African swine fever virus inactivated vaccine for mucosal immunity and preparation method and application thereof
By extracting and inactivateing the treated vaccine from the tissues of the onset pigs and immunizing mucosally, the problem that existing vaccines cannot effectively resist ASFV is solved, and good preventive effects and preparation safety are achieved.
Patent Information
- Application Number
- CN202411880618.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-19
- Publication Date
- 2025-03-18
AI Technical Summary
The existing inactivated African swine fever virus vaccine cannot effectively resist the attack of ASFV, and the specific antibodies produced after injection do not have neutralization effects and have poor protection effect.
The virus that has been inactivated and treated with inactivated is used to ensure the integrity of the capsule protein and capsid protein by extracting the African swine fever virus from the tissues of the onset of pigs and inactivated as a vaccine, and the integrity of the capsule protein and capsid protein is ensured, while the virus DNA is broken by inactivated agents to avoid replication and spread.
The vaccine can produce SIgA in the mucosal area, prevent further invasion of viral particles, achieve good results in preventing African swine fever, and ensure the safety of the preparation.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of vaccines, and in particular to an inactivated African swine fever virus vaccine for mucosal immunization, a preparation method of the inactivated African swine fever virus vaccine, and an application of the prepared inactivated vaccine. Background Art
[0002] African swine fever (ASF) is an acute, febrile, highly contagious animal disease of pigs caused by African swine fever virus (ASFV). It is an animal epidemic disease.
[0003] ASF can be transmitted through the respiratory tract, digestive tract, skin and other routes, with an incubation period of 48 to 72 hours. The disease often presents an outbreak, with extremely high morbidity and mortality rates. The morbidity rate for susceptible pigs is 100%, and the mortality rate can reach 100%. It is one of the most serious diseases that endanger the pig industry.
[0004] After ASFV was introduced into my country in 2018, it continued to spread throughout the country, causing great losses. ASFV is an enveloped double-stranded DNA virus that can cause systemic hemorrhagic disease in pigs. ASFV is the only member of the genus ASFV in the family ASFVidae and is currently the only insect-borne DNA virus. The ASFV virus particle is an icosahedral multi-layer structure with a diameter of about 250nm. From the outside to the inside, it consists of the outer viral envelope, capsid, inner membrane, inner core shell and viral genome.
[0005] ASF is a huge threat to the international pig farming industry and a devastating blow to some pig farming companies. Currently, there is no effective ASF vaccine or effective therapeutic drug developed internationally. It is urgent to develop a safe and effective ASF vaccine.
[0006] Currently, the main vaccines for African swine fever include inactivated vaccines, attenuated vaccines, subunit vaccines, and DNA and live vector vaccines. Among them, inactivated vaccines are the most classic method of vaccine development. However, although the inactivated African swine fever vaccine can partially stimulate pigs to produce antibodies after injection, even with the help of the latest adjuvants, it still cannot resist the attack of ASFV. The reason is that this antibody is a specific antibody and can only play an identification role, not a neutralizing antibody. It cannot neutralize the virus, so the protection effect is poor.
[0007] Attenuated vaccines are naturally or artificially weakened vaccines that have been tested in fields in Portugal and Spain, but with disastrous consequences. Many pigs developed pneumonia, movement disorders, skin ulcers, abortions and deaths after immunization, and most immunized animals developed chronic infections with a final mortality rate of 10%-50%.
[0008] The ASF attenuated vaccine strain prepared by gene knockout has a more stable virulence and a lower chance of reversion to virulence, but it has not been recognized by relevant application units due to its residual virulence.
[0009] Subunit vaccines are vaccines prepared by using recombinant baculovirus to express p30, p54 and p72 proteins. They cannot provide immune protection after immunization, but can only delay the onset of clinical symptoms and reduce the level of viremia. These experiments show that in the face of the numerous antigenic structural proteins and complex immune stimulation process of ASFV, it is difficult to achieve immune prevention effects by relying solely on one or a few proteins.
[0010] DNA and live vector vaccines are the development direction of the new generation of vaccines, and the research on DNA vaccines for ASFV has also begun. JM Argilaguet et al. linked P30 and P54 into the eukaryotic expression plasmid pCMV-PQ. As a result, the DNA vaccine could not resist the attack of strong strains after immunizing pigs. Subsequently, the laboratory further linked the ASFV hemagglutinin protein gene and ubiquitin gene into the above DNA vaccine, but the result still failed to achieve the protective effect; recently, a variety of antigens expressed by adenovirus as a live vector were used to immunize pigs in a "cocktail" manner, but they could not provide protection.
[0011] Although specific antibodies can be produced after infection with African swine fever, there is no effective vaccine for African swine fever virus so far, as there are many genotypes of African swine fever virus and strains of different genotypes cannot produce immune protection. Currently, the prevention and control of the disease mainly relies on strict animal biosafety measures such as immediate slaughter, isolation, and blockade.
[0012] As an ideal immunogen, virus particles can induce highly specific humoral or cellular immune responses after injection, playing a preventive or therapeutic role at the humoral and cellular immunity levels, thus providing a wider range of protection. However, African swine fever virus particles cannot produce satisfactory cellular or humoral immunity. Although inactivated vaccines produce specific antibodies after injection, they do not produce neutralizing antibodies. Therefore, they can only be used as a basis for identifying a certain genotype of virus and cannot be used as a vaccine to protect susceptible animals.
[0013] There is no effective drug for the prevention and treatment of African swine fever. At present, my country mainly relies on biosafety measures to control the disease. Although the development of vaccines has a history of nearly a hundred years, due to some reasons, no safe and satisfactory vaccine has been successfully developed internationally, and the current situation of "unsatisfactory safety or satisfactory effect but unsafe" has emerged. Every year, many pigs in my country suffer from African swine fever, causing huge economic losses to pig farms. The development of a safe and effective African swine fever vaccine has become the key to preventing and controlling the spread of the disease.
[0014] Natural infection with African swine fever virus particles is mainly transmitted through mucosal tissues such as the respiratory tract and digestive tract. Although injection of inactivated vaccines cannot produce sufficient neutralizing antibodies to protect susceptible animals, mucosal contact with inactivated virus particles can produce SIgA, which prevents further invasion of virus particles at the mucosal site, thereby achieving a preventive effect. Summary of the invention
[0015] In view of this, one of the objects of the present invention is to provide an inactivated African swine fever virus vaccine for mucosal immunization; a second object of the present invention is to provide a method for preparing the inactivated African swine fever virus vaccine for mucosal immunization; a third object of the present invention is to provide the use of the inactivated African swine fever virus vaccine for mucosal immunization as a vaccine in the prevention of African swine fever.
[0016] In order to achieve the above object, the present invention provides the following technical solutions:
[0017] 1. An inactivated African swine fever virus vaccine for mucosal immunization, wherein the inactivated vaccine is prepared by inactivating the African swine fever virus.
[0018] Preferably, the African swine fever virus is extracted from tissues of pigs infected with African swine fever.
[0019] Preferably, the tissue is liver tissue, lymphatic tissue, blood or spleen tissue.
[0020] Preferably, the inactivation is carried out by adding a deactivation solution containing 0.1% formaldehyde or β-propiolactone by volume, or 1-2% diethyleneimine by volume.
[0021] Preferably, the vaccine is administered via mucosal immunization, which is nasal spray or oral administration.
[0022] 2. The method for preparing the inactivated African swine fever virus vaccine for mucosal immunization comprises the following steps:
[0023] (1) extracting African swine fever virus from tissues of African swine fever-infected pigs as an antigen;
[0024] (2) adding an inactivation solution to inactivate the antigen extracted in step (1) to obtain an inactivated virus solution; the inactivation solution is a 0.1% by volume formaldehyde or β-propiolactone, or a 1-2% by volume diethyleneimine;
[0025] (3) Add thimerosal to the inactivated virus solution of step (2) until the final thimerosal concentration reaches 0.01% by volume, mix well, and package quantitatively.
[0026] Preferably, in step (1), the tissue is liver tissue, lymphatic tissue, blood or spleen tissue, and the extraction is performed by removing fat and connective tissue from the tissue, chopping and mashing the tissue, collecting the supernatant by centrifugation, and diluting the supernatant by 1-10 times with PBS solution having a pH of 7.2.
[0027] Preferably, in step (2) of the present invention, the inactivation is performed by mixing the antigen with the inactivation solution, shaking well, placing the mixture in a refrigerator at 4°C or 37°C for 48-96 hours, and shaking the mixture every 12 hours to fully inactivate the antigen and prepare an inactivated vaccine.
[0028] 2. The use of the inactivated African swine fever virus vaccine for mucosal immunization as a vaccine in preventing African swine fever.
[0029] The beneficial effects of the present invention are as follows: the present invention provides an inactivated African swine fever virus vaccine for mucosal immunization, which uses the pathogen as a comprehensive antigen component, and the envelope protein and capsid protein are not damaged and maintain complete immunogenicity, but the viral DNA is broken by the inactivator and cannot replicate (no re-strengthening, no spread), thereby ensuring the safety of the preparation; comprehensive and multi-faceted induction of the vaccinated animal population can achieve a good effect in preventing African swine fever. DETAILED DESCRIPTION
[0030] The present invention is further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.
[0031] Example 1. Acquisition of African swine fever virus antigen
[0032] (1) For dead pigs with typical African swine fever symptoms, aseptically remove the liver, spleen, lymph, blood and any other tissues containing African swine fever virus, chop them into pieces, add sterile saline at a ratio of 1:5, then add 100-1000U (μg) / ml of penicillin and streptomycin, place at 4°C overnight, and freeze-thaw repeatedly for 3 times;
[0033] (2) Obtaining virus solution: grind the virus obtained in step (1) and centrifuge at 1000-3000 rpm / min for 5-30 minutes. Take the supernatant and perform sterility test and real-time PCR detection. Dilute with PBS solution with pH 7.2 under sterile conditions for later use;
[0034] Example 2: Sterility test and real-time PCR test of viral load
[0035] (1) Bacteriological examination
[0036] The above supernatant was used to inoculate 2 tubes of fresh blood agar slant culture medium, with 0.2 ml in each tube. The result was negative, proving that there was no bacterial infection.
[0037] (2) African swine fever virus antigen detection (real-time fluorescence quantitative real-time PCR method)
[0038] In Example 1, 1 mL of the prepared sample was inactivated by heating and then tested with a commercially available African swine fever virus antigen detection (real-time fluorescence quantitative real-time PCR method) kit ("African swine fever virus antigen detection kit"). The test result showed a Ct value of ≤35, indicating that the viral load was high.
[0039] Example 3, Formaldehyde Inactivation
[0040] 40% formaldehyde (to a concentration of 0.01-0.02%) was added to the qualified subpackaged suspension of the diseased material obtained in Example 1 and tested in Example 2, and inactivated by shaking at 37°C for 24 hours, and then formaldehyde (to a final concentration of 0.05-0.1%) was added, inactivated by shaking at 37°C for 120 hours, and stored at 4°C.
[0041] Example 4, β-propiolactone inactivation
[0042] 0.1% β-propiolactone was added by volume to the qualified suspension of the subpackaged diseased material obtained in Example 1 and tested in Example 2, and the suspension was inactivated at 4°C for 24 hours. Then, 0.1% β-propiolactone was added and inactivated for 120 hours. The suspension was then placed in a water bath at 37°C for 2 hours and stored at 4°C.
[0043] Example 5, BEI inactivation
[0044] 0.5% BEI was added to the qualified subpackaged suspension of the diseased material obtained in Example 1 and tested in Example 2, and the suspension was inactivated by shaking at 37°C for 24 hours. BEI was then added (to a final concentration of 1-2%), and the suspension was inactivated by shaking at 37°C for 120 hours, and stored at 4°C.
[0045] Example 6: Safety Testing
[0046] (1) Test materials: African swine fever virus inactivated vaccine, batch numbers 202001, 202002, and 202003.
[0047] (2) Experimental animals: 18-22 g Balb / C mice, 20-30 day old healthy piglets (negative for ASFV, PRRSV, CSFV, and PCV2 antibodies detected by ELISA, and negative for ASFV, PRRSV, CSFV, and PCV2 antigens detected by PCR).
[0048] (3) Safety of the vaccine in mice: 18-22 g Balb / C mice were used. Five mice were immunized in each batch of vaccine. A total of 15 mice were immunized in three batches. The immunization method was: 0.5 ml was injected subcutaneously each time. Five blank controls were set up at the same time. The health status of the mice was observed for 20 consecutive days. The results are shown in Table 1.
[0049] The results are shown in Table 1. After immunization, the appetite, mental state and health status of all mice were normal, consistent with the blank control group, and no deaths occurred. It can be seen that the inactivated African swine fever vaccine is safe for mice, see Table 1.
[0050] Table 1. Results of vaccine safety test on mice
[0051]
[0052]
[0053] (4) Safety of the vaccine for piglets
[0054] Select healthy piglets aged 20 to 30 days, and immunize 15 piglets with inactivated African swine fever virus vaccine in three batches of 5 pigs per batch. The immunization method is: inject 2 mL of vaccine into the muscle behind the ear of each pig, and set up 5 blank controls at the same time, and observe clinically for 40 days.
[0055] The results are shown in Table 2. During the entire 40-day observation period, all immunized piglets had normal body temperature, spirit and appetite, and no clinical abnormalities occurred. After the end of the experiment, all 15 piglets were alive and well. The 5 piglets in the blank control group also had no adverse reactions. This shows that the inactivated African swine fever virus vaccine is safe for piglets.
[0056] Table 2. Results of vaccine safety test on piglets
[0057] Group Number of animals body temperature appetite Spirit Abnormal conditions Number of deaths 202001 5 heads normal normal normal none 0 202003 5 heads normal normal normal none 0 202003 5 heads normal normal normal none 0 Control group 5 heads normal normal normal none 0
[0058] Example 7: Effectiveness test
[0059] (1) Experimental animals: 7±1 week old healthy piglets with negative antigen and antibody (negative for ASFV, PRRSV, CSFV, and PCV2 antibodies detected by ELISA, and negative for ASFV, PRRSV, CSFV, and PCV2 antigens detected by PCR).
[0060] (2) Vaccine: African swine fever inactivated vaccine, batch numbers 202001, 202002, and 202003.
[0061] (3) Vaccine efficacy test:
[0062] Grouping and immunization: 20 healthy piglets were randomly divided into 4 groups, 3 vaccine immunization groups and PBS control group, 5 piglets / group. Mucosal immunization 1mL / pig, 7 and 14 days after immunization, the same immunization method was used for booster immunization.
[0063] After immunization, the pigs were kept in the diseased pens to prevent cross-infection with the sick pigs and observed for 30 days.
[0064] In experimental group 1, the vaccine was sprayed into the nose of 5 susceptible pigs at a rate of 1 mL per pig, and the healthy survival of the vaccinated pigs was observed for 30 days.
[0065] In Experimental 2, the vaccine was orally administered to 5 susceptible pigs at a rate of 1 mL per pig, and the healthy survival of the vaccinated pigs was observed for 30 days.
[0066] In experimental group 3, the vaccine was administered to 5 susceptible pigs in drinking water at a rate of 1 mL / pig, and the healthy survival of the vaccinated pigs was observed for 30 days.
[0067] In the control group, PBS was sprayed into the noses of 5 susceptible pigs at a rate of 1 mL per pig. All the inoculated pigs died within 30 days of observation.
[0068] This shows that the inactivated African swine fever virus vaccine is effective for piglets.
[0069] Example 8, method of use
[0070] Grouping and immunization: 30 healthy piglets were randomly divided into 6 groups, 5 vaccine immunization groups and PBS control group, 5 piglets / group. Mucosal immunization 1ml / pig. Once, 7 and 14 days after immunization, the same immunization method was used for booster immunization.
[0071] After immunization, the pigs were kept in the diseased pens to prevent cross-infection with the sick pigs and observed for 30 days.
[0072] In the experimental group 1, five susceptible pigs were sprayed with the vaccine nasally and the healthy survival of the vaccinated pigs was observed for 30 days.
[0073] In Experimental 2, five susceptible pigs were orally vaccinated and their healthy survival was observed for 30 days.
[0074] Five vaccine-drinking susceptible pigs were selected from Experimental 3 group and the healthy survival of the vaccinated pigs was observed for 30 days.
[0075] Five susceptible pigs were selected from the experimental group 4 and all of them died within 30 days of observation.
[0076] In the experimental group 5, the vaccine was injected intramuscularly into 5 susceptible pigs, and all the vaccinated pigs died within 30 days of observation.
[0077] In the control group, five susceptible pigs were sprayed nasally with PBS, and all the inoculated pigs died within 30 days of observation.
[0078] This shows that mucosal immunization of inactivated African swine fever virus vaccine can achieve 100% protection for piglets, but all pigs immunized by mixing with feed or intramuscular injection died during the observation period, indicating that inactivated African swine fever virus vaccine is not suitable for mixing with feed or injection for immunization.
[0079] Example 9: SIgA Detection
[0080] Fecal SIgA antibody levels in piglets
[0081] Determination method: Use the commercially available "Porcine Secretory Immunoglobulin A (SIGA) ELISA Kit" to detect the SIgA antibody level in the sample.
[0082] Specimen collection: All sampled pigs were collected using fecal bags without touching the ground. The specimens were frozen in a -20℃ refrigerator overnight, thawed and crushed, and fully mixed for measurement.
[0083] Detection: According to the instructions of the kit, the OD value at 450nm was measured with an enzyme-linked immunosorbent assay (ELISA) and a control well was set up. The detection results are shown in Table 3.
[0084] Table 3. SIgA antibody levels in piglet feces
[0085] Product batch number Number of experimental pigs Dosage Interval days Mean OD of SIgA Product protection number 202001 5 1+1+1ml 7 925.22±122.15 5 / 5 202002 5 1+1+1ml 7 958.55±125.26 5 / 5 202003 5 1+1+1ml 7 1021.13±115.33 5 / 5 202004 5 1+1+1ml 7 1105.65±121.51 5 / 5 202005 5 1+1+1ml 7 983.87±105.25 5 / 5 Control group 5 PBS 7 306.25±25.38 0 / 5
[0086] The results showed that the mean OD value of SIgA in the feces of the experimental group was significantly higher than that in the control group.
[0087] Example 10, protection period test
[0088] After immunization with the inactivated African swine fever virus vaccine, the protection rates were tested 1 month, 2 months, 3 months and 4 months later. The results are shown in Table 4.
[0089] Table 4. Results of protection period of inactivated African swine fever virus vaccine
[0090]
[0091] The experimental results show that the protection rate of the vaccine reaches 100% within 3 months after mucosal immunization, but drops to 20% in the fourth month. Therefore, it is recommended to revaccinate the vaccine 3 months after use.
[0092] Example 11, 4°C stability test
[0093] The inactivated African swine fever virus vaccine was stored at 4°C, and the protective effect was tested at 3 months, 6 months, 9 months and 12 months. The results are shown in Table 5.
[0094] Table 5. Stability results of inactivated African swine fever virus vaccine
[0095]
[0096] Experimental results show that the vaccine protection rate is 60% when the storage period reaches 9 months, and 20% when the storage period reaches 12 months. Therefore, the vaccine is valid for 6 months when stored at 4°C.
[0097] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.
Claims
1. An inactivated African swine fever virus vaccine for mucosal immunization, characterized in that: The inactivated vaccine is prepared by inactivating the African swine fever virus.
2. The inactivated African swine fever virus vaccine for mucosal immunization according to claim 1, characterized in that: The African swine fever virus is extracted from tissues of pigs infected with African swine fever.
3. The inactivated African swine fever virus vaccine for mucosal immunization according to claim 1, characterized in that: The tissue is liver tissue, lymphatic tissue, blood or spleen tissue.
4. The inactivated African swine fever virus vaccine for mucosal immunization according to claim 1, characterized in that: The inactivation is carried out by adding a deactivation liquid with a volume fraction of 0.1% formaldehyde or beta-propiolactone, or a volume fraction of 1-2% diethyleneimine.
5. The inactivated African swine fever virus vaccine for mucosal immunization according to claim 1, characterized in that: The vaccine is administered via mucosal immunization, which can be nasal spray or oral administration.
6. The method for preparing the inactivated African swine fever virus vaccine for mucosal immunization according to any one of claims 1 to 5, characterized in that: The steps include: (1) extracting African swine fever virus from tissues of African swine fever-infected pigs as an antigen; (2) adding an inactivation solution to inactivate the antigen extracted in step (1) to obtain an inactivated virus solution; the inactivation solution is a 0.1% by volume formaldehyde or β-propiolactone, or a 1-2% by volume diethyleneimine; (3) Add thimerosal to the inactivated virus solution of step (2) until the final thimerosal concentration reaches 0.01% by volume, mix well, and package quantitatively.
7. The preparation method according to claim 6, characterized in that: In step (1), the tissue is liver tissue, lymphatic tissue, blood or spleen tissue, and the extraction is performed by removing fat and connective tissue from the tissue, chopping and mashing the tissue, collecting the supernatant by centrifugation, and diluting the supernatant by 1-10 times with PBS solution having a pH of 7.
2.
8. The preparation method according to claim 6, characterized in that: In step (2), the inactivation is performed by mixing the antigen with the inactivation solution, shaking it thoroughly, placing it in a refrigerator at 4°C or 37°C for 48-96 hours, and shaking it once every 12 hours to fully inactivate it, thereby preparing an inactivated vaccine.
9. Use of the inactivated African swine fever virus vaccine for mucosal immunization according to any one of claims 1 to 5 as a vaccine for preventing African swine fever.