Dengue virus vaccine with attenuated antibody-dependent enhancement

Abstract

The application discloses a dengue virus vaccine with weakened antibody-dependent enhancement effect. The dengue virus nanogen is obtained by combining the D I region and the D III region of the dengue virus E protein fusion protein with the Helicobacter pylori ferritin, and the dengue virus vaccine with weakened antibody-dependent enhancement effect is prepared by using the nanogen. The vaccine overcomes the shortage of the monomer immunogenicity of the E protein D III region, can effectively cause a stronger immune response, produces the antibody for neutralizing the dengue virus invasion of target cells, and can produce a balanced and strong immune protection response to four different serotypes of dengue viruses, so that the ADE effect can be avoided, and the neutralizing antibody level of the host to the dengue virus is significantly improved. In addition, the preparation method of the dengue virus vaccine is simple, easy to purify and high in safety.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine. More particularly, it relates to a dengue virus vaccine with weakened antibody-dependent enhancement effect. BACKGROUND

[0002] Dengue fever is an acute mosquito-borne infectious disease caused by dengue virus (DENV) transmitted by mosquitoes. After dengue virus infection, dengue fever (DF) is generally self-limiting, but if there is no appropriate treatment, combined with secondary dengue virus infection of different serotypes, it may develop into severe complications such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), leading to increased mortality. The widespread transmission of dengue virus poses a threat to human health, but so far, no antiviral drug has been approved, and no vaccine has been widely used for the treatment and prevention of dengue fever.

[0003] Dengue virus belongs to the genus of flavivirus of the family of flaviviridae, and there are mainly four serotypes, i.e. DENV-1, DENV-2, DENV-3 and DENV-4. The genome of dengue virus is a positive single-stranded RNA of 11 kb, including a large open reading frame (ORF), three structural proteins (membrane protein (M), envelope protein (E) and capsid protein (C)) and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5). In the mature dengue virus particle, the C protein wraps the RNA genome and is surrounded by a lipid bilayer membrane, in which the E protein and the M protein are embedded. During the maturation of dengue virus, the prM protein is cleaved to release the pr domain of the M protein, the M protein interacts with the E protein on the mature virus, and the E protein enters the host cell by binding to various receptors (DC-SIGN, mannose receptor, CD207, etc.) and endosomal membrane fusion.

[0004] The E protein of dengue virus contains three domains (D I, D II, D III), among which the D III region is responsible for binding to host receptors and is the main target for inducing highly efficient neutralizing antibodies (NAbs). Therefore, most vaccine strategies are based on the D III domain of dengue virus E protein, but the immunogenicity of the D III domain of E protein is weak and does not contain all the receptor binding sites, and the immunoprotective effect of the vaccine obtained by using the D III domain of E protein as an antigen is not ideal. There are also many vaccines directly based on dengue virus E protein, but studies have shown that the antibodies induced by the FL region of the D II domain of E protein may trigger antibody-dependent enhancement effect (ADE) when infected with different serotypes of dengue virus.

[0005] Antibody-dependent enhancement effect refers to that when there is weak neutralizing antibody or low concentration of antibody, the complex formed by virus binding antibody is not neutralized by the antibody under the mediation of Fc receptor, but promotes the virus to enter and infect the host cell, which may be related to the further aggravation of the disease. The earliest report of ADE comes from dengue fever, and it is of great significance to provide a method for weakening the antibody-dependent enhancement effect of dengue virus and develop a dengue vaccine with high immunogenicity and avoiding the ADE effect during infection. SUMMARY

[0006] The technical problem to be solved by the present application is to overcome the defects and shortcomings of the prior art dengue virus vaccine, such as unsatisfactory immune protection effect and antibody-dependent enhancement effect, and to provide a method for weakening the antibody-dependent enhancement effect of dengue virus, and on this basis, to provide a dengue virus vaccine with weakened antibody-dependent enhancement effect.

[0007] The first object of the present application is to provide a method for weakening the antibody-dependent enhancement effect of dengue virus.

[0008] The second object of the present application is to provide a dengue virus antigen.

[0009] The third object of the present application is to provide a dengue virus nano-antigen.

[0010] The fourth object of the present application is to provide the use of the antigen in the preparation of a drug for resisting dengue virus.

[0011] The fifth object of the present application is to provide a dengue virus vaccine with weakened antibody-dependent enhancement effect.

[0012] The above objects of the present application are achieved by the following technical solutions:

[0013] The present application provides a method for weakening the antibody-dependent enhancement effect of dengue virus, which comprises using a fusion protein obtained by replacing two D II domains in the envelope protein of dengue virus with GGGGS and S as a dengue virus antigen for immunization.

[0014] Specifically, GGGGS replaces the first D II domain from the N terminus of the envelope protein of dengue virus, and S replaces the second D II domain from the N terminus of the surface envelope protein of dengue virus.

[0015] The research shows that the antibodies induced by the FL region of the D II domain of the E protein can cause antibody-dependent enhancement effect when different serotypes of dengue virus infects, and the present application attempts to weaken the antibody-dependent enhancement effect of the dengue virus by cutting off the FL region of the D II domain of the E protein, but since part of the E protein is cut off, the conformation of the remaining part is different from the natural conformation of the E protein, and the immunogenicity is also affected. Through a large amount of research, the present application finds that the D II domain of the E protein of the dengue virus (the induced antibodies have low neutralizing ability) is cut off, and the remaining E protein fragment is connected through GGGGS and S, which not only effectively avoids the antibody-dependent enhancement effect, but also makes the conformation of the obtained fusion protein E13 closer to the natural conformation of the E protein by connecting the D I region and the D III region through GGGGS and S, thereby greatly increasing the induced neutralizing antibody epitopes.

[0016] Based on the above method, the present application also provides several dengue virus antigens:

[0017] A dengue virus antigen, the antigen is a serotype 1 dengue virus antigen, the amino acid sequence of the serotype 1 dengue virus antigen (denoted as E113) is shown in SEQ ID NO. 1.

[0018] A dengue virus antigen, the antigen is a serotype 2 dengue virus antigen, the amino acid sequence of the serotype 2 dengue virus antigen (denoted as E213) is shown in SEQ ID NO. 2,

[0019] A dengue virus antigen, the antigen is a serotype 3 dengue virus antigen, the amino acid sequence of the serotype 3 dengue virus antigen (denoted as E313) is shown in SEQ ID NO. 3,

[0020] A dengue virus antigen, the antigen is a serotype 3 dengue virus antigen, the amino acid sequence of the serotype 4 dengue virus antigen (denoted as E413) is shown in SEQ ID NO. 4.

[0021] The present application also provides a recombinant vector or a transgenic cell line for expressing the above-mentioned antigens (SEQ ID NO. 1-4).

[0022] The present application also provides a dengue virus nano-antigen, which is a nano-antigen obtained by fusing and expressing a fusion protein after the dengue virus antigen is connected with Gvtag shown in SEQ ID NO. 5 through Linker, and then the obtained fusion protein is combined with Sdcatcher-HPF protein shown in SEQ ID NO. 10 through the spontaneous chemical bond of Gv-Sd.

[0023] Specifically, the present application realizes antigen multimerization based on HPF protein by covalently combining the fusion protein obtained after cutting off the D II domain of dengue virus E protein (i.e. the dengue virus antigen) with the GvTagOpti / Sdcatcher (Gv / Sd) system, thereby obtaining a dengue virus nanoantigen. The GvTagOpti / Sdcatcher (Gv / Sd) system can be referred to the Chinese patent with the publication number CN113621031A.

[0024] Specifically, the Gvtag is connected to the C-terminal of the antigen through the Linker.

[0025] Generally, when constructing a nanoantigen using the GvTagOpti / Sdcatcher (Gv / Sd) system, the Gvtag is connected to the N-terminal of the antigen, but for the dengue virus antigen of the present application, the expression amount of the fusion protein is not ideal when the Gvtag is connected to the N-terminal of the antigen. By connecting the Gvtag to the C-terminal of the antigen of the present application, the expression amount of the fusion protein is greatly improved compared to connecting to the N-terminal.

[0026] Specifically, when the Linker is GGS, the amino acid sequences of the fusion proteins obtained by connecting the dengue virus antigens of the present application, i.e. the antigens represented by SEQ ID NO. 1-4, to the Gvtag are represented by SEQ ID NO. 6-9, respectively.

[0027] Specifically, the fusion protein obtained by connecting the dengue virus antigen to the Gvtag has a secretory signal peptide connected to the N-terminal and a His purification tag connected to the C-terminal.

[0028] Specifically, the amino acid sequences of the fusion proteins obtained by connecting the dengue virus antigens (represented by SEQ ID NO. 1-4) of the present application to the secretory signal peptide, the Gvtag and the His purification tag are represented by SEQ ID NO. 11-14, respectively.

[0029] The present application also provides a preparation method of the nanoantibody, which comprises: expressing the fusion protein obtained by connecting the dengue virus antigen of the present application to the Gvtag using a eukaryotic expression system, expressing the Sdcatcher-HPF protein represented by SEQ ID NO. 10 using a prokaryotic expression system, and forming a twenty-fourmer nanoantigen by Gv / Sd covalent action after expression and purification of the fusion protein and the Sdcatcher-HPF protein, which is denoted as E13-HPF twenty-fourmer nanoantigen. If the dengue virus antigen used is a serotype 1 dengue virus antigen (E113), the twenty-fourmer nanoantigen prepared is denoted as E113-HPF.

[0030] The application also protects the use of the antigen in the preparation of a drug against dengue virus, and the drug includes a vaccine.

[0031] The application also provides a dengue virus vaccine with weakened antibody-dependent enhancement effect, and the vaccine is prepared from the dengue virus antigen or dengue virus nano-antigen.

[0032] Specifically, when the antigen used is a nano-antigen, the adjuvant used in the vaccine is an aluminum adjuvant.

[0033] The application also provides a tetravalent dengue virus nano-particle vaccine with weakened antibody-dependent enhancement effect, and the nano-particle vaccine is prepared by uniformly mixing the dengue virus nano-antigens of the four different serotypes in equal proportions and mixing with an aluminum adjuvant.

[0034] The application has the following beneficial effects:

[0035] The application provides a method for weakening the antibody-dependent enhancement effect of dengue virus, and on the basis of the method, a dengue virus antigen, nano-antigen and vaccine are provided. The application takes the fusion of the D I region and the D III region (E13) of the dengue virus E protein as an antigen fragment, and realizes the antigen multimerization by covalently combining Helicobacter pylori ferritin (HPF) through a covalent binding system GvTagOpti / Sdcatcher (Gv / Sd), and adds a secretory signal peptide and a His purification tag, so that the E13-HPF protein can be expressed and self-assembled into a spherical twenty-fourmer nano-particle through a plasmid transfection eukaryotic cell expression system, and then the nano-particle is used as an antigen to prepare a dengue vaccine with weakened antibody-dependent enhancement effect.

[0036] The vaccine of the application overcomes the shortcomings of the monomer immunogenicity of the D III region of the E protein, can effectively cause a stronger immune response, produces antibodies that neutralize the invasion of dengue virus into target cells, and the vaccine can produce a balanced and strong immune protection response against dengue virus of four different serotypes, can avoid the ADE effect, and significantly improves the level of neutralizing antibodies of the host against dengue virus. In addition, the preparation method of the dengue virus vaccine of the application is simple, easy to purify and high in safety. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 It is a structural schematic diagram of the E13-Gvtag-His fusion protein and the Sdcatcher-HPF fusion protein.

[0038] Figure 2 It is a schematic diagram of the construction process of the four serotype dengue virus nano-antigens (E13-HPF protein).

[0039] Figure 3Structure of recombinant expression vector pcDNA3.1-intron-E13-Gvtag-WPRE for expressing E13-Gvtag-His fusion protein.

[0040] Figure 4 SDS-PAGE of purified E13-Gvtag-His fusion protein (about 35KD), E13-Gvtag fusion protein and E13-HPF protein (about 70KD).

[0041] Figure 5 Molecular sieve chart of purified E13-HPF protein.

[0042] Figure 6 Immunization strategy of mice immunized with tetravalent E13-HPF nanoparticle vaccine.

[0043] Figure 7 Detection results of IgG titers specific to four E13 proteins in serum collected from mice immunized with tetravalent E13-HPF nanoparticle vaccine for 10 weeks; **** in the figure indicates extremely significant difference, p<0.0001.

[0044] Figure 8 Detection results of neutralizing activity of antibodies against dengue virus in serum collected from mice immunized with tetravalent E13-HPF nanoparticle vaccine for 10 weeks; **** in the figure indicates extremely significant difference, p<0.0001.

[0045] Figure 9 Detection results of antibody-dependent enhancement effect of antibodies in serum collected from mice immunized with tetravalent E13-HPF nanoparticle vaccine for 10 weeks; *** in the figure indicates extremely significant difference, p<0.001. DETAILED DESCRIPTION

[0046] The present application will be further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and devices used in the present application are conventional reagents, methods and devices in the technical field.

[0047] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0048] Example 1 Construction of dengue virus nanogenes of four serotypes (fusion protein E13-HPF)

[0049] The structure of E13-Gvtag-His fusion protein and Sdcatcher-HPF fusion protein required for constructing dengue virus nanogenes of four different serotypes according to the present application is shown in Figure 1 Figure 1 ​It can be known that, after removing the antibody binding site inducing ADE, i.e., removing the D II domain, the D I domain of the dengue virus E protein becomes three segments, in order to keep the protein conformation of the subsequent obtained fusion protein consistent with the natural conformation of the E protein, the application connects the E protein with the D II domain removed by using GGGGS and S; wherein GGGGS is located at the first gap, which is equivalent to GGGGS replacing the first DII domain of the E protein from the N terminal, and S is located at the second gap, which is equivalent to S replacing the second D II domain of the E protein from the N terminal. The application names the fusion protein obtained by connecting the D II domain removed by GGGGS and S as E13 (if it is the DI and D III fusion protein of the dengue virus of serotype 1, it is recorded as E113), which can be used as an antigen, and in some cases, it is also called antigen E13.

[0050] The amino acid sequence of the dengue virus antigen E113 of serotype 1 is shown in SEQ ID NO. 1, the amino acid sequence of the dengue virus antigen E213 of serotype 2 is shown in SEQ ID NO. 2, the amino acid sequence of the dengue virus antigen E313 of serotype 3 is shown in SEQ ID NO. 3, and the amino acid sequence of the dengue virus antigen E413 of serotype 4 is shown in SEQ ID NO. 4; the amino acid sequences of the above-mentioned dengue virus antigens connected by GGS with the Gvtag shown in SEQ ID NO. 5 are shown in SEQ ID NO. 6-9 respectively; and the amino acid sequences of the above-mentioned antigens connected with the secretory signal peptide (SP), Gvtag and His purification tag are shown in SEQ ID NO. 11-14 respectively.

[0051] The construction process of the dengue virus nanometer antigen of the four serotypes is shown in Figure 2 The prepared E13-Gvtag fusion protein of different serotypes is incubated with the Sdcatcher-HPF fusion protein at 25℃ overnight, the E13-Gvtag fusion protein and the Sdcatcher-HPF fusion protein can be covalently combined by the GvTagOpti / Sdcatcher (Gv / Sd) system, combined by the spontaneous chemical bond of Gv-Sd, and self-assembled to obtain E13-HPF twenty-fourmer, i.e., nanometer antigen, and the four different serotypes of dengue virus nanometer antibodies can be mixed in equal proportions to prepare a tetravalent dengue virus nanometer particle vaccine. The GvTagOpti / Sdcatcher (Gv / Sd) system can be referred to the Chinese patent with the publication number CN113621031A.

[0052] Specifically, the preparation method of the protein E13-Gvtag-His is as follows:

[0053] 1. Construction of recombinant vector expressing protein E13-Gvtag-His

[0054] After adding a translation termination codon at the 3' end of the nucleotide sequence encoding protein E13-Gvtag-His, respectively, it was cloned into the EcoR I and Xba I enzyme cutting sites between the expression vector (pcDNA3.1-Intron-WPRE) added with Intron and WPRE to enhance expression, and the recombinant expression vector pcDNA3.1-intron-E13-Gvtag-WPRE was constructed, and the structural diagram of the recombinant expression vector is shown in Figure 3 .

[0055] The recombinant expression vector constructed was transformed into DH5α competent cells, cultured at 37°C overnight, single colonies were picked and positive clones were identified by PCR; endotoxin-free plasmid (pcDNA3.1-intron-E13-Gvtag-WPRE) was extracted, and the correct pcDNA3.1-intron-E13-Gvtag-WPRE recombinant expression vector was constructed after enzyme digestion and sequencing verification, which was used for expression of nanoantigen protein.

[0056] The verified pcDNA3.1-intron-E13-Gvtag-WPRE recombinant expression vector was transfected into HEK293F cells by liposome transfection protocol, and the cell supernatant was harvested by centrifugation after 5 days of transfection. The target protein E13-Gvtag-His was purified by His purification tag.

[0057] 2. E13-Gvtag-His antigen purification

[0058] The cell supernatant expressing E13-Gvtag-His was filtered through a 0.22 μm filter membrane to remove cell debris; the filtered cell supernatant was passed through a HisTrap excel nickel column for crude purification.

[0059] After crude purification, first wash with PBS (pH = 7.4) buffer and low concentration imidazole buffer (PBS, 20 mM Imidazole, pH = 7.4), 50 mL, remove the flow-through impurities; then, use high imidazole buffer (PBS, 50 or 500 mM Imidazole, pH = 7.4) to elute the target protein; then concentrate the eluate to a small volume of 1 mL; incubate the purified four E13-Gvtag-His proteins with Sdcatcher-HPF at a molar ratio of 1:1 at 25°C overnight.

[0060] The target proteins in the supernatant of transfected cells and the His purification eluent in the preparation of the protein E13-Gvtag-His and the proteins in the polymeric effluent were detected by SDS-PAGE, and the results are shown in Figure 4 As shown in the results in Figure 4 As shown in the results in

[0061] The four kinds of polymeric proteins E13-HPF (nanoparticle antigens) formed after incubation were collected and subjected to molecular sieve chromatography through a Siperose 6 Increase 10 / 300 GL column (GE), and the obtained proteins were purified. The molecular sieve chromatography buffer was PBS, pH 7.4, and the E13-HPF purification molecular sieve chart is shown in Figure 5 As shown in the results in Figure 5 As shown in the results in

[0062] The obtained four kinds of E13-HPF proteins were concentrated and divided into small portions (500 μL), rapidly frozen with liquid nitrogen and stored at -80°C.

[0063] Example 2: ELISA experiment of IgG titer specific to four kinds of E13 proteins

[0064] The four kinds of twenty-fourmer E13-HPF proteins, i.e. the four kinds of E13-HPF (E113-HPF, E213-HPF, E313-HPF, E413-HPF) nanoparticle antigens obtained in Example 1 were mixed in equal proportions by amount of substance, diluted with physiological saline to 100 μg / mL according to the amount shown in Table 1, and emulsified with an equal volume of aluminum adjuvant (adjuvant 2%, item number: vac-alu-250, CAS number: 21645-51-2) to prepare a tetravalent dengue virus nanoparticle vaccine. 6-8 week old Balb / C mice were grouped and immunized. The immunization strategy is shown in As shown in the results in Figure 6 As shown in the results in

[0065] Table 1

[0066]

[0067]

[0068] The ELISA experiment process of IgG titer specific to E13 protein of four different serotypes of dengue virus is as follows:

[0069] 1. Plate coating

[0070] Dilute four antigens E113-Gvtag-His, E213-Gvtag-His, E313-Gvtag-His and E413-Gvtag-His to 5 ng / μL respectively with PBS, 50 μL / well, and add to 96-well ELISA plates (Grenier, 655061) respectively, and tightly paste the plates with adhesive tape to prevent evaporation, and store at 4℃ overnight.

[0071] 2. Blocking

[0072] First, shake off and dry the liquid in the well plate, prepare 5% PBS skimmed milk powder, pour into the sample slot, add 100 μL / well with the gun, and tightly paste the plate with adhesive tape to block at room temperature for 1 h.

[0073] 3. Washing plate

[0074] Shake off and dry the liquid in the well plate, add 200 μL 0.1% PBST solution, and repeat the process for 3 times to completely wash the milk powder solution.

[0075] 4. Adding diluted serum

[0076] Arrange 1.5 ml EP tubes, add 450 ml PBS, and then add 50 μL serum for dilution (10-fold dilution); add 100 μL PBS to the rest of the plate wells with the gun except the first row of plate wells. Dilute by 3 times according to the concentration gradient, add 150 μL diluted serum to the first well, and then add 50 μL to the next row with the gun, repeatedly blow and suck to mix, and repeat the operation until the last row. After mixing, discard the remaining 50 μL of diluted serum. Paste the uppermost plate with adhesive tape to prevent evaporation.

[0077] 5. Incubating primary antibody

[0078] Put the plate with added serum into a 37℃ oven and incubate for 1 h.

[0079] 6. Washing plate

[0080] Shake off and dry the liquid in the well plate, add 200 μL 0.1% PBST solution, and repeat the process for 3 times to completely wash.

[0081] 7. Incubating secondary antibody

[0082] According to the sample species, the enzyme-labeled mouse secondary antibody (Invitrogen, 31430) is prepared at a ratio of 1:4000, 100 μL / well is added by the gun, the uppermost sticker is attached to the plate hole to prevent evaporation, and incubation is performed for 1 h.

[0083] 8. Washing the plate

[0084] The liquid in the hole plate is shaken out and dried, 200 μL of 0.1% PBST solution is added, and the process is repeated 4 times.

[0085] 9. Addition of color developing substrate and termination of color development

[0086] The whole process is operated in the dark, 50 μL of color developing substrate TMB is added by the gun per well, and 50 μL of termination solution is added after color development for 10 min.

[0087] 10. Detection by an enzyme-labeled instrument

[0088] The detection wavelength is set to 450 nm for detection, and each plate is detected 3 times. The raw data and the exported Excel table are saved.

[0089] 11. Data processing

[0090] The detection results of the four E13 protein specific IgG titers in the serum of the mice immunized with the tetravalent E13-HPF nanoparticle vaccine for 10 weeks are shown in Table 1. Figure 7 The tetravalent E13-HPF nanoparticle vaccine is used to immunize Balb / c mice, and the four E13 protein specific IgG titers in the serum of the mice are detected after 10 weeks. The sidak multiple comparison test shows that there is a significant difference between the experimental group and the control group, and the two-tailed probability level is less than 0.05 at a significant level of 0.05. The results show that the tetravalent E13-HPF nanoparticle vaccine can stimulate the mice to produce high levels of IgG titers against the E13 protein of the four serotypes of DENV after 10 weeks from the first immunization.

[0091] Example 3 DENV neutralization test

[0092] The process of the DENV neutralization test is as follows:

[0093] 1. DAY 1: 2*10 4 / well Vero cells are added to a 96-well plate, and the experiment is performed after 12 hours of overnight culture (or after the hole is full)

[0094] 2. DAY 2: Pre-dilution of serum and virus solution

[0095] Each sample serum is pre-diluted as follows:

[0096] 1) First well diluted 20 fold, next 3 fold gradient dilution

[0097] Virus dilution: each virus was prepared at a total of 100 FFU / well

[0098] Mix = 55 μL diluted serum + 55 μL diluted virus, incubate at 37°C for 1 hour

[0099] 3) After washing the cell plate of DAY 1 with 50 μL of PBS, discard the PBS, add the incubated mixture to the cells (100 μL / well), incubate at 37°C for 2 hours, after 2 hours, replace the solution, add 100 μL of 2% DMEM to each well, and do not move the plate after replacing the solution

[0100] 3, DAY 4: collect the plate 48 hours after replacing the solution, discard the supernatant, wash once with 200 μL of PBS, fix the broken membrane with 50 μL of methanol (-20°C storage) for 30 minutes, and wash 3 times with PBS, add 50 μL of primary antibody NS3 (GeneTex, GTX124252) diluted 1:2000 to each well, and incubate at 4°C overnight

[0101] 4, DAY 5: discard the primary antibody, wash 3 times with 0.1% PBST, add 50 μL of fluorescent secondary antibody (ab150077-500 μg, Abeam) diluted 1:1000, incubate at 37°C for 1 hour (avoid light), wash 6 times with 0.1% PBST, gently pat dry after washing 3 minutes on a shaker in the last two times, avoid light, dry for 3 hours or overnight, and then read the fluorescence spot number on a CTL instrument.

[0102] 5, result analysis

[0103] The results of detecting the neutralization activity of the antibodies produced in the serum of the mice immunized with the tetravalent E13-HPF nanoparticle vaccine for 10 weeks against dengue virus are shown in Figure 8 The neutralization activity of DENV-2, DENV-3 and DENV-4 viruses was detected in the serum of the Balb / c mice immunized with the tetravalent E13-HPF nanoparticle for 10 weeks, and the sidak multiple comparison test showed that there was a significant difference between the experimental group and the control group; the two-tailed probability level was less than 0.05 at a significant level of 0.05. The results show that the vaccine prepared by combining E13-HPF with aluminum adjuvant has a high level of neutralizing antibodies that prevent DENV infection in the mouse body fluid 10 weeks after the first immunization, and the neutralizing antibody titer against DENV stimulated by the vaccine is high and significantly different compared with the parallel control.

[0104] Example 4: ADE effect detection

[0105] 1. The serum of the immunized mice obtained in Example 3 was diluted 50 times with serum-free opti medium (GIBCO, Opti MEM), and the serum of the mice infected with DENV-3 was used as a positive control.

[0106] 2. In a 96-well cell culture plate, 54 μL of the pre-diluted serum was added, and 54 μL of DENV-2 virus liquid (5*10 4 FFU / well) was added to each well, and the same amount of virus liquid and serum-free opti medium was added to the virus control well, and incubation was performed at 37°C for 1 hour.

[0107] 3. 8*10 5 cells / well were added to the above-mentioned incubation plate, 10 μL of K562 cells (a cell line expressing Fc receptors, commonly used in the study of the ADE effect of dengue virus) were added, the cells were pre-suspended with serum-free 1640 medium (source culture, L210KJ), and incubation was performed at 37°C for 3 hours, and then 100 μL of 4% 1640 medium was added to each well, and incubation was performed at 37°C for 24 hours.

[0108] 4. The cells were transferred to a 1.5 mL EP tube, centrifuged at 350g for 5 min, the supernatant was discarded, and each tube was added with 400 μL of PBS for washing once, centrifuged at 350g for 5 min, the supernatant was discarded, and the cell pellet was extracted with an RNA extraction kit (EZ Bioscience, BD0004D) to obtain sample RNA. The obtained sample RNA was subjected to reverse transcription by a reverse transcription kit (Vazyme, R211-02) to obtain corresponding sample cDNA.

[0109] 5. The above-obtained cDNA was used as a template, DENV-specific qPCR primers were used, and the CT value of each sample was measured by SYBR fluorescent quantitative PCR at 95°C for 3 min, 95°C for 10 s, and 60°C for 30 s (40 cycles). The viral RNA load of the corresponding sample well was obtained by comparing the standard.

[0110] 6. Result analysis

[0111] The results of the antibody-dependent enhancement effect of the antibodies in the serum of the mice immunized with the tetravalent E13-HPF nanoparticle vaccine for 10 weeks are shown in Figure 9 Compared with the virus control well, the serum of the Balb / c mice immunized with the tetravalent E13-HPF nanoparticle antigen for 10 weeks did not significantly enhance the infection of DENV-2, and the t-test result showed that there was significant difference between the experimental group and the control group; the two-tailed probability level was less than 0.05 at a significant level of 0.05. The results show that the tetravalent E13-HPF nanoparticle vaccine described in the application does not induce a significant ADE phenomenon in the mice 10 weeks after the first immunization, and has good safety compared with the parallel control.

[0112] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A dengue virus antigen, characterized in that, The antigen is any one or several of a serotype 1 dengue virus antigen, a serotype 2 dengue virus antigen, a serotype 3 dengue virus antigen and / or a serotype 4 dengue virus antigen; wherein the amino acid sequence of the serotype 1 dengue virus antigen is shown as SEQ ID NO. 1, the amino acid sequence of the serotype 2 dengue virus antigen is shown as SEQ ID NO. 2, the amino acid sequence of the serotype 3 dengue virus antigen is shown as SEQ ID NO. 3, and the amino acid sequence of the serotype 4 dengue virus antigen is shown as SEQ ID NO.

4.

2. A dengue virus nanoantigen, characterized in that, The nano-antigen is obtained by fusing a dengue virus antigen of claim 1 with a Gvtag shown as SEQ ID NO. 5 through a Linker, and then fusing the obtained fusion protein with a Sdcatcher-HPF protein shown as SEQ ID NO. 10 through a spontaneous chemical bond of Gv-Sd; the Gvtag is connected to the C-terminal of the antigen through the Linker.

3. The nanogen according to claim 2, characterized in that, The Linker is GGS, and the amino acid sequences of the fusion proteins obtained by connecting the antigens shown as SEQ ID NO. 1-4 with the Gvtag are shown as SEQ ID NO. 6-9, respectively.

4. A dengue virus nanoantigen, characterized in that, On the basis of the nano-antigen of claim 2, the N-terminal of the obtained fusion protein is connected with a secretory signal peptide, and the C-terminal is further connected with a His purification tag.

5. The nanoantigen according to claim 4, characterized in that, The amino acid sequences of the antigens shown as SEQ ID NO. 1-4 after being connected with the secretory signal peptide, the Gvtag and the His purification tag are shown as SEQ ID NO. 11-14, respectively.

6. Use of the antigen of claim 1 or the nano-antigen of any one of claims 2-5 in the preparation of a medicament for preventing dengue virus infection.

7. A dengue vaccine with reduced antibody-dependent enhancement effect, characterized in that, The vaccine is prepared from the antigen of claim 1 or the nano-antigen of any one of claims 2-5.

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