Recombinant antigen against zika / dengue virus, and vaccine composition thereof and use thereof

Abstract

A recombinant antigen against Zika / Dengue virus, and a vaccine composition thereof and the use thereof. The recombinant antigen is obtained by means of introducing a specific mutation into the FL fusion loop region and optionally the non-FL fusion loop region of the E protein of a wild-type Zika / dengue virus. By means of introducing the specific mutation, the FL epitope of the recombinant antigen is disrupted, which can avoid inducing the generation of antibodies against the FL epitope; thus, the purpose of reducing or eliminating the ADE effect is achieved. In addition, the recombinant antigen has a correct E protein conformation, has a protein expression level similar to or higher than that of a wild type, and can induce a relatively high titer of specific binding antibodies and neutralizing antibodies in an animal body, so that an effective immune protection can be provided, and good clinical application values and industrialization prospects are achieved.

Description

Recombinant antigen targeting Zika / dengue virus, vaccine composition thereof and application thereof

[0001] Cross-references

[0002] This application claims priority to the Chinese patent application filed on January 5, 2024, with application number 202410024322.3 and invention name “Recombinant antigens against Zika / dengue virus, vaccine compositions thereof and their applications”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application relates to the field of biotechnology, and in particular to recombinant antigens targeting Zika / dengue virus, vaccine compositions thereof, and applications thereof. Background Art

[0004] Zika virus (ZIKV) is a mosquito-borne virus belonging to the Flaviviridae family and the genus Flavivirus. It was first discovered in the jungles of Uganda, Africa. In 2007, Zika virus was limited to sporadic outbreaks in Africa and tropical Asia. It was not until an outbreak in the French Polynesian Islands in 2013, followed by an outbreak in the Americas in 2015-2016, that the virus spread to 84 countries worldwide, including China. However, to date, no vaccine or treatment is available. Although the global incidence of ZIKV infection has decreased, ZIKV still poses a threat to people living in endemic areas, making the development of a ZIKV vaccine urgent.

[0005] Dengue virus (DV), the causative agent of dengue fever (DF), is primarily transmitted by vector insects such as Aedes aegypti and Aedes albopictus. It is widely distributed in tropical and subtropical regions. In some countries, DV infection is a leading cause of hospitalization and death among children. According to the World Health Organization (WHO), dengue fever is currently endemic in over 100 countries and regions, with 2.5 to 3 billion people living in dengue-endemic areas worldwide. Annually, approximately 390 million people are infected, 100 million develop clinical symptoms, over 2 million develop severe DF, and over 20,000 die. The structures of ZIKV and dengue viruses are similar: both are spherical and have an envelope containing the envelope (E) protein. The viral genome within is a single-stranded, positive-strand RNA approximately 11 kb in length, containing only a single open reading frame. The translated polyprotein is cleaved into three structural proteins (C, prM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The C protein forms the nucleocapsid. During viral maturation, the prM protein is enzymatically cleaved to form the membrane protein M, which is anchored to the inner layer of the viral envelope and serves as a crucial component in virally induced protective immunity. It also contributes to the correct folding and structural stability of the E protein. The E protein is the primary glycoprotein of the viral envelope and is involved in viral tropism, erythrocyte agglutination, and the production of anti-erythrocyte agglutination and neutralizing antibodies. It is the primary protective antigen for Zika and dengue viruses.

[0006] The E protein exists as a dimer, and each monomer has three domains, namely DI, DII and DIII. Among them, the DII head (amino acids 98-109) contains a highly conserved fusion loop (FL). The sequences of the FL regions of ZIKV and DV viruses are exactly the same, both D98-R99-G100-W101-G102-N103-G104-C105-G106-L107-F108-G109; the FL region plays a key role in the membrane fusion process of viral invasion. During viral infection, immune cells will produce a large number of antibodies against FL.

[0007] DV belongs to the genus Flavivirus in the family Flaviviridae and is divided into four serotypes: 1, 2, 3, and 4, based on antigenicity. DV infection exhibits a typical phenomenon of antibody-dependent infection enhancement (ADE). When infected with a serotype such as DV-1, immune cells produce corresponding anti-DV-1 antibodies. However, if infected with another dengue virus, such as DV-2, the previously produced anti-DV-1 antibodies not only fail to neutralize the DV-2 virus, but may also aggravate the infection.

[0008] Currently, there are two dengue vaccines: Dengvaxia from Sanofi Pasteur and DVax from Takeda Pharmaceuticals, which was recently approved this year. Both are live attenuated vaccines developed with similar principles. The difference is that Dengvaxia uses the attenuated yellow fever virus strain 17D as its vaccine backbone, while DVax uses the attenuated DV-2 strain PDK-53. DVax enhances immune responses and protection against DV-2, but offers less protection against DV-3 and 4. Dengvaxia has demonstrated significant ADE safety concerns in dengue-naive populations. In the Philippines, more than a dozen children have developed severe dengue fever and even died after receiving Dengvaxia. Consequently, the vaccine's use is strictly restricted to individuals with a history of dengue infection. Five-year follow-up data released this year also revealed potential ADE safety risks for DVax in dengue-naive populations.

[0009] Numerous studies have shown that the hydrophobic fusion loop (FL) of the E protein is the primary epitope that induces the ADE effect and is also a major dominant epitope on the E protein. Among antibodies screened from dengue-infected individuals, 41% (46 / 112) targeted the FL (Dejnirattisai W. et al. Nature Immunology, (2016)). To completely eliminate the ADE effect of a vaccine, the common approach is to mutate the FL epitope so that it cannot induce FL antibodies. At the same time, the mutation should not destroy the remaining epitopes of the E protein or cause a significant decrease in protein expression to ensure that the vaccine can induce sufficient neutralizing antibodies and produce immune protection. However, the FL epitope plays an important function in inducing the fusion of the viral envelope and the host cell membrane when the flavivirus invades the host cell. Its amino acid sequence D98-R99-G100-W101-G102-N103-G104-C105-G106-L107-F108-G109 is extremely highly conserved in the entire flavivirus genus. In the inventors' previous studies, when W101 was mutated to any of the remaining 25 amino acids, the E protein was almost not expressed. In the entire flavivirus genus, only a very small number of variant sequences exist in the insect-specific flavivirus FL, which is very distantly related to the dengue / Zika virus. However, when these variant sequences are used in dengue vaccines, the mutations lead to a significant decrease in E protein expression, and the vaccine cannot provide effective immune protection.

[0010] In view of the above-mentioned problems existing in the existing technology, there is an urgent need to develop a Zika / dengue vaccine that can avoid the occurrence of ADE effect and has high immunogenicity. Summary of the Invention

[0011] Purpose of the Invention

[0012] In response to the problems or needs existing in the prior art, the purpose of this application is to provide a Zika / dengue virus recombinant antigen that can avoid the ADE effect, can be efficiently expressed and has high immunogenicity, a vaccine composition based on the recombinant antigen and its application.

[0013] Solution

[0014] To achieve the above objectives, the present invention introduces FL epitope antibodies during Zika virus culture to induce targeted mutations in the viral FL region. Since the amino acid sequence of the Zika virus FL epitope is identical to that of the dengue virus FL epitope, the inventors subsequently introduced the aforementioned point mutations in the Zika virus FL epitope and other positions into a dengue vaccine, thereby obtaining a recombinant E protein antigen specific for Zika / dengue virus. The resulting recombinant E protein antigen specific for Zika / dengue virus can be efficiently expressed, exhibits high immunogenicity against Zika / dengue virus, and avoids inducing the production of FL epitope antibodies, thereby reducing or eliminating the ADE effect.

[0015] Specifically, this application provides the following technical solutions:

[0016] In a first aspect, the present application provides a recombinant antigen comprising the full-length sequence of the E protein of Zika virus or dengue virus, wherein the E protein has a site mutation selected from the following:

[0017] Mutation at G106 in the FL fusion loop region;

[0018] Double mutations at W101 and G106 in the FL fusion loop region;

[0019] Double mutations at W101 and G106 in the FL fusion loop region and mutation of amino acid 125 in the non-FL fusion loop region;

[0020] Double-site mutations at G102 and G106 in the FL fusion loop region;

[0021] Double-site mutations at N103 and G106 in the FL fusion loop region;

[0022] Three point mutations were found in the FL fusion loop region: W101, N103, and G106.

[0023] In a preferred embodiment, the mutation at G106 in the FL fusion loop region is: G106V mutation;

[0024] In a preferred embodiment, the double-site mutations of W101 and G106 in the FL fusion loop region are selected from the group consisting of: (1) W101R and G106V, (2) W101G and G106V, and (3) W101L and G106V;

[0025] In a preferred embodiment, the double-site mutation of W101 and G106 in the FL fusion loop region and the mutation of amino acid 125 in the non-FL fusion loop region are: double-site mutation of W101R and G106V in the FL fusion loop region and mutation of amino acid 125 in the non-fusion loop region to valine;

[0026] In a preferred embodiment, the double-site mutations of G102 and G106 in the FL fusion loop region are: G102R and G106V double-site mutations;

[0027] In a preferred embodiment, the double-site mutation of N103 and G106 in the FL fusion loop region is selected from the group consisting of: (1) N103T and G106V, (2) N103H and G106V, (3) N103K and G106V, (4) N103P and G106V, and (5) N103Y and G106V;

[0028] In a preferred embodiment, the three point mutations of W101, N103 and G106 in the FL fusion loop region are selected from: (1) W101G, N103T and G106V, and (2) W101G, N103K and G106V.

[0029] In some preferred embodiments, the recombinant antigen has the full-length sequence of the E protein of dengue virus type 1, and the E protein has a site mutation selected from the following:

[0030] G106V mutation;

[0031] W101R and G106V double-site mutations;

[0032] W101G and G106V double-site mutations;

[0033] W101L and G106V double-site mutations;

[0034] Three point mutations: W101R, G106V, and L125V;

[0035] G102R and G106V double-site mutations;

[0036] N103T and G106V double-site mutations;

[0037] N103H and G106V double-site mutations;

[0038] N103K and G106V double-site mutations;

[0039] N103P and G106V double-site mutations;

[0040] N103Y and G106V double-site mutations;

[0041] Three point mutations: W101G, N103T, and G106V;

[0042] Three point mutations: W101G, N103K and G106V.

[0043] In other preferred embodiments, the recombinant antigen has the full-length sequence of the E protein of dengue virus type 2, and the E protein has a site mutation selected from the following:

[0044] G106V mutation;

[0045] W101R and G106V double-site mutations;

[0046] W101G and G106V double-site mutations;

[0047] W101L and G106V double-site mutations;

[0048] Three point mutations: W101R, G106V, and M125V;

[0049] G102R and G106V double-site mutations;

[0050] N103T and G106V double-site mutations;

[0051] N103H and G106V double-site mutations;

[0052] N103K and G106V double-site mutations;

[0053] N103P and G106V double-site mutations;

[0054] N103Y and G106V double-site mutations;

[0055] Three point mutations: W101G, N103T, and G106V;

[0056] Three point mutations: W101G, N103K and G106V.

[0057] In other preferred embodiments, the recombinant antigen has the full-length sequence of the E protein of dengue virus type 3, and the E protein has a site mutation selected from the following:

[0058] G106V mutation;

[0059] W101R and G106V double-site mutations;

[0060] W101G and G106V double-site mutations;

[0061] W101L and G106V double-site mutations;

[0062] Three point mutations: W101R, G106V, and I125V;

[0063] G102R and G106V double-site mutations;

[0064] N103T and G106V double-site mutations;

[0065] N103H and G106V double-site mutations;

[0066] N103K and G106V double-site mutations;

[0067] N103P and G106V double-site mutations;

[0068] N103Y and G106V double-site mutations;

[0069] Three point mutations: W101G, N103T, and G106V;

[0070] Three point mutations: W101G, N103K and G106V.

[0071] In other preferred embodiments, the recombinant antigen has the full-length sequence of the E protein of dengue virus type 4, and the E protein has a site mutation selected from the following:

[0072] G106V mutation;

[0073] W101R and G106V double-site mutations;

[0074] W101G and G106V double-site mutations;

[0075] W101L and G106V double-site mutations;

[0076] Three point mutations: W101R, G106V, and I125V;

[0077] G102R and G106V double-site mutations;

[0078] N103T and G106V double-site mutations;

[0079] N103H and G106V double-site mutations;

[0080] N103K and G106V double-site mutations;

[0081] N103P and G106V double-site mutations;

[0082] N103Y and G106V double-site mutations;

[0083] Three point mutations: W101G, N103T, and G106V;

[0084] Three point mutations: W101G, N103K and G106V.

[0085] Further preferably, when the recombinant antigen has the full-length sequence of the E protein of Zika virus, the recombinant antigen also includes the full-length or partial prM protein sequence or M protein sequence of Zika virus.

[0086] Further preferably, when the recombinant antigen has the full-length sequence of the E protein of the dengue virus, the recombinant antigen also includes the full-length or partial prM protein sequence or M protein sequence of the dengue virus of the corresponding serotype;

[0087] In a further preferred embodiment, when the recombinant antigen has the full-length sequence of the E protein of dengue virus type 1, the recombinant antigen has the amino acid sequence shown in SEQ ID NO: 2-14;

[0088] In a further preferred embodiment, when the recombinant antigen has the full-length sequence of the E protein of dengue virus type 2, the recombinant antigen has the amino acid sequence shown in SEQ ID NO: 16-28;

[0089] In a further preferred embodiment, when the recombinant antigen has the full-length sequence of the E protein of dengue virus type 3, the recombinant antigen has the amino acid sequence shown in SEQ ID NO: 30-42;

[0090] In a further preferred embodiment, when the recombinant antigen has the full-length sequence of the E protein of dengue virus type 4, the recombinant antigen has the amino acid sequence shown in SEQ ID NO: 44-56.

[0091] In a second aspect, the present application provides a method for preparing the recombinant antigen as described in the first aspect above, the preparation method comprising the following steps:

[0092] A Kozak sequence and a signal peptide coding sequence are added to the 5' end of the nucleotide sequence encoding the recombinant antigen according to any one of claims 1 to 8, and a histidine tag coding sequence and a stop codon are added to the 3' end. Cloning and expression are performed, and the correct recombinant is screened. It is then transfected into expression system cells for expression, and the cell culture supernatant is collected to isolate the recombinant antigen.

[0093] Preferably, the expression system cells are mammalian cells, insect cells, yeast cells or bacterial cells;

[0094] Optionally, the mammalian cells are HEK293T cells, 293F series cells or CHO cells; further optionally, the 293F series cells are HEK293F cells, Freestyle293F cells or Expi293F cells;

[0095] Optionally, the insect cells are sf9 cells, Hi5 cells, sf21 cells or S2 cells;

[0096] Optionally, the yeast cell is a Pichia pastoris cell or a yeast cell modified therefrom;

[0097] Optionally, the bacterial cells are Escherichia coli cells.

[0098] In a feasible embodiment, the signal peptide can be any signal peptide commonly used in the art; for example, the signal peptide can be a signal peptide having an amino acid sequence as shown in one of SEQ ID NOs: 57-61.

[0099] In a third aspect, the present application provides a polynucleotide encoding the recombinant antigen as described in the first aspect. The polynucleotide is not limited by the method of producing it, for example, it can be obtained by genetic engineering recombination technology or chemical synthesis method.

[0100] The polynucleotide may be DNA or mRNA, preferably mRNA.

[0101] In a preferred embodiment, the polynucleotide is a DNA having a sequence as shown in one of SEQ ID NOs: 62-113, or an mRNA corresponding to the DNA.

[0102] Specifically, when the recombinant antigen has an amino acid sequence as shown in SEQ ID NOs: 2-14, the polynucleotide encoding the recombinant antigen is a DNA having a sequence as shown in SEQ ID NOs: 62-74, or an mRNA corresponding to the DNA; when the recombinant antigen has an amino acid sequence as shown in SEQ ID NOs: 16-28, the polynucleotide encoding the recombinant antigen is a DNA having a sequence as shown in SEQ ID NOs: 75-87, or an mRNA corresponding to the DNA; and / or, when the recombinant antigen has an amino acid sequence as shown in SEQ ID NOs: 30-42, the polynucleotide encoding the recombinant antigen is a DNA having a sequence as shown in SEQ ID NOs: 88-100, or an mRNA corresponding to the DNA; and / or, when the recombinant antigen has an amino acid sequence as shown in SEQ ID NOs: 44-56, the polynucleotide encoding the recombinant antigen is a DNA having a sequence as shown in SEQ ID NOs: 101-113, or an mRNA corresponding to the DNA.

[0103] The "mRNA corresponding to the DNA" mentioned above refers to an mRNA having a sequence in which all "T" in the specified DNA sequence is replaced by "U".

[0104] In a fourth aspect, the present application provides an expression cassette, a recombinant vector, a transgenic cell line, a recombinant bacterium or a recombinant virus comprising the polynucleotide as described in the third aspect above.

[0105] In a fifth aspect, the present application provides a vaccine or immunogenic composition, which includes the recombinant antigen as described in the first aspect above, or the polynucleotide as described in the third aspect above, or the expression cassette, recombinant vector, transgenic cell line, recombinant bacteria or recombinant virus as described in the fourth aspect above as an active ingredient.

[0106] In specific embodiments, the vaccine or immunogenic composition comprises, in addition to the active ingredient, a physiologically acceptable vehicle, adjuvant, excipient, carrier and / or diluent.

[0107] In some feasible embodiments, the vaccine or immunogenic composition is a recombinant protein vaccine, which includes the recombinant antigen and adjuvant as described in the first aspect above;

[0108] Preferably, the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant and MF59-like adjuvant, CpG adjuvant, QS-21-containing adjuvant, AS series adjuvant (eg AS01 series adjuvant) and nanoparticle adjuvant.

[0109] In other feasible embodiments, the vaccine or immunogenic composition is a DNA vaccine, comprising:

[0110] (i) a eukaryotic expression vector; and

[0111] (ii) a DNA sequence encoding the recombinant antigen as described in the first aspect above, constructed into the eukaryotic expression vector;

[0112] Preferably, the DNA sequence encoding the recombinant antigen as described in the first aspect above is a DNA sequence as shown in any one of SEQ ID NOs: 62-113;

[0113] Preferably, the eukaryotic expression vector is selected from the group consisting of pGX0001, pVAX1, pCAGGS and pcDNA series vectors.

[0114] In other feasible embodiments, the vaccine or immunogenic composition is an mRNA vaccine, and the mRNA vaccine comprises:

[0115] (I) an mRNA sequence encoding the recombinant antigen as described in the first aspect above; and

[0116] (II) lipid nanoparticles;

[0117] Preferably, the mRNA sequence encoding the recombinant antigen as described in the first aspect above is an mRNA sequence corresponding to a DNA sequence as shown in one of SEQ ID NOs: 62-113.

[0118] In other feasible embodiments, the vaccine or immunogenic composition is a viral vector vaccine comprising:

[0119] (1) viral backbone vectors; and

[0120] (2) a DNA sequence encoding the recombinant antigen as described in the first aspect above, constructed into the viral backbone vector;

[0121] Preferably, the DNA sequence encoding the recombinant antigen as described in the first aspect above is a DNA sequence as shown in any one of SEQ ID NOs: 62-113;

[0122] Preferably, the viral backbone vector is selected from one or more of the following viral vectors: adenoviral vector, lentiviral vector, poxvirus vector, influenza virus vector, and adeno-associated virus vector.

[0123] In a feasible implementation, the vaccine or immunogenic composition is in the form of a nasal spray, an oral formulation, a suppository or a parenteral formulation;

[0124] Preferably, the nasal spray is selected from aerosols, sprays and powder sprays;

[0125] Preferably, the oral preparation is selected from tablets, powders, pills, granules, soft / hard capsules, film coatings and ointments;

[0126] Further preferably, the tablet is a sublingual tablet;

[0127] Further preferably, the granules are fine granules;

[0128] Further preferably, the powder is a powder;

[0129] Further preferably, the pills are pellets;

[0130] Preferably, the parenteral preparation is a transdermal preparation, an ointment, a plaster, a liquid for external use, or an injectable preparation; further preferably, the injectable preparation is a push-in preparation.

[0131] In a sixth aspect, the present application provides the use of the recombinant antigen as described in the first aspect above, or the polynucleotide as described in the third aspect above, or the expression cassette, recombinant vector, transgenic cell line, recombinant bacteria or recombinant virus as described in the fourth aspect above in the preparation of a drug for detecting, preventing and / or treating Zika virus or dengue virus infection.

[0132] Preferably, the drug is a vaccine.

[0133] In the seventh aspect, the present application provides a method for preventing and / or treating Zika virus or dengue virus infection, the method comprising: administering to a subject in need thereof a preventive and / or therapeutically effective amount of the following substances: the recombinant antigen as described in the first aspect above, the polynucleotide as described in the third aspect above, the expression cassette, recombinant vector, transgenic cell line, recombinant bacteria or recombinant virus as described in the fourth aspect above and / or the vaccine or immunogenic composition as described in the fifth aspect above.

[0134] The "preventively and / or therapeutically effective amount" may vary depending on the subject of administration, the subject organ, symptoms, the method of administration, etc., and can be determined based on the doctor's judgment, taking into account the type of dosage form, the method of administration, the patient's age and weight, the patient's symptoms, etc. Beneficial effects

[0135] The recombinant antigen against Zika / dengue virus of the present application effectively eliminates the FL epitope in the E protein, avoids the production of FL epitope antibodies that cause ADE, and thus can significantly reduce or eliminate the ADE effect; at the same time, the recombinant antigen can maintain the correct conformation of the E protein and has a protein expression level similar to or higher than that of the wild type. Animal immunization experiments have confirmed that the Zika / dengue vaccine based on the recombinant antigen can induce higher specific antibody titers and neutralizing antibody titers in animals, thereby providing effective immune protection, and the cross-antibody titers induced by it against other serotypes of E proteins are significantly reduced, so the ADE risk of the vaccine is also greatly reduced.

[0136] In addition, the vaccines obtained based on the recombinant antigens provided in this application may include various forms, such as recombinant protein vaccines, nucleic acid vaccines (such as mRNA vaccines), viral vector vaccines (such as adenovirus vector vaccines), virus attenuated or inactivated vaccines based on the antigen sequence, chimeric vaccines with other skeletons, etc., which can be used to prepare Zika and / or dengue vaccines that eliminate the ADE effect, and have good clinical application value and industrialization prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] One or more embodiments are exemplarily illustrated by the accompanying figures, and these exemplary illustrations do not limit the embodiments. The word "exemplary" is used herein to mean "serving as an example, example, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0138] FIG1 shows the results of antigenic epitope detection of the DV1 mRNA vaccine prepared in Example 1, as detected in Example 4.

[0139] FIG2 shows the results of antigenic epitope detection of the DV2 mRNA vaccine prepared in Example 1, as detected in Example 4.

[0140] FIG3 shows the results of antigenic epitope detection of the DV3 mRNA vaccine prepared in Example 1, as detected in Example 4.

[0141] FIG4 shows the results of antigenic epitope detection of the DV4 mRNA vaccine prepared in Example 1, as detected in Example 4.

[0142] FIG5 shows the specific antibody titers against viral E protein induced by each DV mRNA vaccine as detected by enzyme-linked immunosorbent assay, as tested in Example 6.

[0143] FIG6 shows the neutralizing antibody titers against the corresponding serotype DV virus induced by each DV mRNA vaccine as detected by virus microneutralization assay, as tested in Example 7.

[0144] FIG7 shows the cross-antibody titers against DV2 / 3 / 4 and Zika virus E protein induced by the DV1 mRNA vaccine prepared in Example 1, as detected in Example 8.

[0145] Figure 8 shows the cross-antibody titers against DV1 / 3 / 4 and Zika virus E protein induced by the DV2 mRNA vaccine prepared in Example 1, as detected in Example 8.

[0146] Figure 9 shows the cross-antibody titers against DV1 / 2 / 4 and Zika virus E protein induced by the DV3 mRNA vaccine prepared in Example 1, as detected in Example 8.

[0147] FIG10 shows the cross-antibody titers against DV1 / 2 / 3 and Zika virus E protein induced by the DV4 mRNA vaccine prepared in Example 1, as detected in Example 8.

[0148] FIG. 11 shows the neutralizing antibody titers against the corresponding serotype DV viruses induced by DV1-EM3-1 and DV2-EM3 mRNA vaccines as tested by virus microneutralization assay, as described in Example 9.

[0149] FIG12 shows the cross-antibody titers against DV2 / 3 / 4 and Zika virus E protein induced by the DV1-EM3-1 mRNA vaccine prepared in Example 1, as detected in Example 9.

[0150] FIG13 shows the cross-antibody titers against DV1 / 3 / 4 and Zika virus E protein induced by the DV2-EM3 mRNA vaccine prepared in Example 1, as detected in Example 9.

[0151] FIG14 shows the ADE effect against DV2 / 3 / 4 viruses at the cellular level induced by the immune serum of the DV1 mRNA vaccine prepared in Example 1, as detected in Example 10.

[0152] FIG15 shows the ADE effect against DV1 / 3 / 4 viruses at the cellular level induced by the immune serum of the DV2 mRNA vaccine prepared in Example 1, as detected in Example 10.

[0153] FIG16 shows the ADE effect against DV1 / 2 / 4 viruses at the cellular level induced by the DV3 mRNA vaccine immune serum prepared in Example 1, as detected in Example 10.

[0154] FIG17 shows the ADE effect against DV1 / 2 / 3 viruses at the cellular level induced by the immune serum of the DV4 mRNA vaccine prepared in Example 1, as detected in Example 10.

[0155] FIG18 shows the in vivo ADE effect against DV2 virus induced by the immune serum of the DV4 mRNA vaccine prepared in Example 1, as detected in Example 11.

[0156] FIG19 shows the antibody levels against the FL epitope induced by the tetravalent dengue mRNA vaccine using the tetravalent dengue mRNA vaccine mouse serum obtained in Example 12, as detected in Example 14.

[0157] Figure 20 shows the neutralizing antibody titers against four serotypes of DV viruses induced by the quadrivalent dengue mRNA vaccine using the quadrivalent dengue mRNA vaccine mouse serum obtained in Example 12, as detected in Example 15.

[0158] FIG. 21 shows the protective effect of the quadrivalent dengue mRNA vaccine described in Example 12 against challenge with DV1 and DV2 viruses, as tested in Example 16.

[0159] FIG22 shows the antigenic epitope and antigen expression detection results of the DV1 mRNA vaccine prepared in Example 17, as detected in Example 18.

[0160] FIG23 shows the antigenic epitope and antigen expression detection results of the DV2 mRNA vaccine prepared in Example 17, as detected in Example 18.

[0161] Figure 24 shows the antigen epitope and antigen expression detection results of the DV3 mRNA vaccine prepared in Example 17, as detected in Example 18.

[0162] FIG25 shows the results of antigen epitope and antigen expression detection of the DV4 mRNA vaccine prepared in Example 17, as detected in Example 18.

[0163] FIG26 shows the protective effect against DV2 virus challenge induced by the DV2 mRNA vaccine prepared in Example 17, as tested in Example 19.

[0164] FIG27 shows the SDS-PAGE results of the dengue virus-like particles prepared in Example 20, as detected in Example 20.

[0165] FIG28 shows the electron microscopic negative staining results of DV1-EM3-1 virus-like particles prepared in Example 20, as detected in Example 21.

[0166] FIG29 shows the electron microscopic negative staining results of DV2-EM3-1 virus-like particles prepared in Example 20, as detected in Example 21.

[0167] FIG30 shows the electron microscopic negative staining results of the DV3-EM3 virus-like particles prepared in Example 20, as detected in Example 21.

[0168] FIG31 shows the electron microscopic negative staining results of the DV4-EM3 virus-like particles prepared in Example 20, as detected in Example 21.

[0169] Figure 32 shows the neutralizing antibody titers against the corresponding serotype DV virus induced by each DV recombinant protein vaccine as detected by virus microneutralization assay, as tested in Example 22.

[0170] Figure 33 shows the neutralizing antibody titers against four serotypes of DV viruses induced by the tetravalent DV recombinant protein vaccine as tested by virus microneutralization assay, as tested in Example 23. DETAILED DESCRIPTION

[0171] To make the objectives, technical solutions, and advantages of this application more clear, the technical solutions in the embodiments of this application are clearly and completely described below. Obviously, the described embodiments are part of the embodiments of this application, not all of them. Based on the embodiments of this application, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of this application.

[0172] In addition, in order to better illustrate the present application, numerous specific details are provided in the following detailed description. It should be understood by those skilled in the art that the present application can be implemented without certain specific details. In some embodiments, raw materials, components, methods, means, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present application.

[0173] The practice of the present application will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art.

[0174] In addition, unless otherwise explicitly defined elsewhere in this document, the technical terms used herein have the meanings commonly understood by those skilled in the art to which this application belongs. In order to make this application more easily understood, certain technical terms are specifically defined as follows.

[0175] The term “comprise” or variations such as “include” or “comprising” will be understood as including stated elements or components but not excluding other elements or components.

[0176] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as such variations are appropriate for performing the disclosed methods.

[0177] The term "and / or" should be understood to mean any one of the alternatives or a combination of any two or more of the alternatives.

[0178] As used herein, the term "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, that is, including at least one of the number or elements in the list, but also including more than one, and optionally, additional unlisted items.

[0179] Example 1: Preparation of dengue virus mRNA vaccine

[0180] In this example, a series of mRNA constructs encoding the full-length prME protein of dengue virus (i.e., including prM protein + E protein) were designed, including:

[0181] DV1-WT (i.e., DV1 wild type), encoding the prME antigen protein shown in SEQ ID NO: 1;

[0182] DV1-EM3, encoding the prME antigen protein shown in SEQ ID NO: 3;

[0183] DV1-EM3-1, encoding the prME antigen protein shown in SEQ ID NO: 4;

[0184] DV2-WT (i.e., DV2 wild type), encoding the prME antigen protein shown in SEQ ID NO: 15;

[0185] DV2-EM3, encoding the prME antigen protein shown in SEQ ID NO: 17;

[0186] DV2-EM3-1, encoding the prME antigen protein shown in SEQ ID NO: 18;

[0187] DV3-WT (i.e., DV3 wild type), encoding the prME antigen protein shown in SEQ ID NO: 29;

[0188] DV3-EM3, encoding the prME antigen protein shown in SEQ ID NO: 31;

[0189] DV3-EM3-1, encoding the prME antigen protein shown in SEQ ID NO: 32;

[0190] DV4-WT (i.e., DV4 wild type), encoding the prME antigen protein shown in SEQ ID NO: 43;

[0191] DV4-EM3, encoding the prME antigen protein shown in SEQ ID NO:45;

[0192] DV4-EM3-1 encodes the prME antigen protein shown in SEQ ID NO:46.

[0193] Next, according to the codon preference of mammalian cells, the nucleic acid sequence encoding the recombinant dengue virus prME antigen protein was optimized to obtain an optimized nucleic acid coding sequence, wherein the optimized nucleic acid coding sequences of DV1-WT, DV2-WT, DV3-WT, and DV4-WT are shown in SEQ ID NOs: 114-117, respectively, and the optimized nucleic acid coding sequences of DV1-EM3, DV1-EM3-1, DV2-EM3, DV2-EM3-1, DV3-EM3, DV3-EM3-1, DV4-EM3, and DV4-EM3-1 are shown in SEQ ID NOs: 63, 64, 76, 77, 89, 90, 102, and 103, respectively; then, a Kozak sequence and a signal peptide nucleic acid coding sequence (such as SEQ ID NO: 118) were added to the 5' end of each nucleic acid coding sequence, and the amino acid sequence encoded by it was shown in SEQ ID NO: NO:58), added a stop codon at its 3' end, and commissioned GenScript Biotech Co., Ltd. to perform gene synthesis. The synthesized gene fragment was then recombined into the pHRNT vector (invention patent ZL202110224383.0) preserved in the laboratory to obtain the template plasmid for preparing the mRNA vaccine.

[0194] The specific preparation process of mRNA vaccine refers to the method published in invention patent ZL202110224383.0. Briefly, the above template plasmid is linearized by enzyme digestion, and then T7 transcriptase is used to transcribe mRNA in vitro. During the transcription process, modified mRNA is obtained by replacing uracil with pseudouridine or N1-methylpseudouridine to increase the protein expression of mRNA in vivo; subsequently, the mRNA is packaged into nanoparticles using microfluidic technology; during packaging, the aqueous phase is the mRNA solution (50mM sodium acetate buffer solution, pH 4.0), and the ethanol phase is the lipid mixture (prepared with protonated phospholipids, distearoylphosphatidylcholine, cholesterol and PEG-modified phospholipids in a molar ratio of 50:10:38.5:1.5), the total flow rate of the aqueous phase and the ethanol phase is 12ml / min, and the volume ratio of the aqueous phase and the ethanol phase is 3:1; finally, the packaged mRNA vaccine is replaced with PBS using a dialysis bag, and the Quan-iT RiboGreen The RNA assay kit measured the concentrations of encapsulated and free mRNA and calculated the mRNA packaging efficiency. The results showed that the packaging efficiency met the standards of mRNA vaccines. The encapsulated mRNA was then stored at 4°C for use.

[0195] Example 2: Expression and purification of detection antibodies

[0196] In this example, according to the construction methods of antibody heavy chain and light chain sequences and their expression plasmids disclosed in the literature, detection antibodies Z5 (Wang, Q Het al. Sci Transl Med 8, (2016)), Z6 (Dai, L et al. Nat Immunol 22, 958-968, (2021)), 4G2 (Dai, L et al. Nat Immunol 22, 958-968, (2021)), MZ24 (Dussupt, V et al. Nat Med 26, 228-35, (2020)), Ab513 (Robinson, L Net al. Cell 162, 493-504, (2015)), 1F4 (Fibriansah, G et al. Embo Mol Med 6, 358-371, (2014)), 3H5 (Renner, M. et al. Nature Immunology 19, 1248-56, (2018)), MZ-1 (Dussupt, V. et al. Nat Med 26, 228-35, (2020)), MZ-4 (Dussupt, V. et al. Nat Med 26, 228-35, (2020)), 5J7 (Fibriansah, G. et al. Embo Journal 31, 767-779, (2012)) and 5H2 (Cockburn, JJB et al. Embo Journal 31, 767-779, (2012)).

[0197] Among them, Z5, Z6 and 4G2 antibodies recognize the FL epitope of Zika and dengue viruses, and the remaining antibodies (MZ-1, MZ-4, MZ24, Ab513, 1F4, 3H5, 5J7, and 5H2) are all highly neutralizing antibodies that can recognize non-FL epitopes.

[0198] Antibody expression

[0199] 14-16 hours before transfection, split the 293T cells with higher density into plates (e.g., a 10cm dish that is 100% confluent with 293T cells is passaged at a ratio of 1:3). 14-16 hours later, when the cell density reaches over 70%, transfection can be performed. During transfection, the heavy chain and light chain expression plasmids of the antibody constructed above are co-transfected into 293T cells at a ratio of 2:3. 4-6 hours after transfection, the cells are washed twice with PBS and replaced with serum-free DMEM medium for continued culture. Cell supernatants are collected on days 3 and 7 after transfection, centrifuged to remove cell debris, and the antibody supernatants obtained from the two washes are mixed for subsequent antibody protein purification.

[0200] Antibody purification

[0201] A Protein A (5 ml) HP affinity column (purchased from GE) was connected to an AKTA Purifier / Explorer / FPLC / START (GE). The instrument was operated as follows: 20% ethanol in the column was first flushed out with water, and the column was then equilibrated with 20 mM Na3PO4, pH 7.0 buffer. After the conductivity on the instrument stabilized, the above-mentioned antibody supernatant was injected via a 10 ml loop loading method to bind to Protein A at a flow rate of 2 ml / min. After the UV was stable, approximately 0.8 ml of 1 M Tris pH 9.0 buffer was added to the subsequent collection tube (collection volume approximately 3.2 ml). The program was then changed to 100% 0.1 M Gly pH 3.0 to elute the antibody bound to the column. The eluate was collected and then replaced with PBS by concentration and exchange. The resulting antibody solution can be used directly or stored in aliquots at -80°C for later use.

[0202] Example 3: Expression and purification of dengue and Zika virus E proteins

[0203] In this example, dengue and Zika virus E protein expression plasmids were constructed according to the method for soluble expression of dengue and Zika virus E protein dimers disclosed in the literature (Kudlacek, ST et al. Sci Adv 7, (2021)).

[0204] Specifically, the genes encoding amino acids 1-395 of the E protein of DV1 (THSTI-TRC-DV1-08 strain, GenBank: OP310803.1), DV2 (New Guinea C strain, GenBank: KM204118.1), DV3 (YN02 strain, GenBank: KF824903), and DV4 (Guangzhou, China B5 strain, GenBank: AF289029), and the gene encoding amino acids 1-404 of the E protein of ZIKV-SMGC-1 (GenBank: KX266255) (wherein, in order to form a stable dimer of the E protein, the DV1, DV2, and DV4 E proteins contain an A259C mutation, the DV3 E protein contains an A257C mutation, and the ZIKV E protein contains an A264C mutation) were optimized according to the codon preference of mammalian cells to obtain the optimized nucleic acid coding sequences of the above-mentioned viral E protein fragments, as shown in SEQ ID NOs. NO:120-124; then, a Kozak sequence and a signal peptide coding sequence (as shown in SEQ ID NO:119, encoding an amino acid sequence as shown in SEQ ID NO:60) were added to the 5' end of the above nucleic acid coding sequence, and a His tag was added to the 3' end for protein isolation and purification. Gene synthesis was commissioned to GenScript Biotech Co., Ltd., and the synthesized gene fragment was then double-digested with EcoRI at 5' and XhoI at 3' and cloned into the pCAGGS vector to construct E protein expression plasmids pCAGGS-DV1-sE, pCAGGS-DV2-sE, pCAGGS-DV3-sE, pCAGGS-DV4-sE, and pCAGGS-ZV-sE. Protein expression was performed using HEK293F cells, and protein purification was performed using a HisTrap™ Excel affinity chromatography column and a Superdex200 Increase 10 / 300GL gel filtration column.

[0205] Select HEK293F cells in good growth condition and transfect them at a density of 1-1.5x10 6 The cells were inoculated into fresh SMM293-TII medium at a density of 10 cells / ml and cultured in a shaker at 37°C, 5% CO2, and 150 rpm. On the day of transfection, the cell density was adjusted to 2.5 x 10 6Prepare the transfection reagent (using 10ml of cells as an example): dilute 10µg of plasmid with 150mM NaCl to a total volume of 250µl and mix gently. Dilute the Sinofection transfection reagent with 150mM NaCl to a total volume of 250µl and mix gently. Let the diluted plasmid and transfection reagent stand separately for approximately 5 minutes, then gently mix until a total volume of 500µl is achieved. Let stand at room temperature for another 10 minutes. Add the transfection solution dropwise to the cell culture medium, gently shaking the culture flask while adding. After mixing, return the flask to the shaker and continue incubation. Add 350µl of SMS-293-SUPI feed solution 24 hours after transfection, and add 350µl of feed solution every 48 hours thereafter. 5 days after transfection, collect the supernatant for protein purification.

[0206] The supernatant of cells was collected 5 days after transfection, centrifuged at 8000 rpm, 4°C for 1 hour, and then filtered through a 0.22 μm filter. His affinity chromatography purification: First, rinse the HisTrap with water filtered through a 0.22 μm filter. TM Excel affinity chromatography column, rinse for about 3-5 column volumes. Afterwards, rinse the column with HisTrap affinity chromatography equilibration solution until the UV and conductivity detection lines are stable, and the column is considered to have been equilibrated. Then, the cell supernatant is passed through the column at a flow rate of 1-2 mL / min to allow the protein to bind to the column. After all the supernatant has flowed through the column, rinse the column with 5-10 column volumes of affinity chromatography equilibration solution until the UV detection line is stable. Finally, rinse the column with eluents containing 50 mM, 300 mM, and 1 M imidazole in sequence, and prepare the collected protein for SDS-PAGE identification. Further purification is performed using Superdex 200Increase 10 / 300GL gel filtration chromatography. The sample obtained by affinity chromatography is concentrated to less than 800 ul using a 10 kDa ultrafiltration concentration tube and then transferred to a 1.5 mL centrifuge tube. After centrifugation at 12,000 g at 4°C for 20 minutes, ensure that no precipitate is aspirated and transfer the sample to a fresh 1.5 mL centrifuge tube. Remove air bubbles from the sample by centrifugation at 12,000 g at 4°C for 20 minutes and prepare for sample loading. Equilibrate a Superdex 200 Increase 10 / 300 GL gel filtration column with gel filtration chromatography equilibration buffer at a maximum flow rate of 0.5 mL / min until the UV detection line is stable. Load the sample using a 1 mL loop. After loading, flush the column with 20 / 50 equilibration buffer (20 mM Tris, 50 mM NaCl) at a flow rate of 0.5 mL / min. Set the peak collector to collect the sample at the peak. Prepare the collected sample and identify it by SDS-PAGE. Concentrate the identified correctly expressed sample using a 10 kDa ultrafiltration concentrator and store in aliquots at -80°C for later use.

[0207] Example 4: Detection of antigen expression of dengue virus mRNA vaccine

[0208] In this example, we transfected 293T cells with a series of dengue virus prME mRNA vaccines prepared in Example 1, and then used the FL epitope antibody and other neutralizing epitope antibodies prepared in Example 2 to detect the epitope status and protein expression level in each prME.

[0209] Specifically, HEK293T cells were cultured in DMEM medium containing 10% FBS. Before transfection, the cell density was increased to more than 70%. HEK293T cells were transfected with the mRNA vaccine constructed in Example 1. 12 hours after transfection, the cell culture supernatant was discarded, the cells were digested with trypsin and collected, and the cells were washed with PBS and then added with cell fixation and permeabilization solution (BD Cytofix / Cytoperm TM Fixation / Permeabilization Kit), incubate on ice for 20 minutes, and use 1× washing solution (BD Cytofix / Cytoperm TM After washing the cells twice with a Fixation / Permeabilization Kit, antibodies Z5 and Z6 that recognize different epitopes of the dengue prME protein described in Example 2 were added and incubated on ice in the dark for 30 minutes. The cells were washed twice with 1× wash buffer and then FITC-labeled goat anti-human secondary antibody (Abcam, USA) was added and incubated on ice in the dark for 30 minutes. The cells were washed twice with 1× wash buffer and then reselected with PBS. Cell fluorescence was detected on a BD FACSAria III flow cytometer, and the data were analyzed using FlowJo 7.6.1.

[0210] The results are shown in Figure 1-4. Figure 1-4 shows:

[0211] For the DV1 mRNA vaccine, FL epitope antibodies (Z5 and Z6) can bind to DV1-WT but not to the two mutants (DV1-EM3 and DV1-EM3-1), indicating that the FL epitope has been destroyed in the two mutants; other neutralizing epitope antibodies (MZ24, Ab513 and 1F4) can bind to both DV1-WT and the two mutants, indicating that other neutralizing epitopes have not changed in the two mutants, and the protein expression levels of the two mutants are similar to that of the wild type;

[0212] For the DV2 mRNA vaccine, FL epitope antibodies (Z5, Z6, and 4G2) bound to DV2-WT but not to the two mutants DV2-EM3 and DV2-EM3-1, indicating that the FL epitope had been destroyed in the two mutants. Other neutralizing epitope antibodies (3H5, MZ-1, and MZ-4) bound to both DV2-WT and the two mutants, indicating that other neutralizing epitopes had not changed in the two mutants, and the protein expression levels of the two mutants were similar to that of the wild type.

[0213] For the DV3 mRNA vaccine, FL epitope antibodies (Z5 and Z6) bound to DV3-WT but not to the two mutants DV3-EM3 and DV3-EM3-1, indicating that the FL epitope had been destroyed in the two mutants. Other neutralizing epitope antibodies (MZ24, Ab513, and 5J7) bound to both DV3-WT and the two mutants, indicating that other neutralizing epitopes had not changed in the two mutants, and the protein expression levels of the two mutants were similar to that of the wild type.

[0214] For DV4 mRNA vaccine, FL epitope antibodies (Z5 and Z6) bound to DV4-WT but not to the two mutants DV4-EM3 and DV4-EM3-1, indicating that the FL epitope had been destroyed in the two mutants; other neutralizing epitope antibodies (MZ24, Ab513 and 5H2) could bind to both DV4-WT and the two mutants, indicating that other neutralizing epitopes had not changed in the two mutants, and the protein expression levels of the two mutants were similar to those of the wild type.

[0215] The above results show that in all the mutants described above, the FL epitope is destroyed, while other neutralizing antibody epitopes remain unchanged, and the protein expression levels of the mutants are comparable to or higher than those of the wild type. In other words, the FL epitope in the recombinant antigens of the present application is destroyed, which avoids the production of FL epitope antibodies that cause ADE, thereby significantly reducing or eliminating the ADE effect. At the same time, the recombinant antigen is able to bind normally to other neutralizing epitope antibodies, indicating that it has the correct conformation of the E protein. Furthermore, the recombinant antigen has a protein expression level similar to or higher than that of the wild type.

[0216] Example 5: Dengue virus mRNA vaccine mouse immunization experiment

[0217] In this example, female 6-8 week old BALB / c mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were used for vaccine immunization experiments; the experiment was divided into an mRNA vaccine immunization group and a negative control group (i.e., Sham group), with 6 mice in each group; for each immunization group, a placebo (normal saline, as the Sham group) or the dengue mRNA vaccine prepared in Example 1 was injected intramuscularly on day 0 and day 14, respectively, with a dose of 12 μg / mouse each time, and blood was collected on the 4th week after immunization, the serum was separated at 4°C, inactivated at 56°C for 30 minutes, and then stored at -80°C for use.

[0218] Example 6: Detection of specific antibody titers in sera of immunized mice by enzyme-linked immunosorbent assay (ELISA)

[0219] In this example, the specific antibody titer against antigen E protein in the sera of the immunized mice obtained in Example 5 was detected by ELISA experiment.

[0220] Specifically, the following procedures were adopted:

[0221] (1) The dengue virus or Zika virus E protein prepared in Example 3 was diluted to 2 μg / ml using ELISA coating solution (Solarbio, C1050), 100 μl was added to each well of a 96-well ELISA plate (Coring, 3590), and the plate was incubated at 4°C for 12 hours.

[0222] (2) Pour off the coating solution, add PBS, and wash once; add 5% skim milk prepared in PBS as blocking solution to the 96-well plate, 100 μl per well, and leave at room temperature for 1 hour to block; after blocking, wash once with PBS solution;

[0223] (3) During the blocking period in step (2), the mouse serum samples were diluted with blocking solution, starting from 10 times and then diluted in a 2-fold gradient; then, 100 μl of immune serum dilution solution was added to each well of the ELISA plate. The negative control was added with blocking solution, incubated at 37 degrees for 2 hours, and then washed 4 times with PBST;

[0224] (4) Add HRP-conjugated goat anti-mouse secondary antibody (Abcam, ab6789) diluted 1:2000 in blocking solution, incubate at 37°C for 1 hour, and then wash 5-6 times with PBST; add TMB colorimetric solution for color development. After the reaction time is appropriate, add 2M hydrochloric acid to terminate the reaction, and measure the OD450 value on a microplate reader.

[0225] The antibody titer value is defined as the highest serum dilution factor with a reaction value greater than 2.1 times the negative control value. When the reaction value of the lowest dilution factor (detection limit) is still less than 2.1 times the background value, the titer of the sample is defined as half of the lowest dilution factor, i.e. 1:5.

[0226] The results of the ELISA experiment are shown in Figure 5, which shows that: DV1-EM3 can induce DV1 virus E protein-specific antibody titers similar to those of DV1-WT; DV2-EM3-1, DV4-EM3 and DV4-EM3-1 vaccines also induced antibody titers similar to those of the corresponding wild-type vaccines; compared with DV3-WT, the DV3 virus E protein-specific antibody titers induced by DV3-EM3 and DV3-EM3-1 were reduced, but their antibody titer values ​​were still high (>10,000).

[0227] The above results show that the recombinant antigen of the present application can induce high levels of specific binding antibodies in mice, suggesting that it has high immunogenicity and can induce a high level of immune protection effect.

[0228] Example 7: Dengue virus microneutralization assay to detect neutralizing antibody titers in sera of immunized mice

[0229] In this example, the dengue virus microneutralization experiment was performed to quantitatively determine the neutralizing antibody titer against the corresponding serotype of dengue virus in the serum of mice immunized with each mRNA vaccine in Example 5. The specific steps are as follows:

[0230] Vero cells were seeded in 24-well cell culture dishes one day in advance, and the cells were grown to a confluence of more than 70% on the next day for neutralization test; serum was graded diluted with DMEM medium (Invitrogen, C11995500BT) containing 1% FBS (Gibco, 10270-106), and the virus was also diluted with DMEM medium containing 1% FBS. 200ul of serum and virus solution were mixed in equal volumes. At the same time, the diluted virus was mixed with an equal volume of blank medium as a positive control and incubated at 37°C for 30min; after incubation, the supernatant medium in the VERO cell plate was removed, and the above mixture was added to the cells at 350μl / well. After incubation in a 37°C incubator for 1 hour, each well was supplemented with 1% 150 μL of FBS-containing DMEM medium was placed in a 37°C incubator and cultured for further 3 days. After 3 days, the cell culture plate was removed, all supernatants were discarded, and the cells were washed once with PBS. Then, 150 μL of trypsin was added to each well to digest the cells, and the cells were allowed to stand in a 37°C constant temperature incubator for 5 minutes. Then, 150 μL of PBS containing 10% FBS was added to the wells to terminate the trypsin digestion. The digested cells were blown to mix thoroughly, and all cells in the 24-well plate were transferred to a 96-well pointed bottom plate, centrifuged to collect the cells, and then washed once with PBS. Then, 100 μL of Fixation and Permeabilization solution (BD, 554722) was added to each well of the 96-well round bottom plate. After fixation at 4°C in the dark for 30 minutes, the cells were centrifuged at 800 g for 5 minutes to collect the cells and washed twice with 1× Perm / Wash buffer (BD, 554723). 100 μL of AF-488-conjugated Z6 antibody (Z6-AF488) was added to each well and stained at 4°C in the dark for 30 minutes. After staining, cells were collected by centrifugation and washed twice with 1× Perm / Wash buffer. Finally, cells were resuspended in 200 μL of PBS, transferred to a flow cytometer, and FITC fluorescence positivity was determined using a FACS Canto II. GraphPad Prism software was used to perform a nonlinear fit on the data and calculate the neutralization titer (IC50) based on the serum dilution factor required to neutralize 50% of the infected cells. When the lowest dilution of serum failed to neutralize 50% of the infected cells, the IC50 for that sample was defined as half the lowest dilution factor.

[0231] The results are shown in Figure 6, which shows that compared with the corresponding WT vaccine, DV1-EM3, DV2-EM3-1, DV3-EM3, and DV4-EM3 vaccines all induced comparable virus-neutralizing antibody titers, with DV3-EM3-1 and DV4-EM3-1 inducing slightly lower virus-neutralizing antibody titers. In particular, compared with DV3-WT, the DV3-EM3 vaccine induced fewer binding antibodies (Figure 5), but the neutralizing activity of vaccine serum did not decrease (Figure 6), indicating that the DV3-EM3 vaccine induced a higher proportion of high-level neutralizing activity antibodies.

[0232] The above results show that the recombinant antigen of the present application can induce a high level of neutralizing antibody titer in mice, suggesting that it has high immunogenicity and can induce a high level of immune protection effect.

[0233] Example 8: Determination of cross-antibody titer

[0234] Because the FL epitope is highly conserved between dengue and Zika viruses, antibodies against the FL epitope have a broad spectrum, meaning they can cross-bind to the E proteins of all dengue and Zika virus serotypes. However, these antibodies generally have poor virus neutralization activity and are prone to inducing ADE. Antibodies to the FL epitope induced by a dengue virus vaccine of any serotype can cross-bind to the E proteins of the other three serotypes of dengue and Zika viruses. Therefore, assessing the cross-antibody titer of a vaccine can reflect the amount of FL epitope antibodies induced by the vaccine and the ADE risk of the vaccine. Cross-antibody titers are determined by ELISA; see Example 6 for details.

[0235] The results of the cross-antibody titer determination induced by each mRNA vaccine immunization in Example 5 are shown in Figures 7-10. Figures 7-10 show:

[0236] For the DV1 vaccine, DV1-WT induced a large number of antibodies that could cross-bind to DV2 / 3 / 4 and ZIKV virus E protein, while the cross-antibody titer of DV1-EM3 was significantly reduced, and even no cross-binding antibodies were detected in some mice. It can be inferred that the ADE risk of the DV1-EM3 vaccine against DV2 / 3 / 4 and ZIKV viruses is significantly reduced;

[0237] The DV2 / 3 / 4 vaccines also had similar results, that is, compared with the corresponding WT vaccine, the cross-antibody titers induced by each mutant vaccine were significantly reduced, and their ADE risks were also significantly reduced.

[0238] The above results indicate that the recombinant antigens of the present application induce significantly reduced cross-antibodies against E proteins of other serotypes of DV or ZIKV viruses, thereby greatly reducing the risk of vaccine-induced ADE effects and having higher safety.

[0239] Example 9: Determination of neutralizing antibody titers and cross-antibody titers in serum of DV1-EM3-1 and DV2-EM3 vaccines

[0240] In this example, the neutralizing antibody titer and cross-antibody titer of the serum of mice immunized with the DV1-EM3-1 and DV2-EM3 vaccines obtained in Example 5 were measured. The neutralizing antibody titer determination method was referred to Example 7, and the cross-antibody titer determination method was referred to Example 8.

[0241] The results of serum neutralizing antibody titer determination are shown in Figure 11. Figure 11 shows that compared with the corresponding WT vaccine, DV1-EM3-1 induced a considerable virus neutralizing antibody titer. Although the neutralizing antibody titer induced by DV2-EM3 decreased, it was still above 1,000, showing good immunogenicity.

[0242] The results of cross-antibody titer determination are shown in Figures 12-13. Figures 12-13 show that compared with the WT vaccine, the cross-antibody titers induced by DV1-EM3-1 and DV2-EM3 vaccines against different serotypes of dengue virus and Zika virus were significantly reduced. It can be inferred that the ADE risk against different serotypes of dengue virus and Zika virus is also significantly reduced.

[0243] Example 10: Evaluation of vaccine-induced ADE effects in vitro

[0244] In this example, female 6-8 week old BALB / c mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were used for vaccine immunization experiments; the experiment was divided into an mRNA vaccine immunization group and a negative control group (i.e., Sham group), with 8 mice in each group; for each immunization group, a placebo (normal saline, as the Sham group) or the DV1-WT, DV2-WT, DV3-WT, DV4-WT, DV1-EM3-1, DV2-EM3-1, DV3-EM3, DV4-EM3 monovalent dengue mRNA vaccine prepared in Example 1 was injected intramuscularly on day 0 and day 21, respectively, with a dose of 1 μg / mouse each time, and blood was collected in the second week after the booster immunization, the serum was separated at 4°C, inactivated at 56°C for 30 minutes, and then stored at -80°C for use.

[0245] Using a K562 cell model (ATCC, Cat. No. CCL-243) that highly expresses the FcγRIIA receptor, the ADE effects of serum from BALB / c mice immunized with the monovalent mRNA vaccine against the remaining three serotypes of viruses were determined, such as the ADE effects of DV1-WT, DV1-EM3, and DV1-EM3-1 immune sera against DENV2, 3, and 4. The specific steps are as follows:

[0246] Remove the vaccine immune serum and thaw it at 4°C, flick it to mix, and centrifuge briefly. In a clean bench, serially dilute the mouse serum in RPMI 1640 medium (Invitrogen, C11875500BT) containing 1% fetal bovine serum in a 96-well U-shaped plate (Corning 3799) at a 4-fold ratio. Dilute the DENV virus to 2×10 6 FFU / mL, serum dilutions were mixed with equal volumes of the three DENV viruses and incubated at room temperature for 20 minutes. The cultured K562 cells were centrifuged at 800g for 5 minutes, resuspended in RPMI-1640 medium containing 1% FBS, and the cells were counted. The cell density was then adjusted to 4×10 6 / mL, then 10 μL per well was added to the virus and serum mixture and incubated in a 37°C cell culture incubator for 1 hour. RPMI1640 medium containing 1% FBS was added at 100 μL per well and cultured in a 37°C cell culture incubator for another 12-14 hours.

[0247] After 12 hours, cells were transferred to a 96-well plate and centrifuged at 800 g for 5 minutes. The supernatant was removed, and the cells were washed once with PBS and collected by centrifugation. 100 μL of Fixation and Permeabilization solution (BD 554722) was added to each well of the 96-well U-shaped plate and incubated at 4°C for 30 minutes. After centrifugation at 800 g for 5 minutes, the cells were collected and washed twice with 1× Perm / Wash buffer (BD 554723). 100 μL of AF488-conjugated Z6 antibody (Z6-AF488) was added to each well and incubated at 4°C for 30 minutes. The cells were centrifuged at 800 g for 3 minutes, collected, and washed twice with 1× Perm / Wash buffer. The cells were resuspended in 100 μL of PBS per well, and the percentage of virus-positive cells was determined by flow cytometry.

[0248] The results are shown in Figures 14-17, which show that: DENV virus does not infect K562 cells after mixing with Sham immune serum; the immune sera of the four wild-type vaccines (DV1-WT, DV2-WT, DV3-WT and DV4-WT) can detect strong in vitro ADE effects at different dilutions, that is, WT vaccine serum can enhance the infection of dengue virus on K562 cells, and the cell infection rate is increased; compared with the above-mentioned wild-type vaccines, the in vitro ADE effects induced by mouse sera of mutant vaccines (DV1-EM3, DV2-EM3, DV3-EM3, DV4-EM3, DV1-EM3-1, DV2-EM3-1, DV3-EM3-1 and DV4-EM3-1) were significantly reduced, indicating that the risk of ADE effects induced by mutant antigens is significantly reduced.

[0249] Example 11: Evaluation of vaccine-induced ADE effects in vivo

[0250] In this example, the serum of BALB / c mice immunized with the vaccines DV4-WT, DV4-EM3 and DV4-EM3-1 obtained in Example 10 was mixed with DENV2 virus and then subcutaneously injected into AG129 mice to detect the ADE effect of the immune serum of the DENV4 vaccine on the DENV2 virus in the AG129 mouse model.

[0251] BALB / c mice were immunized with the various vaccines or PBS using AG129 mice as the infection model. Four weeks after the booster immunization, blood was collected, serum was separated, and heat-inactivated. The serum from each vaccine group was pooled, aliquoted, and stored at –80°C until use. Before adoptive transfer into AG129 mice, the serum was diluted 1000-fold with PBS to 50 μL. The serum was then mixed with an equal volume of DENV2 virus (6000 FFU). The virus-serum mixture was injected subcutaneously into the AG129 mice. Mice were monitored daily for survival and weight loss, and mice that lost 25% of their initial weight were euthanized.

[0252] The results are shown in Figure 18, which shows that compared with the adoptive PBS immune serum, the wild-type vaccine DV4-WT immune serum induced the death of AG129 mice earlier, showing a significant ADE effect; while the mutant vaccines DV4-EM3 and DV4-EM3-1 immune serum did not cause the death of AG129 mice faster and had no ADE effect.

[0253] Example 12: Immunization of mice with quadrivalent dengue virus mRNA vaccine

[0254] In this example, female 6-8 week old BALB / c mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were used for a quadrivalent dengue virus vaccine immunization experiment; the experiment was divided into a negative control group (Sham group), a quadrivalent wild-type vaccine group (tWT group), and a quadrivalent mutant vaccine group (tMut group), with 8 mice in each group; wherein, the negative control group was immunized with normal saline, and the tWT vaccine was a combination of DV1-WT, DV2-WT, DV3-WT, and DV4-WT prepared in Example 1. The tMut vaccine was prepared by mixing four vaccines, DV1-EM3-1, DV2-EM3-1, DV3-EM3, and DV4-EM3, prepared in Example 1, at 3 μg / dose for each vaccine, i.e., a total of 12 μg / dose. For each immunization group, intramuscular injection was performed on day 0 and day 21, respectively. Blood was collected 6 weeks after the initial immunization, serum was separated at 4°C, inactivated at 56°C for 30 minutes, and then stored at -80°C for use.

[0255] Example 13: Expression and purification of FL antibody scFv

[0256] In this example, according to the antibody heavy chain and light chain sequences disclosed in the literature, dengue virus FL antibodies Z5-scfv (Wang, QHet al. Sci Transl Med 8, (2016)) and Z6-scfv (Dai, L. et al. Nat Immunol 22, 958-968, (2021)) were constructed respectively. After adding a signal peptide and a His tag to the 5' and 3' ends of the scfv antibody amino acid sequence, respectively, the codons were optimized according to mammalian cell codon preference to generate the nucleic acid coding sequences for the Z5-scfv and Z6-scfv antibodies, as shown in SEQ ID NO:125 and SEQ ID NO:126, respectively. A Kozak sequence and a stop codon were then added to the 5' and 3' ends of the nucleic acid coding sequences, respectively, and gene synthesis was performed by GenScript Biotech Co., Ltd. The synthesized gene fragments were then double-digested with EcoRI at the 5' end and XhoI at the 3' end and cloned into the pCAGGS vector to construct the E protein expression plasmids pCAGGS-Z5-scfv and pCAGGS-Z6-scfv. Protein expression was performed using HEK293F cells and purified using HisTrap™ Excel affinity chromatography and Superdex200 Increase 10 / 300GL gel filtration columns.

[0257] Select HEK293F cells in good growth condition and transfect them at a density of 1-1.5x10 6The cells were inoculated into fresh SMM293-TII medium at a density of 10 cells / ml and cultured in a constant temperature shaker at 37°C, 5% CO2, and 150 rpm. On the day of transfection, the cell density was adjusted to 2.5×10 6 Prepare the transfection reagent (using 10 ml of cells as an example): dilute 10 μg of plasmid with 150 mM NaCI to a total volume of 250 μl and mix gently. Dilute the Sinofection transfection reagent with 150 mM NaCI to a total volume of 250 μl and mix gently. Let the diluted plasmid and transfection reagent stand separately for approximately 5 minutes, then gently mix until the total volume is 500 μl. Let stand at room temperature for another 10 minutes. Add the transfection solution dropwise to the cell culture medium, gently shaking the culture flask while adding. After mixing, return the culture to the shaker and continue incubation. Add 350 μl of SMS-293-SUPI feed solution 24 hours after transfection, and add 350 μl of feed solution every 48 hours thereafter. 5 days after transfection, collect the supernatant for protein purification.

[0258] The supernatant of cells was collected 5 days after transfection, centrifuged at 8000 rpm and 4°C for 1 hour, and then filtered through a 0.22 μm filter. His affinity chromatography purification: First, rinse the HisTrap with water filtered through a 0.22 μm filter. TMThe Excel affinity chromatography column was rinsed for approximately 3-5 column volumes. Afterwards, the column was rinsed with HisTrap affinity chromatography equilibration solution until both the UV and conductivity detection lines were stable, indicating that the column was fully equilibrated. Next, the cell supernatant was passed through the column at a flow rate of 1-2 mL / min to allow protein binding. After all the supernatant had passed through the column, the column was rinsed with 5-10 column volumes of affinity chromatography equilibration solution until the UV detection line was stable. Finally, the column was rinsed with eluents containing 50 mM, 300 mM, and 1 M imidazole, respectively. The collected protein was sampled and identified by SDS-PAGE. Further purification was performed using Superdex 200 Increase 10 / 300 GL gel filtration chromatography. The sample obtained from the affinity chromatography was concentrated to less than 800 μl using a 10 kDa ultrafiltration concentrator and transferred to a 1.5 mL centrifuge tube. After centrifugation at 12,000 g at 4°C for 20 minutes, ensure that no precipitate is aspirated and transfer the sample to a fresh 1.5 mL centrifuge tube. Remove air bubbles from the sample by centrifugation at 12,000 g at 4°C for 20 minutes and prepare for sample loading. Equilibrate a Superdex 200 Increase 10 / 300 GL gel filtration column with gel filtration chromatography equilibration buffer at a maximum flow rate of 0.5 mL / min until the UV detection line is stable. Load the sample using a 1 mL loop. After loading, flush the column with 20 / 50 equilibration buffer (20 mM Tris, 50 mM NaCl) at a flow rate of 0.5 mL / min. Set the peak collector to collect the sample at the peak. Prepare the collected sample and identify it by SDS-PAGE. Concentrate the identified correctly expressed sample using a 10 kDa ultrafiltration concentrator and store in aliquots at -80°C for later use.

[0259] Example 14: Competitive enzyme-linked immunosorbent assay (ELISA) detection of FL epitope antibodies induced by quadrivalent dengue mRNA vaccine

[0260] In this example, a competitive ELISA experiment was performed using the serum of mice immunized with the quadrivalent dengue mRNA vaccine obtained in Example 12 to detect the level of antibodies against the FL epitope of the antigen E protein induced by the quadrivalent dengue vaccine.

[0261] Specifically, the following procedures were adopted:

[0262] (1) The dengue virus E protein prepared in Example 3 was diluted to 2 μg / mL using ELISA coating solution (Solarbol, C1050). 100 μL of the solution was added to each well of a 96-well ELISA plate (Coring, 3590) and incubated at 4°C for 12 hours.

[0263] (2) Pour off the coating solution, add PBS, and wash once; add 5% skim milk prepared in PBS as blocking solution to the 96-well plate, 100 μL per well, and leave at room temperature for 1 hour to block; after blocking, wash once with PBS solution;

[0264] (3) During the blocking period in step (2), the scFv competitor antibody mixture (Z5-scfv and Z6-scfv mixed at a concentration ratio of 1:1) was diluted to a concentration of 20 μg / ml with blocking solution. In the non-competitive ELISA assay, 50 μL of blank blocking solution was added. In the competitive ELISA assay, 50 μL of scFv competitor antibody was added. The negative control was the addition of blank blocking solution. The mixture was incubated at 37°C for 1 hour.

[0265] (4) During the blocking period in step (3), the mouse immune serum sample was diluted with blocking solution, starting from 10,000 times and diluted in a 2-fold gradient; then, 50 μL of the diluted immune serum was added to each well of the ELISA plate. The negative control was added with blocking solution, incubated at 37 degrees for 2 hours, and then washed 4 times with PBST;

[0266] (5) Add HRP-conjugated goat anti-mouse secondary antibody (Abcam, ab6789) diluted 1:2000 in blocking solution, incubate at 37°C for 1 hour, and then wash 5-6 times with PBST; add TMB colorimetric solution for color development. After the reaction time is appropriate, add 2M hydrochloric acid to terminate the reaction, and measure the OD450 value on a microplate reader.

[0267] The antibody titer value is defined as the highest serum dilution factor with a reaction value greater than 2.1 times the negative control value. When the reaction value of the lowest dilution factor (detection limit) is still less than 2.1 times the background value, the titer of the sample is defined as half of the lowest dilution factor, i.e. 1:5.

[0268] The results of the competitive ELISA experiment are shown in Figure 19. Figure 19 shows that when the FL epitope scfv competitive antibody is not added (tWT and tMut), all E protein-specific antibodies in the mouse serum can bind to the coated E protein. When the FL epitope scfv competitive antibody is added (tWT-C and tMut-C), the FL epitope antibody in the mouse serum cannot bind to the coated E protein, and the antibodies of the remaining epitopes can bind to the coated E protein normally. Therefore, the difference between the two can represent the number of FL epitope antibodies in the mouse serum; after adding the FL epitope scfv competitive antibody, the tWT vaccine serum antibody titer was significantly reduced, indicating that the tWT vaccine induced a large number of FL epitope antibodies. In comparison, the antibody titer measured in the tMut vaccine serum remained unchanged when the FL epitope scfv competitive antibody was added, indicating that the tMut vaccine did not induce FL epitope antibodies. The above results show that the mutant antigen of the present application does not induce FL antibodies with high ADE activity in mice, suggesting that it has a lower ADE risk.

[0269] Example 15: Detection of neutralizing antibodies induced by dengue quadrivalent vaccine

[0270] Through the dengue virus microneutralization experiment, the neutralizing antibody titers against four serotypes of dengue virus in the serum of mice immunized with the quadrivalent dengue mRNA vaccine obtained in Example 12 were quantitatively determined. The specific operation steps are as follows:

[0271] Vero cells were seeded in 24-well cell culture dishes one day in advance, and the cells were grown to a confluence of greater than 70% the next day for neutralization testing; serum was graded and diluted with DMEM medium (Invitrogen, C11995500BT) containing 1% FBS (Gibco, 10270-106), and the virus was also diluted with DMEM medium containing 1% FBS. 50 μL of serum and virus solution were mixed in equal volumes. At the same time, the diluted virus was mixed with an equal volume of blank medium as a positive control and incubated at 37°C for 30 min; after incubation, the supernatant medium in the VERO cell plate was removed, and the above mixture was added to the cells at 100 μL / well, and the cells were cultured in a 37°C incubator for 3 days; after 3 days, the cell culture plate was removed, all supernatant was discarded, and the cells were washed once with PBS; the medium was removed, the cells were washed once with PBS, and 50 μL of fixation and permeabilization solution (BD Cytofix / Cytoperm Soln Kit) was added to each well. 554714) were fixed at 4°C for 30 minutes; washed twice with PBST, 50 μL of MZ24-HRP diluent (concentration 2 μg / ml) was added to each well, and incubated at 4°C for 1 hour; washed three times with PBST, 50 μL of TMB colorimetric solution was added to each well, and after 8 minutes, the colorimetric assay was terminated with 2M sulfuric acid, and the absorbance was measured at 450 nm using a microplate reader (PerkinElmer). GraphPad Prism software was used to perform nonlinear fitting on the data to calculate the corresponding serum dilution factor that neutralized 50% of the infected cells, which was the neutralization titer (NT) 50 When the serum at the lowest dilution still cannot neutralize 50% of the infected cells, the NT of the sample is defined as 50 Half of the lowest dilution factor.

[0272] The results are shown in Figure 20, which shows that compared with the negative control group, both tWT and tMut vaccines induced high-titer neutralizing antibody immune responses against the four serotypes of viruses. In particular, the tMut vaccine also induced a balanced neutralizing antibody response against the four serotypes of viruses.

[0273] Example 16: Evaluation of dengue quadrivalent vaccine challenge protection

[0274] In this example, the AG129 mouse lethal model was used to detect the protective effect of the quadrivalent dengue mRNA vaccine.

[0275] Specifically, 6-8 week old AG129 mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were used for quadrivalent dengue virus vaccine immunization and challenge experiments. The experiment was divided into a negative control group (Sham group) and a quadrivalent mutant vaccine group (tMut group), with 12 mice in each group; wherein, the negative control group was immunized with normal saline, and the tMut vaccine was a mixture of four vaccines, DV1-EM3-1, DV2-EM3-1, DV3-EM3, and DV4-EM3, prepared in Example 1, with 3 μg / dose of each vaccine, i.e., a total of 12 μg / dose. For each immunization group, intramuscular injection was performed on days 0 and 21, respectively, and on day 49 after the primary immunization, each group of mice was randomly and evenly distributed, half of which were infected with 1×10^6FFU DENV1 virus by subcutaneous injection, and the other half were infected with 1×10^6FFU DENV2 virus by subcutaneous injection. After DENV virus challenge, animals were weighed and monitored daily for clinical signs of disease. Mice that lost 25% of their initial body weight were euthanized for animal welfare reasons, and the mortality rate was finally calculated.

[0276] The results are shown in Figure 21, which shows that after DENV1 and DENV2 virus infection, all mice in the Sham group died, while all mice in the tMut vaccine group survived, indicating that the tMut vaccine can provide excellent immune protection for AG129 mice.

[0277] Example 17: Preparation of dengue virus mRNA vaccine

[0278] In this example, a series of mRNA constructs encoding the full-length prME protein of dengue virus (i.e., including prM protein + E protein) were designed, including:

[0279] DV1-WT (i.e., DV1 wild type), encoding the prME antigen protein shown in SEQ ID NO: 1;

[0280] DV1-EM1, encoding the prME antigen protein shown in SEQ ID NO: 2;

[0281] DV1-EM2, encoding the prME antigen protein shown in SEQ ID NO: 5;

[0282] DV1-EM4, encoding the prME antigen protein shown in SEQ ID NO:6;

[0283] DV1-EM5, encoding the prME antigen protein shown in SEQ ID NO:7;

[0284] DV1-EM6, encoding the prME antigen protein shown in SEQ ID NO:8;

[0285] DV1-EM7, encoding the prME antigen protein shown in SEQ ID NO:9;

[0286] DV1-EM8, encoding the prME antigen protein shown in SEQ ID NO: 10;

[0287] DV1-EM9, encoding the prME antigen protein shown in SEQ ID NO: 11;

[0288] DV1-EM10, encoding the prME antigen protein shown in SEQ ID NO: 12;

[0289] DV1-EM11, encoding the prME antigen protein shown in SEQ ID NO: 13;

[0290] DV1-EM12, encoding the prME antigen protein shown in SEQ ID NO: 14;

[0291] DV2-WT (i.e., DV2 wild type), encoding the prME antigen protein shown in SEQ ID NO: 15;

[0292] DV2-EM1, encoding the prME antigen protein shown in SEQ ID NO: 16;

[0293] DV2-EM2, encoding the prME antigen protein shown in SEQ ID NO: 19;

[0294] DV2-EM4, encoding the prME antigen protein shown in SEQ ID NO: 20;

[0295] DV2-EM5, encoding the prME antigen protein shown in SEQ ID NO: 21;

[0296] DV2-EM6, encoding the prME antigen protein shown in SEQ ID NO: 22;

[0297] DV2-EM7, encoding the prME antigen protein shown in SEQ ID NO: 23;

[0298] DV2-EM8, encoding the prME antigen protein shown in SEQ ID NO: 24;

[0299] DV2-EM9, encoding the prME antigen protein shown in SEQ ID NO: 25;

[0300] DV2-EM10, encoding the prME antigen protein shown in SEQ ID NO: 26;

[0301] DV2-EM11, encoding the prME antigen protein shown in SEQ ID NO: 27;

[0302] DV2-EM12, encoding the prME antigen protein shown in SEQ ID NO: 28;

[0303] DV3-WT (i.e., DV3 wild type), encoding the prME antigen protein shown in SEQ ID NO: 29;

[0304] DV3-EM1, encoding the prME antigen protein shown in SEQ ID NO: 30;

[0305] DV3-EM2, encoding the prME antigen protein shown in SEQ ID NO: 33;

[0306] DV3-EM4, encoding the prME antigen protein shown in SEQ ID NO: 34;

[0307] DV3-EM5, encoding the prME antigen protein shown in SEQ ID NO: 35;

[0308] DV3-EM6, encoding the prME antigen protein shown in SEQ ID NO: 36;

[0309] DV3-EM7, encoding the prME antigen protein shown in SEQ ID NO: 37;

[0310] DV3-EM8, encoding the prME antigen protein shown in SEQ ID NO: 38;

[0311] DV3-EM9, encoding the prME antigen protein shown in SEQ ID NO: 39;

[0312] DV3-EM10, encoding the prME antigen protein shown in SEQ ID NO:40;

[0313] DV3-EM11, encoding the prME antigen protein shown in SEQ ID NO:41;

[0314] DV3-EM12, encoding the prME antigen protein shown in SEQ ID NO:42;

[0315] DV4-WT (i.e., DV4 wild type), encoding the prME antigen protein shown in SEQ ID NO: 43;

[0316] DV4-EM1, encoding the prME antigen protein shown in SEQ ID NO:44;

[0317] DV4-EM2, encoding the prME antigen protein shown in SEQ ID NO:47;

[0318] DV4-EM4, encoding the prME antigen protein shown in SEQ ID NO:48;

[0319] DV4-EM5, encoding the prME antigen protein shown in SEQ ID NO:49;

[0320] DV4-EM6, encoding the prME antigen protein shown in SEQ ID NO: 50;

[0321] DV4-EM7, encoding the prME antigen protein shown in SEQ ID NO:51;

[0322] DV4-EM8, encoding the prME antigen protein shown in SEQ ID NO:52;

[0323] DV4-EM9, encoding the prME antigen protein shown in SEQ ID NO: 53;

[0324] DV4-EM10, encoding the prME antigen protein shown in SEQ ID NO:54;

[0325] DV4-EM11, encoding the prME antigen protein shown in SEQ ID NO:55;

[0326] DV4-EM12 encodes the prME antigen protein shown in SEQ ID NO:56.

[0327] Next, according to the codon preference of mammalian cells, the nucleic acid sequence encoding the recombinant dengue virus prME antigen protein was optimized to obtain an optimized nucleic acid coding sequence, wherein the optimized nucleic acid coding sequences of DV1-WT, DV2-WT, DV3-WT, and DV4-WT are shown in SEQ ID NOs: 114-117, respectively, and the optimized nucleic acid coding sequences of DV1-EM1, DV1-EM2, DV1-EM4, DV1-EM5, DV1-EM6, DV1-EM7, DV1-EM8, DV1-EM9, DV1-EM10, DV1-EM11, and DV1-EM12 are shown in SEQ ID NOs: 114-117, respectively. The optimized nucleic acid coding sequences of DV2-EM1, DV2-EM2, DV2-EM4, DV2-EM5, DV2-EM6, DV2-EM7, DV2-EM8, DV2-EM9, DV2-EM10, DV2-EM11 and DV2-EM12 are shown in SEQ ID NOs: 75 and 78, respectively; the optimized nucleic acid coding sequences of DV3-EM1, DV3-EM2, DV3-EM4, DV3-EM5, DV3-EM6, DV3-EM7, DV3-EM8, DV3-EM9, DV3-EM10, DV3-EM11 and DV3-EM12 are shown in SEQ ID NOs: 75 and 78, respectively. NO:88, 91-100, DV4-EM1, DV4-EM2, DV4-EM4, DV4-EM5, DV4-EM6, DV4-EM7, DV4-EM8, DV4-EM9, DV4-EM10, DV4-EM11, DV4-EM12 optimized nucleic acid coding sequences are shown in SEQ ID NO:101, 104-113, respectively; Then, a Kozak sequence and a signal peptide nucleic acid coding sequence (such as SEQ ID NO:118, which encodes an amino acid sequence such as a signal peptide shown in SEQ ID NO:58) are added to the 5' end of each nucleic acid coding sequence, and after adding a stop codon to its 3' end, GeneScript Biotech Co., Ltd. is commissioned to perform gene synthesis, and the synthesized gene fragments are recombined into the pHRNT vector (invention patent ZL202110224383.0) preserved in the laboratory to obtain a template plasmid for preparing an mRNA vaccine. The mRNA vaccine preparation method is described in Example 1.

[0328] Example 18: Detection of antigen expression of dengue virus mRNA vaccine

[0329] In this example, a series of dengue virus prME mRNA vaccines prepared in Example 17 were transfected into 293T cells. The FL epitope antibodies and other neutralizing epitope antibodies prepared in Example 2 were then used to detect the presence of individual epitopes and protein expression in each prME. The detailed experimental procedures were similar to those in Example 4. Cell fluorescence was measured on a BD FACSAria III flow cytometer. The relative positive rate of each mutant vaccine was calculated, assuming the positive cell rate of the WT vaccine was 100%.

[0330] The results are shown in Figure 22-25, which shows:

[0331] For the DV1 mRNA vaccine, FL epitope antibodies (Z5 and Z6) can bind to DV1-WT, but the binding to all DV1 mutants is reduced or not binding, indicating that the FL epitope has been partially or completely destroyed in all DV1 mutants; the neutralizing epitope antibody Ab513 can bind to both DV1-WT and all DV1 mutants, indicating that the neutralizing epitope still exists in all DV1 mutants, and all DV1 mutants have high protein expression levels.

[0332] Similar results were obtained for DV2 mRNA vaccine, DV3 mRNA vaccine and DV4 mRNA vaccine.

[0333] Example 19: Evaluation of protection against DV2 mutant mRNA vaccine challenge

[0334] In this example, the AG129 mouse lethal model was used to detect the protective effect of the DV2 mRNA vaccine (DV2-EM1, DV2-EM2, DV2-EM4, DV2-EM5, DV2-EM6, DV2-EM7, DV2-EM8, DV2-EM9, DV2-EM10, DV2-EM11, DV2-EM12) of different mutants prepared in Example 17.

[0335] Specifically, 6-8 week old AG129 mice (purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.) were used for vaccine immunization and challenge experiments. The experiments were divided into a negative control group (Sham group) and each mutant vaccine group (DV2-EM1, DV2-EM2, DV2-EM4, DV2-EM5, DV2-EM6, DV2-EM7, DV2-EM8, DV2-EM9, DV2-EM10, DV2-EM11, DV2-EM12 group), with 5 mice in each group. Among them, the negative control group was immunized with normal saline, and the mutant vaccine was 15 μg / mouse per dose. For each immunization group, the mice were injected intramuscularly on days 0 and 21, respectively, and were infected with 1×10^6FFU DENV2 virus by subcutaneous injection on day 49 after the initial immunization. After DENV virus challenge, the animals were weighed and monitored for clinical signs of disease every day. Mice that lost 25% of their initial body weight were euthanized for animal welfare reasons, and the mortality rate was finally calculated.

[0336] The results are shown in Figure 26, which shows that after DENV2 virus infection, all mice in the Sham group died, while all mice in the DV2-EM1, DV2-EM2, DV2-EM4, DV2-EM5, DV2-EM6, DV2-EM7, DV2-EM8, DV2-EM9, DV2-EM10, DV2-EM11, and DV2-EM12 vaccine groups survived; these results indicate that each mutant vaccine can provide excellent immune protection for AG129 mice.

[0337] Example 20: Preparation of dengue virus-like particle vaccine

[0338] For flaviviruses, recombinant expression of the full-length prME protein can form virus-like particles. In this example, a series of mammalian cell expression plasmids encoding the full-length prME protein of dengue virus were designed, including:

[0339] DV1-EM3-1, encoding the prME antigen protein shown in SEQ ID NO: 4;

[0340] DV2-EM3-1, encoding the prME antigen protein shown in SEQ ID NO: 18;

[0341] DV3-EM3, encoding the prME antigen protein shown in SEQ ID NO: 31;

[0342] DV4-EM3, encoding the prME antigen protein shown in SEQ ID NO:45;

[0343] Next, based on the codon preference of mammalian cells, the nucleic acid sequence encoding the recombinant dengue virus prME antigen protein was optimized to obtain optimized nucleic acid coding sequences. The optimized nucleic acid coding sequences of DV1-EM3-1, DV2-EM3-1, DV3-EM3, and DV4-EM3 are shown in SEQ ID NOs: 64, 77, 89, and 102, respectively. Then, a Kozak sequence and a signal peptide nucleic acid coding sequence (such as SEQ ID NO: 118, which encodes an amino acid sequence such as a signal peptide shown in SEQ ID NO: 58) were added to the 5' end of each nucleic acid coding sequence. After adding a stop codon to its 3' end, GenScript Biotech Co., Ltd. was commissioned to perform gene synthesis. The synthesized gene fragment was then recombined into a laboratory-stored pCAGGS vector to obtain a template plasmid for preparing an mRNA vaccine. HEK293T cells were used to express virus-like particles. The virus-like particles were purified using a HiTrap Capto Q ImpRes anion exchange chromatography column and a SuperoseTM6 Increase 10 / 300GL gel filtration chromatography column. The day before transfection, subculture 293T cells in a 15cm dish at a 1:3 ratio. After 14-16 hours, when the cell density reaches 70% or more, transfection can be performed. Before transfection, replace the cell culture medium with fresh non-resistant DMEM medium. Prepare the transfection system: add 30μg of transfection plasmid to 500μl opti-MEM per 15cm dish, mix gently and let stand for 5 minutes; add 150μl Add Transfection Reagent to 350 μl of opti-MEM, mix gently, and let stand for 5 minutes. Then, mix the two and incubate at room temperature for 15 minutes. 4) Mix the transfection system and slowly add it dropwise to the supernatant of the non-resistant cell culture in a 15 cm culture dish. Mix gently and incubate in a 37°C, 5% CO2 incubator. 5) After 4-6 hours, discard the cell culture supernatant containing the transfection reagent and replace it with antibiotic-free and serum-free DMEM medium. Continue incubation for 48 hours, then harvest the cells and expression supernatant.

[0344] Prepare a concentration cup and select a 100 kDa filter based on the size of the VLPs. Collect the cell supernatant 2 days after transfection and centrifuge at 3500 rpm and 4°C for 15 minutes. Pour the supernatant into the concentration cup and slowly open the liquid nitrogen valve to concentrate the supernatant to 5-10 ml. Replace the solution with 20 mM Tris / 150 mM NaCl containing 0.1 mM EDTA, pH 8.0.

[0345] Purification using a HiTrap Capto Q ImpRes anion exchange chromatography column: First, rinse the HiTrap Capto Q ImpRes anion exchange chromatography column with water filtered through a 0.22 μm filter for approximately 3-5 column volumes. Then, rinse the column with 20 mM Tris / 150 mM NaCl equilibration solution until the UV and conductivity lines stabilize, indicating that the column is fully equilibrated. Next, pass the concentrated, eluted cell supernatant through the column at a flow rate of 1 mL / min to allow contaminants to bind to the column, and collect the flow-through. After all the supernatant has passed through the column, rinse the column with 5-10 column volumes of affinity chromatography equilibration solution until the UV line stabilizes. Finally, rinse the column with eluents containing 300 mM, 500 mM, and 1 M NaCl, sequentially. Prepare a sample from the flow-through and characterize it by SDS-PAGE. The sample was further purified using a Superose™6 Increase 10 / 300GL gel filtration column. The sample obtained from the anion exchange chromatography was concentrated to less than 1000 μl using a 100 kDa ultrafiltration concentrator and then transferred to a 1.5 mL centrifuge tube. After centrifugation at 12,000 rpm and 4°C for 20 minutes, ensuring that no precipitate was aspirated, the sample was transferred to a fresh 1.5 mL centrifuge tube. The sample was centrifuged at 12,000 rpm and 4°C for 20 minutes to remove air bubbles and prepare for sample loading. The Superose™6 Increase 10 / 300GL gel filtration column was equilibrated with 20 mM Tris / 150 mM NaCl at a maximum flow rate of 0.5 mL / min until the UV detection line stabilized. The sample was then loaded using a 1 mL loop. After loading, the column was flushed with 20 mM Tris / 150 mM NaCl at a flow rate of 0.5 mL / min. Peak collection was set to collect the sample at the peak. The collected samples were prepared and preliminarily identified by SDS-PAGE:

[0346] 1) Take 20 μl of the purified sample, add 5 μl of 5× protein loading buffer, and heat at 100°C for 3 min;

[0347] 2) Take 10 μl of supernatant and perform SDS-PAGE electrophoresis;

[0348] 3) Coomassie brilliant blue staining;

[0349] 4) Observe the decolorization with decolorizing solution.

[0350] The identification results are shown in Figure 27. Figure 27 shows that the four virus-like particle vaccines DV1-EM3-1, DV2-EM3-1, DV3-EM3 and DV4-EM3 showed only a single band between 70 and 55 kDa in SDS-PAGE gel electrophoresis, which was consistent with expectations, indicating that the sample purity was high.

[0351] Example 21: Characterization of dengue virus-like particle vaccine using transmission electron microscopy

[0352] In this example, the morphology of the dengue virus-like particles prepared in Example 20 was observed and detected by transmission electron microscopy. The specific steps are as follows:

[0353] Prepare copper grid, phosphotungstic acid stain, filter paper, and sample box

[0354] 1) Negative staining sample preparation: First, perform copper grid glow treatment. A glow discharge instrument is usually used to hydrophilize carbon-sprayed copper grids.

[0355] 2) After the copper mesh is glowed, pick it up with pointed tweezers and fix the tweezers with a binder clip.

[0356] 3) Add 6.5 μl of sample onto the copper grid, let it stand for one minute, and then absorb it with filter paper.

[0357] 4) Add 6.5 μl of phosphotungstic acid dropwise onto the copper grid and immediately remove with filter paper.

[0358] 5) Add 6.5 μl of phosphotungstic acid to the copper mesh, let it stand for one minute, and absorb the excess dye with wet filter paper;

[0359] 6) After drying, observe under an electron microscope.

[0360] After the samples purified by the chromatography column were negatively stained with phosphotungstic acid, the samples were observed and analyzed using a 100kV electron microscope. The electron microscopy results are shown in Figures 28-31, which show that the negative staining results of DV1-EM3-1, DV2-EM3-1, DV3-EM3, and DV4-EM3 virus-like particles showed that a large number of uniform virus-like particles with a diameter of about 30 nm could be observed in the field of view.

[0361] Example 22: Immunogenicity testing of monovalent dengue virus recombinant protein vaccine

[0362] Vaccine immunization experiments were performed using female 6-8 week old BALB / c mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.); the experiment was divided into a monovalent dengue virus recombinant protein vaccine immunization group and a negative control group (normal saline, as the Sham group), with 5 mice in each group; for each immunization group, a placebo (or the dengue virus-like particle protein prepared in Example 20, respectively, mixed with a composite adjuvant consisting of Al(OH)3 (Invivogen, vac-alu-250) and CpG 1826 (Invivogen, vac-1826-1) was injected intramuscularly on day 0 and day 21, respectively, and blood was collected in the second week after the booster immunization, serum was separated at 4°C, inactivated at 56°C for 30 minutes, and then stored at -80°C for use. The serum neutralizing antibody titer detection method is described in Example 7.

[0363] The results of the neutralizing antibody titer test are shown in Figure 32, which shows that: no neutralizing antibodies were detected in the serum of the Sham group, and all mutant dengue virus recombinant protein vaccines could detect higher neutralizing antibody titers against their own viruses, with neutralizing antibody titers ranging from 4,000 to 20,000, indicating that the above-mentioned dengue virus recombinant protein vaccine has good immunogenicity.

[0364] Example 23: Immunogenicity testing of quadrivalent dengue virus recombinant protein vaccine

[0365] Vaccine immunization experiments were conducted using female 6-8 week old BALB / c mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.); the experiment was divided into a quadrivalent dengue virus recombinant protein vaccine group (tVLP) and a negative control group (i.e., Sham group), with 5 mice in each group. The tVLP vaccine was prepared as follows: the four virus-like particles DV1-EM3-1, DV2-EM3-1, DV3-EM3, and DV4-EM3 prepared in Example 20 were mixed in a mass ratio of 1:1:1:1, each at 2.5 μg / dose, for a total of 10 μg / dose, and mixed with a composite adjuvant consisting of Al(OH)3 (Invivogen, vac-alu-250) and CpG 1826 (Invivogen, vac-1826-1). Placebo or vaccine was injected intramuscularly on day 0 and day 21, respectively. Blood was collected 2 weeks after booster immunization, serum was separated by centrifugation, and the serum was inactivated by heating at 56°C for 30 minutes and stored at -80°C for use. The serum neutralizing antibody detection method is shown in the examples.

[0366] The results of the neutralization experiment are shown in Figure 33, which shows that: no neutralizing antibodies against the four serotypes of dengue virus were detected in the sham group serum, and the tVLP vaccine can induce a balanced and strong neutralizing antibody immune response against the four serotypes of dengue virus, with a neutralizing antibody titer of 1,100-4,000, indicating that the quadrivalent dengue virus recombinant protein vaccine composed of mutant virus-like particles has good immunogenicity.

[0367] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application. Industrial Applicability

[0368] The recombinant antigen against Zika / dengue virus provided in this application can avoid inducing the production of FL epitope antibodies, thereby achieving the purpose of reducing or eliminating the ADE effect; moreover, the recombinant antigen has the correct E protein conformation and has a protein expression level similar to or higher than the wild type, and can induce higher specific binding antibody titers and neutralizing antibody titers in animals, thereby providing effective immune protection and having good clinical application value and industrialization prospects.

Claims

1. A recombinant antigen, wherein the recombinant antigen has the full-length sequence of the E protein of Zika virus or dengue virus, and the E protein has site mutations selected from the following: A site mutation at position G106 in the FL fusion loop region; Double-site mutations at positions W101 and G106 in the FL fusion loop region; Double-site mutations at positions W101 and G106 in the FL fusion loop region and a mutation at the 125th amino acid in the non-FL fusion loop region; Double-site mutations at positions G102 and G106 in the FL fusion loop region; Double-site mutations at positions N103 and G106 in the FL fusion loop region; Triple-site mutations at positions W101, N103 and G106 in the FL fusion loop region.

2. The recombinant antigen according to claim 1, characterized in that, The site mutation at position G106 in the FL fusion loop region is: G106V mutation; And / or, the double-site mutations at positions W101 and G106 in the FL fusion loop region are selected from: (1) W101R and G106V, (2) W101G and G106V, and (3) W101L and G106V; And / or, the double-site mutations at positions W101 and G106 in the FL fusion loop region and the mutation at the 125th amino acid in the non-FL fusion loop region are: double-site mutations at positions W101R and G106V in the FL fusion loop region and a mutation of the 125th amino acid in the non-fusion loop region to valine; And / or, the double-site mutations at positions G102 and G106 in the FL fusion loop region are: double-site mutations at positions G102R and G106V; And / or, the double-site mutations at positions N103 and G106 in the FL fusion loop region are selected from: (1) N103T and G106V, (2) N103H and G106V, (3) N103K and G106V, (4) N103P and G106V, and (5) N103Y and G106V; And / or, the triple-site mutations at positions W101, N103 and G106 in the FL fusion loop region are selected from: (1) W101G, N103T and G106V, and (2) W101G, N103K and G106V.

3. The recombinant antigen according to claim 2, wherein The recombinant antigen has the full-length sequence of the E protein of dengue virus type 1, and the E protein has site mutations selected from the following: G106V mutation; Double-site mutations at positions W101R and G106V; Double-site mutations at positions W101G and G106V; Double-site mutations at positions W101L and G106V; Triple-site mutations at positions W101R, G106V and L125V; Double-site mutations at positions G102R and G106V; Double-site mutations at positions N103T and G106V; Double-site mutations at positions N103H and G106V; Double-site mutations at positions N103K and G106V; Double-site mutations at positions N103P and G106V; Double-site mutations at positions N103Y and G106V; Triple-site mutations at positions W101G, N103T and G106V; Triple-site mutations at positions W101G, N103K and G106V.

4. The recombinant antigen according to claim 2, wherein The recombinant antigen has the full-length sequence of the E protein of dengue virus type 2, and the E protein has site mutations selected from the following: G106V mutation; Double-site mutations at positions W101R and G106V; Double-site mutations at positions W101G and G106V; Double-site mutations at positions W101L and G106V; Three-site mutations of W101R, G106V, and M125V; Two-site mutations of G102R and G106V; Two-site mutations of N103T and G106V; Two-site mutations of N103H and G106V; Two-site mutations of N103K and G106V; Two-site mutations of N103P and G106V; Two-site mutations of N103Y and G106V; Three-site mutations of W101G, N103T, and G106V; Three-site mutations of W101G, N103K, and G106V.

5. The recombinant antigen according to claim 2, wherein The recombinant antigen has the full-length sequence of the E protein of dengue virus type 3, and the E protein has site mutations selected from the following: G106V mutation; Two-site mutations of W101R and G106V; Two-site mutations of W101G and G106V; Two-site mutations of W101L and G106V; Three-site mutations of W101R, G106V, and I125V; Two-site mutations of G102R and G106V; Two-site mutations of N103T and G106V; Two-site mutations of N103H and G106V; Two-site mutations of N103K and G106V; Two-site mutations of N103P and G106V; Two-site mutations of N103Y and G106V; Three-site mutations of W101G, N103T, and G106V; Three-site mutations of W101G, N103K, and G106V.

6. The recombinant antigen according to claim 2, characterized in that, The recombinant antigen has the full-length sequence of the E protein of dengue virus type 4, and the E protein has site mutations selected from the following: G106V mutation; Two-site mutations of W101R and G106V; Two-site mutations of W101G and G106V; Two-site mutations of W101L and G106V; Three-site mutations of W101R, G106V, and I125V; Two-site mutations of G102R and G106V; Two-site mutations of N103T and G106V; Two-site mutations of N103H and G106V; Two-site mutations of N103K and G106V; Two-site mutations of N103P and G106V; Two-site mutations of N103Y and G106V; Three-site mutations of W101G, N103T, and G106V; Three-site mutations of W101G, N103K, and G106V.

7. The recombinant antigen according to any one of claims 1-6, characterized in that, When the recombinant antigen has the full-length sequence of the E protein of Zika virus, the recombinant antigen further includes the full-length or partial prM protein sequence or M protein sequence of Zika virus.

8. The recombinant antigen according to any one of claims 1-6, characterized in that, When the recombinant antigen has the full-length sequence of the E protein of dengue virus, the recombinant antigen further includes the full-length or partial prM protein sequence or M protein sequence of the corresponding serotype of dengue virus; Preferably, when the recombinant antigen has the full-length E protein sequence of dengue virus type 1, the recombinant antigen has the amino acid sequence shown in SEQ ID NO: 2-14; and / or, when the recombinant antigen has the full-length E protein sequence of dengue virus type 2, the recombinant antigen has the amino acid sequence shown in SEQ ID NO: 16-28; and / or, when the recombinant antigen has the full-length E protein sequence of dengue virus type 3, the recombinant antigen has the amino acid sequence shown in SEQ ID NO: 30-42; and / or, when the recombinant antigen has the full-length E protein sequence of dengue virus type 4, the recombinant antigen has the amino acid sequence shown in SEQ ID NO: 44-56.

9. The preparation method of the recombinant antigen according to any one of claims 1-8, characterized in that, The preparation method comprises the following steps: Adding a Kozak sequence and the coding sequence of a signal peptide to the 5' end of the nucleotide sequence encoding the recombinant antigen according to any one of claims 1-8, adding the coding sequence of a histidine tag and a stop codon to the 3' end, performing clone expression, screening correct recombinants, then transfecting the cells of an expression system for expression, collecting the cell culture supernatant, and isolating and obtaining the recombinant antigen therefrom.

10. The preparation method according to claim 9, characterized in that, The cells of the expression system are mammalian cells, insect cells, yeast cells or bacterial cells; Optionally, the mammalian cells are HEK293T cells, 293F series cells or CHO cells; further optionally, the 293F series cells are HEK293F cells, Freestyle293F cells or Expi293F cells; Optionally, the insect cells are sf9 cells, Hi5 cells, sf21 cells or S2 cells; Optionally, the yeast cells are Pichia pastoris cells or yeast cells modified therefrom; Optionally, the bacterial cells are Escherichia coli cells.

11. A polynucleotide encoding the recombinant antigen according to any one of claims 1-8.

12. The polynucleotide according to claim 11, wherein The polynucleotide is DNA or mRNA, preferably mRNA; Preferably, the polynucleotide is DNA having a sequence shown in any one of SEQ ID NO: 62-113, or the mRNA corresponding to the DNA.

13. An expression cassette, recombinant vector, transgenic cell line, recombinant bacterium or recombinant virus comprising the polynucleotide according to claim 11 or 12.

14. A vaccine composition comprising the recombinant antigen according to any one of claims 1-8, or the polynucleotide according to claim 11 or 12, or the expression cassette, recombinant vector, transgenic cell line, recombinant bacterium or recombinant virus according to claim 13 as an active ingredient.

15. The vaccine composition according to claim 14, wherein It is a recombinant protein vaccine, which comprises the recombinant antigen according to any one of claims 1-8 and an adjuvant; Preferably, the adjuvant is one or more selected from the following adjuvants: aluminum adjuvant, MF59 adjuvant, MF59-like adjuvant, CpG adjuvant, adjuvant containing QS-21, AS series adjuvants and nanoparticle adjuvants.

16. The vaccine composition according to claim 15, characterized in that, It is a DNA vaccine, and the DNA vaccine comprises: (i) a eukaryotic expression vector; and (ii) A DNA sequence encoding the recombinant antigen as described in any one of claims 1-8, which is constructed into the eukaryotic expression vector; Preferably, the DNA sequence encoding the recombinant antigen as described in any one of claims 1-8 is a DNA sequence shown in any one of SEQ ID NO: 62-113; And / or, the eukaryotic expression vector is selected from pGX0001, pVAX1, pCAGGS and pcDNA series vectors.

17. The vaccine composition according to claim 14, wherein It is an mRNA vaccine, and the mRNA vaccine comprises: (I) An mRNA sequence encoding the recombinant antigen as described in any one of claims 1-8; and (II) Lipid nanoparticles; Preferably, the mRNA sequence encoding the recombinant antigen as described in any one of claims 1-8 is an mRNA sequence corresponding to the DNA sequence shown in any one of SEQ ID NO: 62-113.

18. The vaccine composition according to claim 14, wherein It is a viral vector vaccine, which comprises: (1) A viral backbone vector; and (2) A DNA sequence encoding the recombinant antigen as described in any one of claims 1-8, which is constructed into the viral backbone vector; Preferably, the DNA sequence encoding the recombinant antigen as described in any one of claims 1-8 is a DNA sequence shown in any one of SEQ ID NO: 62-113; And / or, the viral backbone vector is selected from one or more of the following viral vectors: adenovirus vector, lentivirus vector, poxvirus vector, influenza virus vector, adeno-associated virus vector.

19. The vaccine composition according to any one of claims 14-18, characterized in that, The vaccine composition is in the form of a nasal spray, oral preparation, suppository or parenteral preparation; Preferably, the nasal spray is selected from aerosols, sprays and powder aerosols; Preferably, the oral preparation is selected from tablets, powders, pills, granules, soft / hard capsules, film-coated tablets and ointments; More preferably, the tablet is a sublingual tablet; More preferably, the granule is a fine granule; More preferably, the powder is a powder; More preferably, the pill is a small pill; Preferably, the parenteral preparation is a transdermal agent, ointment, plaster, external liquid preparation, injectable preparation; more preferably, the injectable preparation is a pushable preparation.

20. Use of the recombinant antigen as described in any one of claims 1-8, the polynucleotide as described in claim 11 or 12, or the expression cassette, recombinant vector, transgenic cell line, recombinant bacterium or recombinant virus as described in claim 13 in the preparation of a drug for detecting, preventing and / or treating Zika virus or dengue virus infection; Preferably, the drug is a vaccine.

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