Genetic engineering subunit vaccine for porcine reproductive and respiratory syndrome virus as well as preparation method and application of genetic engineering subunit vaccine

Abstract

The invention relates to the technical field of veterinary biological products, and discloses a porcine reproductive and respiratory syndrome virus (PRRSV) genetic engineering subunit vaccine and a preparation method and application thereof, the vaccine comprises a PRRSV ZJ266 GP5-M protein complex and an adjuvant, and the adjuvant is a mannose modified chitosan solution. The PRRSV ZJ266 GP5-M protein compound is formed by GP5 protein and M protein of a PRRSV ZJ266 strain expressed and purified by a Bac-to-Bac baculovirus expression system, the nucleotide sequence of the PRRSV ZJ266 GP5-M protein compound is shown as SEQ ID NO: 1, and the amino acid sequence of the PRRSV ZJ266 GP5-M protein compound is shown as SEQ ID NO: 2. According to the invention, a high-level neutralizing antibody can be induced to generate, 100% protection is provided for attack of PRRSV NADC30-like virulent strains, the virus load can be significantly reduced, and detoxification can be prevented. The vaccine provided by the invention is safe and effective, the preparation process is stable, and a new high-quality product choice is provided for preventing the PRRSV.

Description

Technical Field

[0001] This invention relates to the field of veterinary biological products technology, and in particular to a porcine reproductive and respiratory syndrome virus (PRRSV) genetically engineered subunit vaccine, its preparation method, and its application. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS), commonly known as blue ear disease, is a highly contagious and contagious disease. Outbreaks are characterized by abortions (10%-50%), sow mortality (5%-10%), mortality rates exceeding 50% in market pigs, pre-weaning mortality in piglets, and respiratory illness. In recent years, the disease has shown a significant high incidence in China, and its genes are prone to mutation, posing a huge challenge to PRRS control and causing significant losses to the pig industry. It has become one of the most important infectious diseases seriously threatening the development of China's pig industry. The World Organisation for Animal Health (OIE) lists it as a notifiable animal disease, and my country classifies it as a Class II animal disease. The widespread prevalence of PRRS has caused enormous economic losses to the global pig industry. Vaccine research has received attention from various countries, with inactivated vaccines and live attenuated vaccines being developed for production. Subunit vaccines, genetically engineered vaccines, and DNA vaccines are also under active research.

[0003] PRRSV attenuated vaccines, such as Boehringer Ingelvac PRRS MLV (VR-2332 strain) and Syva Pyrsvac-183 (All-183 strain) attenuated vaccines from Spain, as well as attenuated vaccines against highly pathogenic PRRSV (HP-PRRS) developed by the China Animal Disease Prevention and Control Center, the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, and the Special Animal Products Research Institute, respectively, have numerous safety concerns and side effects. For example, Bouwkamp et al. found that immunizing sows with attenuated PRRSV vaccines in pig farms without PRRS prevalence led to mating failure or anestrus. Mortensen et al. reported that in Denmark, after boars and PRRSV-infected pig farms were vaccinated with American-type attenuated vaccines, outbreaks of PRRS caused by American-type strains occurred in unvaccinated farms. The reversion of virulence to attenuated vaccines is another major concern. Nielsen et al. reported that immunizing piglets in pig farms without PRRSV contamination resulted in a significant increase in sow abortion and mortality rates.

[0004] Commercially available inactivated PRRSV vaccines include Boehringer IngelvacPRRS (P120 strain) from Boehringer Ingelheim (Germany) for sows and piglets; SuivacPRRS-IN (VD-E1 / E2 and VD-A1 strains) from Dyntec (Czech Republic) for simultaneous use in boars, sows, and piglets; Progressis from Merial (France); Suipravac-PRRS (5710 strain) from Hybro (Spain); and the CH-1a strain developed by the Harbin Veterinary Research Institute. Studies of commercially available inactivated vaccines have shown that immunization with these vaccines does not induce neutralizing antibodies, and their effect on reducing viremia or clinical symptoms in pigs is not significant. Furthermore, the major antigenic determinants are lost during the inactivation process, resulting in low antigenic content, often requiring multiple vaccinations and leading to higher costs.

[0005] Subunit vaccines contain only the specific antigenic proteins of the pathogen and do not contain viral genetic material, thus exhibiting extremely high safety and representing an important direction in vaccine development. However, the GP5 protein is highly glycosylated and exhibits significant variability among different strains. Selecting widely protective antigens from prevalent strains and utilizing suitable expression systems to achieve correct folding, glycosylation modification, and high-level expression of the GP5-M protein in eukaryotic systems remains a key technological bottleneck in developing highly effective PRRSV subunit vaccines. In particular, effective subunit vaccines targeting currently prevalent NADC30-like strains still require further development. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a porcine reproductive and respiratory syndrome virus (PRRSV) genetically engineered subunit vaccine, its preparation method and application, which can induce the production of high levels of neutralizing antibodies, provide 100% protection against PRRSV NADC30-like virulent strains, significantly reduce viral load and prevent viral shedding.

[0007] The present invention solves the above-mentioned technical problems through the following technical means:

[0008] In a first aspect, the present invention provides a porcine reproductive and respiratory syndrome virus (PRRSV) genetically engineered subunit vaccine, the vaccine comprising a PRRSV ZJ266 GP5-M protein complex and an adjuvant, wherein the adjuvant is a mannose-modified chitosan solution.

[0009] Preferably, the PRRSV ZJ266 GP5-M protein complex is formed from the GP5 protein and M protein of PRRSV ZJ266 strain expressed and purified by the Bac-to-Bac baculovirus expression system, and its nucleotide sequence is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO: 2.

[0010] Preferably, the GP5 protein has a bee signal peptide attached to its N-terminus and a sequence encoding a 6×His tag added to its C-terminus.

[0011] Preferably, the PRRSV ZJ266 GP5-M protein complex in the vaccine is 20-30 μg / mL.

[0012] Preferably, the method for preparing the adjuvant includes the following steps:

[0013] A1. Chitosan was dissolved in an acidic solution, the pH was adjusted to 5.5-5.8, Traut's reagent was added to carry out a thiolation reaction, and then purified to obtain a thiolated chitosan solution.

[0014] A2. The mannose-PEG-NHS activated ester was coupled with the thiolated chitosan solution under pH 5.5-8.0 conditions, and the mixture was purified after the reaction to obtain the adjuvant.

[0015] Preferably, in step A2, the mannose-PEG-NHS activated ester is first dissolved in dimethyl sulfoxide and then added to the thiolated chitosan solution.

[0016] This invention overcomes the technical obstacles of traditional chitosan adjuvants in achieving controlled release, targeting, and stability, as well as the technical problem of mannose-PEG-NHS activated esters being prone to rapid hydrolysis and failure, through a "thiolization-mannylation synergistic modification" strategy, and constructs a novel adjuvant system with three functions: self-crosslinking, active targeting, and steady-state delivery.

[0017] Firstly, thiolization modification achieved a breakthrough in self-crosslinking and controlled-release delivery. By reacting Traut's reagent with the primary amine groups on chitosan molecules, thiol groups were introduced, giving chitosan reversible oxidative crosslinking capabilities. These thiol groups can form disulfide bonds under oxidative conditions, constructing a flexible and tunable three-dimensional network structure, allowing antigens to be stably embedded or coupled within the network. Simultaneously, in the in vivo micro-reducing environment, the disulfide bonds gradually break, enabling the antigen to achieve "self-regulated release." This design overcomes the technical limitations of traditional chitosan, which can only physically adsorb antigens and cannot achieve controlled sustained release, thus significantly extending the antigen release time and the immunostimulation cycle in vivo.

[0018] Then, mannose modification enabled active targeting and signal activation; mannose ligands were successfully introduced through amidation coupling of mannose-PEG-NHS with thiolated chitosan. Mannose specifically recognizes mannose receptors (MRs) on the surface of dendritic cells and macrophages, achieving active targeted delivery via receptor-mediated endocytosis (RME), significantly improving the efficiency of antigen uptake. This binding process also triggers mannose receptor-related signaling pathways, promoting dendritic cell maturation and upregulating the expression of co-stimulatory molecules CD80 / CD86, thus activating cellular immunity. This mechanism overcomes the technical bottleneck of traditional chitosan's inability to achieve active targeting and immune cell activation.

[0019] Secondly, the introduction of PEG spacer arms solves the problems of particle aggregation and system stability; the flexible structure of PEG segments effectively weakens the electrostatic attraction between chitosan molecules, preventing excessive aggregation of nanoparticles, thereby obtaining uniform particle size and good dispersibility. This modification significantly improves the colloidal stability of the adjuvant in the aqueous system, overcoming the defects of turbidity, sedimentation, and uneven antigen distribution in traditional chitosan solutions, providing feasible conditions for industrial preparation and long-term storage.

[0020] Furthermore, by employing a weakly acidic buffer system (pH 5.5–6.0), controlling low-temperature (4–8°C) reaction conditions, and introducing low-polarity organic solvents, long-standing technical challenges such as rapid hydrolysis of mannose-PEG-NHS in aqueous systems, low coupling efficiency, and product instability were effectively overcome. This highly stable modification system ensures batch-to-batch consistency in subsequent vaccine formulations, and the sufficient quantity and uniform distribution of mannose ligands enhance the recognition efficiency of antigen-presenting cells, thereby indirectly strengthening the intensity and persistence of the immune response.

[0021] Finally, multiple modifications achieved a synergistic enhancement of the immune response; thiol crosslinking led to sustained release, mannose ligands provided active targeting, and the PEG spacer arm ensured system stability. These synergistic effects within the same carrier enabled the antigen to achieve high concentrations, prolonged duration, and targeted release at sites of immune cell accumulation. This combination produced a qualitative change: the immune response not only increased in intensity but also significantly prolonged in duration, the Th1 / Th2 response tended to balance, and higher levels of neutralizing antibodies and cellular immune memory effects were generated.

[0022] In summary, this invention not only achieves synergistic delivery through thiol-mannose dual modification in structural design, but also overcomes the key chemical barrier of mannose-PEG-NHS instability at the process implementation level, realizing efficient, reproducible, and stable sugar ligand modification, providing a new technical path for the large-scale preparation of chitosan-based targeted adjuvants.

[0023] Secondly, the present invention provides a method for preparing a porcine reproductive and respiratory syndrome virus (PRRSV) genetically engineered subunit vaccine, used to prepare the aforementioned porcine PRRSV genetically engineered subunit vaccine, the preparation method comprising the following steps:

[0024] S1. Construct a recombinant baculovirus containing genes encoding the GP5 and M proteins of PRRSV ZJ266 strain;

[0025] S2. Infect insect cells with the recombinant baculovirus to express proteins;

[0026] S3. Harvest and purify the expression product to obtain the GP5-M protein complex;

[0027] S4. The purified protein complex is mixed and emulsified with adjuvant to prepare a vaccine.

[0028] Preferably, the purification in step S3 is performed using metal chelate affinity chromatography and molecular sieve chromatography.

[0029] Preferably, the volume mixing ratio of the protein complex to the adjuvant in step S4 is 9:1.

[0030] Thirdly, the present invention provides the application of a porcine reproductive and respiratory syndrome virus (PRRSV) genetically engineered subunit vaccine in the prevention of PRRSV.

[0031] The beneficial effects of this invention are:

[0032] (1) The vaccine component of the present invention is only the purified GP5-M protein complex, which does not contain any viral genetic material. This fundamentally eliminates the risk of "virulence reversion" or gene recombination with wild virus in attenuated vaccines, and also avoids the potential infectivity of inactivated vaccines due to incomplete inactivation.

[0033] (2) This invention selects the GP5 and M proteins of the ZJ266 strain, which have high homology with the currently prevalent NADC30-like strain. The resulting GP5-M heterodimer is the key neutralizing epitope of the virus, ensuring the vaccine's specificity and broad-spectrum protective potential. Using the Bac-to-Bac baculovirus-insect cell expression system, guided by bee signal peptides, high-level secretory expression of the GP5-M protein complex in a eukaryotic environment was achieved, ensuring the correct folding and glycosylation modification of the protein, and preserving its native conformation and immunogenicity to the greatest extent.

[0034] (3) The adjuvant of the present invention introduces thiol and mannose bifunctional groups on the chitosan molecule, thereby achieving the comprehensive technical effects of improving antigen stability, enhancing targeted delivery efficiency and prolonging immune response. Compared with existing adjuvants such as single chitosan and / or mannose-PEG-NHS, the present invention not only achieves quantitative improvement in the intensity and duration of immune response, but also constructs a brand-new "synergistic targeted immune enhancement mechanism". Attached Figure Description

[0035] Figure 1 This is an SDS-PAGE image of the PRRSV GP5-M protein prepared in Example 1;

[0036] Figure 2 These are electrophoresis images of the GP5-M protein content from three batches of vaccines;

[0037] Figure 3 This is a graph showing the particle size analysis results of PRRSV001 batch vaccine;

[0038] Figure 4 This is a graph showing the particle size analysis results of PRRSV002 batch vaccine;

[0039] Figure 5 This is a graph showing the particle size analysis results of PRRSV003 batch vaccine;

[0040] Figure 6 This is a diagram showing the preparation of three batches of products;

[0041] Figure 7 This is a tissue section image of the subunit vaccination site;

[0042] Figure 8 This is a pathological section of pig lung tissue from the vaccine efficacy test of this invention;

[0043] Figure 9 This is a pathological section of inguinal lymph node tissue from pigs used in the immunogenicity test of the vaccine of this invention;

[0044] Figure 10 This describes the tissue lesions in immunized piglets after viral challenge. Detailed Implementation

[0045] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. 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.

[0046] Example 1: Expression and purification of recombinant GP5-M protein

[0047] 1. Construction of recombinant rod particles

[0048] Based on the gene sequences of GP5 (nucleotide sequence as shown in SEQ ID NO: 3) and M (nucleotide sequence as shown in SEQ ID NO: 4) of PRRSV ZJ266 strain, codon optimization was performed and the gene was synthesized. A sequence encoding a bee signal peptide (MKFLVNVALVFMVVYISYIYA) was introduced at the 5' end of the GP5 gene, and a sequence encoding a 6×His tag was introduced at the 3' end of the M gene. The optimized GP5-m gene fragment was cloned into the pFastBac I vector using BamHI and XhoI restriction sites, transformed into DH10Bac competent cells, and positive recombinant bamboo mid-GP5-m was obtained through blue-white screening and PCR identification.

[0049] 2. Generation and amplification of recombinant baculoviruses

[0050] Recombinant Bacmid-GP5-m was transfected into Sf21 insect cells using Cellfectin® II Reagent, and the P0 generation virus was harvested after 72–96 hours of culture. The P0 generation virus was then inoculated into Sf21 suspension cells at an MOI of 0.1 for further culture, yielding P1, P2, and P3 generation viruses sequentially. The P3 generation virus titer reached 1.0 × 10⁻⁶. 8 TCID 50 / mL.

[0051] 3. Protein Expression and Purification

[0052] High-density Sf21 suspension cells were infected with P3 generation virus at MOI=5 (2×10⁶ cells / year). 6 Cells / mL), cultured at 27°C for 72 hours. Cell supernatant was collected by centrifugation. The supernatant was filtered through a 0.22 μm filter and purified using a Ni Sepharose™ Excel affinity chromatography column. The target protein was specifically eluted with elution buffer containing 250 mM imidazole. The eluent was further purified using a Superdex 200 molecular sieve chromatography column to obtain high-purity GP5-M protein (purity >95%, see [link]). Figure 1 Protein concentration was determined using Nanodrop and aliquoted for storage at -80°C.

[0053] Example 2: Preparation of a genetically engineered subunit vaccine against porcine reproductive and respiratory syndrome virus

[0054] 1. Materials and Methods

[0055] 1.1 Experimental animals: 4-5 week old piglets with PRRSV neutralizing antibody titers not higher than 1:4, negative for PRRSV by PCR, purchased from nearby farmers.

[0056] 1.2 Antigen PRRSV GP5-M protein.

[0057] 1.3 Vaccine Preparation The vaccine of this invention is prepared according to the following formula: GP5-M: Prepare 90 mL of the solution, then add 10 mL of adjuvant to ensure a final GP5-M protein concentration of not less than 25 ug / 2 ml, and emulsify for approximately 20 minutes. After emulsification, drop the vaccine onto the water surface; it will diffuse in a white, cloud-like manner.

[0058] The preparation method of the adjuvant includes the following steps:

[0059] A1. Dissolve 0.5 g of chitosan in 50 mL of 1% (v / v) aqueous acetic acid solution, stir until dissolved, adjust pH to 5.6 (using 0.1 M NaOH), and slowly add 50 mg of Traut's reagent (pre-dissolved in PBS pH 7.4 buffer) on ice. React for 30–60 min. Remove small molecule reagents by gel filtration. Obtain a thiolized chitosan solution.

[0060] A2. Dissolve 1g of mannose-PEG-NHS in a trace amount of DMSO, slowly add it to the thiolized chitosan solution, react at room temperature for 2h, remove the free reagent (dialysis) to obtain the adjuvant.

[0061] In addition, the vaccine of Comparative Example 1 was prepared: the difference from the vaccine of the present invention is that the adjuvant is a chitosan solution formed by dissolving 0.5g of chitosan in 50mL of 1% (v / v) acetic acid aqueous solution;

[0062] Comparative Example 2 vaccine: The difference from the vaccine of the present invention is that the adjuvant is a solution formed by mixing 1g of mannose-PEG-NHS and 0.5g of chitosan.

[0063] 1.4 Finished Product Inspection

[0064] 1.4.1 Physical examination: Examine the color, appearance, stability, and viscosity of the vaccine.

[0065] 1.4.2 Quality inspection shall be carried out in accordance with the current Chinese Veterinary Pharmacopoeia.

[0066] 1.4.3 Sterility testing shall be conducted in accordance with the current Chinese Veterinary Pharmacopoeia.

[0067] 1.5 Safety Inspection

[0068] Use 5 healthy and susceptible piglets at 4 - 5 weeks of age. Observe for 2 - 3 days before inoculation. Each piglet is inoculated with 4 ml of the vaccine intramuscularly. Continuously observe for 21 days after inoculation. Measure the body temperature once a day at a fixed time and take the average value as the basal body temperature. Compared with before inoculation, there should be no obvious changes in spirit and appetite, and the body temperature increase should not exceed 1°C; if the body temperature exceeds the basal body temperature by 1°C but does not exceed 1.5°C and remains elevated for no more than 2 temperature readings, it is also judged as qualified. If any accidental death occurs among the animals for safety inspection, this inspection is regarded as having no result and can be retested once. A slight swelling is allowed at the inoculation site, but the diameter of the swelling does not exceed 1 cm and the duration does not exceed 7 days, which is judged as qualified.

[0069] 1.6 Potency Test

[0070] Use 30 healthy and susceptible piglets at 4 - 5 weeks of age, randomly divided into 6 groups with 5 piglets in each group. Groups 1 - 3 are the vaccine groups prepared by the present invention, and groups 5 - 6 are the vaccines of Comparative Example 1 - 2. Each piglet is injected with 2 ml of the vaccine intramuscularly in the neck. Immunize once with the same dose 14 days after immunization. Group 4 is the non - inoculated control group and is isolated and raised under the same conditions. 21 days after the second immunization, all pigs are bled, the serum is separated, and the neutral antibody level is detected; all test pigs are challenged with PRRSV NADC30 like (1.0×10 5.0 TCID 50 / ml), 3 ml is injected intramuscularly and 3 ml is instilled nasally for each pig, and the body temperature is measured and observed daily for 21 days.

[0071] 2 Results

[0072] 2.1 Antigen Concentration Detection Results

[0073] The protein content per dose of GP5 - M in three batches of vaccines was detected by SDS - PAGE, and the results are shown in Figure 2 , lanes 2, 3, and 4. The protein content per dose of GP5 - M in all three batches of vaccines is ≥25 μg / mL.

[0074] 2.2 Vaccine Particle Size Analysis The particle size results of three batches of vaccines are shown in Table 1, and the specific results are shown in Figure 3 , Figure 4 , Figure 5 .

[0075] Table 1 Particle Size Analysis Results of Three Batches of Vaccines

[0076]

[0077] 2.3 Finished Product Inspection Results

[0078] 2.3.1 Appearance Inspection Results

[0079] All three batches of vaccine products were milky white, uniform emulsions. When dropped into water, they diffused in a white, cloud-like manner. The viscosity was below 10 cp, meeting all requirements. Specific results are shown in Table 2, and the three batches of finished products are listed below. Figure 5 .

[0080] 2.3.2 Filling Quantity Inspection Results

[0081] All three batches of vaccines were packaged in 20ml portions, with the volume difference between each batch within 1ml. See Table 2 for specific results.

[0082] 2.3.3 Sterility test results

[0083] Three batches of subunit vaccines were seen Figure 6 All samples tested negative for bacteria. See Table 2 for specific results.

[0084] Table 2 Summary of Product Properties, Fill Weight, and Sterility Test Results for 3 Batches of Products

[0085]

[0086] 2.4 Safety Inspection Results

[0087] The specific results are shown in Table 3.

[0088] Table 3 Summary of Product Safety Inspection Results

[0089]

[0090] 2.5 Results of validity test

[0091] 2.5.1 Serological methods for piglets: At least 4 immunized pigs should have a neutralizing antibody titer ≥1:32, and the neutralizing antibody titer of non-immunized control pigs should all be <1:4. See Table 4 for specific results.

[0092] Table 4. Results of Serological Tests for Piglets

[0093]

[0094] 2.5.2 In the piglet immune challenge method, at least two control pigs showed disease, and at least four immunized pigs should be protected. The specific results are shown in Table 5.

[0095] Table 5 Summary of Product Efficacy Test Results

[0096]

[0097] 3. Conclusion

[0098] 1. The finished product inspection results show that the porcine reproductive and respiratory syndrome virus genetically engineered vaccine developed in the laboratory meets the standards.

[0099] 2. Safety test results showed that no adverse reactions occurred in piglets after injection of 4 mL of the porcine reproductive and respiratory syndrome virus (PRRSV) genetically engineered vaccine, and the three batches of vaccine produced in the laboratory were safe. Efficacy test results showed that the neutralizing antibody levels in pigs immunized with the vaccine prepared in this invention were ≥1:32, and it could protect piglets from disease in 100% of cases. In contrast, Comparative Example 1, under the same conditions, showed a neutralizing antibody ratio below 1:32 in five measurements. While Comparative Example 2 achieved a ratio of 1:32 in some measurements, its overall neutralizing antibody and protection ratio were still significantly lower than those of the group in this invention. The above comparisons show that the adjuvant of this invention significantly improves the level of neutralizing antibodies induced by the vaccine, and this effect cannot be achieved simply by adding mannose-PEG-NHS or chitosan.

[0100] Example 3: Vaccine Safety Trial

[0101] 1. Safety test for piglets

[0102] 1.1 Piglet Safety Testing Protocol

[0103] 1.1.1 Safety Experiment for Single-Dose Vaccination: Ten 4-5 week old piglets were randomly divided into two groups of five each. Group 1, the immunization group, received intramuscular injection of PRRSV001 vaccine, with each pig receiving one dose (2 ml / pig) via intramuscular injection in the neck. Group 2 served as the control group and was not vaccinated. The piglets were isolated and observed for 21 days, with daily monitoring of body temperature, mental state, diet, fecal characteristics, and any abnormalities at the injection site.

[0104] 1.1.2 Safety Experiment of Single-Dose Repeated Vaccination: Ten 4-5 week old piglets were randomly divided into two groups of five each. Group 1, the immunization group, received intramuscular injection of PRRSV002 batch vaccine, with each pig receiving one dose (2ml / pig) via intramuscular injection in the neck. A repeat vaccination was administered two weeks later. Group 2, the control group, was not vaccinated. Pigs were kept in isolation. After the second immunization, piglets were observed for 21 days, with daily monitoring of body temperature, mental state, diet, fecal characteristics, and any abnormalities at the injection site.

[0105] 1.1.3 Safety Trial for Overdose Vaccination: Ten 4-5 week old piglets were randomly divided into two groups of five each. Group 1, the immunization group, received intramuscular injection of PRRSV003 batch vaccine, with each pig receiving two doses (4 ml / pig) via intramuscular injection in the neck. Group 2, the control group, was not vaccinated. Pigs were kept in isolation. They were observed for 21 days post-vaccination, with daily monitoring of body temperature, mental state, diet, fecal characteristics, and any abnormalities at the injection site. On day 21, all piglets were euthanized for pathological examination.

[0106] 1.2 Results of the safety test on piglets

[0107] 1.2.1 Safety Trial of Single-Dose Vaccination: During the clinical observation period, the five piglets immunized with PRRSV001 batch vaccine had normal body temperature (occasionally a few piglets had elevated body temperature, but not exceeding 40.5℃), and their mental state, appetite, feces, and injection site were all normal. Specific results are shown in Table 6.

[0108] Table 6. Results of safety trials for a single dose.

[0109]

[0110] 1.2.2 Safety Trial of Single-Dose Repeated Vaccination: During the clinical observation period, the five piglets immunized with PRRSV002 batch vaccine had normal body temperature, mental state, appetite, feces, and injection site. Specific results are shown in Table 7.

[0111] Table 7. Safety trial results of single-dose repeated vaccination

[0112]

[0113] 1.2.3 Safety Trial of Overdose Vaccination: Five 4-5 week old piglets were immunized with PRRSV003 batch vaccine, 4 ml / pig. During the clinical observation period, some piglets experienced transient fever, but their mental state, appetite, and feces remained normal. Fourteen days after vaccination, no sarcoplasmic dissolution of muscle fibers was observed at the injection site, but a small amount of inflammatory cell infiltration was observed between muscle fibers. Specific results are shown in Table 8. Figure 7 .

[0114] Table 8. Safety test results of overdose vaccination

[0115]

[0116] 1.3 Conclusions of the safety test on piglets

[0117] Piglets were observed for 21 consecutive days after vaccination, with daily records kept of body temperature, mental state, appetite, feces, and reactions at the injection site. Results showed that all immunized piglets had body temperatures within the normal range, normal mental state, appetite, and feces, and no obvious redness, swelling, or suppuration was observed at the injection site. This demonstrates the safety of the vaccine for piglets.

[0118] 2. Safety trials in pregnant sows

[0119] 2.1 Safety Trial Protocol for Pregnant Sows

[0120] 2.1.1 For a single-dose vaccination, 10 pregnant sows were randomly divided into two groups of 5 sows each. Group 1, the immunization group, received an intramuscular injection of PRRSV001 vaccine, with each sow receiving one dose (2 ml / sow) via intramuscular injection in the neck. Group 2, the control group, was not vaccinated and was kept in isolation. The sows were observed until farrowing, and the occurrence of abortion during pregnancy and any difference in litter size compared to the control group were recorded.

[0121] 2.1.2 Single-dose repeated vaccination: Ten pregnant sows were randomly divided into two groups of five each. Group 1, the immunization group, received intramuscular injection of PRRSV002 batch vaccine, with each sow receiving one dose (2ml / sow) via intramuscular injection in the neck. A repeat vaccination was administered two weeks later. Group 2, the control group, was not vaccinated and was kept in isolation. Observations were maintained until farrowing, recording whether abortion occurred during pregnancy and whether the number of piglets born differed from the control group.

[0122] 2.1.3 Overdose Vaccination: Twenty pregnant sows were randomly divided into two groups of five each. Group 1, the immunization group, received intramuscular injections of PRRSV003 batch vaccine, with each sow receiving two doses (4 ml / sow) via intramuscular injection in the neck. Group 2, the control group, was not vaccinated and was kept in isolation. Observations were maintained until farrowing, recording whether abortion occurred during pregnancy and whether there was any difference in litter size compared to the control group.

[0123] 2.2 Results of the safety trial of pregnant sows

[0124] 2.2.1 After a single dose of PRRSV001 vaccine was administered to pregnant sows, no abortions occurred in either the immunized or control sows, and there was no significant difference in litter size. Specific results are shown in Table 9.

[0125] Table 9. Safety trial results of a single dose.

[0126]

[0127] 2.2.2 Single-dose repeated vaccination of pregnant sows: After single-dose repeated vaccination of pregnant sows with PRRSV002 batch vaccine, no abortions occurred in either the vaccinated or control pregnant sows, and there was no significant difference in litter size. Specific results are shown in Table 10.

[0128] Table 10. Safety trial results of single-dose repeated vaccination

[0129]

[0130] 2.2.3 Overdose Vaccination of Pregnant Sows: After overdose vaccination with PRRSV003 batch vaccine, body temperature, mental state, diet, and fecal characteristics were monitored daily until 14 days after repeat vaccination. No abortions occurred in either the immunized or control sows, and there was no significant difference in litter size. Specific results are shown in Table 11.

[0131] Table 11 Safety test results of overdose vaccination

[0132]

[0133] 2.3 Conclusions of the Safety Trial on Pregnant Sows

[0134] The results showed that all immunized sows had normal body temperature, no abortions, and no significant differences in litter size or weak piglet rate compared to the control group. This demonstrates that the vaccine is safe for pregnant sows and has no reproductive toxicity.

[0135] Example 4: Immunogenicity test and immunization period test of vaccine in piglets

[0136] 1. Test Protocol

[0137] Twenty healthy, susceptible piglets aged 4-5 weeks were selected and randomly divided into two groups of 10 each. Each group received 2 ml of the PRRSV genetically engineered vaccine prepared in Example 2. The second group served as a control and was not immunized. Twenty-one days after the second immunization, five piglets from the immunized group and five piglets from the control group were randomly selected and administered PRRSV NADC30-like vaccine (1.0 × 10⁻⁶). 5.0 TCID 50 Piglets were challenged with a viral load of 3 ml ( / ml) via intramuscular injection and 3 ml via nasal drops. Temperature was monitored daily for 21 days, and morbidity and mortality were recorded. Twenty-one days after challenge, three piglets from each group were randomly euthanized, and lymph nodes and lungs were aseptically collected for pathological section preparation. Blood samples were collected from the remaining five piglets in the immunized and control groups at 21 days, 1 month, 2 months, 3 months, and 4 months after the last immunization. Serum was separated, and neutralizing antibody titers were determined.

[0138] 2. Results of immunization efficacy and immunization period trials in piglets

[0139] 2.1 Results of the challenge protection test

[0140] Twenty-eight days after immunization, all piglets were challenged with the virus. They were observed for 14 consecutive days after challenge, and the protection rate was 100% (protection was defined as normal body temperature or a temperature increase of no more than 0.5℃, and the absence of respiratory distress, lethargy, cyanosis, etc.). All piglets in the control group developed the disease after challenge (disease was defined as a temperature increase of more than 1℃, and the presence of clinical symptoms such as respiratory distress, lethargy, cyanosis, etc.). Specific results are shown in Table 12.

[0141] Table 12 Results of the challenge protection test

[0142]

[0143] 2.2 Results of Neutralizing Antibody Level Detection

[0144] Neutralizing antibodies were produced in immunized pigs after the second immunization, and the antibody levels subsequently increased, reaching an average of over 1:50 by the first month post-immunization. Specific results are shown in Table 13.

[0145] Table 13 Neutralizing antibody level test results (after second immunization)

[0146]

[0147] 2.3 Histopathological examination results

[0148] 2.3.1 Lung pathological examination results

[0149] Interstitial pneumonia, interstitial hyperplasia, and inflammatory cell infiltration were clearly visible in the control group; mild interstitial pneumonia was observed in the immunized group. Results are shown below. Figure 8 .

[0150] 2.3.2 Pathological examination results of inguinal lymph nodes

[0151] The control group showed significant subcapsular edema and neutrophil aggregation; no abnormalities were observed in the immunized group. Results are shown below. Figure 9 .

[0152] The results of the challenge test showed that the genetically engineered vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) developed in the laboratory could protect piglets from the disease by 100% after immunization; one month after immunization, the average neutralizing antibody level reached more than 1:50.

[0153] Example 5: Comparison trial with commercially available vaccines

[0154] The vaccine of this invention (25 μg / dose) was compared with two commercially available PRRS inactivated vaccines (Vaccine A and Vaccine B). Piglets were immunized according to the instructions.

[0155] 1. Materials

[0156] 1.1 Porcine Reproductive and Respiratory Syndrome Genetically Engineered Vaccine

[0157] A batch of subunit vaccine with a protein content of 25 μg was prepared according to Example 1 of the present invention.

[0158] 1.2 Commercialized Vaccines

[0159] Purchase one batch each of commercially available inactivated porcine reproductive and respiratory syndrome (PRRS) vaccine A (CH-1a strain) and inactivated porcine reproductive and respiratory syndrome (PRRS) vaccine B (M-2 strain).

[0160] 1.3 Experimental animals: Healthy weaned susceptible piglets aged 28–35 days, negative for porcine reproductive and respiratory syndrome virus antigen and antibody.

[0161] 1.4 Porcine Reproductive and Respiratory Syndrome Virus (Americas type) Indirect ELISA Antibody Detection Kit (Hyperle Biopharmaceuticals, Spain)

[0162] 2 Methods

[0163] Ten piglets were immunized with 1 ml of each vaccine via intramuscular injection behind the ear. Twenty-one piglets were immunized again with 1 ml 21 days later. Ten piglets were used as a control group without injection.

[0164] 2.1 Antibody Detection

[0165] Five pigs were selected for each vaccine immunization and five control pigs were selected. Blood samples were collected on days 7, 21, 28, 35, 49, and 56 after the initial immunization to separate serum. ELISA antibodies were detected according to the kit instructions, and neutralizing antibodies were detected according to the neutralizing antibody detection method.

[0166] 2.2 Virus Challenge Protection Experiment

[0167] Five additional birds from each batch, along with five control birds, were weighed 21 days after the second immunization and then treated with 10... 6.0 TCID 50 The virus was administered at a dose of NADC30 strain / ml, with 2ml administered via nasal drops to each animal. Body temperature was monitored daily until day 18 post-infection. Blood samples were collected on days 3, 7, 11, 15, and 19 post-infection for viral load testing using quantitative real-time PCR. Animals were weighed 21 days after immunization and then euthanized for necropsy. Lung and lymph node lesions were scored based on their severity.

[0168] 3 Results

[0169] 3.1 Antibody Detection

[0170] 3.1.1 ELISA antibody detection results after immunization with different vaccines

[0171] All three vaccines induced the production of specific antibodies after immunization. The subunit vaccine showed detectable specific antibodies as early as 14 days after the initial immunization, with antibody levels rising rapidly and remaining at a high level. In comparison, vaccine A resulted in a relatively slower antibody response, while vaccine B resulted in an even slower response and lower antibody levels. These results indicate that the subunit vaccine developed in this invention produces a better specific immune response than existing commercially available inactivated porcine reproductive and respiratory syndrome (PRRS) vaccines. (See Table 14)

[0172] Table 14 ELISA antibody detection results (IRPC values) after immunization with different vaccines

[0173]

[0174] 3.1.2 Results of neutralizing antibody detection after immunization with different vaccines

[0175] Neutralizing antibodies are detectable and at low levels a considerable time after porcine reproductive and respiratory syndrome virus (PRRSV) infection or vaccination. In this example, both vaccine A and vaccine B produced relatively low levels of neutralizing antibodies, approximately 1:4. In contrast, the subunit vaccine developed according to this invention produced higher levels of neutralizing antibodies 21 days after the second immunization. Experimental results show that the subunit vaccine developed according to this invention induces higher levels of neutralizing antibodies after immunization compared to existing commercially available inactivated PRRS vaccines. See Table 15.

[0176] Table 15 Results of neutralizing antibody detection after immunization with different vaccines (log2)

[0177]

[0178] 3.2 Results of immune challenge

[0179] 3.2.1 Results of body temperature monitoring in immunized piglets after viral challenge

[0180] The body temperature monitoring results showed that the experimental pigs in the control group maintained a higher body temperature for up to 15 days after challenge, while the average body temperature of the pigs in the immunized groups returned to normal 10 days after challenge, and the high fever was not persistent. The subunit vaccine and A vaccine resulted in lower and shorter-lasting temperature increases. The experimental results indicate that subunit and A vaccines can effectively reduce the degree and duration of temperature increase in experimental pigs after virulent viral challenge. (See Table 16.)

[0181] Table 16. Body temperature monitoring results (°C) of immunized piglets after viral challenge.

[0182]

[0183] 3.2.2 Results of viral load detection in immunized piglets after challenge

[0184] The results of whole blood sample testing after challenge showed that the immunization vaccine effectively reduced the viral load in the blood after challenge with a virulent strain. The degree of reduction, from highest to lowest, was as follows: subunit vaccine A and subunit vaccine B developed in this invention. See Table 17.

[0185] Table 17 Results of viral load detection (Ct value) in immunized piglets after challenge.

[0186]

[0187] 3.2.3 Results of weight gain test in immunized piglets after viral challenge

[0188] The weight gain of piglets in each immunization group after viral challenge was significantly better than that of the control group. The order of total weight gain from highest to lowest was: subunit vaccine, A vaccine, and B vaccine. (See Table 18.)

[0189] Table 18 Results of weight gain in immunized piglets after viral challenge

[0190]

[0191] 3.2.4 Tissue lesions and scoring results in immunized piglets after viral challenge

[0192] Depend on Figure 10 It was observed that after viral challenge, control piglets showed reddish-brown mottled lung tissue that did not collapse; enlarged lymph nodes; and an enlarged spleen. Section examination revealed uneven widening of the alveolar diaphragms, with enlarged and proliferating capillary endothelial cells, macrophages, and infiltrated lymphocytes. The severity of lesions in each immunized group was significantly reduced compared to the control group, with the severity of lesions increasing from mild to severe: subunit vaccine, A vaccine, and B vaccine. These results indicate that the subunit vaccine can effectively reduce tissue lesions in animals after viral challenge and is superior to existing commercially available vaccines.

[0193] The results showed that the vaccine of this invention induced ELISA antibodies and neutralizing antibodies at a faster rate and at higher levels. After challenge, the vaccine immunization group of this invention was significantly superior to the two commercial vaccine control groups in terms of clinical symptom relief, viral load reduction, and protection against tissue lesions.

[0194] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. 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 be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A genetically engineered subunit vaccine against porcine reproductive and respiratory syndrome virus, characterized in that, The vaccine comprises a PRRSV ZJ266 GP5-M protein complex and an adjuvant, wherein the adjuvant is a mannose-modified chitosan solution.

2. The porcine reproductive and respiratory syndrome virus (PRRSV) genetically engineered subunit vaccine according to claim 1, characterized in that, The PRRSV ZJ266 GP5-M protein complex is formed from the GP5 protein and M protein of PRRSV ZJ266 strain expressed and purified by the Bac-to-Bac baculovirus expression system. Its nucleotide sequence is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO:

2.

3. The porcine reproductive and respiratory syndrome virus (PRRSV) genetically engineered subunit vaccine according to claim 2, characterized in that, The GP5 protein has a bee signal peptide attached to its N-terminus and a sequence encoding a 6×His tag added to its C-terminus.

4. The porcine reproductive and respiratory syndrome virus (PRRSV) genetically engineered subunit vaccine according to claim 1, characterized in that, The PRRSV ZJ266 GP5-M protein complex in the vaccine is 20-30 μg / mL.

5. The porcine reproductive and respiratory syndrome virus (PRRSV) genetically engineered subunit vaccine according to claim 1, characterized in that, The preparation method of the adjuvant includes the following steps: A1. Chitosan was dissolved in an acidic solution, the pH was adjusted to 5.5-5.8, Traut's reagent was added to carry out a thiolation reaction, and then purified to obtain a thiolated chitosan solution. A2. The mannose-PEG-NHS activated ester was coupled with the thiolated chitosan solution, and the mixture was purified after the reaction to obtain the adjuvant.

6. The porcine reproductive and respiratory syndrome virus (PRRSV) genetically engineered subunit vaccine according to claim 5, characterized in that, In step A2, the mannose-PEG-NHS activated ester is first dissolved in dimethyl sulfoxide and then added to the thiolated chitosan solution.

7. A method for preparing a porcine reproductive and respiratory syndrome virus (PRRSV) genetically engineered subunit vaccine, used to prepare the PRSV genetically engineered subunit vaccine according to any one of claims 1-6, characterized in that, The preparation method includes the following steps: S1. Construct a recombinant baculovirus containing genes encoding the GP5 and M proteins of PRRSV ZJ266 strain; S2. Infect insect cells with the recombinant baculovirus to express proteins; S3. Harvest and purify the expression product to obtain the GP5-M protein complex; S4. The purified protein complex is mixed and emulsified with adjuvant to prepare a vaccine.

8. The method for preparing a porcine reproductive and respiratory syndrome virus (PRRSV) genetically engineered subunit vaccine according to claim 7, characterized in that, In step S3, purification is performed using metal chelate affinity chromatography and molecular sieve chromatography.

9. The method for preparing a porcine reproductive and respiratory syndrome virus (PRRSV) genetically engineered subunit vaccine according to claim 7, characterized in that, In step S4, the volume ratio of the protein complex to the adjuvant is 9:

1.

10. The use of a porcine reproductive and respiratory syndrome virus (PRRSV) genetically engineered subunit vaccine as described in any one of claims 1-6 in the prevention of PRRSV.

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