Porcine circovirus type 2, mycoplasma hyopneumoniae, streptococcus suis triple inactivated vaccine and preparation method thereof

Abstract

This invention discloses a triple inactivated vaccine against porcine circovirus type 2 (PCV2), Mycoplasma hyopneumoniae, and Streptococcus suis, and its preparation method, belonging to the field of veterinary biological products technology. The invention consists of an antigen and a vaccine adjuvant; the antigen is composed of PCV2 antigen, Mycoplasma hyopneumoniae antigen, and Streptococcus suis antigen; the PCV2 antigen is the PCV-2 d-cap protein obtained through genetic engineering; the Mycoplasma hyopneumoniae antigen is inactivated Mycoplasma hyopneumoniae (ES-2) bacterial suspension; the Streptococcus suis is inactivated SS2 and SS9 type Streptococcus suis bacterial suspension; the adjuvant is a novel aqueous adjuvant capable of enhancing cellular and humoral immunity. The triple inactivated vaccine against PCV2, Mycoplasma hyopneumoniae, and Streptococcus suis provided by this invention exhibits significant advantages in preventing these three swine diseases.

Description

Technical Field

[0001] This invention relates to the field of veterinary biological products, and in particular to a triple inactivated vaccine against porcine circovirus type 2, Mycoplasma hyopneumoniae (ES-2), and Streptococcus suis (SS2, SS9) and its preparation method. Background Technology

[0002] Porcine circovirus disease is an infectious disease caused by porcine circovirus type 2. After entering the body, the virus attacks the pig's immune system, causing a decline in immunity and damage to immune organs. Infected pigs suffer from emaciation, stunted growth, anemia, diarrhea, jaundice, and respiratory distress. Sows experience reproductive disorders, and there are extensive pathological changes in internal organs and skin. Furthermore, secondary or concurrent diseases such as Mycoplasma hyopneumoniae and Streptococcus suis may occur.

[0003] Mycoplasma pneumoniae in pigs is a common respiratory infectious disease in pig farming. It mainly causes pigs to exhibit symptoms such as wheezing. In actual farming, the mortality rate of a single infection with mycoplasma pneumoniae is relatively low. However, pigs with mycoplasma pneumoniae usually have a weakened immune system and are susceptible to co-infection with pathogens such as porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, swine influenza virus, and Pasteurella multocida, which exacerbate the disease and lead to death.

[0004] Streptococcal disease in swine is an acute, infectious zoonotic disease, classified as a Class II animal disease in my country. Streptococcus suis is a Gram-positive coccus, classified into 35 different serotypes based on the type of its capsular polysaccharide, among which serotypes 2 and 9 are pathogenic streptococci. Streptococcus suis does not exhibit a clear seasonality, with higher incidence rates in summer and autumn. The disease is often endemic, sometimes occurring in outbreaks. Both morbidity and mortality are relatively high. The source of infection for streptococcal disease in swine includes infected pigs, dead pigs, and carrier pigs. Large numbers of bacteria are found in their blood, muscle, internal organs, saliva, feces, urine, and nasal discharge. Clinical manifestations include septicemia, meningitis, arthritis, and purulent lymphadenitis.

[0005] Currently, only single-dose or dual-dose vaccines targeting the three diseases mentioned above are available on the market. In practice, this leads to excessive immunizations, causing significant stress to pigs. Simultaneously, the pig farming industry suffers from antibiotic overuse, resulting in increased pathogen resistance and rendering drugs largely ineffective. The porcine circovirus type 2, Mycoplasma hyopneumoniae (ES-2), and Streptococcus suis (SS2, SS9) triple inactivated vaccine provided by this invention offers three protections in a single injection, reducing the frequency of immunizations and stress, effectively solving the practical problems encountered in the pig farming industry. Summary of the Invention

[0006] To overcome the shortcomings of existing vaccines, this invention provides a method for preparing a trivalent inactivated vaccine containing porcine circovirus type 2, Mycoplasma hyopneumoniae (ES-2), and Streptococcus suis (SS2, SS9).

[0007] The technical solution of this invention is as follows:

[0008] A triple inactivated vaccine against porcine circovirus type 2 (PCV-2d-cap), Mycoplasma hyopneumoniae, and Streptococcus suis, comprising an inactivated vaccine and a vaccine adjuvant; characterized in that: the inactivated vaccine contains inactivated PCV-2d-cap protein antigen, inactivated Mycoplasma hyopneumoniae ES-2 antigen (CCTCC NO: M2018570), inactivated Streptococcus suis 2-LT antigen (CCTCC NO: M2011282), and inactivated Streptococcus suis 9-YT antigen (CCTCC NO: M2022010); the immune adjuvant is composed of aqueous liquid nanoparticle adjuvant.

[0009] As a preferred embodiment, the preparation process of the PCV-2d-cap protein antigen includes: replacing the promoter Pph of the backbone vector with the promoter SV40 with the nucleotide sequence shown in SEQ ID NO.1; inserting one or more nucleotide sequences of the PCV2d cap protein shown in SEQ ID NO.2 into the multiple cloning site downstream of the backbone vector; then transforming into competent E. coli DH10Bac cells; obtaining recombinant baculovirus plasmids through transposition recombination; plating the revived bacterial solution onto kanamycin, tetracycline, gentamicin, IPTG, and X-gal plates; culturing at 37°C in the dark; selecting white single colonies for expansion culture; transfecting Sf21 insect cells to obtain P0 generation recombinant baculovirus; performing plaque purification; picking empty plaques and passaged in Sf21 cells to obtain recombinant baculovirus; infecting High Five insect cells in suspension culture with recombinant baculovirus with a multiplicity of infection (MOI) of 0.01; centrifuging to collect the supernatant; and then purifying the PCV2d cap protein using a nickel affinity chromatography column. The backbone vector is pFastBAC Dua.

[0010] As a preferred embodiment, the adjuvant is the aqueous liquid nanoparticle adjuvant IMS251C.

[0011] As a preferred option, the inactivated Mycoplasma hyopneumoniae (ES-2) bacterial suspension should have a viable bacterial count of no less than 10⁻⁶ before inactivation. 9 CCU / ml. Inactivated 2-LT and 9-YT type Streptococcus suis bacterial suspensions, with a viable count of not less than 3 × 10⁻⁶ before inactivation. 9 CFU / ml.

[0012] The preparation method of the porcine circovirus type 2, porcine mycoplasma pneumoniae, and porcine streptococcus trivalent inactivated vaccine includes the following steps:

[0013] (1) The recombinant baculovirus and insect cell expression system were cultured in suspension using a bioreactor to obtain the porcine circovirus PCV-2d-cap protein, which was then purified and concentrated; Mycoplasma hyopneumoniae ES-2 and Streptococcus suis 2-LT and 9-YT were cultured in a fermenter, respectively.

[0014] (2) Inactivate Mycoplasma hyopneumoniae ES-2 and Streptococcus suis 2-LT and 9-YT respectively, and then concentrate them;

[0015] (3) Sterilize the adjuvant IMS251C for later use;

[0016] (4) Place the PCV-2d-cap protein obtained in step (1), the inactivated Mycoplasma hyopneumoniae ES-2 and Streptococcus suis 2-LT and 9-YT concentrated bacterial solutions obtained in step (2) into an emulsification tank, slowly add the prepared adjuvant while stirring, and stir evenly; after dispensing, the porcine circovirus type 2, Mycoplasma hyopneumoniae ES-2, Streptococcus suis 2-LT and 9-YT triple inactivated vaccine is obtained.

[0017] As a preferred method, the preparation method of porcine circovirus type 2 antigen is as follows:

[0018] (1) Construct the recombinant plasmid pFastBAC Dual-PCV-2d-cap;

[0019] (2) The recombinant plasmid pFastBAC Dual-PCV-2d-cap was transfected into sf21 insect cells using liposome transfection and the recombinant baculovirus was identified by PCR.

[0020] (3) Recombinant baculovirus was inoculated into High Five insect cells (purchased from Invitrogen) in suspension culture;

[0021] (4) Centrifuge and collect the supernatant after 3-4 days;

[0022] (5) Clarify by ultrafiltration, and then purify by nickel affinity chromatography.

[0023] As a preferred method, the method for obtaining porcine mycoplasma pneumoniae (ES-2) antigen is as follows:

[0024] (1) Inoculate Mycoplasma hyopneumoniae (ES-2) into liquid culture medium. Harvest the Mycoplasma hyopneumoniae (ES-2) bacterial solution when the culture medium turns yellow, becomes slightly turbid, and the pH is 6.8.

[0025] (2) Ultrafiltration concentration of the original Mycoplasma hyopneumoniae (ES-2) bacterial culture resulted in a live count of 10-1. 11 -10 12 CCU / ml, add 0.5 times the original volume of 1×PBS buffer and ultrafilter again to remove the supernatant. Resuspend the bacterial pellet in 1×PBS to 1% of the original volume.

[0026] (3) The concentrated porcine mycoplasma pneumoniae (ES-2) was inactivated.

[0027] As a preferred method, the method for obtaining Streptococcus suis (2-LT and 9-YT) antigens is as follows:

[0028] (1) The revitalized Streptococcus suis 2-LT and 9-YT seed cultures were transferred to TSB liquid medium at an inoculation rate of 1%, and the fermentation tank was set at 37℃ and 200rpm for 18-20h. The bacterial culture was then harvested.

[0029] (2) The bacterial suspensions of Streptococcus suis 2-LT and 9-YT were counted by dilution plate method, respectively;

[0030] (3) The harvested bacterial solution should be inactivated first;

[0031] (4) After the bacterial culture was inactivated by tangential flow ultrafiltration, the bacterial cells were resuspended with an equal volume of 1×PBS buffer, and the supernatant was removed by ultrafiltration again. This process was repeated 5 times. Finally, the cells were resuspended with an appropriate volume of 1×PBS buffer to the required concentration.

[0032] As a preferred embodiment, the inactivation treatment of *Mycoplasma hyopneumoniae* (ES-2) involves adding a thimerosal solution with a final concentration of 0.01%, mixing thoroughly, and inactivating at 37°C for 24 hours, shaking the flask 4-5 times during the process; the inactivation treatment of *Streptococcus suis* (2-LT, 9-YT) involves adding a formaldehyde solution with a final concentration of 0.3%, inactivating at 37°C for 20 hours, shaking the flask 4-5 times during the process.

[0033] The beneficial effects of this invention are as follows: the baculovirus expression system possesses post-translational modification capabilities, enabling correct folding of the expressed protein, and exhibits good biocompatibility. In the trivalent inactivated vaccine of this invention, the porcine circovirus type 2 antigen is the PCV-2d-cap protein expressed and purified by baculovirus, which exhibits good immunogenicity. The porcine circovirus type 2 (PCV2d) in the trivalent inactivated vaccine of this invention is expressed using a baculovirus vector system. The baculovirus vector of this invention replaces the promoter Pph with the promoter SV40, achieving a PCV-2d-cap protein expression level of 0.15 mg / mL, significantly improving the expression level of the target protein in the baculovirus vector. The Cap protein expression content increased from 20-50 ug / ml to approximately 150 ug / ml. Compared with bacterial, yeast, and mammalian cell expression systems, the baculovirus expression system features high expression yield and the ability to undergo post-translational processing of the product. The antigenicity, immunogenicity, and other biological activities of the product expressed by this system are similar to those of the natural protein, and the genetically engineered product can be extracted in large quantities by infecting insect larvae. PCV2d is a variant of PCV2b discovered in 2012, exhibiting higher infectivity and pathogenicity. Although there are bivalent inactivated vaccines against porcine circovirus type 2 and Mycoplasma hyopneumoniae (ES-2), they mainly use the PCV2 type Cap protein antigen, which is expressed after codon optimization based on isolates of PCV2a and PCV2b. There is currently no combination vaccine against PCV2d. Furthermore, current bivalent inactivated vaccines against porcine circovirus type 2 and Mycoplasma hyopneumoniae (ES-2) mainly use aqueous polymer adjuvants such as Gel, ISA206, aluminum salt adjuvants, mineral oil adjuvants, water-soluble adjuvants, and SP oil adjuvants. However, there are no reports on trivalent inactivated vaccines suitable for porcine circovirus type 2, Mycoplasma hyopneumoniae (ES-2), and Streptococcus suis (2-LT, 9-YT). Finding an effective, sustained-release adjuvant with minimal side effects is crucial. Among existing adjuvant materials, aqueous adjuvants are favored for their low side effects, early immune protection, and ease of use. However, their long-lasting, sustained-release effect is inferior to that of oil adjuvants. Therefore, it is essential to combine them with a long-lasting, sustained-release immune enhancer. CN110358742B describes a commercially viable porcine circovirus type 2 (PCV2d) monotherapy vaccine that produces PCV2d type Cap antibody levels after a second immunization, which are weaker than the trivalent vaccine of this invention. Furthermore, in challenge experiments, the viral load in piglets was 1 / 6 higher than that of the trivalent vaccine of this invention.

[0034] The porcine circovirus type 2 (PCV2), Mycoplasma hyopneumoniae (ES-2), and Streptococcus suis (2-LT, 9-YT) trivalent inactivated vaccine provided by this invention demonstrates significant advantages in preventing these three swine diseases and improving pig production performance. Safety tests showed that after intraperitoneal injection of double and overdose doses into Balb / c mice, the mice remained in good condition and showed no mortality after one week of continuous observation. Overdose immunization of pigs also resulted in good condition with no abnormal clinical reactions such as vomiting or redness and swelling at the injection site. Efficacy tests showed that after immunization with the trivalent vaccine, the levels of PCV2, Mycoplasma hyopneumoniae, and Streptococcus suis antibodies all exceeded the antibody titers after single-dose immunization, with no interference between their antigenic components. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. However, the drawings described below are only some embodiments of the present invention, and those skilled in the art can derive other information from these drawings without creative effort.

[0036] Figure 1 This is the purified cap protein;

[0037] Figure 2 For Western blotting validation;

[0038] Figure 3 VLP scanning electron microscopy identification of Cap protein;

[0039] Figure 4 Changes in the CCU of Mycoplasma hyopneumoniae (ES-2) during culture;

[0040] Figure 5 Growth curves of Streptococcus suis SS2 and SS9;

[0041] Figure 6 Flowchart of the preparation process for the triple inactivated vaccine;

[0042] Figure 7 This is a metabolic inhibition assay;

[0043] Figure 8 This represents the virus copy number.

[0044] Figure 9 Survival curve of mice after challenge with Streptococcus suis SS2;

[0045] Figure 10 Survival curve of mice after challenge with Streptococcus suis SS9;

[0046] Figure 11 This is an image of the lungs after the virus attack. Detailed Implementation

[0047] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the implementation of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0048] Example 1: Origin and characteristics of porcine circovirus type 2 Cap protein, Mycoplasma hyopneumoniae (ES-2), and Streptococcus suis (SS2, SS9)

[0049] 1. Origin and characteristics of Cap protein in porcine circovirus type 2

[0050] (1) Source of porcine circovirus type 2 Cap protein

[0051] The fragment (709 bp) shown in SEQ ID NO. 6 (PCV2d-ORF2-1) was artificially synthesized according to the preparation method described in patent CN110358742B as the target gene. In this embodiment, the nucleotide sequence corresponding to SEQ ID NO. 2 was used as the template to clone the target gene. The amplification program was 94℃ for 3 min; 94℃ for 30 s, 52℃ for 30 s, 72℃ for 40 s, 35 cycles; 72℃ for 10 min. The PCR product was recovered and ligated with the vector pMD19-T at 4℃ overnight. The cells were transformed into Trans5α competent cells using the heat shock method. Several single colonies were picked for PCR identification. The correctly identified recombinant plasmid was named pMD19-T-PCV2d-ORF2-1.

[0052] The expression vector pFastBAC Dual promoter P PH Replace with the SV40 promoter, wherein the SV40 promoter fragment is artificially synthesized, such as SEQ ID NO.1;

[0053] pMD19-T-PCV2d-ORF2-1 and the modified expression vector pFastBAC Dual vector were double-digested with BamHI and EcoRI restriction endonucleases, respectively. The digestion process involved 2 μL of 10×Buffer, 1 μL each of BamHI and EcoRI, 1 μL of template, and ddH2O to a final volume of 20 μL. The mixture was incubated at 37°C for 3 hours. The digestion products were then electrophoresed on a 1% agarose gel, and the DNA fragments and pFastBAC were recovered using an agarose gel extraction kit. The dual vector backbone was ligated overnight at 16°C using T4 DNA ligase and transformed into 100 μL of competent E. coli DH10Bac cells. After incubating on ice for 20 min, the cells were heat-shocked at 42°C for 90 s, followed by an ice incubation for 3 min. 500 μL of antibiotic-free LB medium was added, and the cells were incubated at 37°C for 1 h. The cells were then plated on LB agar plates containing triple antibodies (kanamycin, gentamicin, and tetracycline) and incubated at 37°C for 24 h. Positive colonies were purified by blue-white screening. Positive white colonies were aseptically picked and cultured in triple-antibody LB liquid medium for 12-16 h. The recombinant rod cell rBac-PCV2d ORF2 was then extracted.

[0054] Recombinant baculovirus rBac-PCV2d ORF2 was transfected into Sf21 insect cells using liposome transfection. After culturing at 25°C for 4 days, the supernatant was used for plaque purification. Three empty plaques were selected and passaged in Sf21 cells. Baculovirus DNA was extracted from the passaged cells and identified by PCR. The identified recombinant baculovirus was stored for future use.

[0055] High Five insect cells in suspension culture were infected with recombinant baculovirus with a multiplicity of infection (MOI) of 0.01. After suspension culture at 25°C in 250 mL Grace medium for 4 days, the supernatant was collected by centrifugation and then purified by nickel affinity chromatography.

[0056] The purified Cap protein was validated by SDS-PAGE. The results showed that the purity of the purified Cap protein was above 90%, with a concentration of 0.15 mg / mL, which was 0.05 mg / mL higher than that before optimization. Figure 1 As shown.

[0057] The purified Cap protein was validated by Western blotting. The primary antibody was a mouse PCV-2d-cap monoclonal antibody (dilution ratio 1:500), and the secondary antibody was an enzyme-labeled secondary antibody (goat anti-mouse) (dilution ratio 1:2000). The results are as follows: Figure 2 As shown, a specific band appears at 26 kDa.

[0058] The purified Cap protein was identified by scanning electron microscopy (SEM) of VLPs (virus-like particles), and the results are as follows: Figure 3 As shown.

[0059] 2. Sources and characteristics of Mycoplasma hyopneumoniae (ES-2)

[0060] CCU and time relationship of the same generation in ES-2 strains

[0061] Mycoplasma hyopneumoniae ES-2, frozen at -20℃, was removed and placed at room temperature until it reached room temperature. It was then inoculated into Friss medium and incubated at 37℃ for 3 days in a 5% CO2 incubator. The inoculated medium was then transferred at a 1:10 ratio to Friss medium, thoroughly mixed, and placed in a 37℃, 5% CO2 incubator for static culture. CCU was measured every 12 hours. Mycoplasma hyopneumoniae ES-2 was deposited on August 28, 2018, at the China Center for Type Culture Collection (CCTCC) (address: Wuhan University, Wuhan, China), and its taxonomical name is Mycoplasma hyopneumoniae ES-2, with accession number CCTCC NO: M2018570.

[0062] The results showed that the number of Mycoplasma hyopneumoniae (ES-2) in the culture medium gradually increased from day 0 to 2.5, reaching a peak on day 2.5, and then showed a linear decreasing trend. Figure 4 As shown.

[0063] 3. Sources and characteristics of Streptococcus suis (SS2, SS9)

[0064] Growth curves and LD50 values ​​of SS2 and SS9, respectively.

[0065] Streptococcus suis SS2 and SS9, frozen at -20℃, were revived until they reached optimal growth. They were then transferred to TSB medium at a 1% inoculation rate and cultured at 37℃ with shaking at 200 rpm. The OD of the bacterial culture was measured every 2 hours. 600 Streptococcus suis 2-LT, abbreviated as SS2, was deposited on August 9, 2011, at the China Center for Type Culture Collection (CCTCC) (address: Wuhan University, Wuhan, China), with accession number CCTCC NO:

[0066] M2011282. Streptococcus suis serotyp 9SS9YT, abbreviated as Streptococcus suis SS9, was deposited on January 5, 2022 at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China), with accession number CCTCC NO: M2022010.

[0067] The results showed that SS9 had a higher growth rate during the logarithmic growth phase than SS2 and entered the stationary phase at 8 hours, while SS2 entered the stationary phase at 10 hours. Figure 5 As shown.

[0068] The LD50 values ​​(median lethal dose) for SS2 and SS9 are 2.0 × 10⁻⁶ and 2.0 × 10⁻⁶, respectively. 8 CFU, 3.0 × 10 7 CFU.

[0069] Example 2: Preparation process of a trivalent inactivated vaccine against porcine circovirus type 2, Mycoplasma hyopneumoniae (ES-2), and Streptococcus suis (SS2, SS9):

[0070] 1. Preparation of porcine circovirus type 2 semi-finished antigen

[0071] (1) High Five insect cells infected with recombinant baculovirus were transferred to a 500L suspension cell culture bioreactor for suspension culture, and the target protein PCV-2d-cap was obtained as in Example 1.

[0072] (2) The total concentration of the expressed protein was determined by the BCA method after purification:

[0073] (1) Preparation of working solution: Based on the quantity of standard and sample, prepare an appropriate amount of BCA working solution by adding 1 volume of BCA reagent B to 50 volumes of BCA reagent A (50:1).

[0074] (2) Diluting the standard: Take 10 μL of LSA standard and dilute it with PBS to 100 μL, so that the final concentration of the standard is 0.5 mg / mL. Add the standard to the protein standard wells of a 96-well plate at the following ratios: 0, 1, 2, 4, 8, 12, 16, 20 μL, and add PBS to make up to 20 μL.

[0075] (3) Dilute the sample to a suitable concentration, with a total volume of 20 μL;

[0076] (4) Add 200 μL BCA working solution to each well and incubate at 37°C for 15-30 minutes. Measure the absorbance at 562 nm using a microplate reader and calculate the total protein concentration based on the standard curve. The concentration is 1.0 mg / mL.

[0077] 2. Preparation of porcine mycoplasma pneumoniae (ES-2) semi-finished antigen

[0078] (1) Prepare liquid culture medium for Mycoplasma hyopneumoniae (ES-2) according to Friss medium formula (1L) (1×Hanks 282mL, basal solution 376mL, porcine serum 235mL, 50% (w / v) glucose 47mL, 10% (w / v) hydrolyzed milk protein 47mL, 0.4% (w / v) phenol red 8.46mL, 1mol / L NaOH 4.54mL, pH 7.5).

[0079] (2) Mycoplasma hyopneumoniae (ES-2) was inoculated into Friss liquid medium at a ratio of 1:5 (v:v) and cultured in a fermenter at 37°C for 48 hours. The bacterial culture was harvested when the medium turned yellow, became slightly turbid, and the pH dropped to 6.8.

[0080] (3) Ultrafiltration concentration of Mycoplasma hyopneumoniae (ES-2) harvest fluid to achieve a viable count of 10-1. 12 -10 13 CCU / ml; add 1 / 2 volume of 1×PBS buffer and ultrafilter again to remove the supernatant. The bacterial pellet is then broken up and resuspended in 1×PBS to 1% of the original volume.

[0081] (4) Add a thimerosal solution with a final concentration of 0.01% and place it at 2-8℃ for 24 hours for inactivation treatment. Shake well every 2 hours during the period to ensure complete inactivation.

[0082] 3. Preparation of semi-finished antigens of Streptococcus suis (SS2, SS9)

[0083] (1) Transfer Streptococcus suis SS2 and SS9 to TSB liquid medium at an inoculation rate of 1%, and culture in a fermenter at 37°C and 250 rpm for 18 h, and harvest the bacterial solution.

[0084] (2) The bacterial suspensions of Streptococcus suis SS2 and SS9 were counted by dilution plate method, respectively;

[0085] (3) Inactivate the harvested bacterial solution with 0.3% formaldehyde;

[0086] (4) After the bacterial culture was inactivated by tangential flow ultrafiltration, the bacterial cells were resuspended with an equal volume of 1×PBS buffer, and the supernatant was removed by ultrafiltration again. This process was repeated 5 times. Finally, the cells were resuspended with an appropriate volume of 1×PBS buffer to the required concentration.

[0087] 4. Preparation of Trivalent Inactivated Vaccine

[0088] according to Figure 6 The process flow chart for preparing the trivalent inactivated vaccine is shown.

[0089] (1) Sterilize the adjuvant by high-pressure steam at 121°C for 20 minutes.

[0090] (2) Add purified Cap protein (final concentration 20 μg / ml) and Mycoplasma hyopneumoniae (ES-2) (final concentration 1×10⁻⁶). 9 CCU / head dose), Streptococcus SS2 and SS9 (final concentration of 2×10⁻⁶ each) 9 Stir the emulsifier (CFU / ml) in an emulsification tank, and slowly add the prepared adjuvant while stirring. Set the stirring parameters to 0.5 Kr / min for 30 min.

[0091] (3) The porcine circovirus type 2, porcine mycoplasma pneumoniae (ES-2), and porcine streptococcus (SS2, SS9) triple inactivated vaccine is obtained.

[0092] Example 3: Screening of adjuvants for porcine trivalent inactivated vaccine

[0093] 1. Alternative adjuvants

[0094] This invention mainly selects eight adjuvants: Summit Poly Solution (Sps, water adjuvant), CPC (water adjuvant), ISA201 (biphase oil emulsion adjuvant), GEL02 (water-soluble polymerization adjuvant), IMS1313 (water-soluble nano adjuvant), IMS251C (aqueous liquid nanoparticle adjuvant), aluminum hydroxide adjuvant (aluminum glue adjuvant), and white oil 15A adjuvant (mineral oil adjuvant). ISA201 adjuvant is a novel water-in-oil-in-water (W / O / W) biphasic oil emulsion adjuvant. Its main component is highly refined light mineral oil, with small amounts of plant-derived mannitol and oleic acid. Vaccines prepared with this adjuvant differ from traditional mineral oil vaccines in that they have a water-in-oil (O / W) formulation, resulting in reduced viscosity, easier injection, and fewer side effects, while retaining the antigen-presenting efficacy of oil adjuvants. GEL02 is a water-soluble polymer adjuvant (GEL), whose main component is sodium polyacrylate. This adjuvant has the advantages of high antigen loading, stable properties, and simple emulsification process. It can induce humoral immunity and significantly promote T lymphocyte proliferation and differentiation, thereby producing a high level of cellular immunity. IMS1313 is a water-soluble nanoadjuvant that aggregates lymphocytes through micro-permeation of nanoparticles into the lymphatic system, promoting the uptake of antigens by antigen-presenting cells, ultimately acting as antigen-presenting cells. Montanide adjuvant, developed by Seppic in France, includes the Montanide ISA series of adjuvants and Montanide... Montanide IMS series adjuvants are water-soluble complexes containing immunologically active organic compounds and special excipients, which can improve the poor safety profile of traditional oil adjuvants and aluminum salt adjuvants in immunogenic formulations.

[0095] 2. Screening of superior adjuvants

[0096] (1) Using the above 8 alternative adjuvants, trivalent inactivated vaccines were prepared according to Example 2.

[0097] Ninety BALB / c mice weighing 18-22g were acclimatized for one week and then divided into nine groups: eight groups receiving alternative adjuvants and one control group. Each mouse received an intramuscular injection of 0.2ml, while the control group received the same dose of PBS solution. Seven days later, a second immunization was performed using the same method, with a dose of 0.2ml.

[0098] (2) 14 days after the second immunization, blood was collected from the posterior orbital venous plexus in the eight alternative adjuvant groups and the control group. The blood in the centrifuge tube was then placed in a 37°C incubator for 1 hour, and then placed in a 4°C incubator overnight. After the blood clots and shrinks, the tube was centrifuged at 4,000 rpm for 10 minutes. The supernatant was collected in a clean centrifuge tube, which is the serum to be tested, and stored at -20°C.

[0099] (3) The serum titer was detected by indirect ELISA. Cap protein and SS2 were coated on the plate respectively. The amount of Cap protein coated per well was 50 ng, and the amount of SS2 coated per well was 300 ng of the total protein concentration after lysis. The serum to be tested was diluted 40 times, and then enzyme-labeled secondary antibody (goat anti-mouse) was added. Finally, TMB was added for color development, and the absorbance at 450 nm was measured.

[0100] The results showed that the corresponding SS2 antibody levels, from highest to lowest, were ISA201, Sps, CPC, white oil 15A, IMS251C, aluminum hydroxide, GEL02, and IMS1313; the corresponding Cap protein antibody levels, from highest to lowest, were aluminum hydroxide, ISA201, Sps, IMS251C, white oil 15A, GEL02, CPC, and IMS1313, as shown in Table 1.

[0101]

[0102] Table 1. Antibody detection results of Streptococcus SS2 and Cap proteins in mice of different adjuvant groups 14 days after secondary immunization.

[0103] (4) 14 days after the second immunization, SS2 challenge was performed with a dose of 4.54 × 10⁻⁶. 8 CFU / mouse, observe the survival of mice for 5 consecutive days, and calculate the survival rate.

[0104] The results showed that IMS251C had the best protective titer against Streptococcus suis SS2 challenge, reaching 80%, as shown in Table 2.

[0105] Table 2. Protective effect of different adjuvant groups against challenge with Streptococcus suis SS2.

[0106]

[0107] Based on the antibody levels and the protective effect against viral challenge, IMS251C was initially selected as the adjuvant for the subsequent formulation of the trivalent inactivated vaccine.

[0108] Example 4: Safety testing and immunization efficacy evaluation of the porcine trivalent vaccine

[0109] Safety testing of the porcine trivalent vaccine in mice.

[0110] Twenty BALB / c mice weighing 18-22g were acclimatized for one week and then divided into two groups: a triple vaccine group (adjuvant IMS251C, hereinafter the same) and a control group. Each mouse was injected intramuscularly with 0.2ml, while the control group was injected with the same dose of PBS solution. Mice were observed for 14 consecutive days. All mice in both groups were healthy and showed no local or systemic adverse reactions. This result indicates that the porcine triple vaccine prepared in Example 2 is safe for mice.

[0111] Antibody production levels of the porcine trivalent vaccine in mice

[0112] Seventy BALB / c mice weighing 18-22g were acclimatized for one week and then divided into seven groups: Group A (commercial porcine circovirus monotherapy vaccine group (i.e., Keyuanning, provided by Wuhan Keqian Biotechnology Co., Ltd.)), Group B (porcine streptococcus SS2+SS9 inactivated vaccine group (SS2 and SS9 2×10⁻⁶ each)). 9 CFU / mL (the same below), Group C (PCV2dCap (20μg / ml) + SS2 + SS9), Group D (PCV2dCap (20μg / ml) + Mycoplasma hyopneumoniae (ES-2) (1×10) 9 CCU / head dose), Group E (SS2+SS9+ Mycoplasma hyopneumoniae (ES-2) (1×10) 9 The mice were divided into three groups: CCU (one dose), F (pig trivalent vaccine group), and G (control group), with 10 mice in each group. Each mouse was injected intramuscularly with 0.2 ml of the vaccine. The control group was injected with the same dose of PBS solution. Seven days later, the mice were immunized a second time using the same method, with a dose of 0.2 ml. Fourteen days after the second immunization, the antibody levels in each group were measured according to Example 3.

[0113] Table 3 shows the levels of PCV2dCap protein, SS2, and SS9 antibodies produced in different groups.

[0114]

[0115]

[0116] The results showed that after 40-fold dilution of the serum samples, the antibody level corresponding to the PCV2d Cap protein produced by the triple vaccine group (Group F) was slightly higher than that of the porcine circovirus monotherapy group (Group A) and the dual vaccine group (Groups C and D). The antibodies corresponding to SS2 and SS9 produced by the triple vaccine group (Group F) were comparable to those of the SS2+SS9 inactivated vaccine group (Group B) and the dual vaccine group (Groups C and E). The results are shown in Table 3.

[0117] Evaluation of the immunization efficacy of the porcine trivalent vaccine in New Zealand white rabbits

[0118] Twenty-five New Zealand white rabbits weighing 1-1.2 kg were acclimatized for one week and then randomly divided into five groups: Group A (commercial mycoplasma inactivated vaccine group, namely Kechuaning, provided by Wuhan Keqian Biotechnology Co., Ltd.) and Group D (PCV 2d Cap (20μg / ml) + Mycoplasma hyopneumoniae (ES-2) (1×10)). 9 CCU / dosage), Group E (SS2+SS9+Mycoplasma hyopneumoniae (ES-2) (1×10) 9 The vaccine was divided into three groups: CCU (one dose), F (triple vaccine group), and G (control group). Each rabbit received an intramuscular injection of 0.5 ml, while the control group received the same dose of PBS solution. Seven days later, a second immunization was administered using the same method, with a dose of 0.5 ml. Thirty days post-immunization, blood was collected from the marginal ear vein, serum was separated, and IHA (indirect hemagglutination assay) was used to determine the antibody titer for Mycoplasma hyopneumoniae 2 (ES-2).

[0119] IHA specific steps:

[0120] (1) Take 1 mL of sheep red blood cell suspension fixed with 10% dialdehyde, centrifuge at 3000 r / min, discard the supernatant, wash once with 0.1 mol / L pH 4.0 acetate buffer, centrifuge at 3000 r / min, discard the supernatant, add 0.5 mL of concentrated lysed porcine mycoplasma pneumoniae (ES-2) and 9.5 mL of mol / L pH 4.0 acetate buffer, incubate at 37℃ for 1 h, centrifuge at 3000 r / min, discard the supernatant, wash 5 times with 0.1 mol / L pH 7.2 phosphate buffer, centrifuge at 3000 r / min, discard the supernatant, add 10 mL of 5% fetal bovine serum phosphate buffer to obtain sensitized red blood cells, and store at 4℃ for later use.

[0121] Take the sample diluent and add 25 μL / well to each well of a 96-well "V"-shaped microcoagulation plate. Add equal volumes of negative serum, positive serum, and test serum to the first well of each row, performing serial dilutions. Leave the last well as a blank control. Add 25 μL / well of 1% sensitized red blood cell suspension to each well. Incubate at 37℃ for 1 hour and observe the results. Serum titer is expressed as the highest serum dilution at which more than 50% agglutination of sensitized red blood cells occurs in the well. Testing showed that a titer >1:5 was considered positive.

[0122] The results showed that the antibody levels produced by the mycoplasma inactivated vaccine group (group a) were consistent with those of the triple vaccine group (group F). The titer of the triple vaccine group (group F) was greater than 1:20, indicating that the triple vaccine had protective efficacy against pigs. The results are shown in Table 4.

[0123] Table 4. Antibody levels corresponding to mycoplasma in different groups

[0124]

[0125] Metabolic inhibition test

[0126] Take 7 sterile vials containing 2mL Friis liquid culture medium. Add 0.4mL of Mycoplasma hyopneumoniae ES-2 strain culture to the first 3 vials (5, 4, and 3 respectively). 5 10 4 10 3 CCU) and the isolated serum, the isolated serum being the serum from the inactivated single vaccine group or the triple vaccine group, the last 3 animals (5-1, 4-1, 3-1) were given 0.4 ml of Mycoplasma hyopneumoniae ES-2 strain culture respectively (10 ml in each order). 5 10 4 10 3 (CCU), the last (negative) sample was added with 0.4 ml of 1×PBS as a negative control. The samples were incubated at 37°C for 15 days, and the color change of the culture medium was observed.

[0127] The results showed that the addition of serum from the inactivated monotherapy and triple vaccine groups did not change the color of the culture medium, consistent with the negative control. However, the addition of only Mycoplasma hyopneumoniae ES-2 changed the color of the culture medium from red to yellow, indicating that the antibodies produced by the inactivated monotherapy and triple vaccine groups had a metabolic inhibitory effect on Mycoplasma hyopneumoniae ES-2. (See attached figures.) Figure 7 As shown.

[0128] The protective efficacy of the trivalent porcine circovirus (PCV-2d), Streptococcus suis SS2, and Streptococcus suis SS9 against challenge.

[0129] The protective efficacy of the porcine trivalent vaccine against porcine circovirus (PCV)-2d challenge was investigated in a study of 30 18-22g BALB / c mice. These mice were acclimatized for one week and then randomly divided into three groups: a commercial porcine circovirus monotherapy group, a trivalent vaccine group, and a control group. Each mouse received an intramuscular injection of 0.2ml, while the control group received the same dose of PBS solution. Seven days later, a second immunization was administered using the same method, with a dose of 0.2ml. Fourteen days post-immunization, the mice were challenged with the PCV-2d strain (≥10...). 6.5 TCID 50 Each mouse was injected intraperitoneally with 0.5 mL of the virus ( / mL). 21 days after challenge, blood was collected from the retro-orbital venous plexus, serum was separated, and the viral copy number was determined.

[0130] To determine the viral copy number, quantitative real-time PCR (qPCR) is required to obtain the Ct value. The qPCR reaction system includes: 10 μL of 2×SuperRealPreMixPlus, 0.5 μL each of upstream and downstream primers and probe, 2 μL of serum, and ddH2O to a final volume of 20 μL. The upstream primer sequence is PCV2-F2.

[0131] 5'-CGGATATTGTAGTCCTGGTCGTA-3', downstream primer sequence is PCV2-R2:

[0132] 5'-CCTGTCCTAGATTCCCCTATTGATT-3', probe is P2 Probe:

[0133] FAM-5'-CTAGGCCTACGTGGTCTACATTTC-3'-TAMRA. The quantitative PCR program included: 95℃ for 2 min; 95℃ for 10 s, 60℃ for 35 s, 40 cycles. Melting curve analysis was performed from 60℃ to 95℃. The relationship between Ct value and copy number was: Ct = -2.3693 × Log 10 (Copy count) + 33.628 (R) 2 =0.9999).

[0134] The results showed that the viral copy numbers in both the commercial porcine circovirus monotherapy group and the trivalent vaccine group were significantly lower than those in the control group, indicating that the porcine trivalent vaccine had a good protective effect against PCV-2d challenge. Figure 8 As shown.

[0135] The protective effect of the trivalent porcine vaccine against Streptococcus suis SS2 challenge.

[0136] Thirty BALB / c mice weighing 18-22g were acclimatized for one week and then divided into three groups: a Streptococcus suis SS2+SS9 inactivated vaccine group (SS2 and SS9 were 2×10⁻⁶ each). 9(CFU / mL), triple vaccine group, and control group. Each mouse received an intramuscular injection of 0.2 ml, while the control group received the same dose of PBS solution. Seven days later, a second immunization was performed using the same method, with a dose of 0.2 ml. Fourteen days post-immunization, mice were challenged with SS2 virus at a dose of 5.38 × 10⁻⁶ CFU / mL. 8 CFU / mouse, and observe the survival of mice for 5 consecutive days.

[0137] The results showed that after challenging mice with Streptococcus suis SS2, the challenge dose was 5.38 × 10⁻⁶. 8 CFU / animal. In the control group, 100% died after 3 days. The SS2+SS9 inactivated vaccine group showed a protective effect of 40%, and the porcine triple vaccine group showed a protective effect of 60%. This indicates that the porcine triple vaccine provides good protection against challenge with Streptococcus suis SS2. Figure 9 As shown.

[0138] The protective effect of the trivalent porcine vaccine against Streptococcus suis SS9 challenge.

[0139] Thirty BALB / c mice weighing 18-22g were acclimatized for one week and then divided into three groups: a Streptococcus suis SS2+SS9 inactivated vaccine group (SS2 and SS9 were 2×10⁻⁶ each). 9 (CFU / mL), triple vaccine group, and control group. Each mouse received an intramuscular injection of 0.2 ml, while the control group received the same dose of PBS solution. Seven days later, a second immunization was performed using the same method, with a dose of 0.2 ml. Fourteen days post-immunization, mice were challenged with SS9 virus at a dose of 9.6 × 10⁻⁶ CFU / mL. 7 CFU / mouse, and observe the survival of mice for 5 consecutive days.

[0140] The results showed that after challenging mice with Streptococcus suis SS9, the challenge dose was 9.6 × 10⁻⁶. 7 CFU / animal. In the control group, 90% died after 2 days. The SS2+SS9 inactivated vaccine group showed a protective effect of 70%, and the porcine triple vaccine group showed a protective effect of 90%, indicating that the porcine triple vaccine provides better protection against Streptococcus suis SS9 challenge. The results are as follows: Figure 10 As shown.

[0141] Example 5: Secondary screening of adjuvants for porcine trivalent inactivated vaccine

[0142] (1) The three adjuvants (IMS251C, white oil 15A, and GEL02) obtained in the initial screening in Example 3 were used to prepare trivalent inactivated vaccines according to Example 2.

[0143] (2) Eighty-five healthy, susceptible piglets aged 14–21 days were selected and randomly divided into four groups: a triple vaccine IMS251C adjuvant group (20 piglets), a triple vaccine white oil 15A adjuvant group (20 piglets), a triple vaccine white oil GEL02 adjuvant group (20 piglets), a challenge control group (20 piglets), and a circular vaccine blank control group (5 piglets). Each piglet was injected intramuscularly into the neck with 2 ml of PBS solution, while the control group was injected with the same dose. Twenty-one days later, a second immunization was performed using the same method, with a dose of 2 ml.

[0144] (3) Fourteen days after the second immunization, five animals from each group were randomly selected for blood collection from the anterior vena cava to determine the antibody levels corresponding to Cap protein, SS2, SS9, and mycoplasma (detected according to the instructions of the IDEXX kit). When determining the antibody titer corresponding to Cap protein, dilutions of 40, 80, 160, 320, 640, 1280, 2560, and 5120 times were performed.

[0145] The results showed that the levels of Cap protein, SS2, SS9 and mycoplasma antibodies produced by the IMS251C adjuvant group were superior to those of the white oil 15A adjuvant group and the GEL02 adjuvant group. Therefore, IMS251C was selected as the adjuvant for the subsequent preparation of the trivalent inactivated vaccine. The results are shown in Table 5.

[0146] Table 5 shows the antibody levels corresponding to Cap protein, SS2, SS9, and mycoplasma in different groups.

[0147]

[0148] Fourteen days after the second immunization, five piglets from both the triple vaccine group and the challenge control group were simultaneously challenged with PCV2WH strain virus solution (virus content of 1.0 × 10⁷ TCID₅₀ / ml) via intranasal and intramuscular injections. On the day of infection, the weight of all piglets was measured. Twenty-eight days post-infection, the weight of all piglets was measured again; blood was collected from all piglets, serum was separated, and viremia was detected using porcine circovirus type 2 specific PCR. All piglets were euthanized, and inguinal and mesenteric lymph nodes were collected for histological and immunohistochemical (IHC) analysis. The morbidity rate in each experimental group was statistically analyzed.

[0149] Relative weight gain rate = (average daily weight gain of control group - average daily weight gain of piglets in challenge test) / average daily weight gain of piglets in triple vaccine group;

[0150] Average daily weight gain of the control group = (sum of weight of control piglets 28 days after challenge - sum of weight of control piglets on the day of challenge) / (28 days × 5 piglets);

[0151] Average daily weight gain of piglets in the triple vaccine group = (sum of weight of piglets in the triple vaccine group 28 days after challenge - sum of weight of piglets in the triple vaccine group on the day of challenge) / (28 days × 5 piglets);

[0152] Average daily weight gain of piglets in the challenge experiment = (piglet weight 28 days after challenge - piglet weight on the day of challenge) / 28 days.

[0153] The results showed that the incidence rate in the IMS251C adjuvant group was significantly lower than that in the challenge control group, and also lower than that in the white oil 15A adjuvant group and the white oil GEL02 adjuvant group, indicating that the IMS251C adjuvant group had the best protective effect against challenge of PCV2 WH strain (see Table 6).

[0154] Table 6 Results of PCV2 WH strain challenge protection test 14 days after second immunization.

[0155]

[0156]

[0157] Fourteen days after the second vaccination, five piglets from the triple vaccine group and the challenge control group were challenged with the virus simultaneously. Each piglet was injected with 10 ml of Mycoplasma hyopneumoniae ES-2 via the trachea. After 28 days of observation, the piglets were euthanized. The pneumonia lesions of the experimental pigs were scored according to the Goodwin 55-point scale, and the reduction rate of pneumonia lesions was calculated.

[0158] The results showed that the IMS251C adjuvant group had the highest reduction rate of pneumonia lesions, indicating that the IMS251C adjuvant group had the best protective effect against challenge with Mycoplasma pneumoniae ES-2 (see Table 7).

[0159] Table 7 Results of the Mycoplasma hyopneumoniae ES-2 challenge protection test 14 days after the second immunization.

[0160]

[0161] Fourteen days after the second immunization, patients were challenged with SS2 and SS9, with the SS2 challenge dose being 1.638 × 10⁻⁶. 7 CFU / head, SS9 challenge dose is 4×10 9 CFU / head, observe the growth status of piglets for 14 consecutive days.

[0162] The results showed that IMS251C had the best protective titer against Streptococcus suis SS2 challenge, with 100% immune protection. Meanwhile, IMS251C had the best protective effect against Streptococcus suis SS9 challenge, with 60% immune protection. The results are shown in Tables 8 and 9.

[0163] Table 8. Protective effect of different adjuvant groups against challenge with Streptococcus suis SS2.

[0164]

[0165] Table 9. Protective effect of different adjuvant groups against challenge with Streptococcus suis SS9.

[0166]

[0167] Example 6: Evaluation of the safety and immunogenicity of the porcine trivalent vaccine in pigs.

[0168] Safety testing of the trivalent porcine vaccine in piglets

[0169] Ten healthy, susceptible piglets aged 14–21 days were selected and randomly divided into two groups: a triple vaccine group and a control group. Each piglet received a 4 ml intramuscular injection in the neck, while the control group received the same dose of PBS solution. The piglets were observed for 14 consecutive days. Their mental state, respiration, and feed intake were all normal. No redness, swelling, or induration was observed at the vaccine injection site, and there was no local inflammatory reaction.

[0170] No abnormal changes in body temperature were observed in piglets after vaccination with the trivalent vaccine. The temperature only slightly increased one day after immunization (not exceeding 0.5℃ above pre-vaccination temperature), returning to basal body temperature from the second day onwards (see Table 10). The average daily weight gain of piglets 14 days after vaccination was 0.2 kg (see Table 11), indicating that the trivalent vaccine for pigs is safe for piglets.

[0171] Table 10. Results of body temperature measurement in piglets after a single overdose of vaccine.

[0172]

[0173]

[0174] Table 11 Results of piglet weight gain 14 days after vaccination

[0175]

[0176] Evaluation of the immunization efficacy of the porcine trivalent vaccine in piglets by antibody levels

[0177] Thirty healthy, susceptible piglets aged 14–21 days were selected and randomly divided into six groups: a commercial porcine circovirus monotherapy vaccine group (Keyuanning) (5 piglets), a commercial mycoplasma inactivated vaccine group (Kechuanning) (5 piglets), a commercial streptococcus inactivated vaccine group (Kelianning) (5 piglets), a porcine streptococcus SS2+SS9 inactivated vaccine group (5 piglets), a triple vaccine group (5 piglets), and a control group (5 piglets). Each piglet was injected intramuscularly into the neck with 2 ml of PBS solution, while the control group was injected with the same dose. Twenty-one days later, a second immunization was performed using the same method, with a dose of 2 ml. Blood samples were collected before immunization and after immunization (days 14, 21, 28, 35, and 48). The levels of PCV2dCap protein, SS2, SS9, and mycoplasma-related antibodies were measured according to Example 5.

[0178] The results showed that the antibody titers corresponding to PCV2d Cap protein, SS2, and SS9 produced by the triple vaccine group all increased with the extension of time after immunization. By day 48, the average antibody titers were 512±175.27, 0.866±0.133, and 0.733±0.152, respectively. The antibody titer corresponding to mycoplasma reached its maximum value (1.022±0.051) on day 35. Compared with the commercial porcine circovirus monotherapy group, the commercial mycoplasma inactivated vaccine group, and the suicidal streptococcus SS2+SS9 inactivated vaccine group, the average antibody titers produced by the triple vaccine group were all better than those of the inactivated vaccine group. The results are shown in Tables 12, 13, 14, and 15.

[0179] Table 12 Average antibody titers corresponding to PCV2d Cap protein at different time points.

[0180]

[0181] Table 13 Average antibody titers for Streptococcus SS2 at different time points

[0182]

[0183] Table 14 Average antibody titers for Streptococcus SS9 at different time points

[0184]

[0185] Table 15 Average antibody titers corresponding to mycoplasma at different time points.

[0186]

[0187]

[0188] The protective effect of the porcine trivalent vaccine against PCV2 WH strain challenge

[0189] Twenty healthy, susceptible piglets aged 14–21 days were selected and then divided into four groups: Keyuanning (5 piglets), triple vaccine (5 piglets), challenge control (5 piglets), and blank control (5 piglets). Fourteen days after the second immunization, the piglets were challenged according to Example 5, and the incidence of disease in each experimental group was recorded.

[0190] The results showed that the incidence rate in the triple vaccine group was significantly lower than that in the control group, with an incidence rate of 0, which was comparable to that of the commercial single vaccine, indicating that the triple vaccine had a good protective effect against challenge of PCV2 WH strain (see Table 16).

[0191] Table 16 Results of PCV2 WH strain challenge protection test 14 days after secondary immunization.

[0192]

[0193]

[0194] The protective effect of the trivalent porcine vaccine against challenge with Mycoplasma hyopneumoniae (ES-2)

[0195] Fifteen healthy, susceptible piglets aged 14–21 days were selected and then divided into three groups: 5 piglets receiving the second immunization, 5 piglets receiving the triple vaccine, and 5 piglets receiving the challenge control. Fourteen days after the second immunization, the piglets were challenged with the virus according to Example 5, and the reduction rate of pneumonia lesions was calculated.

[0196] The results showed that the reduction rate of pneumonia lesions in the triple vaccine group was over 60%, comparable to that of commercial single vaccines (see Table 17), and there were no significant lesions compared to the control group (see Table 17). Figure 11 The results indicate that the triple vaccine has a good protective effect against challenge with Mycoplasma hyopneumoniae ES-2.

[0197] Table 17 Results of the Mycoplasma hyopneumoniae ES-2 challenge protection test 14 days after the second immunization.

[0198]

[0199] The protective effect of the trivalent porcine vaccine against Streptococcus suis type 2.

[0200] Fifteen healthy, susceptible piglets aged 14–21 days were selected and then divided into three groups: piglets receiving styraxone (5 piglets), piglets receiving triple vaccine (5 piglets), and piglets receiving challenge control (5 piglets). Fourteen days after the second immunization, the piglets were challenged with Streptococcus suis SS2 according to Example 5, and their growth status was observed for 14 consecutive days.

[0201] The results showed that the triple vaccine provided comparable protection against Streptococcus suis SS2 challenge to commercially available single vaccines, with 100% immune protection. The results are shown in Table 18.

[0202] Table 18 Protective effect of different adjuvant groups against challenge with Streptococcus suis SS2

[0203]

[0204] The protective effect of the trivalent porcine vaccine against challenge with Streptococcus suis serotype 9.

[0205] Fifteen healthy, susceptible piglets aged 14–21 days were selected and then divided into three groups: SS2+SS9 (5 piglets), triple vaccine (5 piglets), and challenge control (5 piglets). Fourteen days after the second immunization, the piglets were challenged with Streptococcus suis SS2 according to Example 5, and their growth status was observed for 14 consecutive days.

[0206] The results showed that the triple vaccine had the best protective effect against Streptococcus suis SS9 challenge, with an immune protection rate of 60%, as shown in Table 19.

[0207] Table 19 Protective effect of different adjuvant groups against challenge with Streptococcus suis SS9

[0208] sequence list <110> Wuhan Keqian Biotechnology Co., Ltd. <120> Porcine circovirus type 2, Mycoplasma hyopneumoniae, and Streptococcus suis triple inactivated vaccine and their preparation methods <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 358 <212> DNA <213> Artificial Sequence <400> 1 ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg 60 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg 120 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc 180 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca 240 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc 300 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaa 358 <210> 2 <211> 709 <212> DNA <213> Artificial Sequence <400> 2 gccaccatgg taagcgctat tgttttatat gtgcttttgg cggcggcggc gcattctgcc 60 tttgcggcgg atctaccccg ccaccgttac cgctggagaa ggaaaaatgg catcttcaac 120 acccgcctct cccgcaccat cggttatact gtcaagaaaa ccacagtcag aacgccctcc 180 tggaatgtgg acatgatgag atttaatatt aatgattttc ttcccccagg agggggctca 240 aaccccctca ctgtgccctt tgaatactac agaataagga aggttaaggt tgaattctgg 300 ccctgctccc caatcaccca gggtgacagg ggagtgggct ccactgctgt tattctagat 360 gataactttg taacaaaggc caatgcccta acctatgacc cctatgtaaa ctactcctcc 420 cgccatacca taacccagcc cttctcctac cactcccggt actttacccc gaaacctgtc 480 cttgatagga caatcgatta cttccaaccc aataacaaaa gaaatcaact ctggctgaga 540 ctacaaacta ctggaaatgt agaccatgta ggcctcggca ctgcgttcga aaacagtata 600 tacgaccagg actacaatat ccgtataacc atgtatgtac aattcagaga atttaatctt 660 aaagaccccc cacttaaccc aaagtgacca tcatcaccat caccattaa 709

Claims

1. A triple inactivated vaccine against porcine circovirus type 2, Mycoplasma hyopneumoniae, and Streptococcus suis, comprising an inactivated vaccine and a vaccine adjuvant, characterized in that: The inactivated vaccine contains inactivated porcine circovirus type 2 (PCV) 2d-cap protein antigen, inactivated Mycoplasma hyopneumoniae ES-2 antigen (CCTCC NO: M2018570), inactivated Streptococcus suis 2-LT antigen (CCTCC NO: M2011282), and inactivated Streptococcus suis 9-YT antigen (CCTCC NO: M2022010). The PCV-2d-cap protein antigen was obtained by inserting the PCV-2d-cap protein nucleotide sequence shown in SEQ ID NO.2 into the pFastBAC Dual vector, which replaced the SV40 promoter shown in SEQ ID NO.1, to construct a recombinant baculovirus. The virus was then used to infect High Five insect cells at an MOI of 0.01 and purified by nickel column affinity chromatography. The adjuvant is an aqueous liquid nanoparticle adjuvant, IMS251C.

2. The porcine circovirus type 2, porcine mycoplasma pneumoniae, and porcine streptococcus trivalent inactivated vaccine as described in claim 1, characterized in that: The number of live bacteria of Mycoplasma hyopneumoniae before inactivation should not be less than 10. 9 CFU / ml.

3. The porcine circovirus type 2, porcine mycoplasma pneumoniae, and porcine streptococcus trivalent inactivated vaccine as described in claim 1, characterized in that: Before inactivation, the viable count of Streptococcus suis 2-LT and 9-YT should not be less than 3 × 10⁻⁶. 9 CFU / ml.

4. The method for preparing the porcine circovirus type 2, mycoplasma hyopneumoniae, and streptococcus suis trivalent inactivated vaccine according to any one of claims 1 to 3, characterized in that, Including the following steps: (1) The recombinant baculovirus and insect cell expression system were cultured in suspension using a bioreactor to obtain the porcine circovirus PCV-2d-cap protein, which was then purified and concentrated; Mycoplasma hyopneumoniae ES-2 and Streptococcus suis 2-LT and 9-YT were cultured in a fermenter, respectively. (2) Inactivate Mycoplasma hyopneumoniae ES-2 and Streptococcus suis 2-LT and 9-YT respectively, and then concentrate them; (3) Sterilize the aqueous liquid nanoparticle adjuvant IMS251C for later use; (4) Place the PCV-2d-cap protein obtained in step (1), the inactivated Mycoplasma hyopneumoniae ES-2 and Streptococcus suis 2-LT and SS9 concentrated bacterial solutions obtained in step (2) into an emulsification tank, slowly add the prepared adjuvant while stirring, and stir evenly to obtain the porcine circovirus type 2, Mycoplasma hyopneumoniae ES-2, Streptococcus suis 2-LT and 9-YT triple inactivated vaccine.

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