CN119633106A

CN119633106A - African swine fever virus inactivated vaccine for mucosal immunization and preparation method and application thereof

Info

Publication number: CN119633106A
Application number: CN202411880618.1A
Authority: CN
Inventors: 杨金龙
Original assignee: Individual
Current assignee: Individual
Priority date: 2024-12-19
Filing date: 2024-12-19
Publication date: 2025-03-18

Abstract

本发明公开了用于黏膜免疫的非洲猪瘟病毒灭活疫苗及其制备方法和应用，该疫苗采用病原体为全面的抗原成份，囊膜蛋白和衣壳蛋白不受破坏而保持完整的免疫原性，但病毒DNA受灭活剂作用发生断裂而不能复制(不返强、不扩散)，确保制剂的安全性；对接种动物群体进行全面的、多方面的诱导，能达到预防非洲猪瘟的良好效果。The present invention discloses an inactivated African swine fever virus vaccine for mucosal immunization, and a preparation method and application thereof. The vaccine uses the pathogen as a comprehensive antigen component, and the envelope protein and the capsid protein are not damaged and maintain complete immunogenicity, but the viral DNA is broken by the action of the inactivator and cannot be replicated (no re-strengthening, no spread), thereby ensuring the safety of the preparation; comprehensive and multi-faceted induction is performed on the inoculated animal group, which can achieve a good effect of preventing African swine fever.

Description

African swine fever virus inactivated vaccine for mucosal immunization and preparation method and application thereof

Technical Field

The invention relates to the field of vaccines, in particular to an African swine fever virus inactivated vaccine for mucosal immunization, a preparation method of the African swine fever virus inactivated vaccine and application of the African swine fever virus inactivated vaccine.

Background

African swine fever (AFRICA SWINE FEVER, ASF) is an acute, febrile, highly contagious animal infectious disease of swine caused by African swine fever Virus (AFRICA SWINE FEVER Virus, ASFV), belonging to animal epidemic disease.

ASF can be transmitted through various ways such as respiratory tract, alimentary canal, skin, etc., and the incubation period is 48-72 hours. The disease is usually epidemic, has extremely high morbidity and mortality, has 100 percent of pathogenicity rate to susceptible pigs and 100 percent of mortality, and is one of the most serious diseases endangering the pig industry.

ASFV continuously spreads nationwide after being transferred into China in 2018, and causes great loss. ASFV is a enveloped double-stranded DNA virus that causes systemic hemorrhagic disease in pigs. ASFV is the only member of the African swine fever virus genus of the African swine fever virus family and is currently the only arbovirus DNA. ASFV virions are icosahedral multilayer structures with a diameter of about 250nm, and are, in order from outside to inside, viral outer envelope, capsid, inner membrane, inner core shell and viral genome.

ASF is a huge hazard to the international pig farming industry and a devastating impact to some farming enterprises, and no effective non-pestilence vaccine and no effective therapeutic medicine are yet developed internationally. Development of safe and effective non-pestilence vaccines has been hampered.

The existing african swine fever main vaccine comprises an inactivated vaccine, a low virulent vaccine, a subunit vaccine, DNA and a live carrier vaccine, wherein the inactivated vaccine is used as the most classical vaccine development mode, but after the injection of the african swine fever inactivated vaccine, part of the inactivated vaccine can stimulate pigs to generate antibodies, but even though the live pigs can not resist the attack of ASFV by means of the latest adjuvant, the reason is that the antibodies are specific antibodies and can only play a role in identification, but not neutralizing antibodies, and the effect of neutralizing viruses can not be achieved, so the protection effect is poor.

Attenuated vaccines are natural or artificially attenuated vaccines that have been tested in the field in portugal and spanish but have had disastrous consequences. Many pigs have immunological side effects such as pneumonia, dyskinesia, skin ulcer, abortion, death and the like after immunization, most immunized animals have chronic infection and the final death rate reaches 10% -50%.

The virulence of the ASF attenuated vaccine strain prepared by the gene knockout mode is more stable, the probability of virulence return is smaller, but the residual virulence cannot be affirmed by related application units.

Subunit vaccines are vaccines prepared by expressing p30, p54 and p72 proteins using recombinant baculoviruses, and cannot provide immune protection after immunization, but only delay the occurrence of clinical symptoms and reduce the level of viremia. These experiments demonstrate that it is difficult to achieve immunoprophylaxis against a number of antigen structural proteins and complex immunostimulatory processes of ASFV, relying solely on one or more proteins.

DNA and live vector vaccines have also begun to be studied as a new generation of vaccine development. P30 and P54 were ligated into eukaryotic expression plasmid pCMV-PQ by M.Argilaguet et al, and as a result, the DNA vaccine was unable to resist against virulent strain challenge after immunization of pigs. Subsequently, the laboratory further links ASFV hemagglutinin protein gene and ubiquitin gene into the DNA vaccine, and as a result, the protection effect can not be achieved, and the protection can not be provided after the recent 'cocktail' immunization of live pigs by various antigens expressed by adenovirus as a live vector.

Although specific antibodies can be produced after infection of African swine fever, no effective vaccine for African swine fever virus exists so far because African swine fever virus has numerous genotypes, and strains among different genotypes can not produce immune protection. At present, the prevention and control of the disease are mainly realized by strict animal biosafety measures such as on-the-spot slaughtering, isolation, blocking and the like.

The virus particles are used as ideal immunogens, can induce highly specific humoral or cellular immune response after injection, and have preventive or therapeutic effects on humoral and cellular immune levels, thus being capable of giving wider protectiveness. However, african swine fever virus particles cannot generate satisfactory cellular immunity or humoral immunity, and inactivated vaccines generate specific antibodies but do not generate neutralizing antibodies after injection, so that the virus particles can only be used as a basis for identifying a certain genotype virus and cannot be used as a vaccine for protecting susceptible animals.

African swine fever has no effective preventive and therapeutic drug so far, and the control of the African swine fever in China is mainly limited to biological safety measures. Although vaccine development has been carried out for nearly a hundred years, for some reasons, no safe and satisfactory vaccine development has been achieved internationally, and the current situation of unsatisfactory safe effect or satisfactory effect but unsafe appears, many pig groups in China generate African swine fever each year, huge economic losses are caused for pig farms, and the development of safe and effective African swine fever vaccine becomes a key for preventing and controlling epidemic diseases.

Although natural infection of African swine fever virus particles is mainly transmitted through mucosal tissues such as respiratory tract, digestive tract and the like, and injection of inactivated vaccine cannot generate enough neutralizing antibodies to protect susceptible animals, SIgA can be generated by the virus particles inactivated by mucosal contact, and further invasion of the virus particles is prevented at mucosal parts, so that a prevention effect is achieved.

Disclosure of Invention

In view of the above, one of the purposes of the invention is to provide an African swine fever virus inactivated vaccine for mucosal immunization, the other purpose of the invention is to provide a preparation method of the African swine fever virus inactivated vaccine for mucosal immunization, and the third purpose of the invention is to provide an application of the African swine fever virus inactivated vaccine for mucosal immunization as a vaccine in preventing African swine fever.

In order to achieve the above purpose, the present invention provides the following technical solutions:

1. the African swine fever virus inactivated vaccine for mucosal immunization is prepared by inactivating African swine fever virus.

Preferably, the African swine fever virus is extracted from tissues of swine suffering from African swine fever.

Preferably, the tissue is liver tissue, lymphoid tissue, blood or spleen tissue.

Preferably, the inactivation is that formaldehyde or beta-propiolactone is added with the volume fraction of 0.1 percent, or an inactivation solution with the volume fraction of 1-2 percent of the polyethyleneimine is added for inactivation.

Preferably, the vaccine is immunized through mucous membrane, and the mucous membrane immunization is nasal spray or oral administration.

2. The preparation method of the African swine fever virus inactivated vaccine for mucosal immunization comprises the following steps:

(1) Extracting African swine fever virus from tissues of an African swine fever pig, wherein the African swine fever virus is used as an antigen;

(2) Adding the antigen extracted in the step (1) into an inactivating liquid to inactivate to obtain an inactivating virus liquid, wherein the inactivating liquid is prepared by adding formaldehyde or beta-propiolactone with the volume fraction of 0.1% or diethyl imine with the volume fraction of 1-2%;

(3) And (3) adding the inactivated virus liquid in the step (2) into the thimerosal until the final concentration volume fraction of the thimerosal reaches 0.01%, uniformly mixing, and quantitatively packaging.

In the preferred embodiment of the present invention, in the step (1), the tissue is liver tissue, lymph tissue, blood or spleen tissue, and the extraction is to remove fat and connective tissue from the tissue, to trim the tissue, to mash the crushed tissue, to centrifugally collect the supernatant, and to dilute the supernatant with PBS solution with pH of 7.2 by 1-10 times.

In the preferred embodiment of the invention, in the step (2), the antigen is mixed with the inactivating liquid, fully and uniformly shaken, and placed in a refrigerator at 4 ℃ or 37 ℃ for 48-96 hours, and shaken once every 12 hours to fully inactivate the antigen, thus preparing the inactivated vaccine.

2. The African swine fever virus inactivated vaccine for mucosal immunization is applied to prevention of African swine fever as a vaccine.

The invention has the beneficial effects that the African swine fever virus inactivated vaccine for mucosal immunization is provided, the vaccine adopts pathogens as comprehensive antigen components, envelope proteins and capsid proteins are not destroyed to keep complete immunogenicity, but viral DNA is broken under the action of an inactivating agent and cannot be replicated (not to be strengthened and spread), so that the safety of the preparation is ensured, and the vaccine can achieve good effect of preventing African swine fever by carrying out comprehensive and multi-aspect induction on a vaccinated animal population.

Detailed Description

The present invention will be further described with reference to specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the present invention and practice it.

EXAMPLE 1 African swine fever Virus antigen acquisition

(1) The method comprises the steps of (1) aseptically taking any tissues containing African swine fever virus such as liver, spleen, lymph, blood and the like from a pig dying of typical African swine fever symptoms, adding sterile normal saline according to a ratio of 1:5 after shearing, adding 100-1000U (mug)/ml of streptomycin, standing for 4 ℃ overnight, and repeatedly freezing and thawing for 3 times;

(2) Grinding the virus liquid obtained in the step (1), centrifuging at 1000-3000rpm/min for 5-30 min, collecting supernatant, performing sterile test and real-time PCR detection, and diluting with PBS solution with pH of 7.2 under aseptic condition;

EXAMPLE 2 sterility testing and viral load real-time PCR detection

(1) Bacteriological examination

The supernatant was inoculated into 2 tubes of fresh blood agar slant medium, each tube was inoculated with 0.2ml, and the result was negative, which confirmed no bacterial infection.

(2) African swine fever virus antigen detection (real-time fluorescence quantitative real-time PCR method)

The sample 1mL prepared in the first embodiment is heated and inactivated and then detected by a commercial African swine fever virus antigen detection (real-time fluorescence quantitative real-time PCR) kit (African swine fever virus antigen detection kit), and the Ct value of the detection result is less than or equal to 35, which indicates that the viral load is higher.

EXAMPLE 3 Formaldehyde inactivation

40% Formaldehyde (the concentration of which is 0.01-0.02%) is added into the sub-packaged disease material suspension which is qualified by the detection of the example 2, the sub-packaged disease material suspension is subjected to shaking inactivation for 24 hours at 37 ℃, formaldehyde (the final concentration of which is 0.05-0.1%) is added, and the sub-packaged disease material suspension is subjected to shaking inactivation for 120 hours at 37 ℃ and stored at 4 ℃.

EXAMPLE 4 beta-propiolactone inactivation

The sub-batch disease material suspension obtained in example 1 and qualified in example 2 is added with 0.1% beta-propiolactone by volume, inactivated for 24 hours at 4 ℃, then added with 0.1% beta-propiolactone, inactivated for 120 hours, and then stored in a 37 ℃ water bath for 2 hours at 4 ℃.

Example 5 BEI inactivation

The sub-batch disease material suspension obtained in example 1 and qualified in example 2 was added with 0.5% BEI, and then subjected to shaking inactivation at 37℃for 24 hours, further added with BEI (to make the final concentration 1-2%), and subjected to shaking inactivation at 37℃for 120 hours, and stored at 4 ℃.

Example 6 safety detection

(1) The detection material is African swine fever virus inactivated vaccine, and the lot numbers are 202001, 202002 and 202003.

(2) Test animals are 18-22 g Balb/C mice, and 20-30 day old healthy piglets (ELISA method for detecting ASFV, PRRSV, CSFV, PCV antibody negative and PCR method for detecting ASFV, PRRSV, CSFV, PCV antigen negative).

(3) Safety of vaccine to mice 18-22 g Balb/C mice, immunization of 5 vaccine batches for three batches, immunization of 15 mice, wherein the immunization method comprises the steps of subcutaneously injecting 0.5ml each, setting 5 blank controls, continuously observing for 20 days, and observing the health condition of the mice, and the results are shown in Table 1.

The results are shown in Table 1, and after immunization, all mice have no abnormality in appetite, spirit and health conditions, consistent with the blank control group, no death occurs, and the African swine fever inactivated vaccine is safe for mice, as shown in Table 1.

TABLE 1 safety test results of vaccine on mice

(4) Safety of vaccine to piglets

And selecting healthy piglets of 20-30 days old, wherein 5 pigs per batch are subjected to African swine fever virus inactivated vaccine, and 15 piglets are immunized in three batches. The immunization method comprises intramuscular injection of 2mL of each pig behind the ear, setting up 5 blank controls, and observing clinically for 40 days.

The results are shown in Table 2, and all immunized piglets are normal in body temperature, spirit and appetite within 40 days of the whole observation period of the test, no clinical abnormality phenomenon occurs, and 15 piglets are healthy and alive after the test is finished. The 5 piglets of the blank control group also have no adverse reaction. This indicates that the african swine fever virus inactivated vaccine is safe for piglets.

TABLE 2 safety test results of vaccine on piglets

Group of

Number of animals

Body temperature

Appetite stimulating

Spirit of the invention

Abnormal condition

Number of deaths

202001

5 Heads

Normal state

Without any means for

0

202003

5 Heads

Normal state

Without any means for

0

202003

5 Heads

Normal state

Without any means for

0

Control group

5 Heads

Normal state

Without any means for

0

Example 7 validity detection

(1) Test animals 7+ -1 week old antigen-antibody negative healthy piglets (ELISA method for detecting ASFV, PRRSV, CSFV, PCV-2 antibody negative, PCR method for detecting ASFV, PRRSV, CSFV, PCV-2 antigen negative).

(2) Vaccine, african swine fever inactivated vaccine, and the lot numbers are 202001, 202002 and 202003.

(3) Vaccine efficacy test:

Grouping and immunization healthy piglets 20 were randomly divided into 4 groups, vaccine immunized 3 groups and PBS control group, 5 heads/group. Mucosal immunization was 1 mL/head, 7 and 14 days after immunization, and the same immunization method was used to boost each immunization.

After immunization, the pigs are bred in the disease colony house and are subjected to accurate cross infection with the disease-developing pigs, and the pigs are observed for 30 days.

Experiment 1 group vaccine 5 pigs susceptible to nasal spray per 1mL were observed for healthy survival of 30 days vaccinated pigs.

Experiment 2 group vaccine 5 susceptible pigs were orally administered at 1 mL/day and vaccinated pigs survived for 30 days.

Experiment 3 group vaccine 5 pigs are taken according to 1 mL/drinking water susceptible pig, and the healthy survival of the vaccinated pigs is observed for 30 days.

The control group was prepared from 5 PBS-containing 1 mL/nasal spray susceptible pigs, and all vaccinated pigs died within 30 days of observation.

This demonstrates that african swine fever virus inactivated vaccine is effective for piglets.

Example 8 method of use

Grouping and immunization 30 healthy piglets were randomly divided into 6 groups, vaccine immunization 5 groups and PBS control group, 5 heads/group. Mucosal immunization was 1 ml/first.2 times, 7, 14 days after immunization, with the same immunization method boosting each time.

The vaccine nasal spray susceptible pigs of the experiment 1 group are taken for 5, and the healthy survival of the vaccinated pigs is observed for 30 days.

Experiment 2 groups 5 pigs susceptible to vaccine oral administration were observed for healthy survival of 30 days vaccinated pigs.

The vaccine drinking susceptible pigs are taken from the experiment 3 group, and the healthy survival of the vaccinated pigs is observed for 30 days.

In experiment 4, 5 vaccine-mixed susceptible pigs are taken, and all the vaccinated pigs die within 30 days after observation

The vaccine intramuscular injection susceptible pigs of the experiment 5 groups are 5, and all the vaccinated pigs die within 30 days.

The control group takes 5 PBS nasal spray susceptible pigs, and all the inoculated pigs die after observation for 30 days.

The results show that the African swine fever virus inactivated vaccine mucosal immunity can achieve 100% of protection effect on piglets, but all the piglets die in the observation period of feed mixing and intramuscular injection immunization, and the African swine fever virus inactivated vaccine is not suitable for feed mixing or injection immunization.

Example 9 SIgA detection

Piglet fecal SIgA antibody levels

Assay methods SIgA antibody levels in samples were measured using a commercially available "pig secretory immunoglobulin A (SIGA) ELISA kit".

Sample collection, namely collecting all sampled pigs by using a fecal bag without falling to the ground, freezing the samples in a refrigerator at the temperature of-20 ℃ overnight, freezing, thawing, mashing and fully mixing the samples during measurement.

The detection is carried out according to the instruction of the kit, the OD value at 450nm is detected by an ELISA detector, and a control hole is arranged, and the detection result is shown in Table 3.

TABLE 3 piglet faeces SIgA antibody level

Product lot number	Number of test pigs	Dosage of use	Days of interval	OD mean value 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

The results show that the OD mean of the piglet manure SIgA of the experimental group is very significantly higher than that of the control group.

Example 10 protective phase test

The protection rates after immunization of african swine fever virus inactivated vaccines were measured after 1 month, 2 months, 3 months, and 4 months, respectively, and the results are shown in table 4.

TABLE 4 African swine fever virus inactivated vaccine protection period results

Experimental results show that the protection rate reaches 100% in 3 months after vaccine mucosa immunization, and the protection rate is reduced to 20% in the fourth month. Therefore, it is recommended that the vaccine be re-vaccinated 3 months after use.

Example 11, 4 ℃ stability test

The african swine fever virus inactivated vaccine was stored at 4 ℃ and the protective effect was tested at 3 months, 6 months, 9 months and 12 months, respectively, and the results are shown in table 5.

TABLE 5 stability results of African swine fever virus inactivated vaccine

Experimental results show that the vaccine protection rate is 60% when the storage period is 9 months, and 20% when the storage period is 12 months, so that the effective period of the vaccine at 4 ℃ is 6 months.

The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.

Claims

1. An inactivated African swine fever virus vaccine for mucosal immunization, characterized in that the inactivated vaccine is obtained 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 the tissues of African swine fever-infected pigs.

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 is characterized in that the inactivation is performed by adding an inactivation solution with a volume fraction of 0.1% formaldehyde or β-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 through mucosal immunization, and the mucosal immunization is 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 it comprises the following steps:

(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 solution containing 0.1% by volume of formaldehyde or β-propiolactone, or 1-2% by volume of 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 is 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 then mashing, collecting the supernatant by centrifugation, and diluting it 1-10 times with PBS solution with a pH of 7.2.

8. The preparation method according to claim 6 is characterized in that: in step (2), the inactivation is to mix the antigen with the inactivation solution, shake it thoroughly, place it in a refrigerator at 4°C or 37°C for 48-96 hours, and shake it once every 12 hours to fully inactivate it to prepare 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.