African swine fever virus immunogen combination and application thereof

Abstract

The invention belongs to the field of vaccines, and particularly relates to an African swine fever virus immunogen combination and application thereof. In the present application, provided is an immunogenic composition comprising: (A) an antibody immunogen group comprising an immunogen derived from an African swine fever virus structural protein; and (B) a recombinant T cell immunogen group containing an immunogen derived from an African swine fever virus non-structural protein; also provided are nucleic acid molecules encoding the immunogenic composition, vectors and host cells thereof, vaccines comprising one or more of the foregoing, and related products. The dual-immunogen combination disclosed by the invention can effectively induce binding antibodies aiming at p72, p54, p30 and the like and activate T cell response aiming at a T antigen, p17 and penton, so that an effective prevention effect on the African swine fever virus is achieved.

Description

Technical Field

[0001] The present invention relates to the field of vaccines and biotechnology. Specifically, the present application relates to a dual immunogen composition comprising an antibody immunogen composed of African swine fever virus structural proteins and a T cell immunogen derived from non-structural proteins, a vaccine comprising the immunogen composition and its application. Background Art

[0002] African swine fever is an infectious disease caused by infection with African swine fever virus (ASFV). Its main hosts are domestic pigs and wild boars such as warthogs, bush boars and big forest boars, and it can be transmitted by orthopedic soft ticks.

[0003] ASFV is a large double-stranded DNA enveloped virus with a virus particle diameter of up to 200nm. It is the only member of the African swine fever virus family. Its genome size ranges from 170kb to 194kb, encoding about 160 proteins including structural proteins and host immune regulatory proteins. According to the gene sequence encoding the main capsid protein of the virus (B646L), African swine fever virus can be divided into 24 genotypes. Affected by host differences and strain virulence, African swine fever has a variety of clinical manifestations after infection. Domestic pigs infected with highly virulent strains usually have symptoms such as high fever, purple skin, and multiple organ bleeding, and die within 3-7 days after infection, with a mortality rate close to 100%.

[0004] African swine fever was first spread only in African countries, and has been introduced to Europe, South America, Asia and other regions since the 1960s. After it was introduced to my country in August 2018, it caused huge economic losses and severely hit the domestic breeding industry. According to the data of the World Organization for Animal Health (WOAH), African swine fever has now spread to five continents around the world, and more and more countries have reported outbreaks. [1] .

[0005] Developing a safe and effective African swine fever vaccine is an effective means to prevent the outbreak and spread of African swine fever. However, due to the complexity of the virus structure and the lack of research on the host's immune mechanism of anti-infection, the research on African swine fever vaccine faces huge challenges. Antibody responses to African swine fever virus can be detected in recovered pigs, but healthy pigs with passive immunity cannot resist viral invasion. Oura et al. [2]It was also found that using antibodies to eliminate CD8+T cells in pigs vaccinated with attenuated vaccines would not produce the original protective effect. The inactivated African swine fever virus vaccine cannot stimulate sufficient immune response and has been proven to provide no effective protection. The attenuated live vaccine can replicate in vivo after immunization, simulating viral infection under natural conditions. The attenuated live vaccines obtained through tissue subculture or natural screening, such as NH / P68, OURT88 / 3, etc., can induce immune protection of experimental pigs against the same genotype strains, but lack cross-protection against heterologous genotype strains.

[0006] In recent years, more and more genes related to ASFV virulence and evasion of host immune responses have been identified, and live attenuated vaccines developed by deleting these genes have also emerged. After knocking out the 9GL and UK genes of the Georgia 2007 strain, the experimental pigs were able to resist the Georgia 2007 challenge 14 days after immunization, but knocking out the same genes of the Chinese epidemic strain HLJ / 2008 did not provide protection [3] In addition, there are still several key issues with live attenuated vaccines:

[0007] ——Question (1): Safety. The mechanism by which African swine fever virus resists host cell clearance is very complex and involves multiple signaling pathways. Deleting a limited number of genes cannot guarantee its safety. Many attenuated live vaccines will experience a reversion to virulence during continuous passage, often accompanied by more serious side effects. [4] The developed live attenuated vaccine HLJ / -18-6GD lacks six genes in the MGF306 / 505 family and can provide 100% protection against highly pathogenic homologous strains, but the virulence relapsed in safety experiments. Only after further knocking out the CD2v gene was its virulence further weakened. Vietnam approved two attenuated vaccines for domestic marketing in July 2023. There is a lack of sufficient safety data, and WOAH has issued a warning that "there are risks in using substandard vaccines." [1] .

[0008] ——Question (2): Broad spectrum. Currently, the live attenuated vaccines that provide protection can only target homologous strains, but there are many genotypes and serotypes of African swine fever virus. Only by finding key conserved T cell epitopes can a broad-spectrum response be achieved.

[0009] ——Problem (3): Limited large-scale production. The target cells of African swine fever virus are primary pig macrophages, which are difficult to use for commercial large-scale production.

[0010] With the deepening of research on antigens for viral immune protection, research on screening suitable immunogen combinations and developing vaccines using different vaccine platforms such as subunit vaccines, nucleic acid vaccines, and viral vector vaccines is being widely carried out. Gomez-Puertas et al.[5] It was found that p72, p54, and p30 proteins can induce the body to produce neutralizing antibodies, and there are numerous subunit vaccines and DNA vaccines focusing on the combination of these three immunogens. Immunizing experimental pigs with p54, p30 proteins, or p54 and p30 fusion proteins expressed separately using the baculovirus expression system can induce neutralizing antibodies and resist the E75 strain, but the combination of p72, p54, and p30 proteins cannot protect the experimental pigs against the Pr4 strain. CD2v, as the only protein on the outer membrane of ASFV, can mediate ASFV in vitro red blood cell agglutination and is an important indicator for identifying ASFV serotypes. Immunization with CD2v protein alone can mediate partial protection against the E75 strain, but this result cannot be repeated on other strains. Based on the DNA vaccine platform, the CD2v extracellular region is fused with p54 and p30, respectively, which can induce strong binding antibodies and T cell responses, but there is no protection after the virus attack. Further fusion of ubiquitin protein with immunization was able to induce a stronger T cell response, but was unable to induce the production of binding and neutralizing antibodies, and only produced partial protection after infection: only 2 of the 6 experimental pigs survived (2 / 6).

[0011] Lacasta et al. [6] A random DNA vaccine library containing 80 coding frames of ASFV fused with ubiquitin protein was constructed, but it only produced partial protection. Adenovirus and vaccinia virus are the main viral vectors used to develop African swine fever vaccines, but most studies only detect immune responses associated with viral antigens without evaluating the protective effect of vaccines against challenge. Adenovirus expressing A151R, B119L, B602L, EP402RΔPRR, B438L, K205R, A104R, pp62 and p72 combined with BioMize adjuvant can induce higher binding antibodies, but the challenge results showed that not only was there no protective effect, but the experimental group animals even showed enhanced symptoms. Adenovirus expressing p32, p54, pp62, p72, p37-34-14, p150-I, and p150-II, combined with BioMize adjuvant immunization, can also induce a strong humoral immune response, but only 2 (2 / 10) of the test pigs survived the challenge test. Although the same immunogen combination combined with ZTS-01 adjuvant immunization did not induce high antibody levels, the challenge protection effect was better than the former (5 / 9 survived).

[0012] In addition to single viral vectors, a large number of studies have explored the feasibility of sequential immunization with different viral vectors or viral vectors and subunit vaccines and DNA vaccines. The animals were first primed with adenovirus expressing p72, p54, p30, B602L, E199L, EP153R, F317L, and MGF505-5R, and then boosted with vaccinia virus expressing the same immunogen. The survival rate of experimental animals after OUR T88 / 1 challenge was 100%, but the viral load in the surviving animals remained at a high level 20 days later, and infectiousness could not be ruled out. [7] .

[0013] In summary, this field still needs to further explore the infection mechanism of African swine fever virus and the body's immune protection mechanism, identify and screen immunogens or antigenic epitopes that induce immune responses, and develop safe and effective African swine fever vaccines.

[0014] Vaccinia virus is a widely used vaccine vector that can induce high levels of humoral and cellular immune responses. Vaccinia virus was first developed to protect against smallpox virus, and large-scale population vaccination has proven its high safety. In addition, vaccinia virus can accommodate up to 30kb of foreign genes.

[0015] The African swine fever virus has a complex structure and can resist a variety of extreme physical environments. It can be spread through contaminated utensils, swill, feed, or by attaching to pork products. At present, the main measures for my country to deal with the African swine fever epidemic are still control and culling. Given the huge number of pigs raised in my country, which are mainly raised by individual households and have weak awareness of biosafety prevention, the African swine fever epidemic will exist for a long time and may break out at any time. Therefore, it is urgent to develop a safe and effective African swine fever vaccine.

[0016] References:

[0017] [1]Health WOF A.AFRICAN SWINE FEVER(ASF)Situation Report 55[R].2024.

[0018] [2]Oura CA, Denyer M, Takamatsu H, et al. In vivo depletion of CD8+Tlymphocytes abrogates protective immunity to African swine fever virus[J]. Journal of General Virology, 2005, 86(9): 2445-2450

[0019] [3]Wu K,Liu J,Wang L,et al.Current state of global African swinefever vaccine development under the prevalence and transmission of ASF inChina[J].Vaccines,2020,8(3):531.DOI:10.3390 / vaccines8030531.

[0020] [4]Chen W,Zhao D,He X,et al.A seven-gene-deleted African swine fevervirus is safe and effective as alive attenuated vaccine in pigs[J].Sci ChinaLife Sci,2020,63(5):623-634.DOI:10.1007 / s11427-020-1657-9.

[0021] [5]Gomez-Puertas P,Rodriguez F,Oviedo J M,et al.Neutralizingantibodies to different proteins of African swine fever virus inhibit bothvirus attachment and internalization[J].Journal of virology,1996,70(8):5689-5694

[0022] [6]Lacasta A,Ballester M,Monteagudo P L,et al.Expression libraryimmunization can confer protection against lethal challenge with Africanswine fever virus[J].Journal of virology,2014,88(22):13322-13332

[0023] [7]Goatley LC,Reis AL,Portugal R,et al.Apool of eight virallyvectored African swine fever antigens protect pigs against fatal disease[J].Vaccines,2020,8(2):234 Summary of the invention

[0024] In this application, a new type of African swine fever vaccine is developed to address the technical problems that urgently need to be solved in this field.

[0025] In some aspects of the present application, an immunogenic composition is provided, comprising:

[0026] (A) a panel of antibody immunogens containing structural proteins derived from African swine fever virus; and

[0027] (B) A panel of recombinant T cell immunogens containing non-structural proteins derived from African swine fever virus.

[0028] In some embodiments, the antibody immunogen group comprises one or more full-length or extracellular fragments of structural proteins derived from African swine fever virus, for example, the antibody immunogen group comprises one or more proteins or fragments selected from the following group or any combination thereof: B646L, B602L, H240R, B438L, C204L, D117L, E183L, EP402R; or any combination of the foregoing.

[0029] In some embodiments, the recombinant T cell immunogen group comprises one or more T cell epitopes contained in non-structural proteins selected from the following group and derived from African swine fever virus (ASFV): F334L, H359L, D250R, D339L, F1055L, G1211R, A528 and MGF-505-7R proteins; or any combination of T cell epitopes contained in the aforementioned non-structural proteins; or a fragment having at least 80% sequence identity with the aforementioned T cell epitope combination.

[0030] In some aspects of the present application, a recombinant T cell immunogenic peptide is provided, which comprises a plurality of peptide elements derived from the non-structural protein of African swine fever virus.

[0031] In some aspects of the present application, a nucleic acid molecule encoding the immunogenic composition or recombinant T cell immunogenic peptide of the present application is provided. In some aspects of the present application, a vector or host cell comprising the nucleic acid molecule is provided.

[0032] In some aspects of the present application, an African swine fever vaccine is provided, comprising:

[0033] (I) one or more immunologically active components or their precursors selected from the group consisting of one or more immunogenic compositions, recombinant T cell immunogenic peptides, nucleic acid molecules, vectors or host cells of the present application; and

[0034] (II) one or more pharmaceutically or veterinarily acceptable carriers or excipients or delivery systems.

[0035] In some aspects of the present application, a product is provided, which comprises one or more immunogenic compositions, recombinant T cell immunogenic peptides, nucleic acid molecules, vectors or host cells or vaccines of the present application.

[0036] In some embodiments, the product is a vaccine kit comprising one or more doses of the same or different vaccines of the present application.

[0037] In some aspects of the present application, provided is the use of one or more immunogenic compositions, recombinant T cell immunogenic peptides, nucleic acid molecules, vectors or host cells or vaccines of the present application in the preparation of products for the prevention and / or treatment of African swine fever.

[0038] In some aspects of the present application, a method for preventing and / or treating African swine fever is provided, which comprises administering to a subject in need thereof a preventive and / or therapeutically effective amount of the immunogenic composition, recombinant T cell immunogenic peptide, nucleic acid molecule, vector or host cell, vaccine or product of the present application.

[0039] Those skilled in the art may arbitrarily combine the above technical solutions and technical features without departing from the inventive concept and protection scope of the present invention. Other aspects of the present invention are obvious to those skilled in the art due to the disclosure of this article. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The present invention will be further described below in conjunction with the accompanying drawings, the contents shown in which are only for illustrating the embodiments of the present invention, rather than for limiting the scope of the present invention.

[0041] Figure 1 : Map of the shuttle plasmids used in the examples: A: pD8L-D117L-E183L-TD (wherein T = T antigen, D = DHFR); B: pA56R-H240R-B438L-C204L; C: pSC65-B646L-B602L.

[0042] Figure 2:Construction, verification and expression of rTV-D8L-A56R-Δ-TK: A: Left: Schematic diagram of the 5th round of plaque picking of rTV-D8L screening; Middle: rTV-D8L was verified by D8L-F / D8L-R PCR, showing the size of the target band; Right: rTV-D8L expressed p17, p54 and T antigen. B: Left: Schematic diagram of the 7th round of plaque picking of rTV-D8L-A56R screening; Middle: rTV-D8L-A56R was verified by A56-F / A56-R PCR, showing the size of the target band; Right: rTV-D8L-A56 expressed penton, p49 and p30. C: Left: Schematic diagram of the 10th round of plaque picking in rTV-D8L-A56R-Δ-TK screening; Middle: rTV-D8L-A56R-Δ-TK was verified by TK-F / TK-R PCR, showing the size of the target band; Right: rTV-D8L-A56-Δ-TK expresses p72 and p72 chaperone; D: Left: Schematic diagram of the 6th round of plaque picking in rTV-TK screening; Middle: rTV-TK was verified by TK-F / TK-R PCR, showing the size of the target band; Right: rTV-TK expresses p72 and p72 chaperone.

[0043] Figure 3 :The levels of immune response induced by different recombinant vaccinia virus combinations in mice: A: Schematic diagram of the immunization process and time points; B: The binding antibody titers against p72, p54 and p30 in each group of mice on the 35th, 63rd and 77th days of immunization; C: The levels of cellular immune response induced in each group of mice against T antigen, p17 and penton.

[0044] Figure 4 : The levels of immune response induced by other recombinant vaccinia virus combinations in mice: A: Schematic diagram of the immunization process and time points; B: The binding antibody titers against p72, p54 and p30 in each group of mice on the 56th day of immunization; C: The levels of cellular immune responses induced in each group of mice against T antigen, p17 and penton.

[0045] Figure 5 :Construction, verification and expression of rTV-TK-K8R: A: Map of pK8R-EP402R shuttle plasmid; B: Schematic diagram of the 9th round of plaque picking in rTV-TK-K8R screening; C: rTV-TK-K8R was verified by K8R-F / K8R-R PCR, showing the size of the target band; D: rTV-TK-K8R expresses CD2v.

[0046] Figure 6: The levels of immune response induced by different optimized recombinant vaccinia virus combinations in mice: A: Schematic diagram of the immunization process and time points; B: The binding antibody titers against p72, p54 and p30 in each group of mice on the 70th day of immunization; C: The levels of cellular immune response induced in each group of mice against T antigen, p17 and penton.

[0047] Figure 7 : Protective effect of the recombinant vaccinia virus combination in local pigs: A: Schematic diagram of the immunization process and time points; B top: Changes in clinical scores of feeding status of the control group and the immunized group after challenge; B middle: Changes in clinical scores of mental state of the control group and the immunized group after challenge; B bottom: Changes in clinical scores of lying down degree of the control group and the immunized group after challenge; C top: Changes in body temperature of the control group and the immunized group after challenge; C bottom: Survival curves of the control group and the immunized group after challenge.

[0048] In the figures, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.005. DETAILED DESCRIPTION

[0049] The present disclosure relates to the field of vaccines, and in particular to a dual immunogen composition consisting of an antibody immunogen composed of African swine fever virus structural proteins and a T cell immunogen derived from non-structural proteins, a vaccine containing this immunogen composition, and its application. In this application, a suitable African swine fever virus immunogen is selected, and a conservative T cell epitope is loaded at the same time, and the above-mentioned immunogen is loaded with an appropriate carrier to develop a safe and effective African swine fever vaccine.

[0050] All numerical ranges provided herein are intended to clearly include all values ​​falling between the range endpoints and the numerical ranges therebetween. Features mentioned in the present invention or in the embodiments may be combined. All features disclosed in this specification may be used in combination with any form of composition, and each feature disclosed in the specification may be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0051] As used herein, "about" in the context of a numerical value or a range means ± 10% of the recited or claimed numerical value or range.

[0052] It should be understood that when providing a parameter range, the present invention also provides all integers and decimals within the range. For example, "0.1-2.5 mg / day" includes 0.1 mg / day, 0.2 mg / day, 0.3 mg / day, etc. up to 2.5 mg / day.

[0053] As used herein, “containing,” “having,” or “including” include “comprising,” “mainly consisting of,” “substantially consisting of,” and “consisting of;” “mainly consisting of,” “substantially consisting of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0054] Immunogenic composition

[0055] In the present application, an immunogenic composition is provided, comprising:

[0056] (A) an antibody immunogen panel comprising an immunogen derived from an African swine fever virus structural protein; and

[0057] (B) Recombinant T cell immunogen panel containing immunogens derived from nonstructural proteins of African swine fever virus.

[0058] In some embodiments, the source of the immunogenic fragment in the immunogenic composition of the present application is an African swine fever virus selected from the epidemic strains of African swine fever virus, and / or selected from a plurality of African swine fever virus strains of different genotypes and serotypes.

[0059] In some embodiments, the African swine fever virus is one or more African swine fever virus strains selected from the following group: Pig / HLJ / 18, China / Guangxi / 2019 / domestic pig, CN201801, China / Jilin / 2018 / boar, Pig / Hubei / 628 / 2020, Pig / Liaoning / LC / 2020, Pig / Hebei / Q3 / 2020, and Georgia 2007 / 1.

[0060] In some embodiments, the antibody immunogen group contains one or more full-length or extracellular fragments of structural proteins derived from African swine fever virus.

[0061] In some embodiments, the antibody immunogen group comprises one or more proteins or fragments selected from the following group or any combination thereof: B646L, B602L, H240R, B438L, C204L, D117L, E183L, EP402R; or any combination of the foregoing. In some embodiments, the combination is selected from: a combination of D117L and E183L D117L-E183L, a combination of H240R, B438L and C204L H240R-B438L-C204L, a combination of B646L and B602L B646L-B602L, a combination of D117L, E183L, H240R, B438L and C204L D117L-E183L 3L-H240R-B438L-C204L, the combination of D117L, E183L, H240R, B438L, C204L, B646L and B602L D117L-E183L-H240R-B438L-C204L-B646L-B602L, the combination of B646L, B602L and EP402R B646L-B602L-EP402R. In some embodiments, each protein or fragment in any combination is directly connected or connected by a linker, such as a linker selected from SEQ ID NO: 89-94, IRES, P2A, T2A, E2A and F2A.

[0062] In some embodiments, B646L, B602L, H240R, B438L, C204L, D117L, E183L, and EP402R are peptides having amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 98, respectively, or are peptides having at least 80% sequence identity or homology with peptides having amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 98.

[0063] In some embodiments, B646L, B602L, H240R, B438L, C204L, D117L, E183L, and EP402R are encoded by nucleic acid molecules having nucleotide sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 97, respectively, or by nucleic acid molecules having at least 80% sequence identity or homology with nucleic acid molecules having nucleotide sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 97.

[0064] In some embodiments, the recombinant T cell immunogen group comprises one or more non-structural proteins or fragments thereof derived from African swine fever virus. In some embodiments, T cell epitopes widely recognized by porcine major histocompatibility complex (SLA) molecules are screened by bioinformatics and fused to T antigens, such as the T antigen shown in SEQ ID NO:78. In other embodiments, DHFR is inserted at the 3' end of the fused T antigen to further increase the degradation level of the T antigen in the cell, such as the T antigen with DHFR shown in SEQ ID NO:80.

[0065] In some embodiments, the recombinant T cell immunogen group comprises: (a) one or more T cell epitopes contained in the following group of non-structural proteins: F334L, H359L, D250R, D339L, F1055L, G1211R, A528 and MGF-505-7R proteins; or any combination of T cell epitopes contained in the aforementioned non-structural proteins; or a fragment having at least 80% sequence identity with the aforementioned T cell epitope combination; and

[0066] (b) Optionally, a linker between the peptide elements, such as a linker selected from the group consisting of SEQ ID NOs: 89-94, IRES, P2A, T2A, E2A and F2A.

[0067] In some embodiments, the recombinant T cell immunogen group comprises at least a combination of T cell epitopes contained in F334L, H359L, D250R, D339L, F1055L, G1211R, A528 and MGF-505-7R proteins.

[0068] In some embodiments, the recombinant T cell immunogen set comprises:

[0069] (i) at least 8, 10, 20, 30 or all of the peptides selected from the group consisting of a peptide having an amino acid sequence as shown in SEQ ID NO: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or a peptide having at least 80% sequence identity with any of the foregoing peptides; and / or

[0070] (ii) a peptide having an amino acid sequence as shown in SEQ ID NO: 78 or 80, or a peptide having at least 80% sequence identity with any of the foregoing peptides; and / or

[0071] In some embodiments, the nucleic acid molecule encoding the recombinant T cell immunogen group comprises:

[0072] (i') at least 8, 10, 20, 30 or all of the nucleic acid molecules selected from the following group: a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73 or 75, or a nucleic acid molecule having at least 80% sequence identity with any of the foregoing nucleic acid molecules; and / or (ii') a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 77 or 79, or a nucleic acid molecule having at least 80% sequence identity with any of the foregoing nucleic acid molecules.

[0073] In some embodiments, the recombinant T cell immunogen group is connected or mixed with the antibody immunogen group, for example, the recombinant T cell immunogen group is mixed or connected with B646L, B602L, H240R, B438L, C204L, D117L, E183L, EP402R or any combination of the foregoing (e.g., a combination of D117L and E183L), for example, each independently through a linker selected from SEQ ID NO: 89-94, IRES, P2A, T2A, E2A and F2A.

[0074] Accordingly, the present application also provides a recombinant T cell immunogenic peptide, which comprises:

[0075] (a) one or more T cell epitopes selected from the group consisting of non-structural proteins of African swine fever virus (ASFV): F334L, H359L, D250R, D339L, F1055L, G1211R, A528 and MGF-505-7R proteins; or any combination of T cell epitopes contained in the aforementioned non-structural proteins; or a fragment having at least 80% sequence identity with the aforementioned T cell epitope combination; and

[0076] (b) Optionally, a linker between the peptide elements, such as a linker selected from the group consisting of SEQ ID NOs: 89-94, IRES, P2A, T2A, E2A and F2A.

[0077] In some embodiments, the recombinant T cell immunogenic peptide comprises at least F334L, H359L, D250R, D339L, F1055L, G1211R, A528 and a combination of T cell epitopes contained in the MGF-505-7R protein.

[0078] In some embodiments, the recombinant T cell immunogenic peptide comprises:

[0079] (i) at least 8, 10, 20, 30 or all of the peptides selected from the group consisting of peptides having SEQ ID NO: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,

[0080] 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or a peptide having at least 80% sequence identity with any of the foregoing peptides; and / or

[0081] (ii) a peptide having an amino acid sequence as shown in SEQ ID NO: 78 or 80, or a peptide having at least 80% sequence identity with any of the foregoing peptides.

[0082] In some embodiments, the nucleic acid molecule encoding the recombinant T cell immunogenic peptide comprises:

[0083] (i') at least 8, 10, 20, 30 or all nucleic acid molecules selected from the group consisting of SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,

[0084] 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73 or 75, or a nucleic acid molecule having at least 80% sequence identity with any of the foregoing nucleic acid molecules; and / or

[0085] (ii') a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 77 or 79, or a nucleic acid molecule having at least 80% sequence identity with any of the foregoing nucleic acid molecules.

[0086] The present application also provides a nucleic acid molecule encoding the aforementioned immunogenic composition or recombinant T cell immunogenic peptide or.

[0087] Vectors and host cells

[0088] Also provided in the present application are vectors or host cells comprising the nucleic acid molecules described herein.

[0089] In some embodiments, the vector can be selected from: mRNA vector, DNA plasmid vector (such as shuttle plasmid vector), recombinant virus vector, recombinant bacterial vector; wherein the mRNA vector is selected from linear, circular and self-replicating vectors; the recombinant virus vector is selected from vaccinia virus (such as Tiantan strain, North American vaccine strain, Wyeth-derived strain, Listeria strain, Ankara-derived strain, Copenhagen strain and New York strain), adenovirus (such as adenovirus types 5, 11, 26, 35, 63, 68), adeno-associated virus, herpes simplex virus, measles virus, enterovirus, reovirus, rhabdovirus, flavivirus, influenza virus, parainfluenza virus, respiratory syncytial virus, poliovirus vector.

[0090] In some embodiments, a vaccinia virus comprising an immunogen or a combination thereof of the present application is provided. In some embodiments, one or more sites selected from D8L, A56R, TK, and K8R in the recombinant vaccinia virus (rTV) of the present application are inserted with an immunogen gene. In some embodiments, the recombinant vaccinia virus of the present application includes but is not limited to a recombinant virus into which one or more of the following immunogenic fragments are inserted: D117L-E183L-T antigen, H240R-B438L-C204L, B646L-B602L, D117L-E183L-T antigen-H240R-B438L-C204L, D117L-E183L-T antigen-H240R-B438L-C204L-B646L-B602L, B646L-B602L-EP402R.

[0091] In some embodiments, the recombinant vaccinia virus of the present application can be selected from the following group: rTV-D8L-D117L-E183L-T antigen, rTV-D8L-D117L-E183L-T antigen-A56R-H240R-B438L-C204L, rTV-D8L-D117L-E183L-T antigen-A56R-H240R-B438L-C204L-TK-B646L-B602L, rTV-TK-B646L-B602L, rTV-TK-B646L-B602L-K8R-EP402R.

[0092] In some embodiments, the T antigen in the recombinant vaccinia virus is linked to DHFR to increase the degradation level of the T antigen in the cell. In some embodiments, the recombinant vaccinia virus is selected from the following group: rTV-D8L-D117L-E183L-T antigen-DHFR, rTV-D8L-D117L-E183L-T antigen-DHFR-A56R-H240R-B438L-C204L, rTV-D8L-D117L-E183L-T antigen-DHFR-A56R-H240R-B438L-C204L-TK-B646L-B602L, rTV-TK-B646L-B602L, rTV-TK-B646L-B602L-K8R-EP402R.

[0093] In some embodiments, the vaccinia virus promoter is derived from a strong promoter of vaccinia virus that promotes the expression of viral proteins, such as pE / L, p7.5, pH5, and different truncated forms of each strong promoter. The screening marker includes reporter genes such as LacZ, eGFP, mcherry, BFP, and ZsGreen.

[0094] In some embodiments, the host cell can be a mammalian cell or an insect cell, such as HEK293, HeLa, K562, CHO, NS0, SP2 / 0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19 and MRC-5 cells; High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5 and Ao38.

[0095] Vaccines and products

[0096] The present application provides an African swine fever vaccine, which comprises:

[0097] (I) one or more immunologically active components or their precursors selected from the group consisting of the immunogenic composition, recombinant T cell immunogenic peptide, nucleic acid molecule, vector or host cell described herein; and

[0098] (II) one or more pharmaceutically or veterinarily acceptable carriers or excipients or delivery systems.

[0099] In some embodiments, the African swine fever vaccine is an mRNA vaccine, a DNA vaccine, a viral vector vaccine (such as a vaccinia virus vaccine), or a recombinant protein vaccine.

[0100] In some embodiments, the carrier or excipient or delivery system is selected from: a lipid delivery system, a lipid delivery system, a polymer delivery system or a combination thereof, such as loaded on lipid nanoparticles (e.g., a combination of cationic lipids, structural lipids, auxiliary lipids and stabilizing lipids), polyurethane (PAA), poly-β-amino ester (PBAE), polyethyleneimine (PEI), and lipid-encapsulated polymer micelles.

[0101] In some embodiments, the vaccine further comprises an adjuvant or is used in combination with an adjuvant, for example, the adjuvant is selected from: aluminum adjuvant, cholera toxin and its subunits, oligodeoxynucleotides, manganese ion adjuvants, colloidal manganese adjuvants, Freund's adjuvant, MF59 adjuvant, QS-21 adjuvant, Poly I:C and other TLR ligands, GM-CSF, IL-2, IL-3, IL-7, IL-11, IL-12, IL-18, IL-21.

[0102] In some embodiments, the vaccine is in a form suitable for one or more administration or delivery methods selected from the following groups: respiratory tract atomization inhalation, nasal drops, oral administration, direct injection (e.g., intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection), and mucosal administration.

[0103] In some embodiments, the vaccine is in a form suitable for combined administration of two or more drugs or vaccines, such as co-vaccination or sequential vaccination. In some embodiments, the vaccine is a monovalent or multivalent vaccine.

[0104] In some embodiments, the vaccine is a recombinant vaccinia virus (rTV) vaccine. In some embodiments, the rTV vaccine comprises the following elements: a vaccinia virus vector; a vaccinia virus recombination site; a vaccinia virus shuttle plasmid; a vaccinia virus promoter; and a selection marker.

[0105] The application also provides a product for the prevention and / or treatment of African swine fever, which comprises one or more immunogenic compositions, recombinant immunogenic peptides, nucleic acid molecules, vectors or host cells or vaccines of the present application.

[0106] In some embodiments, the product is a vaccine kit comprising one or more doses of the same or different vaccines described herein. In some embodiments, the product comprises a combination of one or more doses of a recombinant vaccinia virus vaccine selected from the group consisting of rTV-D117L-E183L-T antigen, rTV-H240R-B438L-C204L, rTV-B646L-B602L, rTV-D117L-E183L-T antigen-H240R-B438L-C204L, rTV-D117L-E183L-T antigen-H240R-B438L-C204L-B646L-B602L, rTV-B646L-B602L, rTV-B646L-B602L-EP402R.

[0107] In some embodiments, the product comprises one or more doses of a combination of recombinant vaccinia virus vaccines selected from the group consisting of any combination of rTV-D117L-E183L-T antigen-H240R-B438L-C204L, rTV-D117L-E183L-T antigen-H240R-B438L-C204L-B646L-B602L.

[0108] In some embodiments, the product comprises a combination of one or more doses of a recombinant vaccinia virus vaccine selected from the group consisting of a combination of rTV-D117L-E183L-T antigen-H240R-B438L-C204L and rTV-D117L-E183L-T antigen-H240R-B438L-C204L-B646L-B602L or rTV-B646L-B602L or rTV-B646L-B602L-EP402R, or a combination of two or three doses of rTV-D117L-E183L-T antigen-H240R-B438L-C204L-B646L-B602L or rTV-B646L-B602L-EP402R.

[0109] In some embodiments, the product includes one or more doses of the following recombinant vaccinia virus combinations: a combination of rTV-D117L-E183L-T antigen-H240R-B438L-C204L and rTV-D117L-E183L-T antigen-H240R-B438L-C204L-B646L-B602L or rTV-rTV-B646L-B602L or rTV-B646L-B602L-EP402R; or a combination of rTV-D117L-E183L-T antigen-H240R-B438L-C204L-B646L-B602L and rTV-B646L-B602L or rTV-B646L-B602L-EP402R.

[0110] In some preferred embodiments of the present application, the immunogenic composition is inserted into a vaccinia virus vector to construct an African swine fever virus vaccinia virus vector vaccine, and the constructed recombinant vaccinia virus includes: rTV-D8L (i.e., rTV-D8L-D117L-E183L-T antigen-DHFR), rTV-D8L-A56R (i.e., rTV-D8L-D117L-E183L-T antigen-DHFR-A56R-H240R-B438L-C 204L), rTV-D8L-A56R-Δ-TK (i.e., rTV-D8L-D117L-E183L-T antigen-DHFR-A56R-H240R-B438L-C204L-TK-B646L-B602L), rTV-TK (i.e., rTV-TK-B646L-B602L), and rTV-TK-K8R (i.e., rTV-TK-B646L-B602L-EP402R).

[0111] Immunization method

[0112] The present invention also provides a method for preventing and / or treating African swine fever virus infection and / or its symptoms, which comprises: administering a preventive and / or therapeutically effective amount of one or more vaccines disclosed herein at least once. The vaccination methods that can be used include, but are not limited to: systemic vaccination methods, such as intramuscular injection, subcutaneous injection, and intradermal injection, etc.; respiratory vaccination methods, such as atomization, nasal drops, etc. In some embodiments, the primary immunization adopts systemic vaccination or respiratory vaccination, preferably systemic vaccination.

[0113] In some embodiments of the present disclosure, the interval between each two vaccinations is at least 1 week, such as 2 weeks, 4 weeks, 2 months, 3 months, 6 months or longer.

[0114] In some embodiments, the immunization method of the present disclosure can adopt the method of "prime immunization-boosting" or "prime immunization-boosting-re-boosting", and can adopt a single systemic immunization or respiratory local immunization method, or a combination of the two immunization methods. In some embodiments, the regimen of first immunizing with rTV-D8L-A56R and then boosting with two injections of rTV-D8L-A56R-Δ-TK can induce high titer binding antibodies against p72, p54 and p30 in vivo, and activate T cell responses against T antigen, p17 and penton, thereby playing a preventive role against African swine fever virus.

[0115] In some embodiments, the primary immunization may use rTV-D8L-A56R, and a combination of rTV-D8L-A56R and rTV-TK or rTV-D8L-A56R-TK may be used for one or two booster immunizations. In some embodiments, the primary immunization may use rTV-D8L-A56R, and a combination of rTV-D8L-A56R and rTV-TK or rTV-D8L-A56R-TK may be used for one or two booster immunizations. In some embodiments, both the primary immunization and the booster immunization use a combination of rTV-D8L-A56R and rTV-TK-K8R. In some embodiments, the time interval between the two vaccinations may be independently selected from: 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, half a year or any interval duration during the period. For example, the interval between the primary immunization and the booster immunization may be 2 weeks, 3 weeks or 4 weeks; the interval between the booster immunization and the re-boost immunization may be 2 weeks, 3 weeks or 4 weeks.

[0116] The combination products herein can be provided in the form of a pharmaceutical package or kit, for example, one or more vaccine compositions herein or one or more components thereof can be packaged in one or more containers, for example, in a sealed container such as an ampoule or a sachet indicating the amount of the composition. The vaccine composition can be provided in the form of a liquid, a sterile lyophilized powder or anhydrous concentrate, etc., which can be diluted, reconstituted and / or formulated with an appropriate liquid (e.g., water, saline, etc.) before use to obtain a suitable concentration and form for administration to a subject.

[0117] The combination product disclosed in the present invention can be used to simultaneously induce high levels of neutralizing antibodies and high levels of T cell responses in a subject, and has a dual protective effect, making it have broad application prospects in the prevention and treatment of African swine fever virus infection.

[0118] Example

[0119] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Those skilled in the art may make appropriate modifications and changes to the present invention, and these modifications and changes are within the scope of the present invention.

[0120] The experimental methods for which the specific conditions are not specified in the following examples can be conventional methods in the art, for example, refer to "Molecular Cloning Laboratory Manual" (3rd edition, New York: Cold Spring Harbor Laboratory Press, 1989) or follow the conditions recommended by the supplier. The DNA sequencing method is a conventional method in the art and can also be tested by commercial companies.

[0121] Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used in the text have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only. The specific information of the sequences used in the examples can be found in the appendix and sequence table.

[0122] The experiments in the examples involve the screening, amplification, in vitro purification, animal immunization scheme and detection methods of African swine fever virus vaccinia virus vector vaccine as follows:

[0123] I. Screening of African swine fever virus vaccinia virus vector vaccine

[0124] 1. Preparation of shuttle plasmid:

[0125] Construction of the pD8L-D117L-E183L-TD shuttle plasmid for the recombination of the D8L site: Referring to the instructions of the Novezan Virus Genome Extraction Kit (Cat. No.: RC311-01), the genome of the Tiantan strain of vaccinia virus was extracted, and the genomic DNA was used as a template to PCR amplify about 700 bp at the 5' and 3' ends of the D8L gene. Referring to the instructions of the Novezan Homologous Recombination Kit (C113-02), the original homologous arms of the vector plasmid pSC65 (purchased from BioVector NTCC Type Culture Collection, Cat. No.: BioVector-790265) were replaced by homologous recombination. D117, E183 and T antigen-DHFR (TD) genes were artificially synthesized, D117 and E183 genes were connected by P2A, E183 and T antigen-DHFR genes were connected by IRES, the target gene D117L-P2A-E183L-IRES-TD was amplified by PCR, and connected to the 3' end of the pE / L promoter on the pSC65 vector. The p7.5 promoter and the downstream eGFP gene were inserted into the 3' end of the target gene, and loxp sequences (SEQ ID NO: 82) were inserted into both ends of p7.5-eGFP, and finally the shuttle plasmid pD8L-pE / L-D117L-P2A-E183L-IRES-TD-loxp-p7.5-eGFP-loxp (hereinafter referred to as pD8L-D117L-E183L-TD) was constructed.

[0126] Using the same method as above, Figure 1The plasmid maps shown in B, 1C and 6A were artificially synthesized and the corresponding target genes were inserted to make shuttle plasmids: pA56R-H240R-B438L-C204L (pA56R-pE / L-H240R-P2A-B438L-p7.5-C204L-IRES-loxp-mcherry-loxp), pSC65-B646L-B602L (pSC65-pE / L-B646L-IRES-B602L-loxp-p7.5-eGFP-loxp) and pK8R-EP402R.

[0127] 2. Shuttle plasmid recombinant wild-type vaccinia virus:

[0128] Construction of rTV-D117L-P2A-E183L-IRES-TD recombinant vaccinia virus with recombinant D8L site: Inoculate about 1×10 cells per well of a six-well plate one day in advance. 6 293T cells (purchased from ATCC, catalog number: CRL-3216) were prepared, and the cell density was preferably 80% on the next day. Wild-type vaccinia virus 752-1 (purchased from China Center for Type Culture Collection) was infected at MOI = 0.02. After 2 hours of infection, the complete medium containing the virus supernatant was discarded, 2 mL of fresh maintenance medium was added, and pD8L-D117L-E183L-TD plasmid (transfection reagent purchased from Li Ji Biotechnology) was transfected. The well plate was placed in an incubator at 37°C and 5% CO2 for about 24 hours. The cells were collected on the second day and frozen and thawed three times in a -80°C refrigerator.

[0129] Using essentially the same method as above, recombinant poxviruses were generated using pA56R-H240R-B438L-C204L, pSC65-B646L-B602L or pK8R-EP402R.

[0130] 3. Screening of recombinant vaccinia virus:

[0131] One day in advance, approximately 8 × 10 5143B cells (purchased from ATCC, item number: CRL-8303) were added to ensure that the cell density reached 95% on the second day. The complete culture medium in the well was discarded, 2 mL of fresh maintenance medium was added, 10 μL of the above frozen and thawed cells were infected in each well, and the well plate was placed in an incubator at 37 ° C and 5% CO2 for about 24 hours. The culture medium in the well was discarded on the second day, 2% agarose and 2×DMEM culture medium were mixed in a 1:1 ratio, 1 mL was added to each well, and the well plate was placed in a 4 ° C refrigerator for about 5 minutes, and then fluorescent plaques were selected under a fluorescence microscope, and the plaques were sucked out with a 1 mL pipette tip into a 1.5 mL EP tube containing 500 μL maintenance medium, and frozen and thawed three times in a -80 ° C refrigerator. Repeat this step for about 6-8 rounds until the recombinant vaccinia virus was successfully screened, and its genome was extracted and verified by PCR with primers of homologous arms at both ends. There should be only fragments that meet the size of the target gene, and no wild virus fragments.

[0132] II. Amplification and purification of African swine fever virus vaccinia virus vector vaccine

[0133] 1. Recombinant vaccinia virus amplification:

[0134] The successfully screened recombinant vaccinia virus was amplified in a six-well plate until all cells were infected. The cells were collected and frozen and thawed three times in a -80°C refrigerator. One day in advance, about 1×10 7 DF-1 cells (purchased from ATCC, catalog number: CRL-3586) were added to ensure that the cell density reached 95% on the next day. The complete medium in the dish was discarded, 10 mL of fresh maintenance medium was added, and the virus after freezing and thawing was infected. The cells were placed in an incubator at 37°C and 5% CO2 for culture. The infected cells were collected within 24-48 hours according to the cytopathic effect and frozen and thawed three times in a -80°C refrigerator. About 3×10 7 DF-1 cells, ensuring that the cell density reaches 95% on the second day. Repeat the above steps, collect the infected cells and freeze and thaw three times in a -80°C refrigerator. Expand DF-1 cells to T175 culture bottles, pass them at 1:10 after full growth, and spread the remaining cells to 15cm dishes at 1:10. After 48h, replace the culture medium in the dish with 20mL maintenance medium, infect the seeds collected in the previous round at 1:10 (i.e., 1 15cm dish of cells infects 10 15cm dishes), collect the infected cells and freeze and thaw three times in a -80°C refrigerator. Expand DF-1 cells to 6 bottles of T175 culture bottles, repeat the above steps to spread the remaining cells to 60 15cm dishes at 1:10, infect the seeds collected in the previous round at 1:10, collect the infected cells and freeze and thaw three times in a -80°C refrigerator. Repeat this step 2-3 times according to the required amount of virus, collect the cells after each round of infection and store them in a -80°C refrigerator.

[0135] 2. Recombinant vaccinia virus purification and titer determination:

[0136] The collected cells were frozen and thawed three times in a -80℃ refrigerator, centrifuged at 3000g for 10min, and the supernatant was collected. The precipitate was resuspended with 10mL PBS, centrifuged at 3000g for 10min, and the supernatant was collected. Wash three times in total. Add 12mL 36% sucrose to the ultracentrifuge tube, tilt the ultracentrifuge tube, and slowly add 24mL of virus supernatant in a ratio of 1:2 to ensure the formation of clearly visible stratification. Weigh and balance with an electronic balance, and the error is controlled within 0.1g. Set the ultracentrifuge parameters as follows: speed 18000g, temperature 4℃, speed increase 9 speed decrease 4, time 1.5h. After centrifugation, a clear white precipitate can be seen at the bottom of the centrifuge tube. Carefully discard the supernatant, remove excess supernatant with a pipette tip, resuspend with 200 μL PBS and collect the precipitate, aspirate 3 times with 100 μL PBS and collect the precipitate, aliquot the precipitate into 100 μL / tube, and aliquot 5 μL separately for titer determination and store in a -80°C refrigerator.

[0137] One day in advance, seed each well of a 24-well plate with approximately 2 × 10 5 143B cells, and ensure that the cell density reaches 95% on the next day. Prepare 8 1.5mL EP tubes, add 396μL of maintenance medium to the first EP tube, and add 990μL of maintenance medium to each of the remaining tubes. Take out the 5μL of virus from the -80℃ refrigerator, pipette 4μL and add it to the first EP tube for a 1:100 dilution. After fully pipetting and mixing, pipette 110μL to the second EP tube for a 1:10 dilution, and dilute it to the last EP tube at a 10-fold ratio. Discard the culture medium in the 24-well plate, mix the culture medium of each dilution multiple thoroughly and add it to two wells, 450μL to each well, and add it from high to low according to the dilution multiple. Place the well plate in an incubator at 37℃ and 5% CO2 for incubation. After 48h, count the number of plaques at the end point of the dilution multiple, and calculate the virus titer according to the following formula:

[0138] Virus titer = the sum of the number of plaques in two wells at the same dilution factor × 10 稀释倍数 PFU / mL III. Mouse immunization protocol

[0139] 1. Experimental Animals:

[0140] Female BALB / c mice aged 6 to 8 weeks were purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd.

[0141] 2. Immunization method:

[0142] The mice were injected intramuscularly on the outside of the left and right hind limbs.

[0143] 3. Preparation of immunogens and immunization doses:

[0144] For the preparation of immunogens, see Examples 1 and 4.

[0145] The immunization dose was 1E7 PFU / mouse, 100 μL.

[0146] 4. Immunization interval:

[0147] In Example 2, the primary immunization was performed on day 0, the booster immunization was performed 21 days later, and the booster immunization was performed again 49 days later. In Example 3, the primary immunization was performed on day 0, the booster immunization was performed 14 days later, and the booster immunization was performed again 28 days later. In Example 5, the primary immunization was performed on day 0, the booster immunization was performed 21 days later, and the booster immunization was performed again 42 days later.

[0148] IV. Mouse Testing Protocol

[0149] 1. Blood collection:

[0150] On the 35th, 63rd and 77th days, blood was collected from the orbital venous plexus of mice into 1.5 mL EP tubes and allowed to stand at room temperature until the blood samples coagulated. The samples were centrifuged at 7000 g for 15 min, and the serum was transferred to a clean 1.5 mL EP tube. The serum was centrifuged at 14000 g for 2 min, and the serum was transferred to a clean 1.5 mL EP tube. The serum was inactivated in a 56°C water bath for 30 min.

[0151] 2.ELISA detection of binding antibodies:

[0152] 1) Dilute the antigen protein (p72: purchased from Beijing Sino Biological Technology Co., Ltd.; p54 and p30 purchased from Antibody system) to a final concentration of 1 μg / mL with ELISA diluent, 100 μL per well, and coat overnight;

[0153] 2) On the second day, discard the liquid in the wells and wash three times with PBST containing 0.05% Tween-20, 200 μL per well each time. After the last wash, discard the liquid in the wells and pat dry.

[0154] 3) Add 200 μL of blocking solution (5% skim milk powder, dissolved in PBST) to each well and block for 2 h at room temperature;

[0155] 4) After blocking, discard the liquid in the wells and wash three times with PBST containing 0.05% Tween-20, 200 μL per well each time. After the last wash, discard the liquid in the wells and pat dry.

[0156] 5) Except for the first well, add 100 μL of blocking solution to each well of the remaining sample wells. Dilute the serum 100 times with the blocking solution, mix thoroughly, take 200 μL and add it to the first well of each sample, and then dilute it 2 times, that is, pipette 100 μL into the next dilution well, mix thoroughly and repeat the operation. After the last sample well is fully mixed, discard 100 μL of liquid and incubate at room temperature for 3 hours;

[0157] 6) Discard the liquid in the wells, wash with PBST containing 0.05% Tween-20 5 times, 200 μL per well each time, and discard the liquid in the wells for the last time and pat dry;

[0158] 7) Dilute HRP-labeled secondary antibody (goat anti-mouse, purchased from Yisheng) with blocking solution at 1:5000, 100 μL per well, incubate at room temperature for 1 h;

[0159] 8) Discard the liquid in the wells, wash with PBST containing 0.05% Tween-20 for 5 times, 200 μL per well each time, and discard the liquid in the wells for the last time and pat dry;

[0160] 9) Each pair of gold and silver OPD substrates was dissolved in 20 mL of deionized water, 100 μL was added to each well, and the reaction was allowed to proceed for 5 min in the dark;

[0161] 10) Add 50 μL 2M H2SO4 to each well to terminate the reaction and read the OD on a microplate reader. 492 -OD 630 nm reading;

[0162] 11) Using the negative control group OD 492 Twice the reading was taken as the cut-off value, and the reciprocal of the last dilution multiple of each sample greater than this value was the antibody titer.

[0163] 3.ELISpot detection of T cell response:

[0164] Isolation of single cells from mouse spleen:

[0165] 1) Kill the mouse, place it on its side with the left side facing upward, cut open the skin on the left side of the abdomen, open the peritoneum, remove the spleen of the mouse with sterile forceps, and place it in a 6 cm small dish with 5 mL of complete RPIM1640 medium (R10);

[0166] 2) Wrap the spleen with sterile gauze, hold the end of the gauze with curved forceps, and gently grind the spleen to release splenocytes into the culture medium;

[0167] 3) Transfer the spleen suspension to a 15 mL centrifuge tube and centrifuge at 800 g for 5 min;

[0168] 4) Discard the supernatant, tap the bottom of the centrifuge tube to resuspend the cells, add 5 mL of lysis solution to each tube, invert to mix, and let stand at room temperature for 5 minutes;

[0169] 5) Add 8 mL of R10 to stop red cell lysis and centrifuge at 800 g for 5 min;

[0170] 6) Discard the supernatant, wash once with 5 mL of R10, and centrifuge at 800 g for 5 min;

[0171] 7) Discard the supernatant, resuspend the cells in 5 mL R10, take 20 μL of the cell suspension and add it to 380 μL R10 for 20-fold dilution, count and pipette the required volume of cell suspension according to the experimental design;

[0172] 8) After centrifugation, discard the supernatant of the remaining cells and place them in freezing solution (90% FBS and 10% DMSO) for future use.

[0173] ELISpot detection was performed according to the instructions of the MOUSE IFN-γ kit (purchased from BD, catalog number 551083):

[0174] 1) Dilute IFN-γ antibody (1:200) with sterile PBS, add 100 μL to each well of the Millipore plate, and coat overnight at 4°C;

[0175] 2) Discard the liquid in the plate, wash once with 200μL R10, let stand for 3 minutes, and discard. Add 200μL fresh R10 and block at room temperature for 2 hours;

[0176] 3) Discard the liquid in the plate and add stimulatory peptide library to the plate according to the experimental design, 50 μL per well, where the working concentration of each single peptide is 5 μg / mL, and add 50 μL R10 to each well of the negative control;

[0177] Among them, the stimulatory peptide library was synthesized by Suzhou Qiangyao Biotechnology Co., Ltd., covering T antigen, p17 and penton sequences respectively. Every 10 single peptides constitute a peptide library. Among them:

[0178] ——T antigen contains 31 T cell epitopes (polypeptides shown in SEQ ID NO: 14+2n, n=1-31), each single peptide is 15 amino acids, the first peptide of the first epitope is FMREIKVKEVLYFYT, i.e. aa1-15 in SEQ ID NO: 16, and each subsequent peptide is shifted 4 amino acids compared to the previous peptide. The first peptide of the second epitope is GRVPGFSKEMLFQYI, i.e. aa1-15 in SEQ ID NO: 18, and each subsequent peptide is shifted 4 amino acids compared to the previous peptide, and so on to cover each epitope.

[0179] ——The first peptide of p17 is MDTETSPLLSHNLST, i.e., aa1-15 in SEQ ID NO:12, and each subsequent peptide is shifted by 4 amino acids compared to the previous peptide, i.e., the second peptide is aa5-19 in SEQ ID NO:12, and the third peptide is aa9-23 in SEQ ID NO:12... and so on, the 27th peptide IPSDEQLAELAHS (SEQ ID NO:12aa105-117) is less than 15 amino acids, so the 28th peptide SHIPSDEQLAELAHS is synthesized to make up to 15 amino acids (SEQ ID NO:12aa103-117).

[0180] ——The first peptide of penton is MAANIIATRAVPKMA, i.e. aa1-15 in SEQ ID NO:6, and each subsequent peptide is shifted by 4 amino acids compared to the previous peptide, i.e. the second peptide is aa5-19 in SEQ ID NO:6, the third peptide is aa9-23 in SEQ ID NO:6... and so on, the 58th peptide RIPLYFKSLKTSK (aa229-241 of SEQ ID NO:6) is less than 15 amino acids, so the 59th peptide RIRIPLYFKSLKTSK is synthesized to make it up to 15 amino acids (aa227-241 of SEQ ID NO:6).

[0181] 4) After counting the mouse spleen cells, the cell concentration was adjusted to 4×10 6 cells / mL, add 50 μL (i.e., 2×10 cells / mL) vertically to each well. 5 The Millipore plate was carefully placed in a 37°C, 5% CO2 incubator for 20 h, and the plate was prevented from moving during this period;

[0182] 5) After incubation, remove the Millipore plate, discard the liquid in the plate, add 200 μL of pre-cooled distilled water to each well and wash twice, each time for 5 minutes;

[0183] 6) Discard the liquid in the plate, add 200 μL of PBST containing 0.05% Tween-20 to each well and wash three times, 1 min each time. Pat the remaining liquid dry on absorbent paper for the last time;

[0184] 7) Dilute the biotin-conjugated detection antibody at 1:250 in antibody diluent (PBS + 10% FBS), add 100 μL to each well, and incubate at room temperature for 2 h;

[0185] 8) After incubation, discard the liquid in the plate, add 200 μL of PBST containing 0.05% Tween-20 to each well and wash three times, each time for 1 min. After the last wash, pat the remaining liquid dry on absorbent paper;

[0186] 9) Dilute streptavidin-HRP in antibody diluent (PBS + 10% FBS) at 1:100, add 100 μL to each well, and incubate at room temperature for 1 h;

[0187] 10) Discard the liquid in the plate, add 200 μL of PBST containing 0.05% Tween-20 to each well and wash 4 times;

[0188] 11) Add 200 μL PBS to each well and wash twice. Pat the remaining liquid on absorbent paper for the last wash.

[0189] 12) Prepare the color development solution just before use: add 1 drop of color development substrate to every 1 mL of color development buffer, mix well, add 100 μL to each well, and react at room temperature in the dark for 5-60 minutes;

[0190] 13) Observe the color development carefully. When clear red spots appear, rinse the plate gently under running water for 5 minutes to stop the reaction.

[0191] 14) After drying at room temperature, the plate was counted and the spot forming cell (SFC) was counted and QC analysis was performed using a ChampSpot III enzyme-linked spot image analyzer.

[0192] V. Evaluation scheme for protection against virus attack in experimental pigs

[0193] 1. Experimental Animals:

[0194] Local pigs aged 40-50 days were purchased from Harbin Veterinary Research Institute.

[0195] 2. Immunization method:

[0196] Inject intramuscularly into the triangular area behind the ear of the pig.

[0197] 3. Preparation of immunogens and immunization doses:

[0198] For the preparation of immunogens, see Examples 1 and 5.

[0199] The immunization dose is 2E8-5E8 PFU / pig, 1 mL.

[0200] 4. Immunization interval:

[0201] The first immunization was performed on day 0 of the experiment, and the booster immunization was performed 28 days later.

[0202] 5.Challenge virus strain: HLJ / 18.

[0203] 6. Virus challenge procedure: The virus challenge was carried out in the P4 laboratory of Harbin Veterinary Research Institute. On the 28th day after immunization, each pig was injected intramuscularly with 100HAD of African swine fever virus. 50 The animals were observed for 21 days after the challenge. The body temperature was measured daily and the clinical scores and survival were recorded.

[0204] Example 1. Construction, amplification, purification and expression verification of recombinant vaccinia virus

[0205] In order to construct a safe and effective African swine fever vaccine, we screened out highly immunogenic exogenous genes by consulting literature and data summary. In order to further activate the cellular immune response of pigs, we screened out T cell epitopes widely recognized by the pig major histocompatibility complex (SLA) molecule through bioinformatics and fused them into T antigen (SEQ ID NO:78), and inserted DHFR (SEQ ID NO:80) at its 3' end to increase the degradation level of T antigen in cells. Then, 8 exogenous genes, including B646L, B602L, H240R, B438L, C204L, D117L, E183L, and T antigen, were artificially synthesized and inserted into vaccinia virus vectors to construct African swine fever virus vaccinia virus vector vaccine.

[0206] The eight exogenous genes were divided into three groups: ① D117L, E183L, T antigen; ② H240R, B438L, C204L; ③ B646L, B602L. It is worth noting that this grouping method is only one combination method, and other combinations that can achieve the expression of all exogenous genes are within the scope of protection of this patent. According to experimental method I, shuttle plasmids of the three regions of D8L, A56R, and TK were constructed respectively, and the three groups of exogenous genes were inserted into these three shuttle plasmids in turn, and finally the shuttle plasmid map was constructed as shown in FIG. Figure 1 shown.

[0207] According to the experimental method I, the shuttle plasmid pD8L-D117L-E183L-T antigen DHFR was recombined with 752-1, and the recombinant vaccinia virus was screened by eGFP ( Figure 2 A left), after 5 rounds of screening, rTV-D8L-D117L-E183L-T antigen-DHFR (hereinafter referred to as rTV-D8L) was obtained. After rTV-D8L was amplified in a six-well plate, the genome was extracted as a template and PCR was verified using primers D8L-F / D8L-R (SEQ ID NO: 83 and 84). Figure 2 As shown in A, there is only a single band in PCR, and the size is consistent with the expected size of 6223 bp.

[0208] Further detection of rTV-D8L expression: About 8 × 10 5143B cells were added to ensure that the cell density reached 95% on the second day. The culture medium in the well was replaced with 2 mL of maintenance medium. One well was infected with rTV-D8L at an MOI of 0.2, and the other well was not treated. After 24 hours, the cells were collected to prepare Western Blot samples for protein immunoblotting identification. Figure 2 As shown in A, a large amount of p17 and p54 protein expression can be detected in cells infected with rTV-D8L, while the expression level of T antibodies is relatively low because they carry DHFR.

[0209] Next, based on rTV-D8L, the shuttle plasmid pA56R-H240R-B438L-C204L was recombined. This plasmid carries the mcherry reporter gene and the two-in-one recombinant vaccinia virus ( Figure 2 B left), after 7 rounds of screening, rTV-D8L-D117L-E183L-T antigen-DHFR-A56R-H240R-B438L-C204L (hereinafter referred to as rTV-D8L-A56R) was obtained. After rTV-D8L-A56R was amplified in a six-well plate, the genome was extracted as a template, and PCR verification was performed with primers D8L-F / D8L-R and A56R-F / A56R-R (SEQ ID NO: 85 and 86). The results showed that the D8L region still maintained a stable single band, and the A56R region had only a single band, the size of which was consistent with the expected size of 4925 bp ( Figure 2 B). The expression of rTV-D8L-A56R was further detected by protein immunoblotting. In addition to expressing the originally inserted p17, p54 and T antigen, rTV-D8L-A56R also highly expressed penton, p49 and p30, and the sizes were consistent with expectations ( Figure 2 B (right) only shows the expression of penton, p49 and p30).

[0210] Because pSC65-B646L-B602L carries the eGFP selection marker, before inserting B646L-B602L into rTV-D8L-A56R, the eGFP and mcherry reporter genes carried by rTV-D8L-A56R were knocked out using the Cre-loxP system: about 1×10 6293T cells were added to ensure that the cell density reached 80% on the second day. 4 μg phage-cre (shown in SEQ ID NO: 95) plasmid was transfected using Li Ji. After 6 hours of transfection, rTV-D8L-A56R was infected at MOI=0.02. After 2 hours, the supernatant was discarded and 2 mL of fresh maintenance medium was added. According to the cell infection state, the cells were collected about 24 hours later and frozen and thawed three times in a -80°C refrigerator. According to the experimental method I, the vaccinia virus that does not express eGFP and mcherry was reversely screened by fluorescence microscopy. After 3 rounds of screening, rTV-D8L-A56R-eGFPΔ-mcherryΔ (hereinafter referred to as rTV-D8L-A56R-Δ) was obtained. The genome was extracted as a template and PCR was verified with primers D8L-F / D8L-R and A56R-F / A56R-R. There was a single band at the target band position, and protein immunoblotting detection could detect the expression of the target gene. Next, based on rTV-D8L-A56R-Δ, the shuttle plasmid pSC65-B646L-B602L was recombined. After 10 rounds of screening, rTV-D8L-D117L-E183L-T antigen-DHFR-A56R-H240R-B438L-C204L-TK-B646L-B602L (hereinafter referred to as rTV-D8L-A56R-Δ-TK) was obtained. Figure 2 C left). The genome was extracted as a template and PCR was performed using primers D8L-F / D8L-R (SEQ ID NOs: 83 and 84), A56R-F / A56R-R (SEQ ID NOs: 85 and 86), and TK-F / TK-R (SEQ ID NOs: 87 and 88). The bands in the D8L and A56R regions remained stable, and the target band in the TK region was consistent with the size of 6331 bp ( Figure 2 C), protein immunoblotting detected the expression of p17, p54, T antigen, penton, p49, p30, p72, and p72 chaperone ( Figure 2 C (right) shows only the expression of p72 and p72 chaperone).

[0211] According to the experimental method I, the shuttle plasmid pSC65-B646L-B602L was recombined with 752-1, and the recombinant vaccinia virus was screened by eGFP ( Figure 2 D left), rTV-TK-B646L-B602L (hereinafter referred to as rTV-TK) was obtained after screening, and the insertion and expression of the exogenous gene were detected by PCR and protein immunoblotting ( Figure 2 D center, right).

[0212] The above data indicate that rTV-D8L-A56R, rTV-D8L-A56R-Δ-TK and rTV-TK were successfully screened and constructed, and the target gene was correctly inserted into different recombination sites and expressed normally.

[0213] Example 2. Different combinations of recombinant vaccinia viruses induce immune responses in mice

[0214] The screened rTV-D8L-A56R, rTV-D8L-A56R-Δ-TK and rTV-TK were amplified in large quantities according to experimental method I. After virus purification, the titers were determined to be 5×10 8 PFU / mL, 8×10 8 PFU / mL and 1.1×10 9 PFU / mL. 24 BALB / c mice were randomly divided into 4 groups and immunized as shown in Table 1. Each group was injected intramuscularly. The single immunization volume for each mouse was 100 μL, and the dose was 1E7 PFU. The immunization process was as follows: Figure 3 As shown in A. Peripheral blood was collected from the orbital venous plexus of mice on days 35, 63, and 77, and the levels of binding antibodies against p72, p54, and p30 in mice were evaluated by ELISA. Splenocytes of mice were isolated on day 77, and specific T cells against T antigen, p17, and penton induced by different recombinant vaccinia virus combinations were analyzed by ELISA.

[0215] like Figure 3 As shown in B, with the increase in the number of immunizations, the binding antibody levels of p72, p54, and p30 all increased significantly. Compared with immunization with 2 injections, the binding antibody level at the same time point (35th day vs. 63rd day) after 3 injections increased significantly, with the highest increase of p72, p54, and p30 being 9 times, 25.4 times, and 9 times, respectively. Compared with day 63, the antibody level on day 77 remained basically unchanged or slightly increased. There was no significant difference in the binding antibody level against p72 induced by different immunization combinations. On day 77, the antibody titers of each group were 7611, 25600, and 9051, respectively. The antibody level titer against p54 in group 1 was 7184 on day 35 of immunization, which was significantly higher than that of the other two groups. There was no significant difference in the antibody titers of p54 induced by Group 1 and Group 3 on day 77, while the antibody titer of p54 induced by three injections of rTV-D8L-A56R-Δ-TK on day 77 was 5382, indicating that the induction of binding antibodies was not balanced. The binding antibody titers of p30 induced by each group were generally low, which may be because p30 is driven by the p7.5 promoter, and its promoter activity is weaker than that of pE / L, so its expression level in mice is weaker than that of p72 and p54. The binding antibody titer of Group 3 against p30 on day 77 was 1345, and the titers of other groups were all between 400 and 600.

[0216] T cell immune response results Figure 3 As shown in Figure C, compared with the control group, the recombinant vaccinia virus can induce cellular immune responses against T antigen, p17 and penton in mice. Group 1 had the best induction results, followed by group 3 and finally group 2. The T cell response to the T antigen peptide library was weaker than that to the p17 and penton peptide libraries, which may be because the T antigen immunogen was screened by evaluating the binding to the pig SLA molecule, which has species differences with the mouse MHC molecule.

[0217] The above data suggest that first immunization with rTV-D8L-A56R and then two booster injections with rTV-D8L-A56R-Δ-TK can induce higher levels of p72, p54 and p30 antibodies and T cell responses in mice. The combination of three injections of rTV-D8L-A56R+rTV-TK is superior to three injections of rTV-D8L-A56R-Δ-TK, and is not inferior to the immunization method of group 1 in inducing binding antibody levels.

[0218] Table 1. Immunogenicity of African swine fever virus vaccinia virus vector vaccine in BALB / c mice

[0219]

[0220] Example 3. Optimization of recombinant vaccinia virus combinations to induce immune responses in mice

[0221] Based on the results of Example 2, we further explored a more optimal recombinant vaccinia virus combination. Fifteen BALB / c mice were randomly divided into three groups and immunized as shown in Table 2. The immunization process was as follows: Figure 4 As shown in A. The mice were killed on day 56, and the levels of binding antibodies against p72, p54, and p30 in the mice were evaluated by ELISA. At the same time, the mouse spleen cells were isolated, and the specific T cells against T antigen, p17, and penton induced by different recombinant vaccinia virus combinations were analyzed by ELISA.

[0222] Table 2. Immunogenicity of African swine fever virus-vaccinia virus vector vaccine combination in BALB / c mice

[0223]

[0224] The results are as follows Figure 4 As shown, there was little difference between the two groups in inducing binding antibodies, with Group 1 being slightly better than Group 2 in inducing p72 binding antibodies, while Group 2 was slightly better in inducing p54 binding antibodies. There was no significant difference between the two groups in inducing T cell responses.

[0225] It can be seen from this embodiment that the provided recombinant vaccinia virus vector immune combination for African swine fever virus, such as first immunizing rTV-D8L-A56R, and then using rTV-D8L-A56R+rTV-TK to strengthen two injections, can induce high titer binding antibodies against p72, p54 and p30 in mice, and can also activate T cell responses against T antigen, p17 and penton, and has certain application prospects in the field of preventing African swine fever virus. The more exogenous genes the vaccinia virus is loaded with, the lower its amplification and replication ability will be to a certain extent. Considering the future industrial production cost, the better recombinant vaccinia virus combination is to first immunize rTV-D8L-A56R, and then use rTV-D8L-A56R+rTV-TK to strengthen two injections.

[0226] Example 4. Further optimization of recombinant vaccinia virus

[0227] According to the above examples, we have found out the optimal immune combination of recombinant vaccinia virus vector: first immunize with rTV-D8L-A56R, and then boost with two injections of rTV-D8L-A56R+rTV-TK. Based on this scheme, we inserted a new immune exogenous gene EP402R (SEQ ID NO: 97) into the K8R region of rTV-TK, which encodes CD2v protein (i.e., EP402R polypeptide, SEQ ID NO: 98), and constructed rTV-TK-B646L-B602L-K8R-EP402R (hereinafter referred to as rTV-TK-K8R).

[0228] According to the experimental method I, based on rTV-TK, the shuttle plasmid pK8R-EP402R ( Figure 5 A), the plasmid carries the mcherry reporter gene, and the recombinant vaccinia virus was screened by mcherry. After multiple rounds of screening, rTV-TK-K8R ( Figure 5 B). The genome was extracted as a template and PCR was performed using primers K8R-F and K8R-R (SEQ ID NO: 99 and 100). The results showed that there was only a single band in the K8R region, and the size was consistent with the expected size of 2690 bp ( Figure 5 C). Western blot detected the effective expression of immunogen ( Figure 5 D). It shows that the recombinant vaccinia virus rTV-TK-K8R was successfully constructed, and EP402R was correctly inserted into the K8R region of the vaccinia virus genome and could be expressed normally. In addition, the above results also prove that after further loading the immune exogenous gene EP402R in the vaccinia virus, its amplification and replication ability was not adversely affected, and the obtained recombinant vaccinia virus can be used for the correct and effective loading and expression of the contained immunogen. This combination method not only fully utilizes the advantage of the large capacity of the vaccinia virus, but also enables a single vaccinia virus to load an exogenous gene of appropriate size to ensure its amplification and replication ability.

[0229] Example 5. Further optimized recombinant vaccinia virus combination induces immune response in mice

[0230] In order to explore the effect of the addition of new immunogens on the response to the original immunogens, we set up the following experiment: all experimental groups of mice were first immunized with rTV-D8L-A56R, one group of mice was boosted with a combination of rTV-D8L-A56R and rTV-TK, and the other group of mice was boosted with a combination of rTV-D8L-A56R and rTV-TK-K8R. Antibody and T cell responses were detected 4 weeks after the end of immunization ( Figure 6 A), please refer to the immunization shown in Table 3 for details.

[0231] Table 3. Immunogenicity of two ASFV-vaccinia virus vector vaccine combinations in BALB / c mice

[0232]

[0233] The combined antibody data showed that the addition of the new immunogen did not weaken the original immunogen response. There was no significant difference in the p72, p54, and p30 binding antibodies in the two groups of mice. From the overall trend, the p72 binding antibodies induced by the combination of rTV-D8L-A56R and rTV-TK-K8R were slightly higher ( Figure 6 B) T cell response data also showed that, although there was no significant difference between the two groups, there were more positive cells against each peptide pool in the rTV-D8L-A56R and rTV-TK-K8R combination mice ( Figure 6 C).

[0234] Based on the above embodiments, it can be seen that the present invention provides an optimized recombinant vaccinia virus vector immune combination for African swine fever virus, that is, first immunizing with rTV-D8L-A56R, and then strengthening with two injections of rTV-D8L-A56R+rTV-TK-K8R. It can induce high-titer binding antibodies against p72, p54 and p30 in mice, and can also activate T cell responses against T antigen, p17 and penton, and has broad application prospects in the field of prevention of African swine fever virus.

[0235] Example 6. Evaluation of the protection of recombinant vaccinia virus combination in challenge of pigs in situ

[0236] This vaccine was developed for the prevention and control of African swine fever. An African swine fever virus vaccine was constructed by screening an African swine fever virus immunogen composition. The immune response induced by the recombinant vaccinia virus combination in pigs and the protective effect against virus were further explored.

[0237] Ten local pigs aged 40-50 days were selected and randomly divided into two groups, including 4 in the control group and 6 in the experimental group. They were immunized as shown in Table 4. Each group was injected intramuscularly, and the single immunization volume of each experimental pig was 1 mL. The first injection was 2E8 PFU per experimental pig, and the second injection was 5E8 PFU per experimental pig. After the immunization was completed, the virus was challenged in the fourth week. Each pig was injected intramuscularly with 100 HAD of HLJ / 18 strain. 50 After the virus attack, the patients were observed for 21 days. The experimental procedure was as follows: Figure 7 As shown in A.

[0238] Table 4. Immunogenicity of African swine fever virus vaccinia virus vector vaccine in local pigs

[0239]

[0240] All test pigs were transferred to the P4 laboratory on the 53rd day, challenged with the virus on the 56th day, and observed for 21 days after the challenge. Clinical scores were performed on each pig every day during the observation period. The scoring criteria are shown in Table 5. The test pigs showed typical clinical symptoms after being infected with African swine fever virus: loss of appetite, depression, and lying down. All test pigs gradually developed clinical symptoms from the 4th day after the challenge and continued to worsen. Among them, the clinical score of the control group gradually increased and remained at the highest level from the day of the challenge to the day of onset and death. The clinical score of the vaccine group gradually decreased from about the 10th day, indicating that the clinical symptoms gradually alleviated, and basically recovered to a healthy state 21 days after the challenge ( Figure 7 B). The body temperature of the control group and the vaccine group gradually increased after the virus challenge. The body temperature of the control group was maintained above 40°C from the 4th day after the virus challenge. The body temperature of the vaccine group gradually decreased from the 7th day after the virus challenge, and dropped below 40°C after the 14th day after the virus challenge and gradually returned to the body temperature before the virus challenge ( Figure 7 C). Finally, all 4 test pigs in the control group died within 15 days, while 4 of the 6 test pigs in the vaccine group survived, with a survival rate of about 66.7% ( Figure 7 C below).

[0241] Generally speaking, increasing the number of vaccinations can induce a stronger immune response. Compared with other embodiments, this embodiment only immunizes two injections of the candidate vaccine before the virus challenge, and can achieve a protection rate of 66.7%. Therefore, it is reasonable to speculate that the protective effect of the vaccine combination in other embodiments will be at least not inferior to that of this embodiment.

[0242] Table 5. Clinical scoring system for African swine fever

[0243]

[0244]

[0245] In summary, the recombinant vaccinia virus vector immune combination against African swine fever virus provided by the present invention can effectively induce humoral immune response and cellular immune response in experimental pigs, and provide protection for experimental pigs against attacks by virulent African swine fever.

[0246] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0247] Appendix: Sequence Information

[0248] 1. Immunogenicity Sequence

[0249]

[0250] 2. Other sequences

[0251]

[0252] 3. Specific sequence

[0253] SEQ ID NO: 1: B646L gene

[0254]

[0255] SEQ ID NO:2: B646L polypeptide (p72)

[0256] MASGGAFCLIANDGKADKIILAQDLLNSRISNIKNVNKSYGKPDPEPTLSQIEETHLVHFNAHFKPYVPVGFEYNKVRPHTGTPTLGNKLTFGIPQYGDFFHDMVGHHILGACHSSWQDAPIQGTSQMGAHGQLQTFPRNGYDWDNQTPLEGAVYTLVDPFGRPIVPGTKNAYRNLVYYCEYPGERLYENVRFDVNGNSLDEYSSDVTTLVRKFCIPGDKMTGYKHLVGQEVSVEGTSGPLLCNIHDLHKPHQSKPILTDENDTQRTCSHTNPKFLSQHFPENSHNIQTAGKQDITPITDATYLDIRRNVHYSCNGPQTPKYYQPPLALWIKLRFWFNENVNLAIPSVSIPFGERFITIKLASQKDLVNEFPGLFVRQSRFIAGRPSRRNIRFKPWFIPGVINEISLTNNELYINNLFVTPEIHNLFVKRVRFSLIRVHKTQVTHTNNNHHDEKLMSALKWPIEYMFIGLKPTWNISDQNPHQHRDWHKFGHVVNAIMQPTHHAEISFQDRDTALPDACSSISDISPVTYPITLPIIKNISVTAHGINLIDKFPSKFCSSYIPFHYGGNAIKTPDDPGAMMITFALKPREEYQPSGHINVSRAREFYISWDTDYVGSITTADLVVSASAINFLLLQNGSAVLRYST

[0257] SEQ ID NO:3: B602L gene

[0258]

[0259] SEQ ID NO:4: B602L polypeptide (p72 chaperon)

[0260] MAEFNIDELLKNVLEDPSTEISEETLKQLYQRTNPYKQFKNDSRVAFCSFTNLREQYIRRLIMTSFIGYVFKALQEWMPSYSKPTHTTKTLLSELITLVDTLKQETNDVPSESVVNTILSIADSCKTQTQKSKEAKTTIDSFLREHFVFDPNHAQSAYTCADTNVDTCASMCADTNVDTCASMCADTNVDTCASTCTSTEYTDLADPERIPLHIMQKTLNVPNELQADIDAITQTPQGYRAAHILQNIELHQSIKHMLENPRAFKPILFNTKITRYLSQHIPPQDTFYKWNYIIEDNYEELRAATESIYPEKPDPDLEFAFIIYDVVDSSNQQKVDEFYYKYKKDQIFSEVSSIQLGNWTLLGSFKANRERYNYFNQNNEIIKRILDHRHEEDLKIGKEILRNTIYHKKAKNIQETGPDAPGLSIYNSTFHTDSGIKGLLSFKELKNLEKASGNIKKAREYDFIDDCEEKIKQLLSKENLTPDEESELIKTKKQLDNALEMLNVPDDTIRVDMWVNNNNNKLEKEKEILYTKAEL

[0261] SEQ ID NO:5: H240R gene

[0262] atggctgcaaacattattgcaacaagagccgtgccaaagatggccagcaaaaaagagcatcaatactgtctgctagactcccaggaaaagcgtcatgggcattatcccttttcatttgaattaaagccttatgggcaaacaggcgcaaatatcataggagtacagggctcacttacccatgttatcaaaatgacagtatttccatttatgattccttttcctttacaaaaaactcatatagatgattttattggtggacgcatttatttattttttaaggaactggacatgcaagcagtttctgatgtaaatggaatgcaataccacttcgagttcaaggttgttcctgtaagccccaaccaagtagagcttcttcctgtgaataataaatataaatttacatatgctataccggtagtgcaataccttaccccaatcttttatgatctttcgggaccgctagatttcccattagatactctttcggtccatgtggatatcctctccaatcatatacagcttcctatccaaaaccataacctaacaacgggtgatcgtgtttttatttctggatataaacacctgcaaacgattgaattatgtaaaaataacaagatttttatcaaaaatataccgccgctttcatccgaaaaaataaaactatatatactaaaaaatcgaatcagaattccgctatactttaaatctttaaaaacgtctaagtaa

[0263] SEQ ID NO:6: H240R polypeptide (penton)

[0264] MAANIIATRAVPKMASKKEHQYCLLDSQEKRHGHYPFSFELKPYGQTGANIIGVQGSLTHVIKMTVFPFMIPFPLQKTHIDDFIGGRIYLFFKELDMQAVSDVNGMQYHFEFKVVPVSPNQVELLPVNNKYKFTYAIPVVQYLTPIFYDLSGPLDFPLDTLSVHVDILSNHIQLPIQNHNLTTGDRVFISGYKHLQTIELCKNNKIFIKNIPPLSSEKIKLYILKNRIRIPLYFKSLKTSK

[0265] SEQ ID NO:7: B438L gene

[0266]

[0267] SEQ ID NO:8: B438L polypeptide (p49)

[0268] MYHDYASKLLADYRSDPPLWESDLPRHNRYSDNILNSRYCGNKNGAAPVYNEYTNSPEKAEKGLQLSDLRNFSFMLNPQHKNIGYGDAQDLEPYSSIPKNKLFNHFKNHRPAFSTHTENLIRRNVVRTEKKTFPQVASLKGTQKNCLTQPSSLPSLKNPKNSSVPSTRFSEHTKFFSYEDLPKLRTKGTIKHEQHLGDQMPGQHYNGYIPHKDVYNILCLAHNLPASVEKGIAGRGIPLGNPHVKPNIEQELIKSTSTYTDVPMLGPLPPKDSQHGREYQEFSANRHMLQVSNILHSVFANHSIKPQILEDIPVLNAQLTSIKPVSPFLNKAYQTHYMENIVTLVPRFKSIANYSSPIPNYSKRNSGQAEYFDTSKQTISRHNNYIPKYTGGIGDSKLDSTFPKDFNASSVPLTSAEKDHSLRGDNSACCISSISPSL

[0269] SEQ ID NO:9: C204L gene

[0270] atggattttattttaaatatatccatgaaaatggaggtcatcttcaaaacggatttaagatcatcttcacaagttgtgtttcatgcgggtagcctgtataattggttttctgttgagattatcaatagcggtagaattgttacgaccgctataaaaacattgcttagtactgttaagtatgatattgtgaaatctgctcgtatatatgcagggcaagggtatactgaacatcaggctcaagaagaatggaatatgattctgcatgtgctgtttgaagaggagacggaatcct cagcatcttcggagaacattcatgaaaaaaatgataatgaaaccaatgaatgcacatcctcctttgaaacgttgtttgagcaagagccctcatcggaggtacctaaagactccaagctgtatatgcttgcacaaaagactgtgcaa catattgaacaatatggaaaggcacctgattttaacaaggttattagagcacataattttattcaaaccatttatggaaacccctctaaaggaagaagaaaaagaggtggtaagactcatggttattaaacttttaaaaaaaaaaataa

[0271] SEQ ID NO:10: C204L polypeptide (p30)

[0272] MDFILNISMKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIYGTPLKEEEKEVVRLMVIKLLKKK

[0273] SEQ ID NO:11: D117L gene

[0274] atggacactgaaacgtctccactgctttctcataacctgtcaacccgcgagggaattaaacaaagcacccaaggccttttagcccatacaatcgccaaatatcccggaacaactgcgattctcctgggcattttgattttgctcattattattcttatcatcgttgccatcgtttactataaccggactattgactgcaagtcgagcatacctaaacctcctcctagctactatgtacaacaacctgagcctcaccaccatttcccggtattctttagaaaaaggaaaaactccacctccctgcagtcccacattccaagcgacgaacaattagctgaacttgcgcattcatga

[0275] SEQ ID NO:12: D117L polypeptide (p17) MDTETSPLLSHNLSTREGIKQSTQGLLAHTIAKYPGTTAILLGILILLIIILIIVAIVYYNRTIDCKSSIPKPPPSYYVQQPEPHHHFPVFFRKRKNSTSLQSHIPSDEQLAELAHS

[0276] SEQ ID NO: 13: E183L gene

[0277] atggattctgaattttttcaaccggtttatccgcggcattatggtgagtgtttgtcaccagtcactacaccaagcttcttctccacacatatgtatactattctcattgctatcgtggtcttagtcatcattatcatcgttctaatctatctattctcttcaagaaagaaaaaagctgctgctattgaggaggaagatatacagtttataaatccttatcaagatcagcagtgggtagaagtcactccacaaccaggtacctctaaaccagctggagcgactacagcaagtgtaggcaagccagtcacgggcagaccggcaacaaacagaccagcaacaaacaaaccagttacggacaacccagttacggacagactagtcatggcaactggcgggccggcggccgcacctgcggccgcgagtgctcctgctcatccggctgagccttacacgacagtcactactcagaacactgcttcacaaacaatgtcggctattgaaaatttacgacaaagaaacacctatacgcataaagacctagaaaactccttgtaa

[0278] SEQ ID NO:14: E183L polypeptide (p54)

[0279] MDSEFFQPVYPRHYGECLSPVTTPSFFSTHMYTILIAIVVLVIIIIVLIYLFSSRKKKAAAIEEEDIQFINPYQDQQWVEVTPQPGTSKPAGATTASVGKPVTGRPATNRPATNKPVTDNPVTDRLVMATGGPAAAPAAASAPAHPAEPYTTVTTQNTASQTMSAIENLRQRNTYTHKDLENSL

[0280] SEQ ID NO: 15: T cell epitope-1 gene

[0281] tttatgcgcgaaattaaagtaaaggaagtgttatacttttatacaatgcaggctgcccaagag

[0282] SEQ ID NO: 16: T cell epitope-1 polypeptide

[0283] FMREIKVKEVLYFYTMQAAQE

[0284] SEQ ID NO: 17: T cell epitope-2 gene

[0285] ggaagagttcccggtttttcaaaagaaatgctatttcaatatacgctactttacagataatttgtgcttcatgatgcagtgcaaaagcatttataaggtgggaaacccttttcctcagatgactaaa

[0286] SEQ ID NO: 18: T cell epitope-2 polypeptide

[0287] GRVPGFSKEMLFQYIRYFTDNLCFMMQCKSIYKVGNPFPQMTK

[0288] SEQ ID NO: 19: T cell epitope-3 gene

[0289] acggaaacaatttattagaaaccgcattaatttcattccgatttatgcgaaaaacgagacactggtatttgctctgcggagtttaaacaatagctgtgag

[0290] SEQ ID NO:20: T cell epitope-3 polypeptide

[0291] TEQFIRNRINFIPIYAKNETLVFALRSLNNSCE

[0292] SEQ ID NO:21: T cell epitope-4 gene

[0293] attgaacagtacccaacggataaaaaatatatggcgcttagcggttacaagcaaagctctatgacc

[0294] SEQ ID NO:22: T cell epitope-4 polypeptide

[0295] IEQYPTDKKEYMALSGYKQSSMT

[0296] SEQ ID NO:23: T cell epitope-5 gene

[0297] caaaaataaccaaatagaaactattctggttcaaaaacgttacagccttgctttttcagaatttattcattgtcattactctataaatgctaat

[0298] SEQ ID NO:24: T cell epitope-5 polypeptide

[0299] QNNQIETILVQKRYSLAFSEFIHCHYSINAN

[0300] SEQ ID NO:25: T cell epitope-6 gene

[0301] gaccgcatgtggtatcatatttggattgaaactccagtctacgaactataccacaaaaaataccaaaaatttaggaaa

[0302] SEQ ID NO:26: T cell epitope-6 polypeptide

[0303] DRMWYHIWIETPVYELYHKKYQKFRK

[0304] SEQ ID NO:27: T cell epitope-7 gene

[0305] tttaaaaaatctatgtcatactttgacggtaaaacagaatataagcatatctacttccttgcaatgttatgtaagtcgttggaggaaccc

[0306] SEQ ID NO:28: T cell epitope-7 polypeptide

[0307] FKKSMSYFDGKTEYKHIYFLAMLCKSLEEP

[0308] SEQ ID NO:29: T cell epitope-8 gene

[0309] gtggcccaaggtgaccatgtgcctgaccatatcaacatcctaaaaagaaaccaaatatttccgctgtttgcgcctaccccaagaggcgagggtagattt

[0310] SEQ ID NO:30: T cell epitope-8 polypeptide

[0311] VAQGDHVPDHINILKRNQIFPLFAPTPRGEGRF

[0312] SEQ ID NO:31: T cell epitope-9 gene

[0313] aatagcggaagaggatatatgcatgtaagattttctgctgttgtttcttacttaaatgcctttgatttaatagcggccgttaaaattattaaaaatgacagt

[0314] SEQ ID NO:32: T cell epitope-9 polypeptide

[0315] NSGRGYMHVRFSAVVSYLNAFDLIAAVKIIKNDS

[0316] SEQ ID NO:33: T cell epitope-10 gene

[0317] tcgcaaaataatgttgcagaagtggggcaaattgtgccggtacagcttgctaacagcagcgtgtactatacctggcagacaacag

[0318] SEQ ID NO:34: T cell epitope-10 polypeptide

[0319] SQNNVAEVGQIVPVQLANSSVYYIPGRQQ

[0320] SEQ ID NO:35: T cell epitope-11 gene

[0321] tctaaaaagacttcaagcagatatgctttttgaaaaactttactacctactctataagctcggat

[0322] SEQ ID NO:36: T cell epitope-11 polypeptide

[0323] SKKDFKQICFFEKLYYTYSISSSD

[0324] SEQ ID NO:37: T cell epitope-12 gene

[0325] atgataattaacgtcatcatggatgtattttacgacatattctttaccttaacatcaatctaacccgtgaacaaatt

[0326] SEQ ID NO:38: T cell epitope-12 polypeptide

[0327] MIINVIMDVFYETYSLPYNINLTREQI

[0328] SEQ ID NO:39: T cell epitope-13 gene

[0329] ggattaaataaaaagcgagaacttaaatactccttcatatcatactttatacatactcagtattaaaaataatgaggcc

[0330] SEQ ID NO:40: T cell epitope-13 polypeptide

[0331] GLNKKRELKYSFHIILYTYSVLNNNEA

[0332] SEQ ID NO:41: T cell epitope-14 gene

[0333] gaccaagtgatgacaaagagcatagaggttcatgagtccattctctttgaagaactgcctgatactcaaaaa

[0334] SEQ ID NO:42: T cell epitope-14 polypeptide

[0335] DQVMTKSIEVHESILFEELPDTQK

[0336] SEQ ID NO:43: T cell epitope-15 gene

[0337] gaatcgatttttaatcaattcaatagcggcctgcacaaatactttgctccctcttttgccattttcatgtggatgctcgaaacggcaaattatgttata

[0338] SEQ ID NO:44: T cell epitope-15 polypeptide

[0339] ESIFNQFNSGLHKYFAPSFAIFMWMLETANYVI

[0340] SEQ ID NO:45: T cell epitope-16 gene

[0341] tgcttggacgcaaacctaggaaatcgaacgtataatattctacagcgttttcgcggggatgtccct

[0342] SEQ ID NO:46: T cell epitope-16 polypeptide

[0343] CLDANLGNRTYNILQRFRGDVP

[0344] SEQ ID NO:47: T cell epitope-17 gene

[0345] ttgaccaatgaacaattaatgacgaaactcgttgatgaatttgttcagtatgatttaagcgcgatcagctttgagtttaagcttaaaaagcccaataaaacggac

[0346] SEQ ID NO:48: T cell epitope-17 polypeptide

[0347] LTNEQLMTKLVDEFVQYDLSAISFEFKLKKPNKTD

[0348] SEQ ID NO:49: T cell epitope-18 gene

[0349] attgatattcatgaagtacgatatggagcctacacacttttcatgtatggttccctcgaaaacggttacaaagcagaagtaagg

[0350] SEQ ID NO:50: T cell epitope-18 polypeptide

[0351] IDIHEVRYGAYTLFMYGSLENGYKAEVR

[0352] SEQ ID NO:51: T cell epitope-19 gene

[0353] atggggtaccgcgagaaggaaaaagagtttgcaccatacattcgaatattttttaaaagcctgtatgagcgacgaaaagccattacttacttaaataatatgggctacaac

[0354] acggccgcggac

[0355] SEQ ID NO:52: T cell epitope-19 polypeptide

[0356] MGYREKEKEFAPYIRIFFKSLYERRKAITYLNNMGYNTAAD

[0357] SEQ ID NO:53: T cell epitope-20 gene

[0358] tcccgagaattaaaactacctcttacaagttggatacagcttcagcactattcctacgagcctcgcggcttggta

[0359] SEQ ID NO:54: T cell epitope-20 polypeptide

[0360] SRELKLPLTSWIQLQHYSYEPRGLV

[0361] SEQ ID NO:55: T cell epitope-21 gene

[0362] aatgttatccccgatcgcagagaggtaggaattctgtcatacacctcgctgtatgactgtatctactacgcgggaggacacaaggtatgcaatatgctcattgcctatgcc

[0363] atccatgatgaatacggccgtattgcttgc

[0364] SEQ ID NO:56: T cell epitope-21 polypeptide

[0365] NVIPDRREVGILSYTSLYDCIYYAGGHKVCNMLIAYAIHDEYGRIAC

[0366] SEQ ID NO:57: T cell epitope-22 gene

[0367] cgggatgaggcaaatagcctcatggccaagggtgaatctcttcactacgtctcctttcactttaacaatcgtctcgtg

[0368] SEQ ID NO:58: T cell epitope-22 polypeptide

[0369] RDEANSLMAKGESLHYVSFHFNNRLV

[0370] SEQ ID NO:59: T cell epitope-23 gene

[0371] cacgaaaagtgcttctgtctatgaaagccatgtctacactatgcgccgaggtgaatgaatacctgcga

[0372] SEQ ID NO:60: T cell epitope-23 polypeptide

[0373] HEKVLLSMKAMSTLCAEVNEYLR

[0374] SEQ ID NO:61: T cell epitope-24 gene

[0375] atggtgttaatagagtttttaacaggtttcttctatttatatggaaagagactgttttccatt

[0376] SEQ ID NO:62: T cell epitope-24 polypeptide

[0377] MVLIEFLTGFFYLYGKRLFSI

[0378] SEQ ID NO:63: T cell epitope-25 gene

[0379] tgtcggaagaacattttcttccttccaaatgattttagcaagcataccctacaatggctgggattatattggaaagagcatggctccgtc

[0380] SEQ ID NO:64: T cell epitope-25 polypeptide

[0381] CRKNIFFLPNDFSKHTLQWLGLYWKEHGSV

[0382] SEQ ID NO:65: T cell epitope-26 gene

[0383] ctggccgtgaaaaatcaatcttccaggaaaactttgaacttgttgctatcttacataaattacaaggtgaaaaatgttaaaaagctg

[0384] SEQ ID NO:66: T cell epitope-26 polypeptide

[0385] LAVKNQSSRKTLNLLLSYINYKVKNVKKL

[0386] SEQ ID NO:67: T cell epitope-27 gene

[0387] acagaaagaattacttatctcaaacagatagtgcacaccataaaatatgaaagtggaaggcgatttttggtagacatcattcacagcatttaccaaagttactcactaaaacacgaagatattctt

[0388] SEQ ID NO:68: T cell epitope-27 polypeptide

[0389] TERITYLKQIVHTIKYESGRRFLVDIIHSIYQSYSLKHEDIL

[0390] SEQ ID NO:69: T cell epitope-28 gene

[0391] gcgcaggttaaatatacccttcaagagcttatttcctactgcagcgctctaaccattttacattatgactattcaaccctt

[0392] SEQ ID NO:70: T cell epitope-28 polypeptide

[0393] AQVKYTLQELISYCSALTILHYDYSTL

[0394] SEQ ID NO:71: T cell epitope-29 gene

[0395] gcacgaaacccctttgtatcccaaagtatgtccttgaactattacttttatgaacctgaactaagc SEQ ID NO:72: T cell epitope-29 peptide

[0396] ARNPFVSQSMSLNYYFYEPELS

[0397] SEQ ID NO:73: T cell epitope-30 gene

[0398] gtgtatgaaaagggtaagaccatgcccgtggaattttactatgaacaagagtttgat SEQ ID NO: 74: T cell epitope-30 peptide

[0399] VYEKGKTMPVEFYYEQEFD

[0400] SEQ ID NO:75: T cell epitope-31 gene

[0401] attaccaaggaagaaatcatgcaagcctatgctttggagtatgccatgtat

[0402] SEQ ID NO:76: T cell epitope-31 polypeptide

[0403] ITKEEIMQAYALEYAMY

[0404] SEQ ID NO:77: T antigen gene (without DHFR)

[0405]

[0406] SEQ ID NO:78: T antigen polypeptide (without DHFR)

[0407] MFMREIKVKEVLYFYTMQAAQEGRVPGFSKEMLFQYIRYFTDNLCFMMQCKSIYKVGNPFPQMTKTEQFIRNRINFIPIYAKNETLVFALRSLNNSCEIEQYPTDKKEYMALSGYKQSSMTQNNQIETILVQKRYSLAFSEFIHCHYSINANDRMWYHIWIETPVYELYHKKYQKFRKFKKSMSYFDGKTEYKHIYFLAMLCKSLEEPVAQGDHVPDHINILKRNQIFPLFAPTPRGEGRFNSGRGYMHVRFSAVVSYLNAFDLIAAVKIIKNDSSQNNVAEVGQIVPVQLANSSVYYIPGRQQSKKDFKQICFFEKLYYTYSISSDMIINVIMDVFYETYSLPYNINLTREQIGLNKKRELKYSFHIILYTYSVLNNNEADQVMTKSIEVHESILFEELPDTQKESIFNQFNSGLHKYFAPSFAIFMWMLETANYVICLDANLGNRTYNILQRFRGDVPLTNEQLMTKLVDEFVQYDLSAISFEFKLKKPNKTDIDIHEVRYGAYTLFMYGSLENGYKAEVRMGYREKEKEFAPYIRIFFKSLYERRKAITYLNNMGYNTAADSRELKLPLTSWIQLQHYSYEPRGLVNVIPDRREVGILSYTSLYDCIYYAGGHKVCNMLIAYAIHDEYGRIACRDEANSLMAKGESLHYVSFHFNNRLVHEKVLLSMKAMSTLCAEVNEYLRMVLIEFLTGFFYLYGKRLFSICRKNIFFLPNDFSKHTLQWLGLYWKEHGSVLAVKNQSSRKTLNLLLSYINYKVKNVKKLTERITYLKQIVHTIKYESGRRFLVDIIHSIYQSYSLKHEDILAQVKYTLQELISYCSALTILHYDYSTLARNPFVSQSMSLNYYFYEPELSVYEKGKTMPVEFYYEQEFDITKEEIMQAYALEYAMY

[0408] SEQ ID NO:79: T antigen gene (joined to DHFR)

[0409]

[0410] SEQ ID NO:80: T antigen polypeptide (attached to DHFR)

[0411]

[0412] SEQ ID NO:81: IRES

[0413] tccctcccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataa

[0414] SEQ ID NO:82: Loxp

[0415] ATAACTTCGTATAGCATACATTATACGAAGTTAT

[0416] SEQ ID NO:83: PCR identification primer rTV-D8L-F

[0417] GACGACGACAATAGTGTTCTG SEQ ID NO:84: PCR identification primer rTV-D8L-R

[0418] CATTAGATCCGCCAATACG

[0419] SEQ ID NO:85: PCR identification primer rTV-A56R-F

[0420] TCGAGGTTATTGGTTGTGGATC

[0421] SEQ ID NO:86: PCR identification primer rTV-A56R-R

[0422] TTAGTTTCCTCTGGAAGCAC

[0423] SEQ ID NO:87: PCR identification primer rTV-TK-F

[0424] CAAGGTCCCTATTGTTACAG

[0425] SEQ ID NO:88: PCR identification primer rTV-TK-R

[0426] CGTATATGTGTACCGGGAGC

[0427] SEQ ID NO:89: Peptide linker 1

[0428] GGGGS

[0429] SEQ ID NO:90: ​​Peptide linker 2

[0430] GSAGSAAGSGEFGSG

[0431] SEQ ID NO:91: Peptide linker 3

[0432] EAAAK

[0433] SEQ ID NO:92: Peptide linker 4

[0434] EFPKPSTPPGSSGGAP

[0435] SEQ ID NO:93: Peptide linker 5

[0436] KESGSVSSEQLAQFRSLD

[0437] SEQ ID NO:94: Peptide linker 6

[0438] EGKSSGSGSESKST

[0439] SEQ ID NO:95: cre gene

[0440]

[0441] SEQ ID NO:96: cre polypeptide

[0442] MSNLLTVHQNLPALPVDATSDEVRKNLMDMFDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNVIVMNYIRNLDSETGAMVRLLEDGD

[0443] SEQ ID NO:97: EP402R gene

[0444]

[0445] SEQ ID NO:98: EP402R polypeptide (CD2v)

[0446] MIILIFLIFSNIVLSIDYWVSFNKTIILDSNITNDNNDINGVSWNFFNSFNTLATCGKAGNFCECSNYSTSIYNITNNCSLTIFPHNDVFDTTYQVVWNQIINYTIKLLTPATTPNITYNCTNFLITCKNNGTNTNIYLNINDTFVKYTNESILEYNWNNSNINNFATCIINNTISTSNETTLINCTYLTLSSNYFYTFFKLYYYIPLSIIIGITISILLISIITFLSLRKRKKHVEEIESPPPPESNEEEQCQHDDTTSIHEPSPREPLLPKPYSRYQYNTPIYYMRPSTQPLNPFPPLNKPCPPPKPPPKPCPPPKPPSAESYSPPKPLPSIPLLPNIPPLSTQNISLIHVDRIIGGGGS

[0447] SEQ ID NO:99: PCR identification primer rTV-K8R-F

[0448] GAACATACAGTTTGCACTCATG

[0449] SEQ ID NO: 100: PCR identification primer rTV-K8R-R

[0450] CGCATACTATATGCGATGATTG

Claims

1. An immunogenic composition comprising: (A) an antibody immunogen panel comprising an immunogen derived from an African swine fever virus structural protein; and (B) Recombinant T cell immunogen panel containing immunogens derived from non-structural proteins of African swine fever virus.

2. The immunogenic composition according to claim 1, wherein The African swine fever virus is selected from the epidemic strains of African swine fever virus, and / or selected from a plurality of African swine fever virus strains of different genotypes and serotypes. For example, the African swine fever virus is one or more African swine fever virus strains selected from the following group: Pig / HLJ / 18, China / Guangxi / 2019 / domestic pig, CN201801, China / Jilin / 2018 / boar, Pig / Hubei / 628 / 2020, Pig / Liaoning / LC / 2020, Pig / Hebei / Q3 / 2020, and Georgia 2007 / 1.

3. The immunogenic composition according to claim 1, wherein The antibody immunogen group comprises one or more full-length or extracellular fragments of structural proteins derived from African swine fever virus, For example, the antibody immunogen group comprises one or more proteins or fragments or any combination thereof selected from the group consisting of: B646L, B602L, H240R, B438L, C204L, D117L, E183L, EP402R; or any combination thereof, for example: The combination of D117L and E183L is D117L-E183L. The combination of H240R, B438L and C204L is H240R-B438L-C204L. The combination of B646L and B602L is B646L-B602L. The combination of D117L, E183L, H240R, B438L and C204L is D117L-E183L-H240R-B438L-C204L. The combination of D117L, E183L, H240R, B438L, C204L, B646L and B602L is D117L-E183L-H240R-B438L-C204L-B646L-B602L. The combination of B646L, B602L and EP402R is B646L-B602L-EP402R; Wherein, each protein or fragment in any combination is directly connected or connected through a linker, such as a linker selected from SEQ ID NO: 89-94, IRES, P2A, T2A, E2A and F2A.

4. The immunogenic composition according to claim 3, wherein The B646L, B602L, H240R, B438L, C204L, D117L, E183L, EP402R are peptides having the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 98, or peptides having at least 80% sequence identity or homology with the peptides having the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 98; and / or The B646L, B602L, H240R, B438L, C204L, D117L, E183L, EP402R are encoded by nucleic acid molecules having nucleotide sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 97, respectively, or are encoded by nucleic acid molecules having at least 80% sequence identity or homology with nucleic acid molecules having nucleotide sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 97; and / or The B646L, B602L, H240R, B438L, C204L, D117L, E183L and EP402R optionally further comprise a linker independently selected from the following group: IRES, P2A, T2A, E2A or F2A.

5. The immunogenic combination according to claim 1, wherein The recombinant T cell immunogen group comprises one or more non-structural proteins or fragments thereof derived from African swine fever virus, For example: the recombinant T cell immunogen group comprises: (a) one or more T cell epitopes contained in the following non-structural proteins: F334L, H359L, D250R, D339L, F1055L, G1211R, A528 and MGF-505-7R proteins; or any combination of T cell epitopes contained in the aforementioned non-structural proteins; or a fragment having at least 80% sequence identity with the aforementioned T cell epitope combination; and (b) Optionally, a linker between the peptide elements, such as a linker selected from the group consisting of SEQ ID NOs: 89-94, IRES, P2A, T2A, E2A and F2A.

6. The immunogenic combination according to claim 5, wherein The recombinant T cell immunogen group at least comprises a combination of T cell epitopes contained in F334L, H359L, D250R, D339L, F1055L, G1211R, A528 and MGF-505-7R proteins; and / or The recombinant T cell immunogen group comprises: (i) at least 8, 10, 20, 30 or all of the peptides selected from the group consisting of a peptide having an amino acid sequence as shown in SEQ ID NO: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or a peptide having at least 80% sequence identity with any of the foregoing peptides; and / or (ii) a peptide having an amino acid sequence as shown in SEQ ID NO: 78 or 80, or a peptide having at least 80% sequence identity with any of the foregoing peptides; and / or The encoding nucleic acid molecule of the recombinant T cell immunogen group comprises: (i') at least 8, 10, 20, 30 or all nucleic acid molecules selected from the group consisting of: A nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73 or 75, or a nucleic acid molecule having at least 80% sequence identity with any of the foregoing nucleic acid molecules; and / or (ii') a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 77 or 79, or a nucleic acid molecule having at least 80% sequence identity with any of the foregoing nucleic acid molecules.

7. The immunogenic composition according to claim 3, wherein The recombinant T cell immunogen group is connected or mixed with the antibody immunogen group, for example, the recombinant T cell immunogen group is mixed or connected with B646L, B602L, H240R, B438L, C204L, D117L, E183L, EP402R or any combination of the foregoing (e.g., a combination of D117L and E183L), for example, each independently connected via a linker selected from SEQ ID NO: 89-94, IRES, P2A, T2A, E2A and F2A; and / or wherein the recombinant T cell immunogen group is connected or mixed with the antibody immunogen group to form one or more combinations selected from the group consisting of D117L-E183L-T antigen, D117L-E183L-T antigen-H240R-B438L-C204L, D117L-E183L-T antigen-H240R-B438L-C204L-B646L-B602L, or any combination thereof; and / or, the aforementioned three T antigens An immunogen or a combination thereof and one or more immunogens selected from the group consisting of D117L-E183L, H240R-B438L-C204L, B646L-B602L, D117L-E183L-H240R-B438L-C204L, D117L-E183L-H240R-B438L-C204L-B646L-B602L, B646L-B602L-EP402R; For example: the combination of D117L-E183L-T antigen-H240R-B438L-C204L and D117L-E183L-T antigen-H240R-B438L-C204L-B646L-B602L; the combination of D117L-E183L-T antigen-H240R-B438L-C204L and B646L-B602L; the combination of D117L-E183L-T antigen-H240R-B438L-C204L and B646L-B602L-EP402R.

8. A recombinant T cell immunogenic peptide comprising: (a) one or more T cell epitopes selected from the group consisting of non-structural proteins of African swine fever virus (ASFV): F334L, H359L, D250R, D339L, F1055L, G1211R, A528 and MGF-505-7R proteins; or any combination of T cell epitopes contained in the aforementioned non-structural proteins; or a fragment having at least 80% sequence identity with the aforementioned T cell epitope combination; and (b) Optionally, a linker between the peptide elements, such as a linker selected from the group consisting of SEQ ID NOs: 89-94, IRES, P2A, T2A, E2A and F2A.

9. The recombinant T cell immunogenic peptide according to claim 8, wherein: in, The recombinant T cell immunogenic peptide comprises at least a combination of T cell epitopes contained in F334L, H359L, D250R, D339L, F1055L, G1211R, A528 and MGF-505-7R proteins; and / or The recombinant T cell immunogenic peptide comprises: (i) at least 8, 10, 20, 30 or all of the peptides selected from the group consisting of peptides having SEQ ID NO: 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or a peptide having at least 80% sequence identity with any of the foregoing peptides; and / or (ii) a peptide having an amino acid sequence as shown in SEQ ID NO: 78 or 80, or a peptide having at least 80% sequence identity with any of the foregoing peptides; and / or The encoding nucleic acid molecule of the recombinant T cell immunogenic peptide comprises: (i') at least 8, 10, 20, 30 or all nucleic acid molecules selected from the group consisting of: SEQ ID NO: 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73 or 75, or a nucleic acid molecule having at least 80% sequence identity with any of the foregoing nucleic acid molecules; and / or (ii') a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 77 or 79, or a nucleic acid molecule having at least 80% sequence identity with any of the foregoing nucleic acid molecules.

10. A nucleic acid molecule encoding the immunogenic composition of any one of claims 1 to 7 or the recombinant T cell immunogenic peptide of any one of claims 8 to 9.

11. A vector or host cell comprising the nucleic acid molecule according to claim 10, For example, the vector is selected from: mRNA vector, DNA plasmid vector (such as shuttle plasmid vector), recombinant virus vector, recombinant bacterial vector; wherein the mRNA vector is selected from linear, circular and self-replicating vectors; the recombinant virus vector is selected from vaccinia virus (such as Tiantan strain, North American vaccine strain, Wyeth-derived strain, Listeria strain, Ankara-derived strain, Copenhagen strain and New York strain), adenovirus (such as adenovirus 5, 11, 26, 35, 63, 68), adeno-associated virus, herpes simplex virus, measles virus, enterovirus, reovirus, rhabdovirus, flavivirus, influenza virus, parainfluenza virus, respiratory syncytial virus, poliovirus vector; For example, the host cell is a mammalian cell or an insect cell, such as HEK293, HeLa, K562, CHO, NS0, SP2 / 0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19 and MRC-5 cells; High Five, Sf9, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, BM-N, Ha2302, Hz2E5 and Ao38.

12. An African swine fever vaccine comprising: (I) one or more immunologically active components or their precursors selected from the group consisting of one or more immunogenic compositions as described in any one of claims 1 to 7, recombinant T cell immunogenic peptides as described in any one of claims 8 to 9, nucleic acid molecules as described in claim 10, vectors or host cells as described in claim 11; and (II) one or more pharmaceutically or veterinarily acceptable carriers or excipients or delivery systems.

13. The African swine fever vaccine according to claim 12, wherein The African swine fever vaccine is an mRNA vaccine, a DNA vaccine, a viral vector vaccine (such as a vaccinia virus vaccine), or a recombinant protein vaccine; and / or The carrier or excipient or delivery system is selected from: a lipid delivery system, a lipid delivery system, a polymer delivery system or a combined delivery system thereof, such as loaded on lipid nanoparticles (e.g., a combination of cationic lipids, structural lipids, auxiliary lipids and stabilizing lipids), polyurethane (PAA), poly-β-amino ester (PBAE), polyethyleneimine (PEI), lipid-encapsulated polymer micelles; and / or The vaccine further comprises an adjuvant or is used in combination with an adjuvant, for example, the adjuvant is selected from: aluminum adjuvant, cholera toxin and its subunits, oligodeoxynucleotides, manganese ion adjuvants, colloidal manganese adjuvants, Freund's adjuvant, MF59 adjuvant, QS-21 adjuvant, Poly I:C and other TLR ligands, GM-CSF, IL-2, IL-3, IL-7, IL-11, IL-12, IL-18, IL-21; and / or The vaccine is in a form suitable for one or more administration or delivery methods selected from the following group: respiratory tract aerosol inhalation, nasal drops, oral administration, direct injection (e.g., intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection), mucosal administration; and / or The vaccine is in a form suitable for combined administration of two or more drugs or vaccines, such as combined vaccination or sequential vaccination; and / or The vaccine is a monovalent or multivalent vaccine in a form suitable for sequential or combined immunization.

14. The African swine fever vaccine according to claim 12 or 13, wherein The vaccine is a recombinant vaccinia virus (rTV) vaccine, for example: (a) The rTV vaccine comprises the following elements: a vaccinia virus vector; a vaccinia virus recombination site; a vaccinia virus shuttle plasmid; a vaccinia virus promoter; a selection marker; and / or (b) the recombinant vaccinia virus is selected from one or more recombinant vaccinia viruses into which one or more of the following immunogenic fragments are inserted: rTV-D117L-E183L-T antigen, rTV-H240R-B438L-C204L, rTV-B646L-B602L, rTV-D117L-E183L-T antigen-H240R-B438L-C204L, rTV-D117L-E183L-T antigen-H240R-B438L-C204L-B646L-B602L, rTV-B646L-B602L, or rTV-B646L-B602L-EP402R, wherein T antigen represents the recombinant T cell immunogen group as mentioned in claims 1-7; and / or (c) the recombination site of the vaccinia virus is derived from any one or a combination of several non-replication essential gene sites of the vaccinia strain used, such as J2R, A56R, K8R, C6L, K1L, B13R, A46R, N1L, K8R; and / or (d) The vaccinia virus promoter is a strong promoter from vaccinia virus that promotes the expression of viral proteins, such as pE / L, p7.5, pH5, and different truncated forms of each strong promoter. The screening marker includes reporter genes such as LacZ, eGFP, mcherry, BFP, and ZsGreen.

15. A product for the prevention and / or treatment of African swine fever, comprising one or more of the immunogenic compositions described in any one of claims 1 to 7, the recombinant T cell immunogenic peptides described in any one of claims 8 to 9, the nucleic acid molecule described in claim 10, the vector or host cell described in claim 11, and the vaccine described in any one of claims 12 to 14.

16. The product of claim 15, wherein: The product is a vaccine kit comprising one or more doses of the same or different vaccines as described in any one of claims 12 to 14, For example, the product comprises one or more doses of a combination of recombinant vaccinia virus vaccines selected from the group consisting of rTV-D117L-E183L-T antigen, rTV-H240R-B438L-C204L, rTV-B646L-B602L, rTV-D117L-E183L-T antigen-H240R-B438L-C204L, rTV-D117L-E183L-T antigen-H240R-B438L-C204L-B646L-B602L, rTV-B646L-B602L, rTV-B646L-B602L-EP402R; Preferably, any combination of rTV-D117L-E183L-T antigen-H240R-B438L-C204L, rTV-D117L-E183L-T antigen-H240R-B438L-C204L-B646L-B602L, rTV-B646L-B602L and rTV-B646L-B602L-EP402R; More preferably, a combination of one dose of rTV-D117L-E183L-T antigen-H240R-B438L-C204L and one or two doses of rTV-D117L-E183L-T antigen-H240R-B438L-C204L-B646L-B602L or rTV-rTV-B646L-B602L or rTV-B646L-B602L-EP402R, or a combination of two or three doses of rTV-D117L-E183L-T antigen-H240R-B438L-C204L-B646L-B602L and rTV-B646L-B602L or rTV-B646L-B602L-EP402R.

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