Bovine nodular skin disease subunit vaccine based on P122-Fc fusion protein

Abstract

The invention discloses a bovine nodular skin disease subunit vaccine based on P122-Fc fusion protein, and belongs to the technical field of veterinary biological products. The bovine nodular dermatosis subunit vaccine disclosed by the invention comprises a P122-Fc fusion protein and a water-in-oil-in-water type multiple emulsion adjuvant MONTANIDEISA 206 VG. According to the invention, a genetic engineering subunit vaccine technical route is adopted, the vaccine components are only recombinant proteins and adjuvants and do not contain any forms of live viruses, so that the risks of virulence reversion and vertical transmission are fundamentally eradicated; high safety is successfully combined with a high intensity, Th1 biased immune response. According to the scheme, a high-titer body fluid antibody is excited, and the body fluid antibody is necessary for eliminating virus infected cells. Systematic target animal safety tests (including pregnant cow over-dose inoculation) prove that no abortion and no significant adverse reaction exist, safe immunity to sensitive groups such as pregnant cows is achieved, and the problems that existing mainstream attenuated live vaccines have potential virulence enhancement risks, may cause abortion to pregnant cows, and are poor in immunity to pregnant cows are thoroughly solved. And the application of the feed additive in key livestock groups is strictly limited.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biological products technology, specifically relating to a bovine nodular dermatitis subunit vaccine based on the P122-Fc fusion protein. Background Technology

[0002] Bovine nodular dermatitis (LSD) is an acute, subacute, or chronic infectious disease caused by the bovine nodular dermatitis virus (BNDV), a member of the sheeppoxvirus family. It is characterized by fever, nodules on the skin and mucous membranes, and swollen lymph nodes. LSD can lead to decreased milk production in dairy cows, infertility in bulls, hide damage, and significant economic losses. Currently, global control of LSD primarily relies on vaccination.

[0003] Currently available vaccines for LSD prevention mainly fall into two categories:

[0004] Live attenuated vaccines: These vaccines (such as the Neethling strain live attenuated vaccine) offer good immunoprotection and are currently the most widely used type of vaccine. However, live attenuated vaccines have several inherent drawbacks: Biosafety risks: There is a potential risk of virulence reversion, especially in immunosuppressed animals.

[0005] Adverse reactions: May cause local or systemic adverse reactions, including swelling and fever at the injection site, and may even lead to abortion in pregnant cows.

[0006] Differential diagnosis is difficult: immunized animals may develop clinical symptoms or serological reactions that are difficult to distinguish from wild-type virus infection, interfering with epidemic monitoring and eradication.

[0007] Traditional inactivated vaccines and early subunit vaccines, while offering improved safety, often suffer from weak immunogenicity, short-lasting protection, and the need for multiple immunizations, making them less effective than live vaccines. Furthermore, subunit vaccines prepared from whole viruses or simple recombinant proteins often induce insufficient strength and balance in their immune responses (especially cellular immunity), making it difficult to provide comprehensive protection against intracellular replicating viruses.

[0008] Based on the above background technology, existing LSD vaccine technologies mainly have the following problems: Safety concerns: Live attenuated vaccines pose a risk of miscarriage in sensitive animals such as pregnant cows and also present biosafety risks.

[0009] The immune response is either singular or unbalanced: Traditional inactivated vaccines and some subunit vaccines mainly induce Th2 humoral immunity, while cellular immunity (Th1 / CD8+ T cell response) is weak. However, against intracellular viruses such as LSDV, strong cellular immunity is crucial for clearing virus-infected cells.

[0010] Efficacy and safety are difficult to balance: improvements in safety often come at the cost of immune efficacy, especially in subunit vaccines where it is difficult to simultaneously stimulate strong, Th1-biased humoral and cellular immunity.

[0011] There is a lack of systematic and comprehensive safety and efficacy evaluation data for target animals (including pregnant cows): existing vaccine technologies and publicly available information lack systematic verification of the safety (e.g., single-dose, repeated-dose, overdose reactions and effects on pregnancy outcomes) and efficacy (e.g., ability to induce high levels of neutralizing antibodies) of genetically engineered subunit vaccines in calves, especially pregnant cows, a critical and sensitive population. Summary of the Invention

[0012] In view of this, the purpose of this invention is to provide a bovine nodular dermatitis subunit vaccine based on the P122-Fc fusion protein. This invention successfully combines high safety with a high-intensity, Th1-biased immune response through the innovative design of the P122-Fc fusion protein and a dedicated immune-enhancing adjuvant.

[0013] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a bovine nodular dermatitis subunit vaccine based on the P122-Fc fusion protein, comprising: the P122-Fc fusion protein and the water-in-oil-in-water re-emulsion adjuvant MONTANIDE™ ISA 206 VG.

[0014] Preferably, the P122-Fc fusion protein is the bovine nodular cutaneous virus P122 recombinant protein described in patent CN117229368A.

[0015] Preferably, the antigen content of the P122-Fc fusion protein is 50 μg / mL.

[0016] Preferably, the volume ratio of the water-in-oil-in-water type double emulsion adjuvant MONTANIDE™ ISA 206 VG to the P122-Fc fusion protein dilution is 1:1.

[0017] It contains at least the following beneficial technical effects: This invention employs a genetically engineered subunit vaccine technology route. The vaccine components consist only of recombinant protein and adjuvant, containing no form of live virus, fundamentally eliminating the risks of virulence reversion and vertical transmission. Through the innovative design of "P122-Fc fusion protein + dedicated immune-enhancing adjuvant," it successfully combines high safety with a high-intensity, Th1-biased immune response. This approach elicits high titers of humoral antibodies, which are essential for clearing virus-infected cells. Systematic target animal safety studies (including overdose vaccination in pregnant cows) have confirmed no abortions and no significant adverse reactions, achieving safe immunization in sensitive groups such as pregnant cows. This completely resolves the potential risk of virulence reversion present in existing mainstream attenuated live vaccines, and their potential to cause abortion in pregnant cows, which severely limits their application in critical livestock herds. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0024] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0025] Example 1. Screening of adjuvants and evaluation of immunogenicity for vaccine candidate formulations 1.1 Materials and Methods 1.1.1 Antigens and Adjuvants: 1.1.1.1 Antigen: Recombinant P122-Fc fusion protein (purity >95%), diluted to working concentration using sterile PBS.

[0026] The amino acid sequence of the recombinant P122-Fc fusion protein is as follows: MLRGPGPGLLLAVLCLGTAVRCTEAMLVDIPKSGTETDYDESNNFTAFAGSTIYGYGLKSKKNIKKKEKLINFCIKISIMTSMVSLITITILLAFFNNTCELNQFKEHKPYFLKNPN PTTYSDDDTESELNVYRSCKGIVYSGYCYTFNSEPKSFNDAYDDCEKKNSELPSNNLMNDWISDYLDGTWGEDGNVLFKEKNQELEAIDISDEMRSYYCVRSFFGGGGSGGGGSGGG GSDPTCKPSPCDCCPPPELPGGPSVFIFPPKPKDTLTISGTPEVTCVVVDVGHDDPEVKFSWFVDDVEVNTATTRPREEQFNSTYRVVSALRIQHQDWTGGKEFKCKVHNEGLPAPI VRTISRTKGPAREPQVYVLAPPQEELSKSTVSLTCMVTSFYPDYIAVEWQRNGQPESEDKYGTTPPQLDADSSYFLYSKLRVDRNSWQEGDTYTCVVMHEALHNHYTQKSTSKSAGK.

[0027] 1.1.1.2 Adjuvants: 206 Adjuvant: MONTANIDE™ ISA 206 VG, a water-in-oil-in-water double emulsion adjuvant (SEPPIC, France); 803 Adjuvant: Nanoemulsion type adjuvant MONTANIDE™ ISA 803 VG (SEPPIC, France); Aluminum hydroxide adjuvant: Commercial aluminum gel adjuvant, Alhydrogel® adjuvant 2% (Invitrogen, USA); White oil adjuvant: A classic oil-in-water mineral oil emulsion, formulated according to the "Veterinary Pharmacopoeia of the People's Republic of China". Its main components are light mineral oil (Marcol 52), emulsifiers Span 80 and Tween 80. Negative control: sterile PBS.

[0028] Positive antigen control: Commercial LSD live virus vaccine (attenuated strain).

[0029] 1.1.2 Laboratory Animals and Immunization Procedures: Six- to eight-week-old female BALB / c mice were randomly divided into six groups of 10 each.

[0030] Immunization regimen: Two subcutaneous immunizations were administered on the back on day 0 and day 21.

[0031] Vaccine preparation: Mix the diluted antigen with each adjuvant in a predetermined ratio to achieve a final antigen concentration of 50 μg / mL (where the volume ratio of diluent to adjuvant is 1:1), and thoroughly emulsify or mix. The control group is injected with an equal volume of PBS or a commercially available vaccine.

[0032] Grouping: G1:P122-Fc+206 adjuvant G2:P122-Fc+803 adjuvant G3:P122-Fc + Aluminum hydroxide adjuvant G4:P122-Fc + White Oil Adjuvant G5: Commercial LSD live vaccine (positive control) G6:PBS (negative control) 1.1.3 Sample Collection: Serum was collected via the posterior orbital venous plexus on days 0, 14, 28, and 42 after the initial immunization for antibody detection.

[0033] 1.1.4 Detection Method: 1.1.4.1 Specific antibody titer detection: Indirect ELISA method was used. The enzyme-linked immunosorbent assay (ELISA) plate was coated with purified P122-Fc protein, and the serum specific total IgG antibody against P122 was detected. The endpoint titer was defined as the reciprocal of the highest serum dilution when the absorbance value was 2.1 times higher than the mean of the negative control.

[0034] 1.1.4.2 Antibody subtype analysis: The antibody levels of specific IgG1 (representing Th2 humoral immunity) and IgG2a (representing Th1 cellular immunity) in serum on day 42 were detected using an ELISA subtype detection kit. The results are expressed as OD450nm values.

[0035] 1.2 Test Results 1.2.1 Specific antibody response after immunization Table 1: Dynamic changes in serum GMT (Log10) of anti-P122 specific total IgG antibody in mice of each immunization group

[0036] As shown in Table 1, all adjuvant groups (G1-G4) containing P122-Fc antigen induced specific antibodies 14 days after primary immunization, and antibody titers significantly increased after secondary immunization (enhancing effect). At the experimental endpoint (day 42), the G1 (206 adjuvant) group had the highest antibody titer (GMT Log10 = 5.6), followed by the G3 (aluminum hydroxide) and G2 (803 adjuvant) groups. Although the antibody titer of the G4 (white oil adjuvant) group was lower than the previous three, it was still significantly higher than that of the G5 (commercial live vaccine) group. This indicates that the P122-Fc protein has strong immunogenicity, and when combined with appropriate adjuvants, it can induce a humoral immune response far exceeding that of traditional live vaccines.

[0037] 1.2.2 Antibody subtype analysis (Th1 / Th2 bias) Table 2. Serum anti-P122 specific IgG subtype levels in mice on day 42 (OD450nm)

[0038] Table 2 shows that there were significant differences in the types of immune responses induced by different adjuvants. Groups G1 (206) and G2 (803) exhibited a strong Th1-type immune response bias, with significantly higher IgG2a levels and IgG2a / IgG1 ratios than other groups, consistent with the characteristics of TLR agonists activating cellular immune pathways. Conversely, group G3 (aluminum hydroxide) showed a typical Th2-type response bias, with the highest IgG1 level and the lowest IgG2a / IgG1 ratio (0.54). Group G4 (white oil) showed a relatively balanced Th1 / Th2 mixed response. Group G5 (live vaccine) also showed some Th1 bias. For viral diseases like LSD, inducing strong Th1 and cellular immunity is crucial for clearing intracellular viruses.

[0039] 1.3. Conclusion This adjuvant screening experiment shows that: Immunogenicity strength: The P122-Fc fusion protein, in combination with 206 or 803 adjuvants, can induce the highest level of total antibody titer and produce an immune response that is extremely Th1-biased, which is theoretically most advantageous for defense against LSD virus infection.

[0040] Differences in immune response types: Although aluminum hydroxide adjuvant can induce high-titer antibodies, it mainly produces a Th2-biased response with weak cellular immunity. The immune response induced by white oil adjuvant is more balanced in strength and type.

[0041] Overall Preferred Optimization: Considering the characteristics of LSD virus and the potential need for strong cellular immunity to induce protective immunity, TLR agonist adjuvants (especially 206 adjuvant) demonstrate the best performance in inducing a comprehensive and high-quality immune response, making them the preferred adjuvants for the P122-Fc subunit vaccine of this invention. Aluminum hydroxide adjuvant can be considered as an alternative for inducing strong humoral immunity.

[0042] 2. Vaccine preparation and sample preparation Recombinant P122-Fc fusion protein was prepared strictly according to the process described in patent CN117229368A. Using the selected adjuvant, three batches of bovine nodular dermatitis P122-Fc subunit vaccine were continuously prepared under the same production conditions, with batch numbers SY2301, SY2302, and SY2303. All batches used the same CHO engineered cell line, purification process, adjuvant (MONTANIDE™ ISA 206 VG), and emulsification parameters (the diluted antigen and adjuvant were mixed at a predetermined ratio, with a final antigen concentration of 50 μg / mL, and the volume ratio of diluent to adjuvant was 1:1). A homogeneous milky white vaccine sample was obtained and used for all subsequent tests.

[0043] 3. Safety Inspection 3.1. Experimental Animals Animals: 20 healthy calves aged 3-6 months and 20 healthy pregnant cows in mid-pregnancy (approximately 4-5 months of gestation).

[0044] 3.2 Safety testing of bovine nodular cutaneous virus P122 recombinant protein subunit vaccine in calves and pregnant cows. One batch of the three consecutive batches of trial-produced vaccines (SY2301, SY2302, SY2303) was randomly selected to conduct single-dose vaccination trials, repeated-dose vaccination trials, and overdose vaccination trials on the recommended target animals (calves and pregnant cows).

[0045] Table 3: Grouping of experimental animals

[0046] Twenty calves and twenty pregnant cows were randomly grouped according to Table 3 and then isolated. Observations were conducted continuously until day 14 after the last vaccination, recording the survival, mental state, diet, local and systemic reactions of all experimental cattle daily. Pregnant cows were also closely monitored for signs of abortion or abnormal vaginal discharge. Safety test results are detailed in Table 4. Results showed that all immunized experimental cattle survived during the observation period, with no deaths or significant local (no persistent swelling or necrosis at the injection site) or systemic reactions (no persistent high fever or depression) caused by the vaccine. Their mental state and feed and water intake were normal, with no visible differences compared to the control group. All pregnant cows in the immunized group maintained normal pregnancies without abortion. The results indicate that the three batches of vaccine are safe for calves of the youngest age and pregnant cows in mid-pregnancy, with consistent performance across batches.

[0047] Table 4: Safety Inspection Results of the Genetically Engineered Subunit Trial Vaccine for Bovine Nodular Dermatitis (Batch SY2301)

[0048] Note: "R" indicates a red ear tag; "Y" indicates a yellow ear tag; "B" indicates a blue ear tag; "G" indicates a green ear tag.

[0049] 4. Validity test 4.1. Experimental Animals Animals: 10 healthy calves aged 3-6 months and 10 healthy pregnant cows in mid-pregnancy (approximately 4-5 months of gestation).

[0050] 4.2 Efficacy testing of bovine nodular cutaneous virus P122 recombinant protein subunit vaccine in calves and pregnant cows. One batch of the trial-produced vaccines from three consecutive batches (SY2301, SY2302, and SY2303) was randomly selected to conduct efficacy testing on the recommended target animals (calves and pregnant cows).

[0051] 4.2.1 Experimental grouping and immunization Experimental animals: 3-6 month old LSDV-negative calves and mid-pregnancy LSDV-negative pregnant cows.

[0052] Trial design: Each batch of vaccine was tested independently. Taking one batch as an example: 10 calves were randomly divided into an immunization group (5 calves) and a control group (5 calves); 10 pregnant cows were randomly divided into an immunization group (5 calves) and a control group (5 calves).

[0053] Immunization schedule: Calves / pregnant cows in the immunization group were injected intramuscularly with 1.0 mL of vaccine (1 dose) in the neck, while the control group was injected with an equal volume of physiological saline. A booster immunization was administered 21 days after the initial immunization using the same method.

[0054] Table 5 Grouping of experimental animals

[0055] 4.2.2 Neutralizing antibody detection Blood samples were collected and serum separated before immunization and 21 days after the second immunization (i.e., 42 days after the first immunization). Specific antibodies were detected using an indirect ELISA based on the P122 recombinant protein, and serum neutralizing antibody titers were determined using a virus micro-neutralization assay. Serum toxicity controls and normal cell controls should be normal, and the virus control should be valid. The neutralizing antibody titers in the control group serum should not exceed 1:8.

[0056] 4.2.3 Neutralizing antibody detection results The detailed results of the efficacy test for batch SY2301 vaccine are shown in Table 6, and it meets the qualification standard.

[0057] Table 6 Neutralizing antibody detection results

[0058] The results of neutralizing antibody titers showed that the serum neutralizing antibody titers of calves and pregnant cows after the second immunization reached or exceeded 1:32, while those of the control group were all below 1:8.

[0059] 5. Conclusion The bovine nodular dermatitis genetically engineered subunit vaccine (P122 recombinant protein) prepared by this invention (especially at the recommended dose) has good safety in cattle, with mild and manageable adverse reactions. It has passed efficacy testing, and three consecutive batches of the vaccine have induced high levels of neutralizing antibodies in the target animals (calves and pregnant cows).

Claims

1. A P122-Fc fusion protein-based subunit vaccine for bovine nodular dermatopathy, characterized by, Comprise: P122-Fc fusion protein, water-in-oil-in-water multiple emulsion adjuvant MONTANIDE™ ISA 206 VG.

2. The bovine paratuberculosis subunit vaccine according to claim 1, characterized in that, The P122-Fc fusion protein is a bovine dermal nodular disease virus P122 recombinant protein described in patent CN117229368A.

3. The bovine paratuberculosis subunit vaccine of claim 1, wherein, The antigen content of the P122-Fc fusion protein is 50 μg / mL.

4. The bovine paratuberculosis subunit vaccine of claim 1, wherein, The volume ratio of the water-in-oil-in-water multiple emulsion adjuvant MONTANIDE™ ISA 206 VG to the P122-Fc fusion protein diluent is 1:1.

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