A canine multi-infectious disease vaccine composition, its preparation method and application

Abstract

The application discloses a kind of canine multi-infectious disease vaccine composition and its preparation method and application, belong to the technical field of veterinary biological products, the vaccine composition includes canine distemper virus antigen, canine parvovirus antigen and canine adenovirus type 2 antigen, aluminum adjuvant complex with canine parvovirus antigen is adsorbed, chitosan nanoparticle with immunostimulatory oligonucleotide is wrapped, and polylactic acid-hydroxyacetic acid copolymer sustained-release microsphere with part canine parvovirus antigen is wrapped;Wherein part of canine parvovirus antigen is wrapped in sustained-release microsphere, and the remaining part is adsorbed on aluminum adjuvant.The application uses the above-mentioned one kind of canine multi-infectious disease vaccine composition and its preparation method and application, through the division of labor of three functional units and the differentiation of delivery of the same antigen, can simultaneously efficiently activate humoral immunity and cellular immunity, significantly improve immunogenicity and immunological persistence.

Description

Technical Field

[0001] This invention relates to the field of veterinary biological products technology, and in particular to a canine multi-infectious disease vaccine composition, its preparation method, and its application. Background Technology

[0002] Canine distemper, canine parvovirus, and canine adenovirus infection are the three major viral infectious diseases threatening the health of canines. Multivalent vaccines are an important technological means to achieve multiple protections with a single injection and simplify the immunization schedule. Currently, several multivalent vaccine products targeting these three diseases are available on the market. However, existing canine multivalent vaccines still have the following technical shortcomings: The adjuvant system is singular, making it difficult to fully activate both humoral and cellular immune responses. While the aluminum adjuvant most commonly used in existing vaccines can effectively induce humoral immunity, its ability to activate Th1-type cellular immunity is limited. The immunogenicity and duration of immunity of canine parvovirus subunit vaccines need improvement. Subunit vaccines based on recombinant VP2 protein have good safety profiles, but face technical bottlenecks such as insufficient immunogenicity and short protection period. Attenuated live vaccines pose a risk of residual virulence. Most existing canine vaccines use attenuated live vaccine technology, which, while effective, still poses safety risks to specific groups such as puppies and pregnant dogs. The stability control of multi-component mixtures faces challenges, as the physicochemical compatibility between different components directly affects vaccine stability.

[0003] Therefore, how to develop a canine multivalent vaccine that is highly safe, immunogenic, and has good formulation stability is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0004] The purpose of this invention is to provide a canine multivalent infectious disease vaccine composition, its preparation method, and its application, in order to solve the above-mentioned problems.

[0005] This invention provides a canine multivalent infectious disease vaccine composition comprising canine distemper virus antigen, canine parvovirus antigen, canine adenovirus type 2 antigen, as well as a modular compound adjuvant system and a pharmaceutically acceptable carrier. The modular composite adjuvant system consists of the following three units: (a) Unit 1: Aluminum adjuvant complex adsorbed with canine parvovirus antigen; (b) Second unit: Chitosan nanoparticles loaded with immunostimulatory oligonucleotides; (c) Unit 3: Polylactic acid-glycolic acid copolymer sustained-release microspheres containing partial canine parvovirus antigen; In this embodiment, a portion of the canine parvovirus antigen is encapsulated in the polylactic acid-glycolic acid copolymer sustained-release microspheres, while the remaining portion is adsorbed onto the aluminum adjuvant complex.

[0006] The first unit is an aluminum adjuvant complex adsorbed with canine parvovirus antigen. Aluminum adjuvants are currently the most widely used protein antigen adjuvants, effectively promoting antigen presentation and inducing humoral immune responses. This unit directly adsorbs a portion of the canine parvovirus VP2 protein onto the surface of the aluminum adjuvant, achieving immediate antigen delivery.

[0007] The second unit consists of chitosan nanoparticles loaded with immunostimulatory oligonucleotides (CpG oligonucleotides). CpG oligonucleotides, as TLR9 agonists, can effectively induce a Th1-dominant immune response, compensating for the shortcomings of aluminum adjuvants in activating cellular immunity. Chitosan, a biocompatible polysaccharide material, not only efficiently loads CpG oligonucleotides into its nanoparticles but also promotes mucosal immune responses, enhancing the overall immune efficacy of the system.

[0008] The third unit consists of PLGA sustained-release microspheres encapsulating a portion of canine parvovirus antigen. PLGA (polylactic-co-glycolic acid copolymer) is a biocompatible and biodegradable polymer chemically synthesized from monomers lactic acid and glycolic acid. It possesses a range of characteristics including controlled and sustained drug release, low cytotoxicity, tissue and cell biocompatibility, and targeted delivery. Encapsulating a portion of canine parvovirus antigen in PLGA microspheres enables sustained antigen delivery, prolonging the antigen's exposure time in vivo, thereby enhancing immune memory responses and extending the duration of immune protection.

[0009] A portion of the canine parvovirus antigen is encapsulated in the polylactic acid-glycolic acid copolymer sustained-release microspheres, while the remainder is adsorbed onto the aluminum adjuvant complex. This allows the same antigen to exist in the same vaccine composition in two different delivery forms: a portion is delivered via aluminum adjuvant adsorption for immediate immune activation, and the remaining portion is delivered via PLGA microsphere encapsulation for sustained release, jointly achieving highly effective and durable immune protection.

[0010] Preferably, both the canine distemper virus antigen and the canine adenovirus type 2 antigen are viral antigens obtained through cell culture proliferation and purification, and the content of canine distemper virus antigen in each 1 mL of vaccine is 10. 5.5 ~10 6.5 TCID 50 The content of canine adenovirus type 2 antigen is 10. 5.5 ~10 6.5 TCID 50 .

[0011] Preferably, the canine parvovirus antigen is a recombinant VP2 protein, which is obtained by affinity chromatography purification after soluble expression in Escherichia coli.

[0012] Preferably, the total content of canine parvovirus antigen in each 1 mL of vaccine is 80-150 μg, of which 35%-45% is encapsulated in the polylactic acid-glycolic acid copolymer sustained-release microspheres, and the remaining portion is adsorbed onto the aluminum adjuvant complex; The content of aluminum adjuvant is 1.0–1.5 mg; The content of chitosan nanoparticles is 0.5-1.0 mg, and the content of the immunostimulatory oligonucleotides contained in the chitosan nanoparticles is 20-30 μg; The content of polylactic acid-glycolic acid copolymer sustained-release microspheres is 2-4 mg.

[0013] Preferably, the chitosan nanoparticles are prepared by iontophoresis of chitosan with a degree of deacetylation of 85% to 95% and a molecular weight of 50 to 100 kDa, and the immunostimulatory oligonucleotides are CpG oligonucleotides modified with phosphate thioester.

[0014] Preferably, the particle size of the polylactic acid-glycolic acid copolymer sustained-release microspheres is 2-8 μm, the molar ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 75:25, and the loading rate of the polylactic acid-glycolic acid copolymer sustained-release microspheres on the canine parvovirus antigen is 70%-85%.

[0015] Preferably, it also includes a complex stabilizer, which consists of trehalose, mannitol, gelatin and Tween-80.

[0016] A method for preparing the canine multivalent infectious disease vaccine composition as described above is provided, comprising the following steps: (1) Prepare canine distemper virus antigen, canine parvovirus recombinant VP2 protein antigen and canine adenovirus type 2 antigen respectively for later use; (2) Chitosan nanoparticles loaded with immunostimulatory oligonucleotides were prepared by ionogel method; (3) Polylactic acid-glycolic acid copolymer sustained-release microspheres loaded with part of canine parvovirus antigen were prepared by water-in-oil-in-water double emulsion-solvent evaporation method; (4) The remaining canine parvovirus antigen and aluminum adjuvant are adsorbed under stirring conditions to form the first unit; (5) Under stirring conditions at 4°C, the canine distemper virus antigen, the canine adenovirus type 2 antigen and the chitosan nanoparticles prepared in step (2) are added sequentially to the product of step (4), and the mixture is stirred after each addition of a component. (6) Finally, add the polylactic acid-glycolic acid copolymer sustained-release microspheres prepared in step (3), mix well, add composite stabilizer, adjust the volume, sterilize and dispense to make liquid vaccine.

[0017] Preferably, the stirring speed in steps (5) and (6) does not exceed 200 rpm; the composite stabilizer is composed of trehalose 25-40 g / L, mannitol 15-25 g / L, gelatin 8-12 g / L and Tween-80 0.2-0.4 mL / L.

[0018] The canine multi-infectious disease vaccine composition described above is used to prepare a drug for the prevention of canine distemper, canine parvovirus disease and canine adenovirus type 2 infection.

[0019] Therefore, the present invention, employing the above-mentioned canine multivalent infectious disease vaccine composition, its preparation method, and its application, has the following beneficial effects: (1) Through modular design, the four adjuvant components are organically integrated into three functional units: the aluminum adjuvant complex is responsible for the antigen reservoir effect and humoral immune activation; the chitosan nanoparticles loaded with CpG oligonucleotides are responsible for TLR9 pathway activation and Th1-type cellular immune induction; and the PLGA microspheres are responsible for sustained-release delivery and immune memory enhancement. The three units work together to induce high-titer neutralizing antibodies and stimulate a strong cellular immune response, achieving comprehensive protection against canine distemper, canine parvovirus, and canine adenovirus type 2.

[0020] (2) The canine parvovirus VP2 protein is divided into two parts: one part is adsorbed onto aluminum adjuvant and is rapidly released in the early stage of immunization to activate the immune response and generate early protection; the other part is encapsulated in PLGA microspheres and is slowly released in the later stage of immunization to continuously stimulate the immune system and prolong the period of immune protection, so that the same vaccine can both rapidly generate protective antibodies and maintain long-term immune memory.

[0021] (3) The preparation method adopts a three-unit independent preparation and step-by-step mixing strategy. Chitosan-CpG nanoparticles and PLGA sustained-release microspheres are prepared by iontophoresis and double emulsion-solvent evaporation methods, respectively. After preparation, they are mixed with aluminum adjuvant antigen complex by gentle stirring. The entire mixing process avoids the use of ultrasound and high-speed shearing, and the stirring speed is controlled below 200 rpm, which effectively prevents the destruction of the nanoparticle and microsphere structure and ensures the stability of the physicochemical properties and biological activities of each functional unit.

[0022] (4) The combination of canine distemper virus and canine adenovirus type 2 antigen with canine parvovirus recombinant VP2 protein antigen not only inherits the advantage of strong immunogenicity of viral antigens, but also reduces biosafety risks by substituting live virus with subunit antigens, thus taking into account both immunization efficacy and safety of use.

[0023] (5) The optimal content range of each component was determined, so that the recombinant VP2 protein of canine parvovirus could generate a strong immune response at a low dose of 80-150 μg / dose, and 35%-45% of the antigen was loaded in PLGA microspheres to achieve sustained release and enhanced effect, effectively reducing the demand for total antigen, reflecting economy and sustainability.

[0024] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0025] Figure 1 This is a graph showing the stability data of a canine multivalent infectious disease vaccine composition, its preparation method, and its application example 1, stored at 4°C for 12 months. Detailed Implementation

[0026] To better understand the above technical solutions, a detailed description of the solutions will be provided below in conjunction with the accompanying drawings and specific embodiments. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0027] The terminology used in the embodiments of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The singular forms “a,” “the,” and “the” as used in the embodiments of this invention and the appended claims are also intended to include the plural forms, and “multiple” generally includes at least two unless the context clearly indicates otherwise.

[0028] It should also be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that an article or device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such an article or device. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the article or device that includes said element.

[0029] Example 1 Antigen preparation: Preparation of canine distemper virus antigen: Canine distemper virus Onderstepoort strain was inoculated into Vero cells and cultured at 37°C and 5% CO2. Viral fluid was harvested when cytopathic effects reached 70%–80%. The harvested viral fluid was subjected to three freeze-thaw cycles, centrifuged to remove cell debris, and then filtered through a 0.45 μm filter for sterilization. Ultrafiltration was performed using a 100 kDa molecular weight cutoff membrane for concentration, followed by purification via sucrose density gradient centrifugation at 35,000 rpm for 2 hours. Viral bands were collected, dialysis with PBS to remove sucrose, and total protein content was determined using the BCA method. The viral antigen concentration was adjusted to 60 μg total protein / mL for later use. The half-maximal tissue culture infection dose (MCD) of this viral antigen solution was determined using the Reed-Muench method to be 10. 6.0 TCID 50 / 0.1mL.

[0030] Preparation of recombinant canine parvovirus VP2 protein antigen: The codon-optimized gene sequence encoding the canine parvovirus VP2 protein was cloned into the pET-28a(+) expression vector and transformed into *E. coli* BL21(DE3) competent cells. The gene sequence was codon-optimized for the *E. coli* expression system based on the VP2 gene sequence in GenBank accession number KF149893.1. Positive clones were picked and inoculated into LB medium, and cultured at 37°C with shaking until OD... 600 The concentration of the bacterial cells was 0.6–0.8 mmol / L. IPTG was added to a final concentration of 0.8 mmol / L, and expression was induced at 25°C for 14 hours. The cells were collected and sonicated using a 200W ultrasonicator with a 3-second working time followed by a 4-second interval for a total of 30 minutes. The supernatant was collected by centrifugation at 4°C. The supernatant was purified by nickel affinity chromatography using a HisTrap column. TM FF (5 mL) solution was used. Non-specifically bound proteins were removed by washing with PBS containing 20 mmol / L imidazole, followed by elution of the target protein with PBS containing 250 mmol / L imidazole. The eluent was dialyzed to remove imidazole, and protein concentration was determined using the BCA method, then analyzed by Superdex. TM Further purification was performed using 200 Increase 10 / 300 GL gel filtration chromatography. SDS-PAGE showed that the recombinant VP2 protein had a purity of approximately 98%, and Western blotting confirmed its specific binding to the anti-VP2 monoclonal antibody, indicating retention of the major neutralizing antigenic epitopes of canine parvovirus. Approximately 35 mg of recombinant protein was obtained per liter of fermentation broth. The purified recombinant VP2 protein was diluted to 1.5 mg / mL with PBS and set aside for later use.

[0031] Preparation of canine adenovirus type 2 antigen: Canine adenovirus type 2 Manhattan strain was inoculated into MDCK cells and cultured at 37°C and 5% CO2. The viral fluid was harvested when cytopathic effects reached 80%–90%. After three freeze-thaw cycles, cell debris was removed by centrifugation, followed by sterilization via filtration through a 0.45 μm membrane and ultrafiltration concentration and purification using a 100 kDa molecular weight cutoff membrane. Total protein content was determined by the BCA method, and the viral antigen concentration was adjusted to 60 μg total protein / mL for later use. The half-maximal inhibitory dose (ICD) of the viral antigen solution for tissue culture was determined by the Reed-Muench method to be 10. 6.0 TCID 50 / 0.1mL.

[0032] Preparation of modular composite adjuvant systems: Unit 1: Preparation of aluminum adjuvant complexes adsorbed with canine parvovirus antigen; 1.0 mg of aluminum adjuvant and an appropriate amount of recombinant VP2 protein antigen were stirred and adsorbed at 37°C and 150 rpm for 45 minutes to allow the VP2 protein to be fully adsorbed onto the surface of the aluminum adjuvant, forming the first unit. The aluminum adjuvant was Alhydrogel. TM The aluminum hydroxide content is 10 mg / mL, with Al 3+ count.

[0033] Unit 2: Preparation of chitosan nanoparticles loaded with immunostimulatory oligonucleotides; Chitosan with a degree of deacetylation of 92% and a molecular weight of 80 kDa was dissolved in 1% acetic acid solution to prepare a 2 mg / mL chitosan solution, and the pH was adjusted to 5.5. Phosphothiophosphate-modified CpG oligonucleotides were dissolved in nuclease-free water to prepare a 1 mg / mL CpG solution. Under stirring, the CpG solution was added dropwise to the chitosan solution to achieve a chitosan to CpG mass ratio of 10:1, and stirring was continued for 30 minutes. Subsequently, sodium tripolyphosphate solution (0.5 mg / mL) was added for ionic crosslinking, with a sodium tripolyphosphate to chitosan mass ratio of 1:5, and stirring was continued for 1 hour. The resulting suspension was centrifuged at 10,000 rpm for 30 minutes at 4 °C. The precipitate was washed three times with PBS and resuspended in PBS to obtain chitosan-CpG nanoparticles. Dynamic light scattering analysis showed that the average particle size of the nanoparticles was 320 nm, the Zeta potential was +25 mV, and the CpG loading rate was 82.5%.

[0034] Unit 3: Preparation of polylactic acid-glycolic acid copolymer sustained-release microspheres loaded with some canine parvovirus antigen; The mixture was prepared using a water-in-oil-in-water double emulsion-solvent evaporation method. Polylactic acid-glycolic acid copolymer (PLA-GAC) was dissolved in dichloromethane to prepare an organic phase with a concentration of 80 mg / mL. The molar ratio of lactic acid to glycolic acid in the PLA-GAC was 75:25, and the molecular weight was 20000 Da. The recombinant VP2 protein to be encapsulated was dissolved in PBS to prepare an aqueous phase with a concentration of 1.5 mg / mL. The aqueous phase was added to the organic phase at a volume ratio of 5:1, and the mixture was ultrasonically emulsified for 2 minutes at 200 W under ice bath conditions to form a water-in-oil primary emulsion. The primary emulsion was then added to an aqueous solution containing 2% polyvinyl alcohol (PVA) with a molecular weight of 30000 at a volume ratio of 1:10. The mixture was stirred and emulsified at 800 rpm for 7 minutes at room temperature to form a water-in-oil-in-water double emulsion. Subsequently, the mixture was continuously stirred at 300 rpm for 4 hours at room temperature to allow complete evaporation of the dichloromethane. The microsphere precipitate was collected by centrifugation at 10,000 rpm for 10 minutes, washed three times with PBS, and freeze-dried for 24 hours to obtain polylactic acid-glycolic acid copolymer sustained-release microspheres. The average particle size of the microspheres was determined to be 5.2 μm by dynamic light scattering. The VP2 protein loading rate in the microspheres was determined using the BCA method: 10 mg of microspheres were dissolved in 0.1 M NaOH-1% SDS solution, shaken in a 37°C water bath for 24 hours until completely dissolved, and the protein content was determined by BCA method, with a calculated loading rate of 78.3%. In vitro release assay: The polylactic acid-glycolic acid copolymer sustained-release microspheres were suspended in pH 7.4 PBS buffer and placed in a 37°C constant-temperature shaker. Samples were centrifuged at specific time points to determine the released protein content. The results showed that the in vitro sustained-release time of the microspheres was no less than 28 days, and the cumulative release rate was 92.4% on day 28.

[0035] Vaccine preparation: Take the first, second, and third units of vaccine according to the composition of each 1 mL of vaccine and mix them together: The total content of canine parvovirus antigen is 120 μg, of which about 45 μg is encapsulated in polylactic acid-glycolic acid copolymer sustained-release microspheres and about 75 μg is adsorbed on aluminum adjuvant complex. The canine distemper virus antigen content was 30 μg of total protein; The antigen content of canine adenovirus type 2 was 30 μg of total protein; The aluminum adjuvant content is 1.2 mg, in Al 3+ count; The chitosan nanoparticle content is 0.8 mg, of which the CpG oligonucleotide content is 25 μg; The content of polylactic acid-glycolic acid copolymer sustained-release microspheres is 3.0 mg.

[0036] Under stirring conditions of 4℃ and 80 rpm, the first unit, the aluminum adjuvant-VP2 complex, was first placed in a preparation container. Canine distemper virus antigen, canine adenovirus type 2 antigen, and the second unit, chitosan-CpG nanoparticles, were added sequentially, with stirring for 10 minutes after each addition. Finally, the third unit, polylactic acid-glycolic acid copolymer sustained-release microspheres, was added and gently stirred for 10 minutes. A composite stabilizer, composed of 30 g / L trehalose, 20 g / L mannitol, 10 g / L gelatin, and 0.3 mL / L Tween-80, was added. The mixture was brought to the specified volume with PBS, sterilized through a 0.22 μm filter, and dispensed into 1 mL vials. After passing sterility testing, the vials were stored at 4℃ protected from light for later use.

[0037] Example 2 The vaccine composition was prepared using essentially the same method as in Example 1, except that the amounts of each component were adjusted as follows: In each 1 mL of vaccine, the total content of canine parvovirus antigen was 100 μg, of which 40% was encapsulated in polylactic acid-glycolic acid copolymer sustained-release microspheres and 60% was adsorbed onto aluminum adjuvant; 20 μg of canine distemper virus antigen; 20 μg of canine adenovirus type 2 antigen; and 1.0 mg of aluminum adjuvant, in Al... 3+ The formula included: 0.6 mg chitosan nanoparticles, containing 22 μg CpG oligonucleotides; and 2.5 mg polylactic acid-glycolic acid copolymer sustained-release microspheres. The composite stabilizer used the same formulation as in Example 1. After preparation, the mixture passed sterility testing and was stored at 4°C protected from light for later use.

[0038] Example 3 The vaccine composition was prepared using essentially the same method as in Example 1, except that the amounts of each component were adjusted as follows: In each 1 mL of vaccine, the total content of canine parvovirus antigen was 140 μg, of which 42% was encapsulated in polylactic acid-glycolic acid copolymer sustained-release microspheres and 58% was adsorbed onto aluminum adjuvant; canine distemper virus antigen was 35 μg; canine adenovirus type 2 antigen was 35 μg; and aluminum adjuvant was 1.4 mg, in Al... 3+ The formula included: 0.9 mg chitosan nanoparticles, containing 28 μg of CpG oligonucleotides; and 3.5 mg polylactic acid-glycolic acid copolymer sustained-release microspheres. The composite stabilizer used the same formulation as in Example 1. After preparation, the mixture passed sterility testing and was stored at 4°C protected from light for later use.

[0039] Comparative Example 1 The antigen was prepared according to the method in Example 1, but only aluminum adjuvant was used in the vaccine; chitosan-CpG nanoparticles and polylactic acid-glycolic acid copolymer sustained-release microspheres were not added. Furthermore, 120 μg of the entire canine parvovirus recombinant VP2 protein antigen was adsorbed onto 1.2 mg of aluminum adjuvant. The contents of other antigens were the same as in Example 1. After preparation, the vaccine was sterilized, aliquoted, and stored at 4°C for later use.

[0040] Comparative Example 2 The antigen was prepared according to the method in Example 1, but the vaccine did not contain polylactic acid-glycolic acid copolymer sustained-release microspheres. 120 μg of the complete canine parvovirus recombinant VP2 protein antigen was adsorbed onto 1.2 mg of aluminum adjuvant. Other components, namely 0.8 mg of chitosan-CpG nanoparticles (containing 25 μg of CpG), were the same as in Example 1. After preparation, the vaccine was sterilized, aliquoted, and stored at 4°C for later use.

[0041] Comparative Example 3 The antigen was prepared according to the method in Example 1, but the vaccine did not contain chitosan-CpG nanoparticles. Other components, namely 1.2 mg of aluminum adjuvant, 3.0 mg of polylactic-co-glycolic acid copolymer sustained-release microspheres encapsulating 45 μg of VP2 protein, and the remaining 75 μg of VP2 protein adsorbed onto the aluminum adjuvant, were prepared in the same manner as in Example 1. After preparation, the vaccine was sterilized, aliquoted, and stored at 4°C for later use.

[0042] Comparative Example 4 The antigen and components were prepared according to the method in Example 1, but during vaccine preparation, all components—aluminum adjuvant-VP2 adsorption complex, chitosan-CpG nanoparticles, polylactic acid-glycolic acid copolymer sustained-release microspheres, canine distemper virus antigen, and canine adenovirus type 2 antigen—were directly mixed in one step and stirred at room temperature for 15 minutes without being added in stages. Other processes were essentially the same as in Example 1. After preparation, the vaccine was sterilized, dispensed, and stored at 4°C for later use.

[0043] Comparative Example 5 A commercially available canine trivalent vaccine was selected as a control. This vaccine is a lyophilized powder, reconstituted with the accompanying diluent before use, and each dose contains at least 10 mg of canine distemper virus N-CDV strain. 2.5 TCID 50 At least 10 attenuated Manhattan strains of canine adenovirus type 2. 2.9 TCID 50 At least 10 canine parvovirus NL-35-D strains 7.0 TCID 50 This comparative study used it as a positive control vaccine.

[0044] Comparative Example 6 PBS containing no antigens or adjuvants was used, with sterile PBS solution as a blank control.

[0045] Experimental Example 1 Evaluation of the immunization efficacy of canine multivalent vaccines: Laboratory animals and grouping: Select healthy puppies aged 6-8 weeks, and confirm that they are negative for maternal antibodies against canine distemper virus, canine parvovirus, and canine adenovirus type 2 by ELISA testing. Half of the puppies should be male and half female.

[0046] A total of 70 dogs were randomly divided into 7 groups of 10 dogs each. The immunization schedules for each group are as follows: Example 1 group: 1 mL of the vaccine prepared in Example 1 was injected intramuscularly into each animal; Comparative Example 1: Each animal received an intramuscular injection of 1 mL of the vaccine prepared in Comparative Example 1. Comparative Example 2: Each animal received an intramuscular injection of 1 mL of the vaccine prepared in Comparative Example 2. Comparative Example 3: Each animal received an intramuscular injection of 1 mL of the vaccine prepared in Comparative Example 3. Comparative Example 4: Each animal received an intramuscular injection of 1 mL of the vaccine prepared in Comparative Example 4. Comparative Group 5: Each animal received a commercially available trivalent vaccine via intramuscular injection, followed by reconstitution and one dose per animal; Comparative group 6: each animal was injected intramuscularly with 1 mL of PBS.

[0047] All groups received a second immunization on day 21 after the first immunization, with the same vaccine and dosage as the first immunization.

[0048] Detection method: Serum neutralizing antibody titer assay: Venous blood was collected from dogs in each group on days 14 and 28 (day 7 after the second vaccination), day 42 (day 21 after the second vaccination), day 90, and day 180 after the second vaccination, and serum was separated. The titers of neutralizing antibodies against canine distemper virus, canine parvovirus, and canine adenovirus type 2 were determined using a micro-neutralization assay. Neutralizing antibody titers were defined as complete inhibition of 50 TCID50. 50 The log2 value of the highest serum dilution factor in which the virus causes cytopathic effects is represented, and a neutralizing antibody titer ≥1:32 is considered positive.

[0049] T lymphocyte subset detection: On day 42 after the initial vaccination, three dogs were randomly selected from each group to collect peripheral anticoagulated blood samples, and CD4 counts were measured by flow cytometry. + and CD8 + The proportion of T lymphocytes, and the calculation of CD4. + / CD8 + ratio.

[0050] Virus challenge protection test: Four weeks after the second vaccination (49 days after the first vaccination), five dogs were randomly selected from each group for a virulent virus challenge experiment. The challenge protocol was as follows: virulent canine distemper virus strain SY was administered at 10... 5.0 TCID 50 Each dose was administered via nasal instillation; the virulent canine adenovirus type 2 strain was administered at a dose of 10... 5.0 TCID 50 Each dose was administered via nasal instillation; the virulent canine parvovirus CPV-2a strain was administered at a dose of 10... 5.0 TCID50 Each dog was administered the virus orally. Following challenge, dogs were observed for 21 consecutive days, and the incidence of disease and the number of deaths or near-death euthanasia were recorded for each group. Experimental dogs exhibiting severe clinical symptoms such as persistent high fever, severe hemorrhagic enteritis, or neurological symptoms, and deemed unfit for survival, were promptly euthanized humanely to minimize suffering. Protection rate = (1 - number of deaths / near-death euthanasia / number of challenged animals) × 100%.

[0051] Test results: Table 1. Serum CDV neutralizing antibody titers (Log2) in each group of dogs

[0052] Table 2. Serum CPV neutralizing antibody titers (Log2) in each group of dogs

[0053] Table 3. Serum CVA-2 neutralizing antibody titers (Log2) in each group of dogs

[0054] As shown in Tables 1 to 3, the Example 1 group produced a high level of neutralizing antibodies as early as day 14 after the first immunization, with CDV of 4.2±0.4, CPV of 4.8±0.4, and CAV-2 of 4.0±0.4, significantly higher than that of the Comparative Examples 1 to 6. On days 28 and 42 after the second immunization, the antibody titers of the Example 1 group reached their peak, with CDV of 7.5–8.1, CPV of 8.0–8.6, and CAV-2 of 7.2–7.8, all significantly higher than those of other groups. On days 90 and 180, the neutralizing antibody titers of the Example 1 group remained at a high level, with CDV of 5.3±0.4, CPV of 5.9±0.5, and CAV-2 of 4.9±0.4, while the antibody titers of other groups decreased significantly at the same time points. The vaccine composition of Example 1 was able to induce a higher level of neutralizing antibody response, and the antibody persistence was significantly better than that of Comparative Example 1 containing only aluminum adjuvant, Comparative Example 2 without polylactic acid-glycolic acid copolymer sustained-release microspheres, Comparative Example 3 without chitosan-CpG nanoparticles, and Comparative Example 5 of commercial vaccines.

[0055] Table 4. Proportions of lymphocyte subsets in each group of dogs on day 42 after the first vaccination

[0056] Note: The sample size for each group is 3 dogs, and the values ​​are expressed as the mean plus or minus the standard deviation.

[0057] As shown in Table 4, the CD4+ level in the peripheral blood of dogs in Example 1 group was... + The proportion of T lymphocytes was 35.6 ± 2.8%, CD4+ + / CD8+ The ratio was 1.59 ± 0.12, significantly higher than that of comparative groups 1 to 6. The modular composite adjuvant system of this invention can more effectively activate cellular immune responses, in which chitosan-CpG nanoparticles play a key immunostimulatory role, and CD4... + / CD8 + The increased ratio indicates that the vaccine induced a stronger helper T-cell immune response.

[0058] Table 5 Results of dog challenge tests with virulent strain in each vaccine group

[0059] Note: Protection rate = (1 - number of dead / near-death euthanasia animals / number of challenged animals) × 100%.

[0060] Results Analysis: Table 5 shows that the protection rate of the combined challenge with virulent canine distemper virus, canine parvovirus, and canine adenovirus type 2 in Example 1 reached 100%, and all 5 challenged dogs survived without any clinical symptoms. The protection rate of Comparative Example 2 was 80%, and although some dogs developed the disease, all survived. The protection rate of Comparative Example 5 was 80%, with one dog developing the disease and dying. The protection rates of Comparative Example 1 and Comparative Example 3 were both 60%. The modular composite adjuvant system vaccine of this invention can provide comprehensive and efficient immune protection against three canine infectious diseases, with significantly better protective effects than the comparative examples.

[0061] The above experimental results demonstrate the superiority of the modular composite adjuvant system vaccine established in this invention. Regarding antibody response, the antibody levels in the Example 1 group were significantly higher than those in the control groups on day 14 after the first immunization, and maintained a protective antibody titer at 180 days. The three functional units of this invention—the aluminum adjuvant complex responsible for antigen reservoir effect and humoral immune activation, the chitosan-CpG nanoparticles responsible for TLR9 pathway activation and Th1-type cellular immune induction, and the polylactic-co-glycolic acid copolymer sustained-release microspheres responsible for sustained-release delivery and enhanced immune memory—result in a synergistic immune effect.

[0062] From the perspective of cellular immunity, CD4 in Group 1 of Example 1 + T cell percentage and CD4 + / CD8 + The ratios were significantly higher than those of other groups, indicating that the introduction of chitosan-CpG nanoparticles effectively compensated for the shortcomings of aluminum adjuvant in cellular immune activation. In terms of long-lasting protection, the antibody level in Example 1 remained high on day 180, significantly better than Comparative Example 1 (containing only aluminum adjuvant) and Comparative Example 2 (not containing polylactic-co-glycolic acid copolymer sustained-release microspheres). This demonstrates that the sustained-release delivery mechanism of the third unit, namely the polylactic-co-glycolic acid copolymer sustained-release microspheres, produced a long-lasting immune-enhancing effect.

[0063] Furthermore, Comparative Example 4, which used a conventional one-step mixing process, showed lower antibody levels on days 42 and 90 compared to Example 1. This demonstrates that the step-by-step independent preparation followed by gentle mixing process is of great significance in protecting the integrity and activity of each functional unit.

[0064] Experiment Example 2 Vaccine stability evaluation: The vaccine composition prepared in Example 1 was aliquoted into 1 mL vials and stored at 4°C in the dark. Samples were taken at 0, 3, 6, 9, and 12 months to test various physicochemical properties and biological activities, including pH value, appearance, sterility, canine distemper virus antigen stability, canine parvovirus recombinant VP2 protein antigen stability, canine adenovirus type 2 antigen stability, and in vitro immunogenicity in mice. Antigen stability was determined by ELISA to measure antigen retention. Simultaneously, the presence of visible aggregation or precipitation was observed during vaccine storage.

[0065] Stability results are as follows Figure 1 As shown, during the 12-month storage period at 4°C, the pH value of the vaccine remained between 7.0 and 7.4, and the appearance remained a uniform milky white suspension without obvious stratification, aggregation, or precipitation; the sterility test was qualified; the ELISA antigen retention rates of the three antigens remained above 85%, with the canine distemper virus antigen retention rate at 90.2%, the canine parvovirus recombinant VP2 protein antigen retention rate at 92.5%, and the canine adenovirus type 2 antigen retention rate at 88.6%; the mouse model immunization results showed that the vaccine stored for 12 months could still induce antibody levels that were not statistically different from the vaccine stored for 0 months. This modular compound adjuvant system vaccine has good physicochemical stability and biological activity stability.

[0066] Experimental Example 3 The impact of stepwise mixing process on vaccine performance: The vaccine of Example 1 was compared with the vaccine of Comparative Example 4 to investigate the particle size distribution, antigen adsorption rate and immunogenicity immediately after preparation and after storage at 4°C for 6 months.

[0067] The results showed that immediately after preparation, the polydispersity index (PDI) of the vaccine in Example 1 was 0.18 ± 0.03, while that of the vaccine in Comparative Example 4 was 0.36 ± 0.05. The stepwise mixing process in Example 1 resulted in a more uniform particle size distribution of the functional units in the vaccine. After storage at 4°C for 6 months, the PDI of the vaccine in Example 1 remained relatively stable at 0.22 ± 0.04; however, the PDI of the vaccine in Comparative Example 4 increased to 0.58 ± 0.08, and some microsphere aggregation was observed. Antigen adsorption rate assays showed that the adsorption rate of the aluminum adjuvant for VP2 protein in the vaccine in Example 1 remained at 92.3% after 6 months of storage, while the adsorption rate of the vaccine in Comparative Example 4 decreased to 78.6%. Mouse immunization experiments showed that after 6 months of storage, the antibody levels induced by the vaccine in Example 1 were not significantly different from those induced freshly, while the antibody levels of the vaccine in Comparative Example 4 decreased by approximately 34% compared to freshly prepared vaccines. This demonstrates that the stepwise independent preparation followed by gentle mixing plays a crucial role in protecting the integrity of each functional unit and ensuring the long-term stability of the vaccine.

[0068] Therefore, this invention employs the aforementioned canine multivalent infectious disease vaccine composition, its preparation method, and its application. Through modular design, four adjuvant components are organically integrated into three functional units: an aluminum adjuvant complex is responsible for the antigen reservoir effect and humoral immune activation; chitosan nanoparticles encapsulating CpG oligonucleotides are responsible for TLR9 pathway activation and Th1-type cellular immune induction; and PLGA microspheres are responsible for sustained-release delivery and enhanced immune memory. These three units work synergistically to induce high-titer neutralizing antibodies and stimulate a strong cellular immune response, achieving comprehensive protection against canine distemper, canine parvovirus, and canine adenovirus type 2. The canine parvovirus VP2 protein is divided into two parts: one part is adsorbed onto the aluminum adjuvant and rapidly released in the early stages of immunization, activating the immune response and generating early protection; the other part is encapsulated in PLGA microspheres and slowly released in the later stages of immunization, continuously stimulating the immune system and prolonging the period of immune protection. This allows the same vaccine to both rapidly generate protective antibodies and maintain long-term immune memory.

[0069] The preparation method employs a three-unit independent preparation and step-by-step mixing strategy. Chitosan-CpG nanoparticles and PLGA sustained-release microspheres were prepared using iontophoresis and double emulsion-solvent evaporation methods, respectively. After preparation, they were mixed with the aluminum adjuvant antigen complex by gentle stirring. The entire mixing process avoided the use of ultrasound and high-speed shearing, and the stirring speed was controlled below 200 rpm, effectively preventing damage to the nanoparticle and microsphere structures and ensuring the stability of the physicochemical properties and biological activities of each functional unit. The combination of canine distemper virus and canine adenovirus type 2 antigens with canine parvovirus recombinant VP2 protein antigen not only inherits the strong immunogenicity of viral antigens but also reduces biosafety risks by replacing live viruses with subunit antigens, thus balancing immunogenicity and safety. The optimal content range of each component was determined through optimization, enabling a strong immune response to be generated at a low dose of 80-150 μg / dose of recombinant canine parvovirus VP2 protein. Furthermore, the 35%-45% antigen encapsulation in PLGA microspheres achieved sustained release and enhanced efficacy, effectively reducing the total antigen requirement and demonstrating economic efficiency and sustainability.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A canine multivalent infectious disease vaccine composition, characterized in that, Including canine distemper virus antigen, canine parvovirus antigen, canine adenovirus type 2 antigen, as well as modular compound adjuvant systems and pharmaceutically acceptable carriers; The modular composite adjuvant system consists of the following three units: (a) Unit 1: Aluminum adjuvant complex adsorbed with canine parvovirus antigen; (b) Second unit: Chitosan nanoparticles loaded with immunostimulatory oligonucleotides; (c) Unit 3: Polylactic acid-glycolic acid copolymer sustained-release microspheres containing partial canine parvovirus antigen; In this embodiment, a portion of the canine parvovirus antigen is encapsulated in the polylactic acid-glycolic acid copolymer sustained-release microspheres, while the remaining portion is adsorbed onto the aluminum adjuvant complex.

2. The canine multivalent infectious disease vaccine composition according to claim 1, characterized in that, Both canine distemper virus antigen and canine adenovirus type 2 antigen are viral antigens obtained through cell culture, proliferation, and purification. The content of canine distemper virus antigen in each 1 mL of vaccine is 10. 5.5 ~10 6.5 TCID 50 The content of canine adenovirus type 2 antigen is 10. 5.5 ~10 6.5 TCID 50 .

3. The canine multivalent infectious disease vaccine composition according to claim 1, characterized in that, The canine parvovirus antigen is a recombinant VP2 protein, which is obtained by affinity chromatography purification after soluble expression in Escherichia coli.

4. The canine multivalent infectious disease vaccine composition according to claim 1, characterized in that, The total content of canine parvovirus antigen in each 1 mL of vaccine is 80-150 μg, of which 35%-45% is encapsulated in the polylactic acid-glycolic acid copolymer sustained-release microspheres and the remaining part is adsorbed on the aluminum adjuvant complex. The content of aluminum adjuvant is 1.0–1.5 mg; The content of chitosan nanoparticles is 0.5-1.0 mg, and the content of the immunostimulatory oligonucleotides contained in the chitosan nanoparticles is 20-30 μg; The content of polylactic acid-glycolic acid copolymer sustained-release microspheres is 2-4 mg.

5. The canine multivalent infectious disease vaccine composition according to claim 1, characterized in that, Chitosan nanoparticles are prepared by iontophoresis of chitosan with a degree of deacetylation of 85%–95% and a molecular weight of 50–100 kDa. The immunostimulatory oligonucleotides are CpG oligonucleotides modified with phosphate thioester.

6. The canine multivalent infectious disease vaccine composition according to claim 1, characterized in that, The particle size of the polylactic acid-glycolic acid copolymer sustained-release microspheres is 2-8 μm, the molar ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer is 75:25, and the encapsulation rate of the polylactic acid-glycolic acid copolymer sustained-release microspheres for the canine parvovirus antigen is 70%-85%.

7. The canine multivalent infectious disease vaccine composition according to claim 1, characterized in that, It also contains a complex stabilizer, which consists of trehalose, mannitol, gelatin and Tween-80.

8. A method for preparing a canine multivalent infectious disease vaccine composition as described in any one of claims 1 to 7, characterized in that, Includes the following steps: (1) Prepare canine distemper virus antigen, canine parvovirus recombinant VP2 protein antigen and canine adenovirus type 2 antigen respectively for later use; (2) Chitosan nanoparticles loaded with immunostimulatory oligonucleotides were prepared by ionogel method; (3) Polylactic acid-glycolic acid copolymer sustained-release microspheres loaded with part of canine parvovirus antigen were prepared by water-in-oil-in-water double emulsion-solvent evaporation method; (4) The remaining canine parvovirus antigen and aluminum adjuvant are adsorbed under stirring conditions to form the first unit; (5) Under stirring conditions at 4°C, the canine distemper virus antigen, the canine adenovirus type 2 antigen and the chitosan nanoparticles prepared in step (2) are added sequentially to the product of step (4), and the mixture is stirred after each addition of a component. (6) Finally, add the polylactic acid-glycolic acid copolymer sustained-release microspheres prepared in step (3), mix well, add composite stabilizer, adjust the volume, sterilize and dispense to make liquid vaccine.

9. The method for preparing the canine multivalent infectious disease vaccine composition according to claim 8, characterized in that, The stirring speed in steps (5) and (6) shall not exceed 200 rpm; the composite stabilizer is composed of trehalose 25-40 g / L, mannitol 15-25 g / L, gelatin 8-12 g / L and Tween-80 0.2-0.4 mL / L.

10. The use of the canine multi-infectious disease vaccine composition according to any one of claims 1 to 7 in the preparation of a medicament for the prevention of canine distemper, canine parvovirus infection and canine adenovirus type 2 infection.

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