Rainbow trout nucleic acid vaccine adjuvant and application thereof

Abstract

The invention relates to a rainbow trout nucleic acid vaccine adjuvant. The rainbow trout nucleic acid vaccine adjuvant comprises a polydisulfide compound and cyclic diguanylate delivered by the polydisulfide compound. The invention further relates to application of the rainbow trout nucleic acid vaccine adjuvant in preparation of rainbow trout IHNV nucleic acid vaccines. The rainbow trout nucleic acid vaccine adjuvant, namely a c-di-GMP + CPD compound, is developed on the basis of an STING agonist-c-di-GMP and CPD delivery carrier, so that cost reduction and synergism of rainbow trout nucleic acid vaccines are achieved, the CPD can efficiently deliver cyclic diguanylic acid and has good stability, and the c-di-GMP + CPD compound has the potential of serving as a rainbow trout immunologic adjuvant and can be used for preparing the rainbow trout immunologic adjuvant. The immune protection effect of rainbow trout nucleic acid vaccines can be obviously enhanced, and the use dosage of the vaccines is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of immune prevention and control, and in particular to a rainbow trout nucleic acid vaccine adjuvant and application thereof. Background Art

[0002] Infectious haematopoietic necrosis (IHN) is a viral disease that seriously harms salmon and trout. The pathogen of the disease is IHNV, which can cause up to 80%-100% mortality after an outbreak. It is the main bottleneck facing the development of cold-water fish farming in my country. The applicant has previously designed a DNA vaccine that can effectively enhance the immune protection effect of rainbow trout and resist IHNV virus (WU D, et al. Aquaculture. 2023, 572: 739555, CN118903403A), but the application dose is high and cannot meet the demand for low-cost vaccines in the aquaculture industry. Therefore, it is necessary to develop an adjuvant suitable for rainbow trout nucleic acid vaccine to improve vaccine efficacy and reduce the dosage.

[0003] Cyclic diguanylate (c-di-GMP) is a STING agonist that can bind to the STING protein in the cell body, stimulate the cell to release a variety of cytokines, such as interferon, IL-6 and TNF-α, and can also regulate the body's immune response by activating typical inflammatory pathways such as NF-κB. At present, c-di-GMP has been widely used as an adjuvant in subunit vaccines, inactivated vaccines, etc. in mammals (GALLOVIC MD, et al. Journal of Controlled Release. 2022, 347: 356-368.) (HANSON MC, et al. Journal of Clinical Investigation. 2015, 125 (6): 2532-2546.). However, rainbow trout is a kind of low-level vertebrate with variable temperature. Compared with mammals, its immune response system has certain differences. Therefore, whether c-di-GMP has an immunostimulatory effect on rainbow trout and even fish remains to be studied.

[0004] c-di-GMP is a hydrophilic small nucleic acid molecule that cannot cross the cell membrane to activate the STING pathway under natural conditions. The addition of a delivery vector can help solve this problem. Currently, delivery vectors can be divided into nanoliposome particles (LNP), polymer vectors, protein vectors, viral vectors, etc., among which LNP is the mainstream for nucleic acid delivery. Since fish farming is sensitive to vaccine costs, the more expensive LNP is not applicable, so it is necessary to develop a delivery vector that is suitable for fish and cost-controlled.

[0005] Polydisulfide compound (CPD) is a polymer carrier similar to polyarginine. Its positively charged guanidine group promotes the accumulation of the carrier near the cell surface. The disulfide skeleton can promote the entry of the carrier into the membrane through a disulfide exchange mechanism. After entering the cell, CPD can be rapidly depolymerized under the catalysis of endogenous glutathione to release the cargo (GUO J, et al. ACS Central Science. 2021, 7: 990-1000.). Summary of the invention

[0006] In order to solve the above problems in the prior art, the present invention provides a rainbow trout nucleic acid vaccine adjuvant and application thereof.

[0007] In order to achieve the above-mentioned object, the first aspect of the present invention provides a rainbow trout nucleic acid vaccine adjuvant, the main feature of which is that the adjuvant includes a polydisulfide compound and cyclic diguanylate delivered by the polydisulfide compound.

[0008] Preferably, the mass ratio of the polydisulfide compound to cyclic diguanylate is 4:1 to 10:1.

[0009] Preferably, the rainbow trout nucleic acid vaccine is a rainbow trout DNA vaccine against IHNV virus.

[0010] The second aspect of the present invention provides the use of the rainbow trout nucleic acid vaccine adjuvant in the preparation of rainbow trout IHNV virus nucleic acid vaccine.

[0011] The beneficial effects of the present invention are as follows: based on the STING agonist - c-di-GMP and polydisulfide compound (CPD) delivery vector, a rainbow trout nucleic acid vaccine adjuvant, namely c-di-GMP+CPD complex, is developed, thereby achieving cost reduction and efficiency improvement of rainbow trout nucleic acid vaccine. Polydisulfide compound (CPD) can efficiently deliver cyclic diguanylate and has good stability. The c-di-GMP+CPD complex has the potential to serve as a rainbow trout immune adjuvant, which can significantly enhance the immune protection effect of rainbow trout nucleic acid vaccine and reduce the vaccine dosage. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 A is a CPD synthesis flow chart used in the present invention; Figure 1 B and Figure 1 C is the NMR spectrum and mass spectrum of the CPD monomer synthesized by the present invention; Figure 1 D. Figure 1 E. Figure 1 F are respectively the average molecular weight, H NMR spectrum and average molecular weight distribution curve of the CPD synthesized in the present invention.

[0013] Figure 2 A and Figure 2B shows the results of changes in viral load of c-di-GMP and expression of I-IFN in CHSE-214 cells.

[0014] Figure 3 A and Figure 3 B is a graph showing the results of immune factor determination of c-di-GMP+CPD complex in mouse L929 cells.

[0015] Figure 3 C and Figure 3 D shows the results of viral load changes and I-IFN expression of c-di-GMP+CPD complex in CHSE-214 cells.

[0016] Figure 3 E and Figure 3 F is the stability result diagram of CPD+c-di-GMP complex.

[0017] Figure 4 This is the result of serum neutralizing antibodies 28 days after immunization with DNA vaccine combined with (cdiG+CPD) adjuvant.

[0018] Figure 5 This is the result of the IHNV challenge test using a DNA vaccine combined with (cdiG+CPD) adjuvant 28 days after immunization. DETAILED DESCRIPTION

[0019] In order to more clearly describe the technical content of the present invention, further description is given below in conjunction with specific embodiments.

[0020] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the reagents, materials, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0021] The present invention develops a rainbow trout nucleic acid vaccine adjuvant based on the STING agonist - c-di-GMP and polydisulfide compound (CPD) delivery vector, namely c-di-GMP+CPD complex, thereby achieving cost reduction and efficiency improvement of rainbow trout nucleic acid vaccine and promoting its practical application.

[0022] IHNV isolate GS21 was isolated from diseased rainbow trout in Gansu. The rainbow trout DNA vaccine was designed and preserved by the research group (WU D, et al. Aquaculture. 2023, 572: 739555, CN118903403A). Cyclic diguanylate (c-di-GMP) and polydisulfide compounds (CPD) were synthesized in the laboratory. The pRL-TK plasmid and pGL3-IFN pro-Luc plasmid were preserved in the laboratory. L929 cells and CHSE-214 cells were preserved in the laboratory. The transfection reagent LipofectamineTM 3000 transfection reagent (L3000001) was purchased from Thermo Fisher Scientific; dual-luciferase reporter gene detection kit (RG029S) was purchased from Bio-Time; quantitative PCR kit MonAmpSYBR×green qPCR Mix (MQ10701S) was purchased from Mona.

[0023] Example 1 Development of cyclic diguanylate delivery vector

[0024] Polydisulfide synthesis and cell transfection

[0025] CPD synthesis route Figure 1 As shown in A, the specific synthesis process is as follows:

[0026] CPD monomer preparation

[0027] Add lipoic acid 1 (4.12 g, 20 mmol) and CDI (3.24 g, 20 mmol) to anhydrous DMF (50 mL), and stir the reaction solution under nitrogen protection at room temperature for 2 h to activate lipoic acid. Add L-arginine methyl ester dihydrochloride (2.61 g, 10 mmol) and N, N-diisopropylethylamine (1.74 mL, 10 mmol) to anhydrous DMF (40 ml) solution for 1 h, then add the activated lipoic acid solution and continue stirring at room temperature for 3.5 h. Add 3 times the volume of ether to the reaction solution, mix well and centrifuge (2 min, 3000 rpm), collect the oil, and wash the oil 3 times with a mixed solution of DCM / Et2O (1:2, 30×60 ml), and vacuum dry to obtain a yellow oily CPD monomer (4.22 g, yield 79.6%). The nuclear magnetic spectrum (MS) and mass spectrum (1H NMR) of the CPD monomer are shown as follows: Figure 1 B. Figure 1 As shown in C.

[0028] Preparation of CPD

[0029] Before the reaction, the following solutions were prepared in advance: TEOA buffer (1M, pH = 7.0, H2O), initiator mother solution (-SH, 5mM, TEOA), newly prepared disulfide monomer M1 (1M, TEOA) solution, terminator mother solution (iodoacetamide, 0.5M, H2O). After the above solutions were prepared, they were deoxygenated with nitrogen. The treated solutions were added according to the volume ratio of polymer monomer: initiator: DMF: TEOA = 1:1:1.5:6.5, and the total system was 10mL. During polymerization, the reaction was stirred vigorously under nitrogen protection. After 30 minutes of reaction, 20mL of iodoacetamide stop solution (0.5M) was added to the reaction solution to terminate the reaction. The terminated reaction solution was placed in a dialysis bag with a molecular cutoff of 2000 and then placed in deionized water to dialyze out the unreacted small molecules. The dialysis solution was changed every 4 hours. After dialysis for two days, vacuum freeze-dried to obtain white CPD powder. Nuclear magnetic resonance hydrogen spectrum (1H NMR) Figure 1 E, the average molecular weight and distribution curve of CPD were determined by gel permeation chromatography (see Figure 1 D. Figure 1 F).

[0030] Preparation and transfection of c-di-GMP+CPD complex

[0031] c-di-GMP and CPD were prepared into aqueous solutions using enzyme-free water, where c-di-GMP was 40 μg / mL and CPD was prepared at different concentrations, namely 40, 80, 160, 240, 320, and 400 μg / mL. They were mixed according to the mass ratio (1:1, 1:2, 1:4, 1:6, 1:8, and 1:10) and incubated at room temperature for 30 min to obtain c-di-GMP+CPD complexes with different ratios, which were then added to the cell well plate in equal volumes.

[0032] Immune factor assay

[0033] 24h before transfection, L929 cells were plated in 24-well plates. When the cells grew to a confluence of 60-70%, c-di-GMP+CPD complex was transfected and cultured at 37°C. Cell samples were collected 6h after transfection, and total cell RNA was extracted using an RNA extraction kit. IFN-β and IL6 factors were determined using the MonAmp SYBR×green qPCR Mix quantitative PCR kit.

[0034] like Figure 3 A and Figure 3As shown in B, the test in mouse L929 cells found that CPD can effectively deliver c-di-GMP into cells, promote the upregulation of IFN-β and IL6 expression in mouse cells, and its delivery effect is significantly higher than that of the commercial transfection reagent Lipo3000 (Lp+cdiG group). In addition, the optimal mass ratio of CPD to c-di-GMP is 4:1. Therefore, CPD is a suitable carrier for delivering c-di-GMP.

[0035] Stability determination

[0036] The stability of the CPD+c-di-GMP complex affects its practical application. The CPD+c-di-GMP complex was stored at room temperature (RT), 4°C, and -20°C, and the immune factor activation effect was detected after 1 month, 2 months, and 4 months of storage. The test method for mouse L929 cells was the same.

[0037] like Figure 3 As shown in E and 3F, the mouse L929 cell test found that the CPD+c-di-GMP complex can be stably stored at 4°C for at least 120 days, and can still upregulate the expression of immune factors after long-term storage. Therefore, the complex has good application potential.

[0038] Example 2 Evaluation of cyclic diguanylate in trout cells

[0039] Changes in viral load

[0040] 24 hours before transfection, CHSE-214 cells were plated in 24-well plates. When the cells grew to a confluence of 60-70%, the medium in the well plate was replaced with serum-free medium, and the transfection system was prepared according to the instructions of the transfection reagent to transfect c-di-GMP. 12 hours after transfection, the medium was replaced with 10 4 TCID50 IHNV culture medium, 15 ° C static culture. Collect cell samples 24 hours after infection, extract total cell RNA using RNA extraction kit, and use MonAmp SYBR×greenqPCR Mix for absolute fluorescence quantitative analysis of IHNV N gene. Specific grouping is shown in Table 1 below.

[0041] Table 1

[0042]

[0043] Upregulation of I-IFN expression in fish

[0044] 24h before transfection, CHSE-214 cells were used to plate 24-well plates. When the cells grew to a confluence of 60-70%, the medium in the well plate was replaced with serum-free medium, and the transfection system was prepared according to the instructions of the transfection reagent, and 0.5μg pGL3-IFN pro-Luc plasmid and 0.05μg pRL-TK plasmid were transfected. 24h after transfection, c-di-GMP was transfected in the same way and cultured at 26℃. Cell samples were collected 24h after transfection, and the Fluc expression content was detected using a dual luciferase reporter gene assay kit. The specific grouping is shown in Table 2 below.

[0045] Table 2

[0046]

[0047] like Figure 2 As shown in A, in CHSE-214 cells, c-di-GMP delivered by commercial transfection reagents can significantly inhibit the copying of IHNV virus in trout cells. Figure 2 As shown in Figure B, based on the dual luciferase reporter experiment, c-di-GMP can upregulate the expression of rainbow trout type I IFN in CHSE-214 cells, thereby inhibiting the virus. Combining the results of the two, it can be seen that c-di-GMP has an immunostimulatory effect in trout cells and has the potential to be used as an adjuvant for rainbow trout vaccines.

[0048] like Figure 3 As shown in C and 3D, in salmon and trout cells CHSE-214, the CPD+cdiG group can better inhibit the copy of IHNV in cells and upregulate the expression of rainbow trout type I IFN more significantly. Therefore, the CPD+c-di-GMP complex can be applied to salmon and trout. Therefore, polydisulfide compounds (CPD) can efficiently deliver cyclic diguanylate and have good stability.

[0049] Example 3 Evaluation of cyclic diguanylate in fish

[0050] immunity

[0051] Rainbow trout weighing 5±0.1g were randomly divided into groups of 40 in each group and kept in a circulating water pool (65cm×65cm×70cm) with a water temperature of (16±1)℃ for 14 days. They were injected with the vaccine (injection position was the base of the dorsal fin) and fasted for 2 days before the experiment. The vaccine immunization groups are shown in Table 3 below, and a blank control group (50μL / tail phosphate solution) was set up at the same time.

[0052] Table 3

[0053] Group Vaccine presentation Vaccine components 1 Control group PBS buffer 2 DNA 1 μg DNA vaccine 3 cdiG+CPD 5 μg c-di-GMP+CPD complex 4 DNA+cdiG+CPD 1μg DNA vaccine + 5μg (c-di-GMP + CPD) adjuvant

[0054] Changes in serum neutralizing antibody titer

[0055] Blood was collected from the tail fin of rainbow trout (n=3) 28 days after immunization. Due to the small amount of blood obtained, the blood of three fish was mixed into one sample, placed at 4°C overnight, centrifuged at 3,000×g for 10 min, and the supernatant was taken as a serum sample and frozen at -80°C for later use.

[0056] Take the serum sample stored in the refrigerator and inactivate it in a 56°C water bath for 30 minutes. Dilute the serum sample by 2 times, from 1:20 to 1:40, 1:80, 1:160 and 1:320 respectively. Incubate the above diluted samples with 100TCID50 of IHNV at 15°C for 1 hour. Add the mixed sample after incubation to a single-layer 96-well plate, and make 8 replicates for each gradient. At the same time, set up normal cell blank control, virus control, negative serum control and positive serum control wells. Place it in a 15°C incubator for static culture, and observe whether CPE occurs after 7 to 10 days. The highest serum dilution that makes half of the samples free from infection is used as the neutralizing antibody titer of the sample, and the neutralizing antibody titer <20 is considered a negative sample.

[0057] Serum neutralizing antibody results 28 days after immunization Figure 4 As shown, compared with the DNA immunization group, the DNA+cdiG+CPD immunization group can produce stronger neutralizing antibodies, that is, the DNA vaccine combined with (cdiG+CPD) adjuvant can increase the neutralizing antibody level of fish. This result shows that the c-di-GMP+CPD complex can enhance the specific immune response of rainbow trout, so that a low dose of rainbow trout DNA vaccine can produce a strong immune protection effect.

[0058] Changes in viral load

[0059] Fasting began on day 26 after immunization. On day 28 after immunization, the immune rainbow trout were challenged with 100 μL HNV (10 4 TCID50 / mL), and the injection site was in the abdominal cavity of the fish. Spleen tissues of rainbow trout (n=3) were collected 7 days after the challenge, and total RNA from the spleen was extracted using an RNA extraction kit, and absolute fluorescence quantitative analysis of the IHNV N gene was performed using the MonAmp SYBR×green qPCR Mix quantitative PCR kit.

[0060] IHNV challenge test was performed 28 days after immunization. Figure 5is the viral load of fish 7 days after infection. Compared with the DNA immunization group, the IHNV viral load of the DNA+cdiG+CPD immunization group was lower, that is, the DNA vaccine combined with (cdiG+CPD) adjuvant can significantly reduce the viral load of fish, indicating that the c-di-GMP adjuvant enhances the immune protection effect of the DNA vaccine. Combined with the above results, the c-di-GMP+CPD complex has the potential to be used as an immune adjuvant for rainbow trout, which can significantly enhance the immune protection effect of rainbow trout nucleic acid vaccines and reduce the vaccine dosage.

[0061] In this specification, the present invention has been described with reference to specific embodiments thereof. However, it is apparent that various modifications and variations may be made without departing from the spirit and scope of the present invention. Therefore, the specification and drawings should be regarded as illustrative rather than restrictive.

Claims

1. A rainbow trout nucleic acid vaccine adjuvant, characterized in that: The adjuvant includes a polydisulfide compound and cyclic diguanylate delivered by the polydisulfide compound.

2. The rainbow trout nucleic acid vaccine adjuvant according to claim 1, characterized in that The mass ratio of the polydisulfide compound to cyclic diguanylate is 4:1 to 10:

1.

3. The rainbow trout nucleic acid vaccine adjuvant according to claim 1, characterized in that: The rainbow trout nucleic acid vaccine is a rainbow trout DNA vaccine against IHNV virus.

4. Use of the rainbow trout nucleic acid vaccine adjuvant described in any one of claims 1 to 3 in the preparation of rainbow trout IHNV virus nucleic acid vaccine.

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