Patent Application
20230117978
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.
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.
There is currently no treatment for ASF. Prevention in countries outside Africa has been attempted on a national basis by restrictions on incoming pigs and pork products, compulsory boiling of waste animal products under licence before feeding to pigs and the application of a slaughter policy when the disease is diagnosed. Prevention in Africa is based on measures to keep warthogs and materials contaminated by warthogs away from the herd.
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).
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.
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).
Generally, the invention relates to an attenuated African Swine Fever virus in which expression and/or activity of the genes DP148R, EP153R and EP402R is disrupted, whilst expression and/or activity of particular MGF genes is not disrupted.
The invention provides an attenuated African Swine Fever (ASF) virus in which the expression and/or activity of the genes DP148R, EP153R and EP402R is disrupted; and which 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 DP148R, EP153R and EP402R is disrupted;
and which 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 DP148R, EP153R and EP402R is disrupted;
and which comprises a functional version of one or more of the following genes:
The invention also provides a vaccine comprising an attenuated ASF virus of the invention.
The invention further provides a vaccine of the invention for use in treating and/or preventing ASF in a subject.
The invention further provides a method for treating and/or preventing ASF in a subject which comprises the step of administering to the subject an effective amount of a vaccine according to the invention.
The invention yet further provides a method of attenuating an ASF virus which comprises the step of disrupting the expression and/or activity of the following genes: DP148R, EP153R and EP402R.
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 VIII is confined to four East African countries.
Examples of strains from some of the genotypes are given below:
Genotype I : OURT88/3; Brazil/79; Lisbon/60; BA715; Pret; Benin 97/1; IC/1/96; IC/576; CAM/82; Madrid/62; Malta/78; ZAR85; Katange63; Togo; Dakar59; Ourt88/1; BEN/1/97; Dom_Rep; VAL/76; IC/2/96; Awoshie/99; NIG/1/99; NIG/1/98; ANG/70; BEL/85; SPEC120; Lisbon/57; ASFV-Warm; GHA/1/00; GAM/1/00; Ghana; HOL/86; NAM/1/80; NUR/90/1; CAM/4/85; ASFV-Teng; Tegani; ASFV-E75.
Genotype II: Georgia 2007/1; POL/2015/Podlaskie (Polish strain); Belgium/Etalle/wb/2018; ASFV/Kyiv/2016/131; China/2018/AnhuiXCGQ
Genotype III: BOT 1/99
Genotype IV: ASFV-War; RSA/1/99/W
Genotype VI: MOZ 94/1
Genotype VII: VICT/90/1; ASFV-Mku; RSA/1/98
Genotype VIII: NDA/1/90; KAL88/1; ZAM/2/84; JON89/13; KAV89/1; DEZda; AFSV-Mal; Malawi LIL 20/1
Genotype IX: UGA/1/95
Genotype X: BUR/1/84; BUR/2/84; BUR/90/1; UGA/3/95; TAN/Kwh12; Hindell; ASFV-Ken; Virulent Uganda 65.
The attenuated ASF virus of the present invention may be derivable from a wild-type ASF virus isolate, but includes mutations in its genome such that the expression and/or activity of the genes DP148R, EP153R and EP402R is disrupted.
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” refers to all genes other than the disrupted genes DP148R, EP153R and EP402R. 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 for the genes DP148R, 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 DP148R, EP153R and EP402R.
The disrupted genes DP148R, EP153R and EP402R may also correspond to the wild-type strain. In an embodiment the genes DP148R, EP153R and EP402R correspond to the wild-type strain. In such an embodiment (i.e. where DP148R, EP153R and EP402R correspond to the wild-type strain) expression and/or activity of DP148R, EP153R and EP402R may be disrupted by one or more mutation in an intergenic region such as a promoter. In other words, the DP148R, 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.
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 DP148R, EP153R and EP402R).
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 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 I.
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 may correspond or essentially correspond to that of the Benin 97/1 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.
The complete genome for the African swine fever virus Benin 97/1 pathogenic isolate is given in Genbank Locus: AM712239.1. The complete BA71 isolate genome encodes 151 open reading frames (ORFs), the Benin 97/1 isolate encodes 157 ORFs and the OURT88/3 isolate encodes 151 ORFs.
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.
The 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 DP148R, 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 DP148R, 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 DP148R, 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 DP148R, 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 DP148R, 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 2. 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. 40 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 5 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 30 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 35 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 30 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 40 360 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 5 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. 10 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 35 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 50 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 5 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 10 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 20 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 25 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 5 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 10 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 60 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 25 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 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:
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-κB transcription factor (see
The gene (i.e. nucleotide) sequences and positions in the genome of DP148R genes from different ASFV strains are presented below.
In an embodiment the invention provides an attenuated ASF virus in which the expression and/or activity of the DP148R gene is disrupted. Suitably, the DP148R gene comprises the sequence of SEQ ID No. 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299 or 300. Suitably, the DP148R gene comprises a sequence having at least 70%, at least 80%, at least 90% or at least 95% identity with SEQ ID No. 287, 288, 289, 290, 291, 292, 293, 294, 50 295, 296, 297, 298, 299 or 300. Suitably, the DP148R gene consists of the sequence of SEQ ID No. 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299 or 300.
The amino acid sequence of the Benin 97/1 DP148R protein is depicted below as SEQ ID No. 11:
Suitably, the DP148R gene encodes a protein comprising the sequence of SEQ ID No. 11. Suitably, the DP148R 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. 11. Suitably, the DP148R gene encodes a protein consisting of the sequence of SEQ ID No. 11.
Orthologous DP148R sequences from other genomes share between 74 and 99% amino acid identity. The orthologous DP148R genes from other ASFV isolates are depicted below as SEQ ID Nos 12 to 19, and their location in the ASFV genome is indicated:
Suitably, the DP148R gene encodes a protein comprising the sequence of SEQ ID No. 12.Suitably, the DP148R gene encodes a protein comprising the sequence of SEQ ID No. 13. Suitably, the DP148R gene encodes a protein comprising the sequence of SEQ ID No. 14. Suitably, the DP148R gene encodes a protein comprising the sequence of SEQ ID No. 15. Suitably, the DP148R gene encodes a protein comprising the sequence of SEQ ID No. 16. Suitably, the DP148R gene encodes a protein comprising the sequence of SEQ ID No. 17. Suitably, the DP148R gene encodes a protein comprising the sequence of SEQ ID No. 18. Suitably, the DP148R gene encodes a protein comprising the sequence of SEQ ID No. 19. Suitably, the DP148R gene encodes a protein comprising the sequence of SEQ ID No. 301. Suitably, the DP148R gene encodes a protein comprising the sequence of SEQ ID No. 302. Suitably, the DP148R gene encodes a protein comprising the sequence of SEQ ID No. 303. Suitably, the DP148R gene encodes a protein comprising the sequence of SEQ ID No. 304. Suitably, the DP148R gene encodes a protein comprising the sequence of SEQ ID No. 305.
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 attenuated 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.
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.
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.
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 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. The EP402R protein may be referred to as CD2v due to this similarity.
EP402R is immunogenic (i.e. evokes an immune response) (Netherton et al. 2019 Front. Immunol. 10, 1318). EP402R is required for haemadsorption and may have a role in virus entry or spread. 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 attenuated 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.
The amino acid sequence of the EP402R protein from the Benin 97/1 strain is depicted below as SEQ ID No. 21.
Suitably, the EP402R gene encodes a protein comprising the sequence of SEQ ID No. 21. Suitably, the EP402R 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. 21. Suitably, the EP402R gene encodes a protein consisting of the sequence of SEQ ID No. 21.
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.
Suitably, the ligand-binding domain of the EP402R protein comprises the sequence of SEQ ID No. 34. Suitably, the ligand-binding domain of the EP402R protein comprises a sequence having at least 70%, at least 80%, at least 90% or at least 95% identity with SEQ ID No. 34. Suitably, the ligand-binding domain of the EP402R protein consists of the sequence of SEQ 5 ID No. 34.
The orthologous EP402R genes from other ASFV isolates are depicted below as SEQ ID Nos 22 to 30:
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, such as shown in
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 the sequence of SEQ ID No. 22. Suitably, the EP402R gene encodes a protein comprising the sequence of SEQ ID No. 23. Suitably, the EP402R gene encodes a protein comprising the sequence of SEQ ID No. 24. Suitably, the EP402R gene encodes a protein comprising the sequence of SEQ ID No. 25. Suitably, the EP402R gene encodes a protein comprising the sequence of SEQ ID No. 26. Suitably, the EP402R gene encodes a protein comprising the sequence of SEQ ID No. 27. Suitably, the EP402R gene encodes a protein comprising the sequence of SEQ ID No. 28. Suitably, the EP402R gene encodes a protein comprising the sequence of SEQ ID No. 29. Suitably, the EP402R gene encodes a protein comprising the sequence of SEQ ID No. 30. Suitably, the EP402R gene encodes a protein comprising the sequence of SEQ ID No. 242. Suitably, the EP402R gene encodes a protein comprising the sequence of SEQ ID No. 243. Suitably, the EP402R gene encodes a protein comprising the sequence of SEQ ID No. 244. Suitably, the EP402R gene encodes a protein comprising the sequence of SEQ ID No. 245. Suitably, the EP402R gene encodes a protein comprising the sequence of SEQ ID No. 246.
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 assay such as described herein. For example, 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 which has reduced haemadsorption.
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 attenuated ASFV of the invention or transfected with a mutant, non-functional EP153R or EP402R protein adsorb fewer red blood cells to their surface than cells infected with a wild-type ASFV or 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.
The attenuated African Swine Fever (ASF) virus of one embodiment of the present invention has disrupted expression and/or activity of the genes DP148R, EP153R and EP402R. These genes may be referred to herein as the “disrupted genes”.
In an embodiment the invention provides an attenuated ASFV in which expression of the genes DP148R, 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.
In an embodiment the invention provides an attenuated ASFV in which activity of the genes DP148R, EP153R and EP402R is disrupted.
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 activity 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 attenuated 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.
Gene expression and/or activity are disrupted by mutating the ASFV genome i.e. by mutating 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, deleting nucleotides and/or a combination of these.
In an embodiment the attenuated ASF virus of the invention comprises mutations that disrupt the expression and/or activity of the genes DP148R, 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.
In an embodiment of the attenuated ASFV of the invention, DP148R activity may be disrupted.
In an embodiment the activity of DP148R that may be disrupted is its ability to inhibit type I interferon. In other words, the ability of DP148R to inhibit type I interferon may be decreased. The ability of DP148R to inhibit type I interferon may be decreased by at least 50, 60, 70, 80 or 90%. The ability of DP148R to inhibit type I interferon may be completely abolished.
In an embodiment the activity of DP148R that may be disrupted is its ability to inhibit NF-κB activity. In an embodiment, the invention provides an attenuated ASF virus in which the ability of the DP148R gene to inhibit NFκB activity is disrupted. NFκB activity may be measured using a luciferase reporter assay as described herein.
Disruption of the ability to inhibit NFκB activity means that the ability to inhibit NFκB activity is decreased. In other words, the DP148R gene of the attenuated ASF virus of the invention inhibits NFκB activity less than would a wild-type version of the DP148R gene. The ability to inhibit NFκB activity may be decreased by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In an embodiment the ability of the DP148R gene to inhibit NFκB activity may be abolished i.e. the DP148R gene does not inhibit NFκB activity. In that case, the activity of NFκB would be as high as if no DP148R gene were present. A decrease in inhibition means NFκB activity is higher than it would be with the wild-type version of the DP148R gene. NFκB activity may be twice as high as it would be with the wild type version of the DP148R gene, such as three times as high, four times as high, five times as high or six times as high.
NFκB is a transcription factor that activates transcription of various genes, controlling expression of interferon and pro-inflammatory cytokines as part of the host's innate immune system response to viral infection. Inhibition of NFκB activity means decreasing activation of expression of its target genes. The degree of activation of NFκB target gene expression may be measured using a luciferase reporter assay in which an NFκB-activatable promoter is linked to a luciferase reporter.
In an embodiment the activity of DP148R that may be disrupted is its ability to inhibit translocation of the p65 sub-unit of NFκB to the nucleus. In an embodiment, the invention provides an attenuated ASF virus in which the ability of the DP148R gene to inhibit translocation of the p65 sub-unit of NFκB to the nucleus is disrupted. Translocation of p65 to the nucleus can be measured by immunofluorescence of cells stimulated with NF-κB activators as described herein. The relative and absolute amounts of p65 that translocate to the nucleus may quantified by using differential cell lysis conditions known in the art to either break open the nucleus or leave it intact then quantify the amount of p65 using techniques known in the art such as Western blotting or mass spectrometry. For example, cells expressing a wild type or mutant DP148R protein may be stimulated to activate NF-κB then differentially lysed to produce whole cell lysate, a cytoplasmic fraction and a nuclear fraction. The amount of p65 in each fraction may then be quantified for the different conditions (wild type or mutant DP148R protein).
Disruption of the ability to inhibit translocation of p65 to the nucleus means that the ability to inhibit translocation of p65 to the nucleus is decreased. In other words, the version of the DP148R gene in the attenuated ASF virus of the invention inhibits translocation of p65 to the nucleus less than would a wild-type version of the DP148R gene. The ability to inhibit translocation of p65 to the nucleus may be decreased by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In an embodiment the ability of the DP148R gene to inhibit translocation of p65 to the nucleus may be abolished i.e. the DP148R gene does not inhibit translocation of p65 to the nucleus. In that case, as much p65 would translate to the nucleus as if no DP148R gene were present. A decrease in inhibition means the proportion of p65 that translocates to the nucleus is higher than it would be with the wild-type version of the DP148R gene. The proportion of p65 that translocates to the nucleus may be twice as high as it would be with the wild type version of the DP148R gene, such as three times as high, four times as high, five times as high or six times as high.
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 of the attenuated 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 attenuated 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.
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, 11 L, 12L, 13L, 14L, 20R, 21R and 22R; and MGF 505 1R, 2R and 6R.
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 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.
In the attenuated 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 attenuated 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 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. 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 deletion may be complete, in which case 100% of the coding sequence of the gene is absent when compared to the corresponding genome of the wild-type isolate.
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 “deletion of a gene” (such as “partially deleted”) refers to deletion of a sufficient amount of coding sequence such that expression and/or activity of the gene is disrupted. The amount of coding sequence required to be deleted to disrupt gene expression and/or activity may be very small. For example, deletion of just the start codon (ATG) may be sufficient to disrupt expression and/or activity of the gene to attenuate the virus.
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.
In an embodiment of the attenuated ASF virus of the present invention the genes DP148R, EP153R and EP402R may be at least partially deleted.
The DP148R gene may be at least partially deleted. The EP153R gene may be at least partially deleted. The EP402R gene may be at least partially deleted. The DP148R and EP153R genes may each be at least partially deleted. The DP148R, EP153R and EP402R genes may each be at least partially deleted.
In an embodiment of the attenuated ASF virus of the present invention the genes DP148R, EP153R and EP402R may be completely deleted.
The DP148R gene may be completely deleted. The EP153R gene may be completely deleted. The EP402R gene may be completely deleted. The DP148R and EP153R genes may each be completely deleted. The DP148R, EP153R and EP402R genes may each be completely deleted.
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 attenuated ASFV of the present invention the genes DP148R, EP153R and EP402R may interrupted. The DP148R gene may be interrupted. The EP153R gene may be interrupted. The EP402R gene may be interrupted. The DP148R and EP153R genes may each be interrupted. The DP148R, EP153R and EP402R genes may each 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.
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 a deletion. A gene may comprise multiple mutations that lead to interruption of the gene.
The interruption may be a frame shift mutation. 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 an embodiment of the attenuated 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.
Changing of the one or more amino acids in the ligand-binding domain of the EP402R protein should disrupt expression and/or activity of the EP402R protein. In an embodiment, changing the one or more amino acids in the ligand-binding domain of the EP402R protein may disrupt haemadsorption mediated by the EP402R protein. In an embodiment, changing the one or more amino acids in the ligand-binding domain of the EP402R protein may disrupt ligand binding by the EP402R protein.
In an embodiment, the one or more mutations in the ligand-binding domain 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 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 mutations that change a single amino acid into a different amino acid. Such an amino change may be referred to as a substitution. Changing even a single amino acid may disrupt the activity of the EP402R protein, such as ligand binding activity. For example, changing an amino acid that can be charged to an amino acid that can have the opposite charge may disrupt folding of the EP402R ligand-domain such that a binding pocket is deformed, which prevents ligand binding. Alternatively or additionally, substitution with an amino acid that can have an 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 (VV). 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 E99 and/or Y102 of the Benin 97/1 EP402R protein may be assessed by sequence alignment. For example,
Amino acids corresponding to E99 and Y102 of Benin 97/1 EP402R exist in EP402R proteins from strains other than those shown in
In an embodiment the one or more mutations change an amino acid at position E99 and/or Y102 of Benin 97/1 EP402R protein (SEQ ID NO. 11). In an embodiment the one or more mutations change an amino acid at position E99 of Benin 97/1 EP402R protein (SEQ ID NO. 11). In an embodiment the one or more mutations change an amino acid at position Y102 of Benin 97/1 EP402R protein (SEQ ID NO. 11). In an embodiment the one or more mutations change an amino acid at position Q96 of Georgia 2007/1 EP402R protein (SEQ ID NO. 24).
In an embodiment the one or more mutations change an amino acid at a position in the EP402R protein corresponding to N16, 119, W21, Y76, E99, Y102 and/or N108 of Benin 97/1 EP402R protein (SEQ ID NO. 11). 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 there 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).
In an embodiment the amino acid at the position corresponding to E99 is changed to R and/or the amino acid at the position corresponding to Y102 is changed to D. In an embodiment the amino acid at the position corresponding to E99 is changed to R. In an embodiment the amino acid at the position corresponding to Y102 is changed to D. In an embodiment E99 of Benin 97/1 EP402R is changed to R and/or Y102 of Benin 97/1 EP402R is changed to D. In an embodiment E99 of Benin 97/1 EP402R is changed to R. In an embodiment Y102 of Benin 97/1 EP402R is changed to D. In an embodiment Q96 of Georgia 2007/1 EP402R is changed to R.
The amino acid sequence of the EP402R protein from Benin 97/1 strain comprising the E99R substitution is depicted below as SEQ ID No. 31.
In an embodiment the EP402R gene encodes a protein comprising the sequence of SEQ ID No. 31. Suitably, the EP402R 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. 31, wherein amino acid 99 is R. Suitably, the EP402R gene encodes a protein consisting of the sequence of 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.
In an embodiment the EP402R gene encodes a protein comprising the sequence of SEQ ID No. 32. Suitably, the EP402R 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. 32, wherein amino acid 102 is D. Suitably, the EP402R gene encodes a protein consisting of the sequence of 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.
In an embodiment the EP402R gene encodes a protein comprising the sequence of SEQ ID No. 33. Suitably, the EP402R 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. 33, wherein amino acid 96 is R. Suitably, the EP402R gene encodes a protein consisting of the sequence of SEQ ID No. 33.In an embodiment the EP402R gene encodes a protein comprising a sequence selected from SEQ ID No. 31, 32 or 33.
The mutations that disrupt gene expression and/or activity described herein may be combined in an attenuated ASFV of the invention. In other words, each of the DP148R, EP153R and EP402R genes in an attenuated ASFV of the invention 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 attenuated ASFV of the invention, DP148R may be disrupted by mutation of a promoter sequence, EP153R may be disrupted by complete deletion and EP402R may be disrupted by an amino acid change in its ligand-binding domain. As an alternative example, DP148R may be disrupted by partial deletion, EP153R may be disrupted by interruption and EP402R may be disrupted by complete deletion.
In an embodiment the invention provides an attenuated ASFV in which
the DP148R gene and EP153R gene are each completely deleted, and
the EP402R gene comprises a mutation that changes an amino acid at a position in the EP402R protein which corresponds to E99 of Benin 97/1 EP402R protein (SEQ ID No. 11) to R;
and which comprises a functional version of one or more of the following genes:
The attenuated ASFV may comprise a functional version of one or more of the MGF genes as described herein.
In an embodiment the invention provides an attenuated ASFV in which
The attenuated ASFV may comprise a functional version of one or more of the MGF genes as described herein.
In an embodiment the invention provides an attenuated ASFV in which
The attenuated ASFV may comprise a functional version of one or more of the MGF genes as described herein.
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. 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.
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 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.
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 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 about 102, 103 or 104 HAD50 or TCID50 of the recombinant attenuated virus per pig.
The present invention also provides a method of attenuating an ASF virus which comprises the step of disrupting the expression and/or activity of the genes DP148R, EP153R and EP402R.
Disruption of gene expression and/or activity may be achieved by mutating the ASFV genome in any of the ways described herein.
The DP148R, EP153R and/or EP402R genes may be partially or completely deleted.
The DP148R gene may be at least partially or completely deleted. The EP153R gene may be at least partially or completely deleted. The EP402R gene may be at least partially or completely deleted. The DP148R and EP153R genes may be at least partially or completely deleted. The DP148R, EP153R and EP402R genes may each be partially or completely deleted. The coding sequences of one or more (such as two or more, or all three) of the disrupted genes may be at least partially or completely deleted.
The DP148R, EP153R and/or EP402R genes may be interrupted.
The DP148R gene may be interrupted. The EP153R gene may be interrupted. The EP402R gene may be interrupted. The DP148R and EP153R genes may be interrupted. The DP148R, EP153R and EP402R genes may each be interrupted. The coding sequences of one or more (such as two or more, or all three) of the disrupted genes may be interrupted.
In an embodiment the method of attenuating an ASF virus comprises disrupting the ability of the EP402R gene to mediate haemadsorption.
In an embodiment the method of attenuating an ASF virus comprises comprising introducing one or more mutations in the EP402R gene that disrupt ligand binding by the EP402R protein.
In an embodiment the method of attenuating an ASF virus 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. The mutation may be one or more of the mutation as described herein.
In an embodiment the method of attenuating an ASF virus comprises changing the amino acid at position R96, E99 and/or Y102 of the EP402R protein.
In an embodiment the method of attenuating an ASF virus comprises changing the amino acid at position R96 to Q, the amino acid at position E99 to R and/or the amino acid at position Y102 to D. Suitably the method comprises changing the amino acid at position R96 to Q. Suitably the method comprises changing the amino acid at position E99 to R. Suitably the method comprises changing the amino acid at position Y102 to D.
In an embodiment the method of attenuating an ASF virus comprises completely deleting the DP148R gene, completely deleting the EP153R gene and introducing a mutation in the EP402R gene that changes the amino acid at position R96 of the EP402R protein to Q. In an embodiment the method of attenuating an ASF virus comprises completely deleting the DP148R gene, completely deleting the EP153R gene and introducing a mutation in the EP402R gene that changes the amino acid at position E99 of the EP402R protein to R. In an embodiment the method of attenuating an ASF virus comprises completely deleting the DP148R gene, completely deleting the EP153R gene and introducing a mutation in the EP402R gene that changes the amino acid at position Y102 of the EP402R protein to D.
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.
“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.
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, 11 L, 12L, 13L, 14L, 20R, 21R and 22R; and MGF 505 1R, 2R and 6R.
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 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 and a secondary antibody (
Mutation of residues E99 or Y102 abrogated HAD.
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 equivalent residue 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. 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.
The ability to induce HAD could be restored by infecting WSL cells expressing the CD2vE99R mutant with BeninΔCD2v virus (
However, HAD could not be restored by infecting WSL cells expressing the CD2vE99R mutant with OURT88/3 virus (
Based on these results, two gene deleted Benin viruses were constructed using as backbone the attenuated BeninΔDP148R virus. One virus had in addition to DP148R further deletions of EP153R and EP402R (BeninΔDP148RΔEP153RΔCD2v) and the second had EP153R deleted and the wild-type EP402R gene replaced with the gene expressing CD2vE99R mutant (BeninΔDP148RΔEP153R-CD2vE99R).
The Benin CD2vE99R mutant protein was expressed at the cell surface, as shown in
Cells infected with these viruses did not induce HAD.
HEK293T cells were transfected with an NF-κB dependent luciferase reporter plasmid and either a plasmid encoding the DP148R gene or an empty plasmid (pcDNA3) as a negative control. Twenty four hours post transfection the cells were treated with IL-1β (
In a pair of alternative experiments, NF-κB activation was instead induced by overexpression of the two main kinases of the NF-κB signalling pathway, namely IKKα (
To further elucidate the mechanism by which DP148R inhibits NF-κB activation, the effect of DP148R on nuclear translocation of the p65 sub-unit of NF-κB was investigated. HeLa cells were transfected with a plasmid expressing HA-tagged DP148R and were then stimulated with TNF-alpha or IL-β to activate NF-κB. Unstimulated cells were used as a negative control. Cells were fixed and permeabilised then stained for HA-tagged DP148R using an anti-HA tag antibody and an appropriate secondary antibody. The p65 subunit of NF-κB was stained with a primary antibody and secondary antibody.
Groups of large white/landrace pigs were immunised intramuscularly with 104 TCID50 BeninΔDP148RΔEP153RΔCD2v (Group A—eight pigs) and BeninΔDP148RΔEP153R-CD2vE99R (Group B—six pigs).
Pigs were boosted on day 21 (104 TCID50/m1) and day 28 (106 TCID50/m1) post-immunisation and then challenged with 103 TCID50/m1 parental virulent Benin 97/1 strain on day 45 post-immunisation, in parallel with three non-immunised pigs (control Group E).
The experimental protocol is depicted in
Rectal temperatures were recorded daily for all pigs in Group A and B and the control Group E (
Three pigs in Group B had an increase in temperature above 40.5° C. at day 5 post-immunisation. One pig had a temperature of 41.1° C. by day 3 post-challenge and this was maintained a further 2 days when the animal was culled at the humane endpoint. From the other pigs all survived, one had a single day temperature above 40.5° C. at day 4 post-challenge, three had 2 days increase temperature (days 4 and 5) and one had no increased temperature. All survivors had no further temperature increase until the end of the experiment (day 20 post-challenge) (
Control pigs had temperatures above 40.5° C. from day 4 post-challenge and were culled at the humane endpoint on day 6 post-challenge (
Clinical scores, including increased temperature and other signs such as lethargy and loss of appetite were collected daily (
In Group B mild signs were observed in pigs post-immunization coincident with temperature increases. After challenge mild signs were observed except for the pig which was culled at the humane endpoint and the pig which did not show an increase in temperature which had no clinical signs (
Levels of virus genome in blood were measured by qPCR (
In group B low levels of virus genome were detected in blood from 3 pigs at day 6 post-immunization. After challenge virus genome was detected at low levels (below 10e4 genome copies per ml) from day 7 post-challenge (
ASFV specific antibody responses were measured using a commercially available competitive ELISA based on the VP72 (B646L) major capsid protein (
In pigs in Group B antibody responses were detected by day 14 (1 week before the boost) and increased maintaining a plateau after day 21 to 28 (
Peripheral blood mononuclear cells were collected pre-immunization, boost and challenge from pigs in Groups A and B and stimulated with ASFV Benin virus. Numbers of IFN gamma producing cells were measured by Elispot assay (
Control non-immune pigs (Group E) showed lesions typical of acute ASFV including enlarged and haemmorhagic lymph nodes and spleen. Those pigs which survived challenge had few, if any, ASFV specific lesions (
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/GB2021/050560 | 3/5/2021 | WO |
