Patent Application
20240293525
The present invention relates to attenuated African Swine Fever viruses. The attenuated viruses protect pigs against subsequent challenge with virulent virus. The present invention also relates to the use of such attenuated viruses to treat and/or prevent African Swine Fever. The invention also relates to EP402R proteins of African Swine Fever virus comprising particular amino acid substitutions, as well as polynucleotides encoding such proteins and African Swine Fever viruses comprising such proteins.
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 5-14 days of infection with a mortality rate approaching 100%.
ASFV can infect and replicate in warthogs (Phacochoerus sp.), bushpigs (Potamocherus sp.) and soft ticks of the Ornithodoros species (which are thought to be a vector), but in these species few if any clinical signs are observed and long term persistent infections can be established. ASFV was first described after European settlers brought pigs into areas endemic with ASFV and, as such, is an example of an “emerging infection”. 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 2007, ASFV has spread extensively through neighbouring countries including the Russian Federation. In 2012 the first outbreak was reported in Ukraine and in 2013 the first outbreaks in Belarus. In 2014 further outbreaks were reported in pigs in Ukraine and detection in wild boar in Lithuania and Poland. In 2018 ASFV spread to China and has since spread extensively in China and a number of other Asian countries (Mongolia, Vietnam, Cambodia, Myanmar, N and S Korea, Indonesia, Philippines, Papua New Guinea and Timor-Leste).
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 license 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.
There is thus a need for improved measures to control ASFV infection and prevent spread of the disease.
The complete genome sequences of ASFV isolate Benin 97/1 (a highly pathogenic virus from West Africa, Group1), isolate OURT88/3 (non-pathogenic, attenuated virus from Portugal, Group 1) and isolate BA71V (Vero cell tissue culture adapted non-pathogenic virus, Group 1) have been compared (Chapman et al. 2008 J. Gen. Virol. 89: 397-408). The complete genome of genotype II isolate Georgia 2007/1 has also been sequenced (Chapman et al. 2011 Emerg Infect Dis 17(4): 599-605)
In the OURT88/3 genome, the multigene family (MGF) 360 18R (DP148R) gene, EP153R gene and EP402R gene are each interrupted by frameshift mutations. Additionally, the following MGF genes are absent from the OURT88/3 genome: MGF 110 3L, 6L, 7L, 8L, 10L, 11L and 12L, MGF 300 3L, MGF 360 5L, 6L, 7L, 10L, 11L, 12L, 13L, 14L, 20R, 21R and 22R, and MGF 505 1R, 2R and 6R. The MGF 505 3R gene is also truncated.
The sequences of the high-virulence Lisboa60 strain and the low-virulence NH/P68 strain have also been compared (Portugal et al. 2015 J. Gen. Virol. 96: 408-419).
In the NH/P68 genome, the MGF 360 18R (DP148R) gene, EP153R gene and EP402R gene are each interrupted by a premature stop codon. Additionally, the following MGF genes are absent from the NH/P68 genome: MGF 110 3L, 6L, 7L, 8L, 10L, 11L and 12L, MGF 360 5L, 6L, 7L, 10L, 11L, 12L, 13L, 14L, 20R, 21R and 22R, and MGF 505 1R, 2R and 6R. The MGF 360 9L and MGF 505 3R genes are also truncated.
The sequences of the high-virulence BA71 strain and the low-virulence BA71V strain have also been compared (Rodríguez et al. 2015 PLOS ONE 10(11): e0142889).
Deletion of the DP148R gene from the virulent Benin 97/1 isolate reduced virulence and induced protection against challenge with the parental virus (Reis et al. 2017 J. Virol. 91, 24: e01428-17).
A DIVA vaccine (also referred to as a marker vaccine) allows differentiation of animals that have been infected with a wild type pathogen from animals that have been immunised with the vaccine. DIVA vaccines lack at least one immunogenic antigen (a DIVA marker) which is present in the wild type pathogen. Animals infected with the wild type pathogen produce antibodies against the DIVA marker, whereas vaccinated animals do not. Antibodies to the DIVA marker may be detected using a serological assay. Infected animals (which have antibodies to the DIVA marker) may thus be differentiated from vaccinated animals (which do not have antibodies to the DIVA marker), despite both groups of animals having antibodies to other immunogens of the pathogen.
A DIVA marker should be immunogenic, but deletion of the gene should not affect the vaccine's protective capacity.
Generally, the invention relates to an attenuated African Swine Fever virus in which expression and/or activity of the genes EP153R and EP402R is disrupted, whilst expression and/or activity of particular MGF genes is not disrupted.
The invention also relates to the determination that disruption of expression and/or activity of the EP153R and EP402R genes in combination with a Differentiation of Infected from Vaccinated Animals (DIVA) mutation can attenuate African Swine Fever virus. The invention concerns particularly a DIVA mutation in the K145R gene.
The invention also relates to the determination of particular amino acid changes in the EP402R protein of African Swine Fever virus which disrupt haemadsorption. Accordingly, the invention includes EP402R proteins comprising such amino acid changes, polynucleotides encoding such proteins and African Swine Fever virus comprising such EP402R proteins and polynucleotides.
Furthermore, the invention concerns the combination of the foregoing, in that the amino acid changes in EP402R may be combined with disruption of the activity and/or expression of the EP153R gene and/or a DIVA mutation to attenuate African Swine Fever virus.
The attenuated African Swine Fever viruses of the invention are of particular benefit as when used in a vaccine they provide protection against infection by wild type African Swine Fever virus strains, as demonstrated in the Examples herein.
In one aspect the invention provides an attenuated African Swine Fever (ASF) virus in which the expression and/or activity of the genes EP153R and EP402R is disrupted; and which comprises a functional version of one or more of the following genes:
The invention also provides an attenuated ASF virus in which the expression and/or activity of the genes EP153R and EP402R is disrupted;
The invention also provides an attenuated ASF virus in which the expression and/or activity of the genes EP153R and EP402R is disrupted;
In another aspect the invention provides an attenuated ASF virus in which the expression and/or activity of the genes EP153R and EP402R is disrupted and which comprises a DIVA mutation. In some embodiments the DIVA mutation disrupts expression of the K145R gene.
In another aspect the invention provides an EP402R protein comprising one or more amino acid changes in the ligand-binding domain wherein the amino acid changes disrupt ligand-binding of the EP402R protein.
In another aspect the invention provides an EP402R protein comprising an amino acid change at a position which corresponds to Q96 and/or W99 of the Georgia 2007/1 EP402R protein (SEQ ID No. 24).
The invention also provides a polynucleotide encoding an EP402R protein of the invention.
The invention also provides a vector comprising a polynucleotide of the invention.
In another aspect the invention provides an ASF virus comprising the EP402R protein of the invention. The invention also provides an ASF virus comprising the polynucleotide of the invention.
The invention also provides the ASF virus of the invention for use in treating and/or preventing a disease in a subject, and the use of an ASF virus of the invention for manufacture of a medicament for treating and/or preventing disease in a subject.
The invention also provides a pharmaceutical composition comprising an ASF virus of the invention, and such a pharmaceutical composition for use in treating and/or preventing a disease in a subject.
The invention also provides a vaccine comprising an ASF virus of the invention, and such a vaccine for use in treating and/or preventing African Swine Fever in a subject.
The invention also provides 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 pharmaceutical composition according to the invention or a vaccine according to the invention.
In another aspect the invention provides a method of producing an ASF virus of the invention, the method comprising changing one or more amino acid(s) in the ligand-binding domain of the EP402R protein wherein the amino acid change disrupts ligand-binding of the EP402R protein.
In another aspect the invention provides a method of producing an ASF virus of the invention, the method comprising changing one or more amino acid(s) in the EP402R protein at a position which corresponds to Q96 and/or W99 of the Georgia 2007/1 EP402R protein (SEQ ID No. 24).
In another aspect the invention provides a method of reducing the ability of an ASF virus to induce haemadsorption, the method comprising changing one or more amino acid(s) in the ligand-binding domain of the EP402R protein wherein the amino acid changes disrupt ligand-binding of the EP402R protein.
In another aspect the invention provides a method of reducing the ability of an ASF virus to induce haemadsorption, the method comprising changing one or more amino acid(s) in the EP402R protein at a position which corresponds to Q96 and/or W99 of the Georgia 2007/1 EP402R protein (SEQ ID No. 24).
In another aspect the invention provides a method of attenuating an ASF virus which comprises disrupting the expression and/or activity of the EP153R and EP402R genes.
African swine fever virus (ASFV) is the causative agent of African swine fever (ASF). The genome structure of ASFV is known in the art, as detailed in Chapman et al. 2008 J. Gen. Virol. 89: 397-408. ASFV is a large, icosahedral, double-stranded DNA virus with a linear genome containing at least 150 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, 22 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 II is circulating in a number of countries in Europe and Asia. Genotype IX is confined to several East African countries.
Examples of strains from some of the genotypes are given below:
In an embodiment, the ASF virus of the invention may be attenuated. The attenuated ASF virus of the invention may comprise any of the modifications/mutations described herein, in any combination. The modifications/mutations described herein may attenuate the ASF virus.
The attenuated ASF virus of the present invention may be derivable or be derived from a wild-type ASF virus isolate, by including mutations in its genome such that the expression and/or activity of the genes EP153R and EP402R is disrupted. The virus may also include a DIVA mutation, such as a DIVA mutation that disrupts expression of the K145R 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 warthog. ASFV isolates described to date are summarised in Table 1 below, together with their Genbank Accession numbers.
The genome of the attenuated ASFV of the invention may correspond to any ASFV genotype. The genome of the attenuated ASFV of the invention may essentially correspond to any ASFV genotype.
The term “corresponds to” means that the remainder of the genome of the attenuated ASFV of the invention is the same as a wild-type strain (i.e. a virus that existed at some point in the field). “The remainder of the genome” may refer to all genes other than the genes EP153R and EP402R. “The remainder of the genome” may refer to all genes other than the genes EP153R, EP402R and K145R. In other words, the genes of the attenuated ASFV of the invention may be the same as the genes of the wild-type strain except the genes that are disrupted according to the invention. The genes of the attenuated ASFV of the invention may be the same as the genes of the wild-type strain, except for the genes EP153R and EP402R. The genes of the attenuated ASFV of the invention may be the same as the genes of the wild-type strain, except for the genes EP153R, EP402R and K145R. In an embodiment the genes of the attenuated ASFV of the invention are the same as the genes of the wild-type strain, except for EP153R and EP402R. In an embodiment the genes of the attenuated ASFV of the invention are the same as the genes of the wild-type strain, except for EP153R, EP402R and K145R.
The disrupted genes may also correspond to the wild-type strain. In an embodiment the genes EP153R and EP402R correspond to the wild-type strain. In such an embodiment (i.e. where EP153R and EP402R correspond to the wild-type strain) expression and/or activity of EP153R and EP402R may be disrupted by one or more mutation in an intergenic region and/or non-coding sequence such as a promoter. In other words, the EP153R and EP402R genes are the same as in the wild-type genome but their expression or activity is altered by mutation of a non-genic sequence. Thus all of the genes of the attenuated ASFV of the invention may be the same as the genes of the wild-type strain. In an embodiment all genes of the attenuated ASFV of the invention are the same as the genes of the wild-type strain. In an embodiment all genes of the attenuated ASFV of the invention are the same as the genes of the wild-type strain, except for the K145R gene.
The term “essentially corresponds to” means the same as “corresponds to” with the additional exception that the remainder of the genome may comprise one or more mutations. The one or more mutations may be in other genes (i.e. not in the genes EP153R and EP402R, or not in the genes EP153R, EP402R and K145R).
The genome of the attenuated ASFV may correspond or essentially correspond to genotype I. The genome of the attenuated ASFV may correspond or essentially correspond to genotype II. The genome of the attenuated ASFV may correspond or essentially correspond to genotype III. The genome of the attenuated ASFV may correspond or essentially correspond to genotype IV. The genome of the attenuated ASFV may correspond or essentially correspond to genotype V. The genome of the attenuated ASFV may correspond or essentially correspond to genotype VI. The genome of the attenuated ASFV may correspond or essentially correspond to genotype VII. The genome of the attenuated ASFV may correspond or essentially correspond to genotype VIII. The genome of the attenuated ASFV may correspond or essentially correspond to genotype IX. The genome of the attenuated ASFV may correspond or essentially correspond to genotype X. The genome of the attenuated ASFV may correspond or essentially correspond to genotype XIV.
The genome of the attenuated ASFV may correspond or essentially correspond to genotype I. The genome of the attenuated ASFV may correspond or essentially correspond to genotype II.
Preferably, the genome of the attenuated ASFV may correspond or essentially correspond to genotype II.
The genome of the attenuated ASFV of the invention may correspond or essentially correspond to that of a virulent ASFV strain. Known virulent ASF virus strains include: Georgia 2007/1, Benin 97/1, Kenyan, Malawi Lil20/1, Pretorisuskop/96/4 and Tengani 62. The genome of the attenuated ASFV may correspond or essentially correspond to that of the Benin 97/1 strain.
The genome of the attenuated ASFV may correspond or essentially correspond to that of the Georgia 2007/1 strain.
The genome of the attenuated ASFV of the invention may correspond or essentially correspond to that of an ASFV strain whose virulence is currently unknown, for example: Mkuzi, Warmbaths and Warthog.
In an embodiment the genome of the attenuated ASFV of the invention does not correspond to that of OURT88/3. In an embodiment the genome of the attenuated ASFV of the invention does not correspond to that of NH/P68. In an embodiment the attenuated ASFV of the invention is not OURT88/3. In an embodiment the attenuated ASFV of the invention is not NH/P68. In an embodiment the attenuated ASFV of the invention is neither OURT88/3 nor NH/P68.
In certain aspects, the invention provides an ASF virus in which expression and/or activity of the EP402R gene has been disrupted.
In other aspects, the invention provides an EP402R protein comprising particular amino acid changes.
The EP402R gene encodes a protein which is incorporated in the external layer of the virus and is partly similar to the mammalian T-lymphocyte surface adhesion receptor CD2. In particular, the N-terminal extracellular region of the EP402R protein consists of two immunoglobulin-like (Ig-like) domains similar to the extracellular ligand-binding region of CD2. The EP402R protein may be referred to as CD2v due to this similarity. Accordingly the terms “EP402R” and “CD2v” may be used interchangeably herein. The N-terminal extracellular domain of the EP402R protein may be referred to as the “ligand-binding domain”. The cytoplasmic domain of EP402R protein is dissimilar to CD2.
EP402R is immunogenic (i.e. evokes an immune response) (Netherton et al. 2019 Front. Immunol. 10, 1318). EP402R is required for and directly involved in haemadsorption (Sereda et al. 2018 Slov. Vet. Res, 55(3) 141-150) and may have a role in virus entry or spread. Antibodies from ASFV infected pigs that inhibit haemadsorption can correlate with protection induced against diverse strains supporting a role for antibodies against EP402R in protection of pigs (Malogolovkin et al. 2015 J. Gen. Virol. 96(4) 866-873, Burmakina et al. 2016 J. Gen. Virol. 97(7) 1670-1675). EP402R can bind the host protein AP-1. The functions of EP402R may be mediated by its extracellular, N-terminal, Ig-like domain binding to ligands in the same manner that mammalian CD2 binds extracellular adhesion molecules.
The gene (i.e. nucleotide) sequences and positions in the genome of EP402R genes from different ASFV strains are presented below.
In an embodiment the invention provides an ASF virus in which the expression and/or activity of the EP402R gene is disrupted. Suitably, the EP402R gene comprises the sequence of SEQ ID No. 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240 or 241. Suitably, the EP402R gene comprises a sequence having at least 70%, at least 80%, at least 90% or at least 95% identity with SEQ ID No. 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240 or 241. Suitably, the EP402R gene consists of the sequence of SEQ ID No. 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240 or 241.
In an embodiment of the ASFV of the invention the EP402R gene may be partially or completely deleted. Suitably part or all of the sequence of SEQ ID No. 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240 or 241 is removed from the ASFV genome. Suitably the ASFV genome lacks any of these sequences.
The amino acid sequences of EP402R proteins from different ASFV strains are presented below as SEQ ID Nos 21 to 30 and SEQ ID Nos 242 to 246.
In an embodiment the invention provides an attenuated ASF virus in which the expression and/or activity of the EP402R gene is disrupted. Suitably, the EP402R gene encodes a protein comprising a sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity with any of SEQ ID Nos 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 242, 243, 244, 245 or 246. Suitably, the EP402R gene encodes a protein comprising the sequence of any of SEQ ID Nos 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 242, 243, 244, 245 or 246.
In an embodiment the ASFV of the invention does not express EP402R protein. In other words, the ASFV of the invention does not express any proteins with sequences of SEQ ID Nos 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 242, 243, 244, 245 or 246.
In one aspect the invention provides an EP402R protein comprising one or more amino acid changes in the ligand-binding domain wherein the amino acid changes disrupt ligand-binding of the EP402R protein.
The ligand-binding domain of EP402R is formed by amino acids starting at position 1 (N-terminus) and running to roughly position 200. For example, the ligand-binding domain of Benin 97/1 EP402R is formed by amino acids 1-198 of SEQ ID No. 21 which are presented below as SEQ ID No. 34.
The ligand-binding domain of Georgia 2007/1 EP402R is formed by amino acids 1-203 of SEQ ID No. 24 which are presented below as SEQ ID No. 380.
Suitably, the ligand-binding domain of the EP402R protein comprises the sequence of SEQ ID No. 34 or 380. Suitably, the ligand-binding domain of the EP402R protein comprises a sequence having at least 70%, 80%, 90% or at least 95% identity with SEQ ID No. 34 or 380. The ligand-binding domain of the EP402R protein from other strains can be readily identified by alignment with the sequence of Benin 97/1 EP402R protein and/or Georgia 2007/1 EP402R protein, such as shown in
Suitably one or more amino acids (such as two or more, three or more, four or more or five or more amino acids) in the ligand-binding domain of the EP402R protein are changed compared to the wild type EP402R protein. In an embodiment, the one or more amino acids in the ligand-binding domain are deleted (i.e. removed entirely). In an embodiment, the one or more amino acids in the ligand-binding domain are changed to different amino acids (i.e. replaced). Such an amino change may be referred to as a substitution. Changing one or more amino acids in the ligand-binding domain of the EP402R protein may disrupt expression and/or activity of the EP402R protein. In an embodiment, changing one or more amino acids in the ligand-binding domain of the EP402R protein may disrupt haemadsorption mediated by the EP402R protein.
The amino acid changes in the EP402R protein are caused by one or more mutations in the sequence coding for the EP402R protein. In an embodiment, the one or more mutations may be a deletion or an interruption as described herein. For example, deletion of part of the coding sequence for the ligand-binding domain of the EP402R protein would result in the absence of the encoded amino acids (i.e. changing the amino acids) from the ligand-binding domain, which may disrupt ligand binding.
In an embodiment, one or more of the mutations may be a point mutation. Suitably one or more of the mutations may be point mutation that changes a single amino acid into a different amino acid. Changing even a single amino acid may disrupt expression and/or activity of the EP402R protein, such as ligand binding activity.
Changing one or more amino acids may disrupt folding of the EP402R protein. Disruption of folding may mean the EP402R protein cannot fold at all causing it to be degraded by the cellular protein degradation machinery. Alternatively, the disruption of folding may mean the EP402R protein is folded differently, impairing its function, or the EP402R protein may be folded more slowly and so is not correctly expressed (e.g. it is not expressed at the cell surface).
Changing a charged amino acid to an amino acid with the opposite charge may disrupt folding of the EP402R ligand-domain such that a binding pocket is deformed, which prevents ligand binding due to steric hindrance. Alternatively or additionally, substitution with an amino acid with the opposite charge may prevent electrostatic binding of a ligand. Another possibility is that changing an amino acid with a small side-chain to an amino acid with a bulky side-chain, or vice versa, may disrupt folding of the EP402R ligand-binding domain so that a binding pocket does not form which prevents ligand binding.
Thus in an embodiment the one or more changed amino acids in the ligand-binding domain may comprise a negatively charged amino acid that is changed to a positively charged amino acid. In an embodiment the one or more changed amino acids in the ligand-binding domain may comprise a positively charged amino acid that is changed to a negatively charged amino acid. Positively charged amino acids (i.e. amino acids that can have a positive charge) include histidine (H), lysine (K) and arginine (R). Negatively charged amino acids (i.e. amino acids that can have a negative charge) include aspartic acid (D) and glutamic acid (E).
In an embodiment the one or more changed amino acids in the ligand-binding domain may comprise an amino acid with a small side-chain that is changed to an amino acid with a bulky side-chain. In an embodiment the one or more changed amino acids in the ligand-binding domain may comprise an amino acid with a bulky side-chain that is changed to an amino acid with a small side-chain. Amino acids with a bulky side-chain include tryptophan (W). Amino acids with a small side-chain include glycine (G) and alanine (A).
In an embodiment the one or more changed amino acids in the ligand-binding domain may comprise an amino acid with a hydrophilic side-chain that is changed to an amino acid with a hydrophobic side-chain. In an embodiment the one or more changed amino acids in the ligand-binding domain may comprise an amino acid with a hydrophobic side-chain that is changed to an amino acid with a hydrophilic side-chain. Amino acids with a hydrophobic side-chain include alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y) and tryptophan (W). Amino acids with a hydrophilic side-chain include arginine (R), histidine (H), lysine (K), aspartic acid (D), glutamic acid (E), serine (S), threonine (T), asparagine (N) and glutamine (Q).
Changing amino acid E99 to R or amino acid Y102 to D disrupts the ability of EP402R protein from Benin 97/1 strain to mediate haemadsorption (see
Whether an amino acid corresponds to Q96 and/or W99 of the Georgia 2007/1 EP402R protein (or to E99 and/or Y102 of the Benin 97/1 EP402R protein) may be assessed by sequence alignment. For example,
Amino acids corresponding to Q96 and/or W99 of the Georgia 2007/1 EP402R protein and E99 and Y102 of Benin 97/1 EP402R exist in EP402R proteins from strains other than those shown in
Suitably the amino acid at the position which corresponds to Q96 is changed to R or to an amino acid that is a conservative replacement of R and/or the amino acid at the position which corresponds to W99 is changed to D or to an amino acid that is a conservative replacement of D. An amino acid that is “conservative replacement” has similar characteristics to the other amino acid. For example, the conservative replacement may have similar charge (positive or negative), similar hydrophobicity (hydrophilic or hydrophobic) or similar molecular size, be also aromatic, or have a combination of these characteristics. Suitably the amino acid at the position which corresponds to Q96 is changed to H, K or R and/or the amino acid at the position which corresponds to W99 is changed to D, E, N or Q. Suitably the amino acid at the position which corresponds to Q96 is changed to R and/or the amino acid at the position which corresponds to W99 is changed to D.
In an embodiment the EP402R protein of the invention comprises an amino acid sequence having at least 70% sequence identity, such as at least 75% identity, such as at least 80% identity, such as at least 85% identity, such as at least 90% identity, such as least 95% identity, such as least 96% identity, such as least 97% identity, such as least 98% identity, such as least 99% identity, with any of SEQ ID Nos 21 to 30 or SEQ ID Nos 242 to 246 (i.e. the sequences of EP402R protein from different ASFV strains as described herein). Suitably the EP402R protein of the invention comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID No. 21. Suitably the EP402R protein of the invention comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID No. 22. Suitably the EP402R protein of the invention comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID No. 23. Suitably the EP402R protein of the invention comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID No. 24. Suitably the EP402R protein of the invention comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID No. 25. Suitably the EP402R protein of the invention comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID No. 26. Suitably the EP402R protein of the invention comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID No. 27. Suitably the EP402R protein of the invention comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID No. 28. Suitably the EP402R protein of the invention comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID No. 29. Suitably the EP402R protein of the invention comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID No. 30. Suitably the EP402R protein of the invention comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID No. 242. Suitably the EP402R protein of the invention comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID No. 243. Suitably the EP402R protein of the invention comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID No. 244. Suitably the EP402R protein of the invention comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID No. 245. Suitably the EP402R protein of the invention comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID No. 246.
In certain embodiments, the amino acids that differ between the reference sequence (i.e. SEQ ID Nos 21 to 30 or SEQ ID Nos 242 to 246) and the sequence that is less than 100% identical to the reference sequence are conservative replacements.
The amino acid sequence of the EP402R protein from Benin 97/1 strain comprising the E99R amino acid change is depicted below as SEQ ID No. 31.
The amino acid sequence of the EP402R protein from Benin 97/1 strain comprising the Y102D substitution is depicted below as SEQ ID No. 32.
The amino acid sequence of the EP402R protein from Georgia 2007/1 strain comprising the Q96R substitution is depicted below as SEQ ID No. 33.
The amino acid sequence of the EP402R protein from Georgia 2007/1 strain comprising the W99D substitution is depicted below as SEQ ID No. 379.
In an embodiment the EP402R protein of the invention comprises the amino acid sequence of any of SEQ ID Nos 31, 32, 33 or 379. Suitably the EP402R protein of the invention comprises or consists of the amino acid sequence of SEQ ID No. 31 (Benin 97/1 EP402R protein with E99R amino acid change). Suitably the EP402R protein of the invention comprises or consists of the amino acid sequence of SEQ ID No. 32 (Benin 97/1 EP402R protein with Y102D amino acid change). Suitably the EP402R protein of the invention comprises or consists of the amino acid sequence of SEQ ID No. 33 (Georgia 2007/1 EP402R protein with Q96R amino acid change). Suitably the EP402R protein of the invention comprises or consists of the amino acid sequence of SEQ ID No. 379 (Georgia 2007/1 EP402R protein with W99D amino acid change).
In an embodiment the EP402R protein of the invention comprises one or more amino acid changes at positions in the EP402R protein corresponding to N16, 119, W21, Y76, E99, Y102 and/or N108 of Benin 97/1 EP402R protein (SEQ ID NO. 21). In an embodiment the one or more mutations change the amino acid at position N16, 119, W21, Y76, E99, Y102 and/or N108 of the EP402R protein of the Benin 97/1 strain, or the corresponding position in the EP402R protein of any other ASFV strain. In an embodiment the one or more mutations change an amino acid at a position in the EP402R protein corresponding to S15, W19, Q96, N104, and/or K108D of Georgia 2007/1 EP402R protein (SEQ ID NO. 24). In an embodiment the one or more mutations change the amino acid at position S15, W19, Q96, N104, and/or K108D of the EP402R protein of the Georgia 2007/1 EP402R protein (SEQ ID NO. 24), or the corresponding position in the EP402R protein of any other ASFV strain. In an embodiment the one or more mutations change an amino acid at a position in the EP402R protein corresponding to W20, Q112, N121 and/or R125 of N10 Genotype IX EP402R protein (SEQ ID NO. 27). In an embodiment the one or more mutations change the amino acid at position S15, W19, Q96, N104, and/or K108D of the EP402R protein of the N10 Genotype IX EP402R protein (SEQ ID NO. 27), or the corresponding position in the EP402R protein of any other ASFV strain. These amino acids are in the ligand-binding domain of the EP402R protein and are surface exposed.
Suitably, the mutation is selected from N16R, 119R, W21D, Y 76D, E99R, Y102D and/or N108R at a position corresponding to the position in the Benin 97/1 EP402R protein (SEQ ID NO. 11). Suitably, the mutation is a combination of E99R and N108R at the positions corresponding to the positions in the Georgia 2007/1 EP402R protein (SEQ ID NO. 11).
Suitably, the mutation is selected from S15R, W19D, Q96R, N104R and/or K108D at a position corresponding to the position in the Georgia 2007/1 EP402R protein (SEQ ID NO. 24). Suitably, the mutation is a combination of Q96R and N104R at the positions corresponding to the positions in the Georgia 2007/1 EP402R protein (SEQ ID NO. 24).
Suitably, the mutation is selected from W20D, Q112R, N121R and/or R125D at a position corresponding to the position in the N10 Genotype IX EP402R protein (SEQ ID NO. 27). Suitably, the mutation is a combination of Q112R and N121R at the positions corresponding to the positions in the N10 Genotype IX EP402R protein (SEQ ID NO. 27).
Suitably, where the attenuated ASFV is genotype I, the one or more mutations change an amino acid at a position in the EP402R protein corresponding to Y76, E99, Y102, and/or N108 of Benin 97/1 EP402R protein (SEQ ID NO. 11). Suitably, where the attenuated ASFV is genotype I, the mutation is selected from Y76D, E99R, Y102D, and/or N108R at a position corresponding to the position in the Benin 97/1 EP402R protein (SEQ ID NO. 11). Suitably, where the attenuated ASFV is genotype II, the one or more mutations change an amino acid at a position in the EP402R protein corresponding to S15, W19, Q96, N104, and/or K108D of Georgia 2007/1 EP402R protein (SEQ ID NO. 24). Suitably, where the attenuated ASFV is genotype II, the mutation is selected from S15R, W19D, Q96R, N104R and/or K108D at a position corresponding to the position in the Georgia 2007/1 EP402R protein (SEQ ID NO. 24). Suitably, where the attenuated ASFV is genotype II, the mutation may be a combination of Q96R and N104R at the positions corresponding to the positions in the Georgia 2007/1 EP402R protein (SEQ ID NO. 24).
The invention also provides a polynucleotide encoding the EP402R protein of the invention. Suitably the polynucleotide comprises a sequence having at least 70% identity, such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, with any of SEQ ID Nos 229 to 241 (i.e. the coding sequences of the EP402R genes from different strains of ASFV). Suitably the polynucleotide comprises a sequence having at least 70% identity, such as at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, with SEQ ID No. 229 (i.e. the coding sequences of the Georgia 2007/1 EP402R gene).
The invention also provides a vector comprising the polynucleotide of the invention. Suitable vectors are known in the art and include plasmids for protein expression in cells such as bacteria, yeast or vertebrate cells, among or other cell types known in the art.
In another aspect the invention provides an ASF virus comprising the EP402R protein of the invention, such as described above. In a similar aspect the invention provides an ASF virus comprising the polynucleotide of the invention, such as described above. In another aspect the invention provides an attenuated ASF virus as described herein, wherein the virus comprises an EP402R gene mutated to express an EP402R protein of the invention, such as the EP402R protein described above.
The skilled person will appreciate from the present disclosure that an ASF virus can be mutated to express an EP402R protein comprising any of the amino acid changes described herein by appropriately modifying the coding sequence of the EP402R gene using molecular biology techniques known in the art. Thus the invention encompasses ASF viruses comprising any EP402R protein of the invention disclosed herein. Additionally, the skilled person will appreciate from the present disclosure that any EP402R protein described herein may be combined in an ASF virus of the invention with the other modifications to the ASF virus described herein, in particular modification of the K145R and EP153R genes. Furthermore, the ASFV of the invention comprising the EP402R protein with amino acid changes of the invention may be attenuated.
In an embodiment of the ASF virus of the invention, the EP402R gene comprises one or more mutations that change one or more amino acids in the ligand-binding domain of the EP402R protein. Suitably the EP402R gene comprises one or more mutations (such as two or more, three or more, four or more or five or more mutations) that change one or more amino acids (such as two or more, three or more, four or more or five or more amino acids) in the ligand-binding domain of the EP402R protein.
In embodiments of the ASF virus of the invention, the ASF virus comprises one or more mutations in the EP402R gene that change one or more amino acids in the EP402R protein in any of the ways described herein. For example, in an embodiment the one or more mutations in the EP402R gene change the amino acid at position Q96 and/or W99 of the EP402R protein of the Georgia 2007/1 strain, or the corresponding position in the EP402R protein of any other ASFV strain. In an embodiment the one or more mutations in the EP402R gene change the amino acid at position Q96 of the EP402R protein of the Georgia 2007/1 strain (SEQ ID No. 24) to R.
In an embodiment of the ASFV of the invention, EP402R activity is disrupted.
In an embodiment the activity of EP402R that is disrupted is its ability to mediate haemadsorption. In other words, the ability of EP402R to mediate haemadsorption may be decreased. The ability of EP402R to mediate haemadsorption may be decreased by at least 50, 60, 70, 80 or 90%. The ability of EP402R to mediate haemadsorption may be completely abolished.
The activity of EP402R that is disrupted may be the ability of the EP402R protein to bind ligands via its extracellular N-terminal Ig-like domain (i.e. its ligand-binding domain). Thus in an embodiment the invention provides an ASF virus wherein the EP402R gene comprises one or more mutations that disrupt ligand binding by the EP402R protein. The ability of the EP402R protein to bind one or more ligands may be disrupted. The ability of EP402R to bind one or more ligands may be decreased by at least 50, 60, 70, 80 or 90%. The ability of the EP402R protein to bind one or more ligands may be completely abolished. The ability of the EP402R protein to bind one or more ligands may be completely abolished whilst retaining the ability to bind other ligands. In an embodiment, the invention provides an attenuated ASF virus wherein the EP402R gene comprises one or more mutations that disrupt ligand binding by the EP402R protein. Ligand binding may be measured using assays such as immunoprecipitation, surface plasmon resonance and/or isothermal calorimetry.
In an embodiment the invention provides an ASF virus wherein the changed amino acids in the EP402R protein directly inhibit the interaction between EP402R and its ligand by changing the binding surface on EP402R, as described herein.
In an embodiment the invention provides an ASF virus wherein surface expression of the EP402R protein is reduced compared to a corresponding ASF virus in which the expression and/or activity of the EP402R gene is not disrupted. “Surface expression” of EP402R refers to expression of the EP402R protein on the surface of cells infected with ASF virus. Surface expression of EP402R may be measured by techniques known in the art, such as antibody staining of infected cells (see for example
In certain aspects, the invention provides an ASF virus in which expression and/or activity of the EP153R gene has been disrupted.
The EP153R gene is expressed both early and late in infection. EP153R may also be referred to as 8CR. The EP153R protein is a C-type lectin. C-type animal lectins are found in serum, the extracellular matrix and cell membranes and are thought to act as receptors for carbohydrate ligands. The EP153R protein comprises a C-type lectin domain, a cell attachment sequence (RGD) and a transmembrane domain, and has similarity with CD44 molecules involved in T cell activation.
The gene (i.e. nucleotide) sequences and positions in the genome of EP153R genes from different ASFV strains are presented below.
In an embodiment the invention provides an ASF virus in which the expression and/or activity of the EP153R gene is disrupted. Suitably, the EP153R gene comprises the sequence of SEQ ID No. 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215 or 216. Suitably, the EP153R gene comprises a sequence having at least 70%, at least 80%, at least 90% or at least 95% identity with SEQ ID No. 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215 or 216. Suitably, the EP153R gene consists of the sequence of SEQ ID No. 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215 or 216.
In an embodiment of the ASFV of the invention the EP153R gene may be partially or completely deleted. Suitably part or all of the sequence of SEQ ID No. 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215 or 216 is removed from the ASFV genome. Suitably the ASFV genome lacks any of these sequences.
The accession numbers of EP153R proteins from different strains of ASFV are listed below in Table 3.
The amino acid sequences of the EP153R proteins from different ASFV strains are depicted below.
Suitably, the EP153R gene encodes a protein comprising the sequence of SEQ ID No. 20, 217, 218, 219, 220, 221, 222, 223, 224, 225 226, 227 or 228. Suitably, the EP153R gene encodes a protein comprising a sequence having at least 70%, at least 80%, at least 90% or at least 95% identity with SEQ ID No. 20, 217, 218, 219, 220, 221, 222, 223, 224, 225 226, 227 or 228. Suitably, the EP153R gene encodes a protein consisting of the sequence of SEQ ID No. 20, 217, 218, 219, 220, 221, 222, 223, 224, 225 226, 227 or 228.
In an embodiment the ASFV of the invention does not express EP153R protein. In other words, the ASFV of the invention does not express any proteins with sequences of SEQ ID Nos 20, 217, 218, 219, 220, 221, 222, 223, 224, 225 226, 227 or 228.
EP153R is immunogenic (i.e. evokes an immune response) (Burmakina et al. 2019 J. Gen. Virol. 100: 259-265). EP153R inhibits capsase-3 activation during ASFV infection and thereby has an inhibitory effect on apoptosis. EP153R is required for haemadsorption. EP153R also inhibits MHC-I expression in infected cells.
In an embodiment of the attenuated ASFV of the invention, EP153R activity may be disrupted.
In an embodiment the activity of EP153R that may be disrupted is its ability to mediate haemadsorption. In other words, the ability of EP153R to mediate haemadsorption may be decreased. The ability of EP153R to mediate haemadsorption may be decreased by at least 50, 60, 70, 80 or 90%. The ability of EP153R to mediate haemadsorption may be completely abolished.
In an embodiment the activity of EP153R that may be disrupted is its ability to inhibit caspase-3. In other words, the ability of EP153R to inhibit caspase-3 may be decreased. The ability of EP153R to inhibit caspase-3 may be decreased by at least 50, 60, 70, 80 or 90%. The ability of EP153R to inhibit caspase-3 may be completely abolished. Caspase-3 activity may be measured by assays known in the art, such as described by Hurtado et al. (Hurtado et al. 2004 Virology 326: 160-170). For example, the cleaved active caspase-3 fragment of 17 kDa and the inactive procaspase-3 protein of 34 kDa may be quantified using Western blot or mass spectrometry and compared to ascertain the degree of activation of caspase-3. Alternatively or additionally, the ability of caspase-3 in cell extract to cleave a fluorescent substrate may be measured using high performance liquid chromatography.
In an embodiment the activity of EP153R that may be disrupted is its ability to inhibit MHC-I expression. In other words, the ability of EP153R to inhibit MHC-I expression may be decreased. The ability of EP153R to inhibit MHC-I expression may be decreased by at least 50, 60, 70, 80 or 90%. The ability of EP153R to inhibit MHC-I expression may be completely abolished. MHC-I expression may be measured by assays known in the art, such as described by Hurtado et al. (Hurtado et al. 2011 Arch. Virol. 156(2): 219-234). For example, cell surface expression of MHC-I may be measured using antibody staining of non-permeabilised cells followed by flow cytometry.
In an embodiment the ASFV of the invention comprises a Differentiation of Infected from Vaccinated Animals (DIVA) mutation. A DIVA mutation is a mutation in the ASF virus that enables a vaccine comprising the ASF virus to function as a DIVA vaccine (i.e. subjects vaccinated with the DIVA vaccine can be differentiated from subjects infected with a wild-type ASF virus).
In an embodiment the invention provides an attenuated ASF virus in which the expression and/or activity of the genes EP153R and EP402R is disrupted;
which comprises a functional version of one or more of the following genes:
In an embodiment the invention provides an attenuated ASF virus in which the expression and/or activity of the genes EP153R and EP402R is disrupted;
and which comprises a functional version of one or more of the following genes:
In an embodiment the invention provides an attenuated ASF virus in which the expression and/or activity of the genes EP153R and EP402R is disrupted;
and which comprises a functional version of one or more of the following genes:
In an embodiment the invention provides an ASF virus comprising an EP402R protein comprising one or more amino acid change in the ligand-binding domain wherein the amino acid change disrupts ligand-binding of the EP402R protein (i.e. an EP402 protein of the invention) and/or a polynucleotide encoding said EP402R protein, and further comprising a DIVA mutation.
In an embodiment the DIVA mutation disrupts expression of the K145R gene and/or the B125R gene. Suitably the DIVA mutation disrupts expression of the K145R gene. Suitably the K145R gene is partially deleted. Suitably the K145R gene is completely deleted. Suitably the B125R gene is partially deleted. Suitably the B125R gene is completely deleted.
The K145R gene is a late gene.
The gene (i.e. nucleotide) sequences of K145R genes from different ASFV strains are given below.
In an embodiment an ASFV of the invention comprises a DIVA mutation that disrupts expression of the K145R gene. In other words, the ASFV lacks a functional version of the K145R gene. Suitably the K145R gene comprises the sequence of SEQ ID No. 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338 or 339. Suitably the K145R gene comprises a sequence having at least 70%, at least 80%, at least 90% or at least 95% identity with SEQ ID No. 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338 or 339. Suitably the K145R gene consists of the sequence of SEQ ID No. 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338 or 339.
In an embodiment of the ASFV of the invention the K145R gene may be partially or completely deleted. Suitably part or all of the sequence of SEQ ID No. 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338 or 339 is removed from the ASFV genome. Suitably the ASFV genome lacks any of these sequences.
The accessions numbers of K145R proteins from different strains of ASFV are listed below in Table 4.
The amino acid sequence of K145R proteins from different ASFV strain are depicted below.
In an embodiment the ASFV of the invention does not express K145R protein. In other words, the ASFV of the invention does not express any proteins with sequences of SEQ ID Nos 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351 or 352.
K145R inhibits the host endoplasmic reticulum (ER) stress response (Barber 2015 Stress modulators encoded by African swine fever virus; PHD thesis, St Georges, University of London, 2016). This response is caused by the accumulation of unfolded proteins and may be activated during viral infections due to the substantial amounts of viral proteins being produced. ER stress leads to the increase in expression of the transcription factor CCAAT-enhancer-binding protein homologous protein (CHOP) and its accumulation in the nucleus of the cells. CHOP activity ultimately results in cell apoptosis, thus limiting viral replication.
K145R function may be tested by methods including immunofluorescence using an antibody against CHOP and assessment of its presence in the nucleus of cells following induction of ER stress, and luciferase reporter assay, where the luciferase gene is under control of the CHOP promoter.
The gene (i.e. nucleotide) sequences of B125R genes from different ASFV strains are given below.
In an embodiment an ASFV of the invention comprises a DIVA mutation that disrupts expression of the B125R gene. In other words, the ASFV lacks a functional version of the B125R gene. Suitably the B125R gene comprises the sequence of SEQ ID No. 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364 or 365. Suitably the B125R gene comprises a sequence having at least 70%, at least 80%, at least 90% or at least 95% identity with SEQ ID No. 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364 or 365. Suitably the B125R gene consists of the sequence of SEQ ID No. 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364 or 365.
In an embodiment of the ASFV of the invention the B125R gene may be partially or completely deleted. Suitably part or all of the sequence of SEQ ID No. 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364 or 365 is removed from the ASFV genome. Suitably the ASFV genome lacks any of these sequences.
The accessions numbers of B125R proteins from different strains of ASFV are listed below in Table 5.
The amino acid sequences of B125R proteins from different ASFV strains are depicted below.
In an embodiment the ASFV of the invention does not express B125R protein. In other words, the ASFV of the invention does not express any proteins with sequences of SEQ ID Nos 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377 or 378.
B125R was identified as one of the most abundant viral proteins expressed in infected wild boar cells (WSL-R) (Kaβler et al. 2018 Sci. Rep. 8: 1471). As shown in
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, 110, 300, 360 and 505/530. The N-terminal regions of members of MGFs 300, 360 and 505/530 share significant similarity with each other. It has been shown the MGF 360 and 505 families encode genes essential for host range function that involves promotion of infected-cell survival and suppression of type I interferon response.
An attenuated ASFV according to the present invention comprises a functional version of one or more of the following genes:
In a further aspect the invention provides an attenuated African Swine Fever (ASF) virus in which the expression and/or activity of the genes EP153R and EP402R is disrupted;
and which comprises a functional version of one or more of the following genes:
Suitably, in one embodiment the invention provides an ASFV in which the expression and/or activity of the genes EP153R and EP402R is disrupted;
and which comprises a functional version of one or more of the following genes:
Suitably, in one embodiment the invention provides an ASFV in which the expression and/or activity of the genes EP153R and EP402R is disrupted;
and which comprises a functional version of one or more of the following genes:
Suitably, in one embodiment the invention provides an ASFV in which the expression and/or activity of the genes EP153R and EP402R is disrupted;
and which comprises a functional version of one or more of the following genes:
Suitably, in one embodiment the invention provides an ASFV in which the expression and/or activity of the genes EP153R and EP402R is disrupted;
and which comprises a functional version of one or more of the following genes:
The location of some of these genes in the genomes of a variety of ASFV strains is provided below in Table 6. The sequence identity of each gene to the corresponding Benin 97/1 gene is also provided.
The gene (i.e. nucleotide) sequences of these genes from different strains are given below.
In an embodiment the attenuated ASFV of the invention comprises a functional version of MGF 110 5L. Suitably the functional version of MGF 110 5L comprises the sequence of SEQ ID No. 266, 267, 268, 269, 270, 271, 272, 273, 274 or 275. Suitably the functional version of MGF 110 5L comprises a sequence having at least 70%, at least 80%, at least 90% or at least 95% identity with SEQ ID No. 266, 267, 268, 269, 270, 271, 272, 273, 274 or 275. Suitably the functional version of MGF 110 5L consists of the sequence of SEQ ID No. 266, 267, 268, 269, 270, 271, 272, 273, 274 or 275.
In an embodiment the attenuated ASFV of the invention comprises a functional version of MGF 110 6L. Suitably the functional version of MGF 110 6L comprises the sequence of SEQ ID No. 35, 36, 37, 38, 39, 40, 41, 42 or 43. Suitably the functional version of MGF 110 6L comprises a sequence having at least 70%, at least 80%, at least 90% or at least 95% identity with SEQ ID No. 35, 36, 37, 38, 39, 40, 41, 42 or 43. Suitably the functional version of MGF 110 6L consists of the sequence of SEQ ID No. 35, 36, 37, 38, 39, 40, 41, 42 or 43.
In an embodiment the attenuated ASFV of the invention comprises a functional version of MGF 110 7L. Suitably the functional version of MGF 110 7L comprises the sequence of SEQ ID No. 247, 248, 249, 250, 251, 252, 253, 254, 255 or 256. Suitably the functional version of MGF 110 7L comprises a sequence having at least 70%, at least 80%, at least 90% or at least 95% identity with SEQ ID No. 247, 248, 249, 250, 251, 252, 253, 254, 255 or 256. Suitably the functional version of MGF 110 7L consists of the sequence of SEQ ID No. 247, 248, 249, 250, 251, 252, 253, 254, 255 or 256.
In an embodiment the attenuated ASFV of the invention comprises a functional version of MGF 110 8L. Suitably the functional version of MGF 110 8L comprises the sequence of SEQ ID No. 44, 45, 46, 47, 48, 49 or 50. Suitably the functional version of MGF 110 8L comprises a sequence having at least 70%, at least 80%, at least 90% or at least 95% identity with SEQ ID No. 44, 45, 46, 47, 48, 49 or 50. Suitably the functional version of MGF 110 8L consists of the sequence of SEQ ID No. 44, 45, 46, 47, 48, 49 or 50.
In an embodiment the attenuated ASFV of the invention comprises a functional version of MGF 110 12L. Suitably the functional version of MGF 110 8L comprises the sequence of SEQ ID No. 276, 277, 278, 279, 280, 281, 282, 283, 284, 285 or 286. Suitably the functional version of MGF 110 12L comprises a sequence having at least 70%, at least 80%, at least 90% or at least 95% identity with SEQ ID No. 276, 277, 278, 279, 280, 281, 282, 283, 284, 285 or 286. Suitably the functional version of MGF 110 12L consists of the sequence of SEQ ID No. 276, 277, 278, 279, 280, 281, 282, 283, 284, 285 or 286.
In an embodiment the attenuated ASFV of the invention comprises a functional version of MGF 360 6L. Suitably the functional version of MGF 360 6L comprises the sequence of SEQ ID No. 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or 61. Suitably the functional version of MGF 360 6L comprises a sequence having at least 70%, at least 80%, at least 90% or at least 95% identity with SEQ ID No. 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or 61. Suitably the functional version of MGF 360 6L consists of the sequence of SEQ ID No. 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or 61.
In an embodiment the attenuated ASFV of the invention comprises a functional version of MGF 360 10L. Suitably the functional version of MGF 360 10L comprises the sequence of SEQ ID No. 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 or 74. Suitably the functional version of MGF 360 10L comprises a sequence having at least 70%, at least 80%, at least 90% or at least 95% identity with SEQ ID No. 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 or 74. Suitably the functional version of MGF 360 10L consists of the sequence of SEQ ID No. 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 or 74.
In an embodiment the attenuated ASFV of the invention comprises a functional version of MGF 360 11L. Suitably the functional version of MGF 360 11L comprises the sequence of SEQ ID No. 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 or 87. Suitably the functional version of MGF 360 11L comprises a sequence having at least 70%, at least 80%, at least 90% or at least 95% identity with SEQ ID No. 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 or 87. Suitably the functional version of MGF 360 11L consists of the sequence of SEQ ID No. 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 or 87.
In an embodiment the attenuated ASFV of the invention comprises a functional version of MGF 360 12L. Suitably the functional version of MGF 360 12L comprises the sequence of SEQ ID No. 88, 89, 90, 91, 92, 93, 94 95, 96, 97, 98 or 99. Suitably the functional version of MGF 360 12L comprises a sequence having at least 70%, at least 80%, at least 90% or at least 95% identity with SEQ ID No. 88, 89, 90, 91, 92, 93, 94 95, 96, 97, 98 or 99. Suitably the functional version of MGF 360 12L consists of the sequence of SEQ ID No. 88, 89, 90, 91, 92, 93, 94 95, 96, 97, 98 or 99.
In an embodiment the attenuated ASFV of the invention comprises a functional version of MGF 360 13L. Suitably the functional version of MGF 360 13L comprises the sequence of SEQ ID No. 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 or 111. Suitably the functional version of MGF 360 13L comprises a sequence having at least 70%, at least 80%, at least 90% or at least 95% identity with SEQ ID No. 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 or 111. Suitably the functional version of MGF 360 13L consists of the sequence of SEQ ID No. 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 or 111.
In an embodiment the attenuated ASFV of the invention comprises a functional version of MGF 360 14L. Suitably the functional version of MGF 360 14L comprises the sequence of SEQ ID No. 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, or 123. Suitably the functional version of MGF 360 14L comprises a sequence having at least 70%, at least 80%, at least 90% or at least 95% identity with SEQ ID No. 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, or 123. Suitably the functional version of MGF 360 14L consists of the sequence of SEQ ID No. 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, or 123.
In an embodiment the attenuated ASFV of the invention comprises a functional version of MGF 360 21R. Suitably the functional version of MGF 360 21R comprises the sequence of SEQ ID No. 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133. Suitably the functional version of MGF 360 21R comprises a sequence having at least 70%, at least 80%, at least 90% or at least 95% identity with SEQ ID No. 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133. Suitably the functional version of MGF 360 21R consists of the sequence of SEQ ID No. 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133.
In an embodiment the attenuated ASFV of the invention comprises a functional version of MGF 505 1R. Suitably the functional version of MGF 505 1R comprises the sequence of SEQ ID No. 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145 or 146. Suitably the functional version of MGF 505 1R comprises a sequence having at least 70%, at least 80%, at least 90% or at least 95% identity with SEQ ID No. 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145 or 146. Suitably the functional version of MGF 505 1R consists of the sequence of SEQ ID No. 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145 or 146.
In an embodiment the attenuated ASFV of the invention comprises a functional version of MGF 505 2R. Suitably the functional version of MGF 505 2R comprises the sequence of SEQ ID No. 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157 or 158. Suitably the functional version of MGF 505 2R comprises a sequence having at least 70%, at least 80%, at least 90% or at least 95% identity with SEQ ID No. 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157 or 158. Suitably the functional version of MGF 505 2R consists of the sequence of SEQ ID No. 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157 or 158.
In an embodiment the attenuated ASFV of the invention comprises a functional version of MGF 505 6R. Suitably the functional version of MGF 505 6R comprises the sequence of SEQ ID No. 257, 258, 259, 260, 261, 262, 263, 264 or 265. Suitably the functional version of MGF 505 2R comprises a sequence having at least 70%, at least 80%, at least 90% or at least 95% identity with SEQ ID No. 257, 258, 259, 260, 261, 262, 263, 264 or 265. Suitably the functional version of MGF 505 6R consists of the sequence of SEQ ID No. 257, 258, 259, 260, 261, 262, 263, 264 or 265.
In an embodiment the invention provides an ASFV which comprises one or more, such as all, of the following sequences from the Benin 97/1 strain:
In an embodiment the invention provides an ASFV which comprises one or more, such as all, of the following sequences from the China/2018/AnhuiXCGQ strain:
In an embodiment the invention provides an ASFV which comprises one or more, such as all, of the following sequences from the Georgia 2007/1 strain:
In an embodiment the invention provides an ASFV which comprises one or more, such as all, of the following sequences from the Ken05/Tk1 strain: SEQ ID No. 35 (MGF 110 6L), SEQ ID No. 44 (MGF 110 8L), and
In an embodiment the invention provides an ASFV which comprises one or more, such as all, of the following sequences from the Ken06.Bus strain:
In an embodiment the invention provides an ASFV which comprises one or more, such as all, of the following sequences from the Kenya 1950 strain:
In an embodiment the invention provides an ASFV which comprises one or more, such as all, of the following sequences from the L60 strain:
In an embodiment the invention provides an ASFV which comprises one or more, such as all, of the following sequences from the Malawi Lil-20/1 (1983) strain:
In an embodiment the invention provides an ASFV which comprises one or more, such as all, of the following sequences from the Mkuzi 1979 strain:
In an embodiment the invention provides an ASFV which comprises one or more, such as all, of the following sequences from the Pretorisuskop/96/4 strain:
In an embodiment the invention provides an ASFV which comprises one or more, such as all, of the following sequences from the Tengani 62 strain:
In an embodiment the invention provides an ASFV which comprises one or more, such as all, of the following sequences from the Warmbaths strain:
In an embodiment the invention provides an ASFV which comprises one or more, such as all, of the following sequences from the Warthog strain:
The translation products (i.e. protein sequences) of these genes are given below:
The DP148R gene is located close to the right end of the ASFV genome, at position 177915 to 178679 on the Benin 97/1 genome. The DP148R gene may also be referred to as MGF 360 18R. DP148R is expressed at early times post-infection. The amino acid sequence of the DP148R protein has no significant similarity to other proteins; the secondary structure is predicted to be predominantly helical, but no signal peptide or transmembrane domains are evident.
DP148R inhibits type I interferon. DP148R also inhibits activation of the NF-kB transcription factor (see
The gene (i.e. nucleotide) sequences and positions in the genome of DP148R genes from different ASFV strains are presented below.
The amino acid sequences of DP148R proteins from different ASFV strains is depicted below as SEQ ID Nos 11 to 19 and 301 to 305:
Haemadsorption is the phenomenon whereby cells infected with ASFV adsorb erythrocytes (red blood cells) on their surface. The degree of haemadsorption induced by an ASFV may be measured using a haemadsorption (HAD) assay such as described herein (see for example Examples 1 and 3). For example, cells (such as Vero cells or porcine bone marrow cells) may be transfected with a protein or infected with an ASFV, then red blood cells added and the degree of haemadsorption detected by imaging. In this way, different proteins and viruses can be tested for their effect on haemadsorption.
EP402R and EP153R are involved in mediating haemadsorption of ASFV-infected cells.
In an embodiment the invention provides an attenuated ASF virus wherein the ability of the ASF virus to induce haemadsorption is reduced compared to a corresponding ASF virus in which expression and/or activity of the EP153R and EP402R genes is not disrupted. In an embodiment the invention provides an attenuated African Swine Fever (ASF) virus in which the expression and/or activity of the genes EP153R and EP402R is disrupted;
which comprises a functional version of one or more of the following genes:
The invention also provides an attenuated ASF virus in which the expression and/or activity of the genes EP153R and EP402R is disrupted;
which comprises a functional version of one or more of the following genes:
The invention also provides an attenuated ASF virus in which the expression and/or activity of the genes EP153R and EP402R is disrupted;
which comprises a functional version of one or more of the following genes:
In another aspect the invention provides an ASF virus comprising the EP402R protein of the invention and/or the polynucleotide of the invention wherein the ability of the ASF virus to induce haemadsorption is reduced compared to a corresponding ASF virus which does not comprise the EP402R protein of the invention and/or the polynucleotide of the invention.
In an embodiment of the attenuated ASF virus of the invention, the ability of the EP153R gene and/or the EP402R gene to mediate haemadsorption may be disrupted. In an embodiment, the ability of the EP153R gene to mediate haemadsorption may be disrupted. In an embodiment, the ability of the EP402R gene to mediate haemadsorption may be disrupted.
Reducing haemadsorption or disrupting the ability to mediate haemadsorption means that cells infected with the ASFV of the invention adsorb fewer red blood cells to their surface than cells infected with a wild-type ASFV or with an ASF virus corresponding to, or essentially corresponding to, the ASFV of the invention in which expression and/or activity of the EP153R and EP402R genes has not been disrupted or which does not comprise the EP402R protein of the invention and/or the polynucleotide of the invention. Reducing haemadsorption or disrupting the ability to mediate haemadsorption also means that cells transfected to express a mutant, non-functional EP153R or EP402R protein adsorb fewer red blood cells to their surface than cells transfected with a wild-type EP153R or EP402R protein. The number of red blood cells adsorbed to the surface of the infected/transfected cells may be decreased by at least 50, 60, 70, 80 or 90%. In an embodiment, haemadsorption is abolished i.e. no red blood cells adsorb to the surface of cells infected with the attenuated ASFV of the invention or transfected with a mutant, non-functional EP153R or EP402R protein.
In an embodiment the ASF virus of the present invention has disrupted expression and/or activity of the genes EP153R and EP402R. In another embodiment the ASF virus of the present invention has disrupted expression and/or activity of the genes EP153R, EP402R and K145R. These genes may be referred to herein as the “disrupted genes”.
In an embodiment the invention provides an ASFV in which expression of the genes EP153R and EP402R is disrupted. In an embodiment the invention provides an ASFV in which expression of the genes EP153R, EP402R and K145R is disrupted. Suitably expression of the EP153R gene is disrupted. Suitably expression of the EP402R gene is disrupted. Suitably expression of the K145R gene is disrupted. In an embodiment the invention provides an ASFV in which activity of the genes EP153R and EP402R is disrupted.
The term “expression” with respect to a gene refers to the ability of the ASF virus to produce the product of the gene, such as RNA and/or protein. Disruption of expression of a gene means that production of the gene product is decreased. Expression of the gene may be decreased by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and/or at least 95%. Expression of the gene may be decreased to the extent that production of the gene product, such as RNA and/or protein, is entirely abolished (i.e. the gene product is not produced at all). Disruption of gene expression decreases expression of the gene relative to the expression of the gene when it is not disrupted. For example, a mutated gene may have decreased expression in comparison to a wild-type version of the gene.
A gene the expression of which is disrupted may not be fully transcribed and translated. Transcription of the gene may be decreased by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and/or at least 95%. Transcription of the gene may be abolished (i.e. the gene may not be transcribed). Translation of the gene may be decreased by at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and/or at least 95%. Translation of the gene may be abolished. The gene may be transcribed but not translated. The gene may be transcribed and translated but the protein too rapidly degraded to carry out its function. The gene may be transcribed and translated but the protein may be non-functional.
Gene expression may be measured by techniques known in the art. For example, the amount of mRNA transcribed from a gene may be quantified, such as by using quantitative polymerase chain reaction (qPCR). Alternatively or additionally, the amount of protein may be quantified, such as by using Western blotting or mass spectrometry.
The term “activity” with respect to a gene refers to the ability of the gene to carry out its functions. Different genes have different activities i.e. different functions they fulfil. A given gene may have multiple activities; disruption of gene activity means disruption of one or more of those activities. One or more activities of the gene may be disrupted whilst one or more other activities are not disrupted. Disruption of gene activity decreases the activity of the gene relative to the activity of the gene when it is not disrupted. For example, a mutated gene may have decreased activity in comparison to a wild-type version of the gene. Gene activity may be decreased to the extent that gene activity is entirely abolished.
The ASFV according to the present invention may comprise a non-functional version of the disrupted genes.
Disruption of expression of a gene may also disrupt activity of that gene as the decreased amount of gene product means the gene cannot as effectively carry out one or more of its activities.
In an embodiment the attenuated ASF virus of the invention comprises mutations that disrupt the expression and/or activity of the genes EP153R and EP402R.
Gene expression and/or activity may be disrupted by disrupting transcription of the gene into mRNA i.e. by decreasing gene transcription, such as completely abolishing gene transcription. Gene expression and/or activity may be disrupted by disrupting translation of mRNA into protein. In an embodiment the attenuated ASF virus comprises mutations that decrease transcription and/or translation of the genes. In an embodiment the attenuated ASF virus comprises mutations that cause the genes to not be transcribed and/or translated (i.e. complete abolition of transcription and/or translation).
Gene expression and/or activity may be disrupted by mutating a non-coding sequence associated with the gene, such as a promoter. In an embodiment the attenuated ASF virus comprises mutations in promoters of one or more of the disrupted genes.
Gene expression and/or activity may be disrupted by mutating a coding sequence of one or more of the disrupted genes.
The attenuated ASFV of the invention comprises a functional version of one or more of the following genes:
In some embodiments the attenuated ASFV of the invention may comprise a functional version of one or more of the following genes: MGF 110 3L, 6L, 7L, 8L, 10L, 11L and 12L; MGF 360 5L, 6L, 7L, 10L, 11L, 12L, 13L, 14L, 20R, 21R and 22R; and MGF 505 1R, 2R and 6R. Suitably, the attenuated ASFV comprises functional versions of two or more, such as three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, twenty or more, twenty one, or twenty two of the following genes: MGF 110 3L, 6L, 7L, 8L, 10L, 11L and 12L; MGF 360 5L, 6L, 7L, 10L, 11L, 12L, 13L, 14L, 20R, 21R and 22R; and MGF 505 1R, 2R and 6R. In an embodiment the attenuated ASFV comprises functional versions of all of the following genes: MGF 110 3L, 6L, 7L, 8L, 10L, 11L and 12L; MGF 360 5L, 6L, 7L, 10L, 11L, 12L, 13L, 14L, 20R, 21R and 22R; and MGF 505 1R, 2R and 6R. Suitably the attenuated ASF virus of the invention comprises functional versions of all ASF virus genes other than EP153R and EP402R. Suitably the attenuated ASF virus of the invention comprises functional versions of all ASF virus genes other than EP153R, EP402R and K145R.
The expression “functional version” of a gene refers to a gene the expression and activity of which have not been disrupted. A functional version of a gene may not be mutated in a manner that disrupts gene expression or gene activity. A functional version of a gene may not comprise any mutations. The coding sequence of a functional version of a gene may be complete and uninterrupted. A functional version of a gene may be fully transcribed and translated. A functional version of a gene may comprise the full coding sequence.
A functional version of a gene may correspond to the gene in a wild-type ASFV isolate. A functional version of a gene may correspond to the gene in a virulent ASFV strain. The sequence of a functional version of a gene may be identical to the sequence of the gene in a wild-type ASFV isolate or virulent ASFV strain. The sequence of a functional version of a gene may be identical to the sequence of the gene in the wild-type ASFV isolate from which the attenuated ASFV of the invention is derived. A functional version of a gene may be a natural variant of the gene in a wild-type ASFV isolate.
A functional version of a gene may comprise mutations. However, the mutations should not disrupt the expression or activity of the gene. In other words, the mutations should not affect the function of the gene. A functional version of a gene may comprise one or more synonymous mutations (i.e. mutations which do not alter the amino acid sequence of the protein the gene encodes). A functional version of a gene may comprise one or more silent mutations, which may be synonymous or non-synonymous. A functional version of a gene may comprise deletions that do not disrupt the expression or activity of the gene. A functional version of a gene may comprise one or more single nucleotide polymorphisms (SNPs) that do not disrupt the expression or activity of the gene.
Gene expression and/or activity are disrupted by mutating the ASFV genome i.e. by changing the nucleotide sequence of the ASFV genome. A “mutation” means a change in the nucleotide sequence of the ASFV genome relative to a known ASFV genotype. Mutations include changing one or more nucleotides to different nucleotides (i.e. substitution), adding nucleotides (i.e. insertion), removing nucleotides (i.e. deletion) and/or a combination of these. In an embodiment the ASF virus of the invention comprises one or more mutations that disrupt the expression and/or activity of the genes EP153R and EP402R. In an embodiment the ASF virus of the invention comprises one or more mutations that disrupt the expression of the K145R gene.
Mutations that disrupt gene expression and/or activity may be in non-coding sequence of the ASFV genome and/or in coding sequence of the ASFV genome. The attenuated ASF virus of the invention may comprise one or more mutations in a non-coding region that disrupt the expression and/or activity of the EP153R gene and/or one or more mutations in a non-coding region that disrupt the expression and/or activity of the EP402R gene. The ASF virus of the invention may comprise one or more mutations in a non-coding region that disrupt the expression and/or activity of the K145R gene.
The ASF virus of the invention may comprise one or more mutations in a coding region of the EP153R gene that disrupt the expression and/or activity of the EP153R gene and/or one or more mutations in a coding region of the EP402R gene that disrupt the expression and/or activity of the EP402R gene. The attenuated ASF virus of the invention may comprise one or more mutations in a coding region of the K145R gene that disrupt the expression of the K145R gene.
In the ASFV of the invention, expression and/or activity of genes may be disrupted by deletion. In other words, expression and/or activity of a gene may be disrupted by a mutation that is a deletion. An ASFV of the invention may be made to lack a functional version of a gene by deletion. In other words, the mutation that causes the ASFV to lack a functional version of a gene may be a deletion.
“Deletion” means removal of part of the ASFV genome nucleotide sequence. The deletion may be continuous, or may comprise deletion of a plurality of sections of sequence. Deletion may disrupt gene expression and/or activity in any of the ways described herein. Deletion may cause the ASFV to lack a functional version of the gene in any of the ways described herein.
Deletion may alter the gene product that is produced. Deletion may cause the gene to not be transcribed and/or translated. Deletion may disrupt transcription of the gene into mRNA. For example, deleting a promoter of a gene would disrupt transcription. Deletion may disrupt translation of mRNA into protein. For example, deleting a start codon would disrupt translation.
Gene expression and/or activity may be disrupted by deleting non-coding sequence associated with the gene, such as a promoter.
Gene expression and/or activity may be disrupted by deleting coding sequence of the gene. The ASFV may be made to lack a functional version of the gene by deleting coding sequence of the gene. The expression “deletion of a gene” (such as “partially deleted” or “completely deleted”) refers to deletion of a sufficient amount of coding sequence such that expression and/or activity of the gene is disrupted.
Deletion of coding sequence may be partial (i.e. part of the coding sequence is deleted). The deletion may, for example, remove at least 50, 60, 70, 80 or 90% of the coding sequence of the gene. The amount of coding sequence required to be deleted to disrupt gene expression and/or activity may be very small. For example, partial deletion of a gene may mean deletion of just the start codon (ATG) if this is sufficient to disrupt expression and/or activity of the gene. At the other extreme, the deletion may be complete, in which case 100% of the coding sequence of the gene is deleted (i.e. all of the coding sequence is absent when compared to the corresponding genome of a wild-type isolate). In other words, “completely deleted” means that all of the coding sequence of that gene has been deleted.
Partial and full deletions of a gene can be made using known techniques in the art, such as conditional targeting via Cre-LoxP and Flp-FRT systems, or by inducing a double strand break (DSB) and repair using engineered nucleases such as meganucleases, zinc finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs) and Cas in CRISPR-Cas systems. The DSB repair can be exploited to introduce a desired mutation by providing a vector comprising the desired mutated nucleotide sequence within a sequence that is homologous to the sequences flanking either side of the DSB. This results in the desired mutation being inserted at the site of the DSB. Nucleases such as those above can be engineered to induce DSB at a specific site within the genome. For example, chimeric meganucleases can be readily generated by combining known protein units to recognise a target recognition sequence within a gene or genomic region of interest. ZFNs can also be designed to target specific sequences, for example combining zinc-finger units with known specificities to bind specific regions of DNA. TALENs are artificial restriction enzymes designed by fusing a nuclease domain to DNA-binding TALE (transcription activator-like effector) domains. TALE domains are tandem arrays of amino acid repeats that recognise a single nucleotide and can be designed to target a specific region of DNA. CRISPR-Cas systems consist of a Cas (CRISPR-associated protein) nuclease and a CRISPR (clustered regularly interspaced short palindromic repeat) RNA sequence that guides the Cas protein to recognise and cleave a specific strand of DNA complementary to the CRISPR sequence. Single-stranded guide RNA (sgRNA) can therefore be designed to bind to a specific region of DNA and guide the Cas to introduce a DSB. Accordingly, provided the nucleotide (e.g. DNA or cDNA) sequence of a gene is known, a known nuclease system can be utilised to introduce a partial or full deletion to the gene.
Deletion of coding sequence may be continuous, or may comprise deletion of a plurality of sections of coding sequence. The deletion should remove a sufficient amount of coding sequence such that deletion disrupts the expression and/or activity of the gene i.e. a functional gene product, such as a protein, is no longer produced from the gene.
The expression and/or activity of the genes may be disrupted by interruption of the gene. In other words, the mutation that disrupts expression and/or activity of a gene may be one that interrupts the gene.
In an embodiment of the ASFV of the present invention the genes EP153R, EP402R and K145R may each be interrupted. The EP153R gene may be interrupted. The EP402R gene may be interrupted. The K145R gene may be interrupted.
“Interruption” means the mutation alters the coding sequence of the gene such that a functional gene product, such as a protein, is no longer produced. The term “interruption” may be used herein to refer to a mutation that interrupts a gene. The mutation(s) should interrupt the coding sequence in a manner such that expression and/or activity of the gene is disrupted i.e. a functional gene product, such as a protein, is no longer produced from the gene.
Interruptions may encompass deletions (i.e. removal of one or more nucleotides) within the coding sequence of a gene, but also substitutions (i.e. replacement of one or more nucleotides with different nucleotides) and insertions (i.e. addition of one or more nucleotides) within the coding sequence of a gene.
The interruption may entirely abolish gene product production. For example, where the gene product is a protein, the interruption may render the mRNA nonsensical, causing the mRNA to be degraded and the protein to not be translated, thereby abolishing protein production. The interruption may alter the gene product that is produced. The interruption may cause the gene to not be transcribed and/or translated.
The interruption may be a point mutation (i.e. substitution, insertion or deletion of a single nucleotide). An interruption may be an insertion of one or more nucleotides. An interruption may be a deletion. A gene may comprise multiple mutations that lead to interruption of the gene.
The interruption may be a frame shift mutation, caused by insertion or deletion of nucleotides. A frame shift causes the codons downstream of the frame shift to be read as different amino acids. The protein produced may be non-functional.
The interruption may be mutation of a start codon. A start codon is typically ATG. Mutation of a start codon (e.g. point mutation of one, two or three of the nucleotides) means that translation will not start at that codon. Translation may begin at a subsequent start codon further downstream. If the subsequent start codon is in frame a version of the protein is produced that is N-terminally truncated and so may be non-functional. If the subsequent start codon is not in frame an entirely different or nonsense protein is produced, which would be non-functional. If there is no subsequent start codon, translation is entirely abolished and no protein is produced.
The interruption may be mutation of a stop codon (TAG, TAA or TGA). Mutation of a stop codon (also referred to as a nonstop mutation) causes continued translation of mRNA into a sequence that should not be translated. The resulting protein may be non-functional due to its excessive length.
In embodiments of the ASF virus of the invention, the EP402R gene may comprise one or more mutations that change one or more amino acids in the ligand-binding domain of the EP402R protein. The amino acid changes in EP402R are described in detail elsewhere herein.
The mutations that disrupt gene expression and/or activity described herein may be combined in an ASFV of the invention. In other words, the EP153R and EP402R genes in an ASFV of the invention may each be disrupted by the same type of mutation as any of the other genes or by a different type of mutation as any of the other genes. Furthermore, the K145R gene may be disrupted by the same type of mutation as any of the other genes or by a different type of mutation as any of the other genes.
For example, in an ASFV of the invention, EP153R may be disrupted by complete deletion, EP402R may be disrupted by an amino acid change in its ligand-binding domain and K145R may be disrupted by mutation of a promoter sequence. As an alternative example, EP153R may be disrupted by interruption and EP402R may be disrupted by complete deletion and K145R may be disrupted by partial deletion.
In an embodiment the invention provides an ASFV in which
In an embodiment the invention provides an ASFV in which
In an embodiment the invention provides an ASFV in which
In an embodiment the invention provides an ASFV in which
In an embodiment the invention provides an ASFV in which the EP153R gene and the K145R gene are each completely deleted, and which comprises an EP402R protein comprising the sequence of SEQ ID No. 33.
In an embodiment the invention provides an ASFV in which the EP153R gene, the EP402R gene and the K145R gene are each completely deleted.
In an embodiment the invention provides an ASFV wherein the ASFV genome is the same as that of the Georgia 2007/1 strain, except that
In an embodiment the invention provides an ASFV in which the EP153R gene and the K145R gene are each completely deleted, and which comprises an EP402R protein comprising the sequence of SEQ ID No. 33, wherein the genome of the ASFV corresponds to that of the Georgia 2007/1 strain.
In present invention provides an ASF virus of the invention for use in treating and/or preventing a disease in a subject. The invention also provides use of an ASF virus of the invention for manufacture of a medicament for treating and/or preventing disease in a subject. Suitably the disease is African Swine Fever.
The present invention also provides a vaccine comprising an attenuated ASF virus of the invention.
The term “vaccine” as used herein refers to a preparation which, when administered to a subject, induces or stimulates a protective immune response. In some embodiments the vaccine of the invention induces a partially protective immune response. In some embodiments the vaccine reduces severity and/or duration of ASF symptoms but does not completely abolish ASF symptoms. A vaccine can render an organism immune to a particular disease, in the present case ASF. The vaccine of the present invention thus induces an immune response in a subject which is protective against subsequent ASF virus challenge. A vaccine comprising an attenuated ASFV of the invention may be capable of inducing a cross-protective immune response against a plurality of ASF virus genotypes. In an embodiment a vaccine comprising an attenuated ASFV of the invention of a single genotype may be capable of inducing a cross-protective immune response against a plurality of ASF virus genotypes.
The vaccine may comprise 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 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. Accordingly, the invention provides a vaccine of the invention for use in treating and/or preventing African Swine Fever in a subject.
The present invention also provides a pharmaceutical composition which comprises one or more attenuated ASF virus(es) of the invention. 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) of the invention and optionally one or more adjuvants, excipients, carriers and diluents. The choice of pharmaceutical excipient, carrier or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as (or in addition to) the carrier, excipient or diluent, any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s) and other carrier agents. The pharmaceutical compositions typically should be sterile and stable under the conditions of manufacture and storage. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Sterile injectable formulations may be prepared using a non-toxic parenterally acceptable diluent or solvent. A pharmaceutical composition of the present invention may include pharmaceutically acceptable dispersing agents, wetting agents, suspending agents, isotonic agents, coatings, antibacterial and antifungal agents, carriers, excipients, salts, or stabilizers which are nontoxic to the subjects at the dosages and concentrations employed. Preferably, such a composition can further comprise a pharmaceutically acceptable carrier or excipient for use in the treatment of disease that that is compatible with a given method and/or site of administration, for instance for parenteral (e.g. sub-cutaneous, intradermal, or intravenous injection) or intrathecal administration.
The vaccine or pharmaceutical composition may comprise one or more attenuated ASF virus(es) of the invention in an effective amount.
In an embodiment the invention provides an attenuated ASF virus of the invention which when administered to a subject induces an immune response which is protective against subsequent challenge with virulent ASF virus. In an embodiment the invention provides an attenuated ASF virus of the invention which when administered to a subject induces an immune response which is protective against subsequent challenge with virulent ASF virus of a different genotype to the attenuated ASF virus of the vaccine. In an embodiment the invention provides an attenuated ASF virus of the invention which when administered to a subject induces an immune response which is protective against subsequent challenge with virulent ASF virus of any genotype. Accordingly, the invention provides a vaccine comprising an attenuated ASF virus for use in treating and/or preventing African Swine Fever wherein the African Swine Fever is caused by an ASF virus of a different genotype to the ASF virus of the vaccine. In an embodiment the invention provides a vaccine comprising an attenuated ASF virus for use in treating and/or preventing African Swine Fever wherein the African Swine Fever is caused by an ASF virus of any genotype. In an embodiment the ASF virus of the vaccine corresponds to genotype II, such as Georgia 2007/1 strain and the vaccine is protective against infection with genotype I, II, III, IV, V, VI, VII, VIII, IX, X and/or XIV. In an embodiment the ASF virus of the vaccine corresponds to genotype II, such as Georgia 2007/1 strain and the vaccine is protective against infection with genotype I, IX, X, XIV, and/or VIII. In an embodiment the ASF virus of the vaccine corresponds to genotype II, such as Georgia 2007/1 strain and the vaccine is protective against infection with genotype I, IX, and/or X.
The present invention also provides a method of preventing and/or treating ASF in a subject by administration to the subject of an effective amount of an attenuated virus, vaccine, or pharmaceutical composition of the invention.
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 vaccine may reduce the severity and/or duration of ASF symptoms. The vaccine may completely abolish ASF symptoms.
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 according to the present invention may be a domestic pig.
Suitably, protective immunity as defined herein may be conferred to piglets who are fed colostrum from a vaccinated subject, such as a vaccinated mother.
The vaccine of the invention 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, oral administration comprises adding the vaccine to animal feed or drinking water. In another embodiment, the vaccine may be added to bait for a wild animal, for example bait suitable for wild boar, wild pigs, bushpigs or warthogs.
The dose for pig immunisation may be from about 103 to about 106 HAD50 or TCID50 per pig. The dose for pig immunisation may be from about 103 to about 106 TCID50 per pig. The dose for pig immunisation may be less than 104 HAD50 or TCID50 per pig. For example the dose may be between 102-103 HAD50 or TCID50. The dose may be about 102 HAD50 or TCID50 per pig. The dose may be determined by a veterinary practitioner within the scope of sound veterinary judgment.
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 7, 14, 21 or 28 days) later. Typically the boosting administration is at a higher dose than the priming administration. The boosting dose may be from about 103 to about 106 HAD50 or TCID50 per pig. The boosting dose may be from about 103 to about 106 TCID50 per pig.
The present invention also provides a method of producing an ASF virus of the invention, the method comprising changing one or more amino acid in the ligand-binding domain of the EP402R protein wherein the amino acid change disrupts ligand-binding of the EP402R protein.
The present invention also provides a method of reducing the ability of an ASF virus to induce haemadsorption, the method comprising changing one or more amino acid changes in the ligand-binding domain of the EP402R protein wherein the amino acid changes disrupt ligand-binding of the EP402R protein.
The amino acid changes in the ligand-binding domain of EP402R may be any of the amino acid changes described herein. Such amino acid changes may be made by mutating the ASFV genome as described herein.
In an embodiment, the method comprises changing one or more amino acid in the EP402R protein at a position which corresponds to Q96 and/or W99 of the Georgia 2007/1 EP402R protein (SEQ ID No. 24).
The invention also provides a method of attenuating an ASF virus which comprises disrupting the expression and/or activity of the EP153R and EP402R genes. Suitably the method comprises disrupting the ability of the EP153R gene and/or the EP402R gene to mediate haemadsorption.
Disruption of gene expression and/or activity may be achieved by mutating the ASFV genome in any of the ways described herein.
In an embodiment the method further comprises introducing a DIVA mutation into the ASF virus. Suitably the DIVA mutation disrupts expression of the K145R gene. Suitably the K145R is at least partially deleted, preferably completely deleted. Suitably the K145R gene is interrupted. Suitably the DIVA mutation disrupts expression of the B125R gene. Suitably the B125R is at least partially deleted, preferably completely deleted. Suitably the B125R gene is interrupted.
In an embodiment of the method of producing and/or attenuating an ASFV, the EP153R gene is at least partially deleted, preferably completely deleted. In an embodiment the EP153R gene is interrupted.
In an embodiment of the method of producing and/or attenuating an ASFV, the EP402R gene is at least partially deleted, preferably completely deleted. In an embodiment the EP402R gene is interrupted. Suitably the method comprises introducing one or more mutations in the EP402R gene that reduce surface expression of the EP402R protein compared to a corresponding ASF virus that does not comprise the one or more mutations. Suitably the method comprises introducing one or more mutations in the EP402R gene that disrupt ligand binding by the EP402R protein. Suitably the method comprises introducing one or more mutations in the EP402R gene that change one or more amino acids in the ligand-binding domain of the EP402R protein. Suitably the one or more amino acids are changed to different amino acids. Suitably, changing the amino acids to different amino acids directly inhibits the interaction between EP402R and its ligand by changing the binding surface on EP402R.
In an embodiment the method comprises changing an amino acid in the EP402R protein at a position which corresponds to Q96 and/or W99 of the Georgia 2007/1 EP402R protein (SEQ ID No. 24). Suitably the amino acid in the EP402R protein at a position which corresponds to Q96 of the Georgia 2007/1 EP402R protein (SEQ ID No. 24) is changed to R or to an amino acid that is a conservative replacement of R and/or an amino acid at a position which corresponds to W99 of the Georgia 2007/1 EP402R protein (SEQ ID No. 24) is changed to D or to an amino acid that is a conservative replacement of D. Suitably the amino acid at the position which corresponds to Q96 is changed to H, K or R and/or the amino acid at the position which corresponds to W99 is changed to D, E, N or Q. Suitably the amino acid at the position which corresponds to Q96 is changed to R and/or the amino acid at the position which corresponds to W99 is changed to D.
In an embodiment the invention provides a method of attenuating an ASF virus comprising completely deleting each of the EP153R and K145R genes, changing the amino acid at the position which corresponds to Q96 of the Georgia 2007/1 EP402R protein (SEQ ID No. 24) to R and/or changing the amino acid at the position which corresponds to W99 of the Georgia 2007/1 EP402R protein (SEQ ID No. 24) to D. In an embodiment the invention provides a method of attenuating an ASF virus comprising completely deleting each of the EP153R and K145R genes, and changing the amino acid at the position which corresponds to Q96 of the Georgia 2007/1 EP402R protein (SEQ ID No. 24) to R.
In an embodiment the method of producing and/or attenuating an ASF virus of the invention may be applied to an ASF virus of any genotype (i.e. an ASF virus of any of genotypes I to XXIV). In other words, an ASF virus of any genotype may be the subject of the modifications of the method of the invention. An ASF virus of any genotype may used in the method. In an embodiment the method of producing and/or attenuating an ASF virus of the invention may be applied to an ASF virus of genotype II. In an embodiment the method of producing and/or attenuating an ASF virus of the invention may be applied to an ASF virus of the Georgia 2007/1 strain.
In an embodiment the invention provides a method of attenuating an ASF virus of the Georgia 2007/1 strain comprising completely deleting each of the EP153R and K145R genes, changing Q96 of the Georgia 2007/1 EP402R protein (SEQ ID No. 24) to R and/or changing W99 of the Georgia 2007/1 EP402R protein (SEQ ID No. 24) to D. In an embodiment the invention provides a method of attenuating an ASF virus of the Georgia 2007/1 strain comprising completely deleting each of the EP153R and K145R genes, and changing Q96 of the Georgia 2007/1 EP402R protein (SEQ ID No. 24) to R.
Methods for mutation of viral genes are known in the art. In particular, 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.
In some embodiments, the method of attenuating an ASF virus may comprise retaining the function of one or more of the following genes: MGF 110 3L, 6L, 7L, 8L, 10L, 11L and 12L; MGF 360 5L, 6L, 7L, 10L, 11L, 12L, 13L, 14L, 20R, 21R and 22R; and MGF 505 1R, 2R and 6R. Suitably, the function of two or more, such as three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, twenty or more, twenty one, or twenty two of the following genes are retained: MGF 110 3L, 6L, 7L, 8L, 10L, 11L and 12L; MGF 360 5L, 6L, 7L, 10L, 11L, 12L, 13L, 14L, 20R, 21R and 22R; and MGF 505 1R, 2R and 6R. In an embodiment the method of attenuating an ASF virus may comprise retaining the function of all of the following genes: MGF 110 3L, 6L, 7L, 8L, 10L, 11L and 12L; MGF 360 5L, 6L, 7L, 10L, 11L, 12L, 13L, 14L, 20R, 21R and 22R; and MGF 505 1R, 2R and 6R.
“Retaining the function” of a gene means that expression and activity of the gene is not affected during the attenuation process. The resultant attenuated virus should express a functional version of the gene. Suitably, the genes the function of which is to be retained are unaltered by the method of attenuation. Suitably, the sequences of the genes the function of which is to be retained are unaltered by the method of attenuation.
This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.
The terms “comprising”, “comprises” and “comprised of” as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms “comprising”, “comprises” and “comprised of” also include the term “consisting of”.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.
The present invention also provides further aspects as defined in the following numbered paragraphs (paras).
1. An attenuated African Swine Fever (ASF) virus in which the expression and/or activity of the genes EP153R and K145R is disrupted.
2. An attenuated ASF virus according to para 1 in which the expression and/or activity of the EP402R is not disrupted.
3. An attenuated ASF virus according para 1 or 2 which comprises a functional version of one or more of the following genes:
4. An attenuated ASF virus according to any preceding para wherein the K145R gene is at least partially deleted, preferably completely deleted.
5. An attenuated ASF virus according to para 4 wherein the K145R gene is interrupted.
6. An attenuated ASF virus according to any preceding para wherein the EP153R gene is at least partially deleted, preferably completely deleted.
7. An attenuated ASF virus according to any preceding para wherein the EP153R gene is interrupted.
8. An attenuated ASF virus according to any preceding para which comprises functional versions of all ASF virus genes other than EP153R and K145R.
9. An attenuated ASF virus according to any preceding para wherein the genome of the attenuated ASF virus corresponds to, or essentially corresponds to, genotype II.
10. An attenuated ASF virus according to para 9 wherein the genome of the attenuated ASF virus corresponds to, or essentially corresponds to, that of the Georgia 2007/1 strain.
11. An ASF virus according to any of paras 1 to 10 for use in treating and/or preventing a disease in a subject.
12. Use of an ASF virus according to any of paras 1 to 10 for manufacture of a medicament for treating and/or preventing disease in a subject.
13. A pharmaceutical composition comprising an ASF virus according to any of paras 1 to 10.
14. A pharmaceutical composition according to para 13 for use in treating and/or preventing a disease in a subject.
15. An ASF virus for use according to para 11, use of an ASF virus according to para 12, or a pharmaceutical composition for use according to para 14, wherein the disease is African Swine Fever.
16. A vaccine comprising an ASF virus according to any of paras 1 to 10.
17. A vaccine according to para 16 for use in treating and/or preventing African Swine Fever in a subject.
18. A vaccine for use according to para 17 wherein the African Swine Fever is caused by an ASF virus of a different genotype to the ASF virus of the vaccine.
19. 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 pharmaceutical composition according to para 13 or a vaccine according to para 16.
20. An ASF virus for use according to para 11, use of an ASF virus according to para 12, a pharmaceutical composition for use according to para 14, a vaccine for use according to para 17, or a method according to para 19, wherein the subject is a domestic pig.
21. A vaccine for use according to any of paras 17, 18 or 20, or a method according to claim 19 or 20, in which the vaccine is administered following a prime-boost regime.
22. A method of attenuating an ASF virus which comprises disrupting the expression and/or activity of the EP153R and K145R genes.
23. A method according to para 22 wherein the K145R gene is at least partially deleted, preferably completely deleted.
24. A method according to para 23 wherein the K145R gene is interrupted.
25. A method according to any of paras 22 to 24 wherein the EP153R gene is at least partially deleted, preferably completely deleted.
26. A method according to any of para 22 to 25 wherein the EP153R gene is interrupted.
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.
Mutations were made in African swine fever virus (ASFV) Benin isolate EP402R protein (CD2v) and tested for their effect on haemadsorption (HAD).
A model of the extracellular, N-terminal, IgG-like, ligand-binding domain of CD2v was generated and used to predict the functional amino acid residues involved in binding of CD2v to its ligand. These residues were individually mutated to generate a set of mutant CD2v proteins.
Vero cells were infected with modified vaccinia virus Ankara expressing T7RNA polymerase and transfected with plasmids (pcDNA3) expressing wild-type or mutant CD2v full-length proteins with a C-terminal HA epitope tag. Pig red blood cells were added and cells observed for attachment of red blood cells to the surface. Expression of the wild-type or mutant CD2v proteins was confirmed both by confocal microscopy using permeabilised cells and Western blotting using an antibody recognising the HA tag and a secondary antibody. Cell surface expression of the wild-type or mutant CD2v was also confirmed by staining non-permeabilised cells with sera from pigs immunised with attenuated ASFV containing a wild-type CD2v gene followed by a secondary antibody (
Mutation of residues E99 or Y102 in Benin CD2v abrogated HAD.
Benin CD2v residue E99 is strongly conserved in ASFV, as shown by an alignment of the amino acid sequence of CD2v ligand-binding domain from different ASFV isolates of varying genotypes (
The residue corresponding to Benin CD2v E99 in Georgia CD2v, Q96, was mutated to R and the ability of the mutant protein to induce HAD was tested using the HAD assay described above (wild type and mutant CD2v protein expressed from plasmids in Vero cells). Mutation of Q96 in Georgia CD2v abrogated HAD.
Using the same assay, the following mutations were determined to impact HAD in Benin: N16R, I 19R, W21D, Y 76D, E99R, Y102D. In addition, a combination of E99R+N108R was determined to impact HAD in Benin.
The following mutations were determined to impact HAD in Georgia: S15R, W19D, Q96R, N104R, and K108D. In addition, combinations of S15R+W19D and Q96R+N104R were determined to impact HAD in Georgia.
The following mutations were determined to impact HAD in N10 Genotype IX: W20D, R125D, Q112R+N121R.
For a gene to serve as a Differentiation of Infected from Vaccinated Animals (DIVA) marker, the protein expressed from the gene must be immunogenic. In other words, a subject infected with a virus expressing the DIVA protein must produce antibodies that specifically bind the DIVA protein. In this way, animals vaccinated with a DIVA vaccine (virus lacking the DIVA marker gene) can be differentiated from animals infected with wild type virus (which expresses the DIVA marker gene) because sera of vaccinated animals will not comprise antibodies to the DIVA marker protein, whereas sera of infected animals will comprise antibodies to the DIVA marker protein.
A selection of ASFV genes that might serve as DIVA markers were screened by expressing each gene in cells and testing whether the protein produced could be detected by sera taken from pigs that had previously been infected with ASFV. Detection by the sera would indicate that the protein, expressed by ASFV in the infected pigs, had induced an antibody response in the infected pigs. Such proteins were therefore candidates for DIVA markers.
In particular, 71 plasmids coding for individual ASFV genes (excluding known essential genes) fused to an HA or V5 epitope tag were transfected into Vero cells. The cells were fixed, permeabilised and stained with antisera from pigs that had been infected with different strains of ASFV, followed by a fluorescently labelled secondary antibody. Confocal microscopy was used to assess whether the expressed gene could be detected by the sera. In parallel the cells were stained with an antibody against the HA or V5 tag fused to the ASFV gene and a different fluorescently labelled secondary antibody to confirm expression of the protein.
The pig sera used for staining the cells were from pigs from immunisation studies that had been immunised with the following ASFV strains: BeninΔDP148R (5 pigs), BeninΔMGF (6 pigs), OURT88/3 (5 pigs) and GeorgiaΔMGF (4 pigs). For each pig, a pre-immunisation serum sample (as a control) and a post-immunisation, pre-challenge serum sample were used.
An initial screen of the genes was conducted using sera from BeninΔDP148R immunised pigs (pre-immunisation sera from day 0, post-immunisation sera from day 38 post-immunisation). ASFV genes CP204L, B646L and E183L were used as positive controls and were detected using post-immunisation sera. Pre-immunisation sera did not detect any genes.
Six ASFV genes were detected using BeninΔDP148R post-immunisation sera, as shown below in Table 7 (++ indicates strong detection, + indicates weak detection, − indicates no detection).
The six ASFV genes detected in the initial screen were then tested with pig serum from the other three immunisation studies.
Table 8 below shows detection of ASFV genes using post-immunisation sera from 6 pigs immunised with BeninΔMGF virus (boosted on day 15, post-immunisation serum taken on day 38 post-immunisation; pre-immunisation sera were negative).
Table 9 below shows detection of ASFV genes using post-immunisation sera from 5 pigs immunised with OURT88/3 virus (post-immunisation serum taken on day 20 post-immunisation; pre-immunisation sera were negative except for pig 2).
Table 10 below shows detection of ASFV genes using post-immunisation sera from 4 pigs immunised with GeorgiaΔMGF virus (post-immunisation serum taken on day 34 post-immunisation; pre-immunisation sera taken on day-3 were negative). 2 pigs (A) were immunised with 103 GeorgiaΔMGF; 2 pigs (B) were immunised with 104 GeorgiaΔMGF.
K145R protein was detected by 65% of sera and B125R was detected by 75% of sera. Each of the B125R, B175L, E184L, H339R, K145R and M448R genes was individually deleted. The B175L, E184L, H339R or M448R genes could not be deleted, suggesting that that are essential for virus replication. Thus, the screen identified the K145R and B125R genes as the most promising potential DIVA markers.
Based on the findings described in the Examples above, ASFV was generated in which the K145R and EP153R genes were deleted and the EP402R/CD2v protein was mutated to comprise the Q96R amino acid substitution. Georgia 2007/1 strain (a strain of ASFV genotype II) was used. The ASFV is accordingly designated GeorgiaΔK145RΔEP153RCD2vQ96R.
The ability of GeorgiaΔK145RΔEP153RCD2vQ96R to induce HAD was tested. Porcine bone marrow cells were infected with GeorgiaΔK145RΔEP153RCD2vQ96R or wild type Georgia 2007/1 as control, pig red blood cells were added and the cells observed for attachment of red blood cells to the surface. HAD was observed in cells infected with wild type Georgia 2007/1 at 1 day post-infection (
A group of six Large White/Landrace pigs (Group K) varying in weight from 17 to 19 kg and aged 7 weeks old were immunised by the intramuscular route with 104 TCID50 in 1 ml with GeorgiaΔK145RΔEP153RCD2vQ96R and boosted after 21 days by the same route with the same dose. After a further 18 days the Group K immunised pigs and a control group of 3 non-immune pigs (Group M) were challenged by the intramuscular route with 103 TCID50 in 1 ml with virulent genotype II ASF virus Georgia 2007/1. After a further 20 days pigs were terminated. This experimental protocol is depicted in
Temperatures (
The control group M of non-immune pigs developed an increased temperature (
Two of the pigs in the immunised group K had a transient increase in temperature above 40.6 for 2 days starting at day 11 post-immunisation (
Previous work had shown that deletion of K145R had minimal direct attenuating effects.
Viremia data is shown in
At necropsy macroscopic lesions in different organs and cavities were scored according to a standardised scoring system. In
Sera collected from pigs of group K at different days before immunisation and at different days after immunisation, boost and challenge were tested for levels of antibodies against the major ASFV capsid protein VP72/B646L using a commercial competitive ELISA assay. In
Peripheral blood mononuclear cells (PBMCs) were collected from pigs of group K before immunisation with GeorgiaΔK145RΔEP153RCD2vQ96R, before boost and before challenge with Georgia 2007/1 virus. The PBMCs were stimulated with ASFV and the number of interferon gamma producing cells measured as an indicator of the cellular immune response to ASFV (
The PBMCs were mock stimulated (blue bars) or stimulated with ASFV genotype|Benin97/1 infectious virus (red bars) or ASFV genotype II Georgia 2007/1 virus (green bars). Numbers of interferon gamma producing cells were measured and are shown per 106 cells on the y axis. The pig number is given on the x-axis. The results show very low or no detectable IFN gamma producing cells before immunisation, as expected (
All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in virology, molecular biology or related fields are intended to be within the scope of the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2003289.2 | Mar 2020 | GB | national |
| 2003292.6 | Mar 2020 | GB | national |
| 2005878.0 | Apr 2020 | GB | national |
| 2005880.6 | Apr 2020 | GB | national |
| 2013541.4 | Aug 2020 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2021/050562 | 3/5/2021 | WO |
