Rainbow trout nucleic acid vaccine adjuvants and their applications

Abstract

The present application relates to rainbow trout nucleic acid vaccine adjuvant, the adjuvant includes polydisulfide compound and cyclic di-GMP delivered by polydisulfide compound, the present application also relates to the application of the rainbow trout nucleic acid vaccine adjuvant in the preparation of rainbow trout IHNV virus nucleic acid vaccine.A kind of rainbow trout nucleic acid vaccine adjuvant, i.e.c-di-GMP+CPD complex, is developed based on STING agonist-c-di-GMP and polydisulfide compound (CPD) delivery carrier, so as to realize the cost reduction and efficiency increase of rainbow trout nucleic acid vaccine, polydisulfide compound (CPD) can efficiently deliver cyclic di-GMP, and have good stability, c-di-GMP+CPD complex has the potential as rainbow trout immune adjuvant, can significantly enhance the immune protection effect of rainbow trout nucleic acid vaccine, reduces vaccine dosage.

Description

Technical Field

[0001] This invention relates to the field of immunoprevention and control technology, and in particular to a rainbow trout nucleic acid vaccine adjuvant and its application. Background Technology

[0002] Infectious hematopoietic necrosis (IHN) is a serious viral disease affecting salmon and trout. The pathogen is IHNV, and outbreaks can cause mortality rates as high as 80%-100%, posing a major bottleneck to the development of cold-water fish aquaculture in my country. The applicant previously designed a DNA vaccine that effectively enhances the immune protection of rainbow trout against IHNV (WU D, et al. Aquaculture. 2023, 572:739555, CN118903403A), but the dosage is high and cannot meet the demand for low-cost vaccines in aquaculture. Therefore, it is necessary to develop an adjuvant suitable for rainbow trout nucleic acid vaccines to improve vaccine efficacy and reduce the dosage.

[0003] Cyclic diguanylic acid (c-di-GMP) is a STING agonist that binds to STING proteins in cells, stimulating the release of various cytokines such as interferon, IL-6, and TNF-α. It can also regulate the body's immune response by activating typical inflammatory pathways such as NF-κB. Currently, c-di-GMP is widely used as an adjuvant in mammalian subunit vaccines and inactivated vaccines (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 are poikilothermic lower vertebrates, and their immune response system differs somewhat from that of mammals. Therefore, whether c-di-GMP has an immunostimulatory effect on rainbow trout or even fish remains to be investigated.

[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 helps to solve this problem. Currently, delivery vectors can be divided into liposome nanoparticles (LNPs), polymer vectors, protein vectors, viral vectors, etc., among which LNPs are the mainstream for nucleic acid delivery. Because fish farming is sensitive to vaccine costs, the high cost of LNPs is not suitable. Therefore, it is necessary to develop delivery vectors that are suitable for fish and have controllable costs.

[0005] Polydisulfide compounds (CPDs) are polymer carriers similar to polyarginine. Their positively charged guanidine groups promote the accumulation of the carrier near the cell surface. The disulfide backbone can promote the carrier's entry into the cell membrane through the disulfide exchange mechanism. After entering the cell, CPDs can be rapidly depolymerized under the catalysis of endogenous glutathione, releasing the cargo (GUO J, et al. ACS Central Science. 2021, 7: 990-1000.). Summary of the Invention

[0006] To address the aforementioned problems in the prior art, this invention provides a rainbow trout nucleic acid vaccine adjuvant and its application.

[0007] To achieve the above objectives, a first aspect of the present invention provides a rainbow trout nucleic acid vaccine adjuvant, characterized in that the adjuvant comprises a polydisulfide compound and cyclic diguanosine monophosphate delivered by the polydisulfide compound.

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

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

[0010] A second aspect of the invention provides the application of the aforementioned rainbow trout nucleic acid vaccine adjuvant in the preparation of a rainbow trout IHNV virus nucleic acid vaccine.

[0011] The beneficial effects of this invention are as follows: a rainbow trout nucleic acid vaccine adjuvant, namely the c-di-GMP+CPD complex, has been developed based on the STING agonist---c-di-GMP and the polydisulfide compound (CPD) delivery carrier, thereby achieving cost reduction and efficiency improvement of rainbow trout nucleic acid vaccines. The polydisulfide compound (CPD) can efficiently deliver cyclic diguanosine monophosphate and has good stability. The c-di-GMP+CPD complex has the potential to serve as an immune adjuvant for rainbow trout, which can significantly enhance the immunoprotective effect of rainbow trout nucleic acid vaccines and reduce the dosage of vaccines used. Attached Figure Description

[0012] Figure 1 A is a flowchart of the CPD synthesis process used in this invention; Figure 1 B and Figure 1 C represents the NMR and mass spectra of the CPD monomer synthesized in this invention; Figure 1 D、 Figure 1 E, Figure 1 F represents the average molecular weight, 1H NMR spectrum, and average molecular weight distribution curve of the CPD synthesized in this invention.

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

[0014] Figure 3 A and Figure 3 Figure B shows the results of immunofactor assays of the c-di-GMP+CPD complex in mouse L929 cells.

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

[0016] Figure 3 E and Figure 3 Figure F shows the stability results of the CPD+c-di-GMP complex.

[0017] Figure 4 The image shows the serum neutralizing antibody results 28 days after immunization with a DNA vaccine combined with (cdiG+CPD) adjuvant.

[0018] Figure 5 The image shows the results of an IHNV challenge test conducted 28 days after immunization with a DNA vaccine combined with (cdiG+CPD) adjuvant. Detailed Implementation

[0019] To more clearly describe the technical content of the present invention, the following description is provided in conjunction with specific embodiments.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the reagents and materials used in the following examples are commercially available.

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

[0022] IHNV isolate GS21 was isolated from diseased rainbow trout in Gansu Province. The rainbow trout DNA vaccine was designed and preserved by our research group (WU D, et al. Aquaculture. 2023, 572:739555, CN118903403A). Cyclic diguanylic acid (c-di-GMP) and polydisulfide compound (CPD) were synthesized in the laboratory. The pRL-TK plasmid and pGL3-IFN pro-Luc plasmid were both preserved in the laboratory. L929 cells and CHSE-214 cells were also preserved in the laboratory. The transfection reagent Lipofectamine was used.TM 3000 transfection reagent (L3000001) was purchased from Thermo Fisher Scientific; Dual luciferase reporter gene assay kit (RG029S) was purchased from Beyotime; Quantitative PCR kit MonAmpSYBR×green qPCR Mix (MQ10701S) was purchased from Mona.

[0023] Example 1: Development of Cyclic Diguanylate Delivery Vector

[0024] Polydisulfide compound synthesis and cell transfection

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

[0026] CPD monomer preparation

[0027] Lipoic acid 1 (4.12 g, 20 mmol) and CDI (3.24 g, 20 mmol) were added to anhydrous DMF (50 mL), and the reaction solution was stirred for 2 h at room temperature under nitrogen protection to activate lipoic acid. L-arginine methyl ester dihydrochloride (2.61 g, 10 mmol) and N,N-diisopropylethylamine (1.74 mL, 10 mmol) were added to anhydrous DMF (40 mL) solution and reacted for 1 h. Then, the activated lipoic acid solution was added, and the mixture was stirred for another 3.5 h at room temperature. Three volumes of diethyl ether were added to the reaction solution, and the mixture was centrifuged (2 min, 3000 rpm) to collect the oil. The oil was washed three times with a DCM / Et2O (1:2, 30 × 60 mL) mixture and dried under vacuum to obtain a yellow oily CPD monomer (4.22 g, yield 79.6%). The NMR spectrum (MS) and mass spectrum (1H NMR) of the CPD monomer are shown below. Figure 1 B Figure 1 As shown in C.

[0028] Preparation of CPD

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

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

[0031] c-di-GMP and CPD were separately prepared into aqueous solutions using enzyme-free water, with c-di-GMP at 40 μg / mL and CPD at different concentrations of 40, 80, 160, 240, 320, and 400 μg / mL. These solutions were mixed at mass ratios of 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. These complexes were then added in equal volumes to cell well plates.

[0032] Immune factor assay

[0033] 24 hours before transfection, L929 cells were seeded in 24-well plates. When the cells reached a confluence of 60-70%, they were transfected with the c-di-GMP+CPD complex and incubated at 37°C. Cell samples were collected 6 hours after transfection, and total RNA was extracted using an RNA extraction kit. IFN-β and IL-6 levels were measured using the MonAmp SYBR×green qPCR Mix kit.

[0034] like Figure 3 A and Figure 3As shown in Figure B, tests in mouse L929 cells revealed that CPD effectively delivered c-di-GMP into the cells, promoting the upregulation of IFN-β and IL-6 expression in mouse cells, and its delivery efficiency was significantly higher than that of the commercial transfection reagent Lipo3000 (Lp+cdiG group). Furthermore, the optimal mass ratio of CPD to c-di-GMP was 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℃, and -20℃, respectively, and the activation effect of immune factors was detected after 1 month, 2 months, and 4 months of storage. The same method was used for mouse L929 cells.

[0037] like Figure 3 As shown in E and 3F, tests using mouse L929 cells revealed that the CPD+c-di-GMP complex could be stably stored at 4°C for at least 120 days, and even after prolonged storage, it could still upregulate the expression of immune factors. Therefore, this complex has good application potential.

[0038] Example 2 Evaluation of cyclic diguanosine monophosphate in trout cells

[0039] Viral load changes

[0040] 24 hours before transfection, CHSE-214 cells were seeded in 24-well plates. When the cells reached 60-70% confluence, the medium in the plates was replaced with serum-free medium. The transfection system was prepared according to the transfection reagent instructions, and c-di-GMP cells were transfected. 12 hours after transfection, the medium was replaced with medium containing 10... 4 TCID50 IHNV culture medium was incubated statically at 15°C. Cell samples were collected 24 h after infection, and total RNA was extracted using an RNA extraction kit. Absolute fluorescence quantitative analysis of the IHNV N gene was performed using the MonAmp SYBR×greenqPCR Mix kit. Specific groupings are shown in Table 1 below.

[0041] Table 1

[0042]

[0043] Upregulation of I-IFN expression in fish

[0044] 24 hours before transfection, CHSE-214 cells were seeded in 24-well plates. When the cells reached 60-70% confluence, the medium in the plates was replaced with serum-free medium. The transfection system was prepared according to the transfection reagent instructions, and 0.5 μg of pGL3-IFN pro-Luc plasmid and 0.05 μg of pRL-TK plasmid were transfected. 24 hours after transfection, c-di-GMP was transfected using the same method, and the cells were incubated statically at 26°C. Cell samples were collected 24 hours after transfection, and the expression level of Fluc was detected using a dual-luciferase reporter gene assay kit. Specific groupings are shown in Table 2 below.

[0045] Table 2

[0046]

[0047] like Figure 2 As shown in Figure A, in CHSE-214 cells, c-di-GMP, delivered with a commercial transfection reagent, significantly inhibited the copying of IHNV virus in trout cells. Figure 2 As shown in Figure B, based on the dual luciferase reporter assay, c-di-GMP upregulates the expression of type I IFN in rainbow trout in CHSE-214 cells, thereby inhibiting the virus. Combining these results, it can be concluded that c-di-GMP has an immunostimulatory effect in trout cells and possesses the potential to serve as an adjuvant for rainbow trout vaccines.

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

[0049] Example 3: Evaluation of cyclic diguanosine monophosphate in fish

[0050] immunity

[0051] Rainbow trout weighing 5±0.1g were randomly divided into groups of 40 each. They were temporarily held in a circulating water tank (65cm×65cm×70cm) at a water temperature maintained at (16±1)℃. After 14 days of acclimation, they were vaccinated (injected at the base of the dorsal fin). They were fasted for 2 days before the experiment. The vaccine immunization groups are shown in Table 3 below. A blank control group (50μL / tail phosphate solution) was also set up.

[0052] Table 3

[0053] Group Vaccine representation vaccine components 1 control group PBS buffer 2 DNA 1μg 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 titers

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

[0056] Serum samples stored in a refrigerator were inactivated at 56°C for 30 min. The serum samples were then diluted 2-fold, from 1:20 to 1:40, 1:80, 1:160, and 1:320. These diluted samples were incubated with 100 TCID50 of IHNV at 15°C for 1 h. The incubated mixture was then added to monolayer-coated 96-well plates, with 8 replicates per gradient. Simultaneously, wells were set up for normal cell blank control, virus control, negative serum control, and positive serum control. The plates were incubated statically at 15°C for 7–10 days, observing for CPE. The highest serum dilution that protected half of the samples from infection was taken as the neutralizing antibody titer of that sample; a neutralizing antibody titer <20 was considered a negative sample.

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

[0058] Viral load changes

[0059] Fasting began 26 days post-immunization, and immune rainbow trout were challenged 28 days post-immunization with 100 μL L HNV (10 4 The TCID50 / mL solution was injected into the abdominal cavity of the fish. Spleen tissue was collected from rainbow trout (n=3) 7 days post-infection. Total RNA was extracted from the spleen using an RNA extraction kit, and absolute fluorescence quantitative analysis of the IHNV N gene was performed using the MonAmp SYBR×green qPCR Mix kit.

[0060] IHNV challenge test was performed 28 days after immunization. Figure 5The viral load in fish 7 days after challenge was shown. Compared to the DNA immunization group, the DNA+cdiG+CPD immunization group had a lower IHNV viral load. This indicates that the DNA vaccine combined with the (cdiG+CPD) adjuvant significantly reduced the viral load in fish, demonstrating that the c-di-GMP adjuvant enhanced the immunoprotective effect of the DNA vaccine. Based on these results, the c-di-GMP+CPD complex has the potential to serve as an adjuvant for rainbow trout immunization, significantly enhancing the immunoprotective effect of rainbow trout nucleic acid vaccines and reducing the required vaccine dosage.

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

Claims

1. A rainbow trout nucleic acid vaccine adjuvant, characterized in that, The adjuvant includes a polydisulfide compound and cyclic diguanosine acid delivered by the polydisulfide compound; The polydisulfide compound synthesis process is as follows: Preparation of CPD monomer: Lipoic acid 4.12 g, 20 mmol and CDI 3.24 g, 20 mmol are added in anhydrous DMF 50 mL, and the reaction solution is stirred at room temperature under nitrogen protection for 2 h to activate lipoic acid; L-arginine methyl ester dihydrochloride 2.61 g, 10 mmol and N,N-diisopropyl ethylamine 1.74 mL, 10 mmol are added in anhydrous DMF 40 ml solution and reacted for 1 h, then the activated lipoic acid solution is added and stirred at room temperature for 3.5 h; 3 times the volume of ether is added in the reaction solution, after mixing evenly, centrifugation for 2 min, 3000 rpm, the oil is collected, and the oil is washed with DCM / Et2O, 1:2, 30*60 ml mixture for 3 times, and dried under vacuum to obtain yellow oil CPD monomer; Preparation of CPD: The following solutions are prepared in advance before the reaction: TEOA buffer 1M, pH = 7.0, solvent H2O; initiator stock solution-SH, 5mM, solvent TEOA; CPD monomer solution, 1M, solvent TEOA; terminator stock solution iodoacetamide, 0.5M, solvent H2O; After the solution is configured, it is deoxygenated using nitrogen, and the treated solution is added according to the volume ratio of polymer monomer: initiator: DMF: TEOA = 1: 1: 1.5: 6.5, and the total system is 10 mL; during polymerization, the reaction is stirred vigorously under nitrogen protection; after 30 min of reaction, 20 mL of iodoacetamide termination solution 0.5M is added to the reaction solution to terminate the reaction, and the terminated reaction solution is loaded into a dialysis bag with a molecular weight cut-off of 2000 and then placed in deionized water to dialyze out the unreacted small molecules, and the dialysis solution is changed every 4 h, and after dialysis for two days, vacuum freeze-drying is performed to obtain white CPD powder.

2. The Oncorhynchus mykiss nucleic acid vaccine adjuvant of claim 1, wherein, The mass ratio of the polydisulfide compound and the cyclic diguanosine acid is 4:1 to 10:

1.

3. The Oncorhynchus mykiss nucleic acid vaccine adjuvant of claim 1, wherein, The rainbow trout nucleic acid vaccine is an IHNV virus-resistant rainbow trout DNA vaccine.

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

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