Composite adjuvant for aeromonas salmonicida inactivated vaccine, corresponding application and inactivated vaccine

Abstract

The invention relates to a composite adjuvant for an aeromonas salmonicida inactivated vaccine. The composite adjuvant comprises inulin and an aluminum adjuvant. The invention provides application of the composite adjuvant for the aeromonas salmonicida inactivated vaccine in fish vaccines and the inactivated vaccine. According to the composite adjuvant for the aeromonas salmonicida inactivated vaccine, the corresponding application and the inactivated vaccine, the natural polysaccharide inulin is selected as an immunologic stimulant and is combined with the aluminum adjuvant to prepare the aeromonas salmonicida inactivated vaccine, the composite adjuvant based on the aluminum adjuvant and the inulin has good application potential in turbots, and the composite adjuvant can be used for preparing the aeromonas salmonicida inactivated vaccine. The formalin inactivated vaccine containing the composite adjuvant shows a good protection effect. In addition, the vaccine can significantly enhance innate immune response, thereby enhancing humoral immunity and cellular immunity. Therefore, the composite adjuvant composed of the aluminum adjuvant and the inulin can be used as a candidate adjuvant for developing more fish vaccines.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a composite adjuvant for an inactivated vaccine of Aeromonas salmonicida and corresponding applications, and an inactivated vaccine. Background Art

[0002] Turbot (Scophthalmus maximus) is a marine fish with high economic value, widely distributed in the North Atlantic coast and the Mediterranean region. Artificial breeding of turbot is widely carried out in China, Europe and other places. The breeding cycle of this fish is short, the growth rate is fast, and the adaptability is strong, which is suitable for large-scale breeding. However, under conditions of intensive breeding or poor water quality management, turbot is prone to various diseases during the breeding process, such as vibriosis and salmonicidal Aeromonas infection. Farms usually adopt strict water quality management, isolation of sick fish, use of antibiotics, and development of vaccines.

[0003] Fishery vaccines have made significant progress in recent years, especially in the prevention of bacterial and viral diseases. Traditional inactivated vaccines and live vaccines have been widely used in aquaculture, especially for common bacterial diseases such as vibriosis and Edwardsiella, and mature vaccine products have been developed. For example, the multivalent bacterial inactivated vaccine for turbot can significantly reduce the mortality rate of fish.

[0004] Aluminum adjuvants are a common immunopotentiator in vaccines and are widely used to improve the immune efficacy of vaccines. Aluminum adjuvants are usually used in inactivated vaccines and subunit vaccines, because these vaccines usually require external adjuvants to enhance the immune response. Aluminum adjuvants have been verified for a long time in veterinary and human vaccines and have high safety.

[0005] Inulin, as a natural polysaccharide, not only has good biocompatibility, but also plays an important role in immune enhancement by regulating the immune system. Inulin can stimulate local immune cells such as macrophages and T cells, thereby enhancing local immune defense and helping to resist the invasion of foreign pathogens. Inulin can also reduce the inflammatory response that may be triggered by vaccination by regulating the activity of immune cells. Advax TM Advax is a novel polysaccharide adjuvant based on semi-crystalline delta inulin particles. TM Enhanced vaccine immunogenicity and protection in a range of animal models, including Japanese encephalitis. Although its exact mechanism of action has not been determined, Advax TM The particles bind directly to human monocytes and enhance their co-stimulatory function. Summary of the invention

[0006] In order to solve the above problems in the prior art, the present invention provides a composite adjuvant for an inactivated vaccine of Aeromonas salmonicida and corresponding applications, and an inactivated vaccine.

[0007] In order to achieve the above-mentioned object, the first aspect of the present invention provides a composite adjuvant for an inactivated vaccine of Aeromonas salmonicida, which mainly comprises inulin and aluminum adjuvant.

[0008] The second aspect of the present invention provides the use of the composite adjuvant for the inactivated Aeromonas salmonicida vaccine in fish vaccines.

[0009] The third aspect of the present invention provides an inactivated vaccine of Aeromonas salmonicidae for turbot, the main feature of which is that it comprises the composite adjuvant.

[0010] The composite adjuvant for the inactivated vaccine of Aeromonas salmonids of the present invention and the corresponding application and inactivated vaccine select natural polysaccharide inulin as an immunostimulant, and use it in combination with an aluminum adjuvant to prepare the inactivated vaccine of Aeromonas salmonids. Based on the good application potential of the composite adjuvant of aluminum adjuvant and inulin in turbot, the formalin inactivated vaccine containing the composite adjuvant shows a good protective effect. In addition, the vaccine can significantly enhance the innate immune response, thereby enhancing humoral immunity and cellular immunity. Therefore, the composite adjuvant composed of aluminum adjuvant and inulin can be used as a candidate adjuvant for developing more fish vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a graph showing the survival rate of turbot after inoculation in Example 1.

[0012] Figure 2 This is a graph showing the results of serum specific antibody levels after inoculation with Aeromonas salmonicida in Example 2.

[0013] Figure 3 This is the result diagram of serum total protein level in Example 3.

[0014] Figure 4 This is a graph showing the results of serum lysozyme activity in Example 4.

[0015] Figure 5 A to Figure 5 I is the gene transcription level in spleen tissue of turbot after immunization in Example 5.

[0016] Figure 6 A to Figure 6 C is a graph showing the results of the compound adjuvant Al+I in Example 6 promoting the recruitment of neutrophils at the injection site of zebrafish.

[0017] Figure 7 A to Figure 7 C is a graph showing the results of the composite adjuvant Al+I in Example 6 promoting the recruitment of macrophages at the injection site of zebrafish. DETAILED DESCRIPTION

[0019] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments.

[0020] Inulin, as a natural immune adjuvant, has been used in the research and development of a variety of vaccines. By combining inulin with antigens, the immunogenicity and antibody production of the antigen can be improved. Compared with traditional chemical adjuvants, inulin has lower side effects and better safety. In the present invention, a dose of 10 mg / mL inulin (1 mg per dose) is used as the vaccine formulation. Aluminum adjuvants, as an adjuvant widely used in human vaccines, are rarely used in fishery vaccines. Oil-emulsion adjuvants widely used in fishery vaccines have side effects such as tissue adhesion and granulomatous lesions at the injection site. Vaccines containing a composite adjuvant of aluminum adjuvant + inulin can be used as a new type of water-based vaccine to provide new ideas for disease prevention and treatment.

[0021] Fish rearing conditions in the embodiments of the present invention: Turbot (32.0 g ± 4.0 g) for laboratory evaluation was purchased from a commercial farm in Yantai, China, and the tissue was ground after dissection and examined for bacterial contamination. Healthy turbot was reared in a circulating aerated seawater system (Haisheng, China) with a salinity of 2% ± 0.4%, a temperature of 16.0 ° C ± 1.5 ° C, and a density of about 6 kg / m 3 The fish were fed with commercial fish feed every day during the breeding period and stopped feeding one week before the start of the experiment. All procedures involving fish were in accordance with the policy of the Animal Ethics Committee of East China University of Science and Technology. The fish used for sampling were anesthetized with an appropriate amount of MS-222.

[0022] In the embodiments of the present invention, Graphpad Prism or IBM SPSS software is used for statistical analysis, and two-way analysis of variance or non-parametric test is used to analyze statistical significance.

[0023] Example 1 Effect of the composite adjuvant of the present invention on the protective effect of vaccines

[0024] Vaccine preparation

[0025] The Aeromonas salmonicida used in this study was isolated from turbot infected with granulomatous dermatitis in a fishery and stored in a −80 °C freezer in the laboratory.

[0026] The stored bacterial liquid was streaked on TSA for activation for 24 hours, and then a single colony was picked and cultured on TSB at 22°C for 24 hours. Formaldehyde solution with a final concentration of 0.2% (vol / vol) was added to inactivate the bacteria. After inactivation for 24 hours, the bacteria were streaked on TSA plates to check the inactivation of bacteria. After centrifugation at 8000x g for 5 minutes, the supernatant was discarded and the inactivated cells were resuspended in PBS (FKC).

[0027] Then, an appropriate amount of completely inactivated cells was mixed with Al+I adjuvant to form a formula, with each vaccine group containing 5×10 9 cells / mL FKC (formalin killed cells). Among them, the FKC vaccine contains only FKC inactivated cells and does not contain any adjuvants; the Al+I vaccine contains FKC inactivated cells + Alum + Inu composite adjuvant, the Alum concentration is 250μg / mL (Alhydrogel; InvivoGen, America), the Inu (inulin) concentration is 10mg / mL, and the various components are thoroughly mixed by shaking. The Al vaccine contains FKC inactivated cells + Alum, and the Alum concentration is the same as above. The I vaccine contains FKC inactivated cells + Inu, and the Inu concentration is the same as above.

[0028] Immunization, sampling and challenge

[0029] The fish were divided into 3 groups (45 in each group), namely PBS group, FKC group, and Al+I group. The fish in the PBS group were intraperitoneally injected with 100 μL PBS; the fish in the Al+I group were intraperitoneally injected with 100 μL Al+I vaccine; the fish in the FKC group were intraperitoneally injected with 100 μL FKC vaccine. The fish in the Al group were intraperitoneally injected with 100 μL Al vaccine; and the fish in the I group were intraperitoneally injected with 100 μL I vaccine. The experiment was performed in duplicate.

[0030] Three fish were randomly selected from each group for sampling. Peripheral blood was collected at 7, 14, 21, and 28 days after immunization. After standing at 4°C overnight, serum was collected after centrifugation at 4000 rpm for 10 minutes and stored at -20°C for later use. At the same time, spleens of three fish were collected. The tissues were immersed in RNAstore (Tiangen, China) and stored at -80°C. Before sampling, turbot was immersed in a solution containing an appropriate concentration of tricaine-methanesulfonate (MS-222; Aladdin, China) for anesthesia and euthanasia.

[0031] 28 days after inoculation, each group of fish was injected intramuscularly with 1×10 5 CFU / strip Aeromonas salmonicida. The cumulative mortality of each group after challenge was recorded every day to determine the protective effect of Al+I adjuvant on the vaccine.

[0032] The calculation function of RPS is:

[0033]

[0034] RPS after bacterial infection is an important indicator reflecting the effectiveness of the vaccine. Figure 1The cumulative mortality of each group is shown. In general, the survival rate of vaccinated fish after the challenge was increased compared with the control group. The results of mortality and RPS after the challenge experiment are shown in Table 1 below, where the mortality of the PBS group was 86.7%, the mortality of the FKC group was 63.3%, equivalent to an RPS of 26.9%, the RPS of the Al and I groups were 20.0% and 32.0%, respectively, and the mortality of the vaccine group containing Al+I adjuvant was 33.3%, equivalent to an RPS of 61.5%.

[0035] The results showed that the complex of aluminum adjuvant and inulin had good FKC adjuvant function, protecting turbot from infection with Aeromonas salmonicida, and the immune protection effect was greater than that of single components Al and I.

[0036] Table 1 Mortality and RPS after challenge experiment

[0037]

[0038] Example 2

[0039] Specific antibody detection

[0040] The specific IgM antibody titer of the turbot serum obtained in Example 1 against Aeromonas salmonicida was determined by enzyme-linked immunosorbent assay (ELISA).

[0041] In a high-binding microplate (Corning, USA), 1.0×10 8 CFU / mL of A.salmonicida (100 μL / well) were added and incubated overnight at 4°C. The wells were washed with PBST (PBS containing 0.05% Tween 20) and blocked with PBSTA (PBST containing 1% BSA). Turbot serum was diluted and incubated at room temperature for 3 hours at 100 μL / well. Each sample was evaluated in triplicate. Then the wells were washed three times with 200 μL / well PBST, and 100 μL / well mouse anti-turbot IgM (Aquatic Diagnostics Ltd, british, diluted 1:40 with PBSTA) was added and incubated at room temperature for 1 hour. Wash once more and incubate with 100 μL / well HRP-conjugated goat anti-mouse IgG (Abgent, USA, diluted 1:5000 in PBSTA) for 1 hour. Finally, wash three times, develop color with 100 μL / well TMB at 37°C in the dark for 10 min, stop color development with 50 μL / well H2SO4 (2M), and read the optical density (OD) at 450 nm using an analyzer.

[0042] like Figure 2As shown in the figure, specific IgM antibodies were significantly increased in the vaccination group containing Al+I composite adjuvant at each sampling time point from week 1 to week 4. Interestingly, the serum specific antibody level of the FKC group showed an early high level on day 14, and the increase was slowing down on days 21 and 28. However, the Al+I group still showed a high level on day 21 and continued to maintain this high level on day 28.

[0043] The results showed that the vaccine containing the composite adjuvant Al+I could induce the production of high levels of specific antibodies in the serum of vaccinated turbot.

[0044] Example 3

[0045] Serum total protein

[0046] The total serum protein obtained in Example 1 was determined by biuret reaction using an enhanced BCA protein assay kit (Beyotime, China).

[0047] Prepare an appropriate amount of BCA working solution by adding 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1) and mix thoroughly. Mix 200 μL of BCA working solution with 20 μL of serum sample or standard solution. Incubate at 37°C for 20 minutes and read the optical density (OD) at 562 nm using a microplate.

[0048] Calculate total serum protein according to the formula.

[0049]

[0050] like Figure 3 As shown in the figure, compared with the control group, the total serum protein in the FKC and Al+I adjuvant groups increased significantly after vaccination. In the first 3 weeks, the total serum protein level of turbot in the Al+I group was the highest. And the high level of total serum protein content was still maintained three days after the virus attack. Interestingly, the total serum protein level in the FKC group showed a delayed high level on the 28th day after vaccination. This shows that vaccines containing different adjuvants can more effectively stimulate the innate immune response, and the Al+I composite adjuvant has shown significant effects.

[0051] Example 4

[0052] Lysozyme activity

[0053] The serum obtained in Example 1 was co-incubated with Micrococcus lysodeikticus to determine the activity of lysozyme in the fish serum after inoculation. Specifically, the Micrococcus lysodeikticus was adjusted to OD 570 = 0.5 suspension, take 200 μL suspension and mix with 10 μL serum, shake gently at room temperature and incubate. Read OD every 2 min using a microplate. 570 , through OD 570The slope was calculated over time and the relative activity was calculated by normalization.

[0054] like Figure 4 As shown in the figure, the lysozyme activity of turbot inoculated with Al+I was significantly higher than that of the control group on the 21st day, and the lysozyme activity of the Al+I group also increased significantly three days after the challenge. This also shows that vaccines containing Al+I composite adjuvants can induce a rapid innate immune response.

[0055] Example 5

[0056] Detection of immune-related gene expression

[0057] The method for extracting tissue RNA is as follows:

[0058] (1) The frozen tissue in Example 1 was taken out and thawed, and the preservation solution on the tissue was removed as much as possible with absorbent paper, and then transferred to a clean tissue grinding tube, 1 ml of Trizol was added, and the tissue was fully ground with a high-speed tissue grinder, and allowed to stand at room temperature for 3 minutes;

[0059] (2) Transfer the grinding solution to a clean RNase-free EP tube, add 200 μl of chloroform, vortex for 10 seconds, let stand at room temperature for 3 minutes, and centrifuge at 4°C and 12,000 × g for 15 minutes;

[0060] (3) Carefully pipette 450 μl of supernatant into a new RNase-free EP tube, add 450 μl of isopropanol, mix by inversion, and centrifuge at 4°C, 10,000 × g for 10 min.

[0061] (4) Discard the supernatant, add 1 ml of 75% ethanol (prepared with RNase-free water), invert several times to thoroughly wash the precipitate, and centrifuge at 4°C and 10,000 × g for 5 min;

[0062] (5) Discard the supernatant and let the solution stand at room temperature to allow the liquid to evaporate completely. Dissolve the precipitate with an appropriate amount of RNase-free water and measure the RNA concentration using a Nanodrop instrument.

[0063] Table 2 qPCR primer list

[0064]

[0065] Use the reverse transcription kit FastKing RT Kit for reverse transcription, and the operation steps are the same as the instructions. Take 1μg of total RNA and mix it with gDNase, incubate at 42℃ for 3min to remove genomic DNA; add FastKing RT Enzyme Mix and FQ-RTPrimer, mix well, incubate at 42℃ for 15min, and incubate at 95℃ for 3min to inactivate the reverse transcriptase. Dilute the final cDNA reaction solution (20μl) with 80μl DNase / RNasefree water and store at -20℃ until the next reaction.

[0066] qPCR was performed according to the method recommended in the SYBR green real-time PCR mix manual. Before qPCR, a melting curve was established to confirm the appearance of specific amplification peaks and the absence of primer-dimer formation. The primers for each gene are listed in Table 2. The specificity of each primer pair was verified by NCBI. β-actin was used as the internal reference gene, and the cycle threshold method (2 -ΔΔCt The relative transcription level of each immune-related gene was determined by PCR.

[0067] RT-qPCR was used to detect the transcription levels of inflammatory factors and antigen presentation-related genes in the spleen of turbot after immunization. Figure 5 As shown, the antigen presentation-related genes MHC-I and MHC-II in the compound adjuvant Al+I group reached a peak at 28 days of immunization, and CD4 and CD8 increased significantly at 14 and 28 days of immunization. In addition, IL-1β and IFN-γ were significantly upregulated after 28 days of immunization, IL-12 was significantly upregulated after 28 days of immunization, and TNF-α was significantly upregulated at 7 days and 28 days of immunization. These results show that the Al+I compound adjuvant inactivated vaccine has a good effect on stimulating the cellular immune response of turbot.

[0068] Example 6

[0069] Al+I adjuvant can promote the recruitment of macrophages and neutrophils in zebrafish

[0070] Neutrophils are usually the first responders to acute inflammatory responses, and they play an important role in fish immunity by producing cytokines, regulating the activity of neighboring cells, etc., which promotes the resolution of inflammation and regulates long-term immune responses.

[0071] Macrophages play a vital role in innate immunity and are the body's first line of defense when facing pathogen invasion. Macrophages identify and eliminate pathogens through a variety of mechanisms to prevent the spread of infection.

[0072] (1) The night before, place the neutrophil and macrophage labeled zebrafish male and female broodstock into a breeding tank and separate them with partitions to keep them in a dark environment overnight;

[0073] (2) The next morning, the partition was removed and light stimulation was turned on to allow the zebrafish to mate and lay eggs. After spawning, the eggs were collected using a filter and transferred to fresh culture medium, a small amount of methylene blue was added, and the culture medium was incubated at 28°C. The culture medium was changed every day and dead eggs were removed.

[0074] (3) The larvae were cultured until the third day after hatching, and then transferred to a culture medium containing 200 μg / ml MS-222. After the zebrafish larvae lost their mobility, they were transferred to a 1% agarose gel plate;

[0075] (4) Take 10 μl of the compound adjuvant and transfer it to the microinjection needle. Use the microinjector to inject 1 nl of the adjuvant into the cochlea of ​​the zebrafish larvae.

[0076] (5) After injection, the zebrafish fry were immediately transferred to new culture medium and cultured at 28°C for 3 h and 12 h before fluorescence observation.

[0077] like Figure 6 A to Figure 7 As shown in C, 3 hours after microinjection, significant neutrophil and macrophage aggregation occurred in the cochlea of ​​zebrafish in the Al+I group. Fluorescence quantification of the zebrafish cochlea showed that 6 hours after injection, the number of neutrophils and macrophages in the Al+I group was the highest, while there was basically no obvious neutrophil and macrophage aggregation in the cochlea of ​​the PBS group. These results indicate that the Al+I adjuvant may increase the recruitment of neutrophils and macrophages after vaccine immunization, further triggering innate and adaptive immune responses.

[0078] In summary, the composite adjuvant based on aluminum adjuvant and inulin has good application potential in turbot. The formalin-inactivated vaccine containing the composite adjuvant showed good protection. In addition, the vaccine can significantly enhance the innate immune response, thereby enhancing humoral immunity and cellular immunity. Therefore, the composite adjuvant composed of aluminum adjuvant and inulin can indeed be used as a candidate adjuvant for the development of more fish vaccines.

[0079] 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 composite adjuvant for an inactivated vaccine of Aeromonas salmonicida, characterized in that: Includes inulin and aluminum adjuvants.

2. Use of the composite adjuvant for the inactivated Aeromonas salmonicida vaccine according to claim 1 in fish vaccines.

3. An inactivated vaccine of Aeromonas salmonicida for turbot, characterized in that: Comprising the composite adjuvant according to claim 1.

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