Discovery method for flavivirus vaccine antigen, and use thereof

Abstract

A discovery method for a flavivirus vaccine antigen, and a use thereof. The discovery method is capable of efficiently discovering a flavivirus vaccine antigen in which a FL epitope is destroyed while maintaining proper expression and folding of a prME protein. The obtained flavivirus vaccine antigen can avoid the ADE effect and has good immunogenicity, and can be used for the construction of flavivirus vaccines.

Description

A method for discovering flavivirus vaccine antigens and its application

[0001] Cross-references

[0002] This application claims priority to the Chinese patent application filed on January 5, 2024, with application number 202410024376.X and invention name “A method for discovering a flavivirus vaccine antigen and its application”, 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 a method for discovering a flavivirus vaccine antigen and its application. Background Art

[0004] According to the latest information released by the International Committee on Taxonomy of Viruses (ICTV), the Flaviviridae family comprises four genera: Flavivirus (53 species), Hepacivirus (14 species), Pegivirus (11 species), and Pestivirus (11 species). Based on their mode of transmission, the 53 flavivirus species are divided into three groups: tick-borne flaviviruses (TBFVs), mosquito-borne flaviviruses (MBFVs), and flaviviruses with no known vector (NKVs).

[0005] Dengue virus (DV) and Zika virus (ZIKV) are both mosquito-borne flaviviruses, primarily transmitted by vector insects such as Aedes aegypti and Aedes albopictus. DV, the causative agent of dengue fever (DF), is widely distributed in tropical and subtropical regions. In some countries, DV infection is a leading cause of hospitalization and mortality among children. According to the World Health Organization (WHO), dengue fever is currently endemic in over 100 countries and regions, with 2.5-3 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. ZIKV first broke out in Brazil in 2015 and rapidly spread to 84 countries and regions worldwide, with imported cases of ZIKV infection also occurring in China. ZIKV infection may cause Guillain-Barré syndrome (GBS), but what is more dangerous is that ZIKV infection in pregnant women may cause fetal miscarriage or abnormal brain development of the fetus or newborn, resulting in microcephaly.

[0006] Flaviviruses are enveloped viruses with similar structures. The viral genome encodes three structural proteins (C, PrM, and E). The C protein forms the nucleocapsid. During viral maturation, the PrM protein is enzymatically cleaved to form the membrane protein M, which is then anchored to the inner layer of the viral envelope. It is a crucial component in virally induced protective immunity and 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 erythrocyte agglutination-inhibiting and neutralizing antibodies. It is the primary protective antigen of dengue virus. DV infection exhibits a typical phenomenon of antibody-dependent infection enhancement (ADE). When infected with a serotype of a virus, such as DV-1, immune cells produce antibodies against DV-1. However, upon reinfection with a different dengue virus, such as DV-2, these previously produced anti-DV-1 antibodies not only fail to neutralize the DV-2 virus but also exacerbate infection. ZIKV infection / vaccination may induce cross-reactive antibodies, which can enhance infection with antibody-related DV through ADE. Therefore, developing a dengue / Zika vaccine that has no ADE risk and has good immune protection is a huge challenge.

[0007] Numerous studies have demonstrated that the hydrophobic fusion loop (FL) of the E protein is the primary epitope inducing ADE (antibody degrading) effects and is also a major dominant epitope on the E protein. During vaccine development, if wild-type E protein is used as an immunogen, the FL epitope can induce a large number of antibodies, posing a high risk of ADE. To completely eliminate the risk of vaccine-induced ADE, the FL epitope must be mutated to render it ineffective in inducing FL antibodies. However, the FL epitope plays a crucial role in inducing fusion of the viral envelope with the host cell membrane during flavivirus invasion. Its amino acid sequence, D98-R99-G100-W101-G102-N103-G104-C105-G106-L107-F108-G109, is highly conserved (i.e., the amino acid sequence is identical) throughout the entire flavivirus genus. Mutations in the FL epitope can easily lead to a significant decrease in E protein expression or even complete absence of protein expression. A screening strategy for FL epitope mutants is to make a random mutation saturation library in the FL epitope region of E protein. The FL epitope has 12 amino acids in total, and the theoretical total number of mutants is 12. 19 , the number is huge, and the success rate is low. For example, Meganck et al. used reverse genetics technology to create an amino acid saturation mutation library on the dengue virus FL epitope. After three consecutive generations, only one stable mutant strain N103S-G106L was screened in C6 / 36 cells. However, this mutation strategy only partially eliminated the FL epitope and led to poor protein stability.

[0008] In view of the above, there is an urgent need in the art for a method to develop a flavivirus vaccine antigen with ADE epitope eliminated and good immunogenicity. Summary of the Invention

[0009] Purpose of the Invention

[0010] In response to the problems or needs in the prior art, the purpose of this application is to provide a method for discovering a flavivirus vaccine antigen with ADE epitope eliminated and good immunogenicity and its application.

[0011] Solution

[0012] In order to achieve the above objectives, this application provides the following technical solutions:

[0013] In a first aspect, the present application provides a method for discovering flavivirus vaccine antigens, the method comprising:

[0014] (1) Cultivating flaviviruses in the presence of FL epitope-specific antibodies to induce escape evolution of flaviviruses;

[0015] (2) sequencing the E protein of the escaped evolved flavivirus obtained in step (1) to determine the mutations within the E protein;

[0016] (3) By homologous replacement, the mutation detected in the E protein is introduced into the E protein of the flavivirus to obtain a flavivirus E protein mutant as a flavivirus vaccine antigen.

[0017] In a possible embodiment, step (1) comprises:

[0018] Flaviviruses were screened in multiple rounds in the presence of increasing concentrations of FL epitope-specific antibodies until escape-evolved flaviviruses were obtained.

[0019] Preferably, the escape-evolved flavivirus is a flavivirus that escapes FL epitope-specific antibodies.

[0020] Preferably, the escaped evolved flavivirus comprises a mutation within the FL fusion loop region of the E protein and optionally a mutation within a non-FL fusion loop region.

[0021] Preferably, in the first to third rounds of screening, the FL epitope-specific antibody is added at a concentration that reduces the infection rate of the flavivirus to cells by 20%-80%, preferably by 40%-60%, and more preferably by 50%.

[0022] Preferably, the endpoints of the multiple rounds of screening are: the obtained flavivirus escapes the FL epitope-specific antibody, and optionally, the cell infection ability of the obtained flavivirus remains substantially unchanged compared with the initial virus.

[0023] In a preferred embodiment, step (3) further comprises: combining the obtained flavivirus E protein mutant with the full-length or partial prM protein sequence of the flavivirus as a flavivirus vaccine antigen.

[0024] Further preferably, the discovery method further comprises the step of further screening the flavivirus vaccine antigen obtained in step (3).

[0025] Optionally, the criteria for further screening of flavivirus vaccine antigens are selected from any one or more of the following:

[0026] 1) The obtained flavivirus vaccine antigen does not bind to the FL epitope-specific antibody,

[0027] 2) The binding of the resulting flavivirus vaccine antigen to non-FL neutralizing epitope antibodies is not affected;

[0028] 3) The obtained flavivirus vaccine antigen can be expressed and folded normally; preferably, the expression efficiency of the obtained flavivirus vaccine antigen is equivalent to that of its corresponding wild type.

[0029] In a preferred embodiment, the flavivirus is selected from the group consisting of Zika virus, dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, tick-borne encephalitis virus, and Murray Valley encephalitis virus, preferably Zika, yellow fever virus, or dengue virus;

[0030] Further preferably, the FL epitope-specific antibody is selected from: Z5, Z6, 4G2, 2A10G6, Z7, Z10, Z24, and Z25.

[0031] In a second aspect, the present application provides the application of the method for discovering flavivirus vaccine antigens described in the first aspect in developing flavivirus vaccine antigens or even developing flavivirus vaccines. Beneficial effects

[0032] According to the method for discovering flavivirus vaccine antigens of the present application, escaped-evolved flaviviruses with a destroyed FL epitope can be obtained with high efficiency. Destruction of the FL epitope of the flavivirus means that the epitope that causes the ADE effect is eliminated, thereby avoiding the ADE effect. Furthermore, the resulting escaped-evolved flavivirus can survive normally, indicating that its prME protein is expressed and folded normally. Subsequently, the escaped-evolved flavivirus is sequenced to obtain mutation information in the FL fusion loop region and, optionally, in non-FL fusion loop regions of its E protein. These mutations are then introduced into the flavivirus E protein by homologous replacement to construct the corresponding flavivirus vaccine antigen. Because the E protein mutation information is consistent with that of the escaped-evolved flavivirus, the constructed flavivirus vaccine antigen is expected to also have its FL epitope destroyed and its prME protein properly folded and expressed. In a preferred embodiment, further experimental screening can be used to obtain flavivirus vaccine antigens that avoid the ADE effect and have good immunogenicity. In view of the foregoing, the flavivirus vaccine antigens of the present application have good prospects for industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] 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.

[0034] Figure 1 shows the results of the detection of the infection rate of the ZIKV escape mutation P1 induced by the FL epitope antibody, as described in Example 2;

[0035] FIG2 shows the results of the detection of the infection rate of the ZIKV escape mutant P8 induced by the FL epitope antibody, as described in Example 2;

[0036] FIG3 shows the sequencing results of various ZIKV mutants with FL epitope antibody escape mutations, as described in Example 3;

[0037] FIG4 shows the results of antigenic epitope detection of the DV1 mRNA vaccine prepared in Example 4, as detected in Example 6.

[0038] FIG5 shows the results of antigenic epitope detection of the DV2 mRNA vaccine prepared in Example 4, as detected in Example 6.

[0039] FIG6 shows the results of antigenic epitope detection of the DV3 mRNA vaccine prepared in Example 4, as detected in Example 6.

[0040] FIG7 shows the results of antigenic epitope detection of the DV4 mRNA vaccine prepared in Example 4, as detected in Example 6.

[0041] FIG8 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 8.

[0042] FIG. 9 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 9.

[0043] FIG10 shows the cross-antibody titers against DV2 / 3 / 4 and Zika virus E protein induced by the DV1 mRNA vaccine prepared in Example 4, as detected in Example 10.

[0044] Figure 11 shows the cross-antibody titers against DV1 / 3 / 4 and Zika virus E protein induced by the DV2 mRNA vaccine prepared in Example 4, as detected in Example 10.

[0045] Figure 12 shows the cross-antibody titers against DV1 / 2 / 4 and Zika virus E protein induced by the DV3 mRNA vaccine prepared in Example 4, as detected in Example 10.

[0046] Figure 13 shows the cross-antibody titers against DV1 / 2 / 3 and Zika virus E protein induced by the DV4 mRNA vaccine prepared in Example 4, as detected in Example 10.

[0047] FIG. 14 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 11.

[0048] FIG15 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 11.

[0049] FIG16 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 11.

[0050] FIG17 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 12.

[0051] FIG18 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 12.

[0052] FIG19 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 12.

[0053] FIG20 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 12.

[0054] FIG21 shows the in vivo ADE effect against DV2 virus induced by the immune serum of the DV4 mRNA vaccine prepared in Example 1, as tested in Example 13.

[0055] FIG. 22 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 14, as detected in Example 16.

[0056] Figure 23 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 14, as detected in Example 17.

[0057] FIG. 24 shows the protective effect of the quadrivalent dengue mRNA vaccine described in Example 14 against challenge with DV1 and DV2 viruses, as tested in Example 18.

[0058] FIG. 25 shows the results of antigen epitope and antigen expression detection of the DV1 mRNA vaccine prepared in Example 19, as detected in Example 20.

[0059] FIG26 shows the results of antigen epitope and antigen expression detection of the DV2 mRNA vaccine prepared in Example 19, as detected in Example 20.

[0060] Figure 27 shows the antigen epitope and antigen expression detection results of the DV3 mRNA vaccine prepared in Example 19, as detected in Example 20.

[0061] FIG28 shows the results of antigen epitope and antigen expression detection of the DV4 mRNA vaccine prepared in Example 19, as detected in Example 20.

[0062] FIG29 shows the protective effect against DV2 virus challenge induced by the DV2 mRNA vaccine prepared in Example 19, as tested in Example 21.

[0063] FIG30 shows the SDS-PAGE results of the dengue virus-like particles prepared in Example 22, as detected in Example 22.

[0064] Figure 31 shows the electron microscopic negative staining results of DV1-EM3-1 virus-like particles prepared in Example 22, as detected in Example 23.

[0065] Figure 32 shows the electron microscopic negative staining results of DV2-EM3-1 virus-like particles prepared in Example 22, as detected in Example 23.

[0066] FIG33 shows the electron microscopic negative staining results of the DV3-EM3 virus-like particles prepared in Example 22, as detected in Example 23.

[0067] FIG34 shows the electron microscopic negative staining results of the DV4-EM3 virus-like particles prepared in Example 22, as detected in Example 23.

[0068] Figure 35 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 24.

[0069] Figure 36 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 25. DETAILED DESCRIPTION

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] Example 1: Expression and purification of detection antibodies

[0079] 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, QHet al. Sci Transl Med 8, (2016)), Z6 (Dai, L. et al. Nat Immunol 22, 958-968, (2021)), Z30 (Wang, QHet al. Sci Transl Med 8, (2016)), 4G2 (Dai, L et al. Nat Immunol 22, 958-968, (2021)), MZ24 (Dussupt, V. et al. Nat Med 26, 228-35, (2020)), Ab513 (Robinson, LNet al. Cell 162, 493-504, (2015)), 1F4 (Fibriansah, G et al. Nat Immunol 23, 514-529, (2016)), and 1F6 (Fibriansah, G et al. Nat Immunol 23, 514-529, (2016)) were constructed respectively. 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)) heavy chain and light chain expression plasmids.

[0080] Among them, Z5, Z6 and 4G2 antibodies recognize the FL epitope of Zika and dengue viruses, and the remaining antibodies (Z30, MZ-1, MZ-4, MZ24, Ab513, 1F4, 3H5, 5J7 and 5H2) are all highly neutralizing antibodies that can recognize non-FL epitopes.

[0081] Antibody expression

[0082] 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.

[0083] Antibody purification

[0084] 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.

[0085] Example 2: Escape evolution of ZIKV virus under FL antibody pressure

[0086] In this example, we used the Z5 antibody to induce escape mutations in the ZIKV virus.

[0087] Specifically, 100 μL of P0 virus was added to each of six T25 flasks. Z5 antibody was diluted with RPMI-1640 medium containing 1% FBS and added to the first five T25 flasks to a final concentration of 2 μg / ml, 4 μg / ml, 10 μg / ml, 20 μg / ml, and 40 μg / ml, respectively. The sixth T25 flask was not supplemented with antibody and was replenished with RPMI-1640 medium containing 1% FBS to a final concentration of 5 ml, representing a positive well. The cells were then incubated at 28°C for 30 minutes. Subsequently, C6 / 36 cells prepared in advance were centrifuged, resuspended, and added to each of the six T25 flasks. After 5 days, the cells and supernatant were collected and centrifuged at 4000 rpm for 10 minutes. The supernatant, which was the virus, was aliquoted and frozen. Simultaneously, the cells were resuspended in a 96-well plate and an equal amount of cells from each flask was added to three wells, as triplicates. Afterwards, 100 μL of Fixation and Permeabilization solution (BD, 554722) was added to each well. After fixation at 4°C in the dark for 30 minutes, cells were collected by centrifugation at 800 g for 5 minutes and washed twice with 1× Perm / Wash buffer (BD, 554723). Then, 100 μL of flavivirus FL antibodies (Z5 and Z6) or non-FLE antibody Z30 was used as primary antibodies for staining at 4°C in the dark for 30 minutes. Then, 100 μL of secondary antibody (FITC-conjugated goat anti-human IgG) was added to each well at a 2000-fold dilution 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 flow cytometry tubes, and the FITC fluorescence positivity of cells was detected using a FACS Canto II. The results are shown in Figure 1. In the positive control vial without Z5 antibody, the virus infection rate detected by Z30 antibody was 95%. This rate gradually decreased with increasing Z5 antibody concentration. At a Z5 antibody concentration of 10 μg / ml, the virus infection rate reached 55%, approximately half the infection rate in the positive vial. The virus under these conditions was selected as the P1 virus and entered the next round of screening. In the next round of screening, the first round of screening was repeated, but with increasing Z5 antibody concentrations of 10 μg / ml, 15 μg / ml, 20 μg / ml, 30 μg / ml, and 40 μg / ml. Finally, a virus with an infection rate of approximately 40-50% was selected as the P2 virus.Several rounds of screening were then continued, and the cell infection rate of the viruses obtained in each round of screening was measured. Finally, when the P8 generation virus infection rate was tested (see Figure 2), it was found that when the Z5 antibody concentration reached 200μg / ml and 400μg / ml, the cell infection rate of the virus detected by the Z30 antibody was above 98%, indicating that the infection and replication of the virus were not affected by the Z5 antibody. When the FL epitope Z5 and Z6 antibodies were used for detection, the cell infection rate of the virus was found to be significantly reduced, indicating that the virus replicated under these conditions could not bind well to the Z5 or Z6 antibodies. This shows that under the continuous selection pressure of the Z5 antibody, the Zika virus has begun to escape this antibody. Finally, we selected the virus cultured at a Z5 antibody concentration of 400μg / ml as the P8 generation virus and carried out the next step of viral E protein sequencing on the P8 generation virus to obtain mutation information of the FL epitope and non-FL epitope.

[0088] Example 3: Sequencing of FL Antibody Escape from Zika Virus

[0089] RNA from the P8 generation escape mutant ZIKV described in Example 2 was extracted using the Yimaisai Nucleic Acid Extraction or Purification Reagent Magnetic Bead Viral RNA Extraction Kit. The procedure was performed strictly according to the kit instructions. The following steps were briefly described: 10 μL of Proteinase K and Solution ER were added to each well in the first row (row A) of the kit. 50 μL of viral sample was then added to each well in row A. The cannula was inserted into the cannula slot of the automated nucleic acid extraction instrument. The instrument was started. After 20 minutes, nucleic acid extraction was complete. RNA was carefully aspirated from the sixth row (row F) using a pipette into a clean, RNase-free, eight-tube strip.

[0090] ZIKV cDNA was obtained by reverse transcription using the FastKing One Step RT-PCR Kit. The template in the reaction system was the P8-generation escape mutant ZIKV RNA obtained above, and the primers were specific primers for ZIKV E protein RT-PCR (see Table 1). The 2× FastKing One Step RT-PCR MasterMix and RNase-Free ddH2O in the kit were briefly centrifuged and placed on ice. The RT-PCR reaction system was prepared on ice according to Table 2, and the PCR reaction conditions were set according to Table 3. After the RT-PCR reaction, the reaction products were subjected to agarose gel electrophoresis. The gel was exposed using a gel imager to observe the amplification results and cut out the gel block with the correct band size to recover and purify the target gene.

[0091] Select the TOPO-Blunt Simple Lightning Cloning Kit and connect the recovered target gene to the pTOPO-Blunt Simple vector according to the reaction system in Table 4. Specifically, after adding the reagents according to the reaction system in Table 4, gently pipette to mix or tap the bottom of the tube to mix, and collect all the liquid at the bottom of the centrifuge tube by low-speed instantaneous centrifugation; connect at room temperature (20℃-30℃) for 5 minutes; 100μL competent cells were thawed at room temperature, and after complete thawing, they were gently flicked several times to evenly suspend the cells; 10μL of the ligation product was added, gently mixed, and placed at room temperature for 5 minutes; 200μL LB medium (without antibiotics) was added and shaken at 37℃ and 180rpm for 10 minutes to recover; 200μL of bacterial solution was spread on a plate (containing 50-100mg / ml of ampicillin) and cultured overnight; the plasmid was extracted from the bacterial culture of the single white colony and sent to a sequencing company for sequencing using TOPO vector universal primers. The sequencing results are shown in Figure 3. A total of 13 mutants were screened, including G106V (Mut1), W101R-G106V (Mut2), W101R-G106V-L125V (Mut3), W101G-G106V (Mut4), W101G-N103T-G106V (Mut5), N103T-G106V (Mut6), W101G-N103K-G106V (Mut7), N103H-G106V (Mut8), N103K-G106V (Mut9), N103P-G106V (Mut10), N103Y-G106V (Mut11), G102R-G106V (Mut12), W101L-G106V (Mut13). These mutants can be used to construct Zika / dengue vaccines.

[0092] Table 1. Zika virus E protein sequencing primers

[0093] Table 2. FastKing one-step RT-PCT reaction system

[0094] Table 3. FastKing one-step RT-PCR reaction conditions

[0095] Table 4. Zero-background pTOPO-Blunt Simple Lightning Cloning Kit reaction system

[0096] Example 4: Preparation of dengue virus mRNA vaccine

[0097] The mutation information of the mutants W101R-G106V (Mut2) and W101R-G106V-L125V (Mut3) selected in Example 3 was introduced into the prME proteins of four dengue virus serotypes to construct mRNA vaccines encoding the four serotypes of dengue virus prME mutants. Note: The amino acid 125 of the different dengue virus serotypes varies, but all are mutated to valine. Simultaneously, mRNA vaccines encoding the prME proteins of wild-type DV1-DV4 were constructed as controls.

[0098] Specifically, the constructed mRNA vaccine includes:

[0099] DV1-WT (i.e., DV1 wild type), encoding the prME antigen protein shown in SEQ ID NO: 1;

[0100] DV1-EM3 (i.e., a DV1 prME mutant having a W101R-G106V mutation), encoding the prME antigen protein set forth in SEQ ID NO: 2;

[0101] DV1-EM3-1 (i.e., a DV1 prME mutant having W101R-G106V-L125V mutations), encoding the prME antigen protein set forth in SEQ ID NO: 3;

[0102] DV2-WT (i.e., DV2 wild type), encoding the prME antigen protein shown in SEQ ID NO: 4;

[0103] DV2-EM3 (i.e., a DV2 prME mutant having a W101R-G106V mutation), encoding the prME antigen protein set forth in SEQ ID NO: 5;

[0104] DV2-EM3-1 (i.e., a DV2 prME mutant having W101R-G106V-M125V mutations), encoding the prME antigen protein set forth in SEQ ID NO: 6;

[0105] DV3-WT (i.e., DV3 wild type), encoding the prME antigen protein shown in SEQ ID NO: 7;

[0106] DV3-EM3 (i.e., a DV3 prME mutant having a W101R-G106V mutation), encoding the prME antigen protein set forth in SEQ ID NO: 8;

[0107] DV3-EM3-1 (i.e., a DV3 prME mutant having W101R-G106V-I125V mutations), encoding the prME antigen protein set forth in SEQ ID NO:9;

[0108] DV4-WT (i.e., DV4 wild type), encoding the prME antigen protein shown in SEQ ID NO: 10;

[0109] DV4-EM3 (i.e., a DV4 prME mutant having a W101R-G106V mutation), encoding the prME antigen protein set forth in SEQ ID NO: 11;

[0110] DV4-EM3-1 (ie, a DV4 prME mutant having W101R-G106V-I125V mutations) encodes the prME antigen protein shown in SEQ ID NO:12.

[0111] Next, based on the codon preference of mammalian cells, the nucleic acid sequence encoding the above-mentioned recombinant dengue virus prME antigen protein was optimized to obtain optimized nucleic acid coding sequences, as shown in SEQ ID NO:13-24, respectively; then, the Kozak sequence and the signal peptide nucleic acid coding sequence (as shown in SEQ ID NO:25) were added to the 5' end of each nucleic acid coding sequence, and after adding a stop codon at its 3' end, GeneScript Biotech Co., Ltd. was commissioned to perform gene synthesis, and the synthesized gene fragments were recombined into the pHRNT vector (invention patent ZL202110224383.0) preserved in the laboratory to obtain the template plasmid for preparing the mRNA vaccine.

[0112] 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 in 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 a 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; preferably, the packaged mRNA vaccine is replaced with PBS using a dialysis bag, and the Quan-iT RiboGreen An RNA assay kit was used to measure the concentrations of encapsulated and free mRNA and calculate the mRNA packaging efficiency. The results showed that the packaging efficiency met the standards for mRNA vaccines. The encapsulated mRNA was then stored at 4°C until use.

[0113] Example 5: Expression and purification of dengue and Zika virus E proteins

[0114] 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)).

[0115] 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:27-31; then, a Kozak sequence and a signal peptide coding sequence (as shown in SEQ ID NO:26) were added to the 5' end of the above nucleic acid coding sequence, and a His tag (for protein isolation and purification) and a stop codon were added to the 3' end. Gene synthesis was commissioned to GenScript Biotech Co., Ltd., and the synthesized gene fragment was 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.

[0116] 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.

[0117] 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 The Excel affinity chromatography column was rinsed for approximately 3-5 column volumes. Afterwards, the column was rinsed with HisTrap affinity chromatography equilibration buffer 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 buffer 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 characterized 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 before loading. Equilibrate a Superdex 200 Increase 10 / 300GL gel filtration column with gel filtration 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.

[0118] Example 6: Detection of antigen expression of dengue virus mRNA vaccine

[0119] In this example, we transfected 293T cells with a series of dengue virus prME mRNA vaccines prepared in Example 4, and then used the FL epitope antibody and other neutralizing epitope antibodies prepared in Example 1 to detect the epitope status and protein expression level in each prME.

[0120] 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 4. 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 (Cytofix / Cytoperm from BD). 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 4% FACS Fixation / Permeabilization Kit, antibodies Z5 and Z6, which recognize different epitopes of the dengue prME protein and are prepared in Example 1, 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 reselected with PBS. Cell fluorescence was detected on a BD FACSAria III flow cytometer, and the data were analyzed using FlowJo 7.6.1.

[0121] The results are shown in Figure 4-7. The results in Figure 4-7 show:

[0122] 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;

[0123] 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.

[0124] 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.

[0125] 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.

[0126] These results demonstrate that the FL epitope is disrupted in all of the mutants, while other neutralizing antibody epitopes remain unchanged. Furthermore, the mutants exhibit protein expression levels comparable to or higher than that of the wild-type. Specifically, the FL epitope is disrupted in each of the prME mutants, preventing the production of antibodies against the FL epitope that can cause ADE, thereby significantly reducing or eliminating the ADE effect. Furthermore, each prME mutant antigen is able to bind normally to other neutralizing epitope antibodies, indicating that it possesses the correct conformation of the E protein. Furthermore, each prME mutant antigen exhibits protein expression levels similar to or higher than that of the wild-type.

[0127] Example 7: Dengue virus mRNA vaccine mouse immunization experiment

[0128] 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 4 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.

[0129] Example 8: Detection of specific antibody titers in sera of immunized mice by enzyme-linked immunosorbent assay (ELISA)

[0130] In this example, the specific antibody titer against Antigen E protein in the sera of the immunized mice obtained in Example 7 was detected by ELISA experiment.

[0131] Specifically, the following procedures were adopted:

[0132] (1) The dengue virus or Zika virus E protein prepared in Example 5 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.

[0133] (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;

[0134] (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;

[0135] (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.

[0136] 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.

[0137] The results of the ELISA experiment are shown in Figure 8, 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).

[0138] The above results show that each prME mutant antigen 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.

[0139] Example 9: Dengue virus microneutralization assay to detect neutralizing antibody titers in sera of immunized mice

[0140] 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 7. The specific steps are as follows:

[0141] 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.

[0142] The results are shown in Figure 9, which shows that compared with the corresponding WT vaccine, DV1-EM3, DV1-EM3-1, DV3-EM3, and DV4-EM3 vaccines all induced comparable virus neutralizing antibody titers, while DV3-EM3-1 and DV4-EM3-1 induced slightly lower virus neutralizing antibody titers. In particular, compared with DV3-WT, the DV3-EM3 vaccine induced fewer binding antibodies (Figure 8), but the neutralizing activity of vaccine serum did not decrease (Figure 9), indicating that the DV3-EM3 vaccine induced a higher proportion of high-level neutralizing activity antibodies.

[0143] The above results show that each prME mutant antigen can induce high levels of neutralizing antibody titers in mice, suggesting that it has high immunogenicity and can thus induce a high level of immune protection effect.

[0144] Example 10: Determination of cross-antibody titer

[0145] 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 8 for details.

[0146] The results of the cross-antibody titer determinations induced by immunization with each mRNA vaccine in Example 7 are shown in Figures 10-13. Figures 10-13 show:

[0147] 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;

[0148] 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.

[0149] The above results indicate that the cross-antibodies induced by each prME mutant antigen against the E protein of other serotypes of DV or ZIKV viruses are significantly reduced, thereby greatly reducing the risk of vaccine-induced ADE effects and having higher safety.

[0150] Example 11: Determination of neutralizing antibody titers and cross-antibody titers in serum of DV1-EM3-1 and DV2-EM3 vaccines

[0151] 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 7 were measured. The neutralizing antibody titer determination method was referred to Example 9, and the cross-antibody titer determination method was referred to Example 10.

[0152] The results of serum neutralizing antibody titer determination are shown in Figure 14. Figure 14 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.

[0153] The results of cross-antibody titer determination are shown in Figures 15-16. Figures 15-16 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 are significantly reduced. It is inferred that the ADE risk against different serotypes of dengue virus and Zika virus is also significantly reduced.

[0154] Example 12: Evaluation of vaccine-induced ADE effects in vitro

[0155] 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 4 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.

[0156] 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:

[0157] 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.

[0158] 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.

[0159] The results are shown in Figures 17-20, 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.

[0160] Example 13: Evaluation of vaccine-induced ADE effects in vivo

[0161] In this example, the serum of BALB / c mice immunized with the vaccines DV4-WT, DV4-EM3 and DV4-EM3-1 obtained in Example 12 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.

[0162] Using AG129 mice as an infection model, BALB / c mice were immunized with different vaccines or PBS. Four weeks after the booster immunization, blood was collected, serum was separated, and heat-inactivated. The sera from different mice in each vaccine group were 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. Then, it was mixed with an equal volume of DENV2 virus (6000 FFU). The virus-serum mixture was injected subcutaneously into the AG129 mice. The mice were monitored daily for survival and weight loss, and mice that lost 25% of their initial weight were euthanized.

[0163] The results are shown in Figure 21, 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.

[0164] Example 14: Immunization of mice with quadrivalent dengue virus mRNA vaccine

[0165] 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 4. The tMut vaccine was prepared by mixing four vaccines, DV1-EM3-1, DV2-EM3-1, DV3-EM3, and DV4-EM3, prepared in Example 4, 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.

[0166] Example 15: Expression and purification of FL antibody scFv

[0167] 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:122 and SEQ ID NO:123, respectively. A Kozak sequence and a stop codon were then added to the 5' and 3' ends of the nucleic acid coding sequences, respectively. Gene synthesis was then 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.

[0168] 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.

[0169] 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 buffer until both the UV and conductivity 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 buffer until the UV 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 characterized by SDS-PAGE. Further purification was performed using Superdex 200 Increase 10 / 300 GL gel filtration chromatography. The affinity chromatography sample 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 before loading. Equilibrate a Superdex 200 Increase 10 / 300GL gel filtration column with gel filtration 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.

[0170] Example 16: Competitive enzyme-linked immunosorbent assay (ELISA) detection of FL epitope antibodies induced by quadrivalent dengue mRNA vaccine

[0171] In this example, a competitive ELISA experiment was performed using the serum of mice immunized with the quadrivalent dengue mRNA vaccine obtained in Example 14 to detect the level of antibodies against the FL epitope of the antigen E protein induced by the quadrivalent dengue vaccine.

[0172] Specifically, the following procedures were adopted:

[0173] (1) The dengue virus E protein prepared in Example 5 was diluted to 2 μg / mL using ELISA coating solution (Solarbo, C1050), and 100 μL was added to each well of a 96-well ELISA plate (Coring, 3590) and incubated at 4°C for 12 hours.

[0174] (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;

[0175] (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.

[0176] (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;

[0177] (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.

[0178] 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.

[0179] The results of the competitive ELISA experiment are shown in Figure 22. Figure 22 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.

[0180] Example 17: Detection of neutralizing antibodies induced by dengue quadrivalent vaccine

[0181] The dengue virus microneutralization experiment was used to quantitatively determine 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 14. The specific steps are as follows:

[0182] 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.

[0183] The results are shown in Figure 23, 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.

[0184] Example 18: Evaluation of dengue quadrivalent vaccine challenge protection

[0185] In this example, the AG129 mouse lethal model was used to detect the protective effect of the quadrivalent dengue mRNA vaccine.

[0186] 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 4, with each vaccine at 3 μg / dose, 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 divided 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.

[0187] The results are shown in Figure 24, 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.

[0188] Example 19: Preparation of dengue virus mRNA vaccine

[0189] 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:

[0190] DV1-WT (i.e., DV1 wild type), encoding the prME antigen protein shown in SEQ ID NO: 1;

[0191] DV1-EM1, encoding the prME antigen protein shown in SEQ ID NO:32;

[0192] DV1-EM2, encoding the prME antigen protein shown in SEQ ID NO: 33;

[0193] DV1-EM4, encoding the prME antigen protein shown in SEQ ID NO:34;

[0194] DV1-EM5, encoding the prME antigen protein shown in SEQ ID NO: 35;

[0195] DV1-EM6, encoding the prME antigen protein shown in SEQ ID NO: 36;

[0196] DV1-EM7, encoding the prME antigen protein shown in SEQ ID NO: 37;

[0197] DV1-EM8, encoding the prME antigen protein shown in SEQ ID NO: 38;

[0198] DV1-EM9, encoding the prME antigen protein shown in SEQ ID NO: 39;

[0199] DV1-EM10, encoding the prME antigen protein shown in SEQ ID NO:40;

[0200] DV1-EM11, encoding the prME antigen protein shown in SEQ ID NO:41;

[0201] DV1-EM12, encoding the prME antigen protein shown in SEQ ID NO:42;

[0202] DV2-WT (i.e., DV2 wild type), encoding the prME antigen protein shown in SEQ ID NO: 4;

[0203] DV2-EM1, encoding the prME antigen protein shown in SEQ ID NO:43;

[0204] DV2-EM2, encoding the prME antigen protein shown in SEQ ID NO:44;

[0205] DV2-EM4, encoding the prME antigen protein shown in SEQ ID NO:45;

[0206] DV2-EM5, encoding the prME antigen protein shown in SEQ ID NO:46;

[0207] DV2-EM6, encoding the prME antigen protein shown in SEQ ID NO:47;

[0208] DV2-EM7, encoding the prME antigen protein shown in SEQ ID NO:48;

[0209] DV2-EM8, encoding the prME antigen protein shown in SEQ ID NO:49;

[0210] DV2-EM9, encoding the prME antigen protein shown in SEQ ID NO: 50;

[0211] DV2-EM10, encoding the prME antigen protein shown in SEQ ID NO:51;

[0212] DV2-EM11, encoding the prME antigen protein shown in SEQ ID NO:52;

[0213] DV2-EM12, encoding the prME antigen protein shown in SEQ ID NO:53;

[0214] DV3-WT (i.e., DV3 wild type), encoding the prME antigen protein shown in SEQ ID NO: 7;

[0215] DV3-EM1, encoding the prME antigen protein shown in SEQ ID NO:54;

[0216] DV3-EM2, encoding the prME antigen protein shown in SEQ ID NO:55;

[0217] DV3-EM4, encoding the prME antigen protein shown in SEQ ID NO: 56;

[0218] DV3-EM5, encoding the prME antigen protein shown in SEQ ID NO: 57;

[0219] DV3-EM6, encoding the prME antigen protein shown in SEQ ID NO: 58;

[0220] DV3-EM7, encoding the prME antigen protein shown in SEQ ID NO:59;

[0221] DV3-EM8, encoding the prME antigen protein shown in SEQ ID NO:60;

[0222] DV3-EM9, encoding the prME antigen protein shown in SEQ ID NO:61;

[0223] DV3-EM10, encoding the prME antigen protein shown in SEQ ID NO:62;

[0224] DV3-EM11, encoding the prME antigen protein shown in SEQ ID NO:63;

[0225] DV3-EM12, encoding the prME antigen protein shown in SEQ ID NO:64;

[0226] DV4-WT (i.e., DV4 wild type), encoding the prME antigen protein shown in SEQ ID NO: 10;

[0227] DV4-EM1, encoding the prME antigen protein shown in SEQ ID NO:65;

[0228] DV4-EM2, encoding the prME antigen protein shown in SEQ ID NO:66;

[0229] DV4-EM4, encoding the prME antigen protein shown in SEQ ID NO: 67;

[0230] DV4-EM5, encoding the prME antigen protein shown in SEQ ID NO:68;

[0231] DV4-EM6, encoding the prME antigen protein shown in SEQ ID NO:69;

[0232] DV4-EM7, encoding the prME antigen protein shown in SEQ ID NO:70;

[0233] DV4-EM8, encoding the prME antigen protein shown in SEQ ID NO:71;

[0234] DV4-EM9, encoding the prME antigen protein shown in SEQ ID NO:72;

[0235] DV4-EM10, encoding the prME antigen protein shown in SEQ ID NO:73;

[0236] DV4-EM11, encoding the prME antigen protein shown in SEQ ID NO:74;

[0237] DV4-EM12 encodes the prME antigen protein shown in SEQ ID NO:75.

[0238] 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: 13, 16, 19, and 22, 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: 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: 87-97, 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: 87-97, respectively; NO:98-108, the optimized nucleic acid coding sequences of DV4-EM1, DV4-EM2, DV4-EM4, DV4-EM5, DV4-EM6, DV4-EM7, DV4-EM8, DV4-EM9, DV4-EM10, DV4-EM11, and DV4-EM12 are shown in SEQ ID NO:109-119 respectively; Then, a Kozak sequence and a signal peptide nucleic acid coding sequence (such as SEQ ID NO:120, which encodes an amino acid sequence such as a signal peptide shown in SEQ ID NO:121) are added to the 5' end of each nucleic acid coding sequence, and after adding a stop codon at 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 4.

[0239] Example 20: Detection of antigen expression of dengue virus mRNA vaccine

[0240] In this example, a series of dengue virus prME mRNA vaccines prepared in Example 19 were transfected into 293T cells. The FL epitope antibody and other neutralizing epitope antibodies prepared in Example 1 were then used to detect the presence of various epitopes in each prME and protein expression. The detailed experimental procedures were similar to those in Example 6. 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%.

[0241] The results are shown in Figures 25-28, which show:

[0242] 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.

[0243] Similar results were obtained for DV2 mRNA vaccine, DV3 mRNA vaccine and DV4 mRNA vaccine.

[0244] Example 21: Evaluation of protection against DV2 mutant mRNA vaccine challenge

[0245] 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 19.

[0246] 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.

[0247] The results are shown in Figure 29, which show 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.

[0248] Example 22: Preparation of dengue virus-like particle vaccine

[0249] 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:

[0250] DV1-EM3-1, encoding the prME antigen protein shown in SEQ ID NO: 3;

[0251] DV2-EM3-1, encoding the prME antigen protein shown in SEQ ID NO:6;

[0252] DV3-EM3, encoding the prME antigen protein shown in SEQ ID NO: 8;

[0253] DV4-EM3, encoding the prME antigen protein shown in SEQ ID NO: 11;

[0254] 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: 15, 18, 20, and 23, respectively. Then, a Kozak sequence and a signal peptide nucleic acid coding sequence (such as SEQ ID NO: 120, which encodes an amino acid sequence such as a signal peptide shown in SEQ ID NO: 121) 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.

[0255] 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.

[0256] 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:

[0257] 1) Take 20 μl of the purified sample, add 5 μl of 5× protein loading buffer, and heat at 100°C for 3 min;

[0258] 2) Take 10 μl of supernatant and perform SDS-PAGE electrophoresis;

[0259] 3) Coomassie brilliant blue staining;

[0260] 4) Observe the decolorization with decolorizing solution.

[0261] The identification results are shown in Figure 30. Figure 30 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 is consistent with expectations, indicating that the sample purity is high.

[0262] Example 23: Characterization of dengue virus-like particle vaccine using transmission electron microscopy

[0263] In this example, the morphology of the dengue virus-like particles prepared in Example 22 was observed and detected by transmission electron microscopy. The specific steps are as follows:

[0264] Prepare copper grid, phosphotungstic acid stain, filter paper, and sample box

[0265] 1) Negative staining sample preparation: First, perform glow treatment on the copper mesh. A glow discharge apparatus is usually used to hydrophilize the carbon-sprayed copper mesh.

[0266] 2) After the copper mesh is glowed, pick it up with pointed tweezers and fix the tweezers with a binder clip.

[0267] 3) Add 6.5 μl of sample onto the copper grid, let it stand for one minute, and then absorb it with filter paper.

[0268] 4) Add 6.5 μl of phosphotungstic acid dropwise onto the copper grid and immediately remove with filter paper.

[0269] 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;

[0270] 6) After drying, observe under an electron microscope.

[0271] 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 31-34, 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.

[0272] Example 24: Immunogenicity testing of monovalent dengue virus recombinant protein vaccine

[0273] 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 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 22, 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 9.

[0274] The results of the neutralizing antibody titer test are shown in Figure 35, 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.

[0275] Example 25: Immunogenicity testing of quadrivalent dengue virus recombinant protein vaccine

[0276] 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 22 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 titer detection method is as described in Example 9.

[0277] The results of the neutralizing antibody titer test are shown in Figure 36, which shows that: no neutralizing antibodies against the four serotypes of dengue virus were detected in the serum of the Sham group, 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.

[0278] 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

[0279] The method for discovering flavivirus vaccine antigens provided in the present application can obtain flavivirus vaccine antigens with a destroyed FL epitope and normal prME protein expression and folding with high efficiency. The obtained flavivirus vaccine antigens can avoid the ADE effect and have good immunogenicity, and can be used to prepare flavivirus vaccines; therefore, the method for discovering flavivirus vaccine antigens of the present application can be used for the development of flavivirus vaccines and has excellent industrialization prospects.

Claims

1. A method for discovering a flavivirus vaccine antigen, characterized in that, The discovery method includes: (1) Culturing flavivirus in the presence of an FL epitope-specific antibody to directionally induce the escape evolution of flavivirus; (2) Sequencing the E protein of the flavivirus with escape evolution obtained in step (1) to determine the mutations in its E protein; (3) Introducing the mutations measured in the E protein into the E protein of flavivirus by means of homologous replacement to obtain a flavivirus E protein mutant as a flavivirus vaccine antigen.

2. The discovery method according to claim 1, characterized in that Step (1) includes: Performing multiple rounds of screening on flavivirus in the presence of an FL epitope-specific antibody with gradually increasing concentration until a flavivirus with escape evolution is obtained.

3. The discovery method according to claim 2, characterized in that, The flavivirus with escape evolution is a flavivirus that escapes from the FL epitope-specific antibody.

4. The discovery method according to claim 2 or 3, characterized in that, The flavivirus with escape evolution contains mutations in the FL fusion loop region of the E protein and optionally mutations in the non-FL fusion loop region.

5. The discovery method according to any one of claims 2-4, characterized in that, During the 1st - 3rd rounds of screening, the added concentration of the FL epitope-specific antibody is determined to be: the concentration at which the infection rate of the flavivirus to cells decreases by 20% - 80%, preferably by 40% - 60%, more preferably by 50%.

6. The discovery method according to any one of claims 2-5, characterized in that, The end point of the multiple rounds of screening is: the obtained flavivirus escapes from the FL epitope-specific antibody, and optionally, the cell infection ability of the obtained flavivirus remains basically unchanged compared with the initial virus.

7. The discovery method according to any one of claims 1-6, characterized in that, Step (3) further includes: combining the obtained flavivirus E protein mutant with the full-length or partial prM protein sequence of flavivirus as a flavivirus vaccine antigen.

8. The discovery method according to any one of claims 1-7, characterized in that, The discovery method further includes a step of further screening the flavivirus vaccine antigen obtained in step (3); Preferably, the criteria for further screening the flavivirus vaccine antigen are any one or more selected from the following: 1) The obtained flavivirus vaccine antigen does not bind to the FL epitope-specific antibody, 2) The binding of the obtained flavivirus vaccine antigen to the non-FL neutralizing epitope antibody is not affected; 3) The obtained flavivirus vaccine antigen can be normally expressed and folded; preferably, the expression efficiency of the obtained flavivirus vaccine antigen is comparable to that of its corresponding wild type.

9. The discovery method according to any one of claims 1-8, characterized in that, The flavivirus is selected from: Zika virus, dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, Japanese B encephalitis virus, tick-borne encephalitis virus, and Murray Valley encephalitis virus, preferably Zika virus, yellow fever virus, or dengue virus; Preferably, the FL epitope-specific antibody is selected from: Z5, Z6, 4G2, 2A10G6, Z7, Z10, Z24, Z25.

10. Use of the discovery method according to any one of claims 1 - 9 in developing a flavivirus vaccine antigen, and even in developing a flavivirus vaccine.

Images