African swine fever viral infections

Abstract

Disclosed are compounds which modulates the function, activity and / or expression of a swine leucocyte antigen complex II (SLA II) gene and / or a SLA II protein, for use in medicine, for use as medicaments or for use in treating or preventing ASF or an ASFV infection. The disclosure also provides the use the SLA-DMA, SLA-DMB, RFXANK, RFXAP 5 and CIITA genes for achieving ASFV resistance.

Description

[0001] AFRICAN SWINE FEVER VIRAL INFECTIONS

[0002] FIELD

[0003] The disclosure provides strategies, methods and compounds for use in treating of preventing

[0004] African swine fever and for modulating the host cell entry and / or replication by African swine fever virus.

[0005] BACKGROUND

[0006] African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), a hemorrhagic disease of domestic pigs and wild boar (species Sus scrofa)1-3. ASFV was first identified in Kenya in 1921 , and has been reported since then in most sub-Saharan African countries4’5. By partial sequencing of the gene B646L, which codes for the major capsid protein p72, twenty-four genotypes of ASFV have been specified6J. Currently, only two of them, genotype I and genotype II, have been detected outside Africa, with genotype II being responsible for the current panzootic. Since the introduction of ASFV into Georgia in 2007 outbreaks of ASF have been reported in countries of the European region, the Russian federation, Asia, Oceania and the Americas3,8,9.

[0007] In most affected countries the control of ASF is still limited to biorisk management measures, surveillance approaches and response strategies, which include culling and trade restrictions, and result in significant economic and production losses, as licensed vaccines are currently not commercially available3.

[0008] As such, there is a need for strategies which help target and modulate reproductive ASFV infection and for use in the treatment and / or prevention of ASF.

[0009] SUMMARY

[0010] The present disclosure is based on the finding that a cohort of genes associated with the swine major histocompatibility complex II (MHC II), or swine leucocyte antigen complex II

[0011] (SLA II) are important for productive African swine fever virus (ASFV) infection. The cohort of SLA II genes may include, for example, the SLA-DMA, SLA-DMB, RFXANK, RFXAP and CIITA genes. These genes shall be collectively referred to as the “SLA II genes”. The SLA II genes encode (or express) proteins (referred to as ‘SLA II proteins’) which are exploited by ASFV in order to gain entry to a host cell and / or to facilitate its replication and propagation therein. In one teaching, the SLA II proteins may comprise the proteins encoded or expressed by the SLA-DMA, SLA-DMB, RFXANK, RFXAP and CIITA genes. These SLA II proteins may include the SLA-DMA protein subunit, the SLA-DMB protein subunit, regulatory factor X associated protein (RFXAP) and class II major histocompatibility complex transactivator (CIITA). In particular, this disclosure relates to a newly identified interaction between ASFV and the SLA-DM genes and their protein products, which genes include the SLA-DMA and SLA-DMB genes encoding the SLA-DM sub-unit proteins (namely the SLA-DMA and SLA-DMB MHC protein sub-units). Without wishing to be bound by theory, the data presented in this disclosure has established that by disrupting the interaction between the virus (ASFV) and various factors encoded by the cohort of genes described herein, it is possible to reduce, prevent and / or inhibit (i.e. modulate) ASFV infection or the occurrence of ASF in a swine (or porcine) host. Moreover, it is suggested that targeted modulation (e.g. knockout) of either of any or all of the SLA II genes disclosed herein, may lead to, for example, replication defects, reduced cell-to-cell spread, a reduction in progeny viral titre and a reduced level of viral DNA replication. Any or all of these effects may help prevent ASFV infections and the occurrence of ASF in hosts (e.g. porcine / swine hosts) and host cells. It should be noted that the terms “comprise”, “comprising” and / or “comprises” is / are used to de-note that aspects and embodiments of this invention “comprise” a particular feature or features. It should be understood that this / these terms may also encompass aspects and / or embodiments which “consist essentially of” or “consist of” the relevant feature or features. The term ‘ASFV’ embraces the only member of the genus Asfivirus of the family Asfarviridae and all genotypes / strains. The ASFV linear double stranded DNA genome varies between 170 and 193 kbp in size and contains 150 to 167 predicted protein-encoding open reading frames14-16. ASFV particles contain approximately 82 viral proteins, are about 250 nm in size and multilayered. They comprise an external lipid membrane, an icosahedral outer capsid, an internal lipid membrane, an inner capsid, a thick protein core shell, and a nucleoid containing the genome. ASFV is the causative agent of African swine fever (ASF), a haemorrhagic disease of domestic pigs and wild boar (species Sus scrofa). Accordingly, the disclosure provides: a method of treating or preventing African swine fever (ASF) or an ASFV infection, said method comprising modulating the function, expression and / or activity of a swine leucocyte antigen complex II (SLA II) gene and / or a SLA II protein; a method of treating or preventing ASF or an ASFV infection, said method comprising administering a subject in need thereof, a compound which modulates the function, expression and / or activity of a swine leucocyte antigen complex II (SLA II) gene and / or a SLA II protein; a compound which modulates the function, activity and / or expression of a swine leucocyte antigen complex II (SLA II) gene and / or a SLA II protein, for use in treating or preventing ASF or an ASFV infection; use of a compound which modulates the function, activity and / or expression of a swine leucocyte antigen complex II (SLA II) gene and / or a SLA II protein, in the manufacture of a medicament for use in treating or preventing ASF or an ASFV infection; a compound which modulates the function, activity and / or expression of a swine leucocyte antigen complex II (SLA II) gene and / or a SLA II protein for use in medicine or for use as a medicament. a method of treating or preventing African swine fever (ASF) or an ASFV infection, said method comprising modulating the function, expression and / or activity of the SLA-DMA gene and / or the SLA-DMA protein; a method of treating or preventing ASF or an ASFV infection, said method comprising administering a subject in need thereof, a compound which modulates the function, expression and / or activity of the SLA-DMA gene and / or the SLA-DMA protein; a compound which modulates the function, activity and / or expression of the SLA- DMA gene and / or the SLA-DMA protein, for use in treating or preventing ASF or an ASFV infection; use of a compound which modulates the function, activity and / or expression of the SLA-DMA gene and / or the SLA-DMA protein, in the manufacture of a medicament for use in treating or preventing ASF or an ASFV infection; a method of treating or preventing African swine fever (ASF) or an ASFV infection, said method comprising modulating the function, expression and / or activity of the SLA-DMB gene and / or the SLA-DMB protein; a method of treating or preventing ASF or an ASFV infection, said method comprising administering a subject in need thereof, a compound which modulates the function, expression and / or activity of the SLA-DMB gene and / or the SLA-DMB protein; a compound which modulates the function, activity and / or expression of the SLA- DMB gene and / or the SLA-DMB protein, for use in treating or preventing ASF or an ASFV infection; use of a compound which modulates the function, activity and / or expression of the SLA-DMB gene and / or the SLA-DMB protein, in the manufacture of a medicament for use in treating or preventing ASF or an ASFV infection; In a further teaching, the disclosure provides: a method of modulating productive African swine fever virus (ASFV) infection, said method comprising modulating the function, expression and / or activity of a swine leucocyte antigen complex II (SLA II) gene and / or a SLA II protein; a compound which modulates the function, activity and / or expression of a swine leucocyte antigen complex II (SLA II) gene and / or a SLA II protein, for use in modulating productive African swine fever virus (ASFV) infection; use of a compound which modulates the function, activity and / or expression of a swine leucocyte antigen complex II (SLA II) gene and / or a SLA II protein, in the manufacture of a medicament for use in modulating productive African swine fever virus (ASFV) infection; a method of modulating productive African swine fever virus (ASFV) infection, said method comprising modulating the function, expression and / or activity of the SLA- DMA gene and / or the SLA-DMA protein; a compound which modulates the function, activity and / or expression of the SLA- DMA gene and / or the SLA-DMA protein, for use in modulating productive African swine fever virus (ASFV) infection; use of a compound which modulates the function, activity and / or expression of the SLA-DMA gene and / or the SLA-DMA protein, in the manufacture of a medicament for use in modulating productive African swine fever virus (ASFV) infection; a method of modulating productive African swine fever virus (ASFV) infection, said method comprising modulating the function, expression and / or activity of the SLA- DMB gene and / or the SLA-DMB protein; a compound which modulates the function, activity and / or expression of the SLA- DMB gene and / or the SLA-DMB protein, for use in modulating productive African swine fever virus (ASFV) infection; use of a compound which modulates the function, activity and / or expression of the SLA-DMB gene and / or the SLA-DMB protein, in the manufacture of a medicament for use in modulating productive African swine fever virus (ASFV) infection; Any of the methods described herein may be ‘in vitro’ methods. Accordingly, the disclosure provides: an in vitro method of modulating productive African swine fever virus (ASFV) infection, said method comprising modulating the function, expression and / or activity of a swine leucocyte antigen complex II (SLA II) gene and / or a SLA II protein; an in vitro method of modulating productive African swine fever virus (ASFV) infection, said method comprising contacting a cell with a compound which modulates the function, activity and / or expression of a swine leucocyte antigen complex II (SLA II) gene and / or a SLA II protein. Within the context of this disclosure, the term ‘modulate’ means to reduce, inhibit, prevent or suppress the expression, function and / or activity of any of an SLA II genes described herein and / or the proteins (the SLA II proteins) encoded thereby. As such, a compound which modulates the expression, function or activity of a SLA II gene or SLA II protein, may reduce, inhibit, prevent or suppress the expression, function and / or activity of any of an SLA II genes described herein and / or the proteins (the SLA II proteins) encoded thereby. The phrase ‘modulating productive African swine fever virus (ASFV) infection’ means any change in (for example a reduction, inhibition, suppression and / or prevention of): the ability of ASFV to infect its target (host) cell, spread from cell-to-cell, (progeny) viral titre and / or viral replication. The phrase ‘modulating the expression, function and / or activity of a SLA II gene / protein’ means, for example: decreasing, inhibiting or reducing the level of expression of a SLA II gene; decreasing, inhibiting or reducing the expression or amount of a protein encoded by a SLA II gene (e.g. an SLA II protein); decreasing, inhibiting or reducing the activity of a protein encoded by a SLA II gene (e.g. an SLA II protein); and / or decreasing, inhibiting or reducing the function of a protein encoded by a SLA II gene (e.g. an SLA II protein). Modulating the function or activity of a gene or protein may embrace a reduction or inhibition in the ability of the relevant gene or protein to interact with its usual cell pathways, reactions, and / or ligands. It should be understood that based on the findings presented herein, a reduction (or inhibition) of any aspect of the function or activity of a SLA II gene / protein, may in turn effect (e.g. adversely or negatively effect) the ability of the ASFV to infect a host cell and / or to replicate therein; because, without wishing to be bound by theory, the SLA II genes / proteins described herein (especially the SLA-DMA / B genes / proteins) are important in ASFV infection / replication. The term “cell” (as referred to in the abovementioned in vitro methods) may be any swine or porcine cell which can act as a host for ASFV. The term cell may embrace a Sus scrofa cell. A compound of this disclosure, including compounds for use in treating or preventing an ASFV infection and / or ASF, may modulate the interaction between ASFV and the SLA II genes and / or SLA II proteins described herein. A compound may comprise: A compound which blocks or neutralises the ability of the ASFV to interact with or bind to a SLA II protein, including for example the SLA-DMA and / or SLA-DMB (subunit) protein(s); A compound which binds to an SLA II protein, e.g. the SLA-DMA and / or SLA-DMB (subunit) protein(s) and prevents or inhibits ASFV from binding thereto; A compound which modulates (for example reduces, suppresses, inhibits or prevents) expression of one or more of the SLA II genes described herein, including for example the SLA-DMA and / or SLA-DMB gene(s); A compound which inhibits the function or activity of any of the proteins encoded by the SLA II genes of this disclosure, including for example the SLA-DMA and / or SLA- DMB proteins encoded by the SLA-DMA and / or SLA-DMB gene(s); compounds which prevent a proviral interaction between any of the proteins encoded by any of the SLA II genes described herein (including the SLA-DMA and / or SLA- DMB proteins encoded by the SLA-DMA and / or SLA-DMB genes). A proviral interaction may include, for example, a binding event between ASFV and any of the SLA II gene(s) / protein(s) of this disclosure, which interaction facilitates viral (ASFV) host cell entry and / or replication / propagation. By way of example, the disclosure may provide antibodies which neutralise any (proviral) interaction between ASFV and the product of any of the SLA II genes disclosed herein. As such, useful antibodies may bind to (or have affinity for) any of the SLA II proteins described herein, including, for example the SLA-DMA and / or SLA-DMB protein(s) described herein. Useful antibodies may bind a proviral epitope – that is an epitope within a region of a SLA II protein which usually interacts with ASFV in order to permit viral entry to and / or replication within, a host cell. In one teaching, the antibodies of this disclosure are not used as ‘adjuvants’ and / or are not fused to ASF antigens. The term antibodies may include monoclonal or polyclonal antibodies as well as antibodies of any isotype (IgG, IgM, IgE, IgA and the like). Useful antibodies may include, for example: an anti SLA-DMA antibody (i.e. an antibody with affinity for, or which binds to, the SLA-DMA subunit of the SLA II). The anti-SLA-DMA antibody may bind to a SLA- DMA epitope to prevent any interaction between SLA-DMA and ASFV. By preventing the interaction between SLA-DMA and ASFV, the antibody may act to prevent ASFV entry into a host cell and / or ASFV replication / propagation in a host (e.g. porcine) cell; and / or an anti SLA-DMB antibody (i.e. an antibody with affinity for, or which binds to, the SLA-DMB subunit of the SLA II). The anti-SLA-DMB antibody may bind to a SLA- DMB epitope to prevent any interaction between SLA-DMB and ASFV. By preventing the interaction between SLA-DMB and ASFV, the antibody may act to prevent ASFV entry into a host cell and / or ASFV replication / propagation in a host (e.g. porcine) cell. The disclosure may further provide an anti-RFXANK antibody (i.e. an antibody with affinity for, or which binds to, the protein product of the RFXANK gene), an anti-RFXAP antibody (i.e. an antibody with affinity for, or which binds to, the protein product of the RFXAP gene) and an anti-CIITA antibody (i.e. an antibody with affinity for, or which binds to, the protein product of the CIITA gene). All such antibodies may bind to a proviral epitope of their respective protein targets, which binding event may act to prevent ASFV entry into a host cell and / or ASFV replication / propagation in a host (e.g. porcine) cell. The techniques used to generate antibodies are well established and further information may be found in, 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. Assays for testing antibodies for an effect against ASFV host cell entry and / or replication / propagation may comprise, contacting a cell with ASFV in the presence / absence of a test antibody and observing any difference in the amount of virus which is able to infect and / or propagate / replicate, in the cell. Where the assay shows that in the presence of the test antibody, fewer virus have been able to infect the cell and / or or replicate / propagate within the cell, then the relevant antibody may be useful as an antibody which can neutralise ASFV (or at least its cell entry / replicative functions). The term antibody (as used herein) may be used to embrace all antigen (e.g. SLA-DMA, SLA-DMB, RFXANK, RFXAP and CIITA) binding fragments and variants. There term may include, for example, a single chain antibody, a monoclonal antibody, a chimeric antibody, a domain antibody, a nanobody, a camelid antibody with specificity for any of the disclosed antigens and any antigen-binding fragments of these. The term ‘antibody’ may further encompass bispecific antibodies and fragments and variants known as: Fab, F(ab’)2, monospecific, bispecific Fab2, trispecific Fab3, monovalent, scFv, Bispecific diabody, trispecific diabody, scFv-Fc or minibody (and the like) with specificity for any of the disclosed antigens. While all these antibody fragments and variants may differ, for simplicity, all of these antibody types will be collectively referred to as ‘antibodies’. The disclosure may provide other ‘antibody-like’ molecules with specificity (i.e. an ability to bind and / or neutralise) any of the antigens (e.g. SLA-DMA, SLA-DMB, RFXANK, RFXAP and CIITA) disclosed herein. These ‘antibody-like’ molecules may include, for example, molecules comprising (or consisting essentially of), for example, bicyclic peptides, small affinity ligand (e.g. affibody) type molecules, designed ankyrin repeat proteins (Darpin)-type molecules and anticalin proteins – all designed to have specificity for (or an ability to bind and / or neutralise) any of the antigens of this disclosure (e.g. SLA-DMA, SLA-DMB, RFXANK, RFXAP and CIITA). The present invention further provides nucleic acid sequences encoding any of the disclosed antibodies and / or antibody-like molecules. Moreover, the disclosure provides vectors (e.g. plasmids, viruses, viral vectors and / or other expression cassettes) comprising nucleic acid sequences which encode any of the antibodies and / or antibody-like molecules (or their antigen binding fragments). Such vectors may be designed for expression in mammalian (e.g. human) cells. In one teaching, the disclosure provides an antibody, or antibody-like molecule encoding plasmid / virus. The disclosure further provides a host cell, for example a mammalian, e.g. human cell. The host cell may be a somatic mammalian, e.g. human, cell. An antibody, or antibody-like molecule (or any antigen binding / neutralising fragment) may be administered to a subject in need thereof or to an in vitro cell or tissue by a technique that permits direct physical delivery into a cell. Such techniques may include electroporation and / or microinjection. Any of the disclosed antibodies and / or antibody-like molecules maybe provided in a form which permits intracellular expression and / or targeting to specific intracellular regions and / or structures. An antibody which is expressed intracellularly may be referred to as an intrabody. As such, this disclosure provides intrabodies with specificity (or an ability to bind and / or neutralise) any of the disclosed antigens, including, for example, SLA-DMA, SLA-DMB, RFXANK, RFXAP and CIITA. Expression of an intrabody of this disclosure may be achieved using a vector, for example a plasmid or virus encoding an antibody of this disclosure (or an antigen binding fragment thereof). In one teaching, a cell may be transfected with a plasmid / virus carrying a nucleic acid encoding any of the disclosed antibodies, antibody-like molecules or their antigen- binding (or neutralising) fragments. A vector of this disclosure may further encode a signal peptide or some molecule (perhaps fused or conjugated to the antibody / antibody-like molecule) so that the antibody / antibody-like molecule (or its fragment) is directed to a specific part of the cell (e.g. the nucleus, the cytosol, a mitochondria or the ER lumen). A variety of signal peptides and / or signal / retention molecules are known to the skilled person and many of these can be used to ensure that an antibody, antibody like molecule or antigen-binding (or neutralising fragment thereof) can be expressed in a cell and directed to a specific site or structure (e.g. the cytosol, nucleus or mitochondria). Examples may include, reticulum retention sequences, nuclear localisation sequences, mitochondrial localisation sequences, transduction domains and nanoparticles. Nucleic acids encoding any of these may be included in the various vectors described herein – all for the purpose of ensuring that an antibody or antibody-like molecule which is expressed in a cell, may be correctly targeted so that it can exert its function (in this case modulating the expression, function and / or activity of a SLA II gene / protein. All of this technology is well summarised in Slastnikova et al., 2018 (Targeted Intracellular Delivery of Antibodies: The State of the Art: 2018, 9: 1208) – the entire contents of which are incorporated herein by reference. In one teaching, the disclosure provides a nucleic acid sequence encoding an antibody, antibody-like molecule or antigen (e.g. SLA II protein or SLA-DMA, SLA-DMB, RFXANK, RFXAP and CIITA) binding or neutralising fragment thereof and a signal molecule for ensuring intracellular expression and / or intracellular expression at as specific site or structure. The disclosure may further provide antisense molecules, e.g. antisense oligonucleotides (ASOs) which modulate (for example prevent, reduce, suppress or inhibit) the expression of one or more of the SLA II genes described herein. Antisense oligonucleotides may comprise 10-30, for example 15–20 nucleotides with a sequence which is complementary to a target RNA sequence. The target RNA sequence may be RNA (for example mRNA) generated from any of the SLA II genes described herein (SLA II RNA). Without wishing to be bound by theory, an ASO of this disclosure may bind to a region of the SLA II RNA marking it for degradation and thereby reducing the amount of the corresponding SLA II protein expressed by (or from) the gene. Given the sequence of a gene, an ASO capable of modulating the expression of that gene is relatively straightforward to design and obtain; indeed, one of skill will be aware of the various services and online tools that may be used to generate ASOs for testing. A useful ASO may be selected on the basis of its ability to modulate (e.g. reduce or inhibit) the expression of any of the SLA II proteins of this disclosure – in particular the SLA DMA / B subunits of the SLA II. By way of example a cell exhibiting a known level of SLA II protein (e.g. SLA DMA / B) expression may by contacted with an ASO and the subsequent level of SLA II protein determined in said cell. Any difference between the known level of SLA II protein expression and the expression of the same protein following contact with the ASO may indicate that the ASO has a modulatory effect upon the relevant SLA II gene. An assay of this type may be used to test for ASOs which reduce or inhibit the level of the relevant SLA II protein in the cell. A vector (for example a plasmid, a viral vector or expression cassette) of this disclosure may comprise any of the ASO’s described herein for expression in a cell. The disclosure further provides host cells transformed or transfected with such vectors. Nucleic acid sequences for the SLA-DMA, SLA-DMB, RFXAP and CIITA genes and other sequences associated therewith are provided below as SEQ ID NOS: 1-8. These sequences may be used in the design of ASOs as described herein: SEQ ID NO: 1: NM_001004039.1 Sus scrofa SLA-DM alpha chain (SLA-DMA), mRNA SEQ ID NO: 2: NM_001004039.1 Sus scrofa SLA-DM alpha chain (SLA-DMA), CDS SEQ ID NO: 3: NM_001004039 Sus Scrofa SLA-DM alpha chain precursor translation - back translated, codon optimized for Sus scrofa, used for cloning SEQ ID N: 4: NM_001113707.1 Sus scrofa MHC class II, DM beta (SLA-DMB), mRNA SEQ ID NO: 5: NM_001113707.1 Sus scrofa MHC class II, DM beta (SLA-DMB), CDS SEQ ID NO: 6: NM_001113707 Sus scrofa MHC class II, DM beta (SLA-DMB)- back translated, codon optimized for Sus scrofa, used for cloning SEQ ID NO: 7: XM_013980439.2 PREDICTED: Sus scrofa regulatory factor X associated protein (RFXAP), mRNA

[0012] SEQ ID NO: 8: XM_021085809.1 PREDICTED: Sus scrofa class II major histocompatibility complex transactivator (CIITA), transcript variant X5, mRNA (Protein title MHC class II transactivator isoform X4) The term ‘compounds’ may also include fragments of the proteins encoded by any of the SLA II genes described herein. By way of example, the term compounds may include fragments or portions of the SLA DMA / B subunit proteins. Useful fragments may include parts of the SLA II proteins known to interact with or bind to the ASFV. Such fragments may bind to the ASFV and may serve to block or neutralise any interaction between the ASFV and the native or wild-type SLA II protein. Fragments of the SLA-DMA, SLA-DMB, RFXAP, CIITA proteins described herein may be derived from any of the following sequences: SEQ ID NO: 9: SLA-DMA (accession number: NP_001004039.1: SLA-DM alpha chain precursor [Sus scrofa] – Protein) A useful SLA-DMA fragment may be derived from SEQ ID NO: 9 and may comprise any number of residues from about 5 residues to about 259 residues (and any number therebetween). For example, a fragment may comprise 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 the stated number of residues ± 1, 2, 3 or 4 amino acid residues. As stated, any fragment derived from SEQ ID NO: 9, useful fragments may include parts of the SLA-DMA protein known to interact with or bind to the ASFV. Such fragments may bind to the ASFV and may serve to block or neutralise any interaction between the ASFV and the native or wild-type SLA-DMA protein. SEQ ID NO: 10: SLA-DMB (accession number: NP_001107179.1: MHC class II, DM beta precursor [Sus scrofa], Protein) A useful SLA-DMB fragment may be derived from SEQ ID NO: 10 and may comprise any number of residues from about 5 residues to about 271 residues (and any number therebetween). For example, a fragment may comprise 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 the stated number of residues ± 1, 2, 3 or 4 amino acid residues. As stated, any fragment derived from SEQ ID NO: 10, useful fragments may include parts of the SLA-DMB protein known to interact with or bind to the ASFV. Such fragments may bind to the ASFV and may serve to block or neutralise any interaction between the ASFV and the native or wild-type SLA-DMB protein. SEQ ID NO: 11: (accession number: XP_013835893.2: regulatory factor X-associated protein [Sus scrofa] – Protein) A useful RFXAP fragment may be derived from SEQ ID NO: 11 and may comprise any number of residues from about 5 residues to about 368 residues (and any number therebetween). For example, a fragment may comprise 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, 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 the stated number of residues ± 1, 2, 3 or 4 amino acid residues. As stated, any fragment derived from SEQ ID NO: 11, useful fragments may include parts of the RFXAP protein known to interact with or bind to the ASFV. Such fragments may bind to the ASFV and may serve to block or neutralise any interaction between the ASFV and the native or wild-type RFXAP protein. SEQ ID NO: 12 (accession number: XP_020941468.1: MHC class II transactivator isoform X4 [Sus scrofa], Protein) A useful CIITA fragment may be derived from SEQ ID NO: 12 and may comprise any number of residues from about 5 residues to about 1155 residues (and any number therebetween). For example, a fragment may comprise 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, 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, 570, 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, 780, 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 the stated number of residues ± 1, 2, 3 or 4 amino acid residues. As stated, any fragment derived from SEQ ID NO: 12, useful fragments may include parts of the CIITA protein known to interact with or bind to the ASFV. Such fragments may bind to the ASFV and may serve to block or neutralise any interaction between the ASFV and the native or wild-type CIITA protein. The various SLA II protein fragments described herein may be used to generate antibodies. Antibodies generated in this way (and with affinity for or an ability to bind to a SLA II protein fragment) may be used to neutralise any (proviral) interaction between ASFV and the SLA II protein from which the SLA II protein fragment has been derived. In this regard, fragments for use in a method of generating antibodies (for use in preventing an ASFV infection and / or treating or preventing ASF) may comprise an epitope known to interact with ASFV. In this way, an antibody generated to bind to an SLA II protein fragment of this disclosure may be used to prevent ASFV cell entry to and / or its replication / propagation therein. The disclosure may further provide sequences derived from the nucleic acid sequences presented herein, which sequences encode SLA II protein fragments. This disclosure further provides vectors, for example viral vectors or nucleic acid vectors (e.g. plasmids), which vectors comprise nucleic acid sequences of this disclosure, including for example, sequences which encode any of the antibodies or antibody like molecules disclosed herein, any of the antisense oligonucleotides disclosed herein or any of the SLA II protein fragments described herein. The disclosure further provides host cells, for example cells transformed with any of the vectors described herein. The various compounds (antibodies, antibody-like molecules, SLA II protein fragments or antisense oligonucleotides) disclosed herein may be provided as compositions. A composition of this disclosure may comprise a pharmaceutical composition. A composition of this disclosure may further comprise excipients, buffers and / or diluents. The present disclosure further provides immunogenic compositions, which compositions are intended to raise an immune response for example a protective immune response in a host. Within the context of this disclosure, a protective immune response may comprise a response which neutralises an ASFV or which prevents ASFV from being able to interact with a host cell, enter a host cell and / or replicate and / or propagate within a host cell. An immunogenic composition of this disclosure may comprise any one or more of the SLA II proteins described herein or a functional fragment thereof. Without wishing to be bound by theory, a subject at risk of an ASFV infection (and hence ASF) may be administered an immunogenic composition of this disclosure to induce an immune response, which immune response may serve to prevent a proviral interaction between an SLA II protein and the ASFV. For example, the immunogenic composition may induce an immune response comprising an antibody with affinity for, or specificity to, the SLA II protein of the composition, which antibody binds to the SLA II protein and prevents its interaction with the ASFV. An immunogenic composition of this disclosure may comprise a SLA-DMA protein or fragment thereof and / or a SLA-DMB protein or fragment thereof. In a further teaching, the disclosure provides a vaccine, said vaccine comprising any one or more of the SLA II proteins described herein or a functional fragment thereof. Again, without wishing to be bound by theory, a subject at risk of an ASFV infection (and hence ASF) may be administered a vaccine according to this disclosure to induce an immune response, which immune response may serve to prevent a proviral interaction between an SLA protein and the ASFV. For example, the vaccine may induce an immune response comprising an antibody with affinity for, or specificity to, the SLA II protein of the composition, which antibody binds to the SLA II protein and prevents its interaction with the ASFV. By way of example, a vaccine of this disclosure may comprise a SLA-DMA protein or fragment thereof and / or a SLA-DMB protein or fragment thereof. A vaccine may further comprise an adjuvant and / or one or more additional antigens, for example, one or more additional ASFV antigens and / or one or more antigens from a different pathogen. It should be noted that any of the compounds, antibodies, antibody-like molecules (or their antigen binding / neutralising variants or fragments), SLA II protein fragments, vectors / nucleic acids (which vectors or nucleic acids may encode an antibody, antisense oligonucleotide or SLA II protein fragment) or compositions provided by this disclosure may be for use in medicine, for use a medicament or for use in treating or preventing ASF or an ASFV infection. In one teaching, the present disclosure provides a transgenic (or genetically modified) animal, e.g. a porcine (or swine) animal (e.g. Sus scrofa), which transgenic animal is resistant to ASFV and therefore also resistant to ASF. A genetically modified animal according to this disclosure, may lack a functional copy of any of the SLA-II genes described herein. By way of example, the SLA-DMA gene or the protein encoded thereby may have been rendered non-functional (via some loss of function mutation) or fully knocked out. Additionally or alternatively, the SLA-DMB gene or the protein encoded thereby may have been rendered non-functional (via some loss of function mutation) or fully knocked out. Additionally or alternatively, the RFXANK gene or the protein encoded thereby may have been rendered non-functional (via some loss of function mutation) or fully knocked out. Additionally or alternatively, the RFXAP gene or the protein encoded thereby may have been rendered non-functional (via some loss of function mutation) or fully knocked out. Additionally or alternatively, the CIITA gene or the protein encoded thereby may have been rendered non-functional (via some loss of function mutation) or fully knocked out. Genetically modified animals, such as porcine (or swine) animals (including pigs or wild- boar: Sus scrofa) which have been genetically modified as described above (i.e. modified in some way to modulate (e.g. reduce, prevent or inhibit) the expression of any of the SLA-II genes described herein) may be made using any of the widely available methods. DETAILED DESCRIPTION The present disclosure will now be described in detail by reference to the following figures which show: Figure 1. Genome-wide CRISPR / Cas9 knockout screens identified molecules of the MHC II pathway as relevant for ASFV replication. (a) Diagrams of robust rank aggregation (RRA) scores calculated by the MAGeCK algorithm software of four separate analyses determined in two independent screens. The sgRNA content of the control cells was compared to the sgRNA abundance of cells that survived four subsequent ASFV infections. (b) Mean (-) and single RRA scores of the individual gene hits in the four different analyses. Dark blue dots represent sgRNAs against the indicated genes (x-axis) found in 4 / 4 subsets. Medium light blue dots represent the sgRNAs against gene CCZ1 which were found in 3 / 4 subsets. Light blue dots show sgRNAs which were only found in the 2 subsets of either of the performed screens. (c) Schematic of an MHC II gene locus (e.g. for SLA-DMA or SLA-DMB) with the MHC class II specific regulatory SXY module and specific transcription factors. Proteins which were identified as crucial cellular factor for ASFV infection in the genome-wide CRISPR / Cas9 knockout screen are shown in blue. Figure 2. Parental WSL and WSL knockout cells differ in the expression of MHC II proteins. (a) Indirect immunofluorescence analyses of cell surface expression of SLA-DR in WSL, WSL SLA-DMAKO, WSL SLA-DMBKO, WSL CIITAKOand WSL RFXAPKOcell clones. Bar: 30 µm. (b) Mass spectrometry analysis of quantitative expression levels of the housekeeping gene α-tubulin (TUBA4A-ENSSSCG00000016216), of genes of the MHC II pathway (SLA- DRA-ENSSSCG00000001453, SLA-DRB1-ENSSSCG00000001455, SLA-DQA- ENSSSCG00000001456, SLA-DQB-ENSSSCG00000001457), and of the MHC I pathway (SLA-8-ENSSSCG00000001231, HLA-E-ENSSSCG00000001229) in WSL and indicated knockout cells based on label-free quantitation (LFQ). Data represent means of three replicates. (c) Comparative quantitative analysis of protein expression levels in WSL and individual WSLKO cell clones. Proteins are indicated by dots. Black dots represent proteins involved in antigen processing and presentation. As far as detected, the SLA I / II proteins shown in b are highlighted in red. Figure 3. ASFV replication in WSL knockout cells is impaired. (a) Visualization of ASFV Armenia or ASFV Kenya-infected WSL and WSLKOcells (green) and nucleic acids (blue) by immunofluorescence staining. Representative images of the indicated cell clones infected with different virus dilutions (10-1 to 10-3) to illustrate plating efficiency and plaque sizes. 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 (%) compared to those on WSL cells and standard deviations are shown. Significant differences were calculated by ordinary one-way ANOVA followed by Tukey’s multiple comparison test. **** = <0.0001. (c) For the determination of plaque sizes, areas of fifty plaques per cell line from three independent experiments (n = 150) were measured, and the mean relative areas (%) compared to WSL cells including standard deviations are shown. Significant differences were calculated by Kruskal-Wallis test followed by Dunn’s multiple comparison test. **** = <0.0001. (d) Multi step (MOI 0.02) growth curve analysis of ASFV Armenia or Kenya in WSL and WSLKO cells. Shown are the mean results of three independent experiments (n = 3) with standard deviations. Figure 4: ASFV DNA replication is inhibited in WSL knockout cells. Parental WSL and WSLKO cells were infected with ASFV Armenia at a MOI of 3 and after indicated times the amounts of ASFV DNA were quantified by real-time PCR targeting the viral B646L gene. Genome copy numbers were determined using plasmid standards. Graphs represent means of two biological replicates with standard deviations. Figure 5. ASFV progeny virus particles are detected in infected parental WSL cells but not in knockout cells. (a-d) WSL, (e) WSL SLA-DMAKO, and (f) WSL CIITAKO cells were fixed and analyzed by electron microscopy 16 h after infection with ASFV Armenia at a MOI of 5. Virus factories (arrow), intracellular (*) and extracellular virus particles (#) are indicated. Bars represent 1 µm (a, e, f), or 200 nm (b, c, d). Figure 6. WSL knockout / knockin cells express MHC II transgenes. Lysates of (a) WSL SLA-DMAKOcells and (b) WSL cells expressing indicated SLA-DMA transgenes or GFP, or lysates of (c) SLA-DMBKO cells and (d) WSL cells expressing indicated SLA-DMB transgenes or GFP were separated by SDS-PAGE, transferred to nitrocellulose membranes and probed with antibodies against the indicated proteins or protein tags. Molecular masses of marker proteins (in kDa) are indicated on the left. Figure 7. MHC II transgene expression in WSL knockout / knockin cells restored ASFV replication. (a-b) For the determination of plating efficiency and plaque size ASFV Armenia or ASFV Kenya-infected WSL, WSLKO and WSLKO / KI cells were visualized by immunofluorescence staining. (a) Plating efficiency of ASFV Armenia and ASFV Kenya was calculated by counting ASFV-infected cells or plaques in three independent experiments (n = 6). Shown are the mean relative (%) titers compared to those on WSL cells, and standard deviations. Significant differences were calculated by ordinary one-way ANOVA followed by Tukey’s multiple comparison test. * = <0.05, **** = <0.0001, ns = not significant. (b) For the determination of plaque sizes, areas of fifty plaques per cell line from three independent experiments (n = 150) were measured and the mean relative (%) sizes compared to WSL cells including standard deviations are shown. Significant differences were calculated by Kruskal-Wallis test followed by Dunn’s multiple comparison test. *** = <0.001, **** = <0.0001, ns = not significant. (c-e) Multi step growth (MOI 0.02) analysis of ASFV Armenia and Kenya in untreated and transgene-expressing lentivirus-transduced (c) WSL, (d) WSL- DMAKO, and (e) WSL-DMBKO cells. Shown are the mean results of two independent experiments with two replicates (n = 4) and standard deviations. Materials and Methods Cell lines and viruses Cell lines were received from the cell culture collection for veterinary medicine (CCVM) of the Friedrich-Loeffler-Institut (FLI). The highly passaged wild boar lung cell line (WSL-R-HP, #1346; abbreviated as WSL) was maintained in Ham´s F12 cell culture medium (Ham´s F- 12, 5.32 g / L; IMDM, 8.80 g / L; NaHCO3, 2.45 g / L; pH 7.2) which was supplemented with 10 % fetal calf serum (FCS). Cloning of the cell line was performed by limiting dilution in 96 well plates. Single cells were propagated, and one WSL cell clone with parental phenotype was selected and used for all targeted knockout experiments. A rabbit kidney cell line (RK-13, #0237) was maintained in Minimum Essential Medium (MEM; MEM-Eagle-Hank´s salt, 5.32 g / L; MEM Earle´s salt, 4.76 g / L; NaHCO3, 1.25 g / L; non-essential amino acids, 1 %; Na- Pyruvate, 0.12 g / L; pH 7.2) supplemented with 10 % FCS. The human embryo kidney cell line (HEK293Td4.1, #1539) was also maintained in MEM supplemented with 10 % FCS. All cells were incubated at 37°C and 2.5 % CO2. ASFV Armenia 2008 (ASFV Armenia), a virulent genotype II ASFV isolate from Armenia55, was kindly provided by Sandra Blome (FLI). The virus was adapted to efficient growth in cell culture through 21 serial passages on WSL cells. The genotype IX isolate ASFV Kenya 103355,75was kindly provided by Richard Bishop (International Livestock Research Institute, Nairobi, Kenya). The mutant ASFV Kenya 1033 ΔCD2v dsRed containing a reporter gene expression cassette at the deleted CD2v (EP402R) gene locus55,56was kindly provided by Günther M. Keil (FLI). The plasmid-based PrV mutant PrV-BaΔgGG76was used as heterologous control virus. Porcine CRISPR library The generation and characterization of the porcine CRISPR knockout library (SsCRISPRko.v1) has been described52. Briefly, for the generation of specific single guide RNAs (sgRNA) targeting protein coding genes the genome assembly S. scrofa 10.277was used. Three to four sgRNAs for each gene were selected. In total, the porcine CRISPR library consisted of 83,381 specific sgRNA targeting 20,598 porcine genes and 1001 non- targeting controls, cloned in the pLenti-CRISPRv2 backbone (Addgene #52961). Genome-wide CRISPR / Cas9 knockout screen The genome-wide CRISPR / Cas9 knockout screen was performed as described previously52with slight modifications as described below. In five 20 cm dishes 5 x 106WSL cells each were seeded one day before they were transduced with the lentiviral sgRNA library, that was produced in accordance with the protocol by Joung, et al.78, at a MOT of 0.3 in medium containing 10 µg / ml polybrene. Three days after transduction, cells were split into eight dishes at a density of 5 x 106cells / plate with medium containing 1.25 µg / ml puromycin. At confluency, selected transduced cells were split again into a total of 16 plates at a density of 5 x 106cells / plate. At 12 days post transduction cells were seeded into 30 dishes at a density of 1 x 107cells / dish for infection the next day. At this time at least 6 x 107cells were harvested, sedimented and stored as controls at -20°C for DNA extraction. Infection was performed with ASFV Kenya 1033 ΔCD2v dsRed at a MOI of 0.3 or 0.5 (depending on the available amounts of virus stocks), since this MOI had been shown to be required to kill at least most of infected WSL control cells. The cells were checked daily for fluorescent marker expression and cytopathic effect and medium was added or changed as appropriate. For medium changes 20 % conditioned medium from untreated WSL cells was included. After approx. four to five weeks growing cell colonies from all plates were trypsinized, and divided into two pooled subsets. At least 2 x 107cells of each subset were stored at -20°C for DNA preparation. The remaining cells were reseeded into cell culture dishes and infected as above. Cells surviving the second infection were harvested approx.20 days later. Cell collection for DNA preparation, reseeding and infection was repeated four times in total. The entire screening procedure was conducted twice resulting in two sets of uninfected control cells, and eight survivor populations (two sets per screen). For reliable virus inactivation the sedimented 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 for 1 h at 37°C. After addition of SDS to a final concentration of 0.3 %, samples were incubated further at 75°C for 30 min, before the standard lysis and DNA extraction protocol was executed using sarkosyl buffer, RNase A, pronase, phenol-chloroform extraction, and ethanol precipitation as described before52. To generate sequencing libraries three sequential PCR amplifications of the extracted DNAs were performed. First, four 50 µl reactions per sample containing 5 µg of DNA, 25 fmol each of P5-forward primer (ITA2fwd_P5) and P7-reverse primer (ITA2rev_P7_leCRV), and 3.75 U ExTaq DNA polymerase (Clontech) were prepared according to manufacturer’s specifications. Incubation conditions were as follows: 95°C for 1 min, 28 cycles of 95°C for 30 s, 53°C for 30 s, 72°C for 1 min, 72°C for 10 min. After amplification, the four PCR reactions of each sample were pooled and the 155 bp products were gel purified using a gel extraction kit (Zymo Research). The DNA was eluted in nuclease-free water and 200 ng DNA were used in a second PCR reaction with the same ingredients as before but with 25 fmol each of a sample-specific P5-barcode-forward primer (e.g. ITA2fwd_ID85_P5both) and P7-reverse primer (e.g. ITA2rev_IDxx_P7leCrv2). Reaction conditions were as follows: 95°C for 1 min, 14 cycles of 95°C for 30 s, 57°C for 30 s, 72°C for 1 min, 72°C for 10 min. PCR products were purified with the QIAquick Nucleotide Removal Kit (QIAgen), and eluted in 35 µl nuclease-free water. A third amplification step with the entire eluted DNA was conducted in a volume of 300 µl (6 x 50 µl) using the same compounds as for the second PCR except a tenfold higher concentration of the forward and reverse primers (250 fmol). The PCR reaction was performed as before, but only for one single cycle. The 222 bp amplification products were gel purified and eluted in nuclease-free water. The isolation of DNA and the three consecutive PCR amplifications were performed in parallel for the complete sample sets including the corresponding controls to minimize bias. Samples were sequenced in an IonTorrent Ion S5TMXL System (Invitrogen, Thermo Fisher Scientific) and sequencing data were processed and analyzed on the Galaxy web platform (usegalaxy.eu) using Cutadapt (Galaxy Version 1.16.5), MAGeCK count tool (Galaxy version 0.5.8.4), and MAGeCK test tool (Galaxy version 0.5.8.1) as described52,57,79,80. The sequencing results of two different conditions (‘Control’ vs. ‘Survivor’) were compared using the robust rank aggregation (RRA) method of the MAGeCK test tool57. Generation of WSL knockout cells For the targeted generation of SLA-DMA, SLA-DMB, CIITA, or RFXAP knockout cells one of the four gene specific sgRNAs from the whole genome library was selected and cloned into the multiplex CRISPR / Cas9 vector pX330A-1×4 which was a gift from Takashi Yamamoto (Addgene plasmid # 58768; http: / / n2t. net / addgene: 58768; RRID:Addgene_58768)81and which was modified to express a neomycin resistance gene (neoR; designated as pX330A- 1×4neoRA).To obtain pX330A-1x4neoRA the 8962 bp vector pX330A-1x4 was linearized within a non-functional region by digestion with PciI, and neoR under control of the simian virus 40 (SV40) early promoter was inserted as a 1667 bp PciI fragment isolated from pX330-ΔNLS1 / 2neoR55. In the resulting plasmid this resistance gene was in parallel orientation to the sgRNA and Cas9 genes. Next, complementary DNA oligonucleotides containing the target-specific sequences of the sgRNAs with matching 5’ overhangs were hybridized, phosphorylated, and cloned into BpiI-digested and dephosphorylated pX330A- 1x4neoRA. Accuracy of resulting plasmids was checked by sequencing with the HU6-SF primer. Plasmids pX330A-1x4neoRA-SLA-DMA gR2, -SLA-DMB gR3, -MHCIITA gR2, and - RFXAP gR2, were transfected into cloned WSL cells using the K2®Transfection System (Biontex) according to manufacturer’s instructions. After three days the cells were trypsinized, serially diluted and seeded into 96 well plates using media supplemented with 0.5 mg / ml G418 sulfate (Invitrogen, Thermo Fisher Scientific). Resistant single cell clones were further propagated and checked by immunoblot for Cas9 expression using an anti- FLAG antibody (see below). DNA of Cas9 positive cells was prepared using the QIAamp DNA Mini Kit (QIAgen) according to manufacturer’s instructions, and used for PCR and subsequent sequencing of the PCR products to confirm the integration of the sgRNA sequences as well as insertions or deletions of nucleotides within the targeted genes. Generation of SLA-DM reconstituted cell lines Coding sequences of SLA-DMA (GenBank #NC_010449.5, nt 25133494 to 25137928) and SLA-DMB (GenBank #NC_010449.5, nt 25119278 to 25125089) were spliced in silico and codon optimized. It was also ensured that the binding regions of the selected sgRNA were altered as far as possible by silent base substitutions. The custom-made plasmids (Invitrogen, Thermo Fisher Scientific) pMA-SLA-DMA and -DMB contained 5’-EcoRI and 3’- NotI restriction sites for convenient recloning of the ORFs, and unique BpiI cleavage sites immediately upstream of the termination codons, which permitted in-frame insertion of hybridized oligonucleotides, encoding a StepII-tag (WSHPQFEK) or a Myc-tag (LEQKLISEEDL), respectively, into the BpiI- and NotI-digested constructs. Correct insertions were verified by sequencing using primer M13 Rev (-24). The native and StrepII- or Myc- tagged SLA-DMA ORFs, as well as the native and Myc-tagged SLA-DMB ORFs were recloned as EcoRI / NotI fragments into the correspondingly digested 8140 bp lentivirus vector pLVX-IRES-Puro (TaKaRA / Clontech). As a control the ORF encoding enhanced green fluorescent protein (EGFP) isolated as a 772 bp EcoRI / NotI fragment of pEGFP-N1 (Clontech) was also inserted, resulting in pLVX-EGFP-IRES-Puro. Correct plasmid clones were identified by sequencing using primer CMV promotor-F. Protein expression was confirmed in RK13 cells transfected (X-tremeGENETM HP reagent, Roche) with the newly generated plasmids and immunoblot analysis using the anti-Myc and anti-Strep antibodies. Lentiviruses encoding the SLA-DMA-Strep, SLA-DMA-Myc, SLA-DMB-Myc and EGFP gene, respectively, or empty pLVX-IRES-Puro were generated in HEK-293T cells in accordance with the protocol described by Joung, et al.78. WSL, WSL SLA-DMAKO(11), and WSL SLA- DMBKO(9) cell clones were transduced and knockout / knockin (WSLKO / KI) cells were selected using 1 µg / ml puromycin. Knockout of the native gene and transgene integration was confirmed by PCR and sequencing using primers. Transgene expression was again confirmed by immunoblotting as described below. Sanger sequence analyses The generated plasmids and PCR-amplified (KOD Xtreme Hot Start DNA Polymerase, Merck) relevant genome fragments of recombinant WSL cell lines were sequenced with the indicated primers and the BigDye™ Terminator v1.1 Cycle Sequencing Kit (Thermo Fisher Scientific), in an Applied Biosystems 3500 Genetic Analyzer (Thermo Fisher Scientific). Results were evaluated using the Geneious Prime 2021.0.1 software package (Biomatters, available from https: / / www.geneious.com). Immunoblotting Cells were trypsinized, resuspended in medium containing 10 % FCS, centrifuged, and washed once with phosphate buffered saline (PBS). The sedimented cells were then lysed in sodium dodecyl sulfate-polyacrylamide (SDS) containing sample buffer (0.13 M Tris-HCl, pH 6.8; 4 % SDS; 20 % glycerol; 0.01 % bromophenol blue; 10 % 2-mercaptoethanol), sonicated and incubated for 5 min at 95°C. Proteins were separated in discontinuous SDS polyacrylamide gels and transferred to nitrocellulose membranes. Blots were blocked for 3 h at RT with 5 % skim milk in tris buffered saline with 0.25 % Tween 20 (TBS-T), and probed overnight with specific primary antibodies diluted in 0.5 % skim milk in TBS-T. Binding of monoclonal anti-FLAG (clone M2, #F1804, Sigma-Aldrich), anti α-tubulin (#T5168, Sigma- Aldrich), 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 visualized with secondary fluorophore-labelled donkey anti-rabbit IRDye 800CW (#926-32213, Li-Cor Biosciences) or donkey anti-mouse IRDye 680RD (#926-68072, Li-Cor Biosciences) antibodies in TBS-T. Fluorescent signals were detected with an Odyssey CLx infrared imaging system (CLX-2293; Li-Cor Biosciences). Determination of plaque size and plating efficiency WSL, WSLKO and WSLKO / KI cells were seeded at a density of 4 x 105cells per well in 24 well plates. The next day viruses were serially diluted in cell culture medium supplemented with 5 % FCS and applied to the confluent cell layers. The cells were incubated for 2 h at 37°C and 2.5 % CO2. Subsequently, the inoculum was removed and replaced with methocel medium (6 g / L methyl cellulose in MEM with 5 % FCS). PrV infection was visualized and documented as described below by the inherent GFP expression of PrV-BaΔgGG three days after infection. Four days after ASFV infection the medium was removed and cells were washed once with PBS before they were fixed with 4 % paraformaldehyde in PBS (PFA) for 20 min at room temperature (RT). Formaldehyde fixation was stopped by washing and subsequent incubation with 5 mM NH4Cl in PBS for 30 min at RT. Cells were washed three times with PBS and stored at 4°C until ASFV antigen detection by indirect immunofluorescence (IF) tests (see below). ASFV and PrV-infected cells, foci and plaques were visualized with a Leica DMi8 motorized fluorescence microscope. Using the Leica Application Suite X software whole wells were imaged and resulting mosaic images were merged. For each virus and each cell line the areas of 50 infected cells or plaques were determined using the freehand selection tool of ImageJ (Version 1.53f51; http: / / imagej.nih.gov / ij) in three independent experiments. The mean plaque sizes of respective viruses grown on WSL cells were set to 100 % and mean relative plaque sizes of viruses grown on WSLKOor WSLKO / KIcells as well as standard deviations were calculated using GraphPad Prism (Version 9). For plating efficiency plaques were counted and apparent titers were calculated as PFU / ml. Determination of viral titers and replication kinetics WSL, WSLKO and WSLKOKI cells were seeded at a density of 4 x 105cells (for PrV infection) or 3 x 105cells (for ASFV infection) per well in 24 well plates. One day later PrV-BaΔgGG, ASFV Armenia, or ASFV Kenya, were applied at a MOI of 0.02. After an incubation at RT (PrV) or 37°C (ASFV) for 2 h, cells were washed once with medium and subsequently overlaid with 1 ml medium containing 1 % penicillin / streptomycin (Gibco). PrV-infected cells were frozen three days after infection at -80°C. Single plates of ASFV-infected cells were frozen immediately after the addition of medium, as well as every 24 h until 168 h p.i.. For titration, plates were thawed and lysates were transferred to reaction tubes. After centrifugation at 2655 xg and 4°C for 5 min supernatants were transferred into fresh tubes and stored at -80°C. Virus titrations were performed on confluent RK13 cells (PrV) or WSL cells (ASFV) in 96 well plates. After inoculation with serial dilutions of the virus supernatants (100 µl / well), cells were incubated for 2 h at RT on a rocker (PrV), or centrifuged for 1 h at 689 xg and 37°C (ASFV) before the virus supernatant was removed and cells overlaid with methocel medium. The cells were incubated for 3 days (PrV) or 4 days (ASFV) at 37°C in a 2.5 % CO2 atmosphere. PrV-infected cells were fixed for 1 h by addition of a 3.7 % formaldehyde solution, and subsequently stained with 1 % crystal violet to visualize the plaques. Cells infected with ASFV were washed once with PBS, fixed with ice cold acetone / methanol (1:1, v / v) for 30 min at -20°C, and air dried. Infected cells were visualized by IF staining. DNA replication kinetics For the analysis of viral DNA replication WSL and WSLKO cells were seeded at a density of 3 x 105cells in 24 well plates and infected 24 h later with ASFV Armenia at a MOI of 3 in two replicas for each time. After an incubation of 2 h at 37°C the inoculum was removed, and after a wash, replaced with medium. After 0, 2, 4, 8, 16 and 32 h at 37°C, the medium was aspirated again and the cells were washed once with PBS, pelleted and stored until further analysis at -20°C. DNA was prepared with the NucleoMag Tissue Kit (Macherey-Nagel) according to manufacturer’s recommendations, and eluted in 100 µl elution buffer. Quantitative real-time PCR for DNA detection was performed using the QuantiTect Multiplex PCR NoROX kit (Qiagen) in 12.5 µl reactions containing 2.5 µl of infected cell DNA according to the manufacturer’s instructions. The ASFV B646L gene-specific primer pairs AKB646L-408F and AKB646L-507R, as well as the β-actin gene-specific primer pair ACT- CP-F and ACT-CP-R were included at 800 nM, and the TaqMan probes AKB646L-460P and ACT-CP-P at 160 nM final concentrations. Primer and probes were purchased from Eurogentec. Samples were incubated for 15 min at 95°C followed by 45 cycles of 30 s 95°C, 30 s 55°C, and 30 s 68°C in a Bio-Rad C1000 / CFX96 real-time PCR machine, and results were analyzed using CFX Maestro software (Bio-Rad). ASFV genome copy numbers were determined based on standard curves generated by reactions containing 1010, 108, 106, 104, 102or 100 copies of a p72 expression plasmid (pCAGGS-p72-Georgia, kindly provided by G.M. Keil). Indirect immunofluorescence (IF) analysis After fixation of the cells with PFA as described above, cells were optionally permeabilized with 0.5 % Triton-X 100 in PBS for 15 min at RT. This step was not required for the visualization of surface proteins, or after fixation with acetone / methanol. After washing with PBS, the cells were blocked with 10 % FCS in PBS for 1 h at RT, the polyclonal rabbit anti- ASFV p72 antibody82or the monoclonal mouse anti-pig MHC II (clone MSA 3) antibody (kindly provided by Luise Hartmann and Ulrike Blohm) diluted in blocking buffer were applied for 1 h at RT, and detected 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 antibodies diluted in PBS for another hour. For the detection of p72 antigen in WSLKO / KI cells, the secondary antibody goat anti rabbit Alexa-Fluor 647 (#A21245; Invitrogen, Thermo Fisher Scientific) was used to enable visualization also in the GFP expressing control cell lines. Nucleic acids were stained with Hoechst 33342 (#H3570; Invitrogen, Thermo Fisher Scientific) for 15 min at RT. After each incubation step the cells were washed three times with PBS, and finally analyzed with a Leica DMi8 fluorescence microscope. Mass spectrometry Confluent monolayers of WSL, WSL DMAKO(11), WSL DMBKO(9), WSL CIITAKO(1) and WSL RFXAPKO(6) (n = 3 per clone) were lysed in 2 % SDS in 0.1 M Tris-HCL (pH 8.0) for 10 min at 95°C. Lysates were clarified by centrifugation (14,000 xg, 10 min, RT) and the supernatants were collected. Aliquots containing 100 µg protein (determined by BCA assay) were precipitated by addition of 3 volumes of ice-cold acetone. Protein pellets were recovered by centrifugation at 10,000 xg at 4°C for 15 min and digested into peptides using the EasyPep™ Mini MS Sample Prep Kit (Thermo Scientific) according to the manufacturers protocol. Peptides were resuspended in 0.1 % formic acid (FA) and peptide yields assessed by BCA assay. Peptides (1 µg / sample) were separated on a nanoElute® (Bruker, Bremen, Germany) HPLC equipped with an IonOpticks Aurora column (25 cm x 75 µm ID, 1.6 µm C18) at a temperature of 40°C with a flow rate of 400 nL / min coupled to a timsTOF Pro instrument (Bruker). Solvent A was 0.1 % FA and solvent B 0.1 % FA in acetonitrile. Peptides were eluted with a gradient from 2 % to 15 % solvent B (0-60 min), 15-24 % solvent B (60-90 min), 24 %-34 % solvent B (90-105 min), 34-95 % solvent B (105-107 min). The timsTOF Pro instrument was equipped with a CaptiveSpray nano electrospray ion source (Bruker) and was operated in Parallel Accumulation and Serial Fragmentation (PASEF) mode using the standard DDA method for proteome analysis (1.1 sec cycle time) recommended by the manufacturer. Raw MS-data were processed with Fragpipe83using a data base with porcine sequences downloaded from Ensembl repository84. Qualitative and quantitative analysis of protein identifications was performed using the statistical language R85and Perseus v1.6.15.086. The R-package gprofiler2 version 0.2.187was used to reference porcine protein identifiers to the corresponding genes (HGNC nomenclature) and for enrichment analysis of GO-Terms (GO:BP) and KEGG-pathways. Electron microscopy WSL, WSL SLA-DMAKOand WSL CIITAKOcells were seeded in 6 well plates at a density of 1.5 x 106cells / well.24 h later one complete plate of each type was infected with ASFV Armenia at a MOI of 5. The virus was allowed to penetrate the cells for 2 h at 37°C. Afterwards the virus suspension was removed and replaced with fresh medium containing 1 % penicillin / streptomycin.16 h after application of the virus, cells were scraped into the medium and transferred into a 50 ml centrifuge tube. The cell suspension was centrifuged for 7 min at 350 xg at 4°C. Cells were washed once with 0.1 M sodium cacodylate buffer pH 7.2, before they were fixed with 2.5 % glutaraldehyde in cacodylate buffer (both SERV Electrophoresis) for at least 2 h at 4°C. Fixed cells were centrifuged again (5 min, 1000 xg, 4°C) and the pellet was embedded in low-melting agarose (Sigma Aldrich). After drying the agarose was cut into small pieces (1 mm3), post fixed in 1 % aqueous OsO4 and stained in 2.5 % uranyl acetate (both SERVA Electrophoresis). After a stepwise dehydration in ethanol the samples were cleared in propylene oxide and infiltrated with Glycid Ether 100 (SERVA Electrophoresis). For polymerization, samples were filled in capsules and incubated for 3 days at 60°C. The point of interest was trimmed, and prepared ultrathin sections were transferred to formvar coated nickel grids (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. Statistical analysis All statistical analyses were conducted by using GraphPad Prism (Version 9.0). Statistical significance of differences in plating efficiency was calculated by ordinary one-way ANOVA followed by Tukey’s multiple comparison test. Statistical significance of differences in plaque size was estimated by Kruskal-Wallis test followed by Dunn’s multiple comparison test. The number of times the measurements were repeated is indicated in each figure legend. A p- value < 0.05 was considered significant and is presented in the figures in form of asterisks (*  <  0.05, **  <  0.01, ***  <  0.001, ****  <  0.0001). Data availability Mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http: / / proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD034242. Results A genome-wide CRISPR / Cas9 knockout screen identified cellular factors of the MHC II path-way relevant for ASFV replication. To identify host genes and their respective protein products that are required for ASFV replication in cultured porcine cells, a genome-wide CRISPR / Cas9 screen was performed with the porcine CRISPR / Cas9 knockout library SsCRISPRko.v1 which was previously described and characterized52. The library encoded 83,381 sgRNAs encompassing 1001 non-targeting 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 vector lentiCRISPRv2, which also provides an expression cassette for Cas9, and a puromycin resistance gene for selection. This library was packed into defective lentivirus particles which were then used to transduce highly passaged wild boar lung (WSL) cells, which support efficient replication of many native or adapted ASFV isolates53,54. To ensure the integration of only one sgRNA gene into the genome of a single cell, a low multiplicity of transduction (MOT) of 0.3 was chosen. Puromycin resistant cells that putatively stably expressed Cas9 and single sgRNAs were expanded over two weeks. At that point, about 6 × 107cells of the total number of approximately 4 × 108cells per experiment were stored as uninfected controls for DNA preparation. The remaining cells were reseeded and infected with genotype IX recombinant ASFV Kenya 1033 ΔCD2v dsRed55,56at a multiplicity of infection (MOI) of 0.3 or 0.5. The fluorescent expression marker facilitated the detection of successful infection. A progressive cytopathic effect (CPE) was detectable from 48 h after infection, and after approx. four to five weeks cell colonies became visible that originated from single surviving cells. These cells were pooled in two subsets. Parts of these pools were saved as ‘survivors 1–1’ and ‘survivors 1–2’ for DNA preparation. The other parts were reseeded and infected again, and after approx. three weeks ‘survivors 2–1’ and ‘survivors 2–2’ could be harvested. Seeding and infection of the cells was repeated four times to ensure exposure of all cells to the virus. The repeated infections were necessary, as it was noticed that ASFV Kenya, although inducing a very pronounced CPE in WSL cells, was not always able to lyse all non- transduced control cells during one round of replication. To further exclude false hits from accidentally surviving cells, the screening procedure was performed not only with two cell subsets, but also in two independent experiments. For each screen the DNA of the control (cells before infection) and of the survivors (cells after the infection) of the two subsets was isolated in parallel and the integrated sgRNA gene regions were amplified in three sequential PCRs with suitable primers for Ion Torrent sequencing. Sequencing data was analyzed using the MAGeCK algorithm software which tests whether the abundance of sgRNA genes differs significantly between treated cells (survivors) and controls, and identifies enriched sgRNA sequences targeting specific genomic loci with the calculation of the robust ranking aggregation (RRA)57. With this analysis the sgRNAs targeting SLA-DMB, LOC100736732, RFXAP, SLA-DMA, LOC106509697, RFXANK, and LOC100624181 were found with lowest RRA scores (i.e. most elevated) among the best ten hits of the positively selected genes in all four subsets of the two screens (Fig.1a, b). For all of these genes more than one specific sgRNA was found elevated in the surviving cell pool. In addition, the gene CCZ1 from the best ten hits of the screens was identified in three subsets, whereas the genes LOC102165390, TMEM30A, and VPS33A were found in one of the screens, and the genes MARCO, LYPD4, and VPS18 were under the first ten hits in only one of the subsets (Fig.1a, b). The protein products of most of the latter genes are involved in endocytosis and / or autophagy pathways, and will be further analyzed in future studies. This study focuses on the role of host genes that were identified in all four subsets with very low scores (SLADMB, LOC100736732, RFXAP, SLA- DMA, LOC106509697, RFXANK, and LOC100624181). Remarkably, all of them are related to the major histocompatibility complex II (MHC II / SLA II) (Fig.1c). RFXANK codes for the regulatory factor X associated ankyrin containing protein (RFXANK), and RFXAP encodes the regulatory factor X associated protein (RFXAP). RFXANK, RFXAP and the regulatory factor X 5 (RFX5) assemble and bind to the X box of the SXY module of MHC II gene promoters (Fig.1c). Together with other factors they act as landing pad for the MHC class II transactivator (CIITA)58. CIITA was identified by elevated sgRNAs against LOC100736732, LOC106509697, and LOC100624181. RFXAP, RFXANK and CIITA are highly important for transcriptional activity of MHC class II promoters58. Lastly, the genes SLA-DMA and SLA- DMB were identified in both screens which code for the alpha and beta chains of the non- classical class II swine leucocyte antigen DM (SLA-DM), and are transcribed with the help of the above-mentioned factors. In summary, several lines of evidence point to the MHC II pathway as highly relevant host factor for ASFV replication. Generation of targeted SLA-DMA, SLA-DMB, RFXAP and CIITA knockout cell lines. To verify the importance of the MHC II expression and presentation pathway for the replication cycle of ASFV, targeted gene knockouts were introduced into a newly isolated single cell clone of the WSL cell line. A clone of WSL cells was used to minimize the risk that results might be affected by inherent genetic differences. For the targeted knockout, the genes encoding the non-classical MHC II molecule SLA-DM, SLA-DMA and SLA-DMB, as well as two genes that are important for the transcription of MHC II, RFXAP and CIITA (LOC100736732), were selected. For the knockout, one of the four pig library sgRNA sequences was selected, and cloned into the sgRNA and Cas9 double expression vector pX330A-1 × 4neoRA. WSL cells were transfected with the obtained plasmids, serially diluted and selected for G418 resistance. Resistant single cell clones were propagated and checked for Cas9 expression by immunoblotting. DNA of Cas9 positive cells was then isolated and correct sgRNA gene integration was verified by PCR amplification and sequencing with suitable primers. Furthermore, PCR analyses with primers specific for the targeted gene regions of SLA-DMA, SLA-DMB, RFXAP, and CIITA were performed. After sequence analysis of the amplification products, WSL knockout (WSLKO) cell clones with deleterious 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. The cell clone WSL SLA-DMAKO (11) displayed an 823 nt insertion containing stop codons in all reading frames, which is derived from the transfer vector pX330A-1×4neoRA. Clones WSL SLA-DMAKO (12) and (16), as well as WSL SLA-DMBKO (9) possess deletions of 1 nt leading to frameshifts and premature stop codons. In contrast, WSL SLA-DMBKO(16) and (18) exhibit an identical insertion of 1 nt (T) which directly creates a stop codon (TGA). The cell clones WSL CIITAKO (1), (4), and (8) displayed deletions of 1, 5, and 23 nucleotides, respectively, leading to frameshifts and premature termination. WSL RFXAPKO (6) exhibited an insertion of 1 nt (C), leading to a downstream termination codon (TGA), and in WSL RFXAPKO(8) a stop codon-containing insertion of 140 nt from the vector pX330A-1×4neoRA was found. Remarkably, with all selected cell clones only single PCR products exhibiting unambiguous sequences of the mutated genes were obtained, indicating either identical biallelic changes or large deletions in the other alleles, which included the primer binding sites. Wildtype sequences of the sgRNA target regions were never observed. Previous studies indicated that WSL cells express the MHC II protein SLA-DR at their surface54. This was verified by indirect immunofluorescence (IF) analyses of non- permeabilized cells using an SLA-DR specific monoclonal antibody (mAb) (Fig.2a). Whereas SLA-DR was detectable on the surface of parental WSL cells and the knockout cells WSL SLA-DMAKO and WSL SLA-DMBKO, it was not visible on RFXAP and CIITA knockout cells (Fig.2a). This confirmed that RFXAP and CIITA are essential factors for MHC II transcription. For the characterization of the knockouts on proteome level the protein contents of the knockout cell lines were analyzed by mass spectrometry (MS) (Fig.2b, c). Of the 5495 proteins that were identified and quantified, 4874 were detected in all cell clones. The protein composition of all cell clones was very similar, as indicated by the lack of a clear clustering of the replicates after principal component analysis (PCA). Unfortunately, SLA-DM expression was not detectable in the MS analyses, neither in knockout nor in normal WSL cells. However, single knockout of RFXAP or CIITA suppressed the synthesis of other identified MHC II proteins (SLA-DR, SLA-DQ) below the detection levels, and also affected the expression of MHC I (SLA-8, HLA-E), while the expression of these proteins remained unaffected in WSL SLA-DMAKO and WSL SLA-DMBKO cells (Fig.2b, c). SLA-DM, RFXAP and CIITA are required for efficient ASFV replication in WSL cells. Before testing whether ASFV is able to replicate in cells lacking molecules of the MHC II pathway, unspecific side effects of these knockouts on viability and susceptibility to other virus infections should be excluded. For this purpose, parental WSL, WSL SLA-DMAKO (clones 11, 12, 16), WSL SLA-DMBKO(clones 9, 16, 18), WSL CIITAKO(clones 1, 4, 8), and WSL RFXAPKO(clones 6, 8) cells were infected with a GFP-expressing mutant of the porcine alphaherpesvirus pseudorabies virus (PrV). The studies revealed that neither plating effciency, nor plaque sizes, or progeny virus titers of PrV differed significantly between parental WSL cells and the tested knockout cell lines. This demonstrated that the knockout cells are per se suitable for propagation of a porcine virus. For ASFV infection of the cells two different virus strains were used. Besides the parental genotype IX strain of the virus recombinant used for library screening (ASFV Kenya 1033), a variant of the current panzootic genotype II virus was also included (ASFV Armenia 2008). Parental WSL and knockout cell clones were infected with serial dilutions of both viruses. After 4 days under semi-solid medium the cells were fixed and infected cells and virus plaques were visualized by IF detection of the ASFV capsid protein p72. Fluorescence microscopy revealed that both strains were able to infect considerably more parental WSL cells than any of the knockout cells (Fig.3a). The calculated plating efficiencies on all tested WSLKO cells were significantly reduced to < 4% compared to WSL cells (Fig.3b). In addition, plaque areas were significantly reduced to less than 14% for ASFV Armenia and less than 10% for ASFV Kenya in WSLKO cells compared to plaque sizes of the respective virus on the parental cell line (Fig.3c). A serious impairment of ASFV replication was also seen in multistep (MOI 0.02) growth studies. Whereas ASFV Armenia replicated in WSL cells to maximum titers of 1.8 × 107PFU / ml at 168 h post infection (p.i.), the titers in knockout cells ranged from 1.2 × 104PFU / ml in WSL SLA-DMBKO(18) to 3.8 × 104PFU / ml in WSL RFXAPKO(8) (Fig.3d). The final titer of ASFV Kenya in WSL cells was 2.0 × 107PFU / ml, whereas in knockout cells the titers ranged from 1.2 × 105PFU / ml in WSL SLA-DMAKO (16) to 1.23 × 106PFU / ml in WSL RFXAPKO (8) cells (Fig.3d). Thus, the titers for ASFV Armenia were decreased by 3 logs, and the titers of ASFV Kenya were reduced by at least 1.5 logs on WSL knockout compared to parental cells. Moreover, it was apparent that productive ASFV Armenia replication in knockout cells reached a plateau at 72 h p.i., while the titers of ASFV Kenya increased until 120 h p.i. This might indicate that the few knockout cells successfully infected with ASFV Kenya are able to produce more infectious virus for a longer period of time than ASFV Armenia infected cells, which would be in line with slightly higher titers of ASFV Kenya observed on normal WSL cells. In addition to the analysis of infectious virus progeny in infected cell lysates, viral DNA replication was also investigated (Fig.4). To this end, parental WSL cells and selected WSLKO cell clones were infected with ASFV Armenia at a MOI of 3 and harvested at 0, 2, 4, 8, 16 and 32 h p.i. Total DNA was prepared and used to determine ASFV genome copy numbers by duplex TaqMan qPCR reactions for the detection of the viral B646L gene and the host cell β-actin gene as an internal control (Fig.4). B646L specific probes showed moderately increased DNA amounts after 4 h, and an exponential replication phase until 8 h p.i. in parental WSL cells. At later times, viral DNA replication slowed and resulted in 2.8 × 107genome copies in the analyzed samples (containing DNA of approx.1 × 104cells) at 32 h p.i. A delayed onset of DNA replication was seen in all WSLKO cell clones resulting in a moderate increase of genome copy numbers between 8 h p.i. and 16 h p.i. From 16 h p.i. until the end of the experiment the viral DNA amount remained almost constant and ranged from 1.2 × 105ASFV genomes in SLADMBKO (9) cells to 6.8 × 105ASFV genomes in RFXAPKO(8) cells. Since the ASFV genome copy numbers were considerably lower in all tested WSLKO cells at all analyzed time points the deleted host proteins are obviously important for a step preceding the onset of viral genome replication. To further elucidate which steps of virus replication cycle might be blocked after knockout of the MHC II related genes, WSL, WSL SLA-DMAKO(11), and WSL CIITAKO(1) cells were analyzed by electron microscopy (EM) 16 h after infection with ASFV Armenia at a MOI of 5 (Fig.5). Mature extracellular virus particles, intracellular particles and virus factories were only detected in parental WSL cells, whereas no traces of ASFV replication were found in either of the knockout cells. These results also indicate a function of the knocked-out host cell proteins in initial steps of virus replication, which is in line with the substantially reduced plating effciency and genome copy number of ASFV in WSLKOcells. Susceptibility to ASFV infection can be restored by reintroduction of SLA-DM. To test whether the observed inhibition of ASFV replication in SLA-DM knockout cells is indeed due to the absence of the respective proteins, WSL SLA-DMAKO(11) and SLA-DMBKO(9) cells were stably transformed with SLA- DMA or SLA-DMB expression cassettes, respectively. The nucleotide sequences of the open reading frames were codon-optimized and modified in the sgRNA target regions by the introduction of silent nucleotide alterations to exclude inactivation of the transgenes by the still integrated CRISPR / Cas9 machinery of the knockout cells. The synthetic genes were cloned with and without tags (StrepII, Myc) into lentivirus expression vectors resulting in pLV-SLA-DMA, pLV-SLA-DMA-Myc, pLV-SLA-DMA-Strep, pLV-SLA-DMB, and pLV-SLA- DMB-Myc, and a GFP expression construct (pLV-GFP) serving as control. Parental WSL cells were transduced with all generated vectors; SLA-DMAKO (11) cells were transduced either with pLV-SLA-DMA, pLV-SLA-DMA-Myc, pLV-SLA-DMA-Strep or pLV-GFP, and SLA- DMBKO(9) cells with pLV-SLA-DMB, pLVSLA-DMB-Myc or pLV-GFP and stably transformed cells were selected using puromycin containing medium. Unlike in parental WSL and knockout cells, tagged proteins of the expected sizes were detectable by western blotting in the WSL knockout / knockin (WSLKO / KI) cells (Fig.6). Unfortunately, the untagged SLA-DMA and -DMB proteins were not recognized by the available antibodies raised against human leucocyte antigen (HLA)-DMA and HLA-DMB. Nevertheless, these cell lines were included in the analysis of ASFV infection, since the C-terminally added tags might impair maturation, or heterodimeric complex formation between the transgeneencoded and the unaffected endogenous alpha or beta chains of SLA-DM. In transduced SLA-DMAKO(11) cells plating effciency of ASFV Armenia and Kenya increased to about 77% and 86%, respectively, of the titers in parental WSL cells after reintroduction of authentic SLA-DMA (SLADMAKO-DMAKI) (Fig.7a). In SLA-DMAKO-DMA- MycKIcells 42% (Armenia) and 50% (Kenya), and in SLADMAKO-DMA-StrepKIcells 79% (Armenia) and 90% (Kenya) of the original titers were achieved. In WSL SLADMBKO-DMBKI cells plating efficiencies of 77% (Armenia) and 94% (Kenya), and in SLA-DMBKO-DMB-MycKIcells plating efficiencies of 63% (Armenia) and 56% (Kenya) were observed (Fig.7a). In most cases, the apparent virus titers on different WSLKO / KI cells were not significantly lower than on parental WSL cells (Fig.7a). Thus, the added tags obviously did not (StrepII) or only marginally affect (Myc) the proposed function of SLA-DM for ASFV replication. Stable transduction of WSL SLA-DMAKO and SLA-DMBKO cells with expression cassettes for the corresponding tagged or untagged proteins also restored cell-to-cell spread of ASFV Armenia and ASFV Kenya. In all cases, plaques on different WSLKO / KIcells exhibited similar or even larger sizes than detected on parental WSL cells (Fig.7b). In line with this, growth kinetic studies revealed that on WSLKO / KI cells the progeny virus titers of ASFV Armenia and Kenya were increased by approx.2.5 and 1.5 log, respectively, when compared to those on the parental cells (Fig.7c–e), and final virus titers were again similar to those on original WSL cells. Taken together these results show that the deleterious effects of SLA-DMA / B knockout on susceptibility to ASFV infection can be fully reverted by the expression of corresponding transgenes, demonstrating a crucial specific role of SLADM in ASFV replication. Discussion As an intracellular viral pathogen ASFV, in spite of its complex genome and proteome, still requires many host factors for propagation. Using a genome-wide CRISPR / Cas9 knockout screen in a susceptible porcine cell line (WSL) we identified the genes RFXAP, RFXANK, SLA-DMA, SLA-DMB, and genes encoding CIITA (LOC100736732, LOC106509697, and LOC100624181) as top candidates important for ASFV replication. The proteins encoded by these genes are part of the MHC II (SLA II) expression and presentation pathway58. Targeted inactivation of RFXAP, CIITA, SLA-DMA and SLA-DMB substantially inhibited replication of genotype II and IX ASFV strains, including the current panzootic virus genotype, which confirmed the importance of certain MHC II molecules for ASFV replication in vitro. Moreover, reconstitution of SLA-DMA or SLA-DMB in corresponding knockout cells fully restored their ASFV replication capacity, and points to a crucial specific role of the MHC II molecule SLA-DM during the viral life cycle. The MHC II complex is highly conserved in vertebrates and designated as SLA II in swine and HLA II in humans. Pigs possess four MHC II molecules, SLA-DR, SLA-DQ, SLA-DO and SLA-DM59. SLA-DR and –DQ are classical MHC II transmembrane cell-surface glycoproteins that present exogenous peptides to the antigen receptor of CD4 + helper T cells, whereas SLA-DM and -DO are termed non-classical MHC II molecules. In humans, they are involved in regulation of antigenic peptide loading onto the classical MHC class II molecules. All MHC II proteins are heterodimers that are composed of α and β chains, and in classical MHC II proteins the a1 and b1 domains form the peptide-binding groove. For the human synthesis and presentation pathway it has been described60that the classical MHC II α and β subunits are synthesized in the ER, where they assemble with the specific chaperone CD74 / invariant chain (li). The li promotes MHC II folding, prevents premature peptide binding and sorts the MHC II molecules to late endosomal compartments either directly from the trans-Golgi network or by reinternalization from the cell surface. The acidic environment of the endosomes mostly degrades li, but leaves a specific li fragment, the class II invariant chain associated peptide (CLIP), within the MHC II binding groove. The release of CLIP is induced by the non-classical MHC II molecule DM through the promotion of a conformational change, whereby classical MHC II proteins are loaded with higher affnity antigenic peptides. Those peptides result from antigens that were internalized by clathrin- mediated endocytosis, phagocytosis, or micropinocytosis. ASFV enters its host cells via clathrin-mediated endocytosis or macropinocytosis. In this study, SLA-DMA and SLA-DMB knockout cells displayed a severe defect in susceptibility to ASFV infection and subsequent DNA and virus replication. This effect could be reversed by the reintroduction of the respective SLA-DM subunits, indicating that SLA-DM might be important for ASFV entry, e.g. for efficient uncoating and the release of core particles from the late endosomes. The absence of viral factories and detectable amounts of progeny virus particles in electron microscopy analyses of WSLKOcells, as well as the substantially reduced plating efficiencies and genome copy numbers of ASFV in WSLKOversus parental wildtype cells strongly indicates that infection of the MHC II-deleted cells is blocked very early. In the genome-wide CRISPR / Cas9 knockout screens of WSL cells resistant to ASFV infection, sgRNAs targeting RFXAP, RFXANK, LOC100736732, LOC106509697, and LOC100624181 were also significantly elevated. These genes code for RFXAP, RFXANK, and CIITA (LOC100736732, LOC106509697, LOC100624181). Together with RFX5 these molecules are involved in the expression of all MHC II genes61–65. In CIITAKO or RFXAPKO cells, ASFV infection was inhibited to a similar extent as in knockout cells lacking SLA-DMA or SLA-DMB. Unlike in SLA-DMKO cells, MHC II expression was generally inhibited in cells lacking functional CIITA or RFXAP genes. This was shown by IF and MS analyses which did not detect the classical MHC II molecules SLA-DR and SLA-DQ, although they are present in parental WSL cells. Since CIITA and RFXAP are general regulators of MHC II expression58it is likely that corresponding knockout cells also lack the non-classical MHC II molecules SLA-DO and SLA-DM which could not be detected or quantified with available tools. Although it cannot be excluded that the regulatory proteins CIITA or RFXAP, or SLA- DR, SLA-DQ and SLA-DO are also directly involved in ASFV infection or replication, it appears more likely that SLA-DM is the only relevant MHC II molecule. This is underpinned by the fact that no sgRNA hits targeting SLA-DR, SLA-DQ or SLA-DO were identified in the performed CRISPR / Cas9 knockout screen. Moreover, flow cytometric analysis of susceptible cells indicated that expression of the classical MHC II molecule SLA-DQ is not essential for ASFV infection66. The present results indicate that ASFV-induced downregulation of SLA-DM might also prevent reinfection of host cells and, thereby, enhance the efficiency of virus propagation. 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Claims

Claims 1. A genetically modified or transgenic animal lacking a functional copy of any one or more of the SLA-DMA, SLA-DMB, RFXANK, RFXAP and CIITA genes and / or proteins encoded thereby.

2. The genetically modified or transgenic animal of claim 1, wherein the animal is a swine animal, a porcine animal, a domestic pig or Sus scrofa.

3. The genetically modified or transgenic animal of claim 1 or 2, wherein the SLA-DMA gene and / or the SLA-DMA protein has / have been rendered non-functional or fully knocked out.

4. The genetically modified or transgenic animal of claim 1 or 2, wherein the SLA-DMB gene or the SLA-DMB protein has been rendered non-functional or fully knocked out.

5. A compound which modulates the function, activity and / or expression of a swine leucocyte antigen complex II (SLA II) gene and / or a SLA II protein, for use in medicine, for use as a medicament or for use in treating or preventing ASF or an ASFV infection.

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

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

8. A compound for use of any one of claims 5-7, 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 of any one of claims 5-8, wherein the compound (i) blocks or neutralises the ability of the ASFV to interact with or bind to a SLA II protein; and / or(ii) binds to an SLA II protein to prevents or inhibit ASFV from binding thereto; and / or (iii) modulates (for example reduces, suppresses, inhibits or prevents) expression of one or more of the SLA II genes described herein; and / or (iv) inhibits the function or activity of any of the SLA II proteins; and / or (v) prevents a proviral interaction between an SLA protein encoded by an SLA II gene.

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

11. The compound for use of any one of claims 5-10, wherein the compound is an anti- SLA-DM, anti-SLA-DMA an anti-SLA-DMB antibody or an antigen binding-or neutralising fragment thereof.

12. The compound for use of any one of claims 5-11, wherein the compound is an antisense oligonucleotide which modulates the expression of one or more SLA II genes.

13. The compound for use of any one of claims 5-12, wherein the compound comprises a fragment of an SLA II protein.

14. The compound of claim 13, wherein the compound comprises a fragment of any of the sequences provided as SEQ ID NOS: 9-12.

15. The antibody of claim 10 or 11, wherein the antibody neutralises any proviral interaction between ASFV and a SLA II protein.

16. The antibody of any one of claims 10, 11 or 15, wherein the antibody is an anti- SLA- anti-DMA, anti-SLA-DMB, anti-RFXANK, anti-RFXAP and anti-CIITA antibody.

17. A nucleic acid encoding the antibodies of claims 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 a nucleic acid of any one of claims 17-19.

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

22. The nucleic acid or vector of claims 17-20, for use in medicine, for use as a medicament or for use in treating or preventing ASF or an ASFV infection.

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