A tetravalent dengue DNA vaccine and its application

Abstract

The present invention provides a dengue tetravalent DNA vaccine and its application, belonging to the technical field of dengue vaccines. The nucleotide sequence of the dengue tetravalent DNA vaccine provided by the present invention is as shown in SEQ ID NO: 1. The dengue tetravalent DNA vaccine provided by the present invention is a DNA vaccine against four serotypes of DENV. After inoculating wild-type BALB / c mice with the DNA vaccine against four serotypes of DENV, specific IgG antibodies against four serotypes of dengue virus can be produced, and neutralizing antibodies with protective effects and antiviral specific T cell immunity can be induced.

Description

Technical Field

[0001] The present invention relates to the technical field of dengue vaccines, and in particular to a tetravalent dengue DNA vaccine and its application. Background Art

[0002] The four serotypes of dengue virus (DENV) are the pathogens of dengue fever and dengue hemorrhagic fever, and are divided into four different serotypes, namely dengue serotypes 1 to 4 (DENV-1 to DENV-4). Dengue virus is transmitted by Aedes mosquitoes. Mosquitoes, as well as the virus and vectors, are widely distributed in all tropical and subtropical regions, causing an estimated 300 million new infections per year, about 1 million cases of severe disease, and a case fatality rate of 2-5%. It is worth noting that after a patient is initially infected with a single serotype of DENV, it can induce long-term immunity to the same serotype of DENV, but secondary infection can lead to severe disease, especially after heterotypic infection. The exact cause of this phenomenon is still unclear, but the antibody-dependent enhancement (ADE) phenomenon may lead to increased pathogenicity and virulence. ADE occurs when antibodies from a previous heterotypic infection cannot neutralize a secondary infection with a different subtype, but still bind to the viral protein. This produces a virus-antibody complex that is phagocytosed by cells that do not normally infect through the Fcγ receptor, especially monocytes that infect through the FcγIIa receptor. This leads to the production of viremia. Although only 1% of DENV cases present with severe disease, the mortality rate of severe cases can be as high as 20%. This poses a challenge to vaccine development because a successful vaccine must elicit a balanced, neutralizing, and long-lasting immune response against all four serotypes of DENV.

[0003] The DENV vaccines with the greatest research progress currently include CYD-TDV (Sanofi Pasteur), TAK-003 (DENV-AX; Takeda), and TV-003 (NIAID / NIH), which are three live attenuated vaccines. There is a risk of reversion of virulence and they are prone to causing the ADE effect. And the long-term data of dengue fever clinical trials in recent years have shown that the Sanofi Pasteur vaccine is only effective for those who have been infected with DENV before vaccination. Uninfected individuals who receive the vaccine seem to face a greater risk of severe disease after exposure to the dengue virus, and it is currently recommended to use this vaccine only in people who are immune to the dengue virus. As of now, the research and development of dengue virus vaccines in the field of DNA vaccines is slightly lagging behind. An experimental team has developed nucleic acid vaccines for the four serotypes of dengue virus, which can stimulate strong specific humoral and cellular immunity against one or two serotypes of the virus in mice, but the anti-infection effect on several other serotypes of dengue virus is relatively poor. So far, the research on dengue virus nucleic acid vaccines has only stayed in the pre-clinical stage.

[0004] The DENV genome is a single-stranded positive-sense RNA of 11 Kb, encoding three structural proteins, namely capsid protein C, pre-membrane protein prM, and envelope protein E, and seven non-structural protein domains NS. The EDIII (envelope protein domain III) in the structural protein E has been identified as the main target of highly neutralizing and protective serotype-specific antibodies. Summary of the Invention

[0005] The object of the present invention is to provide a tetravalent dengue DNA vaccine and its application.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a tetravalent dengue DNA vaccine, and the nucleotide sequence of the DNA is as shown in SEQ ID NO: 1.

[0008] Preferably, the vaccine is an expression vector containing the above-mentioned nucleotide sequence.

[0009] Preferably, the expression vector is PVAX1.

[0010] The present invention also provides the use of the above-mentioned vaccine in immunizing four serotypes of dengue virus.

[0011] The tetravalent dengue DNA vaccine provided by the present invention is a DNA vaccine against four serotypes of DENV. After inoculating wild-type BALB / c mice with the DNA vaccine against four serotypes of DENV, specific IgG antibodies against four serotypes of dengue virus can be produced, and neutralizing antibodies with protective effects and antiviral specific T cell immunity can be induced. Brief Description of the Drawings

[0012] Figure 1 PVAX1-ED3 plasmid containing the gene sequence shown in SEQ ID NO: 1 constructed for Example 1.

[0013] Figure 2 The humoral immune response induced by immunizing mice with the DNA vaccine in Example 2; the results are expressed as the standard error (n = 5), ns indicates no significant difference, and asterisks indicate significant differences (t-test, *, P < 0.05; **, P < 0.01).

[0014] Figure 3 The dengue virus-specific T cell immune response produced by immunizing mice with the DNA vaccine in Example 2; the results are expressed as the standard error (n = 3), and asterisks indicate significant differences (t-test, *, P < 0.05; **, P < 0.01; ***, P <0.001).

[0015] Figure 4 In vivo neutralization experiment of DENV-2 conducted for Example 3, (n = 5); asterisks indicate significant differences (*, P < 0.05). Detailed implementation manners

[0016] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be construed as limiting the protection scope of the present invention. Example 1

[0017] The gene sequence shown in SEQ ID NO: 1 was submitted to GenScript Corporation in Shanghai to be constructed onto the pCDNA3.1(+) vector to obtain pCDNA3.1-EDIII. The synthesized pCDNA3.1-EDIII was digested with Nde I and SalI, and the fragment was then constructed onto the PVAX1 expression vector to obtain the plasmid PVAX1-ED3, as Figure 1 shown.

[0018] After the PVAX1-ED3 plasmid was amplified and cultured, an enhanced endotoxin-free plasmid large-scale extraction kit (Tiangen Biochemical, DP120-01) was used to extract the plasmid according to the steps in the instruction manual and purify it. In the last step of purification on the plasmid extraction column, the plasmid was dissolved in RNase-Free water to prepare an immunization vaccine with a concentration of 600 ng / μL. Example 2

[0019] Six-week-old female BALB / c mice (Animal Center of Army Medical University, Chongqing) were randomly divided into 2 groups, with 5 mice in each group, namely the experimental group and the control group. The experimental group was inoculated with the vaccine prepared in Example 1 by electro-pulse intramuscular injection on days 0, 14, and 42. The control group was inoculated with the empty vector PVAX1 by electro-pulse intramuscular injection on days 0, 14, and 42. Each dose of the DNA vaccine contained 30 μg of DNA, 50 μL; each dose of the control group was 50 μL, containing about 30 μg of the empty plasmid.

[0020] One week after the third immunization (i.e., day 49), approximately 100 μL of blood was collected from the tail vein of the mice, centrifuged at 4°C, 10 min, 3000 rpm to separate the serum. The serum was stored at -80°C and analyzed by ELISA; after blood collection, the mice were sacrificed by exsanguination through the eyeball and then decapitated, and spleen cells were taken for ELISPOT analysis.

[0021] (1) Determination of total IgG in mouse serum by ELISA (enzyme-linked immunosorbent assay)

[0022] Dilute the antigens of four serotypes of Dengue virus Envelope Protein-DomainIII (Sino Biological, 40531-V08B, 40471-V08Y1, 40532-V08H1, 40533-V08B2) to 5 μg / mL with coating buffer respectively, coat a 96-well plate with 100 μL per well, and the antigen coating amount is 0.5 μg, and incubate overnight at 4°C. Wash the plate 5 times with PBST solution to remove the uncoated antigens. Add 200 μL of blocking solution (PBST containing 5% BSA) to each well, and incubate at 37°C for 2 h. After blocking, discard the blocking solution, wash the plate 3 times with PBST solution, then add 100 μL of immune serum (using PBST containing 1% BSA as the diluent) and blank control PBS solution to each well, and incubate at 37°C for 2 h; after incubation, wash the plate 3 times with PBST solution, add 100 μL of HRP-goat anti mouse IgG (Solarbio, SE131) diluted 5000-fold (using PBST containing 1% BSA as the diluent) to each well, and incubate at 37°C for 1 h; wash 5 times with PBST solution, add 100 μL of chromogenic solution to each well, place in the dark, develop color for 15 - 30 min, and add 100 μL of stop solution to each well to terminate the color development; read the absorbance value at 450 nm with an enzyme-linked immunosorbent assay reader, and determine the final antibody titer of mouse serum according to the ratio of the OD value to the blank group.

[0023] The results are as Figure 2 shown. Judging from the experimental results, 1 week after the third immunization, mice can produce specific IgG against DENV-1, DENV-2, DENV-3, and DENV-4, indicating that the DNA vaccine can induce specific humoral immunity.

[0024] (2)ELISPOT assay for antigen-specific cellular immunity

[0025] Take the mice 7 days after the third immunization, and use an ELISPOT kit pre-coated with IFN-γ and IL-4 (Dayou, China) to evaluate the cellular immune response of the immunized mice.

[0026] Pre-incubate the culture plate with serum-free RPMI1640 (Thermo Fisher Science) for 15 min. Inoculate the immunized mouse spleen cells at a density of 500,000 cells / well. The positive stimulant in the kit is used as the positive control, and RPMI1640 culture medium is used as the negative control; the experimental group is stimulated with 4 screened antigen (ET1~4) stimulatory peptides.

[0027] ET1:VTQNGRLITANPIVT;

[0028] ET2: RHVLGRLITVNPIVT;

[0029] ET3: KAHNGRLITANPVVT;

[0030] ET4: EKVVGRIISSTPFAE.

[0031] The above sequences were handed over to GenScript Biotech Corporation in Shanghai for polypeptide synthesis.

[0032] The synthetic polypeptides ET1 / ET2 / ET3 / ET4 were mixed in equal amounts and dissolved in deionized water to prepare a mixed polypeptide of 0.8 μg / μL. The mixed polypeptide was added to the experimental group as a stimulant.

[0033] After adding the stimulant, the plate was incubated at 37 °C and 5% CO2 for 60 h, then washed with the washing buffer. Biotin-labeled anti-mouse IFN-γ and IL-4 antibodies were added to each well at 1:100, 100 μL / well, and incubated at room temperature for 1 h; after washing the plate again according to the instructions, diluted enzyme-labeled avidin was added, 100 μL / well, and incubated at room temperature for 1 h; after washing the plate again, the AEC chromogenic solution freshly prepared according to the kit instructions was added to each experimental well, 100 μL / well. After adding the AEC substrate solution, it was developed at 37 °C in the dark for 15 min, and the air-dried plate was read using an automatic ELISPOT reader CTL ImmunoSpot SC. The number of spot-forming cells (SFC) per 500,000 cells was calculated. The results are as Figure 3 shown.

[0034] The results showed that the DNA immunized group of mice secreted significantly higher levels of IFN-γ and IL-4 than the empty vector group. This indicates that the DNA vaccine provided by the present invention successfully induced a dengue virus-specific cellular immune response. Example 3

[0035] In vivo neutralization experiment

[0036] The suckling mice used for the in vivo neutralization experiment were obtained by co-housing 4- to 6-week-old female and male BALB / c mice purchased from the animal center, and the neutralization experiment was carried out on the 3rd day after the suckling mice were born. All the studies on mice in this experiment were approved by the Ethics Committee of Army Medical University and followed the guidelines of Army Medical University and the National Research Council on animal experiments and breeding.

[0037] Take the tail blood of mice in the experimental group and the control group 7 days after the third immunization to separate the serum, inactivate it at 56 °C for 30 min; dilute the serum with deionized water at a ratio of 1:20. Conduct the following operations in a biosafety cabinet in P2 laboratory: Mix 10 μL of the diluted serum with 10 μL of DENV-2 (60 PFU, LD60) virus, incubate at 37 °C for 1 h, and then inject 20 μL of the virus-serum mixture into suckling mice with a micro syringe, and observe the survival of the suckling mice within 15 days after infection. The results are as Figure 4 shown

[0038] The suckling mice injected with the mixture of empty vector vaccine serum and virus began to die on the 7th day after infection, and 60% of the mice died of virus infection within 15 days after infection; all the suckling mice in the DNA immunized serum group survived within 15 days after infection. These data indicate that in virus-free mice, the DNA vaccine can protect mice from lethal DENV-2 challenge.

[0039] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A dengue tetravalent DNA vaccine, characterized in that, The nucleotide sequence of the DNA is shown by SEQ ID NO:

1.

2. The vaccine according to claim 1, characterized in that, The vaccine is an expression vector containing the nucleotide sequence described in claim 1.

3. The vaccine according to claim 2, characterized in that, The expression vector is PVAX1.

4. Use of the vaccine according to any one of claims 1 to 3 in the preparation of a medicament for immunizing against four serotypes of dengue virus.

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