Patent Description:
African swine fever is a devastating haemorrhagic disease of domestic pigs caused by a double-stranded DNA virus, African swine fever virus (ASFV). ASFV is the only member of the Asfarviridae family and replicates predominantly in the cytoplasm of cells. Virulent strains of ASFV can kill domestic pigs within about <NUM>-<NUM> days of infection with a mortality rate approaching <NUM>%.

ASFV can infect and replicate in warthogs (Phacochoerus sp. ), bushpigs (Potamocherus sp. ) and soft ticks of the Ornithodoros species, but in these species few if any clinical signs are observed and long term persistent infections can be established. The disease is currently endemic in many sub-Saharan countries and in Europe in Sardinia. Following its introduction to Georgia in the Trans Caucasus region in <NUM>, ASFV has spread extensively through neighbouring countries including the Russian Federation. In <NUM> the first outbreak was reported in Ukraine and in <NUM> the first outbreaks in Belarus. In <NUM> further outbreaks were reported in pigs in Ukraine and detection in wild boar in Lithuania and Poland.

There is currently no treatment for ASF. Prevention in countries outside Africa has been attempted on a national basis by restrictions on incoming pigs and pork products, compulsory boiling of waste animal products under licence before feeding to pigs and the application of a slaughter policy when the disease is diagnosed.

Prevention in Africa is based on measures to keep warthogs and materials contaminated by warthogs away from the herd.

To date, no effective attenuated or inactivated vaccines have been developed (see http://www. thepigsite. com/pighealth/article/<NUM>/african-swine-fever-asf).

There is thus a need for improved measures to control ASFV infection and prevent spread of the disease.

The ASFV genome encodes five multigene families (MGF <NUM>, MGF <NUM>, MGF <NUM> and MGF <NUM>/<NUM>) located within the left hand 35kb or right hand 15kb terminal variable regions. The MGFs constitute between <NUM> and <NUM>% of the total coding capacity of the ASFV genome. They lack similarity to other known genes. Although the function of individual MGF genes is unknown, it has been shown the MGF <NUM> and <NUM> families encode genes essential for host range function that involves promotion of infected-cell survival and suppression of type I interferon response.

OURT88/<NUM> is a non-pathogenic isolate of ASFV from Portugal. Previous infection with ASFV OURT88/<NUM> has been shown to confer protection against challenge with related virulent viruses (<NPL>; <NPL>).

It has been demonstrated that CD8+ T cells are required for the protection induced by the OURT88/<NUM> strain, since antibody mediated depletion of CD8+ T cells abrogates protection (Oura et al. , <NUM>, as above).

Studies were carried out using the NIH inbred pig lines cc and dd. In these studies, a control group of <NUM> dd pigs and a control group of <NUM> dd pigs were immunised with OURT88/<NUM>. In the first of these experiments, following OURT88/<NUM> immunisation one dd pig developed a transient low viremia of log10 <NUM>-<NUM> TCID <NUM>/ml, but no fever or clinical signs of disease. It was observed that <NUM> of <NUM> of the cc inbred pigs immunised with OURT88/<NUM> were not protected following lethal challenge with OURT88/<NUM> isolate, whereas protective responses were induced in all dd pigs. These results indicate that the genetic background of the pig influences the response to OURT88/<NUM> innoculation.

In subsequent experiments <NUM> dd and <NUM> cc pigs were immunised with OURT88/<NUM> and challenged with OURT88/<NUM> (Takamatsu et al. , <NUM> unpublished results). This confirmed that protective responses were induced in all dd pigs, but not induced in all cc pigs following immunisation with OURT88/<NUM> and that adverse reactions including transient pyrexia, joint swelling and lameness were induced in some of the cc pigs.

Subsequent experiments were carried out in France using the Anses herd of SPF pigs. In these pigs, similar to observations with cc pigs, some pigs developed adverse reactions including transient fever, joint swelling and lameness following immunisation with OURT88/<NUM> (unpublished results).

Although OURT88/<NUM> has been shown to induce a protective immune response in certain animals, this effect does not appear to be universal. Immunisation with OURT88/<NUM> appears to be ineffective in protecting some pigs from subsequent challenge. It is also associated with the induction of adverse immune responses, such as joint swelling, in some pigs.

There is therefore a need for alternative ASFV vaccine candidates with improved efficacy and safety profiles.

<NPL>) demonstrated that pigs immunised with natural low virulence isolates or attenuated viruses produced by passage in tissue culture and by targeted gene deletions can be protected against challenge with virulent viruses.

<NPL>) showed that experimental immunisation of pigs with the non-virulent OURT88/<NUM> genotype I isolate from Portugal followed by the closely related virulent OURT88/<NUM> genotype I isolate could confer protection against challenge with virulent isolates from Africa including the genotype I Benin <NUM>/<NUM> isolate and genotype X Uganda <NUM> isolate.

<NPL>) disclosed that the genomic coding sequences, apart from the inverted terminal repeats and cross-links, have been determined for two African swine fever virus (ASFV) isolates from the same virus genotype, a non-pathogenic isolate from Portugal, OURT88/<NUM>, and a highly pathogenic isolate from West Africa, Benin <NUM>/<NUM>.

<CIT> disclosed a live attenuated antigenically marked classical swine fever vaccine.

<NPL>) analyzed the generation of cell-mediated and humoral immune responses in d/d histocompatible pigs following CSFV infection or vaccination.

The inventors have surprisingly found that deletion of only one gene, namely the DP148R gene, from a region close to the right end of the virulent Benin97/<NUM> genome, resulted in attenuation of a virulent virus and does not reduce virus replication in macrophages. A group of <NUM> pigs immunised with BeninΔDP148R intramuscularly with <NUM><NUM> HAD<NUM> units developed a transient fever and loss of appetite for <NUM> or <NUM> days at day <NUM> post-immunisation. No further clinical signs were observed following boost <NUM> days later or at challenge <NUM> days after the first immunisation. All <NUM> pigs survived challenge.

The invention provides a method of attenuating an ASF virus, which comprises the step of partially or completely deleting, or interrupting the expression of, the DP148R gene, wherein the gene is interrupted such that the gene is not transcribed and/or translated, or wherein the deletion removes at least <NUM>% of the coding portion of the gene.

The method may comprise transfecting virus-infected cells with a transfer vector in which the DP148R gene is missing or interrupted, such that homologous recombination occurs, and selecting recombinant viruses expressing the new portion of sequence.

The present inventors have also surprisingly found that deletion of five multi-gene family (MGF) <NUM> genes <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and three MGF <NUM> genes 1R, 2R, 3R from the left hand end of the ASF virus genome and interruption of two additional genes (MGF360 <NUM> and MGF <NUM>4R) resulted in attenuation of a virulent virus and induction of <NUM>% protection against challenge with parental ASFV virulent virus.

Described herein is an attenuated African Swine Fever (ASF) virus which lacks a functional version of the following genes:.

The following genes may be at least partially (i.e. partially or completely) deleted:.

Described herein is an attenuated African Swine Fever (ASF) virus which lacks a functional version of the DP148R gene. The gene may be partially or completely deleted, or interrupted.

The DP148R mutation may also be made in combination with mutation of the multigene-family <NUM> genes <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> and multigene-family <NUM> genes 1R, 2R, 3R and 4R as described herein. Described herein is an attenuated African Swine Fever (ASF) virus which lacks a functional version of the following genes:.

The virus may be derivable from a virulent ASFV virus isolate. In other words, the genome of the attenuated virus described herein (other than the genes MGF <NUM> genes <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>, and MGF <NUM> genes 1R, 2R, 3R and 4R;and/or DP148R) may correspond to the genome of a virulent ASFV virus isolate. The attenuated virus described herein may be made by deleting/interrupting the genes: MGF <NUM> genes <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> and MGF <NUM> genes 1R, 2R, 3R and 4R; and/or DP148R; from a virulent ASFV virus isolate.

The virus may be derivable from one of the following virulent ASFV virus isolates: Georgia <NUM>/<NUM>, Benin <NUM>/<NUM>, Kenyan, Malawi LiI20/<NUM>, Pretorisuskop/<NUM>/<NUM> and Tengani <NUM>.

The virus may be derivable from Benin <NUM>/<NUM>.

The attenuated ASF virus, when administered to a subject, may induce an immune response which is protective against subsequent challenge with virulent ASF virus.

The attenuated ASF virus, when administered to a subject, may induce a reduced T cell mediated immune response compared to the immune response induced by attenuated virus OURT88/<NUM>. The immune response may involve a lower number of CD8+ positive γδT cells.

Described herein is a vaccine comprising an attenuated ASF virus.

The vaccine may comprise a plurality of attenuated ASF viruses of different genotypes.

Described herein is a vaccine for use in treating and/or preventing African Swine Fever.

The vaccine may induce a cross-protective immune response against a plurality of ASF virus genotypes.

Described herein is a method of attenuating an African Swine Fever (ASF) virus, which comprises the step of partially or completely deleting, or interrupting the expression of, the following genes:.

The following genes may be partially or completely deleted:.

Described herein is a method for treating and/or preventing African Swine Fever in a subject which comprises the step of administering to the subject an effective amount of a vaccine.

The subject may, for example, be a domestic pig.

The vaccine may be administered following a prime-boost regime.

The engineered virus BeninΔMGF described herein has several advantages over OURT88/<NUM>, for example:.

The engineered virus BeninΔDP148R described herein also has several advantages over OURT88/<NUM>, for example:.

The present inventors have furthermore surprisingly found that intranasal administration of an attenuated African Swine Fever (ASF) virus results in improved protection and fewer side effects compared to administration via the intramuscular route, and may require a lower dose to obtain protection.

Described herein is an attenuated ASF virus for use in the treatment and/or prevention of African Swine Fever, wherein the attenuated virus is administered intranasally.

Described herein is a method for treating and/or preventing African Swine Fever in a subject which comprises the step of administering an effective amount of an attenuated ASF virus to the subject by the intranasal route.

Described herein is a vaccine comprising an attenuated ASF virus, wherein the vaccine is formulated for intranasal administration.

Described herein is a kit for delivery of an intranasal vaccine formulation comprising:.

Described herein is an intranasal delivery device comprising an attenuated ASF virus vaccine.

The attenuated ASF virus may be any attenuated ASF virus, such as OURT88/<NUM>, BeninΔMGF or BeninΔDP148R.

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF). The virus causes a haemorrhagic fever with high mortality rates in pigs, but persistently infects its natural hosts, warthogs, bushpigs with no disease signs. It also infects soft ticks of the Ornithodoros genus, which are thought to be used as a vector.

ASFV replicates in the cytoplasm of infected cells, and is the only member of the Asfarviridae family. ASFV is endemic to sub-Saharan Africa and exists in the wild through a cycle of infection between ticks and wild pigs, bushpigs and warthogs. ASFV was first described after European settlers brought pigs into areas endemic with ASFV and, as such, is an example of an 'emerging infection'.

ASFV is a large, icosahedral, double-stranded DNA virus with a linear genome containing at least <NUM> genes. The number of genes differs slightly between different isolates of the virus. ASFV has similarities to the other large DNA viruses, e.g., poxvirus, iridovirus and mimivirus. In common with other viral haemorrhagic fevers, the main target cells for replication are those of monocyte, macrophage lineage.

Based on sequence variation in the C-terminal region of the B646L gene encoding the major capsid protein p72, <NUM> ASFV genotypes (I-XXII) have been identified. All ASFV p72 genotypes have been circulating in eastern and southern Africa. Genotype I has been circulating in Europe, South America, the Caribbean and western Africa. Genotype VIII is confined to four East African countries.

Examples of strains from some of the genotypes are given below:.

ASFV contains five multi-gene families which are present in the left and right variable regions of the genome. The MGFs are named after the average number of codons present in each gene: MGF100, <NUM>, <NUM>, <NUM> and <NUM>/<NUM>. The N-terminal regions of members of MGFs <NUM>, <NUM> and <NUM>/<NUM> share significant similarity with each other. Several genes in MGF360 and <NUM>/<NUM> determine host range and virulence.

The five multigene families of the ASFV genome (MGF <NUM>, MGF <NUM>, MGF <NUM> and MGF <NUM>/<NUM>) are located within the left hand 35kb or right hand 15kb terminal variable regions. The MGFs constititute between <NUM> and <NUM>% of the total coding capacity of the ASFV genome. They lack similarity to other known genes.

The complete genome sequences of isolate Benin <NUM>/<NUM> (a highly pathogenic virus from West Africa, Group1), isolate OURT88/<NUM> (non-pathogenic, attenuated virus from Portugal, Group <NUM>) and isolate BA71V (Vero cell tissue culture adapted non-pathogenic virus, Group <NUM>) have been compared (<NPL>). The isolate OURT88/<NUM> has a deletion of <NUM> bp compared to Benin <NUM>/<NUM> extending from nucleotide number (nt no. ) <NUM> to nt no. <NUM> in the Benin <NUM>/<NUM> genome. In all, five MGF <NUM> genes (<NUM>, <NUM>, <NUM>, <NUM> and <NUM>) are deleted from the OURT88/<NUM> genome. Two MGF <NUM> genes (1R, 2R) are deleted and the MGF 505R 3R gene is truncated in the OURT88/<NUM> genome. These genes are present in the genomes of all eight other pathogenic isolates of ASFV that have been sequenced.

In the attenuated tissue-culture adapted BA71V isolate, the genome contains the MGF <NUM>3R gene but lacks the other seven MGF genes and in addition also has the MGF <NUM><NUM> gene truncated (total deletion of <NUM> bp).

Previously six MGF <NUM> genes (<NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM>) and two MGF <NUM> genes (1R and 2R) were deleted from the highly pathogenic South African isolate Pr4. This deletion markedly reduced viral growth in primary macrophage cell cultures by <NUM>- to <NUM>- fold (Zsak et al <NUM>, as above) and led to attenuation of the virus (cited as unpublished results, <NPL>). However no experiments were carried out to challenge the recovered pigs to determine if they were protected. In fact, at an African swine fever virus workshop at the Biosecurity Research Institute, Manhattan Kansas in May <NUM> it was mentioned that the Pr4 deletion mutant was not protective and induced a chronic form of the disease.

It has also been shown that deletion of three MGF <NUM> genes (<NUM>, <NUM> and <NUM>) and four MGF <NUM> genes (1R, 2R, 3R and 4R truncation) from the pathogenic virus MalawiΔDP71 reduces virus replication in pigs and in addition attenuated the virus (<NPL>). However again, no experiments were reported to determine if the recovered pigs were protected against challenge.

The attenuated ASFV described herein may lack a functional version of the following genes:.

The location of these genes in the genomes of a variety of ASFV strains is provided below (Table <NUM>). The sequence identity of each gene to the corresponding Benin <NUM>/<NUM> gene is also provided.

The translation products of these genes are given below:.

The complete genome for the African swine fever virus Benin <NUM>/<NUM> pathogenic isolate is given in Genbank Locus: AM712239.

From the study described by Chapman et al (<NUM> - as above) it was determined that the complete BA71 isolate genome encodes <NUM> open reading frames (ORFs), the Benin <NUM>/<NUM> isolate encodes <NUM> ORFs and the OURT88/<NUM> isolate encodes <NUM> ORFs.

Described herein is an attenuated ASFV which lacks a functional version of gene DP148R.

The DP148R mutation may be made on its own, or in combination with mutations of the multigene-family <NUM> genes <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> and multigene-family <NUM> genes 1R, 2R, 3R and 4R as described herein. This combination of gene mutations may result in a better safety profile for the attenuated virus.

DP148R is located at genome position <NUM> to <NUM> on the Benin <NUM>/<NUM> genome. The protein sequence is:
Benin <NUM>/<NUM><NUM>-<NUM> - SEQ ID No. <NUM>
<IMG>.

Orthologous DP148R sequences from other genomes share between <NUM> and <NUM>% amino acid identity. The orthologous DP148R genes from other ASFV isolates are located in positions:.

The attenuated ASF virus described herein may be derivable from a wild-type ASF virus isolate, but includes mutations in its genome such that the following genes are completely or partially deleted or otherwise made non-functional: MGF <NUM> genes <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> and MGF <NUM> genes 1R, 2R, 3R and 4R; and/or DP148R gene.

The term "wild-type" indicates that the virus existed (at some point) in the field, and was isolated from a natural host, such as a domestic pig, tick or wart hog. Table <NUM> below lists known ASF virus isolates.

The genome structure of ASFVs is known in the art, as detailed in <NPL>.

The term "corresponds to" means that the remainder of the genome is the same, or substantially the same, as the wild type strain. Thus the genome of the attenuated virus described herein may include the genes of the wild type strain, other than MGF <NUM> genes <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> and MGF <NUM> genes 1R, 2R, 3R and 4R; and/or DP148R gene.

The genome of the attenuated virus may comprise the ORFs conserved in all <NUM> genome sequences available and completely or partially deleted or otherwise made non-functional: MGF <NUM> genes <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> and MGF <NUM> genes 1R, 2R, 3R and 4R; and/or DP148R gene.

The genome of the attenuated recombinant ASF virus described herein may correspond to that of a virulent ASF virus strain (with the exception of the MGF <NUM> genes <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> and MGF <NUM> genes 1R, 2R, 3R and 4R; and/or DP148R gene).

African swine fever virus isolates described to date are summarised in Table <NUM>, together with their Genbank Accession numbers.

Known virulent ASF virus strains include: Georgia <NUM>/<NUM>, Benin <NUM>/<NUM>, Kenyan, Malawi Lil20/<NUM>, Pretorisuskop/<NUM>/<NUM> and Tengani <NUM>.

The genome of the attenuated recombinant ASF virus described herein (with the exception of the MGF <NUM> genes <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> and MGF <NUM> genes 1R, 2R, 3R and 4R; and/or DP148R gene) may correspond to that of the Benin <NUM>/<NUM> isolate.

The genome of the attenuated recombinant ASF virus described herein (with the exception of the MGF <NUM> genes <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> and MGF <NUM> genes 1R, 2R, 3R and 4R, and/or DP148R) may correspond to that of an ASF virus strain whose virulence is currently unknown, for example: Mkuzi, Warmbaths and Warthog.

Described herein is a vaccine composition which comprises a plurality of attenuated ASF viruses. The plurality of attenuated ASF viruses may correspond to a plurality of different isolates, for example, different isolates of high or unknown virulence.

Such a vaccine composition may elicit a cross-protective immune response to several or substantially all ASF viruses.

The attenuated African Swine Fever (ASF) virus described herein lacks a functional version of the following genes:.

The genes may, for example, be wholly or partially deleted.

In particular, the following genes may be at least partially deleted:.

Described herein is an attenuated ASFV which lacks a functional version of DP148R. The gene may be wholly or partially deleted.

The deletion may be continuous, or may comprise a plurality of sections of sequence. The deletion should remove a sufficient amount of nucleotide sequence such that the gene no longer encodes a functional protein. The deletion may, for example, remove at least <NUM>, <NUM> or <NUM>% of the coding portion of the gene.

The deletion may be total, in which case <NUM>% of the coding portion of the gene is absent, when compared to the corresponding genome of the wild-type isolate.

In the attenuated African Swine Fever (ASF) virus of described herein is one or more of the following genes may be interrupted:.

In particular, the following genes may be interrupted:.

Described herein is an attenuated ASF virus wherein the DP148R gene is interrupted.

The gene may be interrupted, for example, by deleting or otherwise modifying the ATG start codon of the gene.

The genome may comprise one or more nucleotide change(s) that ablate expression of the gene. For example, expression of the gene may be ablated by a frame shift or introduction of one or more stop codons in the open reading frame of the gene or a modification of a translational start site.

The interruption may cause the gene to not be transcribed and/or translated.

As mentioned above, the complete genome sequence of the attenuated ASFV strain OURT88/<NUM> has been determined and compared with that of virulent viruses (Chapman et al. , <NUM> as above). In addition to the deletions mentioned above, the OURT88/<NUM> strain also has interruptions in <NUM> other genes: EP402R, EP153R and DP148R (previously referred to as MGF360 18R).

EP402R codes for CD2v protein which is incorporated in the external layer of the virus and may have a role in virus entry or spread. It may also be a target for antibodies that inhibit infection. EP153R is a C-type lectin. DP148R inhibits type I interferon.

The genome of the attenuated virus described herein may comprise complete, uninterrupted and functional versions of one or more of the genes EP402R and EP 153R. In the attenuated virus described herein, both of these genes may be complete, uninterrupted and functional.

Described herein is a vaccine comprising an attenuated ASF virus as described herein.

The term 'vaccine' as used herein refers to a preparation which, when administered to a subject, induces or stimulates a protective immune response. A vaccine can render an organism immune to a particular disease, in the present case ASF. The vaccine described herein thus induces an immune response in a subject which is protective against subsequent ASF virus challenge.

The vaccine may comprise a plurality of attenuated ASF viruses of different genotypes. Such a vaccine may be capable of inducing a cross-protective immune response against a plurality of ASF virus genotypes.

The vaccine may be useful in preventing African Swine Fever.

Described herein is a pharmaceutical composition which comprises one or more attenuated ASF virus(es) described herein. The pharmaceutical composition may be used for treating African Swine Fever.

The vaccine or pharmaceutical composition may comprise one or more attenuated ASF virus(es) described herein and optionally one or more adjuvants, excipients, carriers and diluents.

Immunisation with OURT88/<NUM> induces a high IFNγ response, suggesting that it is mediated by T cells. The attenuated virus described herein appears to induce a reduced T cell mediated response (see <FIG> and <FIG>). The attenuated virus described herein therefore appears to induce a more "useful" (i.e. more protective) cellular immune response than OURT88/<NUM>.

The attenuated virus described herein may induce a reduced T-cell mediated response compared to OURT88/<NUM>. Methods for analysing and comparing T-cell mediated responses are known in the art, for example by assaying T-cell proliferation in response to antigen (e.g. cell counts, thymidine incorporation or BrdU incorporation), assaying cytokine secretion and/or expression in response to antigen (e.g. ELISA, ELISPOT, flow cytometry) or determination of lymphocyte subpopulations present following inoculation of a subject with the virus (e.g. using flow cytometry).

The attenuated virus described herein may induce an immune response comprising a reduced percentage of circulating gamma delta/CD8+ cells and/or a reduced percentage of CD8+/CD4-/γδTCR- (CD8 only cells) and or a reduced percentage of CD8+/CD3- (NK) cells compared to OURT88/<NUM>.

Described herein is a method of preventing and/or treating ASF in a subject by administration of an effective amount of an attenuated virus, vaccine, or pharmaceutical composition as described herein.

The term 'preventing' is intended to refer to averting, delaying, impeding or hindering the contraction of ASF. The vaccine may, for example, prevent or reduce the likelihood of an infectious ASFV entering a cell.

The term "treating" is intended to refer to reducing or alleviating at least one symptom of an existing ASF infection.

The subject may be any animal which is susceptible to ASF infection. ASF susceptible animals include domestic pigs, warthogs, bush pigs and ticks.

The subject vaccinated as described herein may be a domestic pig. Any reference to a method of treatment practised on the human or animal body is interpreted as substances and compositions for use in such treatment.

The vaccine described herein may be administered by any convenient route, such as by intramuscular injection. Other suitable routes of administration include intranasal, oral, subcutaneous, transdermal and vaginal (e.g. during artificial insemination). In one embodiment described herein, oral administration comprises adding the vaccine to animal feed or drinking water. In another embodiment described herein, the vaccine may be added to a bait for a wild animal, for example a bait suitable for wild boar, wild pigs, bushpigs or warthogs.

The present inventors have found the attenuated virus BeninΔMGF described herein is effective at a lower dose compared to OURT88/<NUM>. For example, in Example <NUM>, pigs received <NUM><NUM> HAD of BeninΔMGF which was compared with a dose of <NUM><NUM> TCID<NUM> of the virus OURT88/<NUM>.

The dose for pig immunisation may therefore be less than <NUM><NUM> HAD<NUM> or TCID<NUM> per pig. For example the dose may be between <NUM><NUM>-<NUM><NUM> HAD<NUM> or TCID<NUM>. The dose may be about <NUM><NUM> HAD<NUM> or TCID<NUM> per pig.

The vaccine may be administered following a prime-boost regime. For example, after the first inoculation, the subjects may receive a second boosting administration some time (such as about <NUM>, <NUM>, <NUM> or <NUM> days) later. Typically the boosting administration is at a higher dose than the priming administration. The boosting dose may be about <NUM><NUM>, <NUM><NUM> or <NUM><NUM> HAD<NUM> or TCID<NUM> of the recombinant attenuated virus per pig.

Described herein is a method of attenuating an African Swine Fever (ASF) virus, which comprises the step of at least partially deleting, or interrupting, the expression of the following genes:.

The present invention provides a method of attenuating an ASF virus, which comprises the step of partially or completely deleting, or interrupting the expression of, the DP148R gene, wherein the gene is interrupted such that the gene is not transcribed and/or translated, or wherein the deletion removes at least <NUM>% of the coding portion of the gene.

The DP148R mutation may also be made in combination with mutations of the multigene-family <NUM> genes <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> and multigene-family <NUM> genes 1R, 2R, 3R and 4R as described herein.

Methods for deletion of viral genes are known in the art. For example, homologous recombination may be used, in which a transfer vector is created in which the relevant gene(s) are missing and used to transfect virus-infected cells. Recombinant viruses expressing the new portion of sequence may then be selected. Similar procedures may be used in order to interrupt gene expression, for example by deletion of the ATG start codon.

Therefore, the present invention further provides a method according to the invention, which comprises transfecting virus-infected cells with a transfer vector in which the DP148R gene is missing or interrupted, such that homologous recombination occurs, and selecting recombinant viruses expressing the new portion of sequence.

The present inventors have surprisingly found that intranasal administration of an attenuated African Swine Fever (ASF) virus results in improved protection and fewer side effects compared to administration via the intramuscular route, and may require a lower dose to obtain protection. In particular, intranasal administration of attenuated strain OURT88/<NUM> demonstrated complete protection (<NUM>%) against challenge with parental ASFV virulent virus, which represents a significant improvement over survival rates using the same attenuated virus via the intramuscular route.

Described herein is an intranasal delivery device comprising an attenuated ASF virus vaccine. Suitable devices for intranasal administration of vaccine are well known in the art, for example, a syringe or dropper, an aerosol device (e.g. Omron, Philips respironics InnoSpire Deluxe, Devilbiss mask/nebuliser and compressor BreathEazy), a mucosal atomisation device (e.g. LMA MAD Nasal™, Teleflex VaxlNator™), a single or multidose spray pump, a unit dose powder dispenser or a bidose powder dispenser.

The attenuated ASF virus may be any suitable attenuated ASF virus. In one embodiment described herein, the attenuated ASF virus may be one which is not considered suitable for use in the prevention or treatment of African Swine Fever when administered by a different route of administration, such as the intramuscular route, for example due to an unacceptable safety profile or low efficacy. In particular, the attenuated ASF virus may be OURT88/<NUM>. The attenuated ASF virus may alternatively be an attenuated ASF virus which lacks a functional version of the multigene-family <NUM> genes <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> and multigene-family <NUM> genes 1R, 2R, 3R and 4R, and/or lacks a functional version of DP148R. The attenuated ASF virus may be any suitable attenuated ASF virus as described herein, such as BeninΔMGF or BeninΔDP148R.

Intranasal administration as described herein may be in a droplet, spray or dry powder form, or may be a nebulised or aerosolised vaccine formulation. The dose is typically <NUM>, administered as <NUM> per nostril.

The present inventors have found that intranasal administration of attenuated ASF virus is effective at a lower dose compared to intramuscular administration. For example, in Example <NUM>, pigs immunised intranasally with <NUM><NUM> or <NUM><NUM> of the OURT883 had complete (<NUM>%) protection against lethal challenge, compared to much lower survival rates in pigs immunised with the same doses via the intramuscular route.

The dose for intranasal immunisation may therefore be <NUM><NUM> HAD<NUM> or TCID<NUM> or less per pig. For example the dose may be between <NUM><NUM>-<NUM><NUM> HAD<NUM> or TCID<NUM>. The dose may be about <NUM><NUM> HAD<NUM> or TCID<NUM> per pig.

The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

The MGF <NUM> genes <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and MGF <NUM> genes 1R, 2R, 3R were deleted from the ASFV Benin <NUM>/1isolate. In addition the ATG codons of MGF <NUM><NUM> and of MGF <NUM>4R were deleted to interrupt expression of these genes. The genes deleted were replaced with the GUS gene under control of the ASFV p72 promoter. This was achieved by homologous recombination between plasmid pΔMGFGUS and the virus genome (see Materials and Methods and <FIG>). Recombinant viruses were identified by expression of the GUS gene and purified by infection at limiting dilution. Two independent viruses BeninΔMGFA2 and BeninΔMGFA1 were isolated using this method to reduce the possibility that the phenotype of the gene deletion was associated with mutations which may have occurred elsewhere on the genome.

Recombinant virus BeninΔMGFA2, was further characterised. Genomic DNA was isolated from wild type Benin <NUM>/<NUM> and BeninΔMGFA2 and analysed by PCR to test for the insertion of the GUS marker gene and deletion of the eight MGF genes (<FIG>). Primers BeninD8F and BeninD8R were designed to anneal within the MGF <NUM><NUM> gene, and the MGF360 4R gene, that flank the insertion site. PCR using these primers amplified a <NUM> bp fragment using recombinant virus BeninΔMGFA2 gDNA as template (<FIG> lane <NUM>).

The size of this band is consistent with that of a PCR product in which the eight MGF genes have been deleted and have been replaced by the GUS marker gene under the control of the ASFV vp72 promoter. To confirm that recombinant virus BeninΔMGFA2 contains the GUS gene, a PCR was carried out using an internal GUS gene primer, RGUS, and primer BeninΔ8F. This PCR amplified a fragment consistent with the expected <NUM> bp size (<FIG> lane <NUM>) when DNA from BeninΔMGFA2 was used as template. As expected, no PCR fragment was detected using these primers with wild type Benin <NUM>/<NUM> gDNA as template (<FIG> lane <NUM>). To confirm that the recombinant virus BeninΔMGFA2 did not contain the MGF <NUM><NUM> gene, PCR reactions were carried out using primers BeninΔ8INTF and BeninΔ8INTR located at positions <NUM> and <NUM> within the MGF <NUM><NUM> gene. No PCR fragment was isolated using the recombinant virus BeninΔMGFA2 gDNA as template (<FIG> lane <NUM>), but as expected a <NUM> bp fragment was isolated using wild type Benin <NUM>/<NUM> gDNA as template (<FIG> lane <NUM>). Taken together, the PCR data showed that recombinant virus BeninΔMGFA2 contains the GUS gene in place of the eight MGF genes. To additionally confirm that the eight MGF genes had been deleted, gDNA from virus BeninΔMGFA2 was isolated and the junction at the site of deletion/insertion was sequenced using the primers X and Y. Analysis of the sequence revealed that the eight MGF genes had been deleted and the GUS marker gene had been inserted (<FIG>). The first five bp of the flanking gene MGF <NUM><NUM> and the first seven bp of the MGF <NUM>4R flanking gene had also been deleted. Because the additional deleted sequences each contained the ATG start codon, it can be concluded that the MGF <NUM><NUM> and MGF <NUM>4R genes are not expressed by the recombinant virus BeninΔMGFA2.

To investigate whether deletion of the eight MGF genes affected virus replication, the growth of BeninΔMGFA2 was compared to parental Benin <NUM>/<NUM> virus in primary porcine bone marrow macrophages. Cells were infected at high (<NUM> HAD50/cell) multiplicity of infection (m. ) and total virus was harvested from supernatants at different times post-infection. <FIG> shows that there were no significant differences between the titres of wild type Benin <NUM>/<NUM> and those of BeninΔMGFA2 viruses recovered at any of the time points measured. This shows that deletion of the eight MGF genes did not significantly affect the replication of BeninΔMGFA2 in primary porcine bone marrow macrophages.

One group of three pigs (Group <NUM>) were immunised intramuscularly with <NUM><NUM> HAD of deletion virus BeninΔMGFA2, a second group of two pigs (Group <NUM>) were inoculated with <NUM><NUM> HAD of deletion virus BeninΔMGFA1 and a third group of four pigs (Group <NUM>) were inoculated with <NUM><NUM> TCID<NUM> of attenuated virus OURT88/<NUM>. Clinical scores and temperatures for each pig were recorded every day and blood samples were taken every seven days. The results of these recordings are shown in <FIG> and <FIG> and show that none of the pigs had a clinical score above <NUM> on any day post-inoculation. Four pigs (Group <NUM> pigs <NUM> and <NUM>, Group <NUM> pigs <NUM> and <NUM>) had a temperature above <NUM><NUM>C but this was for one or two days only.

On day <NUM> post-inoculation, <NUM> of blood was taken from each pig, PBMCs were purified and Group <NUM> and <NUM> pig cells were stimulated with BeninΔMGFA2 and BeninΔMGFA1 viruses respectively. PBMCs isolated from Group <NUM> pigs were stimulated with OURT88/<NUM> virus isolate and the numbers of IFNg producing cells from all three Groups were measured by ELIspot. The results show that three out of four pigs from Group <NUM> had a high IFNg response to OURT88/<NUM>, whereas none of the pigs from Groups <NUM> and <NUM> had a high IFNg response to BeninΔMGF or OURT88/<NUM> (<FIG>). At <NUM> days after the first inoculation, pigs in Groups <NUM> and <NUM> were boosted intramuscularly with <NUM><NUM> HAD of viruses BeninΔMGFA2 and BeninΔMGFA1 respectively and pigs in Group <NUM> with <NUM><NUM> TCID<NUM> of OURT88/<NUM>. Clinical observations showed that only one pig (Group <NUM>, pig <NUM>) had a clinical score above score <NUM> (<FIG>) and that only one pig (Group <NUM> pig <NUM>) had a temperature above <NUM><NUM>C for <NUM> day (<FIG>).

On day <NUM> post initial inoculation (<NUM> days post-boost) all three groups of pigs (Groups <NUM>, <NUM> and <NUM>) were challenged intramuscularly with <NUM><NUM> HAD of virulent virus Benin <NUM>/<NUM>. In addition, a control group of three un-inoculated pigs (Group <NUM>) were also challenged with <NUM><NUM> HAD of virulent virus Benin <NUM>/<NUM>. All three pigs from Group <NUM> and one pig (pig <NUM>) from Group <NUM> developed high temperatures and high clinical scores (above score <NUM>) and were terminated at five days post-challenge because the humane end-point on the animal license had been reached (<FIG> and <FIG>). All five pigs from Groups <NUM> and <NUM> and the remaining three pigs from Group <NUM> were protected against challenge with virulent virus Benin <NUM>/<NUM> and continued to be healthy up to day <NUM> when the experiment was terminated (<FIG>).

At day <NUM> post initial inoculation, PBMCs were isolated from blood and cells isolated from spleen and these were stimulated with virus strains and numbers of IFNγ producing cells detected by ELlspot assay. Results showed that all three pigs from Group <NUM> showed high IFNg responses in cells from both blood and spleen, whereas only one pig (pig <NUM>, Group <NUM>) from Groups <NUM> and <NUM> showed high IFNγ responses in blood and spleen (<FIG>).

Infection with BeninΔMGF was shown to decrease the number of circulating gamma delta CD8+ T cells as early as day <NUM> post infection. Higher numbers of circulating CD8+CD4- γδTCR- (CD8 only) and CD8+CD3- (NK cells) cells were observed in pigs infected with OURT88/<NUM> at late days post infection (days <NUM> and <NUM>). No other significant differences between OURT88/<NUM> and BeninΔMGF infected animals were observed for the other cell populations studied (total CD4+, CD4+ CD8+ and total gamma delta T cells) (see <FIG>).

Group <NUM> and <NUM> pigs were inoculated with <NUM><NUM> HAD of the independently isolated recombinant viruses BeninΔMGFA2 and BeninΔMGFA1. None of the pigs showed a clinical score above score <NUM> and no pigs showed a temperature above <NUM>. This showed that deletion viruses BeninΔMGFA2 and BeninΔMGFA1 were attenuated and short transient fever (1or <NUM> days) was detected in <NUM> of the <NUM> pigs but no other clinical signs associated with ASFV infection.

Pigs in Groups <NUM> and <NUM> were boosted intramuscularly with <NUM><NUM> HAD of viruses BeninΔMGFA2 and BeninΔMGFA1. None of the pigs showed a clinical score above score <NUM> and no pigs showed a temperature above <NUM>. Similar results were observed following boost of the pigs in Group <NUM> with the attenuated strain OURT88/<NUM>.

Pigs from Groups <NUM>, <NUM> and <NUM> and three un-inoculated control pigs were challenged with <NUM><NUM> HAD of virulent virus isolate Benin <NUM>/<NUM>. All five pigs (<NUM>%) from Groups <NUM> and <NUM> (BeninΔMGFA2 and BeninΔMGFA1) were protected against virulent virus challenge. <NUM>% of pigs in Group <NUM> and <NUM>% of pigs in Group <NUM> were protected against virulent Benin <NUM>/<NUM> (<FIG>). After challenge with Benin <NUM>/<NUM> no pigs in Groups <NUM> and <NUM> showed a temperature above <NUM> and no pigs had a clinical score above <NUM>. In contrast all pigs in Group <NUM> showed temperatures above <NUM> and had clinical scores above score <NUM>.

The deletion viruses BeninΔMGFA2 and BeninΔMGFA1 showed a better level of protection (<NUM>%) compared to the attenuated strain OURT88/<NUM> (<NUM>%) against challenge with virulent Benin <NUM>/<NUM>.

Non-virulent, non-heamabsorbing ASFV isolate OUR T88/<NUM> was obtained from Ourique in Portgual. Virulent, haemabsorbing isolate Benin <NUM>/<NUM> has been previously described. Both OUR T88/<NUM> and Benin <NUM>/<NUM> are p72 genotype I viruses were grown in primary macrophage cultures derived from bone marrow. Titres of virus were determined as the amount of virus causing haemadsorption (for HAD isolates) or cytopathic effects (for non-HAD isolates) in <NUM>% of infected cultures (HAD<NUM>/ml or TCID<NUM>/ml).

The plasmid vector pΔMGFGUS was constructed to facilitate deletion of the eight MGF genes (MGF <NUM><NUM>, <NUM>, <NUM>, <NUM>, <NUM> and MGF <NUM>1R, 2R, 3R) from the genome of Benin <NUM>/<NUM>. Using Benin <NUM>/<NUM> genomic DNA as template a <NUM> bp fragment (Flank L) located at the <NUM>' terminus of the MGF <NUM><NUM> gene at position <NUM> - <NUM> immediately upstream of the MGF <NUM><NUM> gene was amplified using the primers FlankLF (ACGTTGCAAAGCTTCCATTAATCCCTCCAGTTGTTC) and FlankLR (ACGTTGCAGGTACCCCTCTCTCTGCAGACTCTCACC). Using Benin <NUM>/<NUM> genomic DNA as template, a <NUM> bp fragment (Flank R) located at the <NUM>' terminus of the MGF <NUM>4R gene at position <NUM> - <NUM> immediately downstream of the MGF <NUM>3R gene was amplified using the primer FlankRF (ACGTTGCAGCGGCCGCCTCTCCAAGACATCTGTCGG) and primer FlankRR (ACGTACGTCTCGAGCCTCACATGCCATCTCAAACAATTCC). The FlankL fragment was digested with Hindlll and Kpnl and ligated into vector pP72loxPGUS which was also digested with Hindlll and Kpnl to create plasmid pFlankL-GUS. The FlankR fragment was digested with Notl and Xhol enzymes and ligated into into the vector pFlankL-GUS digested with the same enzymes to create the transfer vector pDMGFGUS. The plasmid pDMGFGUS contains a GUS marker gene flanked on the left hand side by the <NUM>' terminal section of the MGF <NUM><NUM> gene, an ASFV vp72 promoter sequence and a loxP sequence. To the right side of the GUS gene is located the <NUM>' terminal sequence of the MGF <NUM>4R gene.

Primary porcine alveolar macrophages (<NUM> dish, <NUM><NUM> cells) were infected with Benin <NUM>/<NUM> at a multiplicity of infection (m. of <NUM> and incubated at <NUM> for <NUM> hours, and then washed with Earle's saline (<NUM>% porcine serum, penicillin/streptomycin <NUM>,000u mg-<NUM>-<NUM>). A transfection mixture containing <NUM>µl Optimem (Gibco-Life Technologies), <NUM>µg pΔMGFGUS and <NUM>µl TRANS-IT LT-<NUM> (Mirus) transfection reagent was incubated at <NUM> for <NUM> minutes before adding it to the infected cells. Incubation was continued at <NUM> for <NUM> hours before the addition of <NUM> Earle's saline and continued incubation at <NUM>. Virus was harvested from the infected and transfected cells <NUM> hours post-infection and cell debris removed by centrifugation. Aliquots of virus containing supernatant were used to infect bone marrow macrophages on <NUM> well plates. At <NUM> hours post-infection Earle's saline containing <NUM>µg/ml <NUM>-Bromo-<NUM>-chloro-<NUM>-indol-<NUM>-yl β-D-glucopyranosiduronic acid (X-Gluc) was added and wells appearing 'blue' containing recombinant GUS expressing viruses were harvested. Infections at limiting dilution were further carried out on pig bone marrow macrophages containing X-Gluc until only one blue well per <NUM> well plate was observed to indicate infection with the recombinant deletion virus. No evidence of virus infection (cytopathic effect (cpe)) was observed in the other <NUM> wells. Two independent recombinant viruses (BeninΔMGFA2 and BeninΔMGFA1) were isolated using this method. High titre stocks of recombinant viruses BeninΔMGFA2 and BeninΔMGFA1 were grown up on porcine bone marrow macrophages.

The growth of BeninΔMGFA2 was compared to parental Benin <NUM>/<NUM> virus in primary porcine bone marrow macrophages. Cells were infected at high (<NUM> HAD<NUM>/cell) multiplicity of infection (m. ) and total virus was harvested from supernatants at different times post-infection.

Viral genomic DNA from BeninΔMGF and Benin <NUM>/<NUM> virus harvests were purified from 300ul of supernatant from infected pig bone marrow macrophage infected cells using a GE Healthcare Illustra Genomic Prep Mini Spin kit. Analysis of viral genomic DNA was carried out by PCR using the specific DNA primers BeninD8F (GGTGAGAGTCTGCAGAGAGAGG), BeninD8R (GCCCTAGCACTTGTAACG), RGUS (CCTTCTCTGCCGTTTCCAAATCGCCGC), BeninD8INTF (CGATGTATCATTGATGTC), BeninD8INTR (GGATAATCTTAGGGAGGCC).

Viral gDNA was isolated from the harvest of BeninDMGF infected cells and sequenced using the following primers 9LF (ATGACGCATTAAACCGGCG) and 4RR (CAGTATAGCCCTAGCACTTG).

IFN-g ELIspot was carried out as previously described (<NPL>).

Pigs used were cross-bred, large white × Landrace, of average weight <NUM> at the first inoculation. All pigs were maintained in high security SAPO4 facilities throughout and the experiment performed under Home Office licence PPL <NUM>/<NUM>-. One group of three pigs (Group <NUM>, pigs <NUM>, <NUM> and <NUM>) were inoculated intramuscularly with <NUM><NUM>HAD<NUM> of recombinant virus BeninDMGFA2. A second group of two pigs (Group <NUM>, pigs <NUM> and <NUM>) were inoculated intramuscularly with <NUM><NUM>HAD<NUM> of recombinant virus BeninΔMGFA1. A third group of four pigs (Group <NUM>, pigs <NUM>, <NUM>, <NUM> and <NUM>) were inoculated with <NUM><NUM> TCID<NUM> of attenuated strain OURT88/<NUM>. The inventors had previously determined that a dose of <NUM><NUM> TCID<NUM> of OURT88/<NUM> was needed to induce protection and that <NUM><NUM> was less effective.

Three weeks later to boost the ASFV specific adaptive immune responses, pigs in Groups <NUM> and <NUM> were inoculated with <NUM><NUM>HAD<NUM> of recombinant viruses BeninDMGFA2 and BeninDMGFA1 respectively and Group <NUM> pigs were immunised with <NUM><NUM>TCID<NUM> of OURT88/<NUM>. Three weeks post-boost Groups <NUM>, <NUM>, <NUM> and a fourth group of three pigs (Group <NUM>, pigs <NUM>, <NUM> and <NUM>) containing three non-immunised pigs, were challenged intramuscularly with <NUM><NUM>HAD<NUM> of virulent ASFV isolate Benin <NUM>/<NUM>. ASFV-inoculated and challenged pigs were monitored daily for body temperature and clinical signs and these were scored as reported by King et al. All pigs were examined by post-mortem at termination and spleen and lymph tissues were collected.

Blood samples were collected from the infected pigs at different times post infection and the different T cell populations were identified by using the following Zenon (Invitrogen) labelled antibodies: mouse IgG1 anti-porcine CD3 (Alexa Fluor <NUM>), mouse IgG2b anti-porcine CD4 (Alexa Fluor <NUM>), mouse IgG2a anti-porcine CD8αα (R-PE) and mouse IgG2b anti-porcine gamma delta TCR (Alexa Fluor <NUM>). Briefly, 100µl of whole blood was incubated with <NUM>µl of each conjugated antibody for <NUM> minutes at room temperature. Red blood cells were lysed by adding <NUM> of 1x BD FACS lysing solution (BD Biosciences) and vortexing. Cells were pelleted by centrifugation at 1200rpm for <NUM> minutes and the supernatant was discarded. The cells were then fixed with <NUM>% paraformaldehyde for <NUM> minutes, washed twice with PBS and finally analysed by flow cytometry (MACSQuant, Miltenyi Biotec). Lymphocyte subsets were gated based on cell surface markers staining using FCS express software.

The experiments were carried out in level-<NUM> biocontainment facilities (BSL-<NUM>) of Centre de Recerca en Sanitat Animal (CReSA, Barcelona, Spain). All animal experiments were carried out under UK Home Office Licence number <NUM>/<NUM> with the approval of the Ethics Committees for Animal Experiments of the Autonomy University of Barcelona (No. 1189R5) and the Regional Government of Catalonia, Spain (No. <NUM>), and complied fully with the regulated procedures from the Animals (Scientific Procedures) Act <NUM>.

<NUM>-week-old Large White and Pietrain crossbred male piglets in good health, vaccinated against Porcine Circovirus type <NUM> and Mycoplasma hyopneumoniae, average weight <NUM>, from a high health herd tested negative for Porcine Respiratory and Reproductive syndrome (PRRS) and Aujeszky's disease were used. After a <NUM>-day acclimatisation period, three groups of six pigs each were immunised intramuscularly (IM) with <NUM> containing <NUM><NUM> (group A), <NUM><NUM> (group C) and <NUM><NUM> (group E) TCID<NUM>/ml of low virulent ASFV isolate OUR T88/<NUM> respectively. An additional three groups of six pigs each were immunised intranasally (IN), using a mucosal atomization device, with <NUM> per nostril containing <NUM><NUM> (group B), <NUM><NUM> (group D) and <NUM><NUM> (group F) TCID<NUM>/ml of low virulent isolate OUR T88/<NUM>. Three weeks later all immunised groups, together with a control group (group G) containing three non-immunised pigs, were challenged intramuscularly with <NUM> containing <NUM><NUM> TCID<NUM>/ml of the closely related virulent ASFV isolate OUR T88/<NUM>. This control group was housed separately (room <NUM>), while pigs immunised with the same ASFV titre, but by different inoculation routes (IN or IM), were allocated in the same isolation room separated by <NUM> high partition as follows: experiment <NUM> (groups A and B), experiment <NUM> (groups C and D), experiment <NUM> (groups E and F).

Immunisation day was defined as day <NUM> (<NUM> dpi). Rectal temperatures and clinical signs were monitored daily prior to immunisation and throughout the study, following a clinical score previously reported (King et al. EDTA blood and serum samples were collected from all pigs prior to virus immunisation (<NUM> dpi), after immunisation (at <NUM>, <NUM>, <NUM>, <NUM> and <NUM> dpi) and after challenge (at <NUM>, <NUM>, <NUM>, <NUM> and <NUM> dpc).

A post-mortem examination also was carried out in order to evaluate gross lesions of dead pigs or pigs euthanized during the experiment upon reaching a predetermined humane endpoint, as well as at the end of the experiment at <NUM> days post-challenge (dpc). Macroscopic lesions were evaluated in accordance with the standardized pathological framework of ASFV infections (Galindo-Cardiel et al. The humane end point was determined in accordance with the welfare regulations specified in the UK Home Office Licence, so that pigs with a rectal temperature over <NUM> for three consecutive days, or showing three or more clinical signs of disease combined on a single day, were euthanized. Euthanasia was conducted by intravenous injection of pentobarbital sodium.

After collection, EDTA blood samples were frozen at -<NUM> until ASFV detection by quantitative PCR (qPCR) as described previously (King et al. Frozen tissues samples (spleen, tonsil, lung, submandibular, retropharyngeal and gastrohepatic lymph nodes) were also analysed for the presence of ASFV by qPCR. Serum samples were also frozen at -<NUM> until assayed by commercially available ELISA kits for detection of antibodies against Vp72 structural protein of ASFV (INGEZIM PPA Compac, Ingenasa Madrid, Spain). In addition, serum samples were used to evaluate, by commercially available ELISA kits (R&D Systems, Abingdon, UK), porcine cytokines with inflammatory and immunological functions during humoral and cell mediated immunity in pigs (IL-1β, TNFα, IFNγ, IL-<NUM> and IL-<NUM>). Cytokine concentrations were presented as pg/ml.

Data were analysed using the statistical analysis program GraphPad Prism Version <NUM> (GraphPad Software). The values of rectal temperature, clinical signs, macroscopic lesions, viraemia, ASFV antibodies and cytokine levels were assessed to calculate means ± standard deviations (SD). For analysis of temperature and clinical signs, differences between baseline values (day <NUM>) and the values obtained at each time-point in the un-inoculated control group and inoculated groups were analysed using One-way ANOVA with Bonferroni post-test. Furthermore, differences between the un-inoculated control group and infected groups as well as differences between both infected groups at the same time-point were analysed using a two-way ANOVA with Bonferroni post-test. For all comparisons, differences were considered significant at P< <NUM>.

From <NUM> dpc, pigs in control Group G (non-immune) displayed non-specific symptoms such as fever (<NUM>-<NUM>) and apathy (see <FIG>. These clinical signs increased progressively until <NUM> dpc, with rectal temperatures between <NUM>- <NUM>, recumbence and the presence of skin erythema and cyanotic areas on tip of ears, being euthanized for ethical reasons. After euthanasia of pigs at <NUM> dpc, necropsies revealed the presence of gross lesions characteristic of acute forms of ASF such as hemorrhagic lymphadenitis (gastrohepatic and renal lymph nodes being the most severely affected), hyperemic splenomegaly, non-collapsed lungs with interstitial and alveolar edema as well as foam in trachea, petechiae in kidneys (cortex and medulla) and lungs, retroperitoneal edema and moderate hepatic congestion.

Of those pigs immunised with OURT88/<NUM> by the intranasal route <NUM>% (n= <NUM>) of those in groups B (<NUM><NUM> TCID<NUM>) and D (<NUM><NUM> TCID <NUM>) survived challenge. Some of the pigs that survived displayed a transient moderate joint swelling before and after challenge. Two pigs from group B (B3 and B4) and <NUM> from group D (D2, D4 and D5) showed other short transient clinical signs post-challenge (temperature, inappetence, apathy). In group F (<NUM><NUM> TCID<NUM>) <NUM>% survived challenge. The two pigs which did not survive (F3 and F5) were euthanized at day <NUM> post-challenge showing gross lesions characteristic of ASF and other some other clinical signs (elbow swelling and erosion of skin on the nose). Three surviving pigs (F1, F2 and F4) also showed clinical signs and lesions including severe joint swelling, laboured breathing, erythema on ears, conjunctivitis as well as skin erosions/ulcers in nose, flanks and limbs that lasted until the end of the experiment.

Of the pigs immunised by the intramuscular route, <NUM> (E2, E3 and E6) from group E (<NUM><NUM> TCID<NUM>) were euthanized or found dead between <NUM> and <NUM> dpi and before challenge showing signs typical of ASF. Of those pigs challenged <NUM>% (<NUM>/<NUM>) from Group A (<NUM><NUM> TCID<NUM>), <NUM>% (<NUM>/<NUM>) from group C (<NUM><NUM> TCID<NUM>) and <NUM>% (<NUM>/<NUM>) from Group E (<NUM><NUM> TCID<NUM>) survived challenge.

Comparative statistical analysis of clinical scores (<NUM> days before death) developed by pigs that died or were euthanized inside each experimental group (including pigs dead before challenge in group E and non-immunised control pigs), revealed similar kinetics as well as non-significant differences in reached temperatures among groups (<FIG>). In addition, kinetics of clinical scores was similar, significant differences appeared just between pigs dead in group F (IN, <NUM><NUM>) and group A (IM, <NUM><NUM>) (<FIG>). On the other hand, statistical analysis of clinical scores in pigs that survived revealed that after challenge, surviving pigs in group F (IN, <NUM><NUM>) showed significant differences in clinical scores with respect to surviving pigs including in other groups until the end of the study (<FIG>).

During macroscopic evaluation of surviving pigs, lesions were mainly observed in cardiorespiratory system, skin and musculoskeletal system. In general, surviving pigs immunised intramuscularly (<NUM> pigs in all) displayed no lesions or minimal pulmonary lesions (<NUM>/<NUM> pigs), while surviving pigs immunised intranasally (<NUM> pigs in all) showed cardiorespiratory lesions (<NUM>/<NUM> pigs) as well as injuries in skin and joints (<NUM>/<NUM> pigs). In this sense, while lesions were moderate in surviving pigs immunised intranasally with <NUM><NUM> and <NUM><NUM> (groups B and D), surviving pigs inoculated intranasally with <NUM><NUM> (group F) displayed the most intense cardiorespiratory and musculoskeletal injuries (fibrinous pleuritis, fibrinonecrotic pleuropneumonia, fibrinous pericarditis and serofibrinous/purulent periarthritis), cardiorespiratory lesions being compatible with the presence of secondary bacterial infections. Statistical analysis of macroscopic lesion scores confirmed the significant differences among surviving pigs in group F (IN, <NUM><NUM>) and the other groups of surviving animals (<FIG>).

These results have demonstrated a complete protection (<NUM>%) in pigs immunised intranasally with <NUM><NUM> and <NUM><NUM> TCID<NUM>/ml of low virulent ASFV isolate OUR T88/<NUM>. Pigs displayed minimal and transient adverse clinical reactions before and after challenge with the virulent ASFV isolate OUR T88/<NUM> as well as mild lesions in lungs, associated to secondary bacterial infections, and joints mainly. We predict from our results that a lower dose (eg <NUM><NUM> TCID<NUM>) may also induce high levels of protection against lethal challenge.

However, in both the group of pigs immunised intranasally with <NUM><NUM> TCID<NUM>/ml and in all groups immunised intramuscularly, the rate of protection conferred was lower. The lowest survival rates were observed in pigs immunised intramuscularly with <NUM><NUM> TCID<NUM>/ml, where <NUM> of the <NUM> immunised pigs (E2, E3 and E6) died before challenge. The results show that intranasal immunisation of pigs with live attenuated ASFV vaccines is an alternative to the previously reported intramuscular route. Intranasal immunisation induced fewer clinical signs before challenge compared to the intramuscular route and a higher percentage of pigs were protected.

This involved testing delivery of the gene deleted attenuated ASFV BeninΔMGF at three different doses using the IM route (<NUM><NUM>, <NUM><NUM>, <NUM><NUM> HAD<NUM>) and by one dose (<NUM><NUM> HAD<NUM>) using the intranasal route and challenge with parental virulent virus.

The experiments were carried out in SAPO-<NUM> biocontainment facilities at The Pirbright Institute. All animal experiments were carried out under UK Home Office Licence number <NUM>/<NUM> and complied fully with the regulated procedures from the Animals (Scientific Procedures) Act <NUM>.

Large White Landrace crossbred female piglets in good health, average weight <NUM> - <NUM>, from a high health herd were used. After a <NUM>-day acclimatisation period, three groups of six pigs each were immunised intramuscularly (IM) with <NUM> containing <NUM><NUM> (group A), <NUM><NUM> (group B) and <NUM><NUM> (group C) HAD <NUM> of attenuated ASFV gene deleted BeninΔMGF strain respectively. An additional group of six pigs were immunised intranasal (IN), using a mucosal atomization device, with <NUM> per nostril containing <NUM><NUM> BeninΔMGF (group D). Three weeks later all immunised groups were boosted with the same dose of virus by the same route. After a further <NUM> days pigs in groups A to D, together with a control group (group F) containing six non-immunised pigs, were challenged intramuscularly with <NUM> containing <NUM><NUM> TCID<NUM>/ml of the parental virulent ASFV isolate Benin <NUM>/<NUM>. This control group was housed separately, while the other pigs were allocated in the same isolation room separated by <NUM> high partition as follows: room <NUM>(groups A and B), room <NUM> (groups C and D).

Immunisation day was defined as day <NUM> (<NUM> dpi). Rectal temperatures and clinical signs were monitored daily prior to immunisation and throughout the study, following a clinical score previously reported (King et al. EDTA blood and serum samples were collected from all pigs prior to virus immunisation (<NUM> dpi), after immunisation (at <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> dpi) boost and after challenge (at <NUM>, <NUM>, <NUM>, <NUM> and <NUM> dpc).

A post-mortem examination also was carried out in order to evaluate gross lesions of dead pigs or euthanized during the experiment upon reaching a predetermined humane endpoint, as well as at the end of the experiment. Macroscopic lesions were evaluated in accordance with the standardized pathological framework of ASFV infections (Galindo-Cardiel et al. The humane end point was determined in accordance with the welfare regulations specified in the UK Home Office Licence, so that pigs with a rectal temperature over <NUM> for three consecutive days, or showing three or more clinical signs of disease combined on a single day, were euthanized. Euthanasia was conducted by intravenous injection of pentobarbital sodium.

Pigs were immunised and observed daily for clinical signs including temperatures. Results are shown in <FIG>. Of the pigs in groups A (<NUM><NUM>) and B (<NUM><NUM>) <NUM>/<NUM> developed a transient low fever for <NUM> or <NUM> days from day <NUM> or <NUM> post-immunisation. In group C (IM <NUM><NUM>) <NUM>/<NUM> pigs developed a transient low fever for <NUM> or <NUM> days. In group D (IN <NUM><NUM>) <NUM>/<NUM> pigs developed a low transient fever at day <NUM> post-immunisation. No other clinical signs were observed.

The results confirm that increasing the dose of BeninΔMGF does not increase clinical signs post-immunisation by the IM route. <FIG> shows that minimal clinical signs were observed in these and remaining pigs following the boost and until after challenge. Following challenge several pigs were euthanized because they reached moderate severity end-point (<NUM> days fever above <NUM> or <NUM> days without eating). These included <NUM> of the <NUM> pigs from the group immunised with <NUM><NUM> IM, <NUM> pigs immunised with <NUM><NUM> IM, <NUM> pig immunised with <NUM><NUM> IM and <NUM> pigs immunised with <NUM><NUM> IN. At post-mortem, lesions were not observed in any group except for <NUM> pig from group BeninΔMGF <NUM><NUM> IM which had a single lung lesion.

A comparison of mean temperatures of the different groups (<FIG>) showed statistically significant differences between the group immunised with BeninΔMGF <NUM><NUM> IM and other groups post-immunisation at day <NUM> (higher compared to all other groups), day <NUM> (higher compared to BeninΔMGF <NUM><NUM> IM, <NUM><NUM> IN and <NUM><NUM> IM). Post-boost statistically significant differences were observed between BeninΔMGF <NUM><NUM> IM (higher compared to BeninΔMGF <NUM><NUM> IN and BeninΔDP148R). At day <NUM> post-challenge pigs immunised with BeninΔMGF <NUM><NUM> IM had a significantly higher temperature compared to pigs immunised with BeninΔMGF <NUM><NUM> IM.

Pigs immunised with BeninΔMGF <NUM><NUM> IN had significantly different temperatures at days post-immunisation <NUM> (lower compared to BeninΔMGF <NUM><NUM> IM, <NUM><NUM> IM and BeninΔDP148R), post-immunisation day <NUM> (higher compared to BeninΔDP148R), day <NUM> post-challenge (higher compared to BeninΔMGF <NUM> <NUM> IM). Pigs immunised with BeninΔMGF <NUM><NUM> IM had statistically significant temperatures at day <NUM> post-immunisation (higher compared to BeninΔMGF <NUM><NUM> IM, <NUM><NUM> IN) and day <NUM> post-immunisation (higher compared to BeninΔDP148R). Pigs immunised with BeninΔMGF <NUM><NUM> IM had significantly different temperatures at day <NUM> post-immunisation (lower compared to BeninΔMGF <NUM><NUM> IM and <NUM><NUM> IN).

A comparison of mean clinical scores of the different groups (<FIG>) showed statistically significant differences between the group immunised with BeninΔMGF10<NUM> IM and other groups post-immunisation at day <NUM> (higher than BeninΔMGF <NUM><NUM> IM, <NUM><NUM> IN, BeninΔDP148R), day <NUM> (higher than BeninΔMGF <NUM><NUM> IM, <NUM><NUM> IM, <NUM><NUM> IN) and day <NUM> (lower than BeninΔDP148R, higher than BeninΔMGF <NUM><NUM> IM, <NUM><NUM> IN).

In conclusion, BeninΔMGF <NUM><NUM> IM showed significantly higher clinical scores and lower temperatures than other groups post-immunisation.

Porcine alveolar macrophages were infected with ASFV isolates indicated at multiplicity of infection <NUM> (Benin <NUM>/<NUM>, BeninΔMGF, OURT88/<NUM>) or were mock-infected. At different times post-infection (<NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> hours) RNA was harvested from infected cells and levels of mRNA for IFN-β RNA, GAPDH and ASFV B646L gene (VP72) were measured by quantitative reverse transcriptase PCR. <FIG> panel A shows levels of IFN-β compared to control housekeeping gene GAPDH. Panel B shows levels of mRNA for ASFV B646L (VP72) gene.

The deletion mutant BeninΔMGF has a deletion or interruption of <NUM> members of MGF360 and <NUM> members of MGF <NUM> from a region close to the left end of the Benin <NUM>/<NUM> genome. Deletion of these genes increases the induction of IFN -β mRNA in macrophages infected with BeninΔMGF360 compared to parental virulent virus in which IFN-β mRNA was barely detected (see <FIG>). Type I IFNs induce expression of IFN-stimulated genes which are involved in activation of host innate and adaptive immune response pathways and in creating an antiviral state. Our hypothesis is that induction of type I IFN is important for attenuation of virulent ASFV and induction of a protective immune response.

A single gene, DP148R, was deleted from a region close to the right end of the virulent Benin97/<NUM> genome. To achieve this a <NUM> bp fragment from the left and <NUM> bp fragment from the right regions flanking the MGF36018R gene were amplified by PCR. <FIG> shows the primers used to amplify the left flanking region (<NUM>-18RFlankL) and those to amplify the right flanking region (<NUM>-18RFlankR). The sequences amplified are between these primers. Shaded in grey is the sequence of the MGF360 -18R gene which was deleted from the ASFV genome. The start and stop codons are shown in bold. The flanking regions were cloned either side of a reporter gene consisting of the β-Glucorinidase (β-GUS) gene downstream from the ASFV VP72 promoter. This plasmid was transfected into pig macrophages infected with the Benin <NUM>/<NUM> isolate. Progeny virus was tested for expression of the β-GUS gene and recombinant viruses in which the β-GUS gene replaced the MGF360-18R gene was isolated by limiting dilution. The deletion of the gene and location of the insertion was confirmed by PCR analysis using primers that were from the ASFV genome outside of the regions cloned in the transfer plasmid (see <FIG>).

Replication of the BeninΔMGF36018R virus strain in porcine macrophages was compared with parental Benin <NUM>/<NUM> virus. Porcine alveolar macrophages were infected at a multiplicity of <NUM> and at different times post-infection (<NUM>, <NUM>, <NUM>, <NUM>, <NUM> hours) virus from cells and supernatants was harvested and titrated using porcine alveolar macrophages. The results (<FIG>) showed that deletion of the MGF360 18R gene did not reduce virus replication in macrophages.

A group of <NUM> male large white/landrace crossbred pigs (<NUM>-<NUM>) were immunised intramuscularly with <NUM><NUM> HAD<NUM> BeninΔDP148R and observed for clinical signs. All <NUM> pigs displayed transient clinical signs for <NUM> or <NUM> days at days <NUM> or <NUM> post-immunisation. These signs included transient fever, loss of appetite and lethargy.

Pigs were boosted with the same dose of virus by the same route at day <NUM> post-immunisation and were challenged with virulent Benin97/<NUM> in parallel with control non-immunised pigs at day <NUM> post-immunisation. No further clinical signs were observed following the boost and all pigs survived challenge (terminated at <NUM>-<NUM> days post-challenge). No lesions were observed at post-mortem.

A comparison of mean temperatures of the different groups (<FIG>) showed statistically significant differences between the group immunised with BeninΔDP148R and other groups post-immunisation at day <NUM> (lower compared to groups BeninΔMGF <NUM><NUM> IM and <NUM><NUM> IM) and day <NUM> (higher compared to groups BeninΔMGF <NUM><NUM> IM, <NUM><NUM> IM and <NUM><NUM> IN). At days post-boost statistically lower temperatures were observed between BeninΔDP148R and other groups: <NUM>, (BeninΔMGF10<NUM> IM, <NUM><NUM> IM) <NUM> (BeninΔMGF <NUM><NUM> IM), <NUM> (BeninΔMGF <NUM><NUM> IM, <NUM><NUM> IN), <NUM> (BeninΔMGF <NUM><NUM> IM, <NUM><NUM> IN), <NUM> (BeninΔMGF <NUM><NUM> IM, <NUM><NUM> IM, <NUM><NUM> IN), <NUM> (BeninΔMGF <NUM><NUM> IM, <NUM><NUM> IM), <NUM> -<NUM> (BeninΔMGF all groups). Post-challenge significantly lower temperatures were observed between pigs immunised with BeninΔDP148R and other immunised groups at day <NUM> (BeninΔMGF <NUM><NUM> IN) , <NUM> (<NUM><NUM> IM), <NUM> (<NUM><NUM> IM).

A comparison of mean clinical scores of the different groups (<FIG>) showed statistically significant differences between the group immunised with BeninΔDP148R and other groups post-immunisation at day <NUM> (higher than BeninΔMGF <NUM><NUM> IM, <NUM><NUM> IM, <NUM><NUM> IN), day <NUM> (BeninΔMGF <NUM><NUM>IM, <NUM><NUM>IM, <NUM><NUM>IN, <NUM><NUM>IM), day <NUM> (BeninΔMGF <NUM><NUM>IM, <NUM><NUM>IN, <NUM><NUM>IM) and day <NUM> (BeninΔMGF <NUM><NUM>IM, <NUM><NUM>IN, <NUM><NUM>IM).

In conclusion, BeninΔDP148R demonstrated significantly higher clinical scores and lower temperatures than the other groups post-mortem, and no lesions at post-mortem.

Claim 1:
An in vitro method of attenuating an ASF virus, which comprises the step of partially or completely deleting, or interrupting the expression of, the DP148R gene, wherein the gene is interrupted such that the gene is not transcribed and/or translated, or wherein the deletion removes at least <NUM>% of the coding portion of the gene.