Use of SpaA protein as an immunopotentiator in the preparation of subunit vaccines for preventing viral infections

Abstract

The present invention provides the use of a SpaA protein as an immunopotentiator in the preparation of a viral subunit vaccine for preventing infection. The SpaA protein is derived from Erysipelothrix rhizogenes, and the viral subunit vaccine is prepared using a specific viral coat protein, i.e., an antigenic determinant. The vaccine, comprising the Erysipelothrix rhizogenes SpaA protein antigen as an immunopotentiator and a viral subunit protein, can significantly increase the duration of the viral subunit protein blocking rate or enhance the production and duration of neutralizing antibodies against the viral subunit protein, while also shortening the onset of efficacy.

Description

Technical Field

[0001] The present invention belongs to the field of veterinary biological products, and specifically relates to the use of SpaA protein as an immunopotentiator in the preparation of a subunit vaccine for preventing viral infections. The SpaA protein is the SpaA protein of Erysipelothrix rhesus, and the viruses are classical swine fever virus, pseudorabies virus, and porcine circovirus. Background Art

[0002] Vaccine adjuvants are nonspecific immunoenhancers that can promote antigen-specific immune responses by enhancing the immune response and changing the type of immune response. They can usually reduce the amount of antigen used and the number of immunizations, produce faster and more lasting immune responses, and improve the intensity and effectiveness of the vaccine's immune response.

[0003] Subunit vaccines are made by extracting immunologically active fragments of bacterial or viral protein structures through chemical decomposition or controlled proteolysis. Subunit vaccines are made from components containing the primary protective immunogens of pathogenic bacteria, also known as component vaccines. However, subunit vaccines have the disadvantage of low immunogenicity and require adjuvants to produce effective immune responses.

[0004] Erysipelas is an anaerobic bacterium. It was first isolated by Koch in 1878. In 1886, Loeffler identified it as the pathogen of erysipelas. In 1909, Rosenbach isolated this bacterium from a human patient with local skin disease, thus confirming that it is also a human pathogen. Therefore, Erysipelas is the pathogen of a type of zoonotic disease - erysipelas. The main host of Erysipelas is generally domestic pigs, but it can also infect many other birds and rodents. Although fish infection does not cause clear known symptoms, Erysipelas can survive for a long time in the mucus on the surface of fish. In other words, fish can carry Erysipelas for a long time, which greatly increases the risk of humans being infected with Erysipelas.

[0005] Recent studies have revealed that several surface proteins of Erysipelothrix rhizogenes (ERS) with molecular masses of 64, 66, and 43 kDa possess protective immune properties. Makino's group cloned and expressed a 64 kDa surface protein, naming it surface protective antigen A (SPAA). Multiple research groups have conducted studies on the immune function of ERS and confirmed its robust protective efficacy.

[0006] Classical swine fever, also known as hog cholera, is a highly contagious and devastating infectious disease caused by the Classical Swine Fever Virus (CSFV). It has a mortality rate of up to 90%, with symptoms including high fever, skin hemorrhages, general weakness, anorexia, and conjunctivitis. Pseudorabies, also known as pseudorabies, is a viral infectious disease caused by the pseudorabies virus (PRV), a double-stranded DNA herpesvirus that can cause abortion, stillbirth, mummification, and neurological symptoms in piglets, with a mortality rate of up to 70%. Porcine circovirus type 2 (PCV2) is the primary cause of post-weaning multisystemic wasting syndrome (MPWS), which can cause reproductive failure in sows and inflict significant losses to the pig industry. Swine erysipelas is an infectious disease affecting both humans and animals caused by Erysipelothrix rhusiopathiae. It is characterized by high fever, acute systemic septicemia, a purple-red rash on the skin, and a long course of endocarditis and arthritis, often with swelling of the limbs. Clinically, CSFV, PRV, and PCV2 are common co-infections and are the most common pathogens causing PRDC.

[0007] Currently, vaccines against CSFV, PRV, PCV2, and swine erysipelas have been used in clinical immunization, achieving excellent preventive efficacy. However, frequent vaccination not only increases labor costs but also easily induces multiple stresses in the pig herd, leading to slow piglet growth. Consequently, research on mixed vaccine immunization is increasing. Lin Derui et al. (Lin Derui et al. Evaluation of the immune efficacy of several immunization methods with live classical swine fever and pseudorabies vaccines [J]. Chinese Journal of Animal Health) co-administered PRV and CSFV vaccines, demonstrating minimal interference between the two vaccines and no impact on the production of antibodies against each. Zhu Zijian et al. (Zhu Zijian et al. Evaluation of the immune efficacy of simultaneous administration of attenuated laminar-attenuated classical swine fever vaccine and inactivated porcine circovirus type 2 vaccine [J]. Chinese Journal of Preventive Veterinary Medicine) co-administered PCV2 and CSFV vaccines, demonstrating that combined immunization with PCV2 and CSFV produces superior protective efficacy. Tian Xin et al. (Tian Xin et al. Evaluation of the efficacy of co-immunization with an inactivated porcine circovirus type 2 vaccine, live classical swine fever vaccine, and live pseudorabies vaccine [J]. Chinese Journal of Zoonotic Infectious Diseases) co-immunized with an inactivated PCV2 vaccine, live CSFV vaccine, and live PRV vaccine. The results showed that co-immunization did not affect the efficacy of each vaccine. Chinese patent CN 113058032 B reports that co-immunization with CHO cells expressing classical swine fever virus E2 protein, pseudorabies virus gD protein, and pseudorabies virus gB protein, along with insect baculovirus-expressed cap protein, effectively prevents classical swine fever, pseudorabies, and porcine circovirus.

[0008] Since multiple virus co-infection is common in clinical practice, it is difficult for a single vaccine to control the occurrence of infection. Therefore, there is an urgent need to develop a multi-vaccine to prevent viral infection in piglets. Summary of the Invention

[0009] In order to develop a safe and effective multi-vaccine, the inventors unexpectedly discovered during the vaccine combination research that the combination of swine erysipelas subunit protein SpaA and viral subunit protein can significantly increase the duration of the blocking rate of viral subunit protein or increase the duration of viral subunit protein neutralizing antibodies, and can also shorten the onset time.

[0010] To this end, the present invention provides the use of a SpaA protein as an immunopotentiator in the preparation of a viral subunit vaccine for preventing infection. The SpaA protein is the SpaA protein of Erysipelothrix rhizogenes, and the viral subunit vaccine is a viral subunit protein composition. Here, the viral subunit vaccine is a vaccine comprising a specific viral coat protein, i.e., an antigenic determinant.

[0011] In the preferred technical solution of the use of the present invention, preferably, the SpaA protein is the prokaryotically expressed Erysipelothrix rhizogenes SpaA protein, and its amino acid sequence is shown in SEQ No. 1.

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

[0013] In the preferred technical solution of the use of the present invention, preferably, the viral subunit protein is at least selected from the group consisting of the subunit E2 protein of classical swine fever virus, the gD protein of pseudorabies virus, the gB protein of pseudorabies virus, the cap protein of porcine circovirus type 2, the S protein of porcine epidemic diarrhea virus, the S protein of porcine deltacoronavirus, and the VP8 protein of porcine rotavirus.

[0014] In the preferred technical solution of the use of the present invention, preferably, the amino acid sequence of the subunit E2 protein of the classical swine fever virus is shown in SEQ No. 2, the amino acid sequence of the gD protein of the pseudorabies virus is shown in SEQ No. 3, the amino acid sequences of the gB protein of the pseudorabies virus are shown in SEQ No. 4, respectively, the amino acid sequence of the cap protein of the porcine circovirus type 2 is shown in SEQ No. 5, the amino acid sequence of the S protein of the porcine epidemic diarrhea virus is shown in SEQ No. 6, the amino acid sequence of the S protein of the porcine deltacoronavirus is shown in SEQ No. 7, and the amino acid sequence of the VP8 protein of the porcine rotavirus is shown in SEQ No. 8.

[0015] In a preferred embodiment of the present invention, the pharmaceutically acceptable adjuvant is preferably a water-in-oil-in-water biphasic emulsion adjuvant to form the oil phase. More preferably, the pharmaceutically acceptable adjuvant is ISA 201 VG.

[0016] In the preferred technical solution of the use of the present invention, preferably, the mass ratio of the Erysipelothrix rhizogenes SpaA protein to the viral subunit protein is 1:1-2.

[0017] In a preferred technical solution of the use of the present invention, preferably, the protein concentrations of the Erysipelothrix rhizogenes SpaA protein and the viral subunit protein are both 15 μg / head to 75 μg / head.

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

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

[0020] The present invention's combined vaccine of Erysipelothrix rhizogenes SpaA protein antigen and viral subunit protein as an immunopotentiator can significantly increase the duration of the viral subunit protein blocking rate or increase the viral subunit protein neutralizing antibody and duration, and can also shorten the onset time. DETAILED DESCRIPTION

[0021] The present invention will be further described below with reference to the embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention.

[0022] Example 1: Preparation of Classical Swine Fever Virus Subunit E2 Protein, Pseudorabies Virus gD Protein, Pseudorabies Virus gB Protein, Porcine Circovirus Type 2 Cap Protein, Porcine Epidemic Diarrhea Virus S Protein and Porcine Delta Coronavirus S Protein, Porcine Rotavirus VP8 Protein, and Erysipelothrix SpaA Protein

[0023] 1.1 Preparation of the subunit E2 protein of classical swine fever virus: refer to the preparation method and application of a recombinant subunit vaccine of classical swine fever virus in the invention application with publication number CN104826100A of the applicant.

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

[0025] 1.3 Porcine circovirus type 2 cap protein: refer to the invention application of the applicant with publication number CN104873966B, which discloses a method for purifying porcine circovirus type 2 virus-like particles and preparing a vaccine thereof.

[0026] 1.4 Preparation of porcine epidemic diarrhea virus S protein: refer to the porcine epidemic diarrhea virus S protein and its subunit vaccine and its preparation method and application in the invention application No. 201810310540.8 of the applicant.

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

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

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

[0030] Example 2: Preparation of subunit vaccine composition: All consumables and materials used to prepare the vaccine must be sterilized in advance, and the preparation process is completed in a biosafety cabinet or other equipment or environment that can ensure the sterility of the entire preparation process.

[0031] 2.1 Preparation of the Aqueous Phase: The antigenic protein (i.e., viral subunit protein) prepared in Example 1 and the Erysipelothrix rhizogenes SpaA protein were mixed and diluted in sterile PBS at a 1:1 mass ratio to obtain an aqueous phase that met the antigenic protein content requirements. The concentrations of the Erysipelothrix rhizogenes SpaA protein and viral protein were high, so they were diluted with PBS to a final concentration of 15 μg to 75 μg.

[0032] 2.2 Sterilize or sterilize the water-in-oil-in-water biphasic emulsified adjuvant to obtain the oil phase; the water-in-oil-in-water biphasic emulsified adjuvant is ISA 201 VG.

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

[0034] 2.4 Quantitatively package the emulsified liquid to obtain subunit vaccine and label it.

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

[0036] 3.1 Screening of experimental animals: Twelve weaned piglets around 30 days old that were negative for classical swine fever, pseudorabies, porcine circovirus type 2, and erysipelas antigen and antibody were screened.

[0037] 3.2 Experimental Animal Grouping: Twelve weaned piglets were randomly divided into three groups, with four pigs 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 Vaccination: Group 1 pigs were vaccinated with vaccine batch 202311a intramuscularly, while Group 2 was vaccinated with vaccine batch 202311b intramuscularly. Group 3 pigs were kept in separate pens under the same conditions. A second vaccination was administered 21 days after the first vaccination. Antibody titers were monitored 14 days, 21 days, 28 days, and one, two, three, and five months after the first vaccination. See Table 1 for the vaccine formulation.

[0039] Table 1: Vaccine formula

[0040]

[0041] 3.4 Testing for Classical Swine Fever Blocking ELISA Antibodies: Follow the instructions for the IDEXX kit (catalog number) to test antibody titers 14 days, 21 days, 28 days, and two, three, and five months after the first vaccination. The kit's criteria for judging a sample as positive is a blocking rate greater than or equal to 40%, a negative rate less than or equal to 30%, and a questionable rate between 30% and 40%. Twenty-one days after the first vaccination, the antibody titer for batch 202311a vaccine showed an upward trend, with all samples converting to positive. This trend was consistently good, with a blocking rate exceeding 70% for five months. However, some samples from batch 202311b vaccine remained questionable 21 days after the first vaccination, with some not converting to positive. Through five months, the antibody titer remained positive, with some becoming weakly positive. The blocking rates for each group are shown in Table 2.

[0042] Table 2: Blocking ELISA antibody titers after immunization with 202311a / b batch vaccines

[0043]

[0044] According to Examples 2 and 3, experiments were also conducted on combinations of SpaA and gB, gD, SpaA and Cap, E2, etc., and it was found that: compared with the absence of SpaA, the combination of Erysipelothrix rhesus SpaA protein antigen and viral subunit proteins (gB, gD, SpaA and Cap, SpaA, or Cap) can increase the neutralizing antibodies against pseudorabies virus gD protein and pseudorabies virus gB protein, and increase the antibody level of porcine circovirus antibodies and the resistance rate and duration of classical swine fever.

[0045] Example 4: Subunit vaccine composition immune antibody detection and neutralization experiment

[0046] 4.1 Screening of experimental animals: 30 weaned piglets around 30 days old that are negative for classical swine fever, pseudorabies, porcine circovirus type 2, and erysipelas antigen and antibody were screened.

[0047] 4.2 Animal Grouping: Ten weaned piglets were randomly divided into two groups of five each. Group 1 was immunized with a mixture of gD, gB, and SpaA antigens; Group 2 was immunized with a mixture of gD and gB antigens. Twenty weaned piglets were randomly divided into four groups of five each. Groups 1 to 3 were immunized groups; Group 4 served as the unimmunized control group.

[0048] 4.3 Vaccination: Group 1 pigs were vaccinated intramuscularly with vaccine batch 202312a; Group 2 pigs were vaccinated intramuscularly with vaccine batch 202312b; Group 3 pigs were vaccinated with vaccine batch 202312c; 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. Antibody titers were monitored 14 days, 21 days, 28 days, and 2, 3, and 5 months after the first vaccination. See Table 3 for the vaccine combination formula.

[0049] Table 3: Vaccine combination formula

[0050]

[0051] 4.4 Testing for Classical Swine Fever Blocking ELISA Antibodies: Follow the instructions for the IDEXX test kit (catalog number) to test antibody titers 14 days, 21 days, 28 days, and two, three, and five months after the first vaccination. The test kit's criteria for judging samples is that a blocking rate greater than or equal to 40% is considered positive, a blocking rate less than or equal to 30% is considered negative, and a blocking rate between 30% and 40% is considered questionable. For the three batches 202312a / 202312b / 202312c, an upward trend in antibody titers was observed 14 days after the first vaccination. Twenty-one days after the first vaccination, all immunizations with the 202312a / 202312b vaccines turned positive, with good overall uniformity. This trend persisted through five months, with blocking rates exceeding 70%. However, some 202312c vaccine samples remained questionable 21 days after the first vaccination, with some not fully turning positive. Through five months, antibody levels remained positive, with some turning weakly positive. The blocking rates of each group are shown in Table 4.

[0052] Table 4: Blocking ELISA antibody titers of 202312a / b / c batch vaccines after immunization

[0053]

[0054] 4.5 Determination of Neutralizing Antibody Titers against Pseudorabies Virus: Disinfect confluent Vero cell monolayers with 0.08% trypsin (containing 0.02% EDTA) and adjust the cell density to 3×10⁵ cells / ml. Plate 100 μl per well (3×10⁴ cells / well) in a 96-well cell culture plate. Serially dilute the test serum (inactivated at 56°C for 30 minutes) in DMEM (DMEM) from 1:2 to 1:256. Add 300 μl of each dilution to an equal volume of 200 TCID⁵ of Pseudorabies Virus (PRV) and a virus positive control. Mix well and incubate in a 37°C water bath for 1 hour. Inoculate the mixtures of the different dilutions and the positive control onto a 96-well Vero cell monolayer plate. Add the cell maintenance medium as a cell control. Incubate in a 37°C, 5% CO₂ incubator for 96 hours. Observe and record cytopathic effect (CPE) daily. Calculate the titer of PRV neutralizing antibodies in the tested serum using the Reed-Muench method. Cells in the normal cell control wells should all be normal, while cells in the virus control wells should all show cytopathic effect. A neutralization titer >1:70 is considered positive.

[0055] 4.5.1 Neutralizing antibody assay results for the non-immunized control group: The neutralizing antibody titer in the control group was lower than 1:4 throughout the entire immunization test.

[0056] 4.5.2 Neutralizing antibody assay results for the vaccine batch 202312a (SpaA 30 μg): Two weeks after the first vaccination, neutralizing antibody levels reached above 1:70. Three weeks after the first vaccination, neutralizing antibody levels continued to rise, reaching no less than 1:1024. Twenty-eight days after the first vaccination (seven days after the second vaccination), the peak antibody level reached 1:4096. This level persisted until five months after the first vaccination, with neutralizing antibody levels no less than 1:512. See Table 5 for details.

[0057] 4.5.3 Neutralizing antibody assay results for the vaccine batch 202312b group (SpaA 15 μg): Two weeks after the first vaccination, some neutralizing antibodies reached levels above 1:70. Three weeks after the first vaccination, neutralizing antibodies continued to rise, remaining at or above 1:512. Twenty-eight days after the first vaccination (seven days after the second vaccination), the antibody level reached a peak of 1:4096. This level persisted for five months after the first vaccination, with neutralizing antibodies remaining at or above 1:256. See Table 5 for details.

[0058] 4.5.4 Neutralizing antibody assay results for the vaccine batch 202312c (SpaA protein-free) group: Two weeks after the first vaccination, neutralizing antibody levels were below 1:70. Three weeks after the first vaccination, neutralizing antibody levels began to rise, reaching no less than 1:256. Twenty-eight days after the first vaccination (seven days after the second vaccination), the antibody level reached a peak of 1:2048. This level persisted until five months after the first vaccination, with neutralizing antibody levels no less than 1:128. See Table 5 for details.

[0059] Table 5: Results of pseudorabies neutralizing antibody test in pigs

[0060]

[0061] 4.6 Testing for Porcine Circovirus Type 2 Antibody Titers: Antibody titers were measured 14 days, 21 days, 28 days, and two, three, and five months after the first vaccination, according to the instructions for the "Porcine Circovirus Type 2-dCap-ELISA Antibody Detection Kit" produced by Beijing Jinnuo Biotech Co., Ltd. The kit's criteria for evaluation were an S / P value ≥ 0.4 for positive, an S / P value < 0.3 for negative, and a 0.3 ≤ S / P < 0.4 for questionable. All three vaccine batches, 202312a / 202312b, and 202312c, showed positive antibody titers 14 days after the first vaccination. Twenty-one days after the first vaccination, the 202312a / b vaccine batches showed higher titers than the 202312c batch, with improved overall uniformity. This continued through five months after vaccination, with all vaccines demonstrating positive serum antibody titers. See Table 6 for details.

[0062] Table 6: Porcine circovirus type 2 antibody test results

[0063]

[0064] 4.7 Swine Erysipelas Efficacy Test: The experimental animals were group 1 immunized with 202312a, group 2 immunized with 202312b, group 3 immunized with 202312c, and group 4 was a non-immunized control group. All were piglets 5 months old after the second vaccination. The challenge strain was C43-6, the challenge dose was 2 doses of the disease-causing dose, and the viable bacterial count was 2×10 9 CFU.

[0065] 4.7.1 Erysipelas criteria for swine: 1) Death. 2) Appearance of any one or more of the following clinical symptoms: skin erythema, rash, joint swelling, lameness, etc. 3) Body temperature exceeding basal body temperature by 1.5°C for two or more days. The presence of any of the above symptoms constitutes an onset of the disease.

[0066] 4.7.2 Protection Standard: If there are no clinical symptoms such as skin erythema, rash, joint swelling, lameness, death, and fever, the patient is considered protected. If the body temperature rises, it should not exceed 1.5°C of the basal body temperature. If it exceeds 1.5°C but lasts for no more than 2 days, the patient is also considered protected.

[0067] 4.7.3 Erysipelas Challenge and Protection Results: After challenge, none of the three groups (202312a, 201312b, and 202312c) showed skin erythema, rash, joint swelling, lameness, or mortality, and their body temperatures did not rise above 1.5°C above their basal body temperature, indicating complete protection. In contrast, the non-immunized control group developed erythema and rash, and their body temperatures rose above 1.5°C above their basal body temperature for two days, resulting in the disease occurring in all groups.

[0068] In summary, the 202312a / 202312b vaccine group not only has a short onset time and long duration, but also the antigen SpaA protein interacts synergistically with the E2 protein of classical swine fever virus, the gD protein of pseudorabies virus, the gB protein of pseudorabies virus, and the cap protein of porcine circovirus. The amount of protein used is small, and it can also protect against the infection of Erysipelothrix rhizoctonia solani.

[0069] Example 5: Subunit vaccine composition immune neutralization experiment

[0070] 5.1 Screening of experimental animals: 28 weaned piglets around 30 days old that were negative for porcine epidemic diarrhea, porcine delta coronavirus, and porcine rotavirus were screened.

[0071] 5.2 Experimental Animal Grouping: 28 sows were randomly divided into 7 groups, with 4 sows in each group. Groups 1 to 6 were immunized groups; Group 7 was not immunized and served as the control group.

[0072] 5.3 Vaccination: Group 1 pigs were vaccinated intramuscularly with vaccine batch 202401a; Group 2 pigs were vaccinated intramuscularly with vaccine batch 202401b; Group 3 pigs were vaccinated intramuscularly with vaccine batch 202401c; Group 4 pigs were vaccinated intramuscularly with vaccine batch 202401d; Group 5 pigs were vaccinated intramuscularly with vaccine batch 202401e; and Group 6 pigs were vaccinated intramuscularly with vaccine batch 202401f. Group 7 was not vaccinated and served 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 after vaccination. Neutralizing antibody titers were monitored 14 days, 28 days, 3 months, and 5 months after the first vaccination. See Table 7 for the vaccine formulation.

[0073] Table 7: Vaccine formula

[0074]

[0075] 5.4 Neutralizing Antibody Detection

[0076] 5.4.1 Neutralizing Antibody Detection in Porcine Epidemic Diarrhea Piglets: PEDV (GD / HZ, stored in our laboratory) was diluted to 200 TCID in DMEM containing 15 μg / ml trypsin. 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 VERO 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 7.5μg / 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 PEDV-neutralizing antibodies in the test sera was calculated using the Reed-Muench method. Neutralizing antibody levels for both batches 202401a and 202401b showed an upward trend 14 days after the first dose. Neutralizing antibody levels for the 202401a vaccine continued to rise 28 days after the first dose, with overall uniformity being high and maintaining levels above 1:16 for the 202401a vaccine. However, overall antibody levels for the 202401b vaccine were lower than those for the 202401a vaccine 28 days after the first dose, and remained below 1:16 for the first five months. The results for each group are shown in Table 8.

[0077] Table 8: PEDV neutralization titer determination

[0078]

[0079] 5.4.2 Detection of neutralizing antibodies against porcine deltacoronavirus in piglets: Dilute PDCoV (HK / ZJ / 2023, preserved by our laboratory) to 200 TCID in DMEM medium. 50The virus solution (0.1 ml / well) 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 LLC-PK cells grown in a monolayer on a 96-well plate were inoculated with 100 μl per well. Four wells each containing positive controls of unneutralized virus and negative controls inoculated with cell maintenance medium alone were also inoculated with 100 μl 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 (0.01 mol / L, pH 7.2). Serum-free DMEM containing 10 μg / ml trypsin was added at 100 μl per well. The cells were incubated at 37°C in a 5% CO2 incubator for another 5 days. CPE was observed daily, and the PDCoV neutralizing antibody titer in the test sera was calculated using the Reed-Muench method. Neutralizing antibody levels for both batches 202401c and 202401d showed an upward trend 14 days after the first dose. Neutralizing antibody levels for the 202401c vaccine increased significantly 28 days after the first dose, with overall good uniformity maintained, with neutralizing antibody levels above 1:128, up to three months after the first dose. However, overall antibody levels for the 202401d vaccine were lower than those for the 202401c vaccine 28 days after the first dose, and remained below 1:128 for some groups up to five months after the first dose. The results for each group are shown in Table 9.

[0080] Table 9: PDCoV neutralization titer assay

[0081]

[0082] 5.4.3 Neutralizing antibody test for porcine rotavirus: PoRV (HF / ZJ / 2022, stored in this laboratory) was diluted to 200 TCID in DMEM medium. 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.

[0083] Table 10: PoRV neutralization titer determination

[0084]

[0085] Example 6: Subunit vaccine composition immune antibody detection and neutralization experiment

[0086] 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.

[0087] 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.

[0088] 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.

[0089] Table 11: Vaccine formula

[0090]

[0091] 6.4 Neutralizing Antibody Detection

[0092] 6.4.1 Testing for Neutralizing Antibodies in Piglets Infected with Porcine Epidemic Diarrhea: The neutralizing antibody testing procedure is the same as in 5.4.1. The results were essentially consistent with those for batches 202401a / 202401b. Antibody levels for batches 202402a / b / c showed an upward trend 14 days after the first vaccination. Twenty-eight days after the first vaccination, the antibody levels for batches 202402a / 202401b increased significantly, with overall good uniformity. This level persisted for five months, with neutralizing antibody levels above 1:16. For batch 202402c, neutralizing antibody levels 28 days after the first vaccination were lower than those for batches 202402a / 202401b, and remained below 1:16 for five months. The results are shown in Table 12.

[0093] Table 12: PEDV Neutralization Potency Assay

[0094]

[0095] 6.4.2 Testing for Neutralizing Antibody Titers against Swine Delta Coronavirus: Neutralizing antibody testing was performed using the same procedures as in Section 5.4.2. Results were generally consistent with those for batches 202401c / 202401d. Neutralizing antibody titers for vaccines from batches 202402a / 202402b / 202402c showed an upward trend 14 days after the first dose. Twenty-eight days after the first dose, neutralizing antibody titers for vaccines from batches 202402a / 202402b increased significantly, with overall good uniformity. Neutralizing antibody titers remained above 1:128 for five months post-vaccination. However, overall antibody titers for vaccine from batch 202402c were lower than those for vaccines from batches 202402a / b 28 days after the first dose, and remained below 1:128 for five months. Results for each group are shown in Table 13.

[0096] Table 13: PDCoV neutralization titer assay

[0097]

[0098] 6.4.3 Determination of Neutralizing Antibody Titers for Porcine Rotavirus: Neutralizing antibody testing was performed in the same manner as in Section 5.4.3. Results were generally consistent with those for batches 202401e / f. Neutralizing antibody titers for batches 202402a / 202402b / 202402c showed an upward trend 14 days after the first dose. Rotavirus antibody titers for batches 202402a / 202402b increased significantly 28 days after the first dose, with overall good uniformity. This level of neutralizing antibody titers remained above 1:256 for 5 months after the first dose. However, overall antibody levels for batch 202402c were lower than those for batches 202402a / b 28 days after the first dose, and remained below 1:256 for 5 months. Results for each group are shown in Table 14.

[0099] Table 14: PoRV neutralization titer determination

[0100]

[0101] The above results show that when PEDV S protein, PDCoV S protein, PRoV VP8 protein and SpaA protein are mixed, there is no mutual interference among the antigen components after immunization, and sapA enhances the duration of neutralizing antibodies.

[0102] It should be noted that the above invention content and specific embodiments are intended to demonstrate the practical application of the technical solutions provided by the present invention and should not be interpreted as limiting the scope of protection of the present invention. Those skilled in the art will be able to make various modifications, equivalent substitutions, or improvements within the spirit and principles of the present invention.

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 subunit E2 protein of a classical swine fever virus having an amino acid sequence as shown in SEQ No.

2.

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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