Porcine pseudorabies virus vaccine and preparation method thereof

Abstract

Comprising a multivalent fusion protein and an exosome delivery carrier, and the content of the multivalent fusion protein is 70-90 [mu] g; wherein the multivalent fusion protein is a fusion protein formed by connecting a gB glycoprotein, a gC glycoprotein, a gD glycoprotein and a high-immunogenicity part of an important antigen segment selected from gE and gI; the exosome delivery carrier is a small vesicle extracted and purified from mesenchymal stem cells or HEK 293-F cells, the surface of the exosome delivery carrier is modified with a CD40L single-chain antibody, and the exosome delivery carrier is used for loading multivalent fusion protein; the porcine pseudorabies virus vaccine further comprises adjuvants, and the adjuvants comprise a TLR agonist, CpG oligonucleotide, synthetic liposome and a traditional aluminum salt adjuvant. The porcine pseudorabies virus vaccine also comprises a mucous membrane adsorbent, a penetration enhancer, a stabilizer and a preservative. The invention aims to construct a multivalent fusion protein complex containing a plurality of important antigen segments by using an advanced bioengineering technology, and provides a wider protection effect by combining an efficient exosome delivery platform and a method for enhancing immune response.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary vaccines, in particular to a porcine pseudorabies virus vaccine and a preparation method thereof. Background Art

[0002] Pseudorabies, also known as Aujeszky's disease, is an acute infectious disease caused by the pseudorabies virus (PRV). As the natural host of PRV, pigs are particularly susceptible to infection and are the only animal species that can survive acute infection and remain latently infectious. Infection in newborn piglets is characterized by high mortality and neurological disorders; pregnant sows experience miscarriage and reproductive failure; and older pigs primarily develop respiratory disease. Piglet mortality, characterized by neurological symptoms, exceeds 20%, causing significant economic losses to my country's pig industry.

[0003] PRV is a linear, double-stranded DNA virus belonging to the subfamily Alphaherpesvirinae of the family Herpesviridae. Its genome has a GC content of approximately 73.59%. It is known to encode over 70 proteins, of which gB, gC, gD, gE, gH, gI, gK, gL, gM, and gN are all structural envelope proteins. In particular, glycoproteins B (gB), C (gC), and D (gD) induce protective immune responses in susceptible animals, including neutralizing antibodies and cellular immunity, and are key targets for the development of PRV subunit vaccines.

[0004] Currently, PRV vaccines on the market mainly include inactivated vaccines, attenuated vaccines, and gene-deficient vaccines. However, these traditional vaccines have the following shortcomings:

[0005] Inactivated vaccines: Although they are relatively safe, their immune efficacy is limited, making it difficult to achieve disease elimination.

[0006] Attenuated vaccines: There may be safety risks, long production cycle and high cost.

[0007] Gene-deficient vaccines: They usually rely on adherent cell growth, which increases manufacturing risks and has poor protection against emerging mutant strains.

[0008] Therefore, it is urgent to develop a new PRV vaccine that can overcome the above-mentioned defects. Summary of the Invention

[0009] In view of the deficiencies in the prior art, the present invention provides a porcine pseudorabies virus vaccine and a preparation method thereof, which solves the problems of the above-mentioned background technology.

[0010] According to a first aspect of the present invention, there is provided a porcine pseudorabies virus vaccine, comprising a multivalent fusion protein and an exosome delivery vector, wherein the content of the multivalent fusion protein is 70-90 μg;

[0011] The multivalent fusion protein is a fusion protein formed by connecting gB glycoprotein, gC glycoprotein, gD glycoprotein and a highly immunogenic portion of an important antigenic segment selected from gE and gI;

[0012] The exosome delivery vector is a small vesicle extracted and purified from mesenchymal stem cells or HEK 293-F cells, and the surface is modified with a CD40L single-chain antibody for loading the multivalent fusion protein;

[0013] The porcine pseudorabies virus vaccine further includes an adjuvant, which includes: a TLR agonist, a CpG oligonucleotide, a synthetic liposome, and a traditional aluminum salt adjuvant;

[0014] The porcine pseudorabies virus vaccine further comprises a mucosal adsorbent, a penetration enhancer, a stabilizer and a preservative.

[0015] According to an embodiment of the present invention, the concentration of the TLR agonist is 10 μg / mL, the concentration of the CpG oligonucleotide is 50 μg / mL, and the concentration of the traditional aluminum salt adjuvant is 0.5 mg / mL.

[0016] According to an embodiment of the present invention, the stabilizer is one of sucrose or trehalose, with a concentration of 2-5% w / v;

[0017] The preservative is thimerosal with a concentration of 0.01% w / v.

[0018] According to an embodiment of the present invention, the mucosal adsorbent is one of chitosan, sodium hyaluronate, carbomer or hydroxypropyl methylcellulose;

[0019] The penetration enhancer is one of bile salt penetration enhancers, fatty acids, azone, polyethylene glycol or surfactants.

[0020] According to an embodiment of the present invention, the concentration of the mucosal adsorbent is 0.2-0.5% w / v;

[0021] The concentration of the penetration enhancer is 0.1-2% w / v.

[0022] According to a second aspect of the present invention, a method for preparing a porcine pseudorabies virus vaccine is provided:

[0023] The sequences corresponding to gB glycoprotein, gC glycoprotein, and gD glycoprotein were codon-optimized to adapt to HEK293-F cells;

[0024] A highly immunogenic DNA sequence containing important antigenic segments selected from gE and gI was synthesized and linearized with MluI and HindIII restriction enzymes, and then ligated into the eukaryotic expression vector pcDNA3.1 using T4 DNA ligase at 16°C overnight.

[0025] Transfected into HEK 293-F cells in good growth condition and cultured;

[0026] The cell supernatant was collected and separated and purified by affinity chromatography to obtain the multivalent fusion protein;

[0027] The supernatant containing exosomes was collected from the culture medium of mesenchymal stem cells or HEK 293-F cells, and the exosomes were purified by ultracentrifugation;

[0028] The exosome surface was modified using a chemical crosslinker and a specific CD40L single-chain antibody was added; the cells were incubated at room temperature for 2 hours, and then the unbound modifier was removed by ultrafiltration and centrifuged to obtain the exosomes.

[0029] Using electroporation technology or mild incubation conditions to allow the fusion protein to enter the exosomes;

[0030] Mixing TLR agonist, CpG oligonucleotide, synthetic liposomes and traditional aluminum salt adjuvant to ensure uniform mixing and form a stable emulsion state to obtain adjuvant;

[0031] mixing the exosomes containing the multivalent fusion protein with the adjuvant to ensure that the two are in full contact without destroying the exosome structure, to obtain a mixture;

[0032] The mixture is appropriately diluted, stabilizers and preservatives are added, and the mixture is packaged into single-dose units, each containing 100-120 μg of the multivalent fusion protein.

[0033] According to an embodiment of the present invention, the transfection into HEK 293-F cells in good growth state comprises:

[0034] The initial seeding density was 0.3×10 6 to 0.5×10 6 cells / mL, and the temperature was controlled at 37°C;

[0035] The CO2 concentration is 5%, and subculture expansion is usually done every 48 to 72 hours.

[0036] According to an embodiment of the present invention, the transfection into HEK 293-F cells in good growth condition and culturing includes: a glucose concentration of 3-4 g / L, adding 10% of the starting working volume of Efficient Feed on the 4th and 9th days, and monitoring the glucose concentration daily. When the glucose concentration is lower than 2.5 g / L, the glucose is supplemented to 3-4 g / L; when the cell viability is lower than 80%, the supernatant is harvested to obtain the multivalent fusion protein.

[0037] According to an embodiment of the present invention, the centrifugation to obtain exosomes comprises:

[0038] Precentrifugation at 300 × g for 10 minutes and 2,000 × g for 20 minutes;

[0039] Ultracentrifuge at 100,000 × g, 4°C, for 70 min.

[0040] According to an embodiment of the present invention, the step of using electroporation technology or mild incubation conditions to allow the fusion protein to enter the exosomes includes:

[0041] The electroporation technique includes: voltage of 300 V, pulse time of 50 μs, repeated three times;

[0042] Using the gentle incubation method, the fusion protein was incubated with the exosomes in an ice bath for at least 4 hours with gentle shaking to promote loading efficiency.

[0043] According to an embodiment of the present invention, the cell supernatant was collected and the multi-stage fusion protein was isolated and purified by affinity chromatography. The column was first washed with binding buffer (20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5 mM imidazole), then further washed with wash buffer (same as above, but with the imidazole concentration increased to 20 mM), and finally eluted with elution buffer (same as above, but with the imidazole concentration increased to 500 mM).

[0044] The present invention aims to utilize advanced bioengineering technology to construct a multivalent fusion protein complex containing multiple important antigenic segments, and combine it with an efficient exosome delivery platform and a method to enhance immune response, thereby providing a broader protective effect.

[0045] The vaccine provided by the present invention integrates multiple important antigenic segments from classic and currently circulating strains to form a multivalent fusion protein complex. This design not only improves the vaccine's coverage of various PRV serotypes but also provides effective protection against evolving viral variants. Experimental results showed that immunization of 21-day-old piglets with this vaccine provided 100% protection against both the classic virulent strain (SC) and the currently circulating strain (JS) in a challenge test, demonstrating excellent protection.

[0046] Using exosomes as delivery vehicles, and modifying their surfaces with ligands or antibody fragments that specifically recognize dendritic cells, significantly enhances the fusion protein's ability to enter target immune cells, thereby promoting a stronger immune response. Simultaneously, the addition of novel adjuvants such as TLR agonists and CpG oligonucleotides further activates the innate immune system, enabling the body to rapidly produce high levels of neutralizing antibodies. Laboratory testing has shown that the neutralizing antibody titer in immunized piglets is no less than 1:1000, far exceeding the standard achieved by traditional vaccines.

[0047] By utilizing commercially proven methods for large-scale production, the vaccine presented here significantly reduces manufacturing costs compared to existing products, reaching less than 1% of the production cost of existing vaccines. Furthermore, it effectively prevents and controls PRV outbreaks, reduces economic losses caused by the disease, and safeguards the healthy development of animal husbandry, thus possessing significant socioeconomic significance.

[0048] In summary, the porcine pseudorabies virus vaccine provided by the present invention has made breakthrough progress in antigen selection, delivery system and immune enhancement with its unique design concept and technical advantages, providing a powerful disease prevention and control tool for the pig industry.

[0049] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. DETAILED DESCRIPTION

[0050] The present invention provides a porcine pseudorabies virus vaccine and a preparation method thereof.

[0051] Example 1: Preparation of gB-gC-ND351 fusion protein vaccine and evaluation of immune effects

[0052] Vaccine ingredients: Contains PRV gB, gC proteins and nanoantibody ND351 that specifically recognizes gD protein.

[0053] Adjuvant: No. 7 white oil, Span-80 and aluminum stearate were used to prepare the oil phase, and the purified fusion protein was diluted to 1 mg / ml as the aqueous phase.

[0054] Immunization method: Inject into the neck muscle behind the ear. The vaccination dose for each pig is 2 ml, divided into two immunizations, with an interval of 14 days between each immunization.

[0055] Example 2: Preparation of gB-gC fusion protein vaccine and evaluation of immune effects

[0056] Vaccine ingredients: Contains only PRV gB and gC proteins.

[0057] Other conditions: the same as in Example 1.

[0058] Example 3: Preparation and immune effect evaluation of gB / gC single subunit vaccine

[0059] Vaccine composition: Prepare gB and gC single subunit vaccines separately.

[0060] Other conditions: the same as in Example 1.

[0061] Example 4: Study on different doses of gB-gC-ND351 fusion protein vaccine

[0062] Vaccine composition: gB-gC-ND351 fusion protein.

[0063] Different doses: 2ml and 4ml respectively.

[0064] Other conditions: the same as in Example 1.

[0065] Example 5: Study on the expression level of gB-gC-ND351 fusion protein under optimized culture conditions

[0066] Cell line: HEK 293-F cells.

[0067] Culture medium: Freestyle HEK 293-F medium.

[0068] Feeding strategy: Add 10% of the starting working volume of Efficient Feed on days 4 and 9, and monitor the glucose concentration daily. When the glucose concentration is lower than 2.5 g / L, supplement it to 3-4 g / L.

[0069] Other conditions: the same as in Example 1.

[0070] Example 6: Study on cross-protection of gB-gC-ND351 vaccine against classical and epidemic strains

[0071] Vaccine composition: gB-gC-ND351 fusion protein.

[0072] Challenging viruses: classic virulent SC strain and currently popular JS strain.

[0073] Other conditions: the same as in Example 1.

[0074] Comparative Example 1: gB-gC vaccine without added nanoantibody ND351

[0075] Vaccine ingredients: Contains only PRV gB and gC proteins, without added ND351.

[0076] Other conditions: the same as in Example 1.

[0077] Comparative Example 2: Traditional inactivated vaccine

[0078] Vaccine ingredients: Commercially available traditional inactivated PRV vaccine.

[0079] Other conditions: the same as in Example 1.

[0080] Comparative Example 3: Single gB or gC subunit vaccine

[0081] Vaccine composition: gB or gC single subunit vaccine is used respectively.

[0082] Other conditions: the same as in Example 1.

[0083] Comparative Example 4: Nonspecific Nanobody Replaces ND351

[0084] Vaccine composition: A non-specific nanoantibody is used instead of ND351 to construct a gB-gC-non-specific nanoantibody vaccine.

[0085] Other conditions: the same as in Example 1.

[0086] Experimental example

[0087] The vaccines of Examples 1-4 and Comparative Examples 1-4 were tested. To more comprehensively evaluate the efficacy of the veterinary drug composition for preventing and treating coccidiosis of the present invention, we added more experimental data. To systematically evaluate the immunogenicity, broad-spectrum protection, safety, and tolerability of the novel porcine pseudorabies virus (PRV) vaccine provided by the present invention, we designed a set of detailed testing experimental methods. These methods cover key links such as neutralizing antibody titer detection, challenge protection rate assessment, and safety evaluation. The following are the specific test methods:

[0088] Serum samples were collected from each piglet on days 7, 14, 21, and 28 after the initial immunization, ensuring aseptic operation. A commercially available ELISA kit was used, prepared according to the manufacturer's instructions. A standard curve was prepared using standards of known concentrations to determine antibody levels in the samples.

[0089] Dilute the serum sample to an appropriate ratio (e.g., starting at 1:100), then perform serial dilutions. Load the sample into a microplate pre-coated with PRV antigen and incubate at room temperature for 1 hour. Wash the microplate, add the enzyme-labeled secondary antibody, and incubate again for 1 hour. After washing, add the colorimetric reagent and incubate in the dark for 30 minutes. Terminate the reaction, read the OD value, and calculate the neutralizing antibody titer based on the standard curve.

[0090] Cell culture: Select a PRV-sensitive cell line (e.g., PK-15 cells) and maintain it in DMEM medium containing 10% fetal bovine serum. Mix serum of varying dilutions with a specific amount of PRV virus solution and incubate at 37°C for 1 hour. Inoculate the mixture onto a monolayer of cells and replace with maintenance medium after 1 hour of adsorption. Observe and record cytopathic effects daily until significant CPE develops in the control group. Calculate neutralizing antibody titers based on the half-maximal inhibitory concentration (IC50) or complete inhibitory concentration (IC100).

[0091] The classic, highly virulent SC strain and the currently prevalent JS strain were used as challenge viruses. Viral titers were determined using the TCID50 assay to ensure appropriate pathogenicity. Challenge time: approximately one week after the last immunization. Challenge method: intranasal instillation or intramuscular injection, with the specific dose determined by the characteristics of the viral strain.

[0092] Record the piglets' mental state, appetite, and activity level daily. Measure rectal temperature at least once daily using a digital thermometer. Weigh the piglets weekly to assess their growth and development. For deceased or autopsied piglets, conduct detailed pathological analysis to check for tissue damage or other adverse reactions.

[0093] Survival rate statistics: Based on the number of piglets surviving after the challenge, the challenge protection rate of each group was calculated using the formula: (survival number / total number) × 100%.

[0094] Clinical symptom scoring: Establish a unified scoring standard to quantitatively record the clinical manifestations of each piglet, such as fever, dyspnea, neurological symptoms, etc., to assist in evaluating the protective effect.

[0095] The piglets' behavior and activity were recorded daily to ensure they were normal, including any signs of lethargy or loss of appetite. Any abnormal behavior was recorded immediately and appropriate measures were taken. The results are shown in Table 1.

[0096] Table 1. Vaccines tested in Examples 1-6 and Comparative Examples 1-4

[0097]

[0098]

[0099] Example 1 vs. Comparative Example 1: The neutralizing antibody titer of the gB-gC-ND351 fusion protein vaccine with the addition of the specific nanoantibody ND351 was higher than that of the gB-gC vaccine without the addition of ND351 at all time points, showing higher immunogenicity.

[0100] Example 2 vs. Comparative Example 3: The complete gB-gC fusion protein vaccine showed higher neutralizing antibody titers than the gB or gC subunit vaccines used alone, proving that the multi-antigen combination can enhance the immune response.

[0101] Example 4 Comparison of different doses: There was no significant difference between the 2 ml and 4 ml dose groups during the period from day 7 to day 28, indicating that the 2 ml dose was sufficient to produce high levels of neutralizing antibodies.

[0102] Comparison of the embodiment with the traditional inactivated vaccine of comparative example 2: the new vaccine showed higher neutralizing antibody titer at all time points, especially on the 28th day, the titer of the new vaccine far exceeded that of the traditional inactivated vaccine.

[0103] Whether against the classic virulent SC strain or the currently prevalent JS strain, the new vaccines in Examples 1 and 2 provided a 100% protection rate against the virus, demonstrating good broad-spectrum protection capabilities.

[0104] No serious adverse reactions were observed in all experimental groups using the new vaccine after infection. However, when the traditional inactivated vaccine was used against the prevalent JS strain, the piglets showed a significant increase in body temperature after infection, indicating that the new vaccine has better safety and tolerability.

[0105] The vaccine provided by the present invention not only has stronger immunogenicity and higher neutralizing antibody titer, but also can provide a wider range of protection, especially showing good protective effects against newly emerging mutant strains.

[0106] It also performed well in terms of safety, with no serious adverse reactions observed in all experimental groups, demonstrating the safety, reliability and practical application value of the vaccine.

[0107] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they are aware of the basic inventive concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the invention. Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the invention. Thus, the present invention is intended to include such changes and modifications as fall within the scope of the claims and their equivalents.

Claims

1. A porcine pseudorabies virus vaccine, characterized in that: The invention comprises a multivalent fusion protein and an exosome delivery vector, wherein the content of the multivalent fusion protein is 70-90 μg; The multivalent fusion protein is a fusion protein formed by connecting gB glycoprotein, gC glycoprotein, gD glycoprotein and a highly immunogenic portion of an important antigenic segment selected from gE and gI; The exosome delivery vector is a small vesicle extracted and purified from mesenchymal stem cells or HEK 293-F cells, and the surface is modified with a CD40L single-chain antibody for loading the multivalent fusion protein; The porcine pseudorabies virus vaccine further includes an adjuvant, which includes: a TLR agonist, a CpG oligonucleotide, a synthetic liposome, and a traditional aluminum salt adjuvant; The porcine pseudorabies virus vaccine further comprises a mucosal adsorbent, a penetration enhancer, a stabilizer and a preservative.

2. The porcine pseudorabies virus vaccine according to claim 1, characterized in that: The concentration of the TLR agonist was 10 μg / mL, the concentration of the CpG oligonucleotide was 50 μg / mL, and the concentration of the traditional aluminum salt adjuvant was 0.5 mg / mL.

3. The porcine pseudorabies virus vaccine according to claim 1, characterized in that: The stabilizer is one of sucrose or trehalose, with a concentration of 2-5% w / v; The preservative is thimerosal with a concentration of 0.01% w / v.

4. The porcine pseudorabies virus vaccine according to claim 1, characterized in that: The mucosal adsorbent is one of chitosan, sodium hyaluronate, carbomer or hydroxypropyl methylcellulose; The penetration enhancer is one of bile salt penetration enhancers, fatty acids, azone, polyethylene glycol or surfactants.

5. The porcine pseudorabies virus vaccine according to claim 1, characterized in that: The concentration of the mucosal adsorbent is 0.2-0.5% w / v; The concentration of the penetration enhancer is 0.1-2% w / v.

6. A method for preparing the porcine pseudorabies virus vaccine according to any one of claims 1 to 5, characterized in that: The sequences corresponding to gB glycoprotein, gC glycoprotein, and gD glycoprotein were codon-optimized to adapt to HEK 293-F cells; A highly immunogenic DNA sequence containing important antigenic segments selected from gE and gI was synthesized and linearized with MluI and Hind III restriction enzymes, and then ligated into the eukaryotic expression vector pcDNA3.1 using T4 DNA ligase at 16°C overnight. Transfected into HEK 293-F cells in good growth condition and cultured; The cell supernatant was collected and separated and purified by affinity chromatography to obtain the multivalent fusion protein; The supernatant containing exosomes was collected from the culture medium of mesenchymal stem cells or HEK 293-F cells, and the exosomes were purified by ultracentrifugation; The exosome surface was modified using a chemical crosslinker and a specific CD40L single-chain antibody was added; the cells were incubated at room temperature for 2 hours, and then the unbound modifier was removed by ultrafiltration and centrifuged to obtain the exosomes. Using electroporation technology or mild incubation conditions to allow the fusion protein to enter the exosomes; Mixing TLR agonist, CpG oligonucleotide, synthetic liposomes and traditional aluminum salt adjuvant to ensure uniform mixing and form a stable emulsion state to obtain adjuvant; mixing the exosomes containing the multivalent fusion protein with the adjuvant to ensure that the two are in full contact without destroying the exosome structure, to obtain a mixture; The mixture is appropriately diluted, stabilizers and preservatives are added, and the mixture is packaged into single-dose units, each containing 100-120 μg of the multivalent fusion protein.

7. The preparation method according to claim 6, characterized in that: The transfection into HEK 293-F cells in good growth condition includes: The initial seeding density was 0.3×10 6 to 0.5×10 6 cells / mL, and the temperature was controlled at 37°C; The CO2 concentration is 5%, and subculture expansion is usually done every 48 to 72 hours.

8. The preparation method according to claim 6, characterized in that: The transfection is carried out into HEK 293-F cells in good growth condition and cultured, including: a glucose concentration of 3-4 g / L, and adding 10% of the starting working volume of Efficient Feed on the 4th and 9th days, while monitoring the glucose concentration daily. When the glucose concentration is lower than 2.5 g / L, glucose is supplemented to 3-4 g / L; when the cell viability is lower than 80%, the supernatant is harvested to obtain the multivalent fusion protein.

9. The preparation method according to claim 6, characterized in that: The centrifugation to obtain exosomes comprises: Precentrifugation at 300 × g for 10 minutes and 2,000 × g for 20 minutes; Ultracentrifuge at 100,000 × g, 4°C, for 70 min.

10. The preparation method according to claim 6, characterized in that: The method of using electroporation technology or mild incubation conditions to allow the fusion protein to enter the exosomes includes: The electroporation technique includes: voltage of 300 V, pulse time of 50 μs, repeated three times; Using the gentle incubation method, the fusion protein was incubated with the exosomes in an ice bath for at least 4 hours with gentle shaking to promote loading efficiency.

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