Application of SpaA protein as immunopotentiator in preparation of vaccine for preventing viral subunit

Abstract

The invention provides an application of SpaA protein as an immunopotentiator in preparation of a vaccine for preventing viral subunits, the SpaA protein is swine erysipelas filamentous bacillus SpaA protein, and the viral subunit vaccine is a vaccine prepared from a virus shell special protein, namely an antigenic determinant. According to the swine erysipelas filamentous bacillus SpaA protein antigen and viral subunit protein combined vaccine serving as the immunopotentiator, the duration time of the blocking rate of the viral subunit protein can be remarkably prolonged or the neutralizing antibody and duration time of the viral subunit protein can be remarkably prolonged, and the onset time can also be shortened.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of veterinary biological products, and particularly relates to the use of SpaA protein as an immune enhancer in the preparation of a virus subunit vaccine for preventing porcine pestivirus, porcine pseudorabies virus and porcine circovirus. BACKGROUND

[0002] A vaccine adjuvant is a non-specific immune enhancer capable of promoting antigen-specific immune responses by enhancing and changing the type of immune response, which can generally reduce the antigen dosage and the number of immunizations, produce a more rapid and persistent immune response, and improve the intensity and effectiveness of the immune response of the vaccine.

[0003] A subunit vaccine is a vaccine prepared by extracting special protein structures of bacteria and viruses through chemical decomposition or controlled proteolysis, and screening out immunologically active fragments. The subunit vaccine is a vaccine prepared by the main protective immunogen of pathogenic bacteria, also known as a component vaccine. The disadvantage of the subunit vaccine is lower immunogenicity, and it needs to be used with an adjuvant to produce a good immune effect.

[0004] Swine erysipelas is an anaerobic bacterium. It was first isolated by Koch in 1878. In 1886, Loeffler identified it as the pathogen of swine erysipelas disease. In 1909, Rosenbach also isolated the bacterium from patients with local skin diseases in humans, thereby confirming that it is also a human pathogen. Therefore, swine erysipelas bacillus is a pathogen of swine erysipelas, a zoonosis. The main host of swine erysipelas bacillus is generally domestic pigs, but it can also infect other birds and rodents. Although fish infection does not appear clear known symptoms, swine erysipelas bacillus can survive in the mucus on the surface of fish for a long time, that is, fish can carry swine erysipelas bacillus for a long time, which greatly increases the risk of human infection with swine erysipelas bacillus.

[0005] Recent studies have found that several surface proteins with relative molecular masses of 64, 66 and 43 kda in swine erysipelas bacillus have immunoprotective effects. The Makino research group cloned and expressed a surface protein with a size of 64 kda, which was named surface protective antigen a, i.e., swine erysipelas SpaA protein. After multiple research groups studied the immune function of SpaA protein, it was determined that SpaA protein has good immunoprotective effect.

[0006] Swine fever is caused by classical swine fever virus (CSFV), which is a highly contagious and destructive infectious disease, also known as swine cholera, with a mortality rate of up to 90%. The main symptoms are high fever, skin bleeding, general weakness, anorexia, and conjunctivitis. Swine pseudorabies is a viral infectious disease caused by porcine pseudorabies virus (PRV). PRV is a double-stranded DNA herpes virus that can cause abortion in sows, stillbirths, mummies, and neurological symptoms in piglets, with a mortality rate of up to 70%. Porcine circovirus type 2 (PCV2) is the main pathogen causing post-weaning multisystemic wasting syndrome, which can cause reproductive disorders in sows and cause significant losses to the pig industry. Swine erysipelas is an infectious disease caused by Erysipelothrix rhusiopathiae, which is common in humans and animals. The main characteristics are high fever, acute septicemia, purple diamond-shaped rash on the skin, endocarditis, and arthritis with long course and swelling of the limbs. Clinically, CSFV, PRV, and PCV2 are prone to mixed infection, which is a common cause of PRDC.

[0007] Currently, CSFV, PRV, PCV2, and swine erysipelas vaccines have been applied in clinical immunization and have shown good preventive effects. However, frequent immunization not only increases labor costs but also easily causes multiple stress in pig herds, leading to slow growth of piglets. Therefore, research on different mixed vaccines has gradually increased. Lin Dexie et al. (Lin Dexie et al. Evaluation of the immune effect of several immunization methods for swine fever and pseudorabies live vaccines [J]. China Animal Health) mixed PRV vaccine and CSFV vaccine, which had little interference with each other and did not affect the production of their respective immune antibodies. Zhu Zijian et al. (Zhu Zijian et al. Evaluation of the immune effect of simultaneous vaccination with porcine circovirus type 2 inactivated vaccine and swine fever rabbitized attenuated vaccine [J]. Chinese Journal of Preventive Veterinary Medicine) mixed PCV2 and CSFV vaccine, which showed that PCV2 and CSFV mixed immunization could produce good immune protection effect. Tian Xin et al. (Tian Xin et al. Evaluation of the immune effect of mixed immunization of porcine circovirus type 2 inactivated vaccine with swine fever live vaccine and porcine pseudorabies live vaccine [J]. China Journal of Animal Infectious Diseases) mixed PCV2 inactivated vaccine with CSFV live vaccine and PRV live vaccine, which showed that mixed immunization did not affect the immune efficacy of each vaccine. Chinese patent CN 113058032 B reported that the expression of CSFV E2 protein, PRV gD protein, and PRV gB protein in CHO cells and the expression of cap protein in insect baculovirus could prevent swine fever, swine pseudorabies, and porcine circovirus after mixed immunization.

[0008] Since multiple virus co-infection is common in clinic, a single vaccine is difficult to control the occurrence of infection, therefore, it is urgent to develop a multi-vaccine to prevent viral infection of piglets. SUMMARY

[0009] In order to develop a safe and effective multi-vaccine, the inventors unexpectedly found that the subunit protein SpaA of swine erysipelas and the viral subunit protein combined vaccine can significantly improve the duration of the blocking rate of the viral subunit protein, or improve the duration of the neutralizing antibody of the viral subunit protein, and also can shorten the onset time.

[0010] Therefore, the present application provides the use of SpaA protein as an immune enhancer in the preparation of a viral subunit vaccine for preventing viral subunit vaccine, the SpaA protein is the SpaA protein of swine erysipelas, and the viral subunit vaccine is a viral subunit protein composition. Here, the viral subunit vaccine is a vaccine containing viral coat specific proteins, i.e. antigenic determinants.

[0011] In a preferred technical scheme of the use of the present application, preferably, the SpaA protein is a prokaryotic expressed swine erysipelas SpaA protein, and its amino acid sequence is shown in SEQ No. 1.

[0012] In a preferred technical scheme of the use of the present application, preferably, the viral subunit protein composition comprises viral subunit protein and pharmaceutically acceptable adjuvant.

[0013] In a preferred technical scheme of the use of the present application, preferably, the viral subunit protein is at least selected from one of the following: subunit E2 protein of swine fever virus, gD protein of porcine pseudorabies virus, gB protein of porcine pseudorabies virus, cap protein of porcine circovirus type 2, S protein of porcine epidemic diarrhea virus, S protein of porcine delta coronavirus, VP8 protein of porcine rotavirus.

[0014] In a preferred technical scheme of the use of the present application, preferably, the amino acid sequence of the subunit E2 protein of swine fever virus is shown in SEQ No. 2, the amino acid sequence of the gD protein of porcine pseudorabies virus is shown in SEQ No. 3, the amino acid sequence of the gB protein of porcine pseudorabies virus is shown in SEQ No. 4, the amino acid sequence of the cap protein of porcine circovirus type 2 is shown in SEQ No. 5, the amino acid sequence of the S protein of porcine epidemic diarrhea virus is shown in SEQ No. 6, the amino acid sequence of the S protein of porcine delta coronavirus is shown in SEQ No. 7, and the amino acid sequence of the VP8 protein of porcine rotavirus is shown in SEQ No. 8.

[0015] In a preferred technical solution of the use of the present application, preferably, the pharmaceutically acceptable adjuvant is a water-in-oil-in-water double-phase emulsion adjuvant to form an oil phase. More preferably, the pharmaceutically acceptable adjuvant is ISA 201 VG.

[0016] In a preferred technical solution of the use of the present application, preferably, the mass ratio of the porcine Streptococcus suis SpaA protein to the viral subunit protein is 1:1-2.

[0017] In a preferred technical solution of the use of the present application, preferably, the protein concentration of the porcine Streptococcus suis SpaA protein and the viral subunit protein is 15 μg / portion-75 μg / portion.

[0018] In a preferred technical solution of the use of the present application, preferably, the viral subunit protein further comprises sterile PBS to form an aqueous phase.

[0019] In a preferred technical solution of the use of the present application, preferably, the weight ratio of the combination of the SpaA protein and the viral subunit protein and sterile PBS to the pharmaceutically acceptable adjuvant is 1:1, or the volume ratio of the combination of the SpaA protein and the viral subunit protein and sterile PBS to the pharmaceutically acceptable adjuvant is 46:54.

[0020] The porcine Streptococcus suis SpaA protein antigen as an immune enhancer in the combination vaccine of the present application and the viral subunit protein can significantly improve the duration of the blocking rate of the viral subunit protein or improve the neutralizing antibody and duration of the viral subunit protein, and also shorten the onset time. DETAILED DESCRIPTION

[0021] The present application will be further described below in combination with examples, and the examples of the present application are only used to illustrate the technical solutions of the present application, and not to limit the present application.

[0022] Example 1: Preparation of the subunit E2 protein of porcine pestivirus, the gD protein of porcine pseudorabies virus, the gB protein of porcine pseudorabies virus, the cap protein of porcine circovirus type 2, the S protein of porcine epidemic diarrhea virus and porcine delta coronavirus S protein, the VP8 protein of porcine rotavirus and the SpaA protein of porcine Streptococcus suis.

[0023] 1.1 Preparation of the subunit E2 protein of porcine pestivirus: referring to the preparation method and application of a porcine pestivirus recombinant subunit vaccine in the patent application with the publication number CN104826100A of the present applicant.

[0024] 1.2 Preparation of porcine pseudorabies virus gD and porcine pseudorabies virus gB protein: refer to the preparation method of porcine pseudorabies virus gD protein and application of porcine pseudorabies virus subunit vaccine in the invention application with publication number CN109206491A of the present applicant and the preparation method and application of a porcine pseudorabies virus gB protein in the invention application with publication number CN112142827A of the present applicant.

[0025] 1.3 Porcine circovirus type 2 cap protein: refer to the purification of a porcine circovirus type 2 virus-like particle and the preparation method of a vaccine thereof in the invention application with publication number CN104873966B of the present applicant.

[0026] 1.4 Preparation of porcine epidemic diarrhea virus S protein: refer to the porcine epidemic diarrhea virus S protein and subunit vaccine thereof and the preparation method and application thereof in the invention application with application number 201810310540.8 of the present applicant.

[0027] 1.5 Preparation of porcine delta coronavirus S protein: refer to the preparation method and application of a porcine pseudorabies virus gB protein in the invention application with publication number CN109206491A of the present applicant and the invention application with publication number CN112142827A of the present applicant.

[0028] 1.6 Porcine rotavirus VP8 protein: refer to the preparation method in the article Construction and characterization of human rotavirus recombinant VP8* subunit parenteral vaccine candidates by Wen X, Cao D, Jones RW, Li J, Szu S, Hoshino Y. et al. on Vaccine 2012, 30:6121-6.

[0029] 1.7 Swine erysipelas SpaA protein: refer to the preparation method in Truncated Surface Protective Antigen (SpaA) of Erysipelothrix rhusiopathiae Serotype 1a Elicits Protection against Challenge with Serotypes 1a and 2b in Pigs, published by Imada Y, Goji N, Ishikawa H, Kishima M, Sekizaki T. et al. in Infect Immun 1999; 67: 4376-82. The mass ratio of the swine erysipelas SpaA protein to the viral subunit protein is 1:1-2.

[0030] Example 2: Preparation of the subunit vaccine composition: the consumables and materials used for preparing the vaccine are subjected to sterilization in advance, and the preparation process is completed in a biological safety cabinet or other instruments or environments that can ensure the entire preparation process is sterile.

[0031] 2.1 Preparation of the water phase: the antigen proteins (i.e., viral subunit proteins) prepared in Example 1 and the swine erysipelas SpaA protein are mixed and diluted with sterile PBS at a mass ratio of 1:1 to obtain a water phase that meets the antigen protein content requirements. Here, the swine erysipelas SpaA protein and the viral protein have a high concentration, and are diluted with PBS to a final concentration of 15 μg-75 μg.

[0032] 2.2 Sterilization or sterilization of the water-in-oil-in-water double emulsion adjuvant to obtain an oil phase; the water-in-oil-in-water double emulsion adjuvant is ISA 201 VG.

[0033] 2.3 Preheating of the water phase and the oil phase, and then mixing and emulsifying the water phase and the oil phase to obtain an emulsified liquid. The weight ratio of the water phase to the oil phase is 1:1, or the volume ratio of the water phase to the oil phase is 46:54.

[0034] 2.4 Quantitative dispensing of the emulsified liquid to obtain a subunit vaccine, and labeling.

[0035] Example 3: Subunit vaccine antibody detection and neutralization test.

[0036] 3.1 Screening of test animals: 30-day-old weaned piglets that were negative for antibodies against swine fever, porcine pseudorabies, porcine circovirus type 2, and swine erysipelas antigens were selected, and a total of 12 piglets were obtained.

[0037] 3.2 Grouping of test animals: The 12 weaned piglets were randomly divided into 3 groups, with 4 piglets in each group. Group 1 was immunized with a mixture of E2 and SpaA antigens; group 2 was immunized with E2 antigen; and group 3 was not immunized and served as a control.

[0038] 3.3 Immunization: The piglets in group 1 were inoculated with the 202311a batch of vaccine in the neck muscle, the piglets in group 2 were inoculated with the 202311b batch of vaccine in the neck muscle, and the piglets in group 3 were not immunized and served as a control. The piglets were raised in the same conditions. The piglets were immunized twice, 21 days after the first immunization. The antibody titers were tracked at 14 days, 21 days, 28 days, 1 month, 2 months, 3 months, and 5 months after the first immunization. The vaccine formulation table is shown in Table 1.

[0039] Table 1: Vaccine formulation table

[0040] 3.4 Detection of swine fever blocking ELISA antibodies: The antibody titers were detected at 14 days, 21 days, 28 days, 2 months, 3 months, and 5 months after the first immunization according to the operating instructions of the IDEXX kit (item number). The determination criteria of the kit are as follows: the blocking rate of the detected sample is greater than or equal to 40%, which is determined as positive; the blocking rate of the detected sample is less than or equal to 30%, which is determined as negative; and the blocking rate of the detected sample is between 30% and 40%, which is determined as suspicious. The antibody titers of the 202311a batch of vaccine showed an upward trend 21 days after the first immunization, all turned positive, and the overall titers were good and lasted for 5 months after the immunization, with a blocking rate of more than 70%. However, the antibody titers of the 202311b batch of vaccine were still suspicious 21 days after the first immunization, not all turned positive, and lasted for 5 months after the immunization, with some antibodies turning weakly positive. The blocking rates of the groups are shown in Table 2.

[0041] Table 2: Antibody titers of blocking ELISA after immunization with the 202311a / b batch of vaccine

[0042] According to Examples 2 and 3, experiments of SpaA and gB, gD, SpaA and Cap, E2, etc. combinations were also performed, and it was found that, compared with no SpaA, the swine erysipelas filamentous bacterium SpaA protein antigen combined with viral subunit proteins (gB, gD, SpaA and Cap, SpaA, or Cap) could improve the porcine pseudorabies virus gD protein, porcine pseudorabies virus gB protein neutralizing antibody, improve the porcine circovirus antibody level, and improve the swine fever blocking rate and duration.

[0043] Example 4: Antibody detection and neutralization experiment of subunit vaccine composition immunization.

[0044] 4.1 Selection of test animals: 30 piglets of about 30 days old, which are negative for antibodies against CSF, PR, PCV2, and Erysipelas antigens, are selected, in total 30.

[0045] 4.2 Grouping of test animals: 10 weaned piglets are randomly divided into 2 groups, 5 in each group, group 1 is the mixed group of immunized gD, gB, and SpaA antigens; group 2 is the mixed group of immunized gD and gB antigens. 20 weaned piglets are randomly divided into 4 groups, 5 in each group, groups 1-3 are the immunized groups; group 4 is not immunized as the control group.

[0046] 4.3 Immunization: the pigs in group 1 are inoculated with the 202312a batch vaccine in the neck muscle, the pigs in group 2 are inoculated with the 202312b batch vaccine in the neck muscle, and the pigs in group 3 are immunized with the 202312c batch vaccine; the pigs in group 4 are not immunized as the control group; they are raised in the same conditions. The second immunization is performed 21 days after the first immunization, and the antibody titers are tracked at 14 days, 21 days, 28 days, 2 months, 3 months, and 5 months after the first immunization. The vaccine combination formula table is shown in Table 3.

[0047] Table 3: Vaccine combination formula table

[0048] 4.4 Detection of CSF blocking ELISA antibodies: refer to the operation instruction of the IDEXX kit (item number) to detect the antibody titers at 14 days, 21 days, 28 days, 2 months, 3 months, and 5 months after the first immunization. The judgment standard of the kit is that the blocking rate of the detected sample is greater than or equal to 40%, which is judged as positive, the blocking rate of the detected sample is less than or equal to 30%, which is judged as negative, and the blocking rate of the detected sample is between 30-40%, which is judged as suspicious. The antibodies of the 202312a / 202312b / 202312c three batches of vaccines show an upward trend at 14 days after the first immunization, and all the 202312a / 202312b batches of vaccines turn positive at 21 days after the first immunization, and the overall antibody titers are good, which lasts for 5 months after the immunization, and the blocking rates are all above 70%. However, the 202312c batch of vaccines are still suspicious at 21 days after the first immunization, not all of them turn positive, and the antibodies are still positive at 5 months after the immunization, and some antibodies turn weakly positive. The blocking rates of each group are shown in Table 4.

[0049] Table 4: Antibody titers of blocking ELISA after immunization with 202312a / b / c batches of vaccines

[0050] 4.5 Detection of porcine pseudorabies neutralizing antibody titer determination: 0.08% trypsin (containing 0.02 EDTA) was used to digest the monolayer of Vero cells, and the cell density was adjusted to 3x105 cells / ml, and 100 μl (3x104 cells / well) was inoculated into a 96-well cell culture plate. The serum to be tested (56°C inactivation for 30 minutes) was serially diluted from 1:2 to 1:256 with DMEM cell maintenance solution, 300 μl of each dilution was taken, and 200 TCID50 of porcine pseudorabies virus liquid was added, and a virus positive control was set up, mixed and placed in a 37°C water bath for 1 hour. The mixed solution of different dilutions after action and the positive control liquid were inoculated on the 96-well Vero cell culture plate with monolayer, and cell maintenance solution was added as a cell control. It was placed in a 37°C, 5% CO2 incubator for 96 hours. Observe and record the cytopathic effect (CPE) every day, and calculate the titer of porcine pseudorabies virus neutralizing antibody in the tested serum according to the Reed-Muench method. The cells in the normal cell control well should be normal, and the cells in the virus control well should appear cytopathic effect. Neutralization titer > 1:70 is positive.

[0051] 4.5.1 Neutralizing antibody determination results of non-immune control group: The neutralizing antibody titer of the control group was less than 1:4 during the entire immunization test.

[0052] 4.5.2 Neutralizing antibody determination results of 202312a batch vaccine group (SpaA is 30 μg): Two weeks after the first immunization, the neutralizing antibody reached more than 1:70, three weeks after the first immunization, the neutralizing antibody continued to rise, and was not less than 1:1024, 28 days after the first immunization (7 days after the second immunization), the antibody reached 1:4096, and lasted for 5 months after the first immunization, the neutralizing antibody was not less than 1:512. See Table 5 for details.

[0053] 4.5.3 Neutralizing antibody determination results of 202312b batch vaccine group (SpaA is 15 μg): Two weeks after the first immunization, part of the neutralizing antibody reached more than 1:70, three weeks after the first immunization, the neutralizing antibody continued to rise, and was not less than 1:512, 28 days after the first immunization (7 days after the second immunization), the antibody reached 1:4096, and lasted for 5 months after the first immunization, the neutralizing antibody was not less than 1:256. See Table 5 for details.

[0054] 4.5.4 Neutralizing antibody determination results of 202312c batch vaccine group (without SpaA protein): Two weeks after the first immunization, the neutralizing antibody was less than 1:70, three weeks after the first immunization, the neutralizing antibody began to rise, and was not less than 1:256, 28 days after the first immunization (7 days after the second immunization), the antibody reached 1:2048, and lasted for 5 months after the first immunization, the neutralizing antibody was not less than 1:128. See Table 5 for details.

[0055] Table 5: Detection results of porcine pseudorabies neutralizing antibody

[0056] 4.6 Detection of porcine circovirus type 2 antibody titer determination: reference Beijing Jinuobaitai Biotechnology Co., Ltd. Production of "porcine circovirus 2-dCap-ELISA antibody detection kit" operation instruction, detection of antibody titer after 14 days, 21 days, 28 days, 2 months, 3 months, 5 months. The determination standard of the kit is S / P value ≥ 0.4, positive, S / P value < 0.3, negative, 0.3 ≤ S / P < 0.4, suspicious. The antibody of three batches of vaccine 202312a / 202312b / 202312c after one dose is all positive, the titer of 202312a / b batch after one dose is higher than that of 202312c batch, and the whole is better, which lasts for 5 months after immunization, and the antibody titer of all vaccine serum after immunization is positive. See Table 6 for details.

[0057] Table 6: Results of porcine circovirus type 2 antibody detection

[0058] 4.7 Swine erysipelas efficacy test: test animals are group 1 immunized with 202312a group, group 2 immunized with 202312b group, group 3 immunized with 202312c group, and group 4 is non-immunized control group, all are 5 months after the second dose of piglets. The strain for challenge is C43-6 strain, and the challenge dose is 2 times the pathogenic dose, and the number of live bacteria is 2 x 10 9 CFU.

[0059] 4.7.1 Swine erysipelas incidence criteria: 1) death. 2) any one or more of the following clinical symptoms: skin redness, rash, joint swelling, lameness, etc. 3) body temperature above the base temperature by 1.5℃ for 2 days or more. Any of the above is considered to be sick.

[0060] 4.7.2 Protection criteria: no skin redness, rash, joint swelling, lameness, death, elevated body temperature, etc. is considered to be protected; if the body temperature is elevated, it should not exceed the base temperature by 1.5℃, and if it exceeds 1.5℃ but lasts for no more than 2 days, it is also considered to be protected.

[0061] 4.7.3 Swine erysipelas challenge protection results: after challenge, 202312a, 201312b, 202312c groups all have no skin redness, rash, joint swelling, lameness, death, etc. The body temperature is not higher than the base temperature by 1.5℃, and all are protected. The non-immunized control group has redness, rash, and body temperature above the base temperature by 1.5℃ or more, and lasts for 2 days, all are sick.

[0062] In summary, the 202312a / 202312b batch vaccine group not only has a short onset time and long duration, but also has a synergistic effect between the antigen SpaA protein and the porcine fever virus E2 protein, the porcine pseudorabies virus gD protein, the porcine pseudorabies virus gB protein, and the porcine circovirus cap protein. The amount of protein used is less, and it can also protect against infection of porcine erysipelas filamentous bacteria.

[0063] Example 5: Subunit vaccine composition immune neutralization experiment.

[0064] 5.1 Screening of test animals: 30-day-old weaned piglets that are negative for porcine epidemic diarrhea, porcine deltacoronavirus, and porcine rotavirus were screened, and a total of 28 piglets were obtained.

[0065] 5.2 Grouping of test animals: The 28 sows were randomly divided into 7 groups, with 4 piglets in each group. Groups 1-6 were the immune groups, and group 7 was the non-immune control group.

[0066] 5.3 Immunization: The first group of pigs was inoculated with the 202401a batch vaccine in the neck muscle, the second group was inoculated with the 202401b batch vaccine in the neck muscle, and the third group was immunized with the 202401c batch vaccine. The fourth group of pigs was inoculated with the 202401d batch vaccine in the neck muscle, the fifth group was inoculated with the 202401e batch vaccine in the neck muscle, and the sixth group was immunized with the 202401f batch vaccine. The seventh group was not immunized as the control group. They were raised in separate pens under the same conditions. Secondary immunization was performed 21 days after the first immunization, and blood was drawn to isolate serum after immunization. The neutralizing antibody titers were tracked at 14 days, 28 days, 3 months, and 5 months after the first immunization. See Table 7 for the vaccine formulation table.

[0067] Table 7: Vaccine formulation table

[0068] 5.4 Neutralizing antibody detection.

[0069] 5.4.1 Detection of neutralizing antibodies in porcine epidemic diarrhea piglets: PEDV (GD / HZ, preserved by the laboratory) was diluted to 200 TCID 50 / 0.1ml of virus liquid, and the test serum and control serum (PEDV-specific positive serum and negative serum) diluted with DMEM in equal amounts were mixed and placed in a 37°C incubator with 5% CO2 for 1 hour. VERO cells were inoculated on 96-well cell plates with monolayer, 4 wells per hole, 100 μl per hole, and 4 positive control wells of unneutralized virus and 4 negative cell control wells with only cell maintenance solution were set up, 100 μl per hole, adsorbed in a 37°C incubator with 5% CO2 for 1 hour, the inoculum was aspirated, the monolayer cells were washed twice with PBS (0.01 mol / L, pH 7.2), 100 μl of serum-free DMEM containing 7.5 μg / ml trypsin was added per well, and the culture was continued in a 37°C incubator with 5% CO2 for 5 days. CPE was observed daily, and the titer of PEDV neutralizing antibodies in the test serum was calculated according to the Reed-Muench method. The neutralizing antibody showed an upward trend 14 days after the first vaccination with the 202401a / 202401b batches of vaccine, and the neutralizing antibody continued to rise after 28 days of vaccination with the 202401a batch of vaccine, and the overall level was good, and lasted for 5 months after vaccination, and the neutralizing antibody was above 1:16. The antibody level of the 202401b batch of vaccine was lower than that of the 202401a batch after 28 days of vaccination, and the antibody level was lower than 1:16 for 5 months. The results of each group are shown in Table 8.

[0070] Table 8: PEDV neutralization titer determination

[0071] 5.4.2 Pig Delta coronavirus piglet neutralizing antibody detection: PDCoV (HK / ZJ / 2023, preserved by the laboratory) was diluted with DMEM culture medium to 200 TCID 50 / 0.1 ml of virus liquid, and the test serum and control serum (PEDV-specific positive serum and negative serum) diluted with DMEM in equal amounts were mixed and placed in a 37°C incubator with 5% CO2 for 1 hour. Each of the LLC-PK cells inoculated on the 96-well cell plate grew into a monolayer, 4 wells per hole, 100 μl per hole, and 4 positive control wells of unneutralized virus and 4 negative cell control wells with only cell maintenance solution were set up, 100 μl per hole, and placed in a 37°C incubator with 5% CO2 for 1 hour. The inoculum was aspirated, the monolayer cells were washed twice with PBS (0.01 mol / L, pH 7.2), 100 μl of serum-free DMEM containing 10 μg / ml trypsin was added per well, and the incubation was continued in a 37°C incubator with 5% CO2 for 5 days. CPE was observed daily, and the titer of PDCoV neutralizing antibodies in the test serum was calculated according to the Reed-Muench method. The neutralizing antibodies of the 202401c / 202401d two batches of vaccines showed an upward trend 14 days after the first immunization, and the neutralizing antibodies of the 202401c batch of vaccine increased significantly 28 days after the first immunization, and the overall level was good, lasting for 3 months, and the neutralizing antibodies were all above 1:128. The antibody level of the 202401d batch of vaccine was lower than that of the 202401c batch 28 days after the first immunization, and the antibody level was lower than 1:128 for 5 months. The results of each group are shown in Table 9.

[0072] Table 9: PDCoV neutralization titer determination

[0073] 5.4.3 Porcine rotavirus neutralizing antibody detection: PoRV (HF / ZJ / 2022, preserved by the laboratory) was diluted with DMEM culture medium to 200 TCID 50A 0.1ml virus solution was mixed with equal amounts of test serum and control serum (PEDV-specific positive and negative sera) diluted serially in DMEM and incubated at 37°C in a 5% CO2 incubator for 1 hour. Four wells of MA104 cells grown in a monolayer on a 96-well plate were inoculated with 100μl per well. Four wells each containing positive controls of unneutralized virus and negative controls inoculated with cell maintenance medium alone were also inoculated with 100μl per well. The cells were incubated at 37°C in a 5% CO2 incubator for 1 hour. The inoculum was then discarded, and the monolayers were washed twice with PBS (0.01mol / L, pH 7.2). Serum-free DMEM supplemented with 1μg / ml trypsin was added at 100μl per well. The cells were incubated at 37°C in a 5% CO2 incubator for another 5 days. CPE was observed daily, and the titer of PoRV neutralizing antibodies in the test sera was calculated using the Reed-Muench method. Neutralizing antibody levels for both batches 202401e and 202401f showed an upward trend 14 days after the first dose. Twenty-eight days after the first dose, neutralizing antibody levels for the 202401e batch were significantly elevated. This level of neutralizing antibody activity remained high and well-balanced, with levels above 1:256 for all five months post-dose. However, overall antibody levels for the 202401f batch were lower than those for the 202401e batch 28 days after the first dose, and remained below 1:256 for the five-month period. The results for each group are shown in Table 10.

[0074] Table 10: PoRV neutralization titer determination

[0075] Example 6: Detection of immune antibodies and neutralization experiments of subunit vaccine compositions.

[0076] 6.1 Screening of experimental animals: 16 weaned piglets around 30 days old that tested negative for porcine epidemic diarrhea, porcine delta coronavirus, and porcine rotavirus were screened.

[0077] 6.2 Experimental Animal Grouping: 16 piglets were randomly divided into 4 groups, with 4 piglets in each group. Groups 1 to 3 were immunized groups; Group 4 was not immunized and served as the control group.

[0078] 6.3 Vaccination: Group 1 pigs were vaccinated intramuscularly with vaccine batch 202402a; Group 2 pigs were vaccinated intramuscularly with vaccine batch 202402b; Group 3 pigs were vaccinated intramuscularly with vaccine batch 202402c; Group 4 pigs were not vaccinated as a control. Pigs were housed separately under the same conditions. A second vaccination was administered 21 days after the first vaccination. Blood was drawn and serum was isolated. Neutralizing antibody titers were monitored 14 days, 28 days, 3 months, and 5 months after the first vaccination. See Table 11 for the vaccine formulation.

[0079] Table 11: Vaccine formula

[0080] 6.4 Neutralizing antibody detection.

[0081] 6.4.1 Detection of PEDV neutralizing antibody in piglets: The detection steps of neutralizing antibody were the same as 5.4.1. The detection results of 202401a / 202401b were basically consistent. The antibody of 202402a / b / c batches of vaccine rose after 14 days of one-time immunization, and the antibody of 202402a / 202401b batches of vaccine increased significantly after 28 days of one-time immunization, and the overall antibody was good, which lasted for 5 months after immunization, and the neutralizing antibody was more than 1:16. The antibody of 202402c batch of vaccine was lower than that of 202402a / 202401b batch after 28 days of one-time immunization, and the antibody was lower than 1:16 after 5 months. The results are shown in Table 12.

[0082] Table 12: PEDV neutralization titer determination

[0083] 6.4.2 Detection of PDCoV neutralizing antibody titer: The detection steps of neutralizing antibody were the same as 5.4.2. The detection results of 202401c / 202401d were basically consistent. The antibody of 202402a / 202402b / 202402c batches of vaccine rose after 14 days of one-time immunization, and the antibody of 202402a / 202402b batches of vaccine increased significantly after 28 days of one-time immunization, and the overall antibody was good, which lasted for 5 months after immunization, and the neutralizing antibody was more than 1:128. The antibody of 202402c batch of vaccine was lower than that of 202402a / b batch after 28 days of one-time immunization, and the antibody was lower than 1:128 after 5 months. The results of each group are shown in Table 13.

[0084] Table 13: PDCoV neutralization titer determination

[0085] 6.4.3 Detection of PoRV neutralizing antibody titer: The detection steps of neutralizing antibody were the same as 5.4.3. The detection results of 202401e / f were basically consistent. The antibody of 202402a / 202402b / 202402c batches of vaccine rose after 14 days of one-time immunization, and the antibody of 202402a / 202402b batches of vaccine increased significantly after 28 days of one-time immunization, and the overall antibody was good, which lasted for 5 months after immunization, and the neutralizing antibody was more than 1:256. The antibody of 202402c batch of vaccine was lower than that of 202402a / b batch after 28 days of one-time immunization, and the antibody was lower than 1:256 after 5 months. The results of each group are shown in Table 14.

[0086] Table 14: PoRV neutralization titer determination

[0087] The above results show that the PEDV S protein, PDCoV S protein and PRoV VP8 protein and SpaA protein are mixed, and there is no mutual interference of each antigen component after immunization, and sapA enhances the duration of neutralizing antibodies.

[0088] It should be noted that the above summary and detailed description are intended to demonstrate the practical application of the technical solutions provided by the present application, and should not be interpreted as limiting the scope of protection of the present application. Those skilled in the art can make various modifications, equivalent replacements or improvements within the spirit and principles of the present application.

Claims

1. Use of the SpaA protein as an immunopotentiator in the preparation of a viral subunit vaccine for prevention, wherein the SpaA protein is a prokaryotically expressed Erysipelothrix rhizogenes SpaA protein having an amino acid sequence as shown in SEQ No. 1, and the viral subunit vaccine is a porcine rotavirus VP8 protein having an amino acid sequence as shown in SEQ No.

8.

2. The use according to claim 1, characterized in that The viral subunit vaccine comprises viral subunit proteins and a pharmaceutically acceptable adjuvant.

3. The use according to claim 2, characterized in that The pharmaceutically acceptable adjuvant is a water-in-oil-in-water biphasic emulsified adjuvant to form an oil phase.

4. The use according to claim 3, characterized in that The pharmaceutically acceptable adjuvant is ISA 201VG.

5. The use according to claim 1, characterized in that The mass ratio of the Erysipelothrix rhizogenes SpaA protein to the viral subunit protein is 1:1-2.

6. The use according to claim 2, characterized in that The viral subunit proteins also included sterile PBS to form the aqueous phase.

7. The use according to claim 6, characterized in that The weight ratio of the combination of the SpaA protein, the viral subunit protein, the sterile PBS, and the pharmaceutically acceptable adjuvant is 1:1.

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