A novel adjuvant and its use in the preparation of an inactivated vaccine against bovine tuberous skin disease and positive sera thereof

Abstract

The application discloses a squalene-based water-in-oil-in-water vaccine adjuvant and a vaccine composition and application thereof. The adjuvant comprises squalene, tocopherol, lipophilic surfactant and a hydrophilic surfactant combination composed of polyglyceryl dioleate, decaglyceryl monolaurate and polyoxyethylene sorbitan trioleate in a specific mass ratio. The specific combination is the key to forming a stable and uniform squalene multiple emulsion. The vaccine composition prepared by mixing and emulsifying the adjuvant with an antigen shows excellent physical stability, enhanced immune response and good safety in animal experiments, and is especially suitable for preventing bovine nodular dermatosis and preparation of positive serum thereof. The application solves the technical problem that squalene is difficult to stabilize in a complex emulsion system, and provides a replacement scheme superior to a traditional mineral oil adjuvant.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and veterinary vaccine technology, specifically to a novel adjuvant and its application in the preparation of bovine nodular dermatitis inactivated vaccine and its positive serum. Background Technology

[0002] Vaccine adjuvants are key components for enhancing the efficacy of subunit vaccines, inactivated vaccines, and other vaccines with weak immunogenicity. In the field of veterinary vaccines, oil-emulsion adjuvants are widely used due to their powerful immunostimulatory effects. Traditional oil-emulsion adjuvants often use refined white oil (mineral oil) as the oil phase, with representative formulations such as the ISA series from the French company SEPPIC and the technical solution disclosed in Chinese patent CN117942393B. Although these mineral oil-based adjuvants can induce high levels of antibody responses, their inherent biological defects are becoming increasingly prominent: mineral oil is metabolized extremely slowly in animals, and its long-term retention at the injection site can easily trigger persistent inflammatory responses, the formation of foreign body granulomas, or even cysts, seriously affecting animal welfare, carcass quality, and food safety.

[0003] To overcome the safety concerns associated with mineral oil adjuvants, squalene, with its superior biocompatibility, has emerged as a promising alternative. Squalene is a triterpenoid compound naturally found in shark liver oil and human sebum. It is an important intermediate in cholesterol biosynthesis, exhibiting a well-defined metabolic pathway and good biodegradability. The successful application of the human adjuvant MF59 (a squalene oil-in-water emulsion) fully demonstrates squalene's dual advantages in safety and efficacy. However, directly transferring squalene into veterinary vaccines, especially those for commercially viable animals, faces two major industrialization bottlenecks: firstly, cost pressure, requiring efficient formulation design to reduce the cost per dose; secondly, and more critically, the significant differences in the physicochemical properties of squalene (such as viscosity, polarity, and interfacial tension) compared to mineral oil. This often leads to failures when directly applying existing mature emulsification systems optimized for mineral oil (such as the complex surfactant combination of polyglycerol ricinoleate, Span, polyglycerol ester, and Tween disclosed in CN117942393B, which is compounded in specific proportions). Stable and homogeneous emulsions cannot be formed, and it is even more difficult to construct water-in-oil-in-water (W / O / W) dual emulsions, which have more complex structures, significant immunological advantages, but also higher processing difficulty. W / O / W adjuvants, due to their external aqueous phase characteristics, possess advantages such as good injection compliance and the ability to encapsulate active ingredients in the internal aqueous phase, making them an important development direction for veterinary adjuvants.

[0004] Therefore, a long-standing technical challenge in this field is how to develop a novel adjuvant formulation specifically designed for the squalene oil phase, capable of easily and stably forming a W / O / W structure, and while ensuring excellent safety, possessing immune-enhancing efficacy at least equal to, and even surpassing, traditional mineral oil adjuvants. This is not a simple component replacement, but involves a re-exploration and creative adaptation of the entire emulsification system (surfactant types, ratios, phase diagrams). Through extensive experiments, the inventors discovered that a hydrophilic surfactant combination composed of polyglycerol-2-dioleate (PG-2-DO), decaglycerol monolaurate (DGML), and Tween 80 in a specific ratio exhibits an unexpected synergistic stabilizing effect with squalene, and based on this, the present invention was completed. Summary of the Invention

[0005] The primary objective of this invention is to provide a squalene-based W / O / W vaccine adjuvant that is physically stable and easy to prepare. Another objective of this invention is to provide a method for preparing this adjuvant. A further objective of this invention is to provide a vaccine composition comprising this adjuvant and its specific application in the preparation of a vaccine for the prevention of bovine nodular dermatitis or its positive serum.

[0006] Therefore, this invention discloses a vaccine adjuvant comprising squalene, tocopherol as an antioxidant, a lipophilic surfactant, and a hydrophilic surfactant. The hydrophilic surfactant is composed of polyglycerol dioleate, decaglycerol monolaurate, and polyoxyethylene sorbitan trioleate. Through systematic screening, the inventors discovered that when these three components are combined in a specific mass ratio, such as 2:1:1, they can form a synergistic effect with squalene, thereby preparing a water-in-oil-in-water emulsion with uniform particle size distribution and long-term storage stability.

[0007] Preferably, the lipophilic surfactant is a mixture of polyglycerol ricinoleate and sorbitan monooleate, with a mass ratio of 1:1.5 to 2.5. The ratio of the total mass of all surfactants to the total mass of squalene and tocopherol can be 1:2 to 1:3, and the mass ratio of the hydrophilic surfactant to the lipophilic surfactant can be 2:1 to 4:1.

[0008] The vaccine adjuvant can be prepared by a simple method of stirring and mixing the components uniformly under mild heating conditions. The resulting adjuvant is then mixed with an aqueous phase containing the vaccine antigen in an appropriate ratio, and the final vaccine composition can be obtained through a conventional emulsification process.

[0009] The present invention also provides a vaccine composition, which is prepared by mixing the vaccine adjuvant with an aqueous phase containing vaccine antigen at a mass ratio of 1:1 and stirring and emulsifying at 32°C and 400 rpm for 10 minutes.

[0010] Preferably, the vaccine antigen of the present invention is bovine nodular dermatitis virus antigen, and the bovine nodular dermatitis virus antigen is bovine nodular dermatitis virus inactivated virus solution.

[0011] Preferably, the vaccine antigen of the present invention is bovine nodular dermatitis virus antigen, which is a goatpox virus P32-238 mutant protein, wherein the amino acid sequence of the goatpox virus P32-238 mutant protein is shown in SEQ ID NO:1.

[0012] The present invention also provides a method for preparing bovine nodular dermatitis virus positive serum, comprising the following steps: immunizing animals for the first time with a vaccine composition containing the adjuvant and bovine nodular dermatitis virus inactivated viral antigen; 21 days after the first immunization, immunizing animals for the second time with the same vaccine composition; 28 days after the second immunization, separating and testing the animal serum, and obtaining serum that passes the test is bovine nodular dermatitis virus positive serum.

[0013] This serum can be used not only for antigen and antibody detection of bovine nodular dermatosis virus (such as Western blot detection, ELISA detection, chemiluminescence detection, immunofluorescence detection, neutralizing antibody detection, etc.); it is also suitable for preparing products for detecting, identifying or diagnosing viruses; preparing products for research on viral pathogenesis and transmission mechanisms; preparing products for evaluating or monitoring the quality of viral live vaccines; and preparing viral positive serum standards.

[0014] The technical solution provided by this invention has the following beneficial effects: 1. Significantly Improved Physical Stability: By employing a specific combination of hydrophilic surfactants, this invention successfully overcomes the technical obstacles in preparing stable multi-emulsions from squalene. Data shows that this combination is essential for obtaining emulsions with small particle size, low dispersibility, and the ability to pass centrifugal stability tests, and its effect is significantly superior to control schemes that lack any component or use other conventional surfactants.

[0015] 2. Enhanced immune efficacy: In a mouse model of bovine nodular dermatitis, the adjuvant vaccine of this invention induced significantly higher specific antibody titers and exhibited a stronger Th1 immune response bias and cellular immune response, which is superior to the mineral oil adjuvant vaccine formulated based on the closest existing technology.

[0016] 3. Superior safety: Replacing mineral oil with squalene has shown milder local and systemic reactions in animal studies, indicating better potential for biosafety.

[0017] 4. Superior Immunogenicity: Target animal immunization experiments showed that the vaccine using the adjuvant of this invention induced higher levels of neutralizing antibodies against bovine nodular dermatitis virus. Compared to the control group using mineral oil adjuvant, the neutralizing antibody titer of the serum prepared from the vaccine group using the adjuvant of this invention was increased by 3-4 times or more. Furthermore, the use of the goatpox virus P32-238 mutant protein prepared in combination with this adjuvant also achieved excellent immunization effects, comparable to or better than whole-virus inactivated vaccines. It should be noted that the goatpox virus P32-238 mutant protein is a recombinant protein expressed in vitro, which has significant advantages over culturing whole viruses, such as ease of preparation, high expression yield, low production cost, and no risk of virus shedding.

[0018] 5. It can be used to prepare positive sera with high neutralizing antibody titers: Compared with conventional control adjuvants, positive sera for bovine nodular dermatosis virus prepared by immunizing animals with the adjuvant of this invention can increase the neutralizing antibody titer by 3-4 times or more, fully meeting the requirements of product quality control for testing reagents. Therefore, the method of preparing positive sera using the adjuvant of this invention combined with the corresponding antigen provides an innovative approach for preparing polyclonal antibodies against pathogens for which humoral immunity is ineffective.

[0019] In summary, the technical solution of this invention achieves a breakthrough in the stability of squalene W / O / W emulsions by providing a novel and non-obvious combination of surfactants, and on this basis obtains a vaccine adjuvant with excellent comprehensive performance. Attached Figure Description

[0020] Figure 1 The appearance of different formulations.

[0021] Figure 2 Particle size results for different formulations.

[0022] Figure 3 The state of different formulations after centrifugation.

[0023] Figure 4 SDS-PAGE identification results of prokaryotic expression of wild-type P32-282 protein and P32-238 mutant protein. 1 represents whole cells before induction, 2 represents whole cells after induction, 3 represents supernatant after lysis before induction, 4 represents supernatant after lysis after induction, 5 represents precipitate after lysis before induction, 6 represents precipitate after lysis after induction, and 7 represents the marker.

[0024] Figure 5 The results of SDS-PAGE and Western blot analysis of the P32-238 mutant protein are shown in Figure 1, which represents the P32-238 mutant protein. Detailed Implementation

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items. Unless otherwise stated, the reagents, methods, and apparatus used in this invention are conventional reagents, methods, and apparatus in the art. Unless otherwise stated, the reagents and materials used in the following examples are commercially available.

[0026] Among them are polyglycerol polyricinoleate (PGPR), sorbitan monooleate (Span 80), polyglyceryl-2-dioleate (PG-2-DO), and decaglyceryl monolaurate (DGML).

[0027] Example 1: Screening of key surfactant combinations and adjuvant preparation The inventors have discovered that the selection of hydrophilic surfactants is crucial for stabilizing squalene W / O / W emulsions. We systematically screened a variety of common and potentially usable hydrophilic surfactants using emulsion particle size (Dv50), PDI, and centrifugal stability as indicators.

[0028] 1. Screening Experiment Design 1.1 Fixed base formula (1) Squalene (containing 4% tocopherol) 3.6 g; (2) 0.45 g of lipophilic surfactant (PGPR:Span80=1:2).

[0029] 1.2 Variable Groups: Different combinations of hydrophilic surfactants were examined (total mass 1.35g): Group A (this invention): PG-2-DO : DGML : Tween80 = 2 : 1 : 1; Group B (missing DGML): PG-2-DO : Tween80 = 3 : 1 (replace DGML quality with PG-2-DO); Group C (missing PG-2-DO): DGML : Tween80 = 3 : 1 (replace PG-2-DO quality with DGML); Group D (single Tween): Using Tween 80 only; Group E (Conventional Alternative): Use polyglycerol-10-oleate mixed with Tween80 in equal proportions.

[0030] 1.3 Preparation and Evaluation: The components were mixed to prepare an adjuvant. 1 g of adjuvant was emulsified with 1 g of PBS (0.01 M, pH 7.4) at 32 °C and 400 rpm for 10 minutes. The initial particle size and polydispersity index were immediately measured using a particle size analyzer. After standing at 4 °C for 24 hours, the mixture was centrifuged at 4000 g for 15 minutes to observe the layering.

[0031] 2. The screening results are shown in Table 1. Only the specific ternary combination of this invention (Group A) can form a W / O / W emulsion with small particle size, narrow distribution, and centrifugal stability. The stability decreases sharply when either PG-2-DO or DGML is missing (Groups B and C), or when other common surfactants are used (Groups D and E). This demonstrates a synergistic stabilizing effect of PG-2-DO, DGML, and Tween 80 at the proportions specified in this invention; this combination is not obvious. Specific results are shown in Table 1 and... Figures 1-3 As shown.

[0032] Table 1 Summary of screening results for different formulations

[0033] 3. Preparation of the optimal adjuvant (SQ-01): Based on the screening results, the preferred adjuvant of the present invention was prepared as follows: 3.1 Oil phase: Weigh 345.60 g of squalene, add 14.40 g of tocopherol (d-α-tocopherol), and stir to dissolve.

[0034] 3.2 Lipophilic surfactant: Weigh 15.0 g of PGPR and 30.0 g of Span80 and add them to the oil phase above.

[0035] 3.3 Hydrophilic surfactant: Weigh 67.50 g of PG-2-DO, 33.75 g of DGML and 33.75 g of Tween80, and premix them evenly.

[0036] 3.4 Place the container containing the oil phase and the lipophilic surfactant in a water bath at 40±2℃. While stirring magnetically (500 rpm), slowly add the premixed hydrophilic surfactant. After addition, continue stirring at 40℃ for 45 minutes until the mixture is a homogeneous, clear, pale yellow, transparent oily liquid, free of any turbidity or particles. Filter through a 220nm filter membrane and dispense into individual containers. This is adjuvant SQ-01, which should be stored at room temperature away from light.

[0037] Example 2: Control adjuvant and vaccine preparation To conduct a rigorous comparison of efficacy, the following control group was set up: 1. Control adjuvant (M-Oil): Prepared strictly according to the formulation and preparation method of Example 1 in CN117942393B.

[0038] 2. Antigen: Use bovine nodular skin disease virus (LSDV) inactivated virus solution (virus content was 10 before inactivation). 6.0 TCID 50 The whole virus inactivated antigen ( / ml) was prepared by the China Institute of Veterinary Drug Control.

[0039] 3. Vaccine preparation: Adjuvant SQ-01 and M-Oil were mixed with bovine nodular dermatitis virus inactivation solution at a mass ratio of 1:1, and emulsified at 32°C and 400 rpm for 10 minutes to obtain vaccine V-SQ (this invention) and vaccine V-MOil (control). PBS solution (V-Ag) was used as a negative control.

[0040] Example 3: Evaluation of Immunogenic Efficacy (Mouse Model) 1. Immunization regimen: 6-8 week old female BALB / c mice, 12 mice per group, were randomly divided into 3 groups (V-SQ, V-MOil, V-Ag). Each mouse was immunized via hind leg muscle on day 0 and day 21, with 0.1 mL administered.

[0041] 2. Humoral immunity assay: On days 14 and 35 post-secondary immunization, blood was collected from the orbital sinus of 6 mice in each group. Serum was inactivated at 56°C for 30 minutes. The total IgG antibody and its subclasses specific to the P32-238 mutant protein (prepared in Example 4) were detected by indirect ELISA.

[0042] 2.1 Coating: P32-238 mutant protein (1 μg / mL, carbonate coating buffer) overnight at 4°C.

[0043] 2.2 Detection: The standard ELISA procedure was followed, using HRP-labeled goat anti-mouse IgG, IgG1, and IgG2a secondary antibodies. The highest serum dilution with an OD450 value of 0.5 (baseline) was used as the endpoint titer.

[0044] 3. Cellular immune detection: On day 35 after the second immunization, mice were sacrificed and spleens were harvested to prepare single-cell suspensions.

[0045] 3.1 ELISpot detection of IFN-γ: The mouse IFN-γ ELISpot kit was used. Spleen cells (2×10^5 / well) were co-incubated with P32-238 mutant protein (5 μg / mL) for 24 hours, and spot-forming cells (SFC) were counted.

[0046] 3.2 Flow cytometry analysis of T cell response: Another batch of spleen cells was obtained and stimulated for 6 hours with complete culture medium containing P32-238 mutant protein (5 μg / mL) (with the addition of a protein transport inhibitor, such as GolgiStop). After stimulation, cell surface staining (CD3, CD4, CD8) and intracellular staining (IFN-γ, TNF-α, IL-2) were performed and analyzed by flow cytometry.

[0047] 4. The experimental results are shown in Tables 2 and 3.

[0048] The adjuvant vaccine V-SQ of this invention outperforms the existing control V-MOil in inducing total antibody levels, Th1 antibody subclasses (high IgG2a / IgG1 ratio), and antigen-specific CD8+ and CD4+ T cell responses. This indicates that the formulation of this invention not only enhances humoral immunity but also strengthens Th1-biased cellular immunity, a weak point of traditional mineral oil adjuvants.

[0049] Table 2 Serum antibody titers (geometric mean titer, GMT) Note: The differences in IgG GMT between the V-SQ and V-MOil groups at 14 and 35 days were statistically significant (p < 0.001, two-tailed t-test).

[0050] Table 3 Cellular immune response Note: Data are Mean ± SD (n=6). All indicators in the V-SQ group were significantly higher than those in the V-MOil group (p < 0.01).

[0051] Example 4: Construction, expression, and testing of the goat pox virus P32-238 mutant protein The P32 protein is a highly specific immunogenic structural protein of goatpox virus strains. Therefore, this study first analyzed the amino acid sequence of the complete P32 protein (GenBank: ADN44125.1). This protein does not contain a signal peptide sequence, but it is a transmembrane protein with an extracellular region consisting of amino acids 1-282 (aa1-aa282) at the N-terminus. Therefore, researchers in this field generally prefer to directly express the sequence of this extracellular region as a recombinant protein. This study named it P32-282 protein (aa1-aa282). However, during the preparation of this protein, we found that it mainly exists in the form of inclusion bodies and its expression level is extremely low; moreover, this P32-282 protein (aa1-aa282) has a significant cytotoxic effect.

[0052] Meanwhile, based on extensive screening and analysis of protein spatial structures, our researchers selected to truncate the full-length P32 protein of goatpox virus, naming it the P32 truncated protein (aa1-aa238). This protein was prepared using *E. coli*, and the recombinant protein was approximately 28.5 kDa in size, with an expression level of approximately 15 mg / L and a solubility of 40%. While the expression yield of this truncated protein is manageable for use as a detection antigen, it is still too low for use as a vaccine antigen. Therefore, we further developed a point mutation approach for expression. Through mutation and process research, this invention achieved highly efficient expression of the P32-238 mutant protein. Although it remains in inclusion body form, the protein exhibits high expression yield and good immunogenicity, while also eliminating cytotoxicity.

[0053] 1. Construction of expression carriers Using the P32 gene sequence (GenBank accession number ADN44125.1) as a template, the DNA sequence encoding its extracellular region (amino acids 1-282) (containing a 6His tag) was obtained through gene synthesis technology. This sequence was cloned into the Nde I and Xho I restriction sites of the prokaryotic expression vector pET-28a(+) to construct the recombinant expression plasmid pET28a-P32-282 (wild-type). Using this plasmid as a template, overlap extension PCR was employed to site-directedly mutate the codons for lysine (K) at positions 33 and 34 of the sequence to codons for glutamine (Q), constructing the mutant plasmid pET28a-P32-238 (K33Q / K34Q). All plasmids were verified by DNA sequencing. The mutated protein was named the goatpox virus P32-238 mutant protein, and its amino acid sequence is shown in SEQ ID NO:1.

[0054] 2. Expression and Preliminary Observations of Recombinant Proteins The verified recombinant plasmids were transformed into E. coli expression strain BL21(DE3). Single clones were picked and cultured in LB liquid medium containing kanamycin until the OD600 reached approximately 0.6-0.8. Then, 0.5 mM IPTG was added, and expression was induced at 37°C for 4 hours.

[0055] During the induction process, it was observed that the bacterial culture containing the wild-type pET28a-P32-282 plasmid showed significant growth cessation after 2 hours of induction, with an OD600nm value lower than that of the control group (including the empty vector) and the mutant group, indicating that the wild-type protein is toxic to the host cells. In contrast, the strain containing the mutant plasmid pET28a-P32-238(K33Q / K34Q) grew normally, showing no significant difference from the control group.

[0056] 3. Protein expression analysis and inclusion body purification After induction, the bacterial cells were collected by centrifugation and then sonicated. SDS-PAGE analysis was performed on whole bacterial samples before and after induction, as well as the supernatant (soluble components) and precipitate (insoluble components) after induction.

[0057] The results showed that ( Figure 4 Compared to the extremely weak expression band of the wild-type P32-282 protein (not visible in the figure), the P32-238 mutant protein showed a distinct specific band in the whole bacterial protein, with a molecular weight consistent with expectations (approximately 28.5 kDa). This protein was mainly present in the fragmented precipitate, indicating that it still exists in inclusion body form.

[0058] 2.4 Purification and Yield Determination of Inclusion Bodies Bacterial pellets containing mutant proteins were collected and processed according to standard inclusion body purification procedures (washing, denaturation, and dissolution). The proteins were purified using Ni-NTA affinity chromatography under denaturing conditions (8 M urea), and protein concentrations were determined using the Bradford method.

[0059] Results: The average yield of the P32-238 mutant protein reached over 520 mg per liter of fermentation broth, while the yield of the wild-type P32-282 protein was too low to purify the effective protein.

[0060] 2.5 Immunogenicity Validation (Western Blot) The purified P32-238 mutant protein was subjected to SDS-PAGE and then transferred to a PVDF membrane. Immunoblotting analysis was performed using positive serum infected with goatpox virus (primary antibody) and HRP-labeled rabbit anti-goat IgG (secondary antibody).

[0061] The results show that ( Figure 5 The purified P32-238 mutant protein was specifically recognized by positive serum, producing a clear single immunoreaction band, indicating that the introduced K33Q / K34Q double mutation did not destroy the protein's major antigenic epitopes, and the mutant protein maintained good immunoreactivity.

[0062] Example 5: Safety and efficacy test in target animals (cattle) 1. Vaccine and its preparation: Adjuvant SQ-01 and the P32-238 mutant protein prepared in Example 3 (diluted to 100 μg / ml with PBS buffer) were mixed at a mass ratio of 1:1 and emulsified at 32°C and 400 rpm for 10 minutes to obtain vaccine V-SQ-P32 (the concentration of P32-238 mutant protein in the vaccine was 100 μg / ml). Other vaccines were vaccine V-SQ, vaccine V-MOil, and vaccine V-Ag prepared in Example 2.

[0063] 2. Animals and grouping: 20 healthy cattle (8-10 months old) that were negative for LSDV antigen and antibody were randomly divided into 4 groups of 5 animals each.

[0064] Group I: V-SQ (the whole virus inactivated vaccine of this invention) was administered.

[0065] Group II: V-MOil (control vaccine) administered.

[0066] Group III: V-Ag inoculation (antigen control).

[0067] Group IV: V-SQ-P32 (the subunit vaccine of this invention) was administered.

[0068] 2. Immunization and observation: Administer 2 mL / head via intramuscular injection in the neck on days 0 and 21. Record local reactions and rectal temperature within 7 days post-immunization.

[0069] 3. Neutralizing antibody detection: Blood samples were collected from all cattle on days 21, 42, 56 and 70 after the initial immunization. Serum was separated and the level of anti-LSDV neutralizing antibodies in the serum was detected to assess the effectiveness of different adjuvant vaccines.

[0070] 4.4 The experimental results are shown in Tables 4 and 5. Safety: The local and systemic reactions of the squalene adjuvant vaccine of this invention are milder than those of the mineral oil adjuvant vaccine.

[0071] Efficacy: After immunization of target animals (cattle), the neutralizing antibody levels of both groups of vaccines of the present invention were higher than those of the control vaccine group on days 21, 42, 56, and 70 after the first immunization, indicating that the efficacy of the adjuvant combined with the antigen of the present invention surpasses that of the mineral oil adjuvant vaccine. Furthermore, the immunogenicity of the P32 mutant protein prepared by the present invention is comparable to or better than that of the bovine nodular dermatitis virus inactivated solution.

[0072] Table 4 Security Data

[0073] Table 5 Results of efficacy (neutralizing antibody level assay)

[0074] Example 6: Preparation and application of positive serum for bovine nodular dermatitis virus This embodiment provides a method for preparing positive serum for bovine nodular dermatitis virus (LSDV) and its application, including the preparation of an inactivated bovine nodular dermatitis virus vaccine, inoculation and immunization, serum separation steps, and exogenous virus testing for LSDV strain.

[0075] This embodiment is based on Example 5. Cattle immunized with Group I (V-SQ) were selected. 28 days after the second immunization (70 days after the first immunization), and based on the neutralizing antibody titer results 14 days after the second immunization (56 days after the first immunization), cattle with high neutralizing antibody titers were selected. Blood was collected via the jugular vein, and serum was then separated. The separated serum was mixed, inactivated in a 56°C water bath for 30 minutes, filtered through a 0.22μm bacterial filter for sterilization, and then aliquoted into 0.5ml vials and lyophilized for storage. Samples were tested for characteristics, sterility, mycoplasma, exogenous virus, neutralizing antibody titer, and specificity. The results showed that the prepared positive sera were all light red, loose clumps that dissolved rapidly upon addition of diluent; they were free of bacteria, mycoplasma, and exogenous virus contamination; antibodies against bovine foot-and-mouth disease virus, bovine viral diarrhea virus, and bovine infectious rhinotracheitis virus were not detected, indicating good specificity; the neutralizing antibody titer was 1:64.

[0076] The positive serum was used to test for exogenous virus in LSDV NM2020 strain according to Appendix 3305 of the current Chinese Veterinary Pharmacopoeia (2020 edition), Part III. The results showed that the positive serum could neutralize a viral load of 10. 6.0 TCID 50 The LSDV seed culture was diluted to a concentration of / ml, and no incomplete neutralization was observed, nor was any cytotoxicity caused by the positive serum. This indicates that the bovine nodular dermatitis virus positive serum prepared using the process of this invention can meet the requirements for exogenous virus testing of LSDV seed culture.

[0077] In summary, this invention is not simply a matter of replacing mineral oil with squalene. Experimental data demonstrate that the specific PG-2-DO / DGML / Tween80 combination is key to stabilizing the squalene W / O / W system. Ultimately, this unique formulation produces a synergistic technical effect: enhancing both safety and immune efficacy.

[0078] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A vaccine adjuvant, which is a homogeneous, transparent, oily liquid, characterized in that, The adjuvant is composed of an oil phase and a surfactant phase, wherein the mass ratio of the oil phase to the surfactant phase is 2:1; The oil phase is composed of squalene and tocopherol, with tocopherol accounting for 4% of the total mass of the oil phase; The surfactant phase comprises a lipophilic surfactant and a hydrophilic surfactant, wherein the mass ratio of the lipophilic surfactant to the hydrophilic surfactant is 1:3; wherein the lipophilic surfactant comprises polyglycerol ricinoleate and Span 80, and the mass ratio of polyglycerol ricinoleate to Span 80 is 1:2; the hydrophilic surfactant comprises polyglycerol-2-dioleate, decaglycerol monolaurate and Tween 80, and the mass ratio of the three is 2:1:

1.

2. A method for preparing the vaccine adjuvant of claim 1, characterized in that, The method includes the following steps: mixing squalene, tocopherol, lipophilic surfactant and hydrophilic surfactant in proportion, stirring at 40±2℃ until a homogeneous and transparent oily liquid is formed; the stirring is carried out under magnetic stirring at a speed of 500 rpm for 45 minutes.

3. A vaccine composition, characterized in that, The vaccine composition is prepared by mixing the vaccine adjuvant of claim 1 with an aqueous phase containing the vaccine antigen at a mass ratio of 1:1, and stirring and emulsifying at 32°C and 400 rpm for 10 minutes; the vaccine antigen is bovine nodular dermatitis virus antigen, and the bovine nodular dermatitis virus antigen is bovine nodular dermatitis virus inactivated virus solution.

4. The vaccine composition according to claim 3, characterized in that, The bovine nodular dermatitis virus antigen is a goatpox virus P32-238 mutant protein, the amino acid sequence of which is shown in SEQ ID NO:

1.

5. The use of a vaccine adjuvant according to claim 1 or a vaccine adjuvant prepared by the preparation method according to claim 2 in the preparation of animal vaccine compositions or blank W / O / W type emulsions.

6. The application according to claim 5, characterized in that, The blank W / O / W emulsion includes the vaccine adjuvant and an aqueous phase; the aqueous phase is an aqueous reagent capable of mimicking vaccine antigens.

7. A method for preparing bovine nodular dermatitis virus-positive serum, characterized in that, The method includes the following steps: immunizing animals for the first time using the vaccine composition of claim 3; 21 days after the first immunization, continuing to immunize animals for the second time using the vaccine composition of claim 3; 28 days after the second immunization, separating and testing animal serum, and serum that passes the test is considered positive for bovine nodular dermatitis virus.

8. The use of the vaccine composition according to any one of claims 3 to 4 in the preparation of a vaccine for the prevention of bovine nodular dermatitis.

9. The use of the vaccine composition of claim 3 in the preparation of bovine nodular dermatosis virus positive serum.

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