containing the coding sequence of the dengue virus immunogen; (ii) Using the linearized product as a template, the initial mRNA sequence is obtained by in vitro transcription; (iii) Add 5'cap1 (N7mGpppGm) to the 5' end of the mRNA sequence and add a poly(A) tail to the 3' end to obtain the mRNA with translational function.

19. The method as described in claim 18, characterized in that, The mRNA was mixed with empty liposome nanoparticles.

20. The method as described in claim 19, characterized in that, The empty-loaded liposome nanoparticles were prepared using the following steps. (W1) Four lipids were dissolved in ethanol. These four lipids were PEG-modified lipid DMG-PEG 2000, neutral lipid DSPC, cationic lipid D-Lin-MC3-DMA, and cholesterol. (W2) Four lipids were mixed in a predetermined molar ratio and homogenized by a liposome extruder to obtain empty liposome nanoparticles; the molar ratio was DLin-MC3-DMA:DSPC:cholesterol:DMG-PEG2000=(50±10):(10±2):(38.5±7.7):(1.5±0.3).

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