CN111658768A - Multicomponent subunit vaccine for African swine fever and preparation method and application thereof - Google Patents
Multicomponent subunit vaccine for African swine fever and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
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- A61K39/00—Medicinal preparations containing antigens or antibodies
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Abstract
The invention discloses a multicomponent subunit vaccine of African swine fever, a preparation method and application thereof, belonging to the technical field of animal vaccines and veterinary biological products, wherein the multicomponent subunit vaccine comprises an African swine fever virus surface envelope protein CD2V and at least one protein selected from the protein P72 of the African swine fever virus, the protein P30 of the African swine fever virus, the protein P54 of the African swine fever virus membrane structure, and a pharmaceutically acceptable adjuvant; wherein the African swine fever virus surface envelope protein CD2V is an African swine fever virus CD2V protein without a transmembrane region, or a fusion protein of an African swine fever virus CD2V extracellular region protein and a pig antibody Fc, namely CD 2V-Fc. The multicomponent subunit vaccine has strong immunogenicity and good safety, and the vaccine can remarkably induce humoral immune response.
Description
Technical Field
The invention relates to a multicomponent subunit vaccine for African swine fever and a preparation method and application thereof, belonging to the technical field of animal vaccines and veterinary biological products.
Background
African Swine Fever is an acute, febrile, lethal disease caused by African Swine Fever Virus (ASFV). The onset symptoms are similar to those of typical swine fever. Pigs are the only host. The International veterinary Bureau animal health code requires animal epidemics that must be reported. China also ranks the animal epidemic diseases as a type of animal epidemic diseases in the animal epidemic disease list. The disease is introduced into China in 8 months in 2018, and causes great loss to the pig industry in China.
African swine fever virus belongs to African swine fever virus family, members of African virus genus, and is characterized by similarity to iridoviridae family, having some characteristics of both iridovirus and poxvirus. ASFV is a double-stranded linear DNA virus, and is the only arbovirus whose nucleic acid is DNA. The virus particle has an icosahedral structure, and the surface is covered by a capsule membrane. Only one serotype, the viral ASFV genome is about 170-190kb in length, 15 times that of the classical swine fever virus genome, and contains 150-200 large Open Reading Frames (ORFs) encoding more than about 100 proteins. Wherein the P72 protein is the main structural protein of ASFV virus, coded by E138L gene, and is the main part of 20-hedral capsid, and accounts for about 32% of the total protein of African swine fever virus particles. The P72 protein of ASFV strain from different sources has quite conservative antigenic determinants. The P54 protein is also an important structural protein of African swine fever, is coded by an E183L gene, can perform special cross reaction with a cytoplasmic dynein DLC8, plays an important role in the process of virus uptake by cells and the process of virus processing in the cells, and can activate apoptosis so as to induce phagocytosis of the cells and mediate virus transmission. The p30 protein is encoded by the CP204L gene and has a molecular weight of about 30kD to 32kD, and is therefore sometimes referred to as the p32 protein. It was found that the p30 protein is involved in the entry of the virus into the host cell, and that antibodies directed against p30 are able to inhibit the internalization of the virus (Gomez-Puertas et al, 1996). And the p30 protein is the main structural protein of ASFV, has good antigenicity, can induce the organism to generate neutralizing antibody p30 protein, and can induce the neutralizing antibody in the infected animal (Gemoz-Puertas et al, 1996). The CD2V protein is the only protein which can be determined at present and exists on the outer envelope of the virion, and comprises a signal peptide sequence and a transmembrane region. The amino acid sequence of the protein is very similar to that of CD2, and the protein can be specifically combined with a CD2 receptor on the surface of porcine red blood cells. As a result of the study, 90% of virus particles of African swine fever virus having blood-adsorbing property were adsorbed to erythrocytes during infection. Therefore, antibodies against CD2V could well prevent viral adsorption. CD2V can be used as a very good protective antigen against homologous virus infection using unpurified CD2V protein (Ruiz-Gonzalvo, F, 1993).
At present, the African swine fever has been outbreaked in China for many times, and although researchers have conducted a great deal of research on the African swine fever, no effective vaccine for preventing the African swine fever exists at present. Inactivated vaccines do not produce any protective effect. Although the attenuated vaccine can prevent the immunized pig from being infected by the homologous ASFV strain, the immunized pig can have the symptoms of virus carrying, production performance reduction, high temperature, lameness and the like. Although relevant virulence factors of ASFV have been defined and knocked out so far, viruses still have residual virulence, and no effective and safe attenuated vaccine has been successfully developed so far.
In the case where it is currently not possible to prepare inactivated or attenuated vaccines on a large scale, it is of great interest to identify a method for preparing subunit vaccines for the virus in order to study a vaccine capable of preventing the disease or to have subunit proteins capable of preventing the disease. Patent CN103172749B reports a preparation method of an African swine fever protein engineering vaccine, and the vaccine prepared by the method can generate good humoral immunity and cellular immunity for pigs. However, the CD2V prepared by the method is a T cell epitope containing 178aa, is expressed by Escherichia coli, and cannot glycosylate the CD2V protein. Therefore, the immunogenicity of CD2V cannot be guaranteed. In addition, the p72 protein prepared by the method is only a short polypeptide sequence of the p72 protein. The complete immunogenicity of the P72 protein cannot be guaranteed.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: on the basis of realizing the preparation of the extracellular domain of the CD2V protein, the complete P72 protein, the P30 protein and the P54 protein, the subunit vaccine which can be industrially produced in a large scale and has good quality and can prevent the African swine fever virus is developed; secondly, the problem that the African swine fever virus is mutated due to the fact that residual nucleic acid in genomes of the African swine fever virus and other viruses or other vaccines is recombined possibly caused by utilization of the African swine fever attenuated vaccine, so that great biological safety potential hazards and possible immune interference exist is solved; thirdly, the African swine fever virus has more genotypes and complex pathogenic mechanism, and the multivalent vaccine prepared by using the multicomponent protein can overcome the defect of poor immune effect caused by the polymorphism of strains.
According to one aspect of the invention, the invention provides a multicomponent subunit vaccine of African swine fever, which comprises African swine fever virus surface envelope protein CD2V and at least one protein selected from the group consisting of African swine fever virus P72 protein, African swine fever virus P30 protein, African swine fever virus P54 membrane structure protein, and pharmaceutically acceptable adjuvant; wherein the African swine fever virus surface envelope protein CD2V is an African swine fever virus CD2V protein without a transmembrane region, or a fusion protein of an African swine fever virus CD2V extracellular region protein and a pig antibody Fc, namely CD 2V-Fc.
In the technical scheme of the multicomponent subunit vaccine of the present invention, preferably, the membrane structural protein of african swine fever virus P54 is full-length african swine fever virus P54 protein, or african swine fever virus P54 protein with a membrane spanning region removed; the African swine fever virus P72 protein is a full-length African swine fever virus P72 protein or an African swine fever virus P72 protein with chaperone protein; the African swine fever virus P30 protein is a full-length African swine fever virus P30 protein or an African swine fever virus P30 protein with chaperone protein.
In the technical scheme of the multicomponent subunit vaccine, preferably, the chaperone protein is TF protein. Wherein the African swine fever virus P72 protein with chaperonin is TF-P72 protein (TF-P72 for short); the African swine fever virus P30 protein with chaperonin is TF-P30 protein (TF-P30 for short).
In the technical scheme of the multicomponent subunit vaccine of the present invention, preferably, the pharmaceutically acceptable adjuvant is an oil-in-water adjuvant (e.g., 10 # white oil, etc.), a water-in-oil-in-water adjuvant (e.g., ISA201 VG adjuvant, ISA 206 VG adjuvant, etc.), a water-in-oil adjuvant (e.g., ISA 15A VG, ISA 35 VG adjuvant, etc.), or a water adjuvant (e.g., alumina gel adjuvant, IMS 251C VG adjuvant, etc.). More preferably, the pharmaceutically acceptable adjuvant is Montanide ISA201 adjuvant.
According to another aspect of the present invention, there is provided a method of preparing a multicomponent subunit vaccine for african swine fever, the method comprising the steps of: 1) preparing African swine fever virus surface envelope protein CD2V, African swine fever virus P72 protein, African swine fever virus P30 protein and African swine fever virus P54 membrane structural protein; 2) combining the African swine fever virus surface envelope protein CD2V prepared in the step 1) and at least one protein selected from the group consisting of African swine fever virus P72 protein, African swine fever virus P30 protein and African swine fever virus P54 membrane structure protein to prepare an antigen liquid; wherein the African swine fever virus surface envelope protein CD2V is an African swine fever virus CD2V protein without a transmembrane region, or a fusion protein of an African swine fever virus CD2V extracellular region protein and a pig antibody Fc, namely CD 2V-Fc; 3) mixing and stirring the antigen solution prepared in the step 2) and Montanide ISA201 adjuvant according to the mass ratio of 1:1 to obtain the multi-vaccine.
In the technical scheme of the method, preferably, the membrane structural protein of the African swine fever virus P54 is a full-length African swine fever virus P54 protein or an African swine fever virus P54 protein with a transmembrane region removed; the African swine fever virus P72 protein is a full-length African swine fever virus P72 protein or an African swine fever virus P72 protein with chaperone protein; the African swine fever virus P30 protein is a full-length African swine fever virus P30 protein or an African swine fever virus P30 protein with chaperone protein. Preferably, the chaperone protein is a TF protein. Wherein the African swine fever virus P72 protein with chaperonin is TF-P72 protein (TF-P72 for short); the African swine fever virus P30 protein with chaperonin is TF-P30 protein (TF-P30 for short).
In the technical scheme of the method, preferably, in the step 2), each head of the antigen solution contains 15-150 μ g of African swine fever virus surface envelope protein CD2V 15; each head of the antigen solution contains 25-200 mu g of African swine fever virus P72 protein; each head of the antigen solution contains 25-200 mu g of African swine fever virus P30 protein; each head of the antigen solution contains 15-150 mu g of African swine fever virus P54 membrane structural protein.
In the technical scheme of the method, preferably, each head of the antigen solution contains 30 μ g of African swine fever virus surface envelope protein CD 2V-Fc.
In the technical scheme of the method, preferably, each head of the antigen solution contains 50 μ g of African swine fever virus TF-P72 protein.
In the technical scheme of the method, preferably, each head of the antigen solution contains 50 μ g of African swine fever virus TF-P30 protein.
In the technical scheme of the method, preferably, each first portion of the antigen solution contains 30 μ g of African swine fever virus P54 protein.
In the method of the present invention, it is preferable that the antigen liquid further contains a preservative.
In the animal immunization example of the present invention, it was found that vaccines made from proteins, whether the CD2V-Fc protein is combined with a protein or the CD2V protein is combined with other proteins, are comparable in immunogenicity.
According to a further aspect of the invention, the invention provides the use of a multicomponent subunit vaccine of African swine fever for the prevention and treatment of African swine fever infection.
The subunit vaccine capable of preventing the African swine fever simultaneously provided by the invention has the advantages of strong immunogenicity, good safety, no immune interference, no biological potential safety hazard of virus variation, capability of fundamentally purifying the African swine fever virus and the like; the vaccine is used for immunization, so that the pigs can be effectively prevented and protected from being infected by African swine fever viruses; meanwhile, the African swine fever subunit vaccine prepared by the invention can be stored at 4 ℃ for at least more than 6 months, and can meet the industrial application.
Drawings
FIG. 1 shows the results of the detection of antibody levels for different immunization sessions.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and examples, which are only for illustrating the technical solutions of the present invention and are not to be construed as limiting the present invention.
The reagents of the invention are all commercially available products.
Montanide ISA201 adjuvant was purchased from the french seebeck company.
Example 1: preparation of African swine fever virus CD2V protein, P54 protein, P30 protein and P72 protein
1.1 preparation of African swine fever virus CD2V protein: the African swine fever CD2V protein can be prepared by the method for preparing the African swine fever CD2V protein in the patent with the application number of 201910004596.5 or 201910069838.9 or by the method for preparing the African swine fever CD2V protein in other patents or documents, and the full-length African swine fever CD2V protein or CD2V-Fc fusion protein can be prepared according to actual needs.
1.2 preparation of African swine fever virus P54 protein: the full-length P54 protein is prepared by referring to published literature, namely the preparation method of African swine fever P54 protein such as the expression of African swine fever virus P54 protein, the preparation of monoclonal antibody (von Chunyan et al, 2016, Chinese animal quarantine) or the prokaryotic expression of African swine fever virus P54 recombinant protein and the preparation of monoclonal antibody thereof (von Yao, 2014, Youshen university Master thesis) or the preparation method in other patents or literatures.
1.3 preparation of African swine fever virus P72 protein: the recombinant African swine fever virus p72 subunit soluble fusion protein in the patent with the reference application number of 201910142648.5 or the invention patent, the preparation method and the application thereof, or the preparation method in other patents or documents to prepare the African swine fever p72 protein can prepare the full-length African swine fever p72 protein or the truncated p72 fusion protein according to actual needs.
1.4 preparation of African swine fever virus P30 protein: referring to the recombinant soluble African swine fever virus p30 subunit fusion protein in the patent with the application number of 201910142630.5, the preparation method and the application thereof, or the preparation method in other patents or documents to prepare the African swine fever p302 protein, the full-length African swine fever p30 protein or the truncated p30 fusion protein can be prepared according to actual needs.
Example 2: preparation of subunit vaccine for African swine fever
The consumables and materials for preparing the vaccine are all required to be subjected to aseptic processing in advance, and the preparation process is finished in a biological safety cabinet or other instruments or environments capable of ensuring the sterility of the whole preparation process.
2.1 vaccine preparation (example of four protein mixtures)
Preparing an aqueous phase: according to the content of CD2V, P54, TF-P72 and TF-P30 proteins in the vaccine, PBS (or physiological saline) is used for diluting CD2V, P54, TF-P72 and TF-P30 proteins to proper concentration, and the proper concentration is obtained to be an aqueous phase;
preparing an oil phase: according to the total amount of the prepared vaccine, a proper amount of ISA201 VG adjuvant is measured according to the weight ratio of 1:1 and the volume ratio of 46:54 of the antigen phase and the adjuvant;
emulsification: preheating the water phase and the oil phase to 33 ℃, slowly adding the water phase into the oil phase, stirring at 200-500rpm for 20-30min, standing at 20 ℃ for 1h, and standing at 4 ℃ overnight;
subpackaging and storing: subpackaging as required, and storing at 4 deg.C for use after qualified inspection.
Example 3: african swine fever subunit vaccine immunity experiment
3.1 vaccine preparation: proteins and vaccines were prepared according to the methods of examples 1 and 2, with specific vaccine information as shown in table 1 below:
TABLE 1
3.2 immunization experiment: screening 40 piglets (which are negative to CSFV and PCV2 and PRRSV antigen antibody) of 28-35 days old piglets, randomly dividing into 8 groups, 5 piglets in each group, using one group as a blank control group, and respectively immunizing vaccine 1, vaccine 2, vaccine 3, vaccine 4, vaccine 5, vaccine 6 and vaccine 7 by other 7 groups. The blank control group was injected intramuscularly with 1ml of physiological saline each time. In addition, 7 groups of immunization groups were injected intramuscularly with 1ml of the corresponding vaccine each time, three weeks after the priming of the immunization, and 21 days before, before and after the secondary immunization of the group of animals, serum was collected and the antibody titer was examined.
Specific antibodies in porcine serum were detected by conventional ELISA methods. The coating antigen is expressed purified CD2V protein. Diluting the coated antigen to 1 μ g/ml with coating diluent, incubating overnight at 4 ℃, incubating 5% BSA in 37 ℃ incubator for 1 hour, and washing 3 times with PBST; diluting the collected pig serum by 1:500 times, adding the diluted pig serum into a 96-well plate, incubating the pig serum in an incubator at 37 ℃ for 1 hour, and washing the pig serum by PBST for 3 times; adding a goat anti-pig secondary antibody marked by HRP, and incubating for 1 hour in an incubator at 37 ℃; PBST washing 3 times; adding a chromogenic substrate, and incubating for 10 minutes in an incubator at 37 ℃; adding the stop solution. Read 450nm on a microplate reader.
The test results are shown in figure 1, and the results of the ELISA test using the antigen coated with African swine fever CD2V protein show that: the antibody level of each vaccine group and the control group is not obviously different before immunization; the antibody levels of the vaccine 1, the vaccine 2, the vaccine 3, the vaccine 4, the vaccine 5, the vaccine 6 and the vaccine 7 in the first immunization day 14 (before second immunization) are obviously increased and are very different (P is less than 0.01) compared with the control group, wherein the antibody levels of the vaccine (vaccine 1) prepared by the CD2V, the P54, the TF-P30 and the TF-P72 proteins and the vaccine (vaccine 6) prepared by the combination of the CD2V and the TF-P30 in the first immunization day 14 (before second immunization) are slightly higher than those of the other immunization groups, but are not obviously different. At 21 days after the second immunization, the antibody levels of the vaccine 1, the vaccine 2, the vaccine 3, the vaccine 4, the vaccine 5, the vaccine 6 and the vaccine 7 groups are obviously increased ((P <0.01)) compared with 14 days after the first immunization, but the antibody levels among the vaccine groups are basically consistent. The African swine fever subunit vaccine provided by the invention can effectively stimulate the generation of humoral immune response.
The invention is illustrated by the above examples, but it should be understood that the invention is not limited to the particular examples and embodiments described herein. These specific examples and embodiments are included to assist those skilled in the art in practicing the present invention. Further modifications and improvements will readily occur to those skilled in the art without departing from the spirit and scope of the invention and, accordingly, it is intended that the invention be limited only by the terms of the appended claims, along with the full scope of equivalents to which such terms are entitled.
Claims (12)
1. A multicomponent subunit vaccine of African swine fever, the multicomponent subunit vaccine includes African swine fever virus surface envelope protein CD2V and at least one protein selected from the group consisting of African swine fever virus P72 protein, African swine fever virus P30 protein, African swine fever virus P54 membrane structural protein, and pharmaceutically acceptable adjuvant; wherein the African swine fever virus surface envelope protein CD2V is an African swine fever virus CD2V protein without a transmembrane region, or a fusion protein of an African swine fever virus CD2V extracellular region protein and a pig antibody Fc, namely CD 2V-Fc.
2. The multicomponent subunit vaccine of claim 1, wherein the African swine fever virus P54 membrane structural protein is full-length African swine fever virus P54 protein, or a transmembrane-region-deleted African swine fever virus P54 protein; the African swine fever virus P72 protein is a full-length African swine fever virus P72 protein or an African swine fever virus P72 protein with chaperone protein; the African swine fever virus P30 protein is a full-length African swine fever virus P30 protein or an African swine fever virus P30 protein with chaperone protein.
3. The multicomponent subunit vaccine of claim 2, wherein the chaperone protein is a TF protein.
4. The multicomponent subunit vaccine of claim 1, wherein the pharmaceutically acceptable adjuvant is Montanide ISA201 adjuvant.
5. A method of preparing a multicomponent subunit vaccine according to any one of claims 1 to 4, the method comprising the steps of:
1) preparing African swine fever virus surface envelope protein CD2V, African swine fever virus P72 protein, African swine fever virus P30 protein and African swine fever virus P54 membrane structural protein;
2) combining the African swine fever virus surface envelope protein CD2V prepared in the step 1) and at least one protein selected from the group consisting of African swine fever virus P72 protein, African swine fever virus P30 protein and African swine fever virus P54 membrane structure protein to prepare an antigen liquid; wherein the African swine fever virus surface envelope protein CD2V is an African swine fever virus CD2V protein without a transmembrane region, or a fusion protein of an African swine fever virus CD2V extracellular region protein and a pig antibody Fc, namely CD 2V-Fc;
3) mixing and stirring the antigen solution prepared in the step 2) and Montanide ISA201 adjuvant according to the mass ratio of 1:1 to obtain the multi-vaccine.
6. The method according to claim 5, wherein the African swine fever virus P54 membrane structural protein is a full-length African swine fever virus P54 protein or a transmembrane-region-removed African swine fever virus P54 protein; the African swine fever virus P72 protein is a full-length African swine fever virus P72 protein or an African swine fever virus P72 protein with chaperone protein; the African swine fever virus P30 protein is a full-length African swine fever virus P30 protein or an African swine fever virus P30 protein with chaperone protein; wherein the chaperone protein is TF protein.
7. The method according to claim 5 or 6, wherein in step 2), each head of the antigen solution contains 15-150 μ g of African swine fever virus surface envelope protein CD2V 15; each head of the antigen solution contains 25-200 mu g of African swine fever virus P72 protein; each head of the antigen solution contains 25-200 mu g of African swine fever virus P30 protein; each head of the antigen solution contains 15-150 mu g of African swine fever virus P54 membrane structural protein.
8. The method according to claim 7, wherein the antigenic fluid contains 30 μ g of African swine fever virus surface envelope protein CD2V-Fc per head.
9. The method according to claim 7, wherein the antigenic fluid comprises 50 μ g African swine fever virus TF-P72 protein per head.
10. The method according to claim 7, wherein the antigenic fluid comprises 50 μ g African swine fever virus TF-P30 protein per head.
11. The vaccine of claim 7, wherein the antigenic fluid comprises 30 μ g of African swine fever virus P54 protein per head.
12. Use of a multicomponent subunit vaccine according to any one of claims 1 to 4 for the prevention and treatment of African swine fever infection.
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