A method for preparing porcine rotavirus and porcine epidemic diarrhea virus antigens and uses thereof
By simultaneously or sequentially inoculating ST suspension-acclimated cells with porcine rotavirus and porcine epidemic diarrhea virus, the problems of reduced titer and altered antigenicity during co-culture were solved, achieving efficient and safe production of viral antigens, reducing costs and simplifying the process.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-22
- Publication Date
- 2026-06-05
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Abstract
Description
Technical Field
[0001] This invention relates to the fields of biotechnology and veterinary vaccine manufacturing technology, specifically to a method for preparing porcine rotavirus and porcine epidemic diarrhea virus antigens and their applications. Background Technology
[0002] Porcine epidemic diarrhea virus (PoRV) and porcine rotavirus (PEDV) are the main pathogens of viral diarrhea in piglets, and vaccination is the most effective means of prevention. Currently, vaccine antigen production mainly uses traditional adherent cell culture, which has problems such as cumbersome operation, easy contamination, and difficulty in scaling up. In addition, it is necessary to produce two antigens in different cell systems, which is complicated and costly.
[0003] Co-culturing two viruses in the same cell line faces significant technical obstacles: first, viral interference leads to reduced titers; second, co-culturing may induce adaptive mutations or antigenic alterations in the viruses, affecting immunogenicity and safety. Specifically, screening cell lines adapted to both viruses and establishing efficient and stable culture systems, as well as ensuring that co-cultured antigens maintain consistency with single-cultured antigens in key quality attributes such as immunogenicity, neutralizing antibody titers, and pathogenicity / virulence, are unresolved technical challenges in this field. This concern about the unpredictability of antigen quality constitutes a significant technical bias hindering the industrial application of co-culture technology.
[0004] Therefore, in order to solve the above problems, a method for preparing porcine rotavirus and porcine epidemic diarrhea virus antigens and their applications are proposed. Summary of the Invention
[0005] The purpose of this invention is to provide a method for preparing porcine rotavirus and porcine epidemic diarrhea virus antigens and their applications. This method not only ensures that the titers of both viruses are not reduced, but also ensures that the obtained viral antigens are not significantly different from antigens prepared by separate inoculation in terms of key quality attributes, especially in terms of neutralization effect and pathogenicity.
[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing porcine rotavirus and porcine epidemic diarrhea virus antigens includes the following steps: a) Simultaneous or sequential inoculation of porcine rotavirus and porcine epidemic diarrhea virus into ST suspension-acclimated cells; b) Culture to propagate the virus; c) Harvest the viral fluid; Among them, the viral titers of the obtained porcine epidemic diarrhea virus antigen and porcine epidemic diarrhea virus antigen were not lower than those of their respective separately cultured antigens, and the induced neutralizing antibody titers were not significantly different from those of the separately cultured corresponding antigens, and there was no significant difference in pathogenicity to the sensitive models.
[0007] Preferably, the sequential vaccination involves first inoculating with porcine rotavirus, followed by inoculation with porcine epidemic diarrhea virus 12 to 72 hours later, with the latter being preferred.
[0008] More preferably, the interval is 12 to 24 hours.
[0009] Preferably, the multiplicity of infection when inoculated with porcine epidemic diarrhea virus is 0.01 to 0.1.
[0010] Preferably, the multiplicity of infection when inoculated with porcine rotavirus is 0.01 to 1.0.
[0011] Preferably, the density of the ST suspension-acclimated cells at the time of inoculation is from 1.0 × 10⁶ cells / ml to 5.0 × 10⁶ cells / ml.
[0012] A viral antigen composition comprising porcine rotavirus antigen and porcine epidemic diarrhea virus antigen prepared by any one of the methods described herein.
[0013] A swine viral diarrhea vaccine comprising the viral antigen composition and a pharmaceutically acceptable adjuvant and carrier.
[0014] Preferably, the swine viral diarrhea vaccine is an inactivated vaccine or a live attenuated vaccine, preferably an inactivated vaccine, the safety of which has been confirmed through animal experiments.
[0015] An application for preparing porcine rotavirus and porcine epidemic diarrhea virus antigens, comprising any one of the methods or viral antigen compositions for preparing porcine rotavirus and porcine epidemic diarrhea virus antigens.
[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Successfully solved the cell adaptability problem. This invention confirms that ST suspension-acclimated cells can serve as a stable cell matrix for the simultaneous and efficient proliferation of PoRV and PEDV. It enables the stable production of viral antigens in the ST suspension cell co-culture system that are indistinguishable from the gold standard (single-cultured antigens) in terms of immunogenicity (neutralization effect) and safety (pathogenicity), eliminating concerns about the uncertainty of product quality introduced by the co-culture process.
[0017] 2. The safety and efficacy of the vaccine are ensured. Since the co-culture antigen and the separately cultured antigen are equivalent in key quality attributes, the combined vaccine developed based on the antigen of this invention has predictable and guaranteed safety and immunization effect. This is the fundamental premise for the application of this technology to marketed vaccine products.
[0018] 3. While ensuring quality, the method of this invention achieves process innovation. It not only ensures the highest requirement of no difference in antigen quality, but also achieves the excellent effect of no reduction in viral titer. It also simplifies the process of producing two antigens in one batch, which greatly reduces production costs. Compared with separate culture, this method increases production efficiency by 100% (producing two antigens simultaneously in the same reactor), reduces culture medium consumption by 40%, reduces labor costs by 35%, and reduces the production cost of a single batch of vaccine by about 50 yuan per 10,000 doses.
[0019] Appendix Explanation Table 1 shows the comparison of virus titers under different culture methods of the present invention (n=3 replicate experiments, mean ± standard deviation SD). Table 2 shows the comparison of serum neutralizing antibody titers after immunizing pigs with antigens from different sources of the present invention (cultured alone vs. co-cultured) (GMT, n=8 piglets, mean ± SD). Table 3 shows the comparison of pathogenicity scores and qPCR CT values of viruses from different sources in this invention to piglets (n=5 susceptible piglets, mean ± SD). Detailed Implementation
[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying tables. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0021] Please refer to Tables 1 to 3. This invention provides a method for preparing porcine rotavirus and porcine epidemic diarrhea virus antigens and their applications. The technical solution is as follows: Example 1: Culture and maintenance of ST suspension-domesticated cells The ST suspension-acclimated cell line used in this invention was obtained through a conventional serum-free adaptation method in the art. The cells were acclimated at 37°C and 5%... Under 120 rpm conditions, suspension culture and passage were performed in commercial serum-free medium to maintain cell density at 1.0-5.0×106 cells / ml and cell viability at over 90%.
[0022] Example 2: Preparation and titer determination of virus seed strain PEDV HeNZ strain and PoRV HeNP strain seed virus were prepared by ST suspension cell amplification. After virus harvesting, the following methods were used: The method for determining viral titers in 96-well plates ( / ml).
[0023] Example 3: Simultaneous Inoculation Method ST suspension cells in logarithmic growth phase with a density of 3.0 × 10⁶ cells / ml were simultaneously inoculated with PEDV (MOI = 0.03) and PoRV (MOI = 0.1). The cells were then incubated at 37℃ with 5% [resource concentration missing]. Continue suspension culture in a shaker at 120 rpm for 24-48 hours, during which cytopathic effect (CPE) is monitored. When the CPE reaches 85% or higher, the virus solution is harvested and the titers of PEDV and PoRV are determined.
[0024] Example 4: Sequential inoculation method ST suspension cells in logarithmic growth phase with a density of 3.0 × 10⁶ cells / ml were first seeded with PoRV (MOI = 0.1). The cells were then incubated at 37℃ with 5% [presumably a specific concentration of cells]. After culturing for 12 hours in a shaker at 120 rpm, PEDV (MOI=0.03) was inoculated into the same culture system. The virus solution was harvested after culturing for a total of 24-48 hours.
[0025] Example 5: Viral titer determination use The potency of the virus fluids harvested in Examples 3 and 4, as well as the control virus fluids cultured alone, was determined using the method of the present invention. A t-test was performed using GraphPad Prism 9, with P>0.05 indicating no significant difference. The results are shown in Table 1. The potency of PoRV and PEDV obtained by the method of the present invention was no lower than that of their respective control cultures cultured alone.
[0026] Example 6: Comparison of Neutralizing Antibody Titer The co-cultured viral antigens (experimental group) and separately cultured viral antigens (control group) prepared in Examples 3 and 4 of this invention were inactivated with β-propiolactone and then mixed with Freund's adjuvant at a volume ratio of 1:1 to prepare a vaccine. Healthy 3-week-old piglets (8 pigs per group) were immunized in groups, with each piglet receiving 2 ml intramuscularly. A booster immunization was given 14 days later. Serum was collected 14 days after the second immunization.
[0027] The titers of neutralizing antibodies against PoRV and PEDV in serum were determined using a standard virus neutralization assay. The results are shown in Table 2. Statistical analysis (t-test) showed that there was no significant difference in the geometric mean titer (GMT) of neutralizing antibodies against PoRV and PEDV between pigs immunized in the experimental group and those immunized in the control group (p>0.05).
[0028] Example 7: Pathogenicity Comparison Test The co-cultured live virus (experimental group) prepared in Examples 3 and 4 of this invention was compared with the live virus cultured alone (control group) at a concentration of 10⁶. The doses were administered orally to 3-day-old susceptible piglets of PEDV and PoRV (5 piglets per group), with an additional blank control group (injected with culture medium).
[0029] Clinical symptoms of piglets, including mental status, appetite, and diarrhea, were observed for 7 consecutive days, and diarrhea was scored (0 points: normal; 1 point: mild diarrhea; 2 points: moderate diarrhea; 3 points: severe watery diarrhea). Fecal samples were collected on days 3 and 5 post-inoculation, and viral shedding was detected by real-time quantitative PCR. A t-test was performed using GraphPad Prism 9, with P>0.05 considered as no significant difference. The results are shown in Table 3. There were no significant differences between the experimental group and the control group in terms of diarrhea incidence, diarrhea index, and viral shedding titer (p>0.05). The blank group did not experience diarrhea, and the viral shedding titer was below the detection limit, demonstrating that the co-cultured virus did not undergo pathogenic changes. The blank group showed no clinical symptoms, excluding interference from non-specific factors.
[0030] Comparative Example 1: Cultured alone Under the same conditions, PEDV (MOI=0.03) and PoRV (MOI=0.1) were cultured separately in ST suspension cells as a benchmark for comparing titer and quality attributes.
[0031] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
[0032]
[0033] Table 1
[0034] Table 2
[0035] Table 3.
Claims
1. A method for preparing porcine rotavirus and porcine epidemic diarrhea virus antigens, characterized in that, Includes the following steps: a) Simultaneous or sequential inoculation of porcine rotavirus and porcine epidemic diarrhea virus into ST suspension-acclimated cells; b) Culture to propagate the virus; c) Harvest the viral fluid; Among them, the viral titers of the obtained porcine epidemic diarrhea virus antigen and porcine epidemic diarrhea virus antigen were not lower than those of their respective separately cultured antigens, and the induced neutralizing antibody titers were not significantly different from those of the separately cultured corresponding antigens, and there was no significant difference in pathogenicity to the sensitive models.
2. The method for preparing porcine rotavirus and porcine epidemic diarrhea virus antigens according to claim 1, characterized in that: The sequential vaccination involves first inoculating with porcine rotavirus, followed by inoculation with porcine epidemic diarrhea virus 12 to 72 hours later.
3. The method for preparing porcine rotavirus and porcine epidemic diarrhea virus antigens according to claim 2, characterized in that: The interval is 12 to 24 hours.
4. The method for preparing porcine rotavirus and porcine epidemic diarrhea virus antigens according to claim 3, characterized in that: The multiplicity of infection (MLI) when inoculated with porcine epidemic diarrhea virus is 0.01 to 0.
1.
5. The method for preparing porcine rotavirus and porcine epidemic diarrhea virus antigens according to claim 4, characterized in that: The multiplicity of infection (MNI) when swine rotavirus is inoculated is between 0.01 and 1.
0.
6. The method for preparing porcine rotavirus and porcine epidemic diarrhea virus antigens according to claim 5, characterized in that: The density of the ST suspension-acclimated cells at the time of inoculation was 1.0 × 10⁶ cells / ml to 5.0 × 10⁶ cells / ml.
7. A viral antigen composition, characterized in that: This includes porcine rotavirus antigen and porcine epidemic diarrhea virus antigen prepared by the method described in any one of claims 1-6.
8. A vaccine for swine viral diarrhea, characterized in that: It includes the viral antigen composition of claim 7, as well as pharmaceutically acceptable adjuvants and carriers.
9. A swine viral diarrhea vaccine according to claim 8, characterized in that: The swine viral diarrhea vaccine is an inactivated vaccine or a live attenuated vaccine.
10. An application of preparing porcine rotavirus and porcine epidemic diarrhea virus antigens, characterized in that: The method for preparing porcine rotavirus and porcine epidemic diarrhea virus antigens according to any one of claims 1-6 or the viral antigen composition according to claim 7.