CN115990249B - Dengue tetravalent DNA vaccine and application thereof - Google Patents

Abstract

The invention provides a dengue tetravalent DNA vaccine and application thereof, and belongs to the technical field of dengue vaccines. The nucleotide sequence of the dengue tetravalent DNA vaccine provided by the invention is shown as SEQ ID NO. 1. The dengue tetravalent DNA vaccine provided by the invention is a DNA vaccine aiming at four serotypes of DENV. Specific IgG antibodies against the 4 serotypes of dengue virus can be generated after vaccination of DNA vaccines against the 4 serotypes of DENV in wild-type BALB/c mice and neutralizing antibodies with protective effects and antiviral specific T cell immunity are induced.

Description

Dengue tetravalent DNA vaccine and application thereof

Technical Field

The invention relates to the technical field of dengue vaccines, in particular to a dengue tetravalent DNA vaccine and application thereof.

Background

The four serotypes of dengue virus (DENV) are causative agents of dengue and dengue hemorrhagic fever and are divided into four different serotypes, dengue serotypes 1 to 4 (DENV-1 to DENV-4). Dengue virus is transmitted by aedes. Mosquitoes, viruses and media are widely distributed in all tropical and subtropical regions, estimated to cause 3 hundred million new infectors each year, about 100 tens of thousands of serious diseases, and the death rate is 2-5%. Notably, patients can induce long-term immunity to homotypic DENV after primary infection with a single serotype of DENV, but secondary infection can lead to serious disease, particularly after a xeno infection. The exact cause of this phenomenon is not clear, but the Antibody Dependent Enhancement (ADE) phenomenon may lead to increased pathogenicity and virulence. ADE occurs when antibodies from a previous xenotypic infection are unable to neutralize secondary infections of a different subtype, but still bind to viral proteins. This results in a virus-antibody complex that is phagocytosed by cells that are not normally infected by the fcγiia receptor, particularly monocytes that are infected by the fcγiia receptor. This can lead to viremia. Although only 1% of DENV cases present with severe disease, the mortality rate in severe cases can be as high as 20%. This presents challenges for vaccine development, as a successful vaccine must elicit a neutral, long lasting immune response to the equilibrium of all four serotypes of DENV.

The most developed DENV vaccines currently include CYD-TDV (Sainofil-Pasteur), TAK-003 (DENV-AX; wuta) and TV-003 (NIAID/NIH), which are three attenuated live vaccines, and are at risk of virulence reversion and are prone to eliciting ADE effects. And long-term data from recent dengue clinical trials indicate that the sirofine-pasteurized vaccine is effective only on those who had been infected with DENV prior to vaccination. Vaccinated uninfected individuals appear to be at greater risk of serious disease after exposure to dengue virus, and it is currently recommended to use the vaccine only in those who are immune to dengue virus. Up to now, the research and development of dengue virus vaccines in the field of DNA vaccines is slightly lagged, and an experimental team develops 4 serotypes of dengue virus nucleic acid vaccines, which can excite mice to generate stronger specific humoral and cellular immunity against one or two serotypes of viruses, but the anti-infection effect on other serotypes of dengue virus is poor, so that the research of dengue virus nucleic acid vaccines is only remained in preclinical stage.

The DENV genome is a 11Kb single positive strand RNA encoding three structural proteins, capsid protein C, membrane precursor protein prM and envelope protein E, and 7 non-structural protein domains NS, EDIII (envelope protein domain III) of which have been identified as major targets for highly neutralizing and protective serotype specific antibodies.

Disclosure of Invention

The invention aims to provide a dengue tetravalent DNA vaccine and application thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a dengue tetravalent DNA vaccine, and the nucleotide sequence of the DNA is shown as SEQ ID NO. 1.

Preferably, the vaccine is an expression vector comprising the nucleotide sequence.

Preferably, the expression vector is PVAX1.

The invention also provides application of the vaccine in immunization of four serotypes of dengue viruses.

The dengue tetravalent DNA vaccine provided by the invention is a DNA vaccine aiming at four serotypes of DENV. After vaccination of wild-type BALB/c mice with DNA vaccines against 4 serotypes of DENV, specific IgG antibodies against 4 serotypes of dengue virus can be generated and neutralizing antibodies with protective effects and antiviral specific T cell immunity are induced.

Drawings

FIG. 1 is a PVAX1-ED3 plasmid constructed in example 1 and containing the gene sequence shown in SEQ ID NO. 1.

Fig. 2 shows the humoral immune response induced by the DNA vaccine immunized mice in example 2, the results are expressed as standard errors (n=5), ns indicates no significant difference, and asterisks indicate significant differences (t-test, P < 0.05; P < 0.01).

Fig. 3 shows DNA vaccine immunization of mice to generate dengue virus specific T cell immune response in example 2, with the results expressed as standard error (n=3), and asterisks indicate significant differences (T-test, P <0.05, P < 0.01, P < 0.001).

Fig. 4 is an in vivo neutralization experiment of DENV-2 performed in example 3, (n=5), with asterisks indicating significant differences (< 0.05) in P.

Detailed Description

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

The gene sequence shown in SEQ ID NO. 1 is crossed with Shanghai gold St company to construct pCDNA3.1 (+) vector to obtain pCDNA3.1-EDIII. The synthesized pCDNA3.1-EDIII was digested with Nde I and SalI, and the fragment was constructed on a PVAX1 expression vector to give a plasmid PVAX1-ED3, as shown in FIG. 1.

The PVAX1-ED3 plasmid is amplified and cultured, and then the plasmid is extracted and purified by using an enhanced endotoxin-free plasmid big extraction kit (Tiangen biochemistry, DP 120-01) according to the instruction steps, and the plasmid is dissolved in RNASE FREE WATER in the last step of plasmid extraction column purification to prepare the vaccine with the concentration of 600 ng/mu L for immunization.

Example 2

Female BALB/c mice (army university of medical science animal center, chongqing) at 6 weeks of age were randomly divided into 2 groups of 5 animals each, which were experimental and control groups, respectively. The experimental group vaccinated mice with the vaccine prepared in example 1 by means of electric pulse intramuscular injection on days 0, 14 and 42. Control groups mice were vaccinated with empty vector PVAX1 on days 0, 14 and 42 by electric pulse intramuscular injection. The DNA vaccine contained 30. Mu.g of DNA per dose, 50. Mu.L of DNA per dose, and the control contained about 30. Mu.g of empty plasmid per 50. Mu.L of dose.

One week after immunization 3 (i.e., day 49), about 100 μl of blood was collected from the tail vein of the mice, and the serum was isolated by centrifugation at 3000rpm at 4 ℃ for 10 min. Serum was stored at-80℃and analyzed by ELISA, and after blood collection, mice were sacrificed by neck breakage after blood was collected, and spleen cells were collected for ELISPOT analysis.

(1) ELISA method (enzyme-linked immunosorbent assay) for determining total IgG in mouse serum

The four serotypes Dengue virus Envelope Protein-Domain III（Sino Biolgical, 40531-V08B, 40471-V08Y1, 40532-V08H1, 40533-V08B2） antigen were diluted to 5 μg/mL with coating buffer, and 96 well plates were coated 100 μl/well, antigen coated at 0.5 μg, overnight at 4 ℃. The plates were washed 5 times with PBST solution to remove uncoated antigen, 200. Mu.L of blocking solution (PBST with 5% BSA) was added to each well and incubated at 37℃for 2 h. After blocking, the blocking solution was discarded, the plate was washed 3 times with PBST solution, then 100. Mu.L of immune serum (using 1% BSA in PBST as a diluent) and a blank PBS solution were added to each well, incubated at 37℃for 2 hours, after the end of incubation, the plate was washed 3 times with PBST solution, 100. Mu.L/well (using 1% BSA in PBST as a diluent) was added with 5000-fold dilution HRP-goat anti mouse IgG (Solarbio, SE 131), incubated at 37℃for 1 hour, the plate was washed 5 times with PBST solution, 100. Mu.L of color development solution was added to each well, the plate was placed in a dark condition, color development was performed at 15-30 min, 100. Mu.L of stop solution was added to each well to terminate color development, the absorbance at 450 nm was read by an ELISA, and the final antibody titer of mouse serum was determined from the ratio of OD value to blank.

The results are shown in FIG. 2. From the experimental results, 1 week after the third immunization, mice were able to produce specific IgG against DENV-1, DENV-2, DENV-3, DENV-4, indicating that DNA vaccines can induce specific humoral immunity.

(2) ELISPOT assay for antigen-specific cellular immunity

Mice were taken 7 days after immunization at 3 rd time and their cellular immune response was assessed using an IFN-gamma, IL-4 pre-coated ELISPOT kit (Dayou, china).

The plates were pre-incubated with serum-free RPMI1640 (Thermo FISHER SCIENCE) for 15min. The immunized mice spleen cells are inoculated at the density of 500,000/hole, a positive stimulator in the kit is used as a positive control, RPMI1640 culture solution is used as a negative control, and 4 antigens (ET 1-4) after screening are used for stimulating peptides in an experimental group.

ET1:VTQNGRLITANPIVT;

ET2:RHVLGRLITVNPIVT;

ET3:KAHNGRLITANPVVT;

ET4: EKVVGRIISSTPFAE。

The above sequence was submitted to Shanghai gold SpA to synthesize a polypeptide.

The synthetic polypeptide ET1/ET2/ET3/ET4 is mixed in equal quantity and then dissolved in deionized water to prepare 0.8 mug/mu L of mixed polypeptide, and the mixed polypeptide is added into an experimental group to be used as a stimulator.

After addition of the stimulator, after incubation for 60h in 37℃5% CO ₂, the plates were washed with wash buffer, 1:100 biotin-labeled anti-murine IFN-gamma, IL-4 antibody was added per well, incubated for 1h at room temperature, after washing the plates again according to instructions, diluted enzyme-labeled avidin was added, 100. Mu.L per well, incubated for 1h at room temperature, after washing the plates again, ready-prepared AEC color development solution was added to each experimental well according to kit instructions, 100. Mu.L per well, after addition of AEC substrate solution, developed for 15min at 37℃in the dark, and the air-dried plates were 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 shown in FIG. 3.

The results showed that the DNA immunized mice secreted IFN-gamma and IL-4 significantly higher than the empty mice. This shows that the DNA vaccine provided by the invention successfully induces dengue virus specific cellular immune response.

Example 3

In vivo neutralization assay

The suckling mice used for in-vivo neutralization experiments are bred by combining female mice and male mice of 4-6 weeks old BALB/c purchased from animal centers, and the neutralization experiments are carried out on the 3 rd day of birth of the suckling mice. The present study on mice was approved by the ethical committee of the army university of medicine, following guidelines of the army university of medicine and national research committee for animal experiments and feeding.

Serum was isolated from the rat tail blood 7 days after the 3 rd immunization of the experimental group and the control group, inactivated at 56℃for 30min, and diluted with deionized water at 1:20. The diluted 10. Mu.L of serum was mixed with 10. Mu.L of DENV-2 (60 PFU, LD 60) virus in a P2 laboratory biosafety cabinet, incubated at 37℃for 1h, and 20. Mu.L of the virus serum mixture was injected into the milk mice with a microinjector to observe survival of the milk mice within 15 days after infection. The results are shown in FIG. 4

The mice injected with the empty vaccine serum virus mixture began to die at day 7 post-infection, 60% of the mice died from the virus infection within 15 days post-infection, and the mice of the DNA immune serogroup survived all within 15 days post-infection. These data indicate that DNA vaccines can protect mice from lethal DENV-2 in mice that are not infected with the virus.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (4)

1. A dengue tetravalent DNA vaccine is characterized in that the nucleotide sequence of the DNA is shown as SEQ ID NO. 1.

2. The vaccine of claim 1, wherein the vaccine is an expression vector comprising the nucleotide sequence of claim 1.

3. The vaccine of claim 2, wherein the expression vector is PVAX1.

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