African swine fever virus infection

Abstract

Compounds that modulate the function, activity and / or expression of porcine leukocyte antigen complex II (SLA II) genes and / or SLA II proteins, use for medicine, use as medicaments or use for the treatment or prevention of ASF or ASFV infection are disclosed. The present disclosure also provides the use of the SLA-DMA, SLA-DMB, RFXANK, RFXAP5, and CIITA genes to achieve ASFV resistance.

Description

Technical Field

[0001] This disclosure provides strategies, methods, and compounds for treating or preventing African swine fever and for modulating the entry of African swine fever virus into host cells and / or replication. Background Technology

[0002] African swine fever virus (ASFV) is the pathogen of African swine fever (ASF), a disease affecting both domestic and wild boars. Sus scrofa) (type) of hemorrhagic diseases 1-3 The Assamfovir strain was first identified in Kenya in 1921 and has since been reported in most sub-Saharan African countries. 4,5 By partially sequencing the gene B646L encoding the major capsid protein p72, twenty-four ASFV genotypes have been identified. 6,7 Currently, only two genotypes, genotype I and genotype II, have been detected outside of Africa, and the current animal pandemic is caused by genotype II. Since ASFV entered Georgia in 2007, outbreaks have been reported in Europe, the Russian Federation, Asia, Oceania, and the Americas. 3,8,9 .

[0003] In most affected countries, ASF control remains limited to biorisk management measures, monitoring methods, and response strategies, including culling and trade restrictions, resulting in significant economic and production losses due to the lack of licensed vaccines currently available on the market. 3 .

[0004] Therefore, there is a need for strategies to target and modulate ASFV-producing infections and to treat and / or prevent ASF. Summary of the Invention

[0005] This disclosure is based on the finding that gene populations associated with porcine major histocompatibility complex II (MHC II) or porcine leukocyte antigen complex II (SLA II) are important for the production and infection of African swine fever virus (ASFV).

[0006] The SLA II gene population may include, for example, the SLA-DMA, SLA-DMB, RFXANK, RFXAP, and CIITA genes. These genes should be collectively referred to as "SLA II genes".

[0007] The SLA II gene encodes (or expresses) a protein (referred to as the "SLA II protein") that is used by ASFV to enter host cells and / or promote its replication and proliferation in host cells.

[0008] In one teaching, SLA II proteins may include proteins encoded or expressed by the SLA-DMA, SLA-DMB, RFXANK, RFXAP, and CIITA genes. These SLA II proteins may include SLA-DMA protein subunits, SLA-DMB protein subunits, regulatory factor X-related protein (RFXAP), and class II major histocompatibility complex transactivator (CIITA).

[0009] Specifically, this disclosure relates to newly identified interactions between ASFV and SLA-DM genes and their protein products, including the SLA-DMA and SLA-DMB genes encoding SLA-DM subunit proteins (i.e., SLA-DMA and SLA-DMB MHC protein subunits). Not wishing to be theoretically bound, the data presented in this disclosure indicate that by interfering with the interactions between viral ASFV and various factors encoded by the gene population described herein, ASFV infection or ASF occurrence can be reduced, prevented, and / or suppressed (i.e., regulated) in swine (or porcine) hosts. Furthermore, targeted regulation (e.g., knockout) of one, any, or all of the SLA II genes disclosed herein has been shown to result in, for example, replication defects, reduced intercellular transmission, lower progeny viral titers, and reduced viral DNA replication levels. Any or all of these effects may contribute to the prevention of ASFV infection and ASF occurrence in the host (e.g., porcine / pig host) and host cells.

[0010] It should be noted that the terms "comprise," "comprising," and / or "comprises" are used to indicate that aspects and embodiments of the invention "comprise" one or more specific features. It should be understood that these terms may also cover aspects and / or embodiments that are "substantially composed of" or "consist of" one or more associated features.

[0011] The term "ASFV" refers to the African swine fever virus family. Asfarviridae African swine fever virus (genus) Asfivirus The only member and all genotypes / strains of ASFV. The linear double-stranded DNA genome of ASFV ranges in size from 170 to 193 kbp and contains 150 to 167 predicted protein-coding open reading frames. 14-16 ASFV particles contain approximately 82 viral proteins, are about 250 nm in size, and have a multilayered structure. These include an outer lipid membrane, an icosahedral outer capsid, an inner lipid membrane, an inner capsid, a thick protein nucleocapsid, and a nucleoid containing the genome.

[0012] ASFV is the pathogen of African swine fever (ASF), a disease affecting both domestic and wild pigs (wild boars). Sus scrofa) (a type of) hemorrhagic disease.

[0013] Therefore, this disclosure provides: A method for treating or preventing African swine fever (ASF) or ASFV infection, the method comprising regulating the function, expression, and / or activity of the porcine leukocyte antigen complex II (SLA II) gene and / or SLA II protein; A method for treating or preventing ASF or ASFV infection, the method comprising administering to a subject in need a compound that modulates the function, expression, and / or activity of the porcine leukocyte antigen complex II (SLA II) gene and / or SLA II protein; A compound that regulates the function, activity, and / or expression of the porcine leukocyte antigen complex II (SLA II) gene and / or SLA II protein for the treatment or prevention of ASF or ASFV infection; Use of compounds that regulate the function, activity, and / or expression of the porcine leukocyte antigen complex II (SLA II) gene and / or SLA II protein in the preparation of medicaments for the treatment or prevention of ASF or ASFV infection; The use of compounds that regulate the function, activity, and / or expression of porcine leukocyte antigen complex II (SLA II) gene and / or SLA II protein in pharmaceuticals or as pharmaceuticals; A method for treating or preventing African swine fever (ASF) or ASFV infection, the method comprising regulating the function, expression, and / or activity of the SLA-DMA gene and / or SLA-DMA protein; A method for treating or preventing ASF or ASFV infection, the method comprising administering to a subject in need a compound that modulates the function, expression, and / or activity of the SLA-DMA gene and / or SLA-DMA protein; A compound that regulates the function, activity, and / or expression of the SLA-DMA gene and / or SLA-DMA protein for the treatment or prevention of ASF or ASFV infection; Use of compounds that regulate the function, activity and / or expression of the SLA-DMA gene and / or SLA-DMA protein in the preparation of medicaments for the treatment or prevention of ASF or ASFV infection; A method for treating or preventing African swine fever (ASF) or ASFV infection, the method comprising regulating the function, expression, and / or activity of the SLA-DMB gene and / or SLA-DMB protein; A method for treating or preventing ASF or ASFV infection, the method comprising administering to a subject in need a compound that regulates the function, expression, and / or activity of the SLA-DMB gene and / or SLA-DMB protein; A compound that regulates the function, activity, and / or expression of the SLA-DMB gene and / or SLA-DMB protein for the treatment or prevention of ASF or ASFV infection; Use of compounds that regulate the function, activity, and / or expression of the SLA-DMB gene and / or SLA-DMB protein in the preparation of medicaments for the treatment or prevention of ASF or ASFV infection; In further instruction, this disclosure provides: A method for regulating African swine fever virus (ASFV) toxin-producing infection, the method comprising regulating the function, expression and / or activity of porcine leukocyte antigen complex II (SLA II) gene and / or SLA II protein; A compound that regulates the function, activity, and / or expression of the porcine leukocyte antigen complex II (SLA II) gene and / or SLA II protein for use in regulating infection by African swine fever virus (ASFV). Use of compounds that regulate the function, activity, and / or expression of porcine leukocyte antigen complex II (SLA II) gene and / or SLA II protein in the preparation of drugs for regulating infection by toxin-producing African swine fever virus (ASFV); A method for regulating infection by African swine fever virus (ASFV) toxin production, the method comprising regulating the function, expression and / or activity of the SLA-DMA gene and / or SLA-DMA protein; A compound that regulates the function, activity, and / or expression of the SLA-DMA gene and / or SLA-DMA protein for modulating the pathway of African swine fever virus (ASFV) toxin-producing infection. Use of compounds that regulate the function, activity, and / or expression of the SLA-DMA gene and / or SLA-DMA protein in the preparation of drugs for regulating infection by African swine fever virus (ASFV); A method for regulating infection by African swine fever virus (ASFV) toxin production, the method comprising regulating the function, expression and / or activity of the SLA-DMB gene and / or SLA-DMB protein; A compound that regulates the function, activity, and / or expression of the SLA-DMB gene and / or SLA-DMB protein, for use in regulating infection with African swine fever virus (ASFV). Use of compounds that regulate the function, activity, and / or expression of the SLA-DMB gene and / or SLA-DMB protein in the preparation of drugs for regulating infection by African swine fever virus (ASFV); Any method described herein can be an "in vitro" method. Therefore, this disclosure provides: An in vitro method for regulating infection by African swine fever virus (ASFV) toxin production, the method comprising regulating the function, expression and / or activity of porcine leukocyte antigen complex II (SLA II) gene and / or SLA II protein; An in vitro method for regulating infection by African swine fever virus (ASFV) toxin production, the method comprising contacting cells with compounds that regulate the function, activity and / or expression of porcine leukocyte antigen complex II (SLA II) gene and / or SLA II protein; In the context of this disclosure, the term "regulation" means reducing, inhibiting, blocking, or suppressing the expression, function, and / or activity of any SLAII gene and / or the protein encoded therein (SLA II protein) described herein. Therefore, compounds that regulate the expression, function, or activity of SLA II genes or SLA II proteins may reduce, inhibit, block, or suppress the expression, function, and / or activity of any SLA II gene and / or the protein encoded therein (SLA II protein) described herein.

[0014] The phrase “modulation of African swine fever virus (ASFV) toxin-producing infection” refers to any alteration (e.g., reduction, inhibition, suppression, and / or prevention) of ASFV’s ability to infect its target (host) cells, intercellular transmission, (progeny) viral titer, and / or viral replication.

[0015] The phrase "regulating the expression, function, and / or activity of SLA II genes / proteins" means, for example: Reduce, suppress, or lower the expression level of the SLA II gene; Reduce, suppress, or decrease the expression or amount of proteins encoded by the SLA II gene (e.g., SLA II protein); Reduce, inhibit, or decrease the activity of proteins encoded by the SLA II gene (e.g., SLA II proteins); and / or Reduce, suppress, or decrease the function of proteins encoded by the SLA II gene (e.g., SLA II proteins); Regulation of gene or protein function or activity can encompass the ability to reduce or inhibit the interaction of the relevant gene or protein with its normal cellular pathways, responses, and / or ligands. It should be understood that, based on the findings presented herein, any aspect of reducing (or inhibiting) the function or activity of SLA II genes / proteins can consequently affect (e.g., adversely or negatively) the ability of ASFV to infect and / or replicate in host cells; therefore, without wishing to be bound by theory, the SLA II genes / proteins (especially SLA-DMA / B genes / proteins) described herein are important in ASFV infection / replication.

[0016] The term "cell" (as mentioned in the in vitro methods above) can refer to any porcine or suidae cell that can serve as a host for ASFV. The term "cell" may also include wild boar cells.

[0017] The compounds disclosed herein include compounds for treating or preventing ASFV infection and / or ASF, which modulate the interaction between ASFV and the SLA II gene and / or SLA II protein described herein. The compounds may include: Compounds that block or neutralize the ability of ASFV to interact with or bind to SLA II proteins (including, for example, one or more SLA-DMA and / or SLA-DMB (subunit) proteins); Compounds that bind to SLA II proteins (e.g., one or more SLA-DMA and / or SLA-DMB (subunit) proteins) and prevent or inhibit ASFV from binding to them; Compounds that regulate (e.g., reduce, suppress, inhibit, or prevent) the expression of one or more SLA II genes (including, for example, one or more SLA-DMA and / or SLA-DMB genes) described herein; Compounds that inhibit the function or activity of any protein encoded by the SLA II gene in this disclosure (including, for example, SLA-DMA and / or SLA-DMB proteins encoded by one or more SLA-DMA and / or SLA-DMB genes); Compounds that prevent proviral interactions between any proteins encoded by any SLA II genes described herein (including SLA-DMA and / or SLA-DMB proteins encoded by SLA-DMA and / or SLA-DMB genes). Proviral interactions may include, for example, binding events between ASFV and any one or more SLA II genes / proteins disclosed herein, which facilitate viral (ASFV) entry into host cells and / or replication / proliferation.

[0018] For example, this disclosure provides antibodies that neutralize any (proviral) interaction between ASFV and any SLA II gene product disclosed herein. Therefore, available antibodies can bind to any SLA II protein described herein (or have affinity for any SLA II protein described herein), including, for example, one or more SLA-DMA and / or SLA-DMB proteins described herein. Available antibodies can bind to proviral epitopes—that is, epitopes within the SLA II protein region that normally interact with ASFV to allow viral entry into and / or replication within the host cell.

[0019] In one teaching, the antibodies disclosed herein are not used as "adjuvants" and / or are not fused with ASF antigens.

[0020] The term "antibody" can include monoclonal or polyclonal antibodies, as well as antibodies of any isotype (IgG, IgM, IgE, IgA, etc.).

[0021] Available antibodies may include, for example: Anti-SLA-DMA antibodies (i.e., antibodies that have an affinity for or bind to the SLA-DMA subunit of SLA II). Anti-SLA-DMA antibodies bind to the SLA-DMA epitope, thereby preventing any interaction between SLA-DMA and ASFV. By blocking the interaction between SLA-DMA and ASFV, this antibody can prevent ASFV from entering host cells and / or ASFV from replicating / proliferating in host (e.g., suicidal) cells; and / or Anti-SLA-DMB antibodies (i.e., antibodies that have an affinity for or bind to the SLA-DMB subunit of SLA II). Anti-SLA-DMB antibodies bind to SLA-DMB epitopes to prevent any interaction between SLA-DMB and ASFV. By blocking the interaction between SLA-DMB and ASFV, this antibody can prevent ASFV from entering host cells and / or replicating / proliferating in host (e.g., suicidal) cells.

[0022] This disclosure also provides anti-RFXANK antibodies (i.e., antibodies with affinity for or binding to the protein product of the RFXANK gene), anti-RFXAP antibodies (i.e., antibodies with affinity for or binding to the protein product of the RFXAP gene), and anti-CIITA antibodies (i.e., antibodies with affinity for or binding to the protein product of the CIITA gene). All of these antibodies can bind to the proviral epitopes of their respective protein targets, and this binding event can be used to prevent ASFV from entering host cells and / or ASFV from replicating / proliferating in host (e.g., swine) cells.

[0023] The technology for generating antibodies is quite mature. For more information, please refer to, for example, Antibodies: A Laboratory Manual, Second edition (Edited by Edward A. Greenfield, Dana-Farber Cancer Institute: Cold Spring Harbor Laboratory Press), the contents of which are incorporated herein by reference.

[0024] Assays used to test the effect of antibodies on ASFV entry into host cells and / or replication / proliferation may include: exposing cells to ASFV in the presence or absence of the test antibody and observing any differences in the amount of virus in the cells capable of infection and / or replication / proliferation. If the assay shows that less virus is capable of infecting cells and / or replicating / proliferating in cells in the presence of the test antibody, then the relevant antibody may be used as an antibody capable of neutralizing ASFV (or at least its cell entry / replication function).

[0025] The term "antibody" (as used herein) can be used to refer to all antigen-binding fragments and variants, including SLA-DMA, SLA-DMB, RFXANK, RFXAP, and CIITA. This term may include, for example, single-chain antibodies, monoclonal antibodies, chimeric antibodies, domain antibodies, nanobodies, camel antibodies, and any antigen-binding fragments of these antibodies that are specific to any of the disclosed antigens. The term "antibody" may further encompass bispecific antibodies and fragments and variants such as Fab, F(ab')2, monospecific, bispecific Fab2, trispecific Fab3, monovalent, scFv, bispecific biantibodies, trispecific biantibodies, scFv-Fc, or microantibodies (and the like) that are specific to any of the disclosed antigens. Although all these antibody fragments and variants may differ, for simplicity, all of these antibody types are collectively referred to as "antibody".

[0026] This disclosure provides other “antibody-like” molecules that are specific (i.e., capable of binding and / or neutralizing) any of the antigens disclosed herein (e.g., SLA-DMA, SLA-DMB, RFXANK, RFXAP, and CIITA). These “antibody-like” molecules may include, for example, molecules comprising, for example, bicyclic peptides (or molecules substantially composed of, for example, bicyclic peptides), low-affinity ligand (e.g., affinity) type molecules, engineered ankyrin repeat protein (Darpin) type molecules, and anticalcin, all of which are engineered to be specific (or capable of binding and / or neutralizing) any of the antigens disclosed herein (e.g., SLA-DMA, SLA-DMB, RFXANK, RFXAP, and CIITA).

[0027] The present invention also provides nucleic acid sequences encoding any disclosed antibody and / or antibody-like molecule.

[0028] Furthermore, this disclosure provides vectors (e.g., plasmids, viruses, viral vectors, and / or other expression cassettes) comprising nucleic acid sequences encoding any antibody and / or antibody-like molecule (or antigen-binding fragment thereof). Such vectors can be designed for expression in mammalian (e.g., human) cells. In one teaching, this disclosure provides antibodies or antibody-like molecule encoding plasmids / viruses.

[0029] This disclosure also provides a host cell, such as a mammalian cell, like a human cell. The host cell can be a mammalian somatic cell, such as a human cell.

[0030] Antibodies or antibody-like molecules (or any antigen-binding / neutralizing fragments) can be administered to subjects in need or to in vitro cells or tissues via techniques that allow direct physical delivery into cells. Such techniques may include electroporation and / or microinjection.

[0031] Any disclosed antibody and / or antibody-like molecule may be provided in a form that allows for intracellular expression and / or targeting of specific intracellular regions and / or structures. Antibodies expressed intracellularly may be referred to as intrabody antibodies. Therefore, this disclosure provides intrabody antibodies that are specific (or have binding and / or neutralizing capabilities) against any of the disclosed antigens, including, for example, SLA-DMA, SLA-DMB, RFXANK, RFXAP, and CIITA.

[0032] The expression of the intracellular antibodies disclosed herein can be achieved using a vector, such as a plasmid or virus encoding an antibody of this disclosure (or an antigen-binding fragment thereof). In one teaching, cells can be transfected with a plasmid / virus carrying nucleic acid encoding any disclosed antibody, antibody-like molecule, or an antigen-binding (or neutralizing) fragment thereof.

[0033] The vectors disclosed herein may also encode signal peptides or molecules (possibly fused or conjugated with antibodies / antibody-like molecules) to direct antibodies / antibody-like molecules (or fragments thereof) to specific parts of the cell (e.g., the nucleus, cytosol, mitochondria, or ER lumen). Various signal peptides and / or signal / retention molecules are known to those skilled in the art, many of which can be used to ensure the expression of antibodies, antibody-like molecules, or antigen-binding fragments (or fragments thereof) in the cell and to direct them to specific sites or structures (e.g., the cytosol, nucleus, or mitochondria). Examples may include endoplasmic reticulum-retention sequences, nuclear localization sequences, mitochondrial localization sequences, transduction domains, and nanoparticles. Nucleic acids encoding any of the above may be included in the various vectors described herein—all of which are designed to ensure that antibodies or antibody-like molecules expressed in the cell can be properly targeted to perform their function (in this case, to regulate the expression, function, and / or activity of the SLA II gene / protein). All of these techniques are described in Slastnikova. et al., It is well summarized in 2018 (Targeted Intracellular Delivery of Antibodies: The State of the Art: 2018, 9: 1208), and all of its contents are incorporated herein by reference.

[0034] In one teaching, this disclosure provides nucleic acid sequences that encode antibody, antibody-like molecules, or antigens thereof (e.g., SLA II proteins or SLA-DMA, SLA-DMB, RFXANK, RFXAP, and CIITA) binding or neutralizing fragments, as well as signaling molecules for ensuring intracellular expression and / or intracellular expression at specific sites or structures.

[0035] This disclosure may also provide antisense molecules, such as antisense oligonucleotides (ASOs), which regulate (e.g., block, reduce, suppress, or inhibit) the expression of one or more SLA II genes described herein.

[0036] The antisense oligonucleotide may comprise 10 to 30 nucleotides, such as 15 to 20 nucleotides, whose sequence is complementary to the target RNA sequence. The target RNA sequence may be RNA (e.g., mRNA) generated by any SLA II gene (SLA II RNA) described herein. Not wishing to be theoretically constrained, the ASO of this disclosure may bind to a region of SLA II RNA, thereby tagging that region for degradation, thereby reducing the amount of the corresponding SLA II protein expressed by (or from) that gene.

[0037] Given a gene sequence, designing and obtaining an ASO capable of regulating the expression of that gene is relatively straightforward; in fact, those skilled in the art are aware of the various services and online tools available for generating ASOs for testing. Available ASOs can be selected based on their ability to regulate (e.g., reduce or inhibit) the expression of any SLA II protein of this disclosure—particularly the SLA DMA / B subunit of SLA II. For example, cells exhibiting known SLA II protein (e.g., SLA DMA / B) expression levels can be exposed to an ASO, and the subsequent levels of the SLA II protein in said cells can be determined. Any difference between known SLA II protein expression levels and the expression levels of the same protein after exposure to an ASO indicates that the ASO has a regulatory effect on the relevant SLA II gene. This type of assay can be used to test ASOs that reduce or inhibit the levels of the relevant SLA II protein in cells.

[0038] The vectors disclosed herein (e.g., plasmids, viral vectors, or expression cassettes) may include any ASO described herein for expression in cells. This disclosure also provides host cells transformed or transfected with such vectors.

[0039] The nucleic acid sequences of the SLA-DMA, SLA-DMB, RFXAP, and CIITA genes, as well as other related sequences, are provided below as SEQ ID NO: 1-8. These sequences can be used to design the ASOs described herein: SEQ ID NO: 1: NM_001004039.1 Wild boar ( Sus scrofa SLA-DM α chain (SLA-DMA), mRNA SEQ ID NO: 2: NM_001004039.1 Wild boar SLA-DM alpha chain (SLA-DMA), CDS ATGGATCATGAGCTGAGCCAGGGAGCTGCACTGCTACGGCTGCTACACCTTCTGTGGCTGCTGCCCCACTCCTGGACTGCCCCCGAAGCTCCTGCTCCAGGGTGGAGGGATGAGCTGCAAAACCACACGTTCCAGTACACAATGTACTGCCAGGATGGGAATCCCGAAGTGGGACTCTCCGAGGTCTATGATGGGGACCAGCTTTTCTCCTTCAACTTTTCCCAGAACATCCGAGTGCCTCGCCTGCCTGAATTTGCGGACTGGGCTCACCAGATTGAAGACACTCCTGCCATTTTCTTTGACAAAGGATTCTGCCGAGAGATGATCGAAAAAGTTGGCCCACTGTTTGAAGGGAAAATCCCAGTGTCTAGAGGGTTACCCATCGCTGAGGTGTTCACGCTGAAGCCCCTGGAGTTTGGCAAACCCAACACACTGGTCTGTTTTGTCAGTAACCTTTTCCCACCCGCACTGACGGTGACCTGGGAGCATCACTCTGCTCCTGTGGAAGGAATCGGGCCCACTTTCGTCTCCGCCACGGATGACCTCAGCTTCCAGGCCTTTTCTTATTTAAACTTCACACCGACACCCTCTGACCTTTTCTCCTGCGTCGTGACTCATGAGCTCGATGGCTACGTAGCAATCTCTTACTGGGTGCCCCAGAATGCGCTGCCCTCAGACCTCCTGGAGAACGTGCTGTGTGGCGTGGCCTTTGGCCTGGGTGTGCTGGGAATCATTGTTGGCTTAGTCCTCATCATCTACTCTCGGAAGCCTTGCTCAGCCTGA SEQ ID NO: 3: NM_001004039 Reverse translation of wild boar SLA-DM alpha chain precursor, codon-optimized for wild boar, for cloning ATGGACCACGAGCTGTCCCAGGGCGCCGCTTTGCTGCGCTTGCTGCATTTGCTGTGGCTTCTGCCTCACAGTTGGACGGCACCAGAAGCGCCGGCGCCCGGATGGAGGGATGAGCTGCAGAACCATACTTTCCAGTATACCATGTACTGCCAGGACGGTAATCCTGAGGTCGGCCTCAGTGAGGTGTATGACGGCGATCAGCTGTTCTCATTTAATTTCTCCCAGAACATTAGAGTGCCTCGCCTGCCGGAGTTTGCTGATTGGGCCCACCAGATTGAGGATACACCCGCGATCTTCTTTGACAAGGGCTTCTGCCGCGAGATGATTGAGAAGGTGGGCCCACTGTTCGAGGGCAAGATTCCCGTATCTCGCGGCCTCCCAATCGCAGAAGTCTTTACCCTCAAGCCGCTGGAGTTCGGCAAGCCCAATACCTTGGTGTGCTTCGTCTCAAACCTGTTCCCACCCGCCCTGACTGTCACATGGGAGCACCATTCCGCTCCTGTCGAGGGCATTGGACCAACCTTTGTCTCTGCCACCGATGACTTGTCCTTTCAGGCATTCTCCTACCTGAATTTCACTCCTACACCAAGTGACCTGTTCTCCTGCGTCGTGACCCACGAGCTCGACGGCTACGTCGCTATCTCTTACTGGGTCCCTCAGAACGCCTTGCCATCCGACTTGCTCGAGAACGTACTCTGTGGTGTGGCGTTCGGTCTCGGAGTCCTCGGCATTATCGTCGGATTGGTTCTGATTATCTATAGCAGGAAGCCTTGCTCAGCCTGA SEQ ID No: 4: NM_001113707.1 Wild boar class II MHC, DM β (SLA-DMB), mRNA SEQ ID NO: 5: NM_001113707 Wild boar class II MHC, DM β (SLA-DMB), CDS ATGCCCTGTATCTCCCCGGAGCAGAGCATGAGTGCCCTCCTGCAGCTGCTCCTGGGCCTCAGCCTGGGCTGCACCGGAGCAGGTGGCTTTGTGGCCCACGTGGAAAGCACCTGTCTGTTGGATGATGAGGGGACTCCACAGGATTTCACGTATTGCATCTCCTTCAACAAGGACTTGCTGACCTGTTGGGATCCCCAGGAGACCCGTATGGTCCCTTGTGAATTTGGAGCGCTGAATGCGTTGGCCACATATTTCTCTGTTTACCTCAACCAGCAGGAAAAACTGCTCCAGCGCTTGTCCAATGGGCTCCAGAACTGTGCCACACACACCCAACCCTTCTGGAAATCACTGACCCACAGGACACAGCCGCCGTCTGTGCAAGTGGCCAAAACCACTCCTTTTAACACGAGGGAGTCTGTGATGCTGGCCTGCTATGTGTGGGGCTTCTATCCAGCTGATGTGATCATCACGTGGAGGAAGAATGGGCAGCCAGTCCTTCCTCATGGCAAGGCCCATATGATCACCCAGCCCAACGGAGACTGGACATACCAGACCGTCTCCCATTTGGCTACAACCCCCTCTTACGGGGACACCTACACCTGTGTGGTGGAGCACATTGGGGTTCCTGAGCCCATCCTTCAGGACTGGACTTCTGGGCTGTCCCCAGTGCAGACAGTGAAGATTTCTGTGTCTGTGGCGACTCTGGGCCTGGGCCTCATCATCTTCTCCCTTGGTTTGCTCAGCTGCCAGAGATCTGTTGCCCCAGGCTACATTTTCCTCCCGGGGACCACTTATCCAGAAGGTCAGCACATTTCCTAG 翻译内容过长,此处省略,如需完整翻译请告知。 SEQ ID NO: 6: NM_001113707 Wild boar class II MHC, DM β (SLA-DMB) - Reverse translation, codon-optimized for wild boar, for cloning ATGCCATGCATCAGTCCGGAGCAGAGCATGAGCGCTCTTCTGCAGCTCCTGCTCGGCTTGAGCCTCGGATGTACCGGCGCTGGAGGCTTCGTCGCTCACGTGGAGTCTACCTGCCTGCTTGATGACGAAGGCACTCCACAGGACTTCACATATTGCATCTCCTTCAACAAGGATCTGCTTACCTGTTGGGACCCACAGGAGACACGTATGGTGCCCTGCGAGTTCGGCGCTCTCAACGCGCTGGCTACATACTTCTCCGTGTATCTGAATCAACAAGAGAAGCTCCTGCAGCGCTTGTCTAACGGCCTCCAGAACTGCGCCACACATACTCAACCGTTTTGGAAGAGCCTCACTCATCGTACTCAGCCGCCATCCGTGCAGGTGGCCAAGACCACGCCGTTCAACACACGCGAGAGCGTCATGCTGGCTTGCTACGTGTGGGGCTTCTATCCGGCTGACGTCATTATCACCTGGCGCAAGAACGGCCAGCCAGTGCTGCCTCACGGCAAGGCGCACATGATCACACAGCCTAACGGCGATTGGACTTATCAGACAGTGAGCCACCTGGCGACTACACCATCCTATGGAGATACCTACACCTGCGTTGTGGAGCACATTGGCGTACCTGAACCTATCCTGCAGGACTGGACCTCCGGCCTGTCACCTGTCCAAACCGTCAAGATCAGTGTTAGCGTGGCGACTCTCGGCCTTGGCCTGATTATCTTCAGCCTCGGCCTCCTGTCCTGTCAACGCTCAGTGGCTCCAGGTTACATCTTCCTGCCTGGCACCACTTATCCAGAAGGCCAGCACATCTCCTGA SEQ ID NO: 7: Predicted XM_013980439.2: Sus scrofa regulator of calcineurin 1 (PPP3CA), transcript variant X1, mRNA It should be noted that there may be some inaccuracies in the translation of the gene name in the provided content. The original "调节因子X相关蛋白(RFXAP)" is translated as "regulator of calcineurin 1 (PPP3CA)" here, which might need to be further verified according to the specific context and accurate gene information. SEQ ID NO: 8: Predicted XM_021085809.1: Wild boar class II major histocompatibility complex transactivator (CIITA), transcript variant X5, mRNA (protein title MHC class II transactivator isotype X4) The term "compound" may also include fragments of proteins encoded by any SLA II gene described herein. For example, the term "compound" may include fragments or portions of the SLA DMA / B subunit protein.

[0040] Available fragments may include portions of SLA II proteins known to interact with or bind to ASFV. These fragments can bind to ASFV and can be used to block or neutralize any interaction between ASFV and natural or wild-type SLA II proteins.

[0041] The fragments of SLA-DMA, SLA-DMB, RFXAP, and CIITA proteins described in this article may be derived from any of the following sequences: SEQ ID NO: 9: SLA-DMA (Accession number: NP_001004039.1: SLA-DM α-chain precursor [wild boar] - protein) MDHELSQGAALLRLLHLLWLLPHSWTAPEAPAPGWRDELQNHTFQYTMYCQDGNPEVGLSEVYDGDQLFSFNFSQNIRVPRLPEFADWAHQIEDTPAIFFDKGFCREMIEKVGPLFEGKIPVSRGLPIAE VFTLKPLEFGKPNTLVCFVSNLFPPALTVTWEHHSAPVEGIGPTFVSATDDLSFQAFSYLNFTPTPSDLFSCVTHELDGYVAISYWVPQNALPSDLLENVLCGVAFGLGVLGIIVGLVLIIYSRKPCSA Available SLA-DMA fragments may be derived from SEQ ID NO: 9 and may include any number of residues from about 5 residues to about 259 residues (and any number therein). For example, fragments may include about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, or about 255 residues. It should be noted that the term "about" means ±1, 2, 3, or 4 amino acid residues. As stated, any fragment derived from SEQ ID NO: 9 may include portions of SLA-DMA proteins known to interact with or bind to ASFV. These fragments can bind to ASFV and can be used to block or neutralize any interaction between ASFV and natural or wild-type SLA-DMA proteins.

[0042] SEQ ID NO: 10: SLA-DMB (Accession number: NP_001107179.1: MHC class II, DM β precursor [wild boar], protein) MPCISPEQSMSALLQLLLGLSLGCTGAGGFVAHVESTCLLDDEGTPQDFTYCISFNKDLLTCWDPQETRMVPCEFGALNALATYFSVYLNQQEKLLQRLSNGLQNCATHTQPFWKSLTHRTQPPSVQVAKTTPFNT RESVMLACYVWGFYPADVIITWRKNGQPVLPHGKAHMITQPNGDWTYQTVSHLATTPSYGDTYTCVVEHIGVPEPILQDWTSGLSPVQTVKISVSVATLGLGLIIFSLGLLSCQRSVAPGYIFLPGTTYPEGQHIS Available SLA-DMB fragments may be derived from SEQ ID NO: 10 and may include any number of residues from about 5 residues to about 271 residues (and any number therein). For example, fragments may include about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265 or about 270 residues. It should be noted that the term "about" means ±1, 2, 3, or 4 amino acid residues. As stated, any fragment derived from SEQ ID NO: 10 may include portions of SLA-DMB proteins known to interact with or bind to ASFV. These fragments can bind to ASFV and can be used to block or neutralize any interaction between ASFV and natural or wild-type SLA-DMB proteins.

[0043] SEQ ID NO: 11: (Accession number: XP_013835893.2: Regulatory factor X-related protein [wild boar] - protein) MILEQDKLGKSTPRLHRETEEQNTKGKEKALKAAGEKRQGCSCAGAVGVLSRIRPLFSAGRGRWFAQCSWAQQVQKGWGRGPPPGLSSTEVQAVAEGAGPGAASGALRPGAPASAPQALAAAPVPAAASQFTLLVMRPCGGQDEAAAEGVLRQAPALGGSAGTGKPVGYLCEXGGDGEDEAGE DETDLLDTSDPPGGGESTASLEDLEDEETHXGGEGGSGGARRRGSGGTSMSKTCTYEGCSETTSQVAKQRKPWMCKKHRNKMYKDKYKKKKSDQALNCGGAAPAGSAGHVKLEESADNILSIVKQRTGSFGDRPARPTLLEQVLNQKRLSLLRSPEVVQFLQKQQQLLNQQVLEQRQQQIPGTSV The available RFXAP fragments may be derived from SEQ ID NO: 11 and may include any number of residues from about 5 residues to about 368 residues (and any number therein). For example, the fragments may include about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 The number of residues is 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, or about 365 residues. It should be noted that the term "about" means ±1, 2, 3, or 4 amino acid residues. As described, any fragment derived from SEQ ID NO: 11 may include portions of RFXAP proteins known to interact with or bind to ASFV. These fragments can bind to ASFV and can be used to block or neutralize any interaction between ASFV and natural or wild-type RFXAP proteins.

[0044] SEQ ID NO: 12 (Accession number: XP_020941468.1: MHC class II transactivator isotype X4 [wild boar], protein) The available CIITA fragments may be derived from SEQ ID NO: 12 and may include any number of residues from about 5 residues to about 1155 residues (and any number in between).For example, segments may include approximately 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 19 0, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 57 0, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 7 80, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985 990, 950, 1000, 1005, 1010, 1015, 1020, 1025, 1030, 1035, 1040, 1045, 1050, 1055, 1060, 1065, 1070, 1075, 1080, 1085, 1090, 1095, 1100, 1105, 1110, 1115, 1120, 1125, 1130, 1135, 1140, 1145, or about 1150 residues. It should be noted that the term "about" means ±1, 2, 3, or 4 amino acid residues.As described, any fragment derived from SEQ ID NO: 12 may include portions of CIITA proteins known to interact with or bind to ASFV. These fragments may bind to ASFV and may be used to block or neutralize any interaction between ASFV and natural or wild-type CIITA proteins.

[0045] The various SLA II protein fragments described herein can be used to generate antibodies. Antibodies generated in this manner (and having an affinity for or ability to bind to SLA II protein fragments) can be used to neutralize any (proviral) interaction between ASFV and the SLA II protein from which the SLA II protein fragments are derived. Therefore, fragments used in methods for generating antibodies (for the prevention of ASFV infection and / or treatment or prevention of ASF) may include epitopes known to interact with ASFV. Thus, antibodies generated that bind to the SLA II protein fragments of this disclosure can be used to prevent ASFV from entering cells and / or replicating / proliferating within cells.

[0046] This disclosure may also provide sequences derived from the nucleic acid sequences described herein that encode SLA II protein fragments.

[0047] This disclosure further provides vectors, such as viral vectors or nucleic acid vectors (e.g., plasmids), which include nucleic acid sequences of this disclosure, including, for example, sequences encoding any antibody or antibody-like molecule disclosed herein, any antisense oligonucleotide disclosed herein, or any SLA II protein fragment described herein.

[0048] This disclosure also provides host cells, such as cells transformed with any of the vectors described herein.

[0049] The various compounds disclosed herein (antibodies, antibody-like molecules, SLA II protein fragments, or antisense oligonucleotides) can be provided as compositions.

[0050] The compositions disclosed herein may include pharmaceutical compositions.

[0051] The compositions disclosed herein may further include excipients, buffers, and / or diluents.

[0052] This disclosure also provides immunogenic compositions designed to elicit an immune response, such as a protective immune response, in a host. In the context of this disclosure, a protective immune response may include a response that neutralizes ASFV or prevents ASFV from interacting with host cells, entering host cells, and / or replicating and / or proliferating within host cells. The immunogenic compositions of this disclosure may include any or more of the SLA II proteins described herein or functional fragments thereof. Not wishing to be bound by theory, the immunogenic compositions of this disclosure may be administered to subjects at risk of ASFV infection (and therefore ASF) to induce an immune response that can prevent the proviral interaction between the SLA II proteins and ASFV. For example, the immunogenic composition may induce an immune response including an antibody with affinity or specificity for the SLA II protein of the composition, which binds to the SLA II protein and prevents its interaction with ASFV.

[0053] The immunogenic compositions disclosed herein may include SLA-DMA protein or fragments thereof and / or SLA-DMB protein or fragments thereof.

[0054] In further teaching, this disclosure provides a vaccine comprising any or more of the SLA II proteins described herein, or functional fragments thereof. Again, not wishing to be bound by theory, the vaccine of this disclosure may be administered to a subject at risk of ASFV infection (and therefore ASF) to induce an immune response that can prevent the proviral interaction between the SLA proteins and ASFV. For example, the vaccine may induce an immune response including antibodies with affinity or specificity for the SLA II proteins of the composition that bind to the SLA II proteins and prevent their interaction with ASFV. For example, the vaccine of this disclosure may comprise an SLA-DMA protein or a fragment thereof and / or an SLA-DMB protein or a fragment thereof. The vaccine may further comprise an adjuvant and / or one or more other antigens, such as one or more other ASFV antigens and / or one or more antigens from different pathogens.

[0055] It should be noted that any compound, antibody, antibody-like molecule (or its antigen-binding / neutralizing variant or fragment), SLA II protein fragment, vector / nucleic acid (which may encode an antibody, antisense oligonucleotide, or SLA II protein fragment) or composition provided in this disclosure may be used for medical purposes, as a pharmaceutical product, or for the treatment or prevention of ASF or ASFV infection.

[0056] In one teaching, this disclosure provides a transgenic (or genetically modified) animal, such as a suidae (or pig-like animal) (e.g., a wild boar). Sus scrofaThe transgenic animal is resistant to ASFV, and therefore also resistant to ASF.

[0057] According to this disclosure, genetically modified animals may lack a functional copy of any of the SLA-II genes described herein.

[0058] For example, the SLA-DMA gene or the protein it encodes may have become nonfunctional (through some functional mutations) or completely knocked out.

[0059] Alternatively, the SLA-DMB gene or the protein it encodes may have been rendered nonfunctional (through some functional mutation) or completely knocked out.

[0060] Alternatively, the RFXANK gene or the protein it encodes may have become nonfunctional (through some functional mutation) or completely knocked out.

[0061] Alternatively, the RFXAP gene or the protein it encodes may have become nonfunctional (through some functional mutation) or completely knocked out.

[0062] Alternatively, the CIITA gene or the protein it encodes may have become nonfunctional (through some functional mutation) or completely knocked out.

[0063] Genetically modified animals, such as pigs (or swine) (including pigs or wild boars: wild boars) Sus scrofa As described above, gene modifications (i.e., modifications in some way to regulate (e.g., reduce, prevent, or suppress) the expression of any SLA-II gene described herein) can be obtained using any widely available method. Detailed Implementation

[0064] This disclosure will now be described in detail with reference to the following figures, in which: Figure 1. MHC II pathway molecules identified by genome-wide CRISPR / Cas9 knockout screening are associated with ASFV replication. (a) Robust sequencing integration (RRA) scores for four separate analyses identified in two independent screenings, calculated using the MAGeCK algorithm. sgRNA levels in control cells are compared to sgRNA abundance in cells surviving four subsequent ASFV infections. (b) Mean (-) and individual RRA scores for individual gene hits in the four different analyses. Dark blue dots represent sgRNAs targeting a specific gene (x-axis) found in the 4 / 4 subgroup. Medium light blue dots represent sgRNAs targeting the CCZ1 gene found in the 3 / 4 subgroup. Light blue dots indicate sgRNAs found only in the two subgroups selected. (c) Schematic diagram of MHC II loci (e.g., SLA-DMA or SLA-DMB) that possess MHC II class-specific regulatory SXY modules and specific transcription factors. Proteins identified as key cytokines for ASFV infection in genome-wide CRISPR / Cas9 knockout screening are shown in blue.

[0065] Figure 2. Differences in MHC II protein expression between parental WSL and WSL knockout cells. (a) Differences in MHC II protein expression between WSL and WSL SLA-DMA KO WSL SLA-DMB KO WSL CIITA KO and WSL RFXAP KO Indirect immunofluorescence analysis of SLA-DR cell surface expression in cell clones. Scale bar: 30 µm. (b) Quantitative mass spectrometry analysis of the expression levels of housekeeping gene α-tubulin (TUBA4A-ENSSSCG00000016216), MHC II pathway genes (SLA-DRA-ENSSSCG00000001453, SLA-DRB1-ENSSSCG00000001455, SLA-DQA-ENSSSCG00000001456, SLA-DQB-ENSSSCG00000001457), and MHC I pathway genes (SLA-8-ENSSSCG00000001231, HLA-E-ENSSSCG00000001229) in WSL cells and specified knockout cells based on label-free quantification (LFQ). Data represent the mean of three replicates. (c) WSL and a single WSL KO Comparative quantitative analysis of protein expression levels in cell clones. Proteins are represented by dots. Black dots represent proteins involved in antigen processing and presentation. Based on the detection, SLA I / II proteins shown in b are highlighted in red.

[0066] Figure 3. Impaired ASFV replication in WSL knockout cells. (a) WSL cells infected with ASFV Armenia or ASFV Kenya by immunofluorescence staining. KO Cells (green) and nucleic acids (blue) are visualized. Representative images of the cell clones shown, infected with different viral dilutions (10⁻¹ to 10⁻³), illustrate plating efficiency and plaque size. Scale bar: 100 µm. (b) Plating efficiency of ASFV Armenia and ASFV Kenya was calculated by counting ASFV-infected cells or plaques in three independent experiments (n=3). Mean relative apparent titers (%) and standard deviations compared to those on WSL cells are shown. Significance of differences was calculated using ordinary one-way ANOVA and Tukey's multiple comparison test. =<0.0001. (c) To determine plaque size, the area of ​​fifty plaques from each cell line in three independent experiments (n=150) was measured, and the mean relative area (%) compared to WSL cells is shown, including standard deviation. Significance of differences was calculated using the Kruskal-Wallis test and the Dunn multiple comparison test. =<0.0001. (d) ASFV Armenia or Kenya in WSL and WSL KO Multi-step (MOI 0.02) growth curve analysis in cells. Mean results and standard deviations from three independent experiments (n=3) are shown.

[0067] Figure 4 In WSL knockout cells, ASFV DNA replication was suppressed. Parental WSL cells and WSL4 cells were infected with ASFV Armenia at MOI 3. KO Cells were used, and the amount of ASFV DNA was quantified by real-time PCR targeting the viral B646L gene after specified time intervals. Genomic copy number was determined using plasmid standards. Graphs represent the means of two biological replicates with standard deviation.

[0068] Figure 5 ASFV progeny virus particles were detected in infected parental WSL cells, but not in knockout cells. Sixteen hours after infection with Armenian ASFV at MOI 5, (ad)WSL and (e)WSL SLA-DMA were... KO and (f)WSLCIITA KOCells were fixed and analyzed using an electron microscope. The virus factory (arrow) and intracellular (…) were indicated. ) and extracellular viral particles (#). The scale bar indicates 1 µm (a, e, f) or 200 nm (b, c, d).

[0069] Figure 6 WSL knockout / knockin cells express MHC II transgenes. (a)WSL SLA-DMA was isolated by SDS-PAGE. KO Cells and (b) lysates of WSL cells expressing the SLA-DMA transgene or GFP as shown, or (c) SLA-DMB KO Lysates of cells and (d) WSL cells expressing the indicated SLA-DMB transgene or GFP were transferred to a nitrocellulose membrane and detected using antibodies against the indicated proteins or protein tags. The molecular weight (in kDa) of the labeled proteins is shown on the left.

[0070] Figure 7. MHC II transgene expression in WSL knockout / knockin cells restores ASFV replication. (ab) To determine plaque formation efficiency and plaque size, immunofluorescence staining was performed on WSL cells infected with ASFV Armenia or ASFV Kenya. KO and WSL KO / KI Cells were visualized. (a) ASFV plaque formation efficiency for Armenia and Kenya was calculated by counting ASFV-infected cells or plaques in three independent experiments (n=6). Mean relative titers (%) and standard deviations compared to those on WSL cells are shown. Significance of differences was calculated by ordinary one-way ANOVA and Tukey's multiple comparison test. =<0.05, =<0.0001, ns= not significant. (b) To determine plaque size, the area of ​​fifty plaques for each cell line in three independent experiments (n=150) was measured and the mean relative (%) size compared to WSL cells is shown, including standard deviation. Significance of differences was calculated by Kruskal-Wallis test and Duncan multiple comparison test. =<0.001, =<0.0001, ns= not significant. (ce) ASFV Armenia and Kenya in untreated and transgenic expression of lentiviral transduction of (c) WSL, (d) WSL-DMA KO and (e)WSL-DMBKO Multistep growth analysis in cells (MOI 0.02). The mean results (n=4) and standard deviations of two independent experiments with two replicates are shown.

[0071] Materials and methods Cell lines and viruses The cell line was obtained from the Veterinary Cell Culture Collection (CCVM) at Friedrich-Loeffler-Institut (FLI). A highly passaged wild boar lung cell line (WSL-R-HP, #1346; abbreviated WSL) was cultured in Ham's F12 cell medium supplemented with 10% fetal bovine serum (FCS) (Ham-F-12, 5.32 g / L; IMDM, 8.80 g / L; NaHCO3, 2.45 g / L; pH 7.2). Cloning of the cell line was performed by limiting dilution in 96-well plates. Single cells were proliferated, and a WSL cell clone exhibiting the parental phenotype was selected for all targeted knockout experiments. Rabbit kidney cell line (RK-13, #0237) was cultured in minimum essential medium (MEM; MEM Eagle Hank salt, 5.32 g / L; MEM Earle salt, 4.76 g / L; NaHCO3, 1.25 g / L; non-essential amino acids, 1%; sodium pyruvate, 0.12 g / L; pH 7.2) supplemented with 10% FCS. Human embryonic kidney cell line (HEK293Td4.1, #1539) was also cultured in MEM supplemented with 10% FCS. All cells were incubated at 37°C and 2.5% CO2. ASFV Armenia 2008 is a virulence genotype II ASFV isolate from Armenia. 55 Provided courtesy of Sandra Blome (FLI). The virus was adapted for efficient growth in cell culture by passaged continuously for 21 generations in WSL cells. Genotype IX isolate ASFV Kenya 1033 55,75 Provided courtesy of Richard Bishop (International Livestock Research Institute, Nairobi, Kenya). At the missing CD2v (EP402R) locus. 55,56 The mutant ASFV Kenya 1033 ΔCD2v dsRed containing the reporter gene expression cassette was kindly provided by Günther M. Keil (FLI). The plasmid-based PrV mutant PrV-BaΔgGG was also used. 76 Used as a heterologous control virus.

[0072] Pig CRISPR Library The generation and characteristics of the porcine CRISPR knockout library (SsCRISPRko.v1) have been described. 52 In short, to generate specific single guide RNAs (sgRNAs) targeting protein-coding genes, a genomic component from wild boar (S. scrofa) 10.2 was used. 77 Three to four sgRNAs were selected for each gene. Overall, the porcine CRISPR library consisted of 83,381 specific sgRNAs targeting 20,598 porcine genes and 1,001 non-target control molecules, cloned in the pLenti-CRISPRv2 backbone (Addgene#52961).

[0073] Whole-genome CRISPR / Cas9 knockout screening As described above, perform whole-genome CRISPR / Cas9 knockout screening. 52 And with slight modifications as described below. Inoculate 5 x 10⁵ cells into each of five 20 cm culture dishes. 6 One day later, WSL cells were transduced using a lentiviral sgRNA library, which was based on the library by Joung et al. 78 The protocol was used to generate cells in a medium containing 10 µg / ml polybutene with a MOT of 0.3. Three days after transduction, cells were cultured at 5 x 10⁻⁶ cells / mL. 6 Cells were randomly assigned to eight culture dishes at a density of 1.25 µg / ml puromycin. At fusion, the selected transduced cells were cultured at a density of 5 x 10⁻⁶ cells / plate. 6 Cells were redistributed at a density of 1 x 10⁶ cells / plate into a total of 16 plates. At 12 days post-transduction, cells were spaced at a density of 1 x 10⁶ cells / plate. 7 Cells were seeded at a density of 10 cells / dish into 30 culture dishes for infection the following day. At this point, at least 6 x 10⁶ cells / dish were harvested. 7 Cells were pelleted and stored at -20°C as controls for DNA extraction. Infection was performed using ASFV Kenya 1033 ΔCD2v-dsRed at an MOI of 0.3 or 0.5 (depending on available viral stock), as this MOI has been shown to be necessary to kill at least the majority of infected WSL control cells. Cells were examined daily for fluorescent marker expression and cytopathic effects, with medium added or replaced as needed. For medium replacement, 20% conditioned medium from untreated WSL cells was added. After approximately four to five weeks, cell colonies grown from all plates were trypsin-treated and divided into two pooled subgroups. At least 2 x 10⁶ cells from each subgroup were collected. 7Cells were stored at -20°C for DNA preparation. The remaining cells were re-seeded into cell culture dishes and infected as described above. Cells surviving the second infection were harvested approximately 20 days later. Cell collection for DNA preparation, re-seeding, and infection was repeated four times. The entire selection process was performed twice, yielding two groups of uninfected control cells and eight surviving populations (two groups selected each time). To reliably inactivate the virus, the precipitated cells were resuspended in TEN (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl) supplemented with RNase A (500 µg / mL, Serva) and incubated at 37°C for 1 h. After adding SDS to a final concentration of 0.3%, the samples were further incubated at 75°C for 30 min, followed by standard lysis and DNA extraction procedures using sarkosyl lauroyl sarcosyl buffer, RNase A, streptokinase, phenol-chloroform extraction, and ethanol precipitation, as described previously. 52To generate sequencing libraries, the extracted DNA underwent three consecutive PCR amplifications. First, following the manufacturer's instructions, four 50 µl aliquots of reaction mixture were prepared for each sample, each containing 5 µg of DNA, 25 fmol of each P5 forward primer (ITA2fwd_P5) and P7 reverse primer (ITA2rev_P7_leCRV), and 3.75 U of ExTaq DNA polymerase (Clontech). Incubation conditions were as follows: 95°C for 1 min; up to 28 cycles: 95°C for 30 s, 53°C for 30 s, 72°C for 1 min, and 72°C for 10 min. After amplification, the four PCR reactions for each sample were combined, and the 155 bp product was purified using a gel extraction kit (Zymo Research). DNA was eluted in nuclease-free water, and 200 ng of DNA was used for a second PCR reaction with the same composition as before, but with 25 fmol of sample-specific P5-barcode-forward primer (e.g., ITA2fwd_ID85_P5both) and P7 reverse primer (e.g., ITA2rev_IDxx_P7leCrv2). The reaction conditions were as follows: 95°C for 1 min; up to 14 cycles: 95°C for 30 s, 57°C for 30 s, 72°C for 1 min, and 72°C for 10 min. The PCR product was purified using the QIAquick Nucleotide Removal Kit (QIAgen) and eluted in 35 µl of nuclease-free water. A third amplification step was performed using the same compounds as the second PCR, in 300 µl (6 x 50 µl) volumes of the entire eluted DNA, but with ten times higher concentrations of forward and reverse primers (250 fmol). The PCR reaction was performed as described above, but only for a single cycle. The 222 bp amplification product was purified by gel electrophoresis and eluted in nuclease-free water. DNA isolation and three consecutive PCR amplifications were performed in parallel on complete sample sets, including corresponding controls, to minimize bias. (IonTorrent Ion S5) TM Samples were sequenced using the XL system (Invitrogen, Thermo Fisher Scientific), and the sequencing data were processed and analyzed on the Galaxy web platform (usegalaxy.eu) using Cutadapt (Galaxy version 1.16.5), the MAGeCK counting tool (Galaxy version 0.5.8.4), and the MAGeCK testing tool (Galaxy version 0.5.8.1), as described above. 52,57,79,80 The robust sequencing integration (RRA) method of the MAGeCK assay tool was used to compare sequencing results under two different conditions (“control” vs. “survivor”). 57 .

[0074] Generate WSL knockout cells To target the generation of SLA-DMA, SLA-DMB, CIITA, or RFXAP knockout cells, one of four gene-specific sgRNAs was selected from a whole-genome library and cloned into the multiplex CRISPR / Cas9 vector pX330A-1×4, a gift from Takashi Yamamoto (Addgene plasmid #58768; http: / / n2t.net / Addgene:58768; RRID:Addgene_58768). 81 Furthermore, it was modified to express the neomycin resistance gene (neoR; named pX330A-1×4neoRA). To obtain pX330A-1x4neoRA, the 8962 bp vector pX330A-1x4 was linearized in the non-functional region by PciI digestion, and neoR, controlled by the early promoter of simian virus 40 (SV40), was inserted as a 1667 bp PciI fragment, which was isolated from pX330-ΔNLS1 / 2neoR. 55 In the resulting plasmid, the resistance gene is aligned parallel to the sgRNA and Cas9 genes. Next, complementary DNA oligonucleotides containing the sgRNA target-specific sequence and with matching 5' overhangs were hybridized, phosphorylated, and cloned into pX330A-1x4neoRA, which had been digested with BpiI and dephosphorylated. The accuracy of the resulting plasmid was checked by sequencing with HU6-SF primers. K2 was used according to the manufacturer's instructions. ® The transfection system (Biontex) was used to transfect plasmids pX330A-1x4neoRA-SLA-DMA gR2, -SLA-DMB gR3, -MHCIITA gR2, and -RFXAP gR2 into cloned WSL cells. Three days later, cells were trypsinized, serially diluted, and seeded into 96-well plates using medium supplemented with 0.5 mg / ml G418 sulfate (Invitrogen, Thermo Fisher Scientific). Resistant single-cell clones were further proliferated, and Cas9 expression was examined by Western blotting using anti-FLAG antibody (see below). DNA from Cas9-positive cells was prepared using the QIAamp DNA Mini Kit (QIAgen) according to the manufacturer's instructions and used for PCR and subsequent sequencing of PCR products to confirm nucleotide insertions or deletions within the target gene and integration of the sgRNA sequence.

[0075] Generation of SLA-DM recombinant cell lines Computer-simulated splicing and codon optimization were performed on the coding sequences of SLA-DMA (GenBank#NC_010449.5, nt 25133494 to 25137928) and SLA-DMB (GenBank#NC_010449.5, nt 25119278 to 25125089). The binding regions of the selected sgRNAs were also ensured to be altered as much as possible through silent base substitution. Custom plasmids (Invitrogen, Thermo Fisher Scientific) pMA-SLA-DMA and -DMB contain 5'-EcoRI and 3'-NotI restriction sites to facilitate ORF recloning, and a unique BpiI cleavage site located immediately upstream of the stop codon, allowing the insertion of hybrid oligonucleotides encoding Step II tags (WSHPQFEK) or Myc tags (LEQKLISEEDL) into BpiI- and NotI-digested constructs, respectively. Correct insertion was verified by sequencing using primer M13 Rev(-24). Natural and StrepII- or Myc-labeled SLA-DMA ORFs, as well as natural and Myc-labeled SLA-DMB ORFs, were recloned as EcoRI / NotI fragments into the correspondingly digested 8140 bp lentiviral vector pLVX IRESPuro (TaKaRA / Clontech). As a control, an ORF encoding enhanced green fluorescent protein (EGFP) was also inserted (as a 772 bp EcoRI / NotI fragment isolated from pEGFP-N1 (Clontech), resulting in pLVX-EGFP-IRES-Puro). Correct plasmid clones were identified by sequencing using primer CMV promoter-F. Protein expression was confirmed in RK13 cells transfected with the newly generated plasmid (X-tremeGENETM HP reagent, Roche), and immunoblotting analysis was performed using anti-Myc and anti-Strep antibodies. (Based on Joung et al.) 78 The described protocol generates lentiviruses encoding the SLA-DMA-Strep, SLA-DMA-Myc, SLA-DMB-Myc, and EGFP genes, or empty pLVX-IRES-Puro, respectively, in HEK-293T cells. This is effective against WSL and WSL SLA-DMA... KO (11) and WSL SLA-DMB KO (9) Cell clones were transduced, and knockout / knockin selection was performed using 1 µg / ml puromycin (WSL). KO / KI Cells. The knockout of the natural gene and transgene integration were confirmed by PCR and sequencing using primers. Transgene expression was further confirmed by Western blotting, as described below.

[0076] Sanger sequence analysis The resulting plasmids and PCR-amplified genomic fragments from the recombinant WSL cell line (KOD Xtreme Hot Start DNA polymerase, Merck) were sequenced using specified primers and the BigDye™ Terminator v1.1 Cyclic Sequencing Kit (Thermo Fisher Scientific) on an Applied Biosystems 3500 Genetic Analyzer (Thermo Fisher Scientific). Results were evaluated using the Geneious Prime 2021.0.1 software package (Biomarkers, available at https: / / www.geneious.com).

[0077] Immunoblotting Cells were trypsinized, resuspended in medium containing 10% FCS, centrifuged, and washed once with phosphate-buffered saline (PBS). The precipitated cells were then lysed in sample buffer (0.13 M Tris-HCl, pH 6.8; 4% SDS; 20% glycerol; 0.01% bromophenol blue; 10% 2-mercaptoethanol) containing sodium dodecyl sulfate (SDS), sonicated, and incubated at 95°C for 5 min. Proteins were separated in a discontinuous SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The blot was blocked for 3 h at RT with 5% skim milk in Tris-buffered saline (TBS-T) containing 0.25% Tween 20, and detected overnight with a specific primary antibody diluted in 0.5% skim milk in TBS-T. Binding of monoclonal anti-FLAG (clone M2, #F1804, Sigma-Aldrich), anti-α-tubulin (#T5168, Sigma-Adrich), and polyclonal rabbit anti-StrepII (#4217, ProSci), anti-Myc (#PA1-581, Invitrogen, Thermo Fisher Scientific), anti-HLA-DMA (H00003108-D01P, Abnova) and anti-HLA-DMB (H00003109-D01P, Abnova) antibodies was observed using the fluorophore-labeled donkey anti-rabbit secondary antibody IRDye 800CW (#926-32213, Li-Cor Biosciences) or donkey anti-mouse secondary antibody IRDye680RD (#926-68072, Li-Coer Biosciences) in TBS-T was observed. Fluorescence signals were detected using an Odyssey CLx infrared imaging system (CLX-2293; Li-Cor Biosciences).

[0078] Determine plaque size and plaque formation efficiency WSL, WSL KO and WSL KO / KI Cells are spaced at 4 x 10 cells per well 5 Cells were seeded at a density of 1,000 cells per well in 24-well plates. The following day, the virus was serially diluted in cell culture medium supplemented with 5% FCS and applied to confluent cell layers. Cells were incubated at 37°C and 2.5% CO2 for 2 h. Subsequently, the inoculum was removed and replaced with methylcellulose medium (MEM containing 5% FCS, with 6 g / L methylcellulose). PrV infection was observed and recorded by intrinsic GFP expression of PrV-BaΔgGG three days post-infection, as described below. Four days post-ASFV infection, the culture medium was removed, cells were washed once with PBS, and then fixed for 20 min at room temperature (RT) with 4% paraformaldehyde (PFA) in PBS. Formaldehyde fixation was terminated by washing and followed by incubation at RT with 5 mM NH4Cl in PBS for 30 min. Cells were washed three times with PBS and stored at 4°C until ASFV antigen was detected by indirect immunofluorescence (IF) assay (see below). Cells, lesions, and plaques infected with ASFV and PrV were visualized using a Leica DM18 motorized fluorescence microscope. Whole-cell imaging was performed using Leica Application Suite X software, and the resulting stitched images were merged. For each virus and each cell line, the area of ​​50 infected cells or plaques was determined in three independent experiments using ImageJ's free selection tool (version 1.53f51; http: / / imagej.nih.gov / ij). The average plaque size of each virus grown on WSL cells was set to 100%, and WSL was calculated using GraphPad Prism (version 9). KO or WSL KO / KI The mean relative plaque size and standard deviation of viruses grown on cells. For plaque formation efficiency, plaques were counted and the apparent titer was calculated in PFU / ml.

[0079] Determining viral titer and replication kinetics WSL, WSL KO and WSL KOKI Cells are spaced at 4 x 10 cells per well. 5 1 cell (for PrV infection) or 3 x 10 5Cells were seeded at a density of 100 cells (for ASFV infection) in 24-well plates. The following day, PrV-BaΔgGG, ASFV Armenia, or ASFV Kenya were administered at an MOI of 0.02. After incubation at RT (PrV) or 37°C (ASFV) for 2 h, the cells were washed once with medium and then covered with 1 ml of medium containing 1% penicillin / streptomycin (Gibco). PrV-infected cells were frozen at -80°C for three days post-infection. Single plates of ASFV-infected cells were frozen immediately after the addition of medium and frozen every 24 h until 168 hp.i. For titration, the plates were thawed and the lysates were transferred to reaction tubes. After centrifugation at 2655 xg and 4°C for 5 min, the supernatant was transferred to new tubes and stored at -80°C. Virus titration was performed on confluent RK13 cells (PrV) or WSL cells (ASFV) in 96-well plates. After seeding with serially diluted viral supernatant (100 µl / well), cells were incubated at RT on a shaker for 2 h (PrV) or centrifuged at 689 xg and 37°C for 1 h (ASFV). The viral supernatant was then removed, and the cells were covered with methylcellulose medium. Cells were incubated at 37°C and 2.5% CO2 for 3 days (PrV) or 4 days (ASFV). PrV-infected cells were fixed for 1 h with 3.7% formaldehyde solution, followed by staining with 1% crystal violet to visualize the plaques. ASFV-infected cells were washed once with PBS, fixed at -20°C with ice-cold acetone / methanol (1:1, v / v) for 30 min, and then air-dried. Infected cells were visualized by IF staining.

[0080] DNA replication dynamics To analyze viral DNA replication, WSL and WSLKO cells were cultured at 3 x 10⁻⁶ cells / mL. 5Cells were seeded at a density of 1,000 cells per well in 24-well plates and infected with Armenian ASFV at an MOI of 3 for 24 h, with two replicates per incubation. After incubation at 37°C for 2 h, the inoculum was removed, washed, and the culture medium was replaced. After incubation at 37°C for 0, 2, 4, 8, 16, and 32 h, the culture medium was aspirated again, the cells were washed once with PBS, the pellet was collected, and stored at -20°C until further analysis. DNA was prepared using the NucleoMag Tissue Kit (Macherey-Nagel) according to the manufacturer's recommendations and eluted in 100 µl of elution buffer. Quantitative real-time PCR for DNA detection was performed using the QuantiTect Multiplex PCR NoROX Kit (Qiagen) in a 12.5 µl reaction containing 2.5 µl of infected cell DNA, according to the manufacturer's instructions. ASFV B646L gene-specific primer pairs AKB646L-408F and AKB646L-507R, and β-actin gene-specific primer pairs ACT-CP-F and ACT-CP-R were added at 800 nM, and TaqMan probes AKB646L-460P and ACT-CP-P were added at a final concentration of 160 nM. Primers and probes were purchased from Eurogentec. Samples were incubated at 95°C for 15 min, followed by up to 45 cycles in a Bio-Rad C1000 / CFX96 real-time PCR instrument: 30 s at 95°C, 30 s at 55°C, and 30 s at 68°C. Results were analyzed using CFX Maestro software (Bio-Rad). The results were based on samples containing 10... 10 10 8 10 6 10 4 10 2 The ASFV genome copy number can be determined by a standard curve generated from the reaction of a 100-copy p72 expression plasmid (pCAGGS-p72-Georgia, courtesy of GM Keil).

[0081] Indirect immunofluorescence (IF) analysis After fixing cells with PFA as described above, cells can optionally be permeabilized for 15 min at RT with 0.5% Triton-X 100 in PBS. This step is not required for visualization of surface proteins or after fixation with acetone / methanol. After washing with PBS, cells are blocked at RT with 10% FCS in PBS for 1 h, followed by application of polyclonal rabbit anti-ASFVp72 antibody diluted in blocking buffer at RT. 82Alternatively, monoclonal mouse anti-pig MHC II (clonal MSA 3) antibody (kindly provided by Luise Hartmann and Ulrike Blohm) was incubated for 1 h, followed by further detection for 1 h with goat anti-rabbit Alexa Fluor 488 (#A11008, Invitrogen, Thermo Fisher Scientific) or goat anti-mouse Alexa Fluor 488 (#A11001, Invitrogen, Thermo Fisher Scientific) secondary antibody diluted in PBS. For detection of the p72 antigen in WSLKO / KI cells, visualization was also achieved using goat anti-rabbit Alexa Fluor 647 secondary antibody (#A21245; Invitrogen, Thermo Fisher Scientific) in a GFP-expressing control cell line. Nucleic acids were stained with Hoechst 33342 (#H3570; Invitrogen, Thermo Fisher Scientific) at RT for 15 min. After each incubation step, cells were washed three times with PBS and finally analyzed using a Leica DMi8 fluorescence microscope.

[0082] mass spectrometry WSL and WSL DMA were added to 2% SDS in 0.1 M Tris-HCl (pH 8.0) at 95°C. KO (11) WSLDMB KO (9) WSL CIITA KO (1) and WSL RFXAP KO(6) The fusion monolayer (n=3 per clone) was lysed for 10 min. The lysate was clarified by centrifugation (14,000 xg, 10 min, RT), and the supernatant was collected. Aliquots containing 100 µg of protein (determined by BCA assay) were precipitated by adding 3 volumes of ice-cold acetone. The protein particles were recovered by centrifugation at 10,000 xg for 15 min at 4 °C and digested into peptides using the EasyPep™ Mini MS Sample Preparation Kit (ThermoScientific) according to the manufacturer’s protocol. The peptides were resuspended in 0.1% formic acid (FA), and peptide yield was assessed by BCA assay. Peptides (1 µg / sample) were separated on a nanoElute® (Bruker, Bremen, Germany) HPLC system equipped with an IonOpticks Aurora column (25 cm x 75 µm ID, 1.6 µm C18) at a flow rate of 400 nL / min, coupled to a timsTOFPro instrument (Bruker). Solvent A was 0.1% FA, and solvent B was 0.1% FA in acetonitrile. Peptides were eluted using a gradient of 2%–15% solvent B (0–60 min), 15–24% solvent B (60–90 min), 24%–34% solvent B (90–105 min), and 34–95% solvent B (105–107 min). The timsTOF Pro instrument is equipped with a CaptiveSpray nanoelectrospray ion source (Bruker) and operates using the manufacturer-recommended standard DDA method for proteomics analysis (1.1-second cycle time) in parallel accumulation and sequential fragmentation (PASEF) modes. A database with pig sequences downloaded from the Ensembl repository is used. 84 Use Fragpipe 83 Process the raw MS data. Use the statistical language R. 85 And Perseus v1.6.15.0 86 Qualitative and quantitative analysis was performed for protein identification. The R package gprofiler version 2.2.1 was used. 87 Used to map porcine protein identifiers to corresponding genes (HGNC nomenclature) and for enrichment analysis of GO terms (GO:BP) and the KEGG pathway.

[0083] electron microscope WSL, WSL SLA-DMA KO and WSL CIITA KO Cells at 1.5 x 10 6Cells were seeded at a density of 10 cells / well in 6-well plates. After 24 h, one complete plate of each type was infected with Armenian ASFV at an MOI of 5. The virus was allowed to penetrate the cells at 37 °C for 2 h. Subsequently, the virus suspension was removed and replaced with fresh medium containing 1% penicillin / streptomycin. 16 h after virus application, cells were scraped into the medium and transferred to 50 ml centrifuge tubes. The cell suspension was centrifuged at 350 x g for 7 min at 4 °C. The cells were washed once with 0.1 M sodium dimethylarsinate buffer (pH 7.2) and then fixed at 4 °C with 2.5% glutaraldehyde in dimethylarsinate buffer (both SERV Electrophoresis) for at least 2 h. The fixed cells were centrifuged again (5 min, 1000 x g, 4 °C) and the precipitate was embedded in low-melting-point agarose (Sigma-Aldrich). After drying, the agarose was cut into small pieces (1 mm³), post-fixed in a 1% OsO₄ aqueous solution, and stained in 2.5% uranyl acetate (both SERVA Electrophoresis). After stepwise dehydration in ethanol, the sample was clarified in propylene oxide and infiltrated with glycidyl ether 100 (SERVA Electrophoresis). For polymerization, the sample was encapsulated and incubated at 60°C for 3 days. Target points were trimmed, and the prepared ultrathin sections were transferred to a Formvar-coated nickel grid (Plano, Wetzlar, Germany). All grids were counterstained with uranyl acetate and lead citrate before examination with a Tecnai Spirit transmission electron microscope (FEI, Eindhoven, The Netherlands) at an accelerating voltage of 80 kV.

[0084] Statistical analysis All statistical analyses were performed using GraphPad Prism (version 9.0). Statistical significance of differences in plaque formation efficiency was calculated using ordinary one-way ANOVA and the GraphPad multiple comparison test. Statistical significance of differences in plaque size was assessed using the Kruskal-Wallis test and the Duncan multiple comparison test. The number of repeated measures is indicated in each graph legend. A p-value < 0.05 was considered significant and is indicated by an asterisk in the graph. <0.05, <0.01, <0.001, <0.0001).

[0085] Data availability Mass spectrometry proteomics data have been deposited into the ProteomeXchange consortium (http: / / proteomecentral.proteomexchange.org) via the PRIDE partner repository using the dataset identifier PXD034242.

[0086] result Genome-wide CRISPR / Cas9 knockout screening identified cytokines associated with the MHC II pathway for ASFV replication. To identify the host genes required for ASFV replication and their respective protein products in cultured porcine cells, the previously described and characterized porcine CRISPR / Cas9 knockout library SsCRISPRko.v1 was used. 52 Genome-wide CRISPR / Cas9 screening was performed. The library encodes 83,381 sgRNAs, including 1,001 non-targeted control sgRNAs and 82,380 specific sgRNAs targeting 20,598 porcine genes, with three to four sgRNAs per gene. The sgRNA sequences were cloned into the lentiCRISPRv2 vector, which also provides Cas9 expression cassettes and a puromycin resistance gene for selection.

[0087] The library was packaged into defective lentiviral particles and then used to transduce highly passaged wild boar lung (WSL) cells, which support efficient replication of many natural or adaptive ASFV isolates. 53,54 To ensure that only one sgRNA gene was integrated into the genome of a single cell, a low multiple transduction (MOT) of 0.3 was selected. Puromycin-resistant cells assuming stable expression of Cas9 and a single sgRNA were amplified over a two-week period. At this point, the total number of cells in each experiment was approximately 4 × 10⁻⁶. 8 Approximately 6 × 10⁶ cells 7 One cell was stored as an uninfected control for DNA preparation. The remaining cells were reseeded and genotype IX recombinant ASFV Kenya 1033 ΔCD2v dsRed at a multiplicity of infection (MOI) of 0.3 or 0.5. 55,56Infection was performed. Fluorescent expression labeling aided in the detection of successful infection. Progressive cytopathic effect (CPE) was detectable 48 h post-infection, and cell colonies originating from single surviving cells were visible approximately four to five weeks later. These cells were merged into two subgroups. A portion of these merged cells was preserved as “Survivor 1-1” and “Survivor 1-2” for DNA preparation. The remaining portions were re-inoculated and reinfected, and “Survivor 2-1” and “Survivor 2-2” could be harvested approximately three weeks later. Inoculation and infection of cells were repeated four times to ensure all cells were exposed to the virus. Repeated infection was necessary because it was noted that while ASFV Kenya induces very pronounced CPE in WSL cells, it does not always lyse all untransduced control cells during a single round of replication. To further rule out false hits from unexpectedly surviving cells, the screening procedure was performed not only with two cell subgroups but also in two separate experiments.

[0088] For each screening, DNA was isolated in parallel from the control group (pre-infection cells) and the survival group (post-infection cells) in two subgroups, and the integrated sgRNA gene region was amplified in three consecutive PCRs using primers suitable for Ion Torrent sequencing. Sequencing data were analyzed using the MAGeCK algorithm software to test whether there was a significant difference in sgRNA gene abundance between the treated cells (surviving cells) and the control group, and robust sorting aggregation (RRA) was calculated. 57 To identify enriched sgRNA sequences that target specific genomic sites.

[0089] This analysis revealed that, across all four subgroups in both screenings, the sgRNAs targeting SLA-DMB, LOC100736732, RFXAP, SLA-DMA, LOC106509697, RFXANK, and LOC100624181 exhibited the lowest RRA scores (i.e., most elevated) among the top ten hits for the positively selected genes. Figure 1a (b) For all these genes, more than one specific sgRNA was significantly enriched in the surviving cell pools. Furthermore, the top ten hit gene CCZ1 from the screening was identified in three subgroups, while genes LOC102165390, TMEM30A, and VPS33A appeared in only one screening, and genes MARCO, LYPD4, and VPS18 were among the top ten hits in only one subgroup. Figure 1a(b) Most of the protein products in the latter group of genes are involved in endocytosis and / or autophagy pathways and will be further analyzed in further studies. This study focuses on the roles of host genes (SLADMB, LOC100736732, RFXAP, SLA-DMA, LOC106509697, RFXANK, and LOC100624181) identified in all four very low-scoring subgroups. Notably, all of these are associated with major histocompatibility complex II (MHC II / SLA II). Figure 1c RFXANK encodes a regulatory factor X-related protein containing ankyrin (RFXANK), and RFXAP encodes a regulatory factor X-related protein (RFXAP). RFXANK, RFXAP, and regulatory factor X5 (RFX5) assemble and bind to the X box of the SXY module of the MHC II gene promoter. Figure 1c Together with other factors, they act as a landing pad for MHC class II transactivators (CIITA). 58 CIITA was identified by elevated sgRNA levels in LOC100736732, LOC106509697, and LOC100624181. RFXAP, RFXANK, and CIITA are crucial for transcriptional activity of MHC class II promoters. 58 Finally, SLA-DMA and SLA-DMB genes were identified in both the α and β chains encoding non-classical porcine leukocyte antigen DM (SLA-DM) and were transcribed with the assistance of the aforementioned factors. In summary, some evidence suggests that the MHC II pathway is a highly relevant host factor for ASFV replication.

[0090] Generate cell lines targeting SLA-DMA, SLA-DMB, RFXAP, and CIITA knockout.

[0091] To validate the importance of MHC II expression and presentation pathways for the ASFV replication cycle, targeted gene knockout was introduced into newly isolated single-cell clones from the WSL cell line. Using WSL cell clones minimized the risk of results being influenced by inherent genetic differences. For targeted knockout, genes encoding non-canonical MHC II molecules SLA-DM, SLA-DMA, and SLA-DMB, as well as two genes important for MHC II transcription, RFXAP and CIITA (LOC100736732), were selected.

[0092] For knockout, one of four porcine library sgRNA sequences was selected and cloned into the sgRNA and Cas9 dual expression vector pX330A-1×4neoRA. WSL cells were transfected with the obtained plasmid, serially diluted, and G418 resistance was screened. Resistant single-cell clones were proliferated, and Cas9 expression was checked by Western blotting. DNA from Cas9-positive cells was then isolated, and the correct integration of the sgRNA gene was verified by PCR amplification and sequencing using appropriate primers. Furthermore, PCR analysis was performed using primers specific to the target gene regions of SLA-DMA, SLA-DMB, RFXAP, and CIITA. After sequence analysis of the amplified products, WSL knockout (WSL knockout) sequences with unwanted nucleotide insertions or deletions (INDELs) in SLA-DMA (clones 11, 12, 16), SLA-DMB (clones 9, 16, 18), CIITA (clones 1, 4, 8), or RFXAP (clones 6, 8) were selected. KO Cell cloning. Cell cloning WSLSLA-DMA KO (11) An 823 nt insert containing a stop codon is displayed in all reading boxes. This insert originates from the transfer vector pX330A-1×4neoRA. Cloning WSL SLA-DMA KO (12) and (16) and WSL SLA-DMB KO (9) It has a 1-nt deletion that leads to frameshift and premature termination of the codon. In contrast, WSL SLA-DMB KO (16) and (18) exhibited the same 1 nt (T) insertion, which directly produced a stop codon (TGA). Cell clones WSL CIITAKO (1), (4), and (8) showed deletions of 1, 5, and 23 nucleotides, respectively, resulting in frameshift and premature termination. WSL RFXAPKO (6) exhibited a 1 nt (C) insertion, resulting in a downstream stop codon (TGA), while WSL RFXAP... KO An insertion of 140 nt from the vector pX330A-1×4neoRA was found in (8), the insertion containing a stop codon. Notably, only a single PCR product showing a well-defined sequence of the mutant gene was obtained from all selected cell clones, suggesting that either the same biallelic change or a large deletion occurred in the other alleles (including the primer binding site). Wild-type sequences of the sgRNA target region were never observed.

[0093] Previous studies have shown that WSL cells express the MHC II protein SLA-DR on their surface. 54This was validated by indirect immunofluorescence (IF) analysis of non-permeable cells using an SLA-DR-specific monoclonal antibody (mAb). Figure 2a Although SLA-DR is present in both parental WSL cells and knockout WSL SLA-DMA KO and WSL SLA-DMB KO It can be detected on the surface, but not on RFXAP and CIITA knockout cells. Figure 2a This confirms that RFXAP and CIITA are important factors in MHC II transcription.

[0094] To characterize the knockout at the proteomic level, the protein content of the knockout cell line was analyzed by mass spectrometry (MS). Figure 2b (c) Of the 5495 proteins identified and quantified, 4874 were detected in all cell clones. The protein composition of all cell clones was very similar, as principal component analysis (PCA) showed a lack of obvious clustering among the replicates. Unfortunately, SLA-DM expression was not detected in either knockout or normal WSL cells in MS analysis. However, a single knockout by RFXAP or CIITA suppressed the synthesis of other identified MHC II proteins (SLA-DR, SLA-DQ) below detection levels and also affected the expression of MHC I proteins (SLA-8, HLA-E), which are present in WSL LLA-DM. KO and WSL SLA-DMB KO Expression in cells remained unaffected. Figure 2b c).

[0095] SLA-DM, RFXAP, and CIITA are essential for efficient ASFV replication in WSL cells. Before testing whether ASFV can replicate in cells lacking MHC II pathway molecules, the non-specific side effects of these knockouts on viability and susceptibility to other viral infections should be ruled out. For this purpose, parental WSL cells and WSL SLA-DM were infected with a GFP-expressing porcine alpha herpesvirus pseudorabies virus (PrV) mutant. KO (Clones 11, 12, 16), WSL SLA-DMB KO (Clone 9, 16, 18), WSL CIITA KO (Clone 1, 4, 8) and WSL RFXAP KO (Clon 6, 8) cells. Studies showed no significant differences in plaque formation efficiency, plaque size, or PrV progeny virus titer between parental WSL cells and the tested knockout cell lines. This indicates that knockout cells themselves are well-suited for porcine virus proliferation.

[0096] Two different viral strains were used for ASFV infection of cells. In addition to the parental genotype IX strain (ASFV Kenya 1033) used for library screening of recombinants, a variant of genotype II virus currently experiencing a pandemic in animals (ASFV Armenia 2008) was also included. Parental WSL and knockout cell clones were infected with serial dilutions of both viruses. After 4 days in semi-solid medium, cells were fixed, and infected cells and viral plaques were observed by IF assay of the ASFV capsid protein p72. Fluorescence microscopy showed that both strains were able to infect significantly more parental WSL cells than any knockout cell clone. Figure 3a ).

[0097] Compared to WSL cells, all tested WSL cells KO The computational plaque formation efficiency of cells was significantly reduced to <4% ( Figure 3b Furthermore, compared to the plaque size of the corresponding virus on the parental cell line, the plaque size in WSL... KO In cells, the plaque area of ​​ASFV Armenia was significantly reduced to less than 14%, and the plaque area of ​​ASFV Kenya was significantly reduced to less than 10%. Figure 3c In multi-step (MOI 0.02) growth studies, severely impaired ASFV replication was also observed. Armenian ASFV replicated to a maximum titer of 1.8 × 10⁻⁶ in WSL cells 168 h post-infection. 7 PFU / ml (pi), while the titer in knockout cells ranged from WSL SLA-DMB. KO (18) 1.2×10 4 PFU / ml to WSL RFXAP KO (8) 3.8×10 4 PFU / ml Figure 3d The final titer of ASFV Kenya in WSL cells was 2.0 × 10⁻⁶. 7 PFU / ml, while in knockout cells, the titer ranged from WSL SLA-DMA. KO (16) 1.2 × 10⁻⁶ in cells 5 PFU / ml to WSL RFXAP KO (8) 1.23 × 10⁻⁶ in the cells 6 PFU / ml Figure 3dTherefore, compared to parental cells, WSL knockout resulted in a 3-log reduction in ASFV Armenia titer and at least a 1.5-log reduction in ASFV Kenya titer. Furthermore, it was evident that productive replication of ASFV Armenia in knockout cells plateaued at 72 hpi, while ASFV Kenya titer increased until 120 hpi. This may suggest that the few knockout cells successfully infected with ASFV Kenya were able to produce more infectious virus over a longer period compared to cells infected with ASFV Armenia, consistent with the slightly higher ASFV Kenya titer observed in normal WSL cells.

[0098] In addition to analyzing infectious viral progeny in infected cell lysates, viral DNA replication was also studied. Figure 4 Therefore, parental WSL cells and selected WSL cells were infected with Armenian ASFV at an MOI of 3. KO Cell clones were established and harvested at 0, 2, 4, 8, 16, and 32 hp.i. Total DNA was prepared, and the ASFV genome copy number was determined by double-stranded TaqMan qPCR to detect the viral B646L gene and the host cell β-actin gene (used as an internal control). Figure 4 In parental WSL cells, the B646L-specific probe showed a moderate increase in DNA quantity after 4 h, indicating an exponential replication phase until 8 hpi. Subsequently, viral DNA replication slowed down, and at 32 hpi, the analyzed sample (containing approximately 1 × 10⁻⁶ DNA) showed a decrease in viral DNA content. 4 2.8 × 10⁻⁶ cells of DNA are produced. 7 One genome copy number. A delayed onset of DNA replication was observed in all WSLKO cell clones, resulting in a modest increase in genome copy number between 8 hpi and 16 hpi. From 16 hpi to the end of the experiment, the viral DNA amount remained almost constant, ranging from SLADMB KO (9) 1.2 × 10⁻⁶ in cells 5 ASFV genome to RFXAP KO (8) 6.8 × 10⁻⁶ in the cells 5 ASRV genomes are unequal. Because at all time points of analysis, all tests were performed using WSL... KO The ASFV genome copy number was significantly reduced in all cells, indicating that the missing host proteins are clearly important for a step before viral genome replication begins. To further elucidate which steps of the viral replication cycle might be blocked after MHC II-related gene knockout, WSL and WSL SLA-DMA were analyzed by electron microscopy (EM) 16 h after infection with Armenian ASFV at an MOI of 5. KO(11) and WSL CIITA KO (1) Cells ( Figure 5 Mature extracellular viral particles, intracellular particles, and viral factories were detected only in parental WSL cells, while no trace of ASFV replication was found in any knockout cells. These results also suggest that the knockout host cell proteins play a role in the initial steps of viral replication, which is consistent with WSL. KO The significantly reduced ASFV plaque formation efficiency and genome copy number in cells were consistent. Susceptibility to ASFV infection could be restored by reintroducing SLA-DM. To test whether the ASFV replication inhibition observed in SLA-DM knockout cells was indeed due to the lack of the corresponding protein, WSL cells were stably transformed with SLA-DMA or SLA-DMB expression cassettes, respectively. KO (11) and SLA-DMB KO (9) Cells. By introducing silencing nucleotide alterations, codon optimization and modification of the nucleotide sequence in the open reading frame of the sgRNA target region were performed to eliminate the inactivation of the transgene by the CRISPR / Cas9 mechanism still integrated in knockout cells. Synthetic genes with and without tags (StrepII, Myc) were cloned into lentiviral expression vectors to obtain pLV SLA DMA, pLV SLA DMA-Myc, pLV SLA-DMA-Strep, pLV-SLA DMB, and pLV SLA DMB Myc, as well as a GFP expression construct (pLV-GFP) used as a control. Parental WSL cells were transduced with all generated vectors; SLA-DMA KO (11) Cells were transduced with pLV-SLA-DMA, pLV-SLA-DMA-Myc, pLV-SLA-DMA-Strep, or pLV-GFP, and SLA-DMB was used. KO (9) Cells were transduced using pLV-SLA-DMB, pLVSLA-DMB-Myc, or pLV-GFP, and stably transformed cells were selected using puromycin-containing medium. Unlike parental WSL and knockout cells, in WSL knockout / knockin (WSL... KO / KI In cells, marker proteins of expected size can be detected by Western blotting. Figure 6 Unfortunately, the unlabeled SLA-DMA and -DMB proteins were not recognized by existing antibodies against human leukocyte antigen (HLA)-DMA and HLA-DMB. However, these cell lines were included in the ASFV infection analysis because the C-terminal tagging could impair maturation or the formation of heterodimeric complexes between the transgene-encoded SLA-DM and the unaffected endogenous α or β chains.

[0099] Reintroducing the original SLA-DMA (SLADMA) KO -DMA KI After that, in the transduced SLA-DMA KO (11) In cells, the plaque formation efficiency of ASFV Armenia and Kenya increased to approximately 77% and 86% of the titer in parental WSL cells, respectively. Figure 7a ). In SLA-DMA KO -DMA-Myc KI In cells, the original titers were reached at 42% (Armenia) and 50% (Kenya) in SLADMA. KO -DMA-Strep KI In cells, the original titers were achieved at 79% (Armenia) and 90% (Kenya). In WSLLADMB KO -DMB KI In cells, plaque formation efficiency of 77% (Armenia) and 94% (Kenya) was observed in SLA-DMB. KO -DMB-Myc KI In cells, plaque formation efficiency was observed at 63% (Armenia) and 56% (Kenya). Figure 7a In most cases, different WSLs KO / KI The apparent viral titer on the cells was not significantly lower than that on the parental WSL cells. Figure 7a Therefore, the added tag clearly had no or only slightly affected the proposed function of (StrepII)SLA-DMA in ASFV replication. WSL SLA-DMA was stably transduced using expression cassettes with the correspondingly labeled or unlabeled proteins. KO and SLA-DMB KO Cellular transmission also facilitated the restoration of intercellular transmission of ASFV in Armenia and ASFV in Kenya. In all cases, different WSLs KO / KI The plaques on the cells showed a size similar to or even larger than those detected on parental WSL cells. Figure 7b Consistent with this, growth kinetic studies have shown that, compared to those on parental cells, growth in WSL... KO / KI On cells, the ASFV progeny virus titers from Armenia and Kenya increased by approximately 2.5 and 1.5 log units, respectively. Figure 7c -e), the final viral titer was again similar to that on the original WSL cells. In summary, these results indicate that the detrimental effects of SLA-DMA / B knockout on susceptibility to ASFV infection can be completely reversed by the expression of the corresponding transgene, suggesting that SLADM plays a crucial specific role in ASFV replication.

[0100] discuss Despite its complex genome and proteome, ASFV, as an intracellular viral pathogen, still requires numerous host factors for replication. Using whole-genome CRISPR / Cas9 knockout screening in susceptible porcine cell lines (WSLs), we identified RFXAP, RFXANK, SLA-DMA, SLA-DMB genes, and genes encoding CIITA (LOC100736732, LOC106509697, and LOC100624181) as important candidate genes for ASFV replication. The proteins encoded by these genes are part of the MHC II (SLAII) expression and presentation pathway. 58 Targeted inactivation of RFXAP, CIITA, SLA-DMA, and SLA-DMB significantly inhibited the replication of genotype II and IX ASFV strains, including those currently involved in the animal pandemic, confirming the importance of certain MHC II molecules for ASFV replication in vitro. Furthermore, reconstitution of SLA-DMA or SLA-DMB in the corresponding knockout cells fully restored their ASFV replication capacity, demonstrating that the MHC II molecule SLA-DM plays a crucial and specific role in the viral life cycle.

[0101] MHC II complexes are highly conserved in vertebrates, named SLA II in pigs and HLA II in humans. Pigs possess four MHC II molecules: SLA-DR, SLA-DQ, SLA-DO, and SLA-DM. 59 SLA-DR and -DQ are classic MHC II transmembrane cell surface glycoproteins that present exogenous peptides to antigen receptors on CD4+ helper T cells, while SLA-DM and -DO are considered non-classical MHC II molecules. In humans, they are involved in regulating the loading of antigen peptides onto classic MHC II molecules. All MHC II proteins are heterodimers composed of α and β chains; in classic MHC II proteins, the α1 and β1 domains form peptide-binding grooves. The synthetic and presentation pathways in humans have been described. 60The classic MHC II α and β subunits are synthesized in the ER, where they assemble with specific chaperones, CD74 / constant chain (li). li promotes MHC II folding, prevents premature peptide binding, and sorts MHC II molecules into late endosome compartments either directly from the trans-Golgi network or via reinternalization from the cell surface. The acidic environment of the endosome almost entirely degrades li, but leaves specific li fragments, class II constant chain-related peptides (CLIPs), within the MHC II binding groove. CLIP release is induced by non-classical MHC II molecules (DMs) through conformational changes, where classic MHC II proteins are loaded with antigenic peptides of higher affinity. These peptides are produced from antigens internalized via clathrin-mediated endocytosis, phagocytosis, or microphagocytosis.

[0102] ASFV enters host cells via clathrin-mediated endocytosis or macrophagocytosis. In this study, SLA-DMA and SLA-DMB knockout cells exhibited severe defects in susceptibility to ASFV infection and subsequent DNA and viral replication. This effect could be reversed by reintroducing the corresponding SLA-DM subunits, suggesting that SLA-DM may be important for ASFV entry, for example, for efficient uncoating and release of the core particle from late endosomes. WSL KO Electron microscopy analysis of the cells revealed the absence of viral factories and detectable amounts of progeny viral particles, and compared to parental wild-type cells, WSL cells showed no viral defects. KO The significantly reduced plaque formation efficiency and genome copy number of ASFV strongly suggest that infection of MHC II-deficient cells was blocked at a very early stage.

[0103] In genome-wide CRISPR / Cas9 knockout screening of anti-ASFV-infected WSL cells, sgRNAs targeting RFXAP, RFXANK, LOC100736732, LOC106509697, and LOC100624181 were also significantly elevated. These genes encode RFXAP, RFXANK, and CIITA (LOC100736732, LOC106509697, and LOC100624181). These molecules, along with RFX5, are involved in the expression of all MHC II genes. 61-65 At CIITA KO or RFXAP KO In cells, the degree of suppression of ASFV infection was similar to that in knockout cells lacking SLA-DMA or SLA-DMB. (Compared to SLA-DMB) KOCellular differences exist; in cells lacking functional CIITA or RFXAP genes, MHC II expression is typically suppressed. This was confirmed by IF and MS analyses, which failed to detect the canonical MHC II molecules SLA-DR and SLA-DQ, despite their presence in parental WSL cells. Since CIITA and RFXAP are universal regulators of MHC II expression, this is not the case. 58 Therefore, the corresponding knockout cells are also likely to lack the non-canonical MHC II molecules SLA-DO and SLA-DM, which cannot be detected or quantified using existing tools. While the regulatory proteins CIITA or RFXAP, or SLA-DR, SLA-DQ, and SLA-DO, cannot be ruled out as directly involved in ASFV infection or replication, SLA-DM appears to be the only relevant MHC II molecule. This is supported by the fact that no sgRNA targeting SLA-DR, SLA-DQ, or SLA-DO was identified in the CRISPR / Cas9 knockout screening. Furthermore, flow cytometry analysis of susceptible cells indicates that expression of the canonical MHC II molecule SLA-DQ is not essential for ASFV infection. 66 .

[0104] Current results suggest that ASFV-induced downregulation of SLA-DM may also prevent reinfection of host cells, thereby increasing the efficiency of viral replication.

[0105] The data presented in this paper are the first to clearly demonstrate that components of the MHC II system, particularly the non-classical membrane protein SLA-DM, are crucial for ASFV replication.

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Claims

1. A genetically modified animal or transgenic animal that lacks a functional copy of any one or more of the SLA-DMA gene, SLA-DMB gene, RFXANK gene, RFXAP gene, and CIITA gene and / or the proteins encoded by them.

2. The genetically modified animal or transgenic animal according to claim 1, wherein, The animal in question is a pig, a suidae animal, a domestic pig, or a wild boar. Sus scrofa ).

3. The genetically modified animal or transgenic animal according to claim 1 or 2, wherein, This has resulted in the SLA-DMA gene and / or SLA-DMA protein becoming nonfunctional or being completely knocked out.

4. The genetically modified animal or transgenic animal according to claim 1 or 2, wherein, This has resulted in the SLA-DMB gene or SLA-DMB protein becoming nonfunctional or being completely knocked out.

5. A compound that regulates the function, activity, and / or expression of the porcine leukocyte antigen complex II (SLA II) gene and / or SLA II protein, for medical use, for use as a medicine, or for the treatment or prevention of ASF or ASFV infection.

6. The compound for the use according to claim 5, wherein, The compound regulates the function, activity, and / or expression of the SLA-DMA gene and / or the SLA-DMA protein.

7. The compound for the use according to claim 5, wherein, The compound regulates the function, activity, and / or expression of the SLA-DMB gene and / or the SLA-DMB protein.

8. The compound for use according to any one of claims 5 to 7, wherein, The compound reduces, inhibits, prevents, or suppresses the expression, function, and / or activity of the SLA II gene / protein.

9. The compound for use according to any one of claims 5 to 8, wherein, The compound (i) The ability of the ASFV to block or neutralize the interaction or binding of the ASFV with the SLA II protein; and / or (ii) binding to SLA II proteins to prevent or inhibit ASFV binding; and / or (iii) Regulation (e.g., reduction, suppression, inhibition, or prevention) of the expression of one or more SLA II genes described herein; and / or (iv) Inhibit the function or activity of any SLA II protein; and / or (v) Preventing proviral interactions between SLA proteins encoded by the SLA II gene.

10. The compound for use according to any one of claims 5 to 9, wherein, The compound is an antibody.

11. The compound for use according to any one of claims 5 to 10, wherein, The compound is an anti-SLA-DM, anti-SLA-DMA, anti-SLA-DMB antibody or its antigen-binding or neutralizing fragment.

12. The compound for use according to any one of claims 5 to 11, wherein, The compound is an antisense oligonucleotide that regulates the expression of one or more SLA II genes.

13. The compound for use according to any one of claims 5 to 12, wherein, The compound includes a fragment of the SLA II protein.

14. The compound according to claim 13, wherein, The compounds include fragments of any sequence provided in SEQ ID NO:9 to 12.

15. The antibody according to claim 10 or 11, wherein, The antibody neutralizes any proviral interaction between ASFV and the SLA II protein.

16. The antibody according to any one of claims 10, 11, or 15, wherein, The antibodies are anti-SLA-DMA, anti-SLA-DMB, anti-RFXANK, anti-RFXAP, and anti-CIITA antibodies.

17. A nucleic acid encoding the antibody of claim 10, 11, 15 or 16.

18. A nucleic acid encoding the antisense oligonucleotide of claim 12.

19. A nucleic acid encoding the SLA II protein fragment of claim 13.

20. A vector comprising the nucleic acid of any one of claims 17 to 19.

21. A host cell, said host cell being transformed or transfected with or comprising the vector according to claim 20.

22. The nucleic acid or vector according to claims 17 to 20, for medical use, for use as a medicine, or for use in the treatment or prevention of ASF or ASFV infection.

Images