Preparation method and application of G2a and G2c porcine epidemic diarrhea virus bivalent inactivated vaccine

Abstract

The invention discloses a preparation method and application of a G2a and G2c porcine epidemic diarrhea virus bivalent inactivated vaccine. The vaccine comprises a G2a subtype strain KQ01 (the preservation number is CCTCC NO: V202005) and a G2c subtype strain LN202305 (the preservation number is CCTCC NO: V202588). The specific operation process comprises the following steps: (1) screening two epidemic subtype dominant strains of PEDV (porcine epidemic diarrhea virus) G2a and G2c; (2) preparing PEDV G2a and G2c virus liquid; (3) preparing a bivalent inactivated vaccine of the porcine epidemic diarrhea virus G2a / G2c; (4) carrying out safety inspection on the porcine epidemic diarrhea virus G2a / G2c bivalent inactivated vaccine; and (5) testing the potency of the porcine epidemic diarrhea virus G2a / G2c bivalent inactivated vaccine. The vaccine induces high-titer neutralizing antibodies to G2a and G2c subtype PEDV, realizes cross protection, and is used for preventing current epidemic PEDV infection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of veterinary biological products, in particular to a bivalent inactivated vaccine against Porcine epidemic diarrhea virus (PEDV) G2a / G2c dual genotype, including antigen isolation and screening, vaccine preparation and application. BACKGROUND

[0002] Porcine Epidemic Diarrhea (PED) is a highly contagious enteric disease caused by PEDV, with vomiting, severe diarrhea, dehydration and extremely high mortality (up to 100% for suckling piglets) as the main characteristics, causing huge economic losses to the global pig industry. PEDV belongs to the Coronaviridae family of Coronaviruses, with a single-stranded positive-sense RNA genome, and has the characteristics of high mutation rate and easy recombination. According to the genetic differences of the highly variable spike protein (S protein) gene, PEDV can be divided into two major genotypes, G1 and G2, which can be further divided into G1a, G1b, G1c, G2a, G2b and G2c subtypes. Since 2010, highly pathogenic G2 genotype strains (especially G2a and G2c subtypes) have become the dominant strains globally.

[0003] Existing studies have shown that the cross-protection between different genotypes / subtypes is limited. Although there are some PEDV vaccines (including inactivated vaccines and attenuated vaccines) on the market, most of them are based on early G1 type strains or a single G2 type strain, and the vaccines based on G1 type strains do not provide complete protection against the currently prevalent G2a / G2c strains, with unsatisfactory immune effect; vaccines based on a single strain (whether G2a or G2c) may not be able to effectively cover all prevalent G2 subtypes. G2a and G2c strains have certain amino acid differences in key antigen epitopes (such as the S protein COE domain), which may lead to limited neutralization ability of antibodies induced by a single vaccine strain, and thus cannot provide comprehensive protection against all prevalent strains, so it is urgent to develop a vaccine that can simultaneously target the two major prevalent subtypes (G2a and G2c) and provide more extensive cross-protection and stronger immune efficacy. SUMMARY

[0004] To overcome the problem that existing vaccines cannot effectively prevent and control the two major prevalent subtypes (PEDV G2a and G2c), the present application provides a bivalent inactivated vaccine against PEDV G2a and G2c genotypes, as well as a preparation method and application thereof.

[0005] To achieve the object of the present application, the first aspect of the present application provides a porcine epidemic diarrhea virus PEDV G2a strain KQ01, which is deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, and has a preservation number of CCTCC NO: V202005.

[0006] Further, the S gene nucleotide sequence thereof is shown as SEQ ID NO: 1.

[0007] The second aspect of the present application provides a porcine epidemic diarrhea virus PEDV G2c strain LN202305, which is deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, and has a preservation number of CCTCC NO: V202588.

[0008] Further, the S gene nucleotide sequence thereof is shown as SEQ ID NO: 2.

[0009] The third aspect of the present application provides a vaccine comprising the porcine epidemic diarrhea virus PEDV G2a strain KQ01 and the porcine epidemic diarrhea virus PEDV G2c strain LN202305, and a pharmaceutically acceptable adjuvant and buffer; Further, the content of the two strains is 10 6.0 -10 8.0 TCID 50 / ml; the antigen ratio of the G2a and G2c strains is 1:0.5-1:2; and the adjuvant is one of squalene adjuvant, oil-in-water adjuvant or aluminum adjuvant.

[0010] Further, the content of the two strains is 10 7.0 TCID 50 / ml; the antigen ratio of the G2a and G2c strains is 1:1; the adjuvant is one of squalene adjuvant, preferably ISA201 VG; and the mass ratio of the adjuvant to the antigen is 1:1.

[0011] The fourth aspect of the present application provides a preparation method of a G2a and G2c porcine epidemic diarrhea virus bivalent inactivated vaccine, comprising the following steps: (1) Strain screening: screening of two epidemic subtype dominant strains of PEDV G2a and G2c; (2) Virus culture: inoculating the G2a strain and the G2c strain into suspension type ST cells respectively, with a MOI of 0.001-0.1, culturing in a bioreactor at 36-37℃ for 48-72h, and harvesting virus liquid; after clarification, ultrafiltration concentration and purification operations, virus liquid with a virus content of ≥10 7.0 TCID 50 / ml is obtained; (3) Virus inactivation: the purified virus liquid stock or dilution is added with formaldehyde at a final concentration of 0.1-0.3% (v / v), inactivated at 37±1℃ for 36-48h, and the complete inactivation is verified by cell culture method; (4) Preparation of vaccine: the inactivated virus liquid is diluted with PBS to a vaccine preparation concentration of 10 7.0 TCID 50 / ml, mixed and emulsified with an adjuvant at a volume ratio of 1:1-1:1.5 (v / v) according to 1:1 volume ratio, quantitatively packaged, capped and labeled after detection, and the vaccine is obtained.

[0012] The application provides application of the PEDV G2a strain KQ01 and the G2c strain LN202305 in claim 2 in preparation of a bivalent inactivated vaccine for preventing infection of porcine epidemic diarrhea virus G2a and G2c subtypes.

[0013] Compared with the prior art, the application has the following beneficial effects: (1) A bivalent inactivated vaccine for PEDV G2a / G2c dual gene subtypes is first proposed and constructed: the existing vaccines are mostly single strains, and cannot effectively cover the two major subtypes G2a and G2c currently prevalent; the application first combines the G2a strain KQ01 and the G2c strain LN202305, filling the technical blank of the existing vaccine in insufficient cross protection of G2a / G2c dual subtypes.

[0014] (2) An advantageous strain combination with broad-spectrum cross immunogenicity is obtained through systematic screening: from 14 clinical isolates, through multidimensional screening of virus content, virulence, immunogenicity and cross neutralization reaction spectrum, KQ01 (G2a) and LN202305 (G2c) are finally selected; the combination can induce neutralizing antibodies against different G2a and G2c strains.

[0015] (3) A high-efficiency virus amplification and inactivation process is established, ensuring the feasibility of industrial production of the vaccine: the ST cells are large-scale cultured by using a bioreactor, the virus inoculation time, culture conditions and virus harvesting nodes are optimized, and the virus titer is ensured to be stable at ≥10 7.0 TCID 50 / ml; the 0.2% formaldehyde inactivation and ISA201 VG adjuvant emulsification process is stable, easy to scale up, suitable for industrial production, has no virus diffusion risk and high biological safety.

[0016] (4) Provides broader-spectrum immune protection: After immunizing sows, the neutralizing antibody titers against both G2a and G2c strains were ≥1:128, significantly higher than the immunization range of existing single-strain vaccines; piglet challenge experiments showed that 100% protection (5 / 5) was achieved against both G2a and G2c strains, while all control groups developed the disease. The vaccine had no adverse reactions in pregnant sows, did not cause fever, abortion, stillbirth, or weak piglets, and did not affect reproductive performance.

[0017] (5) Solving the key problems in the current prevention and control of PED: It overcomes the problem of immunization failure caused by the incompatibility of existing vaccines with strains, and is especially suitable for areas where G2a / G2c are mixed. It provides pig farms with an efficient and safe immunization tool with clear industrial application value and market prospects. Attached Figure Description

[0018] Figure 1 Results of PEDV S gene phylogenetic analysis.

[0019] Figure 2 Immunogenicity evaluation results of different PEDV strains.

[0020] Figure 3 Clinical observation results of some piglets. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0022] Example 1: Screening of dominant strains of two prevalent PEDV subtypes, G2a and G2c PEDV strains were isolated using Vero cells by incubation with high concentrations of trypsin and supplementation with trypsin-phosphate broth and trypsin. The isolated PEDV strains were then subjected to phylogenetic analysis, viral load determination, virulence experiments, and immunogenicity experiments to screen out strains with relatively high viral load, strong virulence, and good immunogenicity.

[0023] (1) PEDV virus isolation Sample processing: Take the homogenate of small intestine tissue from piglets, centrifuge at 12000 r / min for 2 minutes at 4℃, take the supernatant and filter it through a 0.22μM filter membrane for sterilization, add trypsin with a final concentration of 40 μg / mL to the filtrate and incubate at 37℃ for 30 min to activate the virus.

[0024] Cell inoculation: 400 μL of the activated virus treatment solution was inoculated into the Vero cells that had grown into a single layer (the cell culture solution was discarded before inoculation, and the cells were washed twice with PBS), and 1 mL of cell maintenance solution (10 μg / mL trypsin, 0.3% trypsin phosphate broth-free DMEM culture solution) was added. The cells were adsorbed at 37°C for 1 hour, the inoculum was aspirated, the cells were washed twice with PBS, 5 mL of cell maintenance solution was added, and the cells were continued to be cultured.

[0025] Virus harvesting and passage: The cells were observed daily, and trypsin was added to a final concentration of 10 μg / mL. If cytopathic effect was observed, the culture was harvested. If no cytopathic effect was observed, the culture supernatant was harvested after 72 hours of culture, and the blind passage was continued. If no cytopathic effect was observed after 3 generations of blind passage, and no specific band was detected by PCR, the passage was stopped.

[0026] Strain screening: A total of 14 PEDV strains were isolated from 2019 to 2025. As shown in the phylogenetic tree analysis results, 7 strains were located in the G2a branch, and 7 strains were located in the G2c branch. Figure 1

[0027] (2) PEDV virus content determination The virus solution collected by repeated freezing and thawing 3 times was centrifuged at 3000 r / min for 3 minutes at 4°C, and the supernatant was taken. The virus was diluted by 10 times in series with cell maintenance solution (10 μg / mL trypsin-free DMEM culture solution), i.e., 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , and 10 -7 . Each dilution was inoculated into a good Vero cell monolayer that had been washed 3 times with serum-free DMEM medium, with one vertical row of 8 holes (96-well cell culture plate) for each dilution, and 100 μl was added to each hole. Eight normal cell control holes were also set up, and the cell monolayer of the cell control holes was also washed 3 times with serum-free DMEM medium, and 100 μL of serum-free DMEM medium containing 10 μg / mL trypsin was added. The cells were continued to be cultured in a 37°C, 5% CO2 incubator, observed continuously for 4 days, and the cytopathic effect was recorded. The normal cell control holes should not show cytopathic effect. The TCID 50 was calculated by the Reed-Muench method, and the results were expressed as the TCID 50 value per 100 μL of virus solution.

[0028] As shown in Table 1, among the 7 G2a subtype strains, the virus titer of KQ01 was the highest, which was 10 7.88 TCID 50 ​ / mL, higher than other strains; among 7 G2c subtypes, LN202305 titer was 10 7.80 TCID 50 / mL higher than other strains.

[0029] Table 1 PEDV virus content determination

[0030] (3) PEDV virulence test Screen 3-5 day-old piglets produced by sows with PEDV neutralizing antibody not higher than 1:4. Use 7 G2a type PEDV and 7 G2c type PEDV virus liquid (virus content is 10 5.0 TCID 50 / ml) for virulence test, orally administer 2.0 ml virus liquid to each piglet in the challenge group, and orally administer 2.0 ml PBS to each piglet in the blank control group, continuously observe for 7 days after challenge, and record the clinical performance and incidence of the piglets.

[0031] The statistical results are shown in Tables 2 and 3, and the virulence of G2a is KQ01, SY2555, MZ1205, ZL20201117 in turn, and the virulence of G2c is LN202305, GX2504098-7, HNXX250223, HBXZ2401-19 in turn.

[0032] Table 2 Statistical results of virulence test of 7 G2a type PEDV

[0033] Table 3 Statistical results of virulence test of 7 G2c type PEDV

[0034] (4) Immunogenicity experiment Inactivate 3 G2a type PEDV strains KQ01, SY2555, MZ1205 and 3 G2c type PEDV strains LN202305, HNXX250223, HBXZ2401-19 virus liquid with high virus content and strong virulence, emulsify them uniformly with MONTANIDETM ISA201VG adjuvant to prepare PEDV inactivated vaccine, and the PEDV virus content in each dose (2 ml) of the vaccine is 10 7.0 TCID 50 / ml. Divide 24-26 day-old piglets with PEDV neutralizing antibody not higher than 1:4 into groups at random, and inoculate each piglet with one dose. Collect blood 21 days after immunization, isolate serum, and determine the neutralizing antibody titers in the serum against different PEDV strains.

[0035] The detection results are as follows Figure 2As shown, in the G2a strain, the PEDV KQ01 immune serum not only showed a higher neutralizing antibody titer to the strain, but also showed a higher neutralizing antibody level to the SY2555 and MZ1205 strains; in contrast, the SY2555 and MZ1205 strains only showed a higher neutralizing antibody response to themselves. In addition, the PEDV KQ01 strain also showed a higher neutralizing antibody titer to the G2c strain than the SY2555 and MZ1205 strains.

[0036] In the G2c strain, the PEDV LN202305 immune serum also induced a higher neutralizing antibody level to the strain and the HNXX250223 and HBXZ2404-19 strains; while the HNXX250223 and HBXZ2404-19 strains only showed a higher neutralizing activity to themselves. At the same time, the PEDV LN202305 strain also showed a significantly higher neutralizing antibody titer to the G2a strain than the HNXX250223 and HBXZ2404-19 strains.

[0037] Based on the evaluation results of the above cross-neutralization reaction spectrum, the present application selects the PEDV G2a strain KQ01 and the PEDV G2c strain LN202305 as candidate vaccine strains. The S gene nucleotide sequences thereof are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0038] The porcine epidemic diarrhea virus G2a KQ01 was deposited in the China Center for Type Culture Collection on December 19, 2019, with a preservation number of CCTCC NO: V202005, and a preservation address of Wuhan, China, Wuhan University.

[0039] The porcine epidemic diarrhea virus PEDV G2c strain LN202305 was deposited in the China Center for Type Culture Collection on November 26, 2025, with a preservation number of CCTCC NO: V202588, and a preservation address of Wuhan, China, Wuhan University.

[0040] Example 2 Preparation of PEDV G2a and G2c virus liquid (1) Expansion culture of the bioreactor: when the ST cells in the seed tank are cultured for 72 h or 96 h and the cell viability is more than 95%, the cells are transferred to the bioreactor. Before inoculating the cells, the dissolved oxygen (DO) electrode, pH electrode, and temperature electrode are calibrated, and the tank body is sterilized. Pump 20% of the total volume of the bioreactor into the growth solution, and inoculate the cells in the bioreactor at a cell density of 0.8 x 10 6 6 / ml, and set the optimal culture conditions to 37°C, pH 7.0, and DO 40%, 40 r / min.

[0041] (2) Virus inoculation: when the density of ST cells in the bioreactor reaches 6.0-7.0 x 10 6 cells / ml, the cell density is diluted to 3 x 10 6 cells / ml with growth solution, and the final concentration of trypsin added is 10 ug / ml, and the virus is inoculated at a dose of 0.1 MOI of porcine epidemic diarrhea virus variant 2a or 2c, and the optimal culture conditions are set as 37℃, pH 7.0, DO 40%, and 40 r / min.

[0042] (3) Virus liquid harvesting: sample detection of TCID50 24h after inoculation, detection of virus content and purity, and after meeting the requirements, quantitative dispensing, and labeling of name, production date, passage, and titer.

[0043] (4) Virus content determination and sterility and foreign matter testing: virus titer determination and testing are carried out according to the current Appendix of Chinese Veterinary Pharmacopoeia, and the virus content is not less than 10 7.0 TCID 50 / ml, and should be sterile and free of specified foreign matter.

[0044] (5) Virus liquid purification: the first step is to clarify the antigen after centrifugation by microfiltration with a 0.45 μm hollow fiber column, the second step is to concentrate by ultrafiltration with a 300KD hollow fiber column, and the final step is to further purify the concentrated virus liquid by molecular sieve chromatography.

[0045] Example 3 Preparation of porcine epidemic diarrhea virus G2a / G2c bivalent inactivated vaccine (1) Inactivation: the purified virus liquid stock or diluted virus liquid is added to the inactivation tank, formaldehyde is added for inactivation, the final concentration of formaldehyde is 0.2%, and the mixture is stirred while adding, and after uniform mixing, it is inactivated at 37℃ for 48h, and during this period, stirring is carried out every 2-4 hours, and the inactivation is verified by cell culture method.

[0046] (2) Water phase preparation: the inactivated virus liquid is diluted to the vaccine concentration (10 7.0 TCID 50 / ml) with PBS, and then mixed uniformly at a ratio of 1:1 (volume ratio), and preheated to 32℃.

[0047] (3) Emulsification, dispensing: the water phase is mixed with ISA201 VG adjuvant at a volume ratio of 1:1, emulsified, and after detection, it is quantitatively dispensed, capped, and labeled.

[0048] Example 4 Safety test of porcine epidemic diarrhea virus G2a / G2c bivalent inactivated vaccine (1) Test scheme Ten sows in the 5th-6th week of gestation were selected, 5 of which were inoculated with the porcine epidemic diarrhea virus G2a / G2c bivalent inactivated vaccine, and 5 of which were set as blank control group without inoculation, 4.0 ml per pig was injected in the neck muscle, and the same method was used for inoculation again after 21 days. All the pregnant sows were measured for temperature for 10 days: the average value of the average temperature on the 2nd day before the second inoculation and the day of inoculation was taken as the basal temperature, and the temperature was measured continuously for 7 days after inoculation. The observation of the inoculation site, clinical manifestations, whether abortion or birth of piglets (number of stillbirths, number of weak piglets and number of healthy piglets) and the like of the sows were recorded continuously until the sows gave birth.

[0049] (2) Test results After inoculation, all the sows were normal in spirit, behavior, respiration and feeding, the inoculation site was normal, and there was no redness, induration, ulceration, and no systemic adverse reactions; the results are shown in Tables 4 and 5 After inoculation, the body temperature of all the pregnant sows did not exceed 1℃ of the basal temperature; there was no significant difference in the birth of piglets between the vaccine immunization group and the blank control group, and there was no abortion, stillbirth, weak piglet and the like. The above test results show that the porcine epidemic diarrhea virus G2a / G2c bivalent inactivated vaccine has no effect on the performance of pregnant sows, and is safe for pregnant sows.

[0050] Table 4 Test results of body temperature of pregnant sows

[0051] Table 5 Statistical results of the birth of piglets of pregnant sows

[0052] Example 5 Potency test of porcine epidemic diarrhea virus G2a / G2c bivalent inactivated vaccine (1) Test scheme Ten pregnant sows in the 5th-6th week of gestation with negative porcine epidemic diarrhea neutralizing antibody (PEDV neutralizing antibody titer not higher than 1:4) were selected and randomly divided into 2 groups, 5 sows in each group, 1 dose of porcine epidemic diarrhea virus G2a / G2c bivalent inactivated vaccine was inoculated in group 1, and the same amount of normal saline was injected intramuscularly in group 2 as a control group, and the same method was used for inoculation again after 21 days, blood was collected 14 days after the second immunization, and the neutralizing antibody level was measured. After the sows gave birth, 10 healthy piglets of 3-5 days old were randomly selected from the immunization group and the control group respectively. Among them, the immunization group and the control group were further divided into two subgroups: each group had 5 piglets orally gavaged with G2a strain, and the other 5 piglets were challenged with G2c strain. The challenge dose was uniformly 2 ml (virus titer was 10 5.0 TCID 50 / ml). After the challenge, the clinical manifestations and symptom changes of the piglets were observed continuously for 7 days and recorded.

[0053] (2) The test results are shown in Table 6. On the 14th day after the second immunization, the neutralizing antibody titers of the immunized sows against G2a and G2c strains were both ≥1:128, while the neutralizing antibody titers of the blank control sows were both <1:4. The results of the challenge test are shown in Tables 7 and Figure 3 Table 7. The results of the challenge test are shown in Tables 7 and

[0054] Table 6. The results of the challenge test are shown in Tables 7 and

[0055] Table 7. The results of the challenge test are shown in Tables 7 and

[0056] Although the above examples have described the present application and its embodiments in detail, it should be noted that, for those skilled in the art, changes, modifications, substitutions, combinations, simplifications, etc. of the corresponding conditions, etc. without departing from the technical principles of the present application should be considered as equivalent replacement manners, and these improvements should be considered as the protection scope of the present application.

Claims

1. A porcine epidemic diarrhea virus (PEDV) G2c strain LN202305, characterized in that, The deposit was preserved in China Center for Type Culture Collection, and the preservation number was CCTCC NO: V202588.

2. A bivalent inactivated vaccine against G2a and G2c porcine epidemic diarrhea virus, characterized in that, The vaccine comprises the porcine epidemic diarrhea virus 2a KQ01 strain with the preservation number CCTCC NO: V202005 and the porcine epidemic diarrhea virus PEDV G2c strain LN202305 of claim 1, and a pharmaceutically acceptable adjuvant and buffer; The two strains are present in a ratio of 10 6.0 -10 8.0 TCID 50 / ml; the G2a and G2c strains are present in a ratio of 1 :0.5 to 1 :2; and the adjuvant is one of a squalene adjuvant, an oil-in-water adjuvant, or an aluminum adjuvant.

3. The vaccine of claim 2, characterized in that, The two strains are present in a ratio of 10 7.0 TCID 50 / ml; the G2a and G2c strains are present in a ratio of 1:1; and the adjuvant is one of squalene adjuvants, preferably ISA 201 VG.

4. A method for the preparation of a bivalent inactivated vaccine against G2a and G2c porcine epidemic diarrhea virus, characterized in that, The method comprises the following steps: (1) Strain screening: screening of two epidemic subtypes of PEDV G2a and G2c dominant strains; (2) Virus culture: The PEDV G2a strain KQ01 described in claim 2 and the G2c strain described in claim 1 were respectively inoculated into suspension ST cells with an MOI of 0.001-0.1 and cultured in a bioreactor at 36-37℃ for 48-72 h. The virus solution was harvested and purified by clarification, ultrafiltration concentration and purification to obtain a virus content ≥10 7.0 TCID 50 / ml of virus solution; (3) Virus inactivation: adding formaldehyde with a final concentration of 0.1-0.3% (v / v) to the purified virus liquid stock or dilution, inactivating at 37±1℃ for 36-48h, and verifying the complete inactivation by cell culture method; (4) Preparation of vaccine: the inactivated two virus liquids are respectively diluted to the vaccine concentration of 10 7.0 TCID 50 / ml with PBS, mixed uniformly according to the volume ratio of 1:1, then mixed and emulsified with the adjuvant according to the volume ratio of 1:1-1:1.5 (v / v), and after detection, the vaccine is obtained by quantitative packaging, cover rolling and labeling.

5. Use of the PEDV G2a strain KQ01 of claim 2 and the G2c strain LN202305 of claim 1 in the preparation of a bivalent inactivated vaccine for preventing infection of porcine epidemic diarrhea virus G2a and G2c subtypes.

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