Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/20—Antivirals for DNA viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/525—Virus
  • A61K2039/5256—Virus expressing foreign proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/53—DNA (RNA) vaccination
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
  • A61K2039/541—Mucosal route
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
  • A61K2039/552—Veterinary vaccine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/70—Multivalent vaccine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10041—Use of virus, viral particle or viral elements as a vector
  • C12N2710/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/12011—Asfarviridae
  • C12N2710/12022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/12011—Asfarviridae
  • C12N2710/12034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/24011—Poxviridae
  • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
  • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
  • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• the present invention relates to a vaccine for the treatment and/or prevention of 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 which 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%.
• OURT88/3 is a non-pathogenic isolate of ASFV from Portugal. Previous infection with ASFV OURT88/3 has been shown to confer protection against challenge with related virulent viruses (Boinas et al (2004) J Gen Virol 85:2177-2187; Oura et al (2005) J. Gen. Virol. 86:2445-2450). In addition some gene-deleted viruses are also effective in inducing protection against challenge (eg BeninAMGF and BeninADP148R).
• ASFV has also been investigated. However, these studies have determined that the ability of an ASFV polypeptide to induce an immunogenic response does not necessarily predict an ability to induce protection to ASFV challenge (see Neilan et al.] Virology; 319(2); 337-342; 2004). Further, ASFV polypeptides may only induce protection when used in specific combinations. For example, Neilan et al. (as above) reported that a combination of p30, p54, p72 and p22 was not sufficient to induce protection. In contrast, Argilaguet et al. (Antiviral Res; 98(1); 61-65; 2013) reported that a combination of p30, p54 and EP402R gave partial protection against ASFV in DNA vaccination experiments.
• the present inventors have determined a minimal combination of three ASFV polypeptides that are capable of inducing repeatable protection against ASFV when administered prior to ASFV challenge.
• the inventors have determined that protection is not induced when any one of the three ASFV polypeptides is absent. Further, the inventors have demonstrated that this minimal combination of three ASFV polypeptides may be combined with additional ASFV polypeptides to provide improved vaccine compositions for inducing reliable protection against ASFV challenge and treatment and/or prevention of ASF.
• the present invention provides an ASF virus subunit vaccine which comprises: (i) one or more recombinant polynucleotides which encode polypeptides shown as SEQ ID NO: 1 , 2 and 3 or an immunogenic fragment thereof; or a variant with at least 70% sequence identity to one of SEQ ID NO: 1 , 2 or 3 or an immunogenic fragment thereof; wherein the total number of different ASF virus (ASFV) polypeptides encoded by the one or more recombinant polynucleotides is 10 or fewer; or (ii) recombinant polypeptides shown as SEQ ID NO: 1 , 2 and 3 or an immunogenic fragment thereof; or a variant with at least 70% sequence identity to one of SEQ ID NO: 1 , 2 and 3 or an immunogenic fragment thereof; wherein vaccine comprises 10 or fewer different ASFV polypeptides.
• ASFV ASF virus
• subunit vaccine is used herein to refer to a vaccine which comprises individual polypeptides or polynucleotides encoding said polypeptides; in contrast to vaccines which comprise whole virus particles (such a live, attenuated virus and dead virus).
• the number of different ASFV polypeptides encoded by the one or more recombinant polynucleotides may be 9 or fewer, 8 or fewer, 7 or fewer, or 6 or fewer polypeptides ASFV polypeptides; or the vaccine may comprise 9 or fewer, 8 or fewer, 7 or fewer, or 6 or fewer.
• the vaccine may comprise: (i) one or more further polynucleotides encoding an ASFV polypeptide selected from SEQ ID NO: 4-8 or an immunogenic fragment thereof, or a variant with at least 70% sequence identity to one of SEQ ID NO: 4-8 or an immunogenic fragment thereof; or (ii) one or more further polypeptides selected from SEQ ID NO: 4-8 or an immunogenic fragment thereof; or a variant with at least 70% sequence identity to one of SEQ ID NO: 4-8 or an immunogenic fragment thereof.
• the vaccine may comprise: a) recombinant polynucleotides which encode polypeptides comprising SEQ ID NO: 1-8 or an immunogenic fragment thereof or a variant with at least 70% sequence identity to SEQ ID NO: 1-8 or immunogenic fragments thereof; or recombinant polypeptides comprising SEQ ID NO: 1-8 or an immunogenic fragment thereof or variants with at least 70% sequence identity to SEQ ID NO: 1-8 or immunogenic fragments thereof; (b) recombinant polynucleotides which encode polypeptides comprising SEQ ID NO: 1-3 and 6-8 or an immunogenic fragment thereof or variants with at least 70% sequence identity to SEQ ID NO: 1-3 and 6-8 or immunogenic fragments thereof; or recombinant polypeptides comprising SEQ ID NO: 1-3 and 6-8 or an immunogenic fragment thereof or variants with at least 70% sequence identity to SEQ ID NO: 1-3 and 6-8 or immunogenic fragments thereof; or (c) recombinant polynucleotides which encode
• the recombinant polynucleotides may comprise SEQ ID NO: 9, 10 and 11 or a variant thereof with at least 70% sequence identity.
• the further polynucleotides may comprise one or more of SEQ ID NO: 12-16 or a variant thereof with at least 70% sequence identity.
• the polynucleotide may be present in a vector.
• the vector may be selected from an adenovirus, a modified vaccinia Ankara vector and a pseudorabies virus vector.
• Each of the recombinant polynucleotides may be provided in the same vector.
• the present invention provides a vaccine according to the first aspect of the invention for use in treating and/or preventing African swine fever in a subject.
• the present invention further relates to a method for treating and/or preventing African swine fever in a subject which comprises administering a therapeutically effective amount of a vaccine according to the invention to the subject.
• the present invention relates to the use of a recombinant polynucleotide, vector or recombinant polypeptide as defined in the first aspect of the invention in the manufacture of a medicament for the treatment and/or prevention of African swine fever in a subject.
• the subject may be a swine subject.
• the subject may be a domestic pig.
• the vaccine may be administered according to a prime-boost procedure.
• the priming composition may comprise one or more adenovirus vectors and the boosting composition may comprise one or more modified vaccinia Ankara vectors.
• the vaccine may be administered by oral, intravenous, intramuscular, subcutaneous, intranasal or intradermal administration.
• FIG. 2 Representative amino acid sequences for ASFV polypeptides for use in the present invention (SEQ ID NO: 1-8 and 17)
• Figure 3 Representative nucleic acid sequences encoding ASFV polypeptides for use in the present invention (SEQ ID NO: 9-16)
• the present inventors have determined a minimal combination of three ASFV polypeptides that are capable of inducing repeatable protection against ASFV.
• African swine fever virus is the causative agent of African swine fever (ASF).
• the virus causes a haemorrhagic fever with high mortality rates in pigs, but persistently infects its natural hosts, warthogs, bushpigs with no disease signs. It also infects soft ticks of the Ornithodoros genus, which are thought to be used as a vector.
• ASFV replicates in the cytoplasm of infected cells, and is the only member of the Asfarviridae family to do so.
• ASFV is endemic to sub-Saharan Africa and exists in the wild through a cycle of infection between ticks and wild pigs, bushpigs and warthogs.
• ASFV was first described after European settlers brought pigs into areas endemic with ASFV and, as such, is an example of an 'emerging infection'.
• ASFV is a large, icosahedral, double-stranded DNA virus with a linear genome containing at least 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.
• ASFV genotypes Based on sequence variation in the C-terminal region of the B646L gene encoding the major capsid protein p72, 24 ASFV genotypes (l-XXIX) have been identified. All ASFV p72 genotypes have been circulating in eastern and southern Africa. Genotype I is been circulating in Sardinia and western Africa. Genotype II is circulating in Europe, China, Vietnam and Camboida. Genotype VIII is confined to four East African countries.
• strains from some of the genotypes are given below. Strains shown in bold were used in the identity analysis shown in Table 2.
• Genotype I OURT88/3; Brazil/79; Portugal/60; BA715; ASFV-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; Portugal/57; ASFV-Warm; GHA/1/00; GAM/1/00; Ghana; HOL/86; NAM/1/80; NUR/90/1 ; CAM/4/85; ASFV-Teng; Tengani; ASFV-E75.
• Genotype II Georgia 2007/1 , Belgium 2018/1 , China/2018/AnhuiXCGQ
• Genotype III BOT 1/99
• Genotype IV ASFV-Warthog; 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; Malawi LIL 20/1
• Genotype IX UGA/1/95; Ken06.Bus
• Genotype X BUR/1/84; BUR/2/84; BUR/90/1 ; UGA/3/95; TAN/Kwh12; Hindell; ASFV-Ken; Virulent Kenya 65; Ken05/Tk1.
• the present inventors have determined that the composition comprising a minimal combination of ASFV polypeptides SEQ ID NO: 1-3 are capable of inducing protection against ASFV challenge.
• polypeptide is used in the normal sense to mean a series of residues, typically L- amino acids, connected one to the other typically by peptide bonds between the a-amino and carboxyl groups of adjacent amino acids.
• the term is synonymous with "protein”.
• recombinant polypeptide is used to mean that the polypeptide is isolated from its natural environment, for example, extracted from or produced outside of an ASFV.
• the recombinant polypeptide is not provided in the context of an ASFV and is expressed from a recombinant nucleic acid, i.e. DNA or RNA that is created artificially.
• the vaccine of the present invention may comprise a combination of ASFV polypeptides, as defined herein.
• the present vaccine may comprise recombinant polypeptides shown as SEQ ID NO: 1 , 2 and 3 or an immunogenic fragment thereof; or a variant with at least 70% sequence identity to one of SEQ ID NO: 1 , 2 and 3 or an immunogenic fragment thereof.
• polypeptide of SEQ ID NO: 3 or an immunogenic fragment thereof; or a variant with at least 70% sequence identity to one of SEQ ID NO: 3 or an immunogenic fragment thereof may be substituted with SEQ ID NO: 17 or an immunogenic fragment thereof; or a variant with at least 70% sequence identity to one of SEQ ID NO: 17 or an immunogenic fragment thereof.
• SEQ ID NO: 17 is a representative EP153R sequence from a Genotype II strain.
• the variant may comprise an amino acid sequence which is at least 70, 80, 85, 90, 95, 98 or 99% identical to one of SEQ ID NO: 1 , 2 or 3; or an immunogenic fragment thereof.
• the variant may comprise an amino acid sequence which is at least 70, 80, 85, 90, 95, 98 or 99% identical to SEQ ID NO: 1 or an immunogenic fragment thereof.
• the variant may comprise an amino acid sequence which is at least 70, 80, 85, 90, 95, 98 or 99% identical to SEQ ID NO: 2 or an immunogenic fragment thereof.
• the variant may comprise an amino acid sequence which is at least 70, 80, 85, 90, 95, 98 or 99% identical to SEQ ID NO: 3 or an immunogenic fragment thereof.
• the present vaccine may comprise recombinant polypeptides shown as SEQ ID NO: 1 , 2 and 3; or a variant with at least 70% sequence identity to one of SEQ ID NO: 1 , 2 and/or 3.
• the vaccine may consist of recombinant polypeptides shown as SEQ ID NO: 1 , 2 and 3; or a variant with at least 70% sequence identity to one of SEQ I D NO: 1 , 2 and/or 3.
• the recombinant polypeptides may consist of amino acid sequences shown as SEQ ID NO: 1 , 2 and 3 or a variant thereof which shares at least 70% sequence identity
• the vaccine may further comprise one or more polypeptides selected from SEQ ID NO: 4-8 or an immunogenic fragment thereof; or a variant with at least 70% sequence identity to one of SEQ ID NO: 4-8 or an immunogenic fragment thereof.
• the variant may comprise an amino acid sequence which is at least 70, 80, 85, 90, 95, 98 or 99% identical to one of SEQ ID NO: 4-8; or an immunogenic fragment thereof.
• the variant may comprise an amino acid sequence which is at least 70, 80, 85, 90, 95, 98 or 99% identical to SEQ ID NO: 4 or an immunogenic fragment thereof.
• the variant may comprise an amino acid sequence which is at least 70, 80, 85, 90, 95, 98 or 99% identical to SEQ ID NO: 5 or an immunogenic fragment thereof.
• the variant may comprise an amino acid sequence which is at least 70, 80, 85, 90, 95, 98 or 99% identical to SEQ ID NO: 6 or an immunogenic fragment thereof.
• the variant may comprise an amino acid sequence which is at least 70, 80, 85, 90, 95, 98 or 99% identical to SEQ ID NO: 7 or an immunogenic fragment thereof.
• the variant may comprise an amino acid sequence which is at least 70, 80, 85, 90, 95, 98 or 99% identical to SEQ ID NO: 8 or an immunogenic fragment thereof.
• the vaccine may further comprise one or more polypeptides selected from SEQ ID NO: 4-8 or a variant with at least 70% sequence identity to one of SEQ ID NO: 4-8.
• the vaccine may comprise at least 2, at least 3, at least 4 or at least 5 polypeptides selected from SEQ ID NO: 4-8 or an immunogenic fragment thereof; or a variant with at least 70% sequence identity to one of SEQ ID NO: 4-8.
• the vaccine may comprise 2, 3, 4 or 5 polypeptides selected from SEQ ID NO: 4-8 or an immunogenic fragment thereof; or a variant with at least 70% sequence identity to one of SEQ ID NO: 4-8 or an immunogenic fragment thereof.
• the vaccine may comprise recombinant polypeptides comprising SEQ ID NO: 1-8 or an immunogenic fragment thereof; or variants with at least 70% sequence identity to SEQ ID NO: 1-8 or immunogenic fragments thereof.
• the vaccine may comprise recombinant polypeptides comprising SEQ ID NO: 1-3 and 6-8 or an immunogenic fragment thereof; or variants with at least 70% sequence identity to SEQ ID NO: 1-3 and 6-8 or immunogenic fragments thereof.
• the vaccine may comprise recombinant polypeptides comprising SEQ ID NO: 1-5 or an immunogenic fragment thereof; or variants with at least 70% sequence identity to SEQ ID NO: 1-5 or immunogenic fragments thereof.
• variants can be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express a variant in terms of sequence identity.
• Sequence comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These publicly and commercially available computer programs can calculate sequence identity between two or more sequences.
• Sequence identity may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (for example less than 50 contiguous amino acids).
• the sequence identity is determined across the entirety of the sequence selected from SEQ ID NO: 1-8 or SEQ ID NO: 9-16. In one embodiment, the sequence identity is determined across the entirety of the candidate sequence being compared to a sequence selected from SEQ ID NO: 1-8 or SEQ ID NO: 9-16.
• a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.
• An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). It is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
• variable includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acids from or to the sequence providing the resultant amino acid sequence retains substantially the same activity as the unmodified sequence.
• the variant should be capable of inducing an immune response.
• the induction of an immune response may be determined by the demonstration of a recall response in peripheral immune cells or splenocytes isolated from a subject previously immunised with the polypeptide or immunogenic fragment thereof.
• a recall response may be demonstrated by interferon production (e.g. IFNy) and/or a proliferative response following an in vitro challenge with an antigen or polypeptide previous used to immunise a subject.
• the variant should be capable of inducing a protective immune response in a subject, against subsequent challenge with ASFV when administered in combination with a polypeptide selected from SEQ ID NO: 1-3 or a variant or immunogenic fragment thereof as described herein.
• the polypeptide may comprise an immunogenic fragment of an amino acid sequence selected from SEQ ID NO: 1-8 or a variant thereof which shares at least 70% sequence identity.
• immunogenic fragment it is meant a portion or a part of SEQ ID NO: 1-8 which is capable of inducing an immune response.
• the immunogenic fragment should be capable of inducing a protective immune response in a subject against subsequent challenge with ASFV when administered in combination with a polypeptide selected from SEQ ID NO: 1-3 or a variant or immunogenic fragment thereof as described herein
• Immunogenic fragments of SEQ ID NO: 1-8, or a variant thereof may for example be between 8 and 200 amino acids, for example 8 to 150 amino acids, 8 to 100 amino acids, 8 to 75 amino acids, 8 to 50 amino acids, 8 to 25 amino acids, 8 to 20 amino acids, 8 to 15 amino acids or 8 to 12 amino acids.
• an immunogenic fragment of SEQ ID NO: 1-8, or a variant thereof may be 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids in length.
• the polynucleotide may be any suitable type of polynucleotide, such as a synthetic RNA/DNA sequence, a cDNA sequence or a partial genomic DNA sequence.
• Polynucleotide generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
• Polynucleotides include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
• polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
• the term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
• Modified bases include, for example, tritylated bases and unusual bases such as inosine.
• polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
• Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides.
• recombinant polynucleotide is used to mean that the polynucleotide is isolated from its natural environment, for example, extracted from or produced outside of an ASFV.
• the recombinant polynucleotide is not provided in the context of an ASFV and is provided as a polynucleotide, i.e. DNA or RNA that is created artificially.
• variant is used to mean a naturally occurring nucleic acid sequence which differs from a subject sequence.
• the variant may have at least 70% sequence identity with the subject sequence or up to and including 20 mutations.
• the present vaccine may comprise one or more polynucleotides which encode a combination of recombinant polypeptides according to the vaccine of the present invention.
• the present vaccine may comprise one or more polynucleotides which encode recombinant polypeptides shown as SEQ ID NO: 1 , 2 and 3 or an immunogenic fragment thereof; or a variant with at least 70% sequence identity to one of SEQ ID NO: 1 , 2 and 3 or an immunogenic fragment thereof.
• the present vaccine may comprise one or more polynucleotides which encode recombinant polypeptides shown as SEQ ID NO: 1 , 2 and 3; or a variant with at least 70% sequence identity to one of SEQ ID NO: 1 , 2 and/or 3.
• the vaccine may consist of one or more polynucleotides which encode recombinant polypeptides shown as SEQ ID NO: 1 , 2 and 3; or a variant with at least 70% sequence identity to one of SEQ ID NO: 1 , 2 and/or 3.
• the vaccine may further comprise one or more polynucleotides which encode one or more polypeptides selected from SEQ ID NO: 4-8 or an immunogenic fragment thereof; or a variant with at least 70% sequence identity to one of SEQ ID NO: 4-8 or an immunogenic fragment thereof.
• the vaccine may further comprise one or more polynucleotides which encode one or more polypeptides selected from SEQ ID NO: 4-8 or a variant with at least 70% sequence identity to one of SEQ ID NO: 4-8.
• the vaccine may comprise one or more polynucleotides which encode at least 2, at least 3, at least 4 or at least 5 polypeptides selected from SEQ ID NO: 4-8 or an immunogenic fragment thereof; or a variant with at least 70% sequence identity to one of SEQ ID NO: 4-8.
• the vaccine may comprise one or more polynucleotides which encode 2, 3, 4 or 5 polypeptides selected from SEQ ID NO: 4-8 or an immunogenic fragment thereof; or a variant with at least 70% sequence identity to one of SEQ ID NO: 4-8 or an immunogenic fragment thereof.
• the vaccine may comprise one or more polynucleotides which encode recombinant polypeptides comprising SEQ ID NO: 1-8 or an immunogenic fragment thereof; or variants with at least 70% sequence identity to SEQ ID NO: 1-8 or immunogenic fragments thereof.
• the vaccine may comprise one or more polynucleotides which encode recombinant polypeptides comprising SEQ ID NO: 1-3 and 6-8 or an immunogenic fragment thereof; or variants with at least 70% sequence identity to SEQ ID NO: 1-3 and 6-8 or immunogenic fragments thereof.
• the vaccine may comprise one or more polynucleotides which encode recombinant polypeptides comprising SEQ ID NO: 1-5 or an immunogenic fragment thereof; or variants with at least 70% sequence identity to SEQ ID NO: 1-5 or immunogenic fragments thereof.
• a polynucleotide encoding the polypeptide shown as SEQ ID NO: 1-3 or a variant thereof may comprise SEQ ID NO: 9-11 , respectively; or a variant thereof which shares at least 70% sequence identity to one of SEQ ID NO: 9-11.
• a polynucleotide encoding the polypeptide shown as SEQ ID NO: 1 or a variant thereof may comprise SEQ ID NO: 9; or a variant thereof which is at least 70, 80, 85, 90, 95, 98 or 99% identical to SEQ ID NO: 9.
• a polynucleotide encoding the polypeptide shown as SEQ ID NO: 2 or a variant thereof may comprise SEQ ID NO: 10; or a variant thereof which is at least 70, 80, 85, 90, 95, 98 or 99% identical to SEQ ID NO: 10.
• a polynucleotide encoding the polypeptide shown as SEQ ID NO: 3 or a variant thereof may comprise SEQ ID NO: 11 ; or a variant thereof which is at least 70, 80, 85, 90, 95, 98 or 99% identical to SEQ ID NO: 11.
• a polynucleotide encoding the polypeptide shown as SEQ ID NO: 4-8 or a variant thereof may comprise SEQ ID NO: 12-16, respectively; or a variant thereof which shares at least 70% sequence identity to one of SEQ ID NO: 12-16.
• a polynucleotide encoding the polypeptide shown as SEQ ID NO: 4 or a variant thereof may comprise SEQ ID NO: 12; or a variant thereof which is at least 70, 80, 85, 90, 95, 98 or 99% identical to SEQ ID NO: 12.
• a polynucleotide encoding the polypeptide shown as SEQ ID NO: 5 or a variant thereof may comprise SEQ ID NO: 13; or a variant thereof which is at least 70, 80, 85, 90, 95, 98 or 99% identical to SEQ ID NO: 13.
• a polynucleotide encoding the polypeptide shown as SEQ ID NO: 6 or a variant thereof may comprise SEQ ID NO: 14; or a variant thereof which is at least 70, 80, 85, 90, 95, 98 or 99% identical to SEQ ID NO: 14.
• a polynucleotide encoding the polypeptide shown as SEQ ID NO: 7 or a variant thereof may comprise SEQ ID NO: 15; or a variant thereof which is at least 70, 80, 85, 90, 95, 98 or 99% identical to SEQ ID NO: 15.
• a polynucleotide encoding the polypeptide shown as SEQ ID NO: 8 or a variant thereof may comprise SEQ ID NO: 16; or a variant thereof which is at least 70, 80, 85, 90, 95, 98 or 99% identical to SEQ ID NO: 16.
• the vaccine may further comprise one or more polypeptides selected from SEQ ID NO: 4-8 or a variant with at least 70% sequence identity to one of SEQ ID NO: 4-8.
• the vaccine may comprise one or more polynucleotides comprising SEQ ID NO: 9-16 or variants with at least 70% sequence identity to SEQ ID NO: 9-16.
• the vaccine may comprise one or more polynucleotides comprising SEQ ID NO: 9-11 and 14-16; or variants with at least 70% sequence identity to SEQ ID NO: 9-11 and 14-16.
• the vaccine may comprise one or more polynucleotides comprising SEQ ID NO: 9-13; or variants with at least 70% sequence identity to SEQ ID NO: 9-13 or immunogenic fragments thereof.
• the present invention is based, at least in part, on the inventors’ determination of a minimal combination of polypeptides, or immunogenic fragments thereof, which is capable of inducing protection against ASFV.
• references to polypeptides or combinations of polypeptides provided herein are intended to apply equally to corresponding polynucleotides or combinations of polynucleotides which encode said polypeptides.
• the total number of different ASFV polypeptides (or encoded by the one or more recombinant polynucleotides) in the present vaccine is 10 or fewer.
• the number of different ASFV polypeptides is 9 or fewer, 8 or fewer, 7 or fewer, or 6 or fewer.
• the number of different ASFV polypeptides is 3, 4, 5, 6, 7 or 8.
• the number of different ASFV polypeptides is 3.
• the number of different ASFV polypeptides is 5.
• the number of different ASFV polypeptides is 6.
• the number of different ASFV polypeptides is 8.
• a“vector” may be any agent capable of delivering or maintaining nucleic acid in a host cell, and includes viral, bacterial and eukaryotic vectors, plasmids, naked nucleic acids, nucleic acids complexed with polypeptide or other molecules and nucleic acids immobilised onto solid phase particles.
• Polynucleotides in accordance with the present invention may be delivered by viral or non-viral techniques.
• Non-infectious delivery systems include but are not limited to DNA transfection methods.
• transfection includes a process using a non-infectious vector to deliver nucleic acid of the invention to a target cell.
• Typical transfection methods include electroporation, nucleic acid biolistics, lipid-mediated transfection, compacted nucleic acid-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated, cationic facial amphiphiles (CFAs), multivalent cations such as spermine, cationic lipids or polylysine, 1 , 2, -bis (oleoyloxy)-3-(trimethylammonio) propane (DOTAP)-cholesterol complexes and combinations thereof.
• CFAs cationic facial amphiphiles
• multivalent cations such as spermine, cationic lipids or polylysine, 1 , 2, -bis (oleoyloxy)-3-(trimethylammonio) propane (DOTAP)-cholesterol complexes and combinations thereof.
• Non-viral delivery systems may also include, but are not limited to, bacterial delivery systems. Bacteria have previously been used as anticancer agents and as delivery agents for anticancer drugs.
• Cell adhesion molecules are a large group of molecules involved in a variety of cell-to-cell and cell-to-extra-cellular matrix (ECM) interactions and are exploited by a number of pathogenic micro-organisms as receptors for cell entry. These molecules may be used for the targeting and uptake of both gene and drug delivery systems.
• ECM cell-to-cell and cell-to-extra-cellular matrix
• a gene gun delivery system may also be used for the delivery of DNA.
• Viral delivery systems include but are not limited to adenovirus vectors, modified vaccinia Ankara vectors, adeno-associated viral (AAV) vectors, herpes viral vectors such as pseudorabies virus vector, retroviral vectors, lentiviral vectors or baculoviral vectors, Venezuelan equine encephalitis virus (VEE), poxviruses such as: canarypox virus, entomopox virus, penguine alphavirus, and alphavirus based DNA vectors.
• AAV adeno-associated viral
• herpes viral vectors such as pseudorabies virus vector, retroviral vectors, lentiviral vectors or baculoviral vectors
• VEE Venezuelan equine encephalitis virus
• poxviruses such as: canarypox virus, entomopox virus, penguine alphavirus, and alphavirus based DNA vectors.
• the vector may be an adenovirus vector.
• the adenovirus is a double-stranded, linear DNA virus that does not replicate through an RNA intermediate.
• RNA intermediate There are over 50 different human serotypes of adenovirus divided into 6 subgroups based on their genetic sequence.
• Adenoviruses are double-stranded DNA non-enveloped viruses that are capable of in vivo, ex vivo and in vitro transduction of a broad range of cell types of human and non-human origin. These cells include respiratory airway epithelial cells, hepatocytes, muscle cells, cardiac myocytes, synoviocytes, primary mammary epithelial cells and post-mitotically terminally differentiated cells such as neurons. Adenoviral vectors are also capable of transducing non dividing cells.
• Adenoviruses have been used as vectors for gene therapy and for expression of heterologous genes.
• the large (36 kb) genome can accommodate up to 8 kb of foreign insert DNA and is able to replicate efficiently in complementing cell lines to produce very high titres of up to 1012 transducing units per ml.
• Adenovirus is thus one of the best systems to study the expression of genes in primary non-replicative cells.
• the expression of viral or foreign genes from the adenovirus genome does not require a replicating cell.
• Adenoviral vectors enter cells by receptor mediated endocytosis. Once inside the cell, adenovirus vectors rarely integrate into the host chromosome. Instead, they function episomally (independently from the host genome) as a linear genome in the host nucleus.
• the vector may be a modified vaccinia Ankara (MVA) vector.
• the MVA virus is related to Vaccinia virus, a member of the genera Orthopoxvirus in the family of Poxviridae.
• the MVA virus has been generated by 516 serial passages on chicken embryo fibroblasts of the Chorioallantois Vaccinia Ankara (CVA) virus.
• CVA Chorioallantois Vaccinia Ankara
• the MVA virus has lost approximately 15% of the genomic DNA at multiple sites (Mayr and Munz 1964 in Monol Orig 195, 24-35; Meyer et al. 1991 in J Gen Virol 72 ( Pt 5), 1031-1038).
• the MVA virus has been analysed to determine alterations in the genome relative to the wild-type CVA strain.
• Six major deletions of genomic DNA (deletion I, II, III, IV, V, and VI), totalling 31.000 base pairs, have been identified (Meyer, et al. 1991 in J Gen Virol 72 (Pt 5), 1031-1038). MVA does not replicate in human and non-human primate cells.
• vaccinia vectors include avipox vectors such as fowlpox or canarypox known as ALVAC and strains derived therefrom which can infect and express recombinant proteins in human cells but are unable to replicate.
• avipox vectors such as fowlpox or canarypox known as ALVAC and strains derived therefrom which can infect and express recombinant proteins in human cells but are unable to replicate.
• ex vivo delivery systems include ex vivo delivery systems, which include but are not limited to DNA transfection methods such as electroporation, DNA biolistics, lipid-mediated transfection and compacted DNA-mediated transfection.
• the vector may be a plasmid DNA vector.
• plasmid refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof.
• the vector may comprise more than one nucleic acid sequences, each of which encodes a different polypeptide as described for use in the present vaccine.
• one or more vectors may comprise multiple nucleic acid sequences which between them encode a combination of polypeptide as described for use in the present vaccine
• the vector may comprise more than one nucleic acid sequence, such that the vector comprises nucleic acid sequences which between them encode a combination of polypeptide as described for use in the present vaccine.
• an internal ribosome entry site is used to initiate translation of the second (and subsequent) coding sequence(s) in a polycistronic message (Adam et al 1991 J. Virol. 65, 4985).
• IRES elements Insertion of IRES elements into retroviral vectors is compatible with the retroviral replication cycle and allows expression of multiple coding regions from a single promoter (Adam et al (as above); Koo et al (1992) Virology 186:669-675; Chen et al 1993 J. Virol 67:2142-2148).
• IRES elements were first found in the non-translated 5’ ends of picornaviruses where they promote cap-independent translation of viral proteins (Jang et al (1990) Enzyme 44: 292-309). When located between open reading frames in an RNA, IRES elements allow efficient translation of the downstream open reading frame by promoting entry of the ribosome at the IRES element followed by downstream initiation of translation.
• IRES encephalomyocarditis virus
• IRES includes any sequence or combination of sequences which work as or improve the function of an IRES.
• the IRES(s) may be of viral origin (such as EMCV IRES, PV IRES, or FMDV 2A-like sequences) or cellular origin (such as FGF2 IRES, NRF IRES, Notch 2 IRES or EIF4 IRES).
• viral origin such as EMCV IRES, PV IRES, or FMDV 2A-like sequences
• cellular origin such as FGF2 IRES, NRF IRES, Notch 2 IRES or EIF4 IRES.
• the IRES In order for the IRES to be capable of initiating translation of each polynucleotide it should be located between or prior to the polynucleotides in the vector genome.
• the polynucleotide may encode a polypeptide which comprises two or more immunogenic polypeptides for use in the present vaccine, wherein each polypeptide is joined by a cleavage site.
• the cleavage site may be self-cleaving, such that when the polypeptide is produced, it is immediately cleaved into the individual immunogenic polypeptides without the need for any external cleavage activity.
• FMDV Foot-and-Mouth disease virus
• a ‘self-cleaving peptide’ refers to a peptide which functions such that when the nascent polypeptide comprising two or more polypeptides for use in the present vaccine and the self cleaving peptide is produced, it is immediately“cleaved” or separated into distinct and discrete ASFV polypeptides without the need for any external cleavage activity.
• the polypeptide comprises multiple ASFV polypeptides for use in the present vaccine, for example, three or more ASFV polypeptides, there may be a self-cleaving polypeptide present between each of the ASFV polypeptides.
• the self-cleaving peptide may be a 2A self-cleaving peptide from an aphtho- or a cardiovirus.
• the primary 2A/2B cleavage of the aptho- and cardioviruses is mediated by 2A“cleaving” at its own C-terminus.
• apthoviruses such as foot-and-mouth disease viruses (FMDV) and equine rhinitis A virus
• the 2A region is a short section of about 18 amino acids, which, together with the N-terminal residue of protein 2B (a conserved proline residue) represents an autonomous element capable of mediating“cleavage” at its own C-terminus.
• control sequences which include promoters/enhancers and other expression regulation signals.
• Prokaryotic promoters and promoters functional in eukaryotic cells may be used.
• Tissue specific or stimuli specific promoters may be used.
• Chimeric promoters may also be used comprising sequence elements from two or more different promoters.
• Suitable promoting sequences are strong promoters including those derived from the genomes of viruses, such as polyoma virus, adenovirus, fowlpox virus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), retrovirus and Simian Virus 40 (SV40), or from heterologous mammalian promoters, such as the actin promoter, EF1a, CAG, TK, SV40, ubiquitin, PGK or ribosomal protein promoter.
• viruses such as polyoma virus, adenovirus, fowlpox virus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), retrovirus and Simian Virus 40 (SV40), or from heterologous mammalian promoters, such as the actin promoter, EF1a, CAG, TK, SV40, ubiquitin, PGK or
• tissue-specific promoters such as rhodopsin (Rho), rhodopsin kinase (RhoK), cone-rod homeobox containing gene (CRX), neural retina-specific leucine zipper protein (NRL), Vitelliform Macular Dystrophy 2 (VMD2), Tyrosine hydroxylase, neuronal-specific neuronal-specific enolase (NSE) promoter, astrocyte-specific glial fibrillary acidic protein (GFAP) promoter, human a1-antitrypsin (hAAT) promoter, phosphoenolpyruvate carboxykinase (PEPCK), liver fatty acid binding protein promoter, Flt-1 promoter, INF-b promoter, Mb promoter, SP-B promoter, SYN1 promoter, WASP promoter, SV40 / hAlb promoter, SV40 / CD43, SV40 / CD45, NSE / RU5' promoter, ICAM-2
• Enhancers are relatively orientation- and position-independent; however, one may employ an enhancer from a eukaryotic cell virus, such as the SV40 enhancer on the late side of the replication origin (bp 100-270) and the CMV early promoter enhancer.
• the enhancer may be spliced into the vector at a position 5' or 3' to the promoter, but is preferably located at a site 5' from the promoter.
• the promoter can additionally include features to ensure or to increase expression in a suitable target cell.
• the features can be conserved regions e.g. a Pribnow Box or a TATA box.
• the promoter may contain other sequences to affect (such as to maintain, enhance or decrease) the levels of expression of a nucleotide sequence. Suitable other sequences include the Sh1-intron or an ADH intron. Other sequences include inducible elements, such as temperature, chemical, light or stress inducible elements. Also, suitable elements to enhance transcription or translation may be present.
• vaccine 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, for example in the present case ASF.
• the vaccine of the present invention thus induces an immune response in a subject that is protective against subsequent ASFV challenge.
• the present vaccine may comprise a vector, recombinant polypeptide and/or recombinant polynucleotide as described herein.
• the present vaccine comprises a plurality of vectors, recombinant polypeptides and/or recombinant polynucleotides as defined herein.
• plurality is used herein to mean more than one vector, recombinant polypeptide and/or recombinant polynucleotide as described herein.
• a plurality may mean two, three, four, five, six, seven, eight, nine, or ten recombinant polypeptides and/or recombinant polynucleotides as defined herein.
• the vaccine may be useful in preventing African Swine Fever.
• the vaccine composition may optionally comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
• the choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
• the vaccine composition 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 that may aid or increase the delivery or immunogenicity of the vaccine.
• Such a formulation may, for example, be in a form suitable for oral, intravenous, intramuscular, subcutaneous, intranasal, intradermal administration or suppository routes or implanting (e.g. using slow release molecules).
• the vaccine may additionally comprise an adjuvant.
• adjuvants include but are not limited to aluminium salts, oil emulsions and bacterial components (e.g. LPS and liposomes).
• the present invention also provides a method of preventing and/or treating ASF in a subject by administration of an effective amount of a vaccine of the invention.
• the present invention further provides a vaccine of the first aspect of the present invention for use in treating and/or preventing ASF.
• 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.
• treating is intended to refer to reducing or alleviating at least one symptom of an existing ASF infection.
• the vaccine may be administered in a convenient manner such as by the oral, intravenous, intramuscular, subcutaneous, intranasal, intradermal or suppository routes or implanting (e.g. using slow release molecules).
• a veterinarian or producer will determine the actual dosage which will be most suitable for an individual subject or group of subjects and it may vary, for example, with the age, weight and response of the particular subject(s).
• the dosage is such that it is sufficient to reduce and/or prevent disease symptoms.
• route of delivery e.g., oral vs intravenous vs subcutaneous vs intratumoural, etc
• dose amount may impact route of delivery.
• focused delivery e.g., in this example, intratumoural delivery
• intratumoural delivery may be desired and/or useful.
• Other factors to be considered when optimizing routes and/or dosing schedule for a given therapeutic regimen may include, for example, the particular cancer being treated (e.g., type, stage, location, etc.), the clinical condition of a subject (e.g., age, overall health, etc.), the presence or absence of combination therapy, and other factors known to medical practitioners.
• the dosage is such that it is sufficient to stabilise or improve symptoms of the disease.
• the vaccine may be administered following a prime-boost regime.
• the subject may receive a second boosting administration some time (e.g. 3 to 21 , 3 to 14, 5 to 14 or 5 to 7 days) later.
• the subject may receive a second boosting administration about 3, 5, 7, 14 or 21 days after the first inoculation.
• the first inoculation may be administered as one or more adenovirus vectors as described herein; and the second inoculation may be administered as one or more MVA vectors as defined herein.
• the boosting administration is at a higher dose than the priming administration.
• the vector, immunogenic composition or vaccine may be administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and oral formulations. Where the immunogenic composition or vaccine is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. as a suspension.
• 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 subject may be susceptible to ASF infection.
• the subject may have been diagnosed as positive for the disease and/or show one or more symptom(s) associated with the infection.
• 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.
• Example 1 Immunisation of pigs with pools of subunit ASFV polypeptides and challenge with ASFV
• Various polynucleotides encoding different ASFV polypeptides were individually cloned in recombinant adenovirus and modified vaccinia Ankara (MVA) vectors.
• the cloned sequences were from the genotype I OURT88/3 strain except for EP153R and MGF360-11 L which were from the genotype I strain Benin 1997/1.
• Ten groups of pigs were immunised as shown in Table 1.
• Pigs in experiments AR000737 and AR000742 were primed with 5E9 IU of adenovirus and 7.5E7 pfu of MVA. Pigs in experiments AR000750 and AR000858 were primed with 1.5 c 10 10 infectious units of each recombinant adenovirus vector and boosted with 2 c 10 8 plaque forming units of each recombinant MVA vector. Details of the different combinations of antigens, vectors used for the priming and boosting doses and induction of protection are provided in Table 1.
• B602L, E183L and EP153R are present in all of the groups in which protection was observed after challenge. Protection was determined when animals did not reach the predetermined humane endpoints of the study.
• the OUR T88/1 isolate was grown in primary macrophages from pig bone marrow.
• Virus stocks were prepared from a spleen suspension from an infected pig and titres were determined by limiting dilution using haemadsorption to detect virus infected cells and titres were calculated by the Spearman- Karber method
• Pigs were either outbred cross-bred Large White and Landrace or outbred cross-bred White, Landrace and Hampshire from a high health status farm or. Pigs were of average size 20 kg at the start of experiments.
• Recombinant adenoviruses (rAds) and modified vaccinia Ankara viruses were delivered by intramuscular injection in at most 5 ml total volume at two separate sites.
• MVA or rAd boosts were delivered to pigs 4 weeks after the initial rAd prime. Pigs were challenged intramuscularly with 10 4 HAD 5O Georgia 2007/1 virus four to eight weeks after the boost. Pigs were observed for development of clinical signs and these were scored using the scoring system used previously. Blood and tissue samples were collected to measure levels of virus replication.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Virology (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Organic Chemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Molecular Biology (AREA)
• Microbiology (AREA)
• Mycology (AREA)
• Immunology (AREA)
• Epidemiology (AREA)
• Genetics & Genomics (AREA)
• Biophysics (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Oncology (AREA)
• Communicable Diseases (AREA)
• Biotechnology (AREA)
• Engineering & Computer Science (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Biochemistry (AREA)
• Gastroenterology & Hepatology (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Peptides Or Proteins (AREA)

Priority Applications (11)

Applications Claiming Priority (2)

Publications (1)

Family

ID=67990353

Family Applications (1)

Country Status (15)

Cited By (1)

Families Citing this family (2)

Citations (2)

Family Cites Families (7)

• 2019
  • 2019-07-29 GB GBGB1910794.5A patent/GB201910794D0/en not_active Ceased
• 2020
  • 2020-07-28 TW TW109125471A patent/TW202118771A/zh unknown
  • 2020-07-28 CN CN202080062696.4A patent/CN114375199A/zh active Pending
  • 2020-07-28 WO PCT/GB2020/051808 patent/WO2021019232A1/en not_active Ceased
  • 2020-07-28 MX MX2022001028A patent/MX2022001028A/es unknown
  • 2020-07-28 US US17/628,286 patent/US20220273788A1/en active Pending
  • 2020-07-28 AU AU2020322191A patent/AU2020322191A1/en active Pending
  • 2020-07-28 BR BR112022000865A patent/BR112022000865A2/pt unknown
  • 2020-07-28 KR KR1020227006051A patent/KR20220041144A/ko active Pending
  • 2020-07-28 EP EP20751252.6A patent/EP4003412A1/en active Pending
  • 2020-07-28 CA CA3148586A patent/CA3148586A1/en active Pending
  • 2020-07-28 PH PH1/2022/550063A patent/PH12022550063A1/en unknown
  • 2020-07-28 JP JP2022505456A patent/JP2022542174A/ja active Pending
• 2022
  • 2022-01-24 CL CL2022000161A patent/CL2022000161A1/es unknown
  • 2022-02-21 CO CONC2022/0001782A patent/CO2022001782A2/es unknown

Patent Citations (2)

Also Published As

Similar Documents

Legal Events