IMMUNOSING COMBINATION OF AFRICAN SWINE FEVER VIRUS

Abstract

The invention proposes an immunogenic combination of African swine fever virus. The immunogenic composition includes: (A) an antibody immunogenic group containing immunogenic genes derived from the structural proteins of African swine fever virus; and (B) a recombinant T cell immunogenic group containing immunogenic genes derived from the non-structural proteins of African swine fever virus. The invention also proposes the coding nucleic acid molecule of the immunogenic composition, its vector and host cell, a vaccine containing one or more of the above components, and related products. The two immunogenic combinations can generate effective binding antibodies against p72, p54, and p30, as well as activate T cell responses against T antigen, p17, and penton, thus providing effective protection against African swine fever virus.

Description

African swine fever virus immunogen combination and its application Technical Field

[0001] The present invention relates to the fields 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 applications thereof. 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 ornithological 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 and encodes 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 develop symptoms such as high fever, purple skin, and bleeding in multiple organs, and die within 3-7 days after infection, with a mortality rate close to 100%.

[0004] African swine fever initially spread only within African countries, but it was introduced into Europe, South America, Asia and other regions in the 1960s. After it was introduced into my country in August 2018, it caused huge economic losses and severely hit the domestic breeding industry. According to 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] The development of 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 vaccines faces huge challenges. Antibody responses to African swine fever virus can be detected in recovered pigs, but healthy pigs with passive immunity are unable to resist viral invasion. Oura et al. [2]It has also been found that using antibodies to eliminate CD8+ T cells in pigs vaccinated with attenuated vaccines will not produce the original protective effect. Inactivated African swine fever virus vaccines fail to stimulate a sufficient immune response and have been shown to provide ineffective protection. Live attenuated vaccines can replicate in vivo after immunization, simulating natural viral infection. Live attenuated vaccines obtained through tissue culture or natural screening, such as NH / P68 and OURT88 / 3, can induce immune protection in experimental pigs against the same genotype strain, but lack cross-protection against heterologous genotypes.

[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 virus 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] ——Problem (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 live attenuated vaccines will experience a reversion to virulence during continuous passage, often accompanied by severe side effects. [4] The developed live attenuated vaccine, HLJ / -18-6GD, lacks six genes within the MGF306 / 505 family and provides 100% protection against highly pathogenic homologous strains. However, in safety experiments, virulence relapsed and was only further attenuated by knocking out the CD2v gene. Vietnam approved two live attenuated vaccines for domestic marketing in July 2023, but these lack sufficient safety data, prompting the World Health Organization (WOAH) to warn of the risks of 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 macrophages of pigs, which are difficult to use for commercial large-scale production.

[0010] With the deepening of research on antigens that protect against viral immunity, 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] The p72, p54, and p30 proteins have been shown to induce neutralizing antibodies, and numerous subunit and DNA vaccines have focused on combinations of these three immunogens. Immunization of pigs with p54, p30, or a fusion of p54 and p30 expressed separately using a baculovirus expression system has been shown to induce neutralizing antibodies and protect against the E75 strain. However, the combination of p72, p54, and p30 proteins failed to protect against the Pr4 strain. CD2v, the only protein on the ASFV outer membrane, mediates in vitro erythrocyte agglutination and is a key indicator for identifying ASFV serotypes. Immunization with CD2v alone can induce partial protection against the E75 strain, but this result has not been replicated with other strains. Expressing the CD2v extracellular domain fused to p54 and p30, respectively, using a DNA vaccine platform, has been shown to induce strong binding antibodies and T cell responses, but has not provided protection following challenge. 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 antibodies and neutralizing antibodies. Only partial protection was produced after infection: only 2 of the 6 experimental pigs survived (2 / 6 survived).

[0011] Lacasta et al. [6] A randomized DNA vaccine library containing 80 ASFV coding sequences fused to ubiquitin was constructed, but this yielded only partial protection. Adenovirus and vaccinia virus are the primary viral vectors used to develop African swine fever vaccines, but most studies have examined immune responses to viral antigens without assessing vaccine protection against challenge. Adenovirus expressing A151R, B119L, B602L, EP402RΔPRR, B438L, K205R, A104R, pp62, and p72, combined with BioMize adjuvant, induced high levels of binding antibodies. However, challenge results revealed no protection and even enhanced symptoms in animals. 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) test pigs survived the challenge test. Although the same immunogen combination combined with ZTS-01 adjuvant immunization induced a low level of antibodies, 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. First, adenovirus expressing p72, p54, p30, B602L, E199L, EP153R, F317L, and MGF505-5R was used as the primary immunization, and then vaccinia virus expressing the same immunogen was used to boost the immune system. 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 after 20 days, and infectiousness could not be ruled out. [7] .

[0013] In summary, further research is still needed in this field to 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, a widely used vaccine vector, can induce high levels of humoral and cellular immune responses. Originally developed to protect against smallpox virus, vaccinia virus has proven highly safe through large-scale population vaccination. Furthermore, vaccinia virus can accommodate exogenous genes up to 30kb in length.

[0015] The African swine fever virus (ASF) has a complex structure and is resistant to a variety of extreme physical environments. It can be spread through contaminated utensils, pig swill, feed, and by attaching itself to pork products. my country's current primary response to ASF remains control and culling. Given my country's massive pig farming sector, primarily based on individual households and weak biosecurity awareness, the ASF epidemic is expected to persist and could erupt at any time. Therefore, the development of a safe and effective ASF vaccine is urgent.

[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+T lymphocytes 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 swine fever vaccine development under the prevalence and transmission of ASF in China[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 fever virus is safe and effective as a live attenuated vaccine in pigs[J].Sci China Life Sci,2020,63(5):623-634.DOI:10.1007 / s11427-020-1657-9.

[0021] [5]Gomez-Puertas P,Rodriguez F,Oviedo J M,et a1. Neutralizing antibodies to different proteins of African swine fever virus inhibit both virus attachment and internalization[J].Journal of virology,1996,70(8):5689-5694

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

[0023] [7]Goatley LC, Reis AL, Portugal R, et al. A pool of eight virally vectored African swine fever antigens protect pigs against fatal disease[J]. Vaccines, 2020, 8(2): 234

[0024] Summary of the Invention

[0025] 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.

[0026] In some aspects of the present application, an immunogenic composition is provided, comprising:

[0027] (A) a panel of antibody immunogens containing African swine fever virus structural proteins; and

[0028] (B) A panel of recombinant T cell immunogens containing nonstructural proteins derived from African swine fever virus.

[0029] 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 or any combination thereof selected from the following group: B646L, B602L, H240R, B438L, C204L, D117L, E183L, EP402R; or any combination of the foregoing.

[0030] In some embodiments, the recombinant T cell immunogen group comprises one or more T cell epitopes contained in the non-structural proteins selected from the group consisting of F334L, H359L, D250R, D339L, F1055L, G1211R, A528 and MGF-505-7R proteins derived from African swine fever virus (ASFV); 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.

[0031] In some aspects of the present application, a recombinant T cell immunogen peptide is provided, which comprises multiple peptide elements derived from the non-structural protein of African swine fever virus.

[0032] 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.

[0033] In some aspects of the present application, an African swine fever vaccine is provided, comprising:

[0034] (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

[0035] (II) one or more pharmaceutically or veterinarily acceptable carriers or excipients or delivery systems.

[0036] 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.

[0037] In some embodiments, the product is a vaccine kit comprising one or more doses of the same or different vaccines of the present application.

[0038] 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.

[0039] In some aspects of the present application, a method for preventing and / or treating African swine fever is provided, comprising 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.

[0040] 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 will be obvious to those skilled in the art due to the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The present invention will be further described below with reference to the accompanying drawings, the contents of which are only for illustrating the embodiments of the present invention and are not intended to limit the scope of the present invention.

[0042] 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.

[0043] Figure 2: Construction, Validation, and Expression of rTV-D8L-A56R-Δ-TK: A: Left: Schematic diagram of the fifth round of plaque picking for rTV-D8L screening; Center: PCR verification of rTV-D8L using D8L-F / D8L-R, showing the target band size; Right: rTV-D8L expresses p17, p54, and T antigen. B: Left: Schematic diagram of the seventh round of plaque picking for rTV-D8L-A56R screening; Center: PCR verification of rTV-D8L-A56R using A56-F / A56-R, showing the target band size; Right: rTV-D8L-A56 expresses penton, p49, and p30. C: Left: Schematic diagram of the 10th round of plaque picking in rTV-D8L-A56R-Δ-TK screening; Middle: Verification of rTV-D8L-A56R-Δ-TK using 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: Verification of rTV-TK using TK-F / TK-R PCR, showing the size of the target band; Right: rTV-TK expresses p72 and p72 chaperone.

[0044] Figure 3: Immune response levels induced by different recombinant vaccinia virus combinations in mice: A: Schematic diagram of the immunization process and time points; B: Binding antibody titers against p72, p54, and p30 in each group of mice on days 35, 63, and 77 of immunization; C: Cellular immune response levels induced against T antigen, p17, and penton in each group of mice.

[0045] Figure 4: Immune response levels induced by other recombinant vaccinia virus combinations in mice: A: Schematic diagram of the immunization process and time points; B: Binding antibody titers against p72, p54, and p30 in each group of mice on the 56th day of immunization; C: Cellular immune response levels induced against T antigen, p17, and penton in each group of mice.

[0046] Figure 5: Construction, verification and expression of rTV-TK-K8R: A: Map of the pK8R-EP402R shuttle plasmid; B: Schematic diagram of the 9th round of plaque picking for rTV-TK-K8R screening; C: Verification of rTV-TK-K8R using K8R-F / K8R-R PCR, showing the size of the target band; D: rTV-TK-K8R expresses CD2v.

[0047] Figure 6: Immune response levels induced in mice by different optimized recombinant vaccinia virus combinations: A: Schematic diagram of the immunization process and time points; B: Binding antibody titers against p72, p54, and p30 in each group of mice on the 70th day of immunization; C: Cellular immune response levels induced in each group of mice against T antigen, p17, and penton.

[0048] Figure 7: Protective effect of the recombinant vaccinia virus combination in field 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 the virus challenge; B (middle): Changes in clinical scores of mental state of the control group and the immunized group after the virus challenge; B (bottom): Changes in clinical scores of lying-down degree of the control group and the immunized group after the virus challenge; C (top): Changes in body temperature of the control group and the immunized group after the virus challenge; C (bottom): Survival curves of the control group and the immunized group after the virus challenge.

[0049] In the figures, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.005. DETAILED DESCRIPTION

[0050] This disclosure relates to the field of vaccines, particularly to a dual immunogen composition consisting of an antibody immunogen composed of structural proteins of the African swine fever virus and a T cell immunogen derived from non-structural proteins, as well as vaccines containing this immunogen composition and their applications. In this application, a safe and effective African swine fever vaccine was developed by selecting a suitable African swine fever virus immunogen, simultaneously loading it with conserved T cell epitopes, and using an appropriate carrier to deliver the immunogen.

[0051] All numerical ranges provided herein are intended to expressly include all values ​​falling between the endpoints of the ranges and ranges therebetween. Features described herein or in the embodiments may be combined. All features disclosed herein may be used in any combination, and any feature disclosed herein may be replaced by any alternative feature that serves the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the features disclosed are intended only to be general examples of equivalent or similar features.

[0052] 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.

[0053] It should be understood that when a parameter range is provided, the present invention also provides all integers and decimals thereof 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.

[0054] As used herein, “containing,” “having,” or “including” encompasses “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.”

[0055] Immunogenic composition

[0056] In the present application, an immunogenic composition is provided, comprising:

[0057] (A) an antibody immunogen panel comprising immunogens derived from African swine fever virus structural proteins; and

[0058] (B) Recombinant T cell immunogen panel containing immunogens derived from nonstructural proteins of African swine fever virus.

[0059] In some embodiments, the source of the immunogenic fragment in the immunogenic composition of the present application is African swine fever virus selected from the epidemic strains of African swine fever virus, and / or selected from multiple African swine fever virus strains of different genotypes and serotypes.

[0060] 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, Georgia 2007 / 1.

[0061] In some embodiments, the antibody immunogen panel comprises one or more full-length or extracellular fragments of structural proteins from African swine fever virus.

[0062] In some embodiments, the antibody immunogen group comprises one or more proteins or fragments selected from the group consisting of B646L, B602L, H240R, B438L, C204L, D117L, E183L, EP402R, or any combination thereof. In some embodiments, the combination is selected from the group consisting of: 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), and a combination of D117L, E183L, H240R, B438L and C204L (D117L-E183L). In some embodiments, the proteins or fragments in any combination are directly linked or linked via a linker, such as a linker selected from the group consisting of SEQ ID NOs: 89-94, IRES, P2A, T2A, E2A, and F2A.

[0063] 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, respectively.

[0064] In some embodiments, B646L, B602L, H240R, B438L, C204L, D117L, E183L, and EP402R are encoded by nucleic acid molecules having the 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 to nucleic acid molecules having the nucleotide sequences shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 97, respectively.

[0065] In some embodiments, the recombinant T cell immunogen panel comprises one or more nonstructural proteins or fragments thereof derived from African swine fever virus. In some embodiments, T cell epitopes broadly recognized by porcine major histocompatibility complex (SLA) molecules are screened through bioinformatics and fused to form a T antigen, such as the T antigen set forth in SEQ ID NO: 78. In other embodiments, DHFR is inserted at the 3' end of the fused T antigen to further enhance intracellular degradation of the T antigen, such as the T antigen with DHFR set forth in SEQ ID NO: 80.

[0066] In some embodiments, 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

[0067] (b) Optionally, a linker positioned 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.

[0068] In some embodiments, the recombinant T cell immunogen group comprises at least F334L, H359L, D250R, D339L, F1055L, G1211R, A528 and a combination of T cell epitopes contained in the MGF-505-7R protein.

[0069] In some embodiments, the recombinant T cell immunogen panel comprises:

[0070] (i) at least 8, 10, 20, 30 or all of the peptides selected from the group consisting of peptides having an amino acid sequence as set forth 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 to any of the foregoing peptides; and / or

[0071] (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

[0072] In some embodiments, the nucleic acid molecule encoding the recombinant T cell immunogen group comprises:

[0073] (i') at least 8, 10, 20, 30 or all of the 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 to 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 to any of the foregoing nucleic acid molecules.

[0074] In some embodiments, the recombinant T cell immunogen group is linked or mixed with the antibody immunogen group, for example, the recombinant T cell immunogen group is mixed or linked 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 NOs: 89-94, IRES, P2A, T2A, E2A and F2A.

[0075] Accordingly, the present application also provides a recombinant T cell immunogenic peptide comprising:

[0076] (a) one or more T cell epitopes selected from the group consisting of F334L, H359L, D250R, D339L, F1055L, G1211R, A528 and MGF-505-7R proteins contained in non-structural proteins of African swine fever virus (ASFV); or any combination of T cell epitopes contained in the foregoing non-structural proteins; or a fragment having at least 80% sequence identity with the foregoing T cell epitope combination; and

[0077] (b) Optionally, a linker positioned 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.

[0078] 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.

[0079] In some embodiments, the recombinant T cell immunogenic peptide comprises:

[0080] (i) at least 8, 10, 20, 30 or all of the peptides selected from the group consisting of peptides having an amino acid sequence as set forth 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 to 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 of the 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 to any of the foregoing nucleic acid molecules; and / or

[0084] (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.

[0085] The present application also provides nucleic acid molecules encoding the aforementioned immunogenic compositions or recombinant T cell immunogenic peptides.

[0086] Vectors and host cells

[0087] Also provided in the present application are vectors or host cells comprising the nucleic acid molecules described herein.

[0088] In some embodiments, the vector can be selected from: mRNA vector, DNA plasmid vector (such as shuttle plasmid vector), recombinant viral vector, recombinant bacterial vector; wherein the mRNA vector is selected from linear, circular and self-replicating vectors; the recombinant viral 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.

[0089] In some embodiments, a vaccinia virus comprising the immunogen of the present application or a combination thereof is provided. In some embodiments, the immunogen gene is inserted into one or more sites selected from D8L, A56R, TK, and K8R in the recombinant vaccinia virus (rTV) of the present application. In some embodiments, the recombinant vaccinia virus of the present application includes, but is not limited to, recombinant viruses 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.

[0090] 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.

[0091] In some embodiments, the T antigen in the recombinant vaccinia virus is linked to DHFR to increase the level of T antigen degradation in cells. In some embodiments, the recombinant vaccinia virus is selected from the group consisting of 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, and rTV-TK-B646L-B602L.

[0092] In some embodiments, the vaccinia virus promoter is derived from a strong vaccinia virus promoter that drives viral protein expression, such as pE / L, p7.5, pH5, and different truncated forms of each strong promoter. Screening markers include reporter genes such as LacZ, eGFP, mcherry, BFP, and ZsGreen.

[0093] 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.

[0094] Vaccines and products

[0095] This application provides an African swine fever vaccine, which comprises:

[0096] (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

[0097] (II) one or more pharmaceutically or veterinarily acceptable carriers or excipients or delivery systems.

[0098] 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.

[0099] 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), lipid-encapsulated polymer micelles.

[0100] 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.

[0101] In some embodiments, the vaccine is in a form suitable for one or more administration or delivery methods selected from the group consisting of respiratory aerosol inhalation, nasal drops, oral administration, direct injection (e.g., intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection), and mucosal administration.

[0102] In some embodiments, the vaccine is in a form suitable for combined administration of two or more drugs or vaccines, such as co-administration or sequential vaccination. In some embodiments, the vaccine is a monovalent or multivalent vaccine.

[0103] 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.

[0104] 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.

[0105] 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 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.

[0106] 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.

[0107] In some embodiments, the product comprises 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 two or three doses of rTV-D117L-E183L-T antigen-H240R-B438L-C204L-B646L-B602L or rTV-B646L-B602L-EP402R.

[0108] In some embodiments, the product includes one or more doses of the following recombinant vaccinia virus combinations: rTV-D117L-E183L-T antigen-H240R-B438L-C204L in combination with rTV-D117L-E183L-T antigen-H240R-B438L-C204L-B646L-B602L or rTV-rTV-B646L-B602L or rTV-B646L-B602L-EP402R; or rTV-D117L-E183L-T antigen-H240R-B438L-C204L-B646L-B602L and rTV-B646L-B602L or rTV-B646L-B602L-EP402R.

[0109] In some preferred embodiments of the present application, the immunogen 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).

[0110] Immunization methods

[0111] Also provided herein is a method for preventing and / or treating African swine fever virus infection and / or its symptoms, comprising administering at least one preventatively and / or therapeutically effective amount of one or more vaccines disclosed herein. Possible vaccination methods include, but are not limited to, systemic immunization methods, such as intramuscular, subcutaneous, and intradermal injections; and intrarespiratory immunization methods, such as aerosolization and intranasal drops. In some embodiments, the primary immunization is administered systemically or intrarespirator-directed, with systemic vaccination being preferred.

[0112] 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.

[0113] In some embodiments, the immunization method of the present disclosure can adopt a "prime-boost" or "prime-boost-reboost" approach, and can adopt a single systemic immunization or respiratory tract local immunization approach, or a combination of the two immunization approaches. In some embodiments, a regimen of first immunizing with rTV-D8L-A56R and then boosting with two injections of rTV-D8L-A56R-Δ-TK can induce high titers of binding antibodies against p72, p54, and p30 in vivo, while activating T cell responses against T antigen, p17, and penton, thereby playing a preventive role against African swine fever virus.

[0114] In some embodiments, the primary immunization may be performed with rTV-D8L-A56R, and one or two booster immunizations may be performed with a combination of rTV-D8L-A56R and rTV-TK, or rTV-D8L-A56R-TK. In some embodiments, the primary immunization may be performed with rTV-D8L-A56R, and one or two booster immunizations may be performed with a combination of rTV-D8L-A56R and rTV-TK, or rTV-D8L-A56R-TK. In some embodiments, both the primary and booster immunizations are performed with 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 the group consisting of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, or any interval therebetween. For example, the interval between the primary and booster immunizations may be 2 weeks, 3 weeks, or 4 weeks; and the interval between the booster immunization and the re-boost immunization may be 2 weeks, 3 weeks, or 4 weeks.

[0115] The combination products herein can be provided in the form of a pharmaceutical pack 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 sealed containers such as ampoules or sachets 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., and can be diluted, reconstituted and / or formulated with an appropriate liquid (e.g., water, saline, etc.) before use to obtain an appropriate concentration and form for administration to a subject.

[0116] The combination product disclosed herein can be used to simultaneously induce high levels of neutralizing antibodies and high levels of T cell responses in a subject, having a dual protective effect, making it have broad application prospects in the prevention and treatment of African swine fever virus infection.

[0117] Example

[0118] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only 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 variations to the present invention, and these modifications and variations are within the scope of the present invention.

[0119] For experimental procedures in the following examples where specific conditions are not specified, conventional methods in the art may be employed, for example, as described in Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, New York, 1989) or according to the conditions recommended by the supplier. DNA sequencing methods are conventional in the art and are also available from commercial companies.

[0120] Unless otherwise indicated, percentages and parts are by weight. Unless otherwise defined, all technical and scientific terms used herein 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 embodiments and materials described herein are for illustrative purposes only. For detailed information on the sequences used in the examples, please refer to the Appendix and Sequence Listing.

[0121] 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:

[0122] I. Screening of African swine fever virus-based vaccinia virus-vectored vaccines

[0123] 1. Preparation of shuttle plasmid:

[0124] Construction of the pD8L-D117L-E183L-TD shuttle plasmid for recombination of the D8L site: Referring to the instructions of the Novozan Virus Genome Extraction Kit (Cat. No. RC311-01), the genome of the Tiantan strain of vaccinia virus was extracted. Using the genomic DNA as a template, approximately 700 bp of the 5' and 3' ends of the D8L gene were PCR amplified. Referring to the instructions of the Novozan 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. The D117, E183, and T antigen-DHFR (TD) genes were artificially synthesized. The D117 and E183 genes were linked by P2A, and the E183 and T antigen-DHFR genes were linked by IRES. The target gene, D117L-P2A-E183L-IRES-TD, was amplified by PCR and ligated to the 3' end of the pE / L promoter in the pSC65 vector. The p7.5 promoter and the downstream eGFP gene were inserted at the 3' end of the target gene. LoxP sequences (SEQ ID NO: 82) were inserted at both ends of p7.5-eGFP, ultimately constructing the shuttle plasmid pD8L-pE / L-D117L-P2A-E183L-IRES-TD-loxP-p7.5-eGFP-loxP (hereafter referred to as pD8L-D117L-E183L-TD).

[0125] Using essentially the same method as above, the corresponding target genes were artificially synthesized and inserted according to the plasmid maps shown in Figures 1B, 1C, and 6A, respectively, to prepare the 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.

[0126] 2. Shuttle plasmid recombinant wild-type vaccinia virus:

[0127] Construction of rTV-D117L-P2A-E183L-IRES-TD recombinant vaccinia virus with recombinant D8L site: One day in advance, inoculate about 1×10 6 293T cells (purchased from ATCC, catalog number: CRL-3216) were prepared, ensuring a cell density of 80% on the next day. The cells were infected with wild-type vaccinia virus 752-1 (purchased from China Center for Type Culture Collection) at an MOI of 0.02. Two hours after infection, the complete medium containing the viral supernatant was discarded, and 2 mL of fresh maintenance medium was added. The cells were transfected with the pD8L-D117L-E183L-TD plasmid (transfection reagent purchased from Liji Biotechnology) and incubated at 37°C, 5% CO2 for approximately 24 hours. The cells were harvested the next day and frozen and thawed three times in a -80°C freezer.

[0128] Using essentially the same method as above, recombinant poxviruses were generated using pA56R-H240R-B438L-C204L, pSC65-B646L-B602L, or pK8R-EP402R.

[0129] 3. Recombinant Vaccinia Virus Screening:

[0130] One day in advance, approximately 8 × 10 5143B cells (purchased from ATCC, catalog number: CRL-8303) were plated, ensuring a cell density of 95% on the next day. The complete medium in the wells was discarded, and 2 mL of fresh maintenance medium was added. Each well was infected with 10 μL of the freeze-thawed cells. The plate was incubated at 37°C with 5% CO₂ for approximately 24 hours. The next day, the medium was discarded, and 1 mL of a 1:1 mixture of 2% agarose and 2× DMEM was added to each well. The plate was placed in a 4°C refrigerator for approximately 5 minutes. Fluorescent plaques were then selected under a fluorescence microscope and aspirated with a 1 mL pipette tip into a 1.5 mL EP tube containing 500 μL of maintenance medium. The plaques were then frozen and thawed three times in a -80°C refrigerator. This step was repeated for approximately 6-8 rounds until recombinant vaccinia virus was successfully screened. The genome was extracted and verified using PCR with primers using both homology arms. Only fragments matching the target gene size should be present, with no wild-type virus fragments present.

[0131] II. Amplification and Purification of African Swine Fever Virus Vaccine Vector Vaccine

[0132] 1. Recombinant vaccinia virus amplification:

[0133] The recombinant vaccinia virus that was successfully screened 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℃ refrigerator. One day in advance, about 1×10 7 DF-1 cells (purchased from ATCC, catalog number: CRL-3586) were grown to ensure that the cell density reached 95% on the next day. Discard the complete medium in the dish and add 10 mL of fresh maintenance medium. Infect the virus after freezing and thawing. Place in an incubator at 37°C and 5% CO2. Collect the infected cells within 24-48 hours based on the cytopathic effect and freeze-thaw them three times at -80°C. One day in advance, inoculate approximately 3×10 cells in a 15 cm dish. 7 DF-1 cells were added to ensure that the cell density reached 95% on the next day. Repeat the above steps, collect the infected cells and freeze-thaw three times in a -80℃ refrigerator. Expand DF-1 cells into T175 culture flasks, and after confluence, passage them at a ratio of 1:10. The remaining cells were plated into 15cm dishes at a ratio of 1:10. After 48 hours, the culture medium in the dish was replaced with 20mL of maintenance medium. The seeds collected in the previous round were infected at a ratio of 1:10 (i.e., 1 15cm dish of cells infected 10 15cm dishes). The infected cells were collected and freeze-thawed three times in a -80℃ refrigerator. Expand DF-1 cells into 6 bottles of T175 culture flasks, repeat the above steps and plate the remaining cells into 60 15cm dishes at a ratio of 1:10. The seeds collected in the previous round were infected at a ratio of 1:10. The infected cells were collected and freeze-thawed three times in a -80℃ refrigerator. Repeat this step 2-3 times according to the required amount of virus, and the cells collected after each round of infection were stored in a -80℃ refrigerator.

[0134] 2. Purification and titer determination of recombinant vaccinia virus:

[0135] The collected cells were frozen and thawed three times in a -80°C refrigerator, centrifuged at 3000g for 10 minutes, and the supernatant was collected. The pellet was resuspended in 10mL PBS, centrifuged at 3000g for 10 minutes, and the supernatant was collected. Wash three times in total. Add 12mL of 36% sucrose to the ultracentrifuge tube, tilt the ultracentrifuge tube, and slowly add 24mL of viral supernatant in a ratio of 1:2 to ensure the formation of clearly visible layers. Weigh with an electronic balance and balance the tube, with the error controlled within 0.1g. Set the ultracentrifuge parameters as follows: speed 18000g, temperature 4°C, 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.

[0136] 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 maintenance medium to the first EP tube, and add 990μL maintenance medium to each of the remaining tubes. Take out the 5μL of virus from the -80℃ refrigerator, aspirate 4μL and add it to the first EP tube for a 1:100 dilution. After thoroughly pipetting and mixing, aspirate 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 48 hours, count the number of plaques at the end point of the dilution multiple, and calculate the virus titer according to the following formula: Virus titer = the sum of the number of plaques in the two wells of the same dilution multiple × 10 稀释倍数 PFU / mL

[0137] III. Mouse Immunization Protocol

[0138] 1. Experimental Animals

[0139] Female BALB / c mice aged 6 to 8 weeks were purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd.

[0140] 2. Immunization method:

[0141] The mice were injected intramuscularly into the lateral sides of the left and right hind limbs.

[0142] 3. Immunogen preparation and immunization dose:

[0143] For the preparation of immunogens, see Examples 1 and 4.

[0144] The immunization dose was 1E7 PFU / mouse, 100 μL.

[0145] 4. Immunization interval:

[0146] 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.

[0147] IV. Mouse Testing Protocol

[0148] 1. Blood collection:

[0149] On days 35, 63, and 77, blood was collected from the orbital venous plexus of mice into 1.5 mL EP tubes and allowed to stand at room temperature until blood coagulated. The samples were centrifuged at 7000 g for 15 minutes, and the serum was transferred to a clean 1.5 mL EP tube. The serum was then centrifuged at 14000 g for 2 minutes, 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 minutes.

[0150] 2.ELISA detection of bound antibodies:

[0151] 1) Dilute the antigen protein (p72: purchased from Beijing Sino-Bio Technologies Co., Ltd.; p54 and p30 purchased from Antibody system) to a final concentration of 1 μg / mL using ELISA diluent. Add 100 μL per well and coat overnight.

[0152] 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. Discard the liquid in the wells for the final wash and pat dry.

[0153] 3) Add 200 μL of blocking solution (5% skim milk powder dissolved in PBST) to each well and block at room temperature for 2 h;

[0154] 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. Discard the liquid in the wells for the final wash and pat dry.

[0155] 5) Add 100 μL of blocking solution to each well except the first well. Dilute the serum 100-fold with blocking solution, mix thoroughly, and add 200 μL to the first well of each sample. Then perform a 2-fold dilution, i.e., pipette 100 μL into the next dilution well, mix thoroughly, and repeat this operation. After thoroughly mixing the last sample well, discard 100 μL of liquid and incubate at room temperature for 3 hours.

[0156] 6) Discard the liquid in the wells and wash five times with PBST containing 0.05% Tween-20, 200 μL per well each time. Discard the liquid in the wells after the last wash and pat dry.

[0157] 7) Dilute HRP-labeled secondary antibody (goat anti-mouse, purchased from Yisheng) at a dilution of 1:5000 in blocking solution, add 100 μL per well, and incubate at room temperature for 1 h.

[0158] 8) Discard the liquid in the wells and wash five times with PBST containing 0.05% Tween-20, 200 μL per well each time. Discard the liquid in the wells for the last wash and pat dry.

[0159] 9) Dissolve each pair of gold and silver OPD substrates in 20 mL of deionized water, add 100 μL to each well, and react in the dark for 5 minutes;

[0160] 10) Add 50 μL 2M H2SO4 to each well to terminate the reaction and read the OD value on a microplate reader. 492 -OD 630 nm reading;

[0161] 11) The OD of the negative control group 492 Twice the reading was taken as the cut-off value, and the reciprocal of the last dilution factor of each sample greater than this value was the antibody titer.

[0162] 3.ELISpot detection of T cell response:

[0163] Isolation of single cells from mouse spleen:

[0164] 1) Sacrifice the mouse and place it in lateral recumbency with the left side facing upward. Cut open the skin on the left side of the abdomen, open the peritoneum, and remove the spleen with sterile forceps. Place the spleen in a 6 cm dish containing 5 mL of complete RPIM1640 medium (R10).

[0165] 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;

[0166] 3) Transfer the spleen suspension to a 15 mL centrifuge tube and centrifuge at 800 g for 5 minutes;

[0167] 4) Discard the supernatant, tap the bottom of the centrifuge tube to resuspend the cells, add 5 mL of lysis buffer to each tube, invert to mix, and let stand at room temperature for 5 minutes;

[0168] 5) Add 8 mL of R10 to stop red cell lysis and centrifuge at 800 g for 5 min;

[0169] 6) Discard the supernatant, wash once with 5 mL of R10, and centrifuge at 800 g for 5 min;

[0170] 7) Discard the supernatant and resuspend the cells in 5 mL of R10. Take 20 μL of the cell suspension and add it to 380 μL of R10 for a 20-fold dilution. After counting, pipette the required volume of cell suspension according to the experimental design.

[0171] 8) After centrifugation, discard the supernatant of the remaining cells and place them in freezing solution (90% FBS and 10% DMSO) for future use.

[0172] ELISpot detection was performed according to the instructions of the MOUSE IFN-γ kit (purchased from BD, cat. no. 551083):

[0173] 1) Dilute IFN-γ antibody (1:200) with sterile PBS, add 100 μL to each well of a Millipore plate, and coat overnight at 4°C.

[0174] 2) Discard the liquid in the plate, wash once with 200 μL of R10, let stand for 3 minutes, and discard. Add 200 μL of fresh R10 and block at room temperature for 2 hours.

[0175] 3) Discard the liquid in the plate and add 50 μL of the stimulatory peptide library to each well according to the experimental design, where the working concentration of each single peptide is 5 μg / mL. For the negative control, add 50 μL of R10 to each well.

[0176] The stimulatory peptide library was synthesized by Suzhou Qiangyao Biotechnology Co., Ltd., covering T antigen, p17 and penton sequences. Each library consists of 10 single peptides.

[0177] The T antigen contains a total of 31 T cell epitopes (polypeptides represented by SEQ ID NO: 14+2n, where n=1-31). Each single peptide consists of 15 amino acids. The first peptide of the first epitope is FMREIKVKEVLYFYT, i.e., aa1-15 in SEQ ID NO: 16. Each subsequent peptide is shifted by 4 amino acids relative to the previous peptide. The first peptide of the second epitope is GRVPGFSKEMLFQYI, i.e., aa1-15 in SEQ ID NO: 18. Each subsequent peptide is shifted by 4 amino acids relative to the previous peptide. This process is repeated to cover every epitope.

[0178] ——The first peptide of p17 is MDTETSPLLSHNLST, which is aa1-15 in SEQ ID NO: 12. Each subsequent peptide is shifted by 4 amino acids compared to the previous peptide, that is, the second peptide is aa5-19 in SEQ ID NO: 12, the third peptide is aa9-23 in SEQ ID NO: 12... and so on. The 27th peptide IPSDEQLAELAHS (SEQ ID NO: 12 aa105-117) is less than 15 amino acids, so the 28th peptide SHIPSDEQLAELAHS (SEQ ID NO: 12 aa103-117) is synthesized to make it up to 15 amino acids.

[0179] ——The first peptide of penton is MAANIIATRAVPKMA, which is aa1-15 in SEQ ID NO: 6. Each subsequent peptide is shifted by 4 amino acids compared to the previous peptide, that is, 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).

[0180] 4) After counting the mouse spleen cells, the cell concentration was adjusted to 4×10 6 cells / mL, 50 μL (i.e., 2×10 cells / mL) was added vertically to each well. 5 The Millipore plate was carefully placed in a 37°C, 5% CO2 incubator and incubated for 20 h. During this period, the plate was prevented from moving.

[0181] 5) After incubation, remove the Millipore plate, discard the liquid in the plate, and add 200 μL of pre-cooled distilled water to each well and wash twice, each time for 5 minutes;

[0182] 6) Discard the liquid in the plate and add 200 μL of PBST containing 0.05% Tween-20 to each well. Wash three times for 1 minute each time. Pat the remaining liquid dry on absorbent paper.

[0183] 7) Dilute the biotin-conjugated detection antibody at a ratio of 1:250 in antibody diluent (PBS + 10% FBS), add 100 μL to each well, and incubate at room temperature for 2 h.

[0184] 8) After incubation, discard the liquid in the plate and add 200 μL of PBST containing 0.05% Tween-20 to each well. Wash three times for 1 minute each time. Pat the remaining liquid dry on absorbent paper.

[0185] 9) Dilute streptavidin-HRP 1:100 in antibody diluent (PBS + 10% FBS), add 100 μL to each well, and incubate at room temperature for 1 hour;

[0186] 10) Discard the liquid in the plate and add 200 μL of PBST containing 0.05% Tween-20 to each well and wash four times;

[0187] 11) Add 200 μL PBS to each well and wash twice. Pat the remaining liquid on absorbent paper for the last wash.

[0188] 12) Prepare the color development solution immediately before use: add 1 drop of color development substrate per 1 mL of color development buffer, mix thoroughly, add 100 μL to each well, and incubate at room temperature in the dark for 5-60 minutes.

[0189] 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.

[0190] 14) After drying at room temperature, the plate was counted and the spot forming cells (SFC) were counted and QC analysis was performed using a ChampSpot III enzyme-linked spot image analyzer.

[0191] V. Evaluation of protection against virus attack in experimental pigs

[0192] 1. Experimental Animals

[0193] 40-50 day old local pigs were purchased from Harbin Veterinary Research Institute.

[0194] 2. Immunization method:

[0195] Inject intramuscularly into the triangular area behind the ear of the pig.

[0196] 3. Immunogen preparation and immunization dose:

[0197] For the preparation of immunogens, see Examples 1 and 5.

[0198] The immunization dose is 2E8-5E8 PFU / pig, 1 mL.

[0199] 4. Immunization interval:

[0200] The primary immunization was performed on day 0 of the experiment, and the booster immunization was performed 28 days later.

[0201] 5.Challenge virus strain: HLJ / 18.

[0202] 6. Virus challenge procedure: The virus challenge was carried out in the P4 laboratory of Harbin Veterinary Research Institute. On the 28th day after the completion of immunization, each pig was injected intramuscularly with 100 HAD of African swine fever virus. 50 The animals were observed for 21 days after challenge. Body temperature was measured daily and clinical scores and survival were recorded.

[0203] Example 1. Construction, amplification, purification and expression verification of recombinant vaccinia virus

[0204] To develop a safe and effective African swine fever vaccine, we screened for highly immunogenic exogenous genes through literature review and data analysis. To further activate the pig's cellular immune response, we used bioinformatics to identify a T cell epitope broadly recognized by the porcine major histocompatibility complex (SLA) molecule and fused it to a T antigen (SEQ ID NO: 78). DHFR (SEQ ID NO: 80) was inserted at its 3' end to enhance intracellular degradation of the T antigen. We then artificially synthesized eight exogenous genes—B646L, B602L, H240R, B438L, C204L, D117L, E183L, and T antigen—and inserted them into a vaccinia virus vector to construct an African swine fever vaccine.

[0205] The eight exogenous genes were divided into three groups: ① D117L, E183L, and T antigen; ② H240R, B438L, and C204L; and ③ B646L and B602L. It is important to note that this grouping method is only one combination; other combinations that achieve expression of all exogenous genes are within the scope of this patent. Following Experimental Method I, shuttle plasmids were constructed for the D8L, A56R, and TK regions, respectively. The three groups of exogenous genes were sequentially inserted into these three shuttle plasmids. The resulting shuttle plasmid map is shown in Figure 1.

[0206] Following experimental method 1, the shuttle plasmid pD8L-D117L-E183L-T antigen DHFR was recombined with 752-1. Recombinant vaccinia viruses were screened using eGFP (Figure 2A, left). After five rounds of screening, rTV-D8L-D117L-E183L-T antigen-DHFR (hereinafter referred to as rTV-D8L) was obtained. After small-scale amplification in six-well plates, the genome was extracted and used as a template for PCR verification using primers D8L-F / D8L-R (SEQ ID NOs: 83 and 84). As shown in Figure 2A, the PCR yielded a single band, consistent with the expected size of 6223 bp.

[0207] Further detection of rTV-D8L expression: one day in advance, approximately 8×10 5143B cells were plated, ensuring a cell density of 95% on the next day. The culture medium in each well was replaced with 2 mL of maintenance medium. One well was infected with rTV-D8L at an MOI of 0.2, while the other well remained untreated. After 24 hours, cells were harvested and Western blot samples were prepared for protein immunoblotting. As shown in Figure 2A (right), significant expression of p17 and p54 proteins was detected in cells infected with rTV-D8L, while expression levels were relatively low in T cells due to their DHFR carrier status.

[0208] Next, based on rTV-D8L, the shuttle plasmid pA56R-H240R-B438L-C204L was recombined. This plasmid carries the mcherry reporter gene, and two-in-one recombinant vaccinia virus was screened using mcherry (Figure 2B, left). After seven rounds of screening, rTV-D8L-D117L-E183L-T antigen-DHFR-A56R-H240R-B438L-C204L (hereinafter referred to as rTV-D8L-A56R) was obtained. After small-scale amplification of rTV-D8L-A56R in six-well plates, the genome was extracted and used as a template for PCR verification using primers D8L-F / D8L-R and A56R-F / A56R-R (SEQ ID NOs: 85 and 86). The results showed that the D8L region still maintained a stable single band, while the A56R region had only a single band, the size of which was consistent with the expected 4925 bp (Figure 2B, center). Western immunoblotting was used to further examine the expression of rTV-D8L-A56R. In addition to expressing the originally inserted p17, p54, and T antigen, rTV-D8L-A56R also expressed high levels of penton, p49, and p30, all at levels consistent with expectations (Figure 2B, right, shows only the expression of penton, p49, and p30).

[0209] 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 plated, ensuring a cell density of 80% on the second day. 4 μg of phage-cre (SEQ ID NO: 95) plasmid was transfected using a LiJi assay. Six hours after transfection, rTV-D8L-A56R was infected at an MOI of 0.02. Two hours later, the supernatant was discarded and 2 mL of fresh maintenance medium was added. Depending on the infection status of the cells, the cells were harvested approximately 24 hours later and frozen and thawed three times at -80°C. Following experimental method I, vaccinia viruses lacking eGFP and mcherry expression were screened using fluorescence microscopy. After three rounds of screening, rTV-D8L-A56R-eGFPΔ-mcherryΔ (hereinafter referred to as rTV-D8L-A56R-Δ) was obtained. The genome was extracted and used as a template. PCR verification using primers D8L-F / D8L-R and A56R-F / A56R-R revealed a single band at the target band position. Western blotting also detected expression of the target gene. Next, the shuttle plasmid pSC65-B646L-B602L was recombined based on rTV-D8L-A56R-Δ. 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 2C, left). The genome was extracted as a template, and PCR verification 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 2C , middle). Protein immunoblotting detected the expression of p17, p54, T antigen, penton, p49, p30, p72, and p72 chaperone (only the expression of p72 and p72 chaperone is shown in Figure 2C , right).

[0210] Following experimental method I, the shuttle plasmid pSC65-B646L-B602L was recombined with 752-1, and the recombinant vaccinia virus was screened using eGFP ( Figure 2D , left). After screening, rTV-TK-B646L-B602L (hereinafter referred to as rTV-TK) was obtained. PCR and protein immunoblotting confirmed correct insertion and expression of the exogenous gene ( Figure 2D , center and right).

[0211] The above data show that rTV-D8L-A56R, rTV-D8L-A56R-Δ-TK and rTV-TK were successfully screened and constructed, and the target genes were correctly inserted into different recombination sites and expressed normally.

[0212] Example 2. Different combinations of recombinant vaccinia viruses induce immune responses in mice

[0213] The rTV-D8L-A56R, rTV-D8L-A56R-Δ-TK and rTV-TK obtained by screening 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. Twenty-four BALB / c mice were randomly divided into four groups and immunized as shown in Table 1. Each group received 1E7 PFU intramuscularly, with a single immunization volume of 100 μL per mouse. The immunization schedule is shown in Figure 3A. Peripheral blood was collected from the orbital venous plexus of the mice on days 35, 63, and 77, and the levels of antibodies binding to p72, p54, and p30 in the mice were assessed by enzyme-linked immunosorbent assay. On day 77, splenocytes were isolated from the mice, and ELISpot analysis was performed to analyze the expression of T cells specific to T antigen, p17, and penton induced by different recombinant vaccinia virus combinations.

[0214] As shown in Figure 3B, 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 immunization with 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 level of binding antibodies 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. On day 77, there was no significant difference in p54 antibody titers induced between Groups 1 and 3. However, after three injections of rTV-D8L-A56R-Δ-TK, the p54 antibody titer was 5382 on day 77, suggesting uneven induction of binding antibodies. The p30 binding antibody titers were generally low across all groups. This may be because p30 is driven by the p7.5 promoter, whose promoter activity is weaker than that of the pE / L promoter, resulting in lower expression levels in mice than p72 and p54. The p30 binding antibody titer in Group 3 on day 77 was 1345, while the titers in the other groups were between 400 and 600.

[0215] Figure 3C shows the results of T cell immune responses. Compared with the control group, the recombinant vaccinia virus induced cellular immune responses against T antigen, p17, and penton in mice. Group 1 induced the best results, followed by Group 3, and finally Group 2. T cell responses to the T antigen peptide library were weaker than those to the p17 and penton peptide libraries. This may be because the T antigen immunogen was selected by evaluating its binding to porcine SLA molecules, which differ from the species of mouse MHC molecules.

[0216] These data suggest that immunization with rTV-D8L-A56R followed by a two-dose booster with rTV-D8L-A56R-Δ-TK induced higher levels of p72, p54, and p30 antibodies and T cell responses in mice. The three-dose rTV-D8L-A56R + rTV-TK combination was superior to the three-dose rTV-D8L-A56R-Δ-TK regimen and comparable to Group 1 in inducing binding antibody levels.

[0217] Table 1. Immunogenicity of African swine fever virus vaccinia virus vector vaccine in BALB / c mice

[0218] Example 3. Optimization of recombinant vaccinia virus combinations to induce immune responses in mice

[0219] Based on the results of Example 2, we further explored optimal recombinant vaccinia virus combinations. Fifteen BALB / c mice were randomly divided into three groups and immunized as shown in Table 2. The immunization process is shown in Figure 4A. Mice were sacrificed on day 56, and the levels of binding antibodies against p72, p54, and p30 were assessed using enzyme-linked immunosorbent assay (ELISA). Splenocytes were isolated and analyzed by ELISA for the expression of specific T cells against T antigen, p17, and penton induced by different recombinant vaccinia virus combinations.

[0220] Table 2. Immunogenicity of African swine fever virus and vaccinia virus vector vaccine combination in BALB / c mice

[0221] The results are shown in Figure 4. There was little difference between the two groups in terms of induction of binding antibodies. Group 1 was 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.

[0222] As can be seen from this example, the provided recombinant vaccinia virus vector immune combination for African swine fever virus, such as the first immunization with rTV-D8L-A56R, followed by two booster injections of rTV-D8L-A56R + rTV-TK, can induce high titers of binding antibodies against p72, p54, and p30 in mice, and can also activate T cell responses against T antigen, p17, and penton, showing certain application prospects in the field of preventing African swine fever virus. The more exogenous genes a vaccinia virus carries, the lower its amplification and replication ability will be. Considering the future cost of industrial production, the more optimal recombinant vaccinia virus combination is to first immunize with rTV-D8L-A56R, followed by two booster injections of rTV-D8L-A56R + rTV-TK.

[0223] Example 4. Further Optimization of Recombinant Vaccinia Virus

[0224] Based on the above examples, we identified an optimal immunization combination for recombinant vaccinia virus vectors: an initial immunization with rTV-D8L-A56R, followed by two booster doses of rTV-D8L-A56R and rTV-TK. Based on this strategy, we inserted a novel exogenous immune gene, EP402R (SEQ ID NO: 97), encoding the CD2v protein (i.e., EP402R polypeptide, SEQ ID NO: 98), into the K8R region of rTV-TK to construct rTV-TK-B646L-B602L-K8R-EP402R (hereinafter referred to as rTV-TK-K8R).

[0225] Following experimental method 1, the shuttle plasmid pK8R-EP402R (Figure 5A) was reconstructed based on rTV-TK, which carries the mCherry reporter gene. Recombinant vaccinia viruses were screened using mCherry, and after multiple rounds of screening, rTV-TK-K8R (Figure 5B) was obtained. The genome was extracted as a template, and PCR verification using primers K8R-F and K8R-R (SEQ ID NOs: 99 and 100) revealed a single band in the K8R region, approximately 2690 bases in size. p As expected (Figure 5C). Protein immunoblotting detected the effective expression of the immunogen (Figure 5D). This 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 was able to express normally. In addition, the above results also prove that after further loading the immune exogenous gene EP402R into the vaccinia virus, its amplification and replication ability was not adversely affected, and the resulting recombinant vaccinia virus can be used for the correct and effective carrying 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, ensuring its amplification and replication ability.

[0226] Example 5. Further optimized recombinant vaccinia virus combination induces immune response in mice

[0227] To investigate the effect of the addition of a new immunogen on the response to the original immunogen, we set up the following experiment: all experimental groups of mice were first primed 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 6A), with specific reference to the immunization shown in Table 3.

[0228] Table 3. Immunogenicity of two African swine fever virus vaccinia virus vector vaccine combinations in BALB / c mice

[0229] The 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 between the two groups of mice. From the overall trend, the rTV-D8L-A56R and rTV-TK-K8R combination induced slightly higher levels of p72 binding antibodies (Figure 6B). T cell response data also showed that although there was no significant difference between the two groups, the rTV-D8L-A56R and rTV-TK-K8R combination mice had more cells positive for each peptide pool (Figure 6C).

[0230] From the above examples, 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 boosting with two injections of rTV-D8L-A56R+rTV-TK-K8R. This scheme 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 preventing African swine fever virus.

[0231] Example 6. Evaluation of protection against challenge with recombinant vaccinia virus combinations in live pigs

[0232] This vaccine is developed for the prevention and control of African swine fever. By screening the African swine fever virus immunogen composition, an African swine fever virus vaccine is constructed. Therefore, the immune response and anti-virus protection effect of the recombinant vaccinia virus combination inducing pigs are further explored.

[0233] Ten 40-50 day old local pigs were selected and randomly divided into two groups, with 4 pigs in the control group and 6 pigs in the experimental group. They were immunized as shown in Table 4. Each group was injected intramuscularly, and the single immunization volume for each experimental pig was 1 mL. The first injection of each experimental pig was 2E8 PFU, and the second injection of each experimental pig was 5E8 PFU. After the immunization was completed, the virus was challenged 4 weeks later. Each pig was injected intramuscularly with 100 HAD of HLJ / 18 strain. 50 , observation was continued for 21 days after the challenge. The experimental process is shown in Figure 7A.

[0234] Table 4. Immunogenicity of African swine fever virus vaccinia virus vectored vaccine in local pigs

[0235] All pigs were transferred to the P4 laboratory on day 53 and challenged on day 56. They were observed for 21 days after challenge. Clinical scores were assessed daily during the observation period, as outlined in Table 5. Typical clinical symptoms of African swine fever virus infection in the experimental pigs included decreased appetite, depression, and a tendency to lie down. All pigs developed clinical symptoms starting on day 4 post-challenge, with symptoms progressively worsening. Clinical scores in the control group gradually increased and remained at their highest level from the day of challenge until the day of onset and death. Clinical scores in the vaccine group gradually decreased from around day 10, indicating a gradual alleviation of clinical symptoms. By day 21 post-challenge, the pigs had essentially recovered to a healthy state (Figure 7B). Body temperature in both the control and vaccine groups gradually increased after challenge. Starting on day 4 post-challenge, the control group maintained a temperature above 40°C. The vaccine group experienced a gradual decrease in temperature from day 7 post-challenge, dropping below 40°C from day 14 post-challenge and gradually returning to pre-challenge temperatures (Figure 7C, top). Finally, all four test pigs in the control group died within 15 days, while four of the six test pigs in the vaccine group survived, with a survival rate of approximately 66.7% (Figure 7C, bottom).

[0236] Generally speaking, increasing the number of vaccinations can induce a stronger immune response. Compared to other examples, this example achieved a 66.7% protection rate with only two doses of the candidate vaccine before challenge. Therefore, it is reasonable to speculate that the protective efficacy of the vaccine combinations in other examples will be at least as good as that of this example.

[0237] Table 5. African swine fever clinical scoring system

[0238] 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 virulent African swine fever.

[0239] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

[0240] Appendix: Sequence Information

[0241] 1. Immunogenic Sequence

[0242] 2. Other sequences

[0243] 3. Specific sequence

[0244] SEQ ID NO: 1: B646L gene

[0245] SEQ ID NO: 2: B646L polypeptide (p72)

[0246] SEQ ID NO: 3: B602L gene

[0247] SEQ ID NO: 4: B602L polypeptide (p72 chaperon)

[0248] SEQ ID NO: 5: H240R gene

[0249] SEQ ID NO: 6: H240R polypeptide (penton)

[0250] SEQ ID NO: 7: B438L gene

[0251] SEQ ID NO: 8: B438L polypeptide (p49)

[0252] SEQ ID NO: 9: C204L gene

[0253] SEQ ID NO: 10: C204L polypeptide (p30)

[0254] SEQ ID NO: 11: D117L gene

[0255] SEQ ID NO: 12: D117L polypeptide (p17)

[0256] SEQ ID NO: 13: E183L gene

[0257] SEQ ID NO: 14: E183L polypeptide (p54)

[0258] SEQ ID NO: 15: T cell epitope-1 gene

[0259] SEQ ID NO: 16: T cell epitope-1 polypeptide

[0260] SEQ ID NO: 17: T cell epitope-2 gene

[0261] SEQ ID NO: 18: T cell epitope-2 polypeptide

[0262] SEQ ID NO: 19: T cell epitope-3 gene

[0263] SEQ ID NO: 20: T cell epitope-3 polypeptide

[0264] SEQ ID NO: 21: T cell epitope-4 gene

[0265] SEQ ID NO: 22: T cell epitope-4 polypeptide

[0266] SEQ ID NO: 23: T cell epitope-5 gene

[0267] SEQ ID NO: 24: T cell epitope-5 polypeptide

[0268] SEQ ID NO: 25: T cell epitope-6 gene

[0269] SEQ ID NO: 26: T cell epitope-6 polypeptide

[0270] SEQ ID NO: 27: T cell epitope-7 gene

[0271] SEQ ID NO: 28: T cell epitope-7 polypeptide

[0272] SEQ ID NO: 29: T cell epitope-8 gene

[0273] SEQ ID NO: 30: T cell epitope-8 polypeptide

[0274] SEQ ID NO: 31: T cell epitope-9 gene

[0275] SEQ ID NO: 32: T cell epitope-9 polypeptide

[0276] SEQ ID NO: 33: T cell epitope-10 gene

[0277] SEQ ID NO: 34: T cell epitope-10 polypeptide

[0278] SEQ ID NO: 35: T cell epitope-11 gene

[0279] SEQ ID NO: 36: T cell epitope-11 polypeptide

[0280] SEQ ID NO: 37: T cell epitope-12 gene

[0281] SEQ ID NO: 38: T cell epitope-12 polypeptide

[0282] SEQ ID NO: 39: T cell epitope-13 gene

[0283] SEQ ID NO: 40: T cell epitope-13 polypeptide

[0284] SEQ ID NO: 41: T cell epitope-14 gene

[0285] SEQ ID NO: 42: T cell epitope-14 polypeptide

[0286] SEQ ID NO: 43: T cell epitope-15 gene

[0287] SEQ ID NO: 44: T cell epitope-15 polypeptide

[0288] SEQ ID NO: 45: T cell epitope-16 gene

[0289] SEQ ID NO: 46: T cell epitope-16 polypeptide

[0290] SEQ ID NO: 47: T cell epitope-17 gene

[0291] SEQ ID NO: 48: T cell epitope-17 polypeptide

[0292] SEQ ID NO: 49: T cell epitope-18 gene

[0293] SEQ ID NO: 50: T cell epitope-18 polypeptide

[0294] SEQ ID NO: 51: T cell epitope-19 gene

[0295] SEQ ID NO: 52: T cell epitope-19 polypeptide

[0296] SEQ ID NO: 53: T cell epitope-20 gene

[0297] SEQ ID NO: 54: T cell epitope-20 polypeptide

[0298] SEQ ID NO: 55: T cell epitope-21 gene

[0299] SEQ ID NO: 56: T cell epitope-21 polypeptide

[0300] SEQ ID NO: 57: T cell epitope-22 gene

[0301] SEQ ID NO: 58: T cell epitope-22 polypeptide

[0302] SEQ ID NO: 59: T cell epitope-23 gene

[0303] SEQ ID NO: 60: T cell epitope-23 polypeptide

[0304] SEQ ID NO: 61: T cell epitope-24 gene

[0305] SEQ ID NO: 62: T cell epitope-24 polypeptide

[0306] SEQ ID NO: 63: T cell epitope-25 gene

[0307] SEQ ID NO: 64: T cell epitope-25 polypeptide

[0308] SEQ ID NO: 65: T cell epitope-26 gene

[0309] SEQ ID NO: 66: T cell epitope-26 polypeptide

[0310] SEQ ID NO: 67: T cell epitope-27 gene

[0311] SEQ ID NO: 68: T cell epitope-27 polypeptide

[0312] SEQ ID NO: 69: T cell epitope-28 gene

[0313] SEQ ID NO: 70: T cell epitope-28 polypeptide

[0314] SEQ ID NO: 71: T cell epitope-29 gene

[0315] SEQ ID NO: 72: T cell epitope-29 polypeptide

[0316] SEQ ID NO: 73: T cell epitope-30 gene

[0317] SEQ ID NO: 74: T cell epitope-30 polypeptide

[0318] SEQ ID NO: 75: T cell epitope-31 gene

[0319] SEQ ID NO: 76: T cell epitope-31 polypeptide

[0320] SEQ ID NO: 77: T antigen gene (without DHFR)

[0321] SEQ ID NO: 78: T antigen polypeptide (without DHFR)

[0322] SEQ ID NO: 79: T antigen gene (followed by DHFR)

[0323] SEQ ID NO: 80: T antigen polypeptide (linked to DHFR)

[0324] SEQ ID NO: 81: IRES

[0325] SEQ ID NO: 82: Loxp

[0326] SEQ ID NO: 83: PCR identification primer rTV-D8L-F

[0327] SEQ ID NO: 84: PCR identification primer rTV-D8L-R

[0328] SEQ ID NO: 85: PCR identification primer rTV-A56R-F

[0329] SEQ ID NO: 86: PCR identification primer rTV-A56R-R

[0330] SEQ ID NO: 87: PCR identification primer rTV-TK-F

[0331] SEQ ID NO: 88: PCR identification primer rTV-TK-R

[0332] SEQ ID NO: 89: Peptide linker 1

[0333] SEQ ID NO: 90: Peptide linker 2

[0334] SEQ ID NO: 91: Peptide linker 3

[0335] SEQ ID NO: 92: Peptide linker 4

[0336] SEQ ID NO: 93: Peptide linker 5

[0337] SEQ ID NO: 94: Peptide linker 6

[0338] SEQ ID NO: 95: cre gene

[0339] SEQ ID NO: 96: cre polypeptide

[0340] SEQ ID NO: 97: EP402R gene

[0341] SEQ ID NO: 98: EP402R polypeptide (CD2v)

[0342] SEQ ID NO: 99: PCR identification primer rTV-K8R-F

[0343] SEQ ID NO: 100: PCR identification primer rTV-K8R-R

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 nonstructural 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 via 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, respectively, or are 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, EP402R can also be selected The protein comprises a linker independently selected from the group consisting of 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 of the 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 NOs: 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 following group: 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, a combination of the aforementioned three T antigen-containing immunogens or a combination thereof with 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 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 immunogenic peptide comprises: (i') at least 8, 10, 20, 30 or all of the 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.

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.