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WO2021161189A1 - Assemblage extracellulaire de particules de type virus - Google Patents

Assemblage extracellulaire de particules de type virus Download PDF

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Publication number
WO2021161189A1
WO2021161189A1 PCT/IB2021/051085 IB2021051085W WO2021161189A1 WO 2021161189 A1 WO2021161189 A1 WO 2021161189A1 IB 2021051085 W IB2021051085 W IB 2021051085W WO 2021161189 A1 WO2021161189 A1 WO 2021161189A1
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ahsv
host cell
structural proteins
transforming
cell population
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Ann Elizabeth Meyers
Edward Peter Rybicki
Inga Isabel Hitzeroth
Susan Jennifer DENNIS
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University of Cape Town
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University of Cape Town
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Priority to EP21705618.3A priority Critical patent/EP4103721A1/fr
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Priority to SA522440104A priority patent/SA522440104B1/ar
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    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/04—Inactivation or attenuation; Producing viral sub-units
    • 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
    • A61K39/15—Reoviridae, e.g. calf diarrhea virus
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C12N15/8258—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon for the production of oral vaccines (antigens) or immunoglobulins
    • 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
    • A61K2039/5258—Virus-like particles
    • 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
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12111—Orbivirus, e.g. bluetongue virus
    • C12N2720/12123—Virus like particles [VLP]
    • 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
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12111—Orbivirus, e.g. bluetongue virus
    • C12N2720/12134—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
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/12011—Reoviridae
    • C12N2720/12111—Orbivirus, e.g. bluetongue virus
    • C12N2720/12151—Methods of production or purification of viral material

Definitions

  • the African horse sickness virus (AHSV) genome consists of 10 segments of linear double-stranded RNA, encoding seven structural and five non-structural proteins.
  • the virion is non-enveloped and is composed of three distinct protein layers.
  • the inner core is formed by VP3, and the outer core layer is formed by the protein VP7, which is the group-specific antigen used in ELISA-based diagnostics.
  • VP7 assembles into trimers that attach to the VP3 surface.
  • these two layers enclose the subcore, which comprises 10 dsRNA segments together with the transcription complex, to form a stable icosahedral core particle around 78 nm in diameter.
  • the outer capsid is composed of globular trimers of protein VP5, surrounded by trimeric spikes of protein VP2, which is the protein containing the antigenic determinants that induce serotype-specific neutralizing antibodies.
  • African horse sickness is an infectious, non-contagious illness of horses, which is frequently fatal in susceptible hosts. It is recognized as one of the most lethal viral diseases of horses worldwide. The disease is caused by a number of distinct serotypes of African horse sickness virus (AHSV), a group of non-enveloped isometric dsRNA viruses from the genus Orbivirus, family Reoviridae, the virus being transmitted by biting midges of the Culicoides genus. The virus is endemic to sub- Saharan Africa. South Africa is one of the few countries where all nine serotypes of the virus have been isolated.
  • AHSV African horse sickness virus
  • live-attenuated vaccines Disease control in South Africa has largely been effected by immunization with live-attenuated vaccines.
  • the currently used live-attenuated vaccine is supplied in two polyvalent vials containing three and four AHSV serotypes each, however neither AHSV-5 nor AHSV-9 is included in the vaccine.
  • the live-attenuated vaccine is currently the best option in the fight against AHS, its use has raised concerns with regard to possible reversion to virulence, gene segment re-assortment between outbreak and vaccine strains, and the absence of its ability to Differentiate between Infected and Vaccinated Animals (DIVA).
  • DIVA Differentiate between Infected and Vaccinated Animals
  • the live- attenuated vaccine is not licensed or authorised for use outside of the African subcontinent.
  • DIVA vaccines which would primarily address the concerns of the South African equestrian community, as well as being acceptable prophylactic or rapid response vaccines in the European and other emerging outbreak contexts.
  • AHSV research Due to raised international awareness and local dissatisfaction with the current vaccine, AHSV research has focussed in recent years on the development of recombinant vaccines based on selected antigenic AHSV proteins, particularly the outer capsid proteins VP2 and VP5.
  • Baculovirus expression systems and poxvirus vectors have been used to produce vaccines that induce protective immunity against various AHSV antigens.
  • VLPs Virus-like particles
  • Such vaccines present no risk of reversion to virulence nor of dsRNA segment re-assortment with wild virus strains because they do not contain viral RNA or non-structural proteins, which also makes it possible to distinguish between vaccinated and infected animals using molecular diagnostic techniques.
  • the present invention relates to in vitro methods for extracellular production of African Horse Sickness Virus (AHSV) virus like particles (VLPs) and to uses of said VLPs.
  • AHSV African Horse Sickness Virus
  • VLPs virus like particles
  • AHSV African Horse Sickness Virus
  • AHSV structural proteins may be selected from structural proteins from different AHSV serotypes, resulting in the production of chimaeric VLPs that may be used in the production of a multivalent vaccine composition.
  • nucleic acid sequences may be cloned into a single expression vector.
  • two of the nucleic acid sequences could be cloned into one expression vector and two nucleic acid sequences could be cloned into a second expression vector.
  • each nucleic acid is capable of being expressed as an independent protein.
  • each nucleic acid sequence could be individually cloned into its own expression vector.
  • the host cell populations may be combined after the step of expressing the structural proteins but before the step of recovering the expressed structural proteins.
  • the expressed structural proteins are first recovered from their respective host cell populations and then combined.
  • the step of transforming is performed on at least two host cell populations, since co-transformation and co-expression of all of the structural proteins in a single host cell population would not provide evidence of extracellular assembly of the VLPs.
  • the inventors however contend that co transformation of a single cell population with expression vectors encoding the four structural proteins, co-expression of the structural proteins, recovery and subsequent incubation of the structural proteins for between 20 to 48 hours at 4 °C would also result in extracellular assembly of the VLPs.
  • nucleotide sequences may be codon optimised sequences.
  • the host cell may be selected from any suitable cell used for the expression of proteins such as the group consisting of plant cells, insect cells, mammalian cells, algal cells, yeast cells or bacterial cells.
  • the host cell is a plant cell and most preferably the plant cell is a Nicotiana benthamiana cell.
  • the four structural proteins are all from a single AHSV serotype.
  • at least one of the structural proteins is from a first AHSV serotype and wherein at least one of the structural proteins is from a second AHSV serotype.
  • the resultant VLP will be a chimaeric VLP comprising structural proteins from different AHSV serotypes.
  • AHSV African Horse Sickness Virus
  • VLP virus like particle
  • a method of preventing African Horse Sickness in a subject comprising administering a therapeutically effective amount of the AHSV VLP produced according to the method of the invention to a subject.
  • the subject being is selected from the group consisting of horses, mules, donkeys and zebras.
  • Figure 1 After incubating overnight at 4 °C, crude plant lysates were centrifuged through discontinuous OptiprepTM gradients and fractions collected from the bottom of the tubes were separated by denaturing SDS-PAGE followed by Coomassie blue staining. Leaves were singly infiltrated with the 4 AHSV serotype 5 recombinant Agrobacterium strains and then homogenized together - fractions 6 (lane 1) and 7 (lane 2) are shown.
  • AHSV viral proteins VP2 SEQ ID NO:1
  • VP3 SEQ ID NO:2
  • VP5 SEQ ID NO:3
  • VP7 SEQ ID NO:4
  • Figure 2 After incubating overnight at 4 °C, crude plant lysates were centrifuged through discontinuous OptiprepTM gradients and fractions collected from the bottom of the tubes were separated by denaturing SDS-PAGE followed by Coomassie blue staining. Leaves co-infiltrated with the AHSV-5 VP3, AHSV-5 VP5 and AHSV-5 VP7 recombinant Agrobacterium strains were homogenized together with leaves infiltrated with the AHSV-5 VP2 recombinant Agrobacterium strain - fractions 6 (lane 3), 7 (lane 2) and 8 (lane 1) are shown. The location of the AHSV viral proteins VP2, VP3, VP5 and VP7 are indicated to the right of the gel, while the molecular weight marker sizes are shown on the left.
  • FIG. 3 Gradient fraction 7 of the homogenate obtained from the leaves singly infiltrated with the 4 AHSV serotype 5 recombinant Agrobacterium strains was imaged by TEM, revealing the presence of fully assembled VLPs together with some assembly intermediates. Scale bars, 200 nm.
  • FIG. 4 Gradient fraction 7 of the homogenate obtained from the leaves co-infiltrated with the AHSV-5 VP3, AHSV-5 VP5 and AHSV-5 VP7 Agrobacterium strains and the leaves separately infiltrated with the AHSV-5 VP2 recombinant Agrobacterium strain was imaged by TEM, revealing the presence of fully assembled VLPs together with some assembly intermediates. Scale bars, 200 nm.
  • Figure 5 After incubating overnight at 4 °C, crude plant lysates were centrifuged through discontinuous OptiprepTM gradients and fractions collected from the bottom of the tubes were separated by denaturing SDS-PAGE followed by Coomassie blue staining. Leaves co-infiltrated with the AHSV-5 VP3, AHSV-4 VP5 (SEQ ID NO:6) and AHSV-5 VP7 Agrobacterium strains were homogenized together with leaves infiltrated with the AHSV-4 VP2 (SEQ ID NO:5) recombinant Agrobacterium strain - fractions 6 (lane 1), 7 (lane 2) and 8 (lane 3) are shown. The location of the AHSV viral proteins VP2, VP3, VP5 and VP7 are indicated to the left of the gel, while the molecular weight marker sizes are shown on the right.
  • Figure 6 After incubating overnight at 4 °C, crude plant lysates were centrifuged through discontinuous OptiprepTM gradients and fractions collected from the bottom of the tubes were separated by denaturing SDS-PAGE followed by Coomassie blue staining.
  • Lane 1 shows the results from leaves co-infiltrated with the AHSV-5 VP2, AHSV-5 VP3, AHSV-4 VP5 and AHSV-5 VP7 Agrobacterium strains , while in lane 2, leaves co-infiltrated with the AHSV-5 VP3 and AHSV-5 VP7 Agrobacterium strains were homogenized together with leaves co-infiltrated with the AHSV-4 VP2 and AHSV-4 VP5 recombinant Agrobacterium strain - fraction 7 is shown in each case.
  • the location of the AHSV viral proteins VP2, VP3, VP5 and VP7 are indicated to the right of the gel, while the molecular weight marker sizes are shown on the left.
  • FIG. 7 Gradient fraction 7 of the homogenate obtained from the leaves co-infiltrated with the AHSV-5 VP3, AHSV-4 VP5 and AHSV-5 VP7 Agrobacterium strains and the leaves separately infiltrated with the AHSV-4 VP2 recombinant Agrobacterium strain was imaged by TEM, revealing the presence of fully assembled VLPs together with some assembly intermediates.
  • Figure 8 Gradient fraction 7 of the homogenate obtained from the leaves co-infiltrated with the AHSV-5 VP2, AHSV-5 VP3, AHSV-4 VP5 and AHSV-5 VP7 Agrobacterium strains was imaged by TEM, revealing the presence of fully assembled VLPs together with some assembly intermediates and demonstrating that AHSV-5 VP2 will assemble with AHSV-4 VP5 to form the outer capsid layer.
  • FIG. 9 Gradient fraction 7 of the homogenate obtained from the leaves co-infiltrated with the AHSV-5 VP3 and AHSV-5 VP7 Agrobacterium strains and the leaves separately co-infiltrated with the AHSV-4 VP5 and AHSV-4 VP2 recombinant Agrobacterium strains was imaged by TEM, revealing the presence of fully assembled VLPs together with some assembly intermediates, however far fewer fully assembled VLPs were observed when leaves co-infiltrated with the AHSV-5 VP3 and AHSV-5 VP7 Agrobacterium strains were homogenized together with leaves co infiltrated with the AHSV-4 VP2 and AHSV-4 VP5 recombinant Agrobacterium strains. Scale bars, 200 nm.
  • Figure 11 Amino acid sequence of the AHSV5 VP3 protein (SEQ ID NO: 1
  • Figure 12 Amino acid sequence of the AHSV5 VP5 protein (SEQ ID NO: 1
  • Figure 14 Amino acid sequence of the AHSV4 VP2 protein (SEQ ID NO: 1
  • Figure 15 Amino acid sequence of the AHSV4 VP5 protein (SEQ ID NO: 1
  • Figure 16 Codon optimised nucleotide sequence encoding the AHSV5 VP2 protein (SEQ ID NO:7).
  • Figure 17 Codon optimised nucleotide sequence encoding the AHSV5 VP3 protein (SEQ ID NO:8).
  • VP5 protein (SEQ ID NOS).
  • VP7 protein (SEQ ID NO:10).
  • Figure 20 Codon optimised nucleotide sequence encoding the AHSV4 VP2 protein (SEQ ID NO:11).
  • Figure 21 Codon optimised nucleotide sequence encoding the AHSV4 VP5 protein (SEQ ID NO:12).
  • the present invention relates to the production of African horse sickness virus-like particles (AHSV VLPs) by separately expressing or expressing in different combinations in plants the 4 individual AHSV capsid proteins (VP2, VP3, VP5 and VP7) which make up the virus particle and then mixing the clarified plant lysates to allow assembly of the AHS VLPs in vitro.
  • AHSV VLPs African horse sickness virus-like particles
  • the inventors have shown that following this approach the formation of serotype specific VLPs is possible, further this method also allows for the production of chimeric AHSV VLPs by mixing the capsid proteins from different African horse sickness virus (AHSV) serotypes to make particles.
  • the present invention allows for the production of a multivalent VLP vaccine that provides protection against multiple serotypes of AHSV.
  • This invention allows manufacturers to rapidly produce multi-capsid protein component viruses from available stocks.
  • the individual proteins may be produced at large scale and stored long term. Then once an outbreak of AHS virus occurs VLPs can be produced in vitro by combining the relevant proteins from the relevant serotype.
  • chimeric particles may be produced by the methods disclosed herein in order to produce vaccines that protect against multiple serotypes. The methods also ensure that variable expression is managed. Variable expression occurs when multiple vectors are transformed into a single plant. If one of the expression vectors does not produce the intended target protein, then there is no way of knowing this. This could also result in incomplete formation of the capsid particles.
  • the present invention solves that problem directly by allowing the amounts of each protein to be determined prior to the structural proteins being combined in order to produce a AHSV VLP.
  • the structural proteins may be combined in a ratio of 1 :1 :1 :1 or a ratio of 1 :1 :2:1 or a ratio of 2:1 :2:1 of the structural proteins VP2:VP3:VP5:VP7.
  • This invention gives a manufacturer additional control in the assembly of a candidate chimaeric AHS vaccine. It enables the improved assembly of the AHS chimaeric VLPs, by mixing different combinations of structural proteins from different serotypes of AHSV. The value proposition is therefore the additional control that the method provides the manufacturer and the possibility of using the method within a quality management system towards the production of AHS vaccine.
  • African horse sickness or “AHS” is meant the disease itself.
  • the virus is referred to herein as “African horse sickness virus” or “AHSV” belongs to a group of approximately 9 related but genetically distinct “serotypes”.
  • AHSV is a double stranded ribonucleic acid (dsRNA) virus that causes an infectious, non-contagious disease of equids. It is classified as an Orbivirus in the family Reoviridae. The virus is transmitted by biting midges of the Culicoides species.
  • dsRNA double stranded ribonucleic acid
  • the AHS virion is an icosahedral, non-enveloped particle, composed of three concentric layers surrounding the segmented double-stranded RNA genome.
  • the AHS virion has been reported to be approximately 80 nm in diameter.
  • the subcore composed of structural protein VP3, encloses 10 linear genome segments and enzymatic minor proteins VP1 , VP4 and VP6.
  • the subcore is covered by a layer of VP7 trimers forming the core particle.
  • the core is surrounded by the outermost layer composed of structural proteins VP5 and VP2, with VP2 being the neutralizing antigen and serotype determinant.
  • There are nine known serotypes of AHSV and all are present within South Africa and most parts of sub-Saharan Africa.
  • AHSV VLPs and compositions according to the invention may be used to treat or prevent AHSV infection or conditions associated with AHSV infection.
  • condition associated with AHSV infection is meant any condition, disease or disorder that has been correlated with the presence of an existing AHSV infection and includes secondary effects.
  • AHSV infects equid species, such as horses, donkeys, mules and zebra, amongst others.
  • Zebras are considered the natural vertebrate host of AHSV and rarely exhibit clinical signs of infection. Respiratory and circulatory functions are impaired in diseased animals and result in oedema of subcutaneous and intermuscular tissues, of lungs and haemorrhages of serosal surfaces. These animals also exhibit pyrexia and loss of appetite.
  • the AHSV VLPs may be produced by either:
  • the structural proteins may be expressed as set out in cell populations transformed according to Table 1 .
  • an AHSV VLP made according to the method of the invention may be a chimaeric AHSV VLP wherein the capsid proteins are produced from different AHSV serotypes.
  • the VP3, VP5 and VP7 structural proteins are from one AHSV and the VP2 structural protein is from a second AHSV sertotype.
  • a “protein,” “peptide” or “polypeptide” is any chain of two or more amino acids, including naturally occurring or non-naturally occurring amino acids or amino acid analogues, irrespective of post-translational modification (e.g., glycosylation or phosphorylation).
  • nucleic acid or “nucleic acid molecule” encompass both ribonucelotides (RNA) and deoxyribonucleotides (DIMA), including cDNA, genomic DNA, and synthetic DNA.
  • the nucleic acid may be double-stranded or single- stranded. Where the nucleic acid is single-stranded, the nucleic acid may be the sense strand or the antisense strand.
  • a nucleic acid molecule may be any chain of two or more covalently bonded nucleotides, including naturally occurring or non- naturally occurring nucleotides, or nucleotide analogs or derivatives.
  • RNA is meant a sequence of two or more covalently bonded, naturally occurring or modified ribonucleotides.
  • DNA refers to a sequence of two or more covalently bonded, naturally occurring or modified deoxyribonucleotides.
  • nucleic acid molecule refers to two nucleic acids molecules, e.g., DNA or RNA, which are capable of forming Watson-Crick base pairs to produce a region of double-strandedness between the two nucleic acid molecules. It will be appreciated by those of skill in the art that each nucleotide in a nucleic acid molecule need not form a matched Watson-Crick base pair with a nucleotide in an opposing complementary strand to form a duplex. One nucleic acid molecule is thus “complementary” to a second nucleic acid molecule if it hybridizes, under conditions of high stringency, with the second nucleic acid molecule.
  • a nucleic acid molecule according to the invention includes both complementary molecules.
  • a “substantially identical” sequence is an amino acid or nucleotide sequence that differs from a reference sequence only by one or more conservative substitutions, or by one or more non-conservative substitutions, deletions, or insertions located at positions of the sequence that do not destroy or substantially reduce the antigenicity of one or more of the expressed polypeptides or of the polypeptides encoded by the nucleic acid molecules. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the knowledge of those with skill in the art. These include using, for instance, computer software such as ALIGN, Megalign (DNASTAR), CLUSTALW or BLAST software.
  • polypeptide or polynucleotide sequence that has at least about 80% sequence identity, at least about 90% sequence identity, or even greater sequence identity, such as about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the sequences described herein.
  • two nucleic acid sequences may be “substantially identical” if they hybridize under high stringency conditions.
  • the “stringency” of a hybridisation reaction is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation which depends upon probe length, washing temperature, and salt concentration. In general, longer probes required higher temperatures for proper annealing, while shorter probes require lower temperatures.
  • Hybridisation generally depends on the ability of denatured DNA to re anneal when complementary strands are present in an environment below their melting temperature.
  • a typical example of such “stringent” hybridisation conditions would be hybridisation carried out for 18 hours at 65°C with gentle shaking, a first wash for 12 min at 65°C in Wash Buffer A (0.5% SDS; 2XSSC), and a second wash for 10 min at 65°C in Wash Buffer B (0.1% SDS; 0.5% SSC).
  • polypeptides, peptides or peptide analogues can be synthesised using standard chemical techniques, for instance, by automated synthesis using solution or solid phase synthesis methodology. Automated peptide synthesisers are commercially available and use techniques known in the art. Polypeptides, peptides and peptide analogues can also be prepared from their corresponding nucleic acid molecules using recombinant DNA technology.
  • the nucleic acid molecules of the invention may be operably linked to other sequences.
  • operably linked is meant that the nucleic acid molecules encoding the VP2, VP3, VP5 and/or VP7 polypeptides of the invention and regulatory sequences are connected in such a way as to permit expression of the proteins when the appropriate molecules are bound to the regulatory sequences.
  • operably linked sequences may be contained in vectors or expression constructs which can be transformed or transfected into host cells for expression. It will be appreciated that any vector or vectors can be used for the purposes of expressing the VP2, VP3, VP5 and/or VP7 of the invention.
  • the term “recombinant” means that something has been recombined.
  • nucleic acid construct When used with reference to a nucleic acid construct the term refers to a molecule that comprises nucleic acid sequences that are joined together or produced by means of molecular biological techniques.
  • the term “recombinant” when used in reference to a protein or a polypeptide refers to a protein or polypeptide molecule which is expressed from a recombinant nucleic acid construct created by means of molecular biological techniques.
  • Recombinant nucleic acid constructs may include a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature.
  • a recombinant nucleic acid construct indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e. by human intervention.
  • Recombinant nucleic acid constructs may be introduced into a host cell by transformation.
  • Such recombinant nucleic acid constructs may include sequences derived from the same host cell species or from different host cell species.
  • vector refers to a means by which polynucleotides or gene sequences can be introduced into a cell.
  • vectors There are various types of vectors known in the art including plasmids, viruses, bacteriophages and cosmids. Generally polynucleotides or gene sequences are introduced into a vector by means of a cassette.
  • cassette refers to a polynucleotide or gene sequence that is expressed from a vector, for example, the polynucleotide or gene sequences encoding the VP2, VP3, VP5 and/or VP7 polypeptides of the invention.
  • a cassette generally comprises a gene sequence inserted into a vector, which in some embodiments, provides regulatory sequences for expressing the polynucleotide or gene sequences.
  • the vector provides the regulatory sequences for the expression of the VP2, VP3, VP5 and/or VP7 polypeptides.
  • the vector provides some regulatory sequences and the nucleotide or gene sequence provides other regulatory sequences. “Regulatory sequences” include but are not limited to promoters, transcription termination sequences, enhancers, splice acceptors, donor sequences, introns, ribosome binding sequences, poly(A) addition sequences, and/or origins of replication.
  • AHSV VLPs or compositions of the invention can be provided either alone or in combination with other compounds (for example, nucleic acid molecules, small molecules, peptides, or peptide analogues), in the presence of an adjuvant, or any carrier, such as a pharmaceutically acceptable carrier and in a form suitable for administration to mammals, for example, humans, cattle, sheep, etc.
  • compounds for example, nucleic acid molecules, small molecules, peptides, or peptide analogues
  • an adjuvant such as a pharmaceutically acceptable carrier and in a form suitable for administration to mammals, for example, humans, cattle, sheep, etc.
  • a “pharmaceutically acceptable carrier” or “excipient” includes any and all antibacterial and antifungal agents, coatings, dispersion media, solvents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • a “pharmaceutically acceptable carrier” may include a solid or liquid filler, diluent or encapsulating substance which may be safely used for the administration of the AHSV VLPs or vaccine composition to a subject.
  • the pharmaceutically acceptable carrier can be suitable for intramuscular, intraperitoneal, intravenous, subcutaneous, oral or sublingual administration.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions, dispersions and sterile powders for the preparation of sterile solutions.
  • Suitable formulations or compositions to administer the AHSV VLPs and compositions to subjects who are to be prophylactically treated for an African horse sickness infection, who are suffering from an African horse sickness infection or subjects which are presymptomatic for a condition associated with African horse sickness infection fall within the scope of the invention.
  • Any appropriate route of administration may be employed, such as, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracistemal, intraperitoneal, intranasal, aerosol, topical, or oral administration.
  • subject includes wild and domestic ruminants, equids or any specified target animal
  • an effective amount of the AHSV VLPs or compositions of the invention can be provided, either alone or in combination with other compounds, with immunological adjuvants, for example, aluminium hydroxide dimethyldioctadecylammonium hydroxide or Freund’s incomplete adjuvant.
  • immunological adjuvants for example, aluminium hydroxide dimethyldioctadecylammonium hydroxide or Freund’s incomplete adjuvant.
  • the AHSV VLPs or compositions of the invention may also be linked with suitable carriers and/or other molecules, such as bovine serum albumin or keyhole limpet hemocyanin in order to enhance immunogenicity.
  • the AHSV VLPs produced according to the method of the invention invention may be provided in a kit, optionally with a carrier and/or an adjuvant, together with instructions for use.
  • an “effective amount” of a compound according to the invention includes a therapeutically effective amount, immunologically effective amount, or a prophylactically effective amount.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as treatment of an African horse sickness infection or a condition associated with such infection. The outcome of the treatment may for example be measured by a decrease in viremia, inhibition of viral gene expression, delay in development of a pathology associated with the African horse sickness infection, stimulation of the immune system, or any other method of determining a therapeutic benefit.
  • a therapeutically effective amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects.
  • any of the AHSV VLPs produced according to the methods of the present invention will vary depending on the symptoms, age and body weight of the subject, the nature and severity of the disorder to be treated or prevented, the route of administration, the African horse sickness infection being treated and the form of the composition. Any of the compositions of the invention may be administered in a single dose or in multiple doses. The dosages of the compositions of the invention may be readily determined by techniques known to those of skill in the art or as taught herein.
  • immunologically effective amount is meant an amount effective, at dosages and for periods of time necessary, to achieve a desired immune response.
  • the desired immune response may include stimulation or elicitation of an immune response, for instance a T or B cell response.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result, such as prevention of onset of a condition associated with an African horse sickness infection.
  • a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount.
  • Dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the judgment of the person administering or supervising the administration of the AHSV VLPs or compositions of the invention. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected. The amount of active compound(s) in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single dose may be administered, or multiple doses may be administered over time. It may be advantageous to formulate the compositions in dosage unit forms for ease of administration and uniformity of dosage.
  • preventing when used in relation to an infectious disease, or other medical disease or condition, is well understood in the art, and includes administration of a composition which reduces the frequency of or delays the onset of symptoms of a condition in a subject relative to a subject which does not receive the composition.
  • Prevention of a disease includes, for example, reducing the number of diagnoses of the infection in a treated population versus an untreated control population, and/or delaying the onset of symptoms of the infection in a treated population versus an untreated control population.
  • prophylactic or therapeutic treatment is well known to those of skill in the art and includes administration to a subject of one or more of the compositions of the invention. If the composition is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the subject) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).
  • the unwanted condition e.g., disease or other unwanted state of the subject
  • Toxicity and therapeutic efficacy of compositions of the invention may be determined by standard pharmaceutical procedures in cell culture or using experimental animals, such as by determining the LD50 and the ED50. Data obtained from the cell cultures and/or animal studies may be used to formulating a dosage range for use in a subject.
  • the dosage of any composition of the invention lies preferably within a range of circulating concentrations that include the ED50 but which has little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose may be estimated initially from cell culture assays.
  • VLP or “virus-like particle” refers to the capsid-like structure which results from the assembly of AHSV VP2, VP3, VP5 and VP7 polypeptides. These particles are antigenically and morphologically similar to native AHS virus virions but do not include viral genetic material; accordingly, these particles are not replicating nor infectious.
  • the invention also relates in part to a method of eliciting an immune response in a subject comprising administering to a subject in need thereof a prophylactically effective amount of the AHSV VLPs produced according to the method of present invention.
  • AHSV serotype 5 AHSV-5 capsid protein genes
  • VP2 SEQ ID NO:7
  • VP3 SEQ ID NO:8
  • VP5 SEQ ID NO:9
  • VP7 SEQ ID NO:10
  • AHSV serotype 4 AHSV-4 capsid protein genes
  • VP2 SEQ ID NO:11
  • VP5 SEQ ID NO:12
  • a consensus gene sequence for each of the AHSV-5 viral capsid proteins VP2, VP3, VP5, and VP7 and for the AHSV-4 viral capsid proteins VP2 and VP5 was obtained by aligning sequences (7-12 available) for these genes listed in GenBank, using CLC Mainbench bioinformatics software (Qiagen Bioinformatics, Aarhus, Denmark). Consensus sequences were codon optimized for expression in N. benthamiana and synthesized by GenScript Biotech Corporation (Nanjing, China) with flanking Age I and Xho ⁇ restriction enzyme sites.
  • Restriction enzyme cloning was used to insert the genes into the pEAQ-HT expression vector obtained from George Lomonossoff, John Innes Centre, UK (Sainsbury et at., 2009), to produce pEAQ- AHS5-VP2, pEAQ-AHS5-VP3, pEAQ-AHS5-VP5, pEAQ-AHS5-VP7, pEAQ-AHS4- VP2 and pEAQ-AHS4-VP5.
  • AHSV-5 and AHSV-4 plasmid constructs were electroporated into Agrobacterium radiobacter AG L1-ATCC BAA-101 as described previously (Maclean et at., 2007), and recombinant clones were selected at 27 °C on Luria-Bertani (LB) media plates containing 25 pg/rnL carbenicillin and 50 pg/rnL kanamycin.
  • LB Luria-Bertani
  • AHSV-4 and AHSV-5 capsid proteins were achieved by agroinfiltration of 5-week-old N. benthamiana plants.
  • Agrobacterium transformants each carrying one of the AHSV capsid protein genes were subcultured and grown overnight with agitation at 27 °C in Luria-Bertani broth (LBB) base supplemented with 50 pg/mL kanamycin, 20 pM acetosyringone and 2 mM MgS0 4 .
  • LBB Luria-Bertani broth
  • leaves from plants individually infiltrated with strains carrying AHSV-5 VP2, AHSV-5 VP3, AHSV-5 VP5 or AHSV-5 VP7 were harvested and homogenized together in two volumes of Bicine buffer pH 8.4 containing 1 x Complete Mini, EDTA-free protease inhibitor cocktail (Roche) - PI buffer.
  • leaves co-infiltrated with strains carrying AHSV-5 VP3, AHSV-5 VP7 and either AHSV-4 VP5 or AHSV-5 VP5 were homogenized together with leaves infiltrated with the Agrobacterium strain carrying the pEAQ-AHSV-4 VP2 or with AHSV-5 VP2 construct in two volumes of PI buffer.
  • the homogenates were incubated at 4 °C for 60 min with gentle agitation and then filtered through four layers of MiraclothTM (Merck).
  • the crude plant filtrates were clarified by centrifugation at 13000 rpm for 20 min at 4 °C in a JAM rotor (Beckman) and the supernatants incubated for 20 - 48 hours at 4 °C.
  • Fractions of plant extracts prepared from leaves co-infiltrated with AHSV-5 VP2, AHSV-5 VP3, AHSV-4 VP5 and AHSV-5 VP7 are shown in Figure 6, lane 1
  • fractions of plant extracts prepared from leaves co-infiltrated with AHSV-5 VP3 and AHSV-5 VP7 homogenised together with plant extract from leaves co-infiltrated with AHSV-4 VP2 and AHSV-4 VP5 are shown in Figure 6, lane 2.

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Abstract

La présente invention concerne des procédés in vitro pour la production extracellulaire de particules de type virus (VLP) du virus de la maladie du cheval africain (AHSV). L'invention concerne des particules de type virus AHSV per se et des procédés de prévention de la maladie du cheval africain. L'invention concerne également l'utilisation de VLP de l'AHSV dans la prévention de la maldie du cheval africain.
PCT/IB2021/051085 2020-02-10 2021-02-10 Assemblage extracellulaire de particules de type virus Ceased WO2021161189A1 (fr)

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WO2016042059A1 (fr) * 2014-09-18 2016-03-24 Glaxosmithkline Biologicals S.A. Vaccin

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DENNIS SUSAN J. ET AL: "Immunogenicity of plant-produced African horse sickness virus-like particles: implications for a novel vaccine", PLANT BIOTECHNOLOGY JOURNAL, vol. 16, no. 2, 1 February 2018 (2018-02-01), GB, pages 442 - 450, XP055799384, ISSN: 1467-7644, DOI: 10.1111/pbi.12783 *
DENNIS SUSAN J. ET AL: "Safety and immunogenicity of plant-produced African horse sickness virus-like particles in horses", VETERINARY RESEARCH, vol. 49, no. 1, 1 December 2018 (2018-12-01), pages 105, XP055800171, Retrieved from the Internet <URL:https://veterinaryresearch.biomedcentral.com/track/pdf/10.1186/s13567-018-0600-4.pdf> DOI: 10.1186/s13567-018-0600-4 *
MACLEAN, J.KOEKEMOER, M.OLIVIER, A.STEWART, D.HITZEROTH, I.RADEMACHER, T.FISCHER, R. ET AL.: "Optimization of human papillomavirus type 16 (HPV-16) L1 expression in plants: comparison of the suitability of different HPV-16L1 gene variants and different cell-compartment localization", J. GEN. VIROL., vol. 88, 2007, pages 1460 - 1469, XP002490006, DOI: 10.1099/vir.0.82718-0
RUTKOWSKA DARIA A. ET AL: "Plant-produced chimeric virus-like particles - a new generation vaccine against African horse sickness", BMC VETERINARY RESEARCH, vol. 15, no. 432, 3 December 2019 (2019-12-03), pages 1 - 15, XP055786107, Retrieved from the Internet <URL:http://link.springer.com/article/10.1186/s12917-019-2184-2/fulltext.html> DOI: 10.1186/s12917-019-2184-2 *
SAINSBURY, F.THUENEMANN, E.C.LOMONOSSOFF, G.P.: "pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants", PLANT BIOTECHNOL. J., vol. 7, 2009, pages 682 - 693

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