EP1951876A2 - Antigenes chimeriques et vaccins - Google Patents

Antigenes chimeriques et vaccins

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Publication number
EP1951876A2
EP1951876A2 EP06821354A EP06821354A EP1951876A2 EP 1951876 A2 EP1951876 A2 EP 1951876A2 EP 06821354 A EP06821354 A EP 06821354A EP 06821354 A EP06821354 A EP 06821354A EP 1951876 A2 EP1951876 A2 EP 1951876A2
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EP
European Patent Office
Prior art keywords
nucleic acid
seq
polypeptide
protein
acid sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP06821354A
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German (de)
English (en)
Inventor
Henk Huismans
Francois Maree
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University of Pretoria
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University of Pretoria
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Publication of EP1951876A2 publication Critical patent/EP1951876A2/fr
Withdrawn legal-status Critical Current

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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion 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
    • A61K39/12—Viral antigens
    • 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
    • A61K39/125—Picornaviridae, e.g. calicivirus
    • A61K39/135—Foot- and mouth-disease virus
    • 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
    • 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
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
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    • 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
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6075—Viral 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
    • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N2720/12122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
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    • 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
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32122—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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • THIS INVENTION relates to the technical field of immunology. More particularly, this invention relates to a nucleic acid vector, an antigen display system, a vaccine, a method for presenting or displaying an antigen, a method for increasing the solubility of a desired peptide, polypeptide, protein or protein multimer, a method of inducing an immunogenic response in a host, a method for producing an antigen display unit, a method of increasing the solubility of a desired peptide, and a recombinant DNA molecule.
  • the aetiological agent of African horsesickness, a highly infectious noncontagious disease of equines, is African horsesickness virus (AHSV), a member of the genus Orbivirus in the family Reoviridae.
  • AHSV African horsesickness virus
  • BTV bluetongue virus
  • Orbiviruses include ten double-stranded (ds)RNA genome segments packaged in a highly ordered icosahedral core particle including two major proteins, VP3 and VP7, and also including three minor structural proteins, VP1 , VP4 and VP6, with enzymatic functions related to the transcription and processing of virus mRNA.
  • the core is surrounded by an outer capsid layer, comprised of two viral proteins VP2 and VP5, of which VP2 is the serotype-specific antigen able to induce a protective humoral immune response.
  • the 3-dimensional structure of the top domain of AHSV VP7 trimers has been confirmed by X-ray crystallography to a resolution of 2.6 A.
  • the VP7 monomer is composed of two distinct domains - a top domain composed of amino acids 121 -249 folded into an anti- parallel ⁇ -sandwich, and a lower domain containing both the first 120 amino acids at the N-terminus and the last 99 residues at the C-terminus arranged in the form of nine ⁇ - helices with extended loops in-between.
  • Each VP7 has a short C-terminal arm, which may tie trimers together during capsid formation. Deletion of five amino acids at the C-terminus of BTV VP7 abolishes core-like particle (CLP) formation after co-expression of VP7 and VP3, presumably due to lack of trimer-thmer interactions.
  • CLP core-like particle
  • AHSV VP7 is a highly hydrophobic and insoluble protein.
  • the hydrophobic VP7 trimers spontaneously aggregate into large, flat, hexagonal disc-shaped crystals with a dimension of up to about 6 ⁇ m.
  • the pool of soluble VP7 trimers in infected cells is therefore very small and this has traditionally been considered to be a problem in their use in particulate or sub-unit vaccines.
  • a chimeric antigen including an orbivirus VP7 polypeptide or part thereof with a foreign peptide inserted into a top domain region of said orbivirus VP7 polypeptide, or part thereof.
  • orbivirus refers generally to African horsesickness virus or bluetongue virus.
  • a chimeric antigen refers to peptides, or proteins derived from any organism or species including sequences derived from disease-causing organisms, as well as peptides encoded by synthetic nucleic acids. The use of this term also includes peptides or proteins derived from structural or non-structural proteins of AHSV or BTV or any of the other oribiviruses. According to a further aspect of the invention, there is provided a method of making a chimeric antigen, the method including the step of inserting a foreign peptide into a top domain of an orbivirus VP7 polypeptide, or part thereof.
  • an immunogenic composition comprising the chimeric antigen of the invention, and a pharmaceutically acceptable carrier or diluent.
  • a method of inducing an immune response in a subject comprising the step of administering an effective amount of the immunogenic composition of the invention to the subject thereby to elicit or induce said immune response.
  • the invention provides a method of prophylactic treatment or method of inducing a protective immune response in a subject, the method comprising administering an immunologically effective amount of a chimeric antigen according to the invention to the subject.
  • the foreign peptide may be a peptide or epitope from any organism, and may include peptides or epitopes from disease-causing agents, such as a viral, bacterial, fungal, or parasitic disease-causing agents.
  • disease-causing agents such as a viral, bacterial, fungal, or parasitic disease-causing agents.
  • any suitable immunogen, antigen, epitope, or other desired proteinaceous compound may be suitable for inclusion in the antigen display system of the invention, whether synthetically manufactured, or derived from an organism or biological material.
  • the foreign peptide may be a polypeptide or protein.
  • the foreign peptide may be inserted into the top domain of the orbivirus VP7 polypeptide by providing an isolated nucleic acid sequence encoding the foreign peptide and inserting it into the appropriate region of the VP7 gene sequence encoding the top domain, or a region proximal thereto, thereby providing a recombinant nucleic acid molecule encoding the chimeric VP7 antigen of the invention.
  • the invention further comprises a recombinant DNA molecule which includes a nucleic acid sequence encoding an orbivirus VP7 polypeptide or part thereof and a nucleic acid sequence encoding a foreign peptide such that the foreign peptide is inserted into a top domain of said orbivirus VP7 polypeptide, or part thereof, when the gene encoding the recombinant VP7 polypeptide is expressed and translated.
  • the invention includes, as another aspect thereof, a nucleic acid vector including a nucleic acid sequence encoding an orbivirus VP7 polypeptide or part thereof, the nucleic acid sequence having at least one cloning site inserted therein at a position corresponding to, or proximal to, a top domain of the encoded VP7 polypeptide when the gene encoding the recombinant VP7 polypeptide is expressed and translated.
  • a single orbivirus VP7 protein may combine with two other VP7 proteins to form a VP7 protein trimer.
  • the orbivirus VP7 trimers may aggregate to form VP7 protein particles.
  • the VP7 orbivirus protein may be African horsesickness virus (AHSV) VP7 or bluetongue virus (BTV) VP7. More particularly, the VP7 protein may be the VP7 protein of AHSV serotype 9.
  • the invention extends to a purified or isolated AHSV VP7 polypeptide comprising at least one of the amino acid sequence of SEQ. ID. NO. 7, a fragment thereof; and sequences having at least 60% homology, preferably at least 70% homology, most preferably at least 80% homology thereto.
  • the AHSV VP7 polypeptide may be encoded by an isolated nucleic acid sequence selected from at least one of the nucleotide sequence of SEQ. ID. NO. 1 , the complement thereof, and sequences having at least 60% homology, preferably at least 70% homology, most preferably at least 80% homology thereto.
  • the insertion site or sites of the AHSV VP7 protein vector may be provided by inserting multiple nucleic acid cloning sites between the nucleic acid codons corresponding to amino acids 144 and 145 of the VP7 protein, between amino acids 177 and 178 of the VP7 protein, between amino acids 200 and 201 of the VP7 protein, or between any one or more combinations of such codons.
  • the AHSV VP7 polypeptide or protein may be encoded by an isolated nucleic acid sequence comprising at least one of the nucleotide sequences selected from SEQ. ID. NO. 2, SEQ. ID. NO.
  • SEQ. ID. NO. 4 SEQ ID. No. 5, SEQ. ID. NO. 6, the complement thereof, and nucleic acid sequences having at least 60% homology, preferably at least 70% homology, most preferably at least 80% homology, to such nucleic acid sequences.
  • the encoded AHSV VP7 polypeptide may have substantial sequence homology with at least one of the polypeptides selected from SEQ. ID. NO. 8, SEQ. ID. NO. 9, SEQ. ID. NO. 10, SEQ. ID. NO. 11 , or SEQ. ID. NO. 12, fragments thereof, and polypeptide sequences having at least 60% homology, preferably at least 70% homology, most preferably at least 80% homology thereto.
  • the nucleic acid sequence of the VP7 vector may have inserted therein a foreign nucleic acid sequence encoding a desired foreign peptide, thereby forming a recombinant nucleic acid sequence in accordance with the invention.
  • the recombinant nucleic acid may produce a chimeric protein or antigen following expression of the gene and translation of the mRNA thereof.
  • the foreign nucleic acid sequence may comprise of the nucleic acid sequences selected from SEQ. ID. NO. 17, SEQ. ID. NO. 18, SEQ. ID. NO. 19, SEQ. the complement thereof, fragments thereof, and nucleic acid sequences having at least 60% homology, preferably at least 70% homology, most preferably at least 80% homology thereto.
  • cloning site 144/145 may have inserted therein a nucleic acid sequence encoding, in tandem, two copies of a first antigen, while site 177/178 in the same vector may have a single copy of a second antigen.
  • the nucleic acid vector may operably be linked to a cis control element, such as a promoter.
  • the nucleic acid vector may include a prokaryotic or eukaryotic origin of replication.
  • the nucleic acid vector may include nucleic acid sequences encoding selection attributes, such as antibiotic resistance selection attributes, or other marker genes.
  • an antigen presentation system for presenting a desired antigen to the immune system of a subject, the antigen presentation system including: any one or more of the nucleic acid vectors of the invention, said nucleic acid vector having at least one of the foreign nucleic acid sequences mentioned herein inserted into at least one of the cloning sites provided in the nucleic acid vector.
  • a purified chimeric protein or antigen including: a protein sequence encoded by at least one of the nucleic acid vectors of the invention; and a foreign peptide sequence inserted into a top domain of the protein sequence encoded by the nucleic acid vector.
  • a vaccine composition which comprises: a chimeric protein or antigen of the invention including a peptide derived from a disease-causing agent; and a suitable pharmaceutically acceptable carrier or adjuvant.
  • an antigen display unit comprising a trimer of the orbivirus VP7 polypeptide of the invention having inserted into a top domain thereof a foreign peptide or epitope.
  • the antigen display unit of the invention may include trimers of the VP7 protein as well as associative structures comprised of complexes of two, three, or any higher level of associated or aggregated VP7 trimers.
  • the invention extends further to any one or more of the purified chimeric proteins or antigen display units of the invention, in crystalline or semi-crystalline form.
  • a eukaryotic or prokaryotic host cell including the nucleic acid vector of the invention.
  • the nucleic acid vector may have inserted therein a nucleic acid sequence encoding a desired foreign polypeptide or protein.
  • prophylactic treatment of an animal against a disease-causing agent may also be accomplished by providing a DNA vaccine composition including any one or more of the isolated recombinant nucleic acid sequences of the invention, under control of a suitable promoter, the recombinant nucleic acid sequence having included therein a nucleic acid sequence coding for a peptide derived from, or similar to, a peptide encoded by the disease-causing agent.
  • a method of increasing the solubility of a desired peptide including the steps of: providing an isolated nucleic acid sequence encoding an orbivirus VP7 polypeptide of the invention, or part thereof; inserting an isolated nucleic acid sequence encoding the desired peptide into a region of the nucleic acid sequence of the orbivirus VP7 gene encoding a top domain loop or a region proximal to a top domain loop of the structural protein, thereby forming a recombinant nucleic acid molecule; and causing the resultant recombinant nucleic acid molecule to be expressed as a chimeric protein or antigen.
  • the chimeric protein or antigen may be subjected to at least one of sonification or repeated freeze/thaw cycles.
  • a method of increasing the immunogenicity of a desired peptide including the steps of: providing a nucleic acid sequence encoding an orbivirus VP7 polypeptide of the invention, or part thereof; inserting a nucleic acid sequence encoding the desired peptide into the orbivirus VP7 encoding nucleic acid sequence to form a recombinant nucleic acid sequence; and causing the resulting recombinant nucleic acid sequence to be expressed in a suitable expression system to produce a chimeric protein or antigen; and subjecting the chimeric protein or antigen to at least one of sonification or repeated freeze/thaw cycles.
  • a method of eliciting an immune response in an subject including the steps of: providing a nucleic acid sequence encoding the orbivirus VP7 polypeptide of the invention, or part thereof; inserting a nucleic acid sequence encoding the desired foreign polypeptide into the orbivirus VP7 nucleic acid sequence to form a recombinant nucleic acid sequence; causing the resultant recombinant nucleic acid sequence to be expressed in a suitable expression system to produce a chimeric protein or antigen; and introducing the resultant chimeric protein or antigen into the body of the subject thereby to elicit an immune response.
  • the method may include a further step of introducing one or more booster doses or shots of the chimeric protein into the body of the animal at suitable periods following the initial introduction of the chimeric protein.
  • Such periods may include a first booster shot administered between 9 and 16 days following initial introduction into the animal, and a second booster shot administered between 18 and 32 days following initial introduction of the chimeric protein into the animal.
  • the nucleic acid sequences and recombinant nucleic acid sequences of the invention may be expressed in eukaryotic or prokaryotic cells, preferably in insect cells. More specifically, the nucleic acid sequences may be expressed as recombinant baculoviruses in insect cells, such as insect cells derived from Spodoptera frugiperda. However, it is within the contemplation of the invention that any suitable expression system may be used to produce the chimeric proteins of the invention.
  • the chimeric protein may be complexed with or attached to an adjuvant, immunogen, or other product suitable for eliciting or promoting an immune response against the chimeric protein or desired peptide.
  • the chimeric protein may be purified prior to introduction into the body of the animal.
  • a method of administering antigens into the body of a subject including the steps of: providing a nucleic acid sequence encoding the orbivirus VP7 polypeptide of the invention, or part thereof; inserting a nucleic acid sequence encoding a desired antigen or foreign peptide into the orbivirus VP7 nucleic acid sequence to form a recombinant nucleic acid sequence; causing the resultant recombinant nucleic acid sequence to be expressed in a suitable expression system to produce a chimeric antigen; and administering into the body of the subject a whole cell fraction or crude lysate containing a sufficient amount of the chimeric antigen to elicit at least a humoral immune response in the subject.
  • a substance or composition for use in a method of treating or preventing a disease in a subject the substance or composition comprising a chimeric protein or antigen in accordance with the invention, and said method comprising administering an effective amount of said substance or composition to said subject.
  • a chimeric protein or antigen in accordance with the invention in the manufacture of a medicament or preparation for treating or preventing a disease caused by a disease- causing agent.
  • a recombinant DNA molecule in accordance with the invention in the manufacture of a medicament or preparation for treating or preventing a disease caused by a disease- causing agent.
  • a method for producing a chimeric protein or chimeric antigen comprises growing a host cell or organism containing the recombinant DNA molecule in accordance with the invention, such that the DNA molecule is expressed by the host cell or organism, and isolating the expressed chimeric protein or chimeric antigen.
  • the disease may be selected from, but is not limited to, foot and mouth disease, African horsesickness, bluetongue disease.
  • kits for producing a chimeric protein or antigen in accordance with the invention including: a plasmid vector having inserted therein an isolated nucleic acid orbivirus VP7 sequence of the invention, or part thereof; and instructions for the insertion and/or expression of a desired polypeptide into the plasmid vector.
  • the invention includes, as another aspect thereof, antibodies produced against an antigen delivered or displayed using the system, methods, or antigens of the invention.
  • the antibodies may be polyclonal antibodies, monospecific polyclonal antibodies, or monoclonal antibodies.
  • the foreign peptide or polypeptide may be an epitope, an antigen, a hapten, an immunogen, a structural protein, a non-structural protein, or a polypeptide having an enzymatic function. More particularly, the polypeptide may be selected from at least one of HIV proteins, foot-and-mouth disease virus antigens, green fluorescent protein, enhanced green-fluorescent protein, one of the influenza virus proteins and/or orbiviral structural or non-structural proteins, e.g. such as from AHSV or BTV.
  • the invention extends further to an orbivirus VP7 polypeptide including a non-polar residue located within about 20 residues of the C-terminal end of the protein substituted by a polar residue. More specifically, the invention extends to an AHSV VP7 L345R polypeptide having a leucine residue at position 345 mutated to an arginine residue.
  • the invention includes a polypeptide sequence selected from at least one of amino acid SEQ. ID. No. 15, a fragment thereof, and polypeptides having at least 60% homology, preferably at least 70% homology, most preferably at least 80% homology thereto.
  • the invention includes a polynucleotide sequence selected from at least one of nucleotide SEQ. ID. NO. 13, the complement thereof, fragments thereof, and sequences having at least 60% homology, preferably at least 70% homology, most preferably at least 80% homology thereto.
  • an AHSV VP7 L345R having inserted into a top domain thereof a cloning site.
  • the invention extends thus to include a polypeptide selected from at least one of amino acid SEQ. ID. No. 16, a fragment thereof, and sequences having at least 60% homology, preferably at least 70% homology, most preferably at least 80% homology thereto.
  • the invention extends also to an isolated polynucleotide sequence selected from at least one of nucleic acid sequence SEQ. ID. NO. 14, the complement thereof, a fragment thereof, and nucleic acid sequences having at least 60% homology, preferably at least 70% homology, most preferably at least 80% homology thereto.
  • Figure 1 shows a model of the three-dimensional structure of VP7 obtained using the PyMOL program
  • Figure 2 shows SDS-PAGE analysis of recombinant VP7 proteins in insect cells.
  • C Molecular markers; Lane 1 : Mock infected cells; Lane 2: Wild-type baculovirus infected cells; Lane 3: Cells infected with recombinant baculoviruses that express wild- type AHSV VP7; Lane 4: Cells expressing VP7-177; Lane 5: Cells expressing VP7-200;
  • Figure 3 show sucrose density gradient analysis of total cell lysates of Sf9 insect cells infected with the different insertion vector constructs listed in Table 1.
  • Cells were harvested 72 hours after infection, lysed in buffer containing NP40 as detergent, layered onto 50-70% sucrose gradients and centrifuged for 14 hours at 30 000 rpm in an SW 50.1 rotor in a Beckmann ultracentrifuge. The gradients were fractionated by drop collection from the bottom and an equal portion of each fraction, including the pellet analyzed by SDS-PAGE.
  • VP7-wild-type is shown in 3A, the insertion mutant proteins with single insertions VP7-200, VP7-177 and VP7-144 in 3B, 3C and 3D.
  • FIG. 3E The insertion mutant VP7 protein with the double insertion VP7-144-200 is shown in Figure 3E and with the triple insertion VP7-144-177-200 in Figure 3F.
  • Figure 4 (4A to 4F) 4A shows a light microscopic view of the needle-like structures formed in cells infected with recombinant baculoviruses expressing VP7-200.
  • Figures 4B to 4F show Scanning Electron Microscopy photographs of the particulate structures in cells infected with recombinant baculoviruses expressing VP7 derivatives.
  • Figure 4B shows the hexagonal crystals formed by wild-type VP7
  • Figures 4C and 4D show similar looking structures formed by VP7-177 and VP7-200 respectively.
  • Figures 4E and 4F show the particles from protein VP7-144-177-200 at both a lower magnification ( Figure 4E) and a higher magnification (Figure 4F).
  • Figures 5 shows sucrose density gradient analyses of the total cell lysates of Sf9 insect cells infected with L345R mutated WT AHSV VP7 and AHSV VP7- 200 constructs.
  • Cells were harvested 72 hours after infection, lysed in buffer containing NP40 as detergent, layered onto 50-70 % sucrose gradients and centhfuged for 14 hours at 30 000 rpm in an SW 50.1 rotor in a Beckmann ultracentrifuge. The gradients were fractionated by drop collection from the bottom and an equal portion of each fraction, including the pellet, analyzed by SDS-PAGE.
  • the VP7-WT is shown in Figure 5A, mutated VP7 in Figure 5B, the VP7-177 control in Figure 5C and the mutated VP7- 177 in Figure 5D.
  • Figure 6 shows a trimerization assay of VP7 wild-type and protein VP7-177. Boiled as well as unboiled samples of the soluble fractions of AHSV VP7-L345R and VP7-177-L345R were loaded onto a 10% polyacrylamide gel and run for 2 hours at 110kV. The first lane shows molecular weight size marker. The positions of the trimers and monomers are as indicated.
  • Figure 7 shows sucrose gradient analyses of four different constructs expressed in Sf9 insect cells, viz. P4 (SEQ. ID. NO. 17) and P5 (SEQ. ID. NO. 18) inserted, respectively into site 144 of pFB-VP7-144, and the P5 (SEQ. ID. NO. 18) and P6 (SEQ. ID. NO. 19) peptides inserted into site 177 of pFB-VP7-177.
  • P4 SEQ. ID. NO. 17
  • P5 SEQ. ID. NO. 18
  • P6 SEQ. ID. NO. 19
  • Figure 8 shows a graph of the amount of fluorescence of each fraction containing VP7-eGFP following sucrose gradient analysis.
  • Figure 9 shows a graph of soluble VP7-eGFP production and formation of insoluble aggregates. Soluble VP7-eGPF in the medium represents cells that have broken up.
  • Figure 10 shows a graph of differences between long (18 hour) and short (1 hour) periods of centhfugation on particle distribution for VP7-eGFP.
  • Figure 11 shows the results of sucrose fractionations of VP7mt177 containing the P6 and P7 inserts.
  • Figure 12 shows a representation of the antibody responses against VP7mt177 containing the P6 insert.
  • Figure 13 shows a representation of the antibody responses against VP7mt177 containing the P7 insert
  • Figure 14 shows a representation of antibody responses against a crude lysate, soluble fraction from the crude lysate, and the particulate fraction from the crude lysate of Sf9 insect cells expressing VP7mt177 with the P6 insert.
  • Figure 15 shows an immune blot of an NS1 fusion protein containing an VP2 insert that overlaps with P4 against guinea pig serum raised against VP7-144-P4 NS1.
  • This invention relates to the use of a structural orbiviral protein, VP7, as an antigen for the presentation of epitopes or other desired peptides, so as to elicit an immune response from host animals against such epitopes or peptides.
  • epitopes, immunogens or haptens are not soluble, severely impairing their use as vaccine candidates when used by themselves.
  • Conjugating such epitopes, immunogens, or haptens to carrier molecules facilitates the generation of antibodies against such molecules, but is frequently unsuccessful due to poor or inadequate display of such molecules to the host immune system.
  • CLP core-like particle
  • the third strategy which is the strategy pursued by the Inventors hereof, is to use soluble chimeric AHSV VP7 trimers with or without associated small particles comprised of complexes of two, three, or more aggregated trimers for immune display.
  • the inherent insolubility of the AHSV VP7 posed a significant barrier to the use of AHSV VP7 protein in antigen delivery, display, or presentation.
  • AHSV VP7 solubility can be manipulated within a range of between 2.5% to about 70% of the total amount of AHSV VP7 expressed. These effects appear to be dependent on the peptide that is inserted, as well as the site of insertion into the VP7 protein. Even more surprisingly, the Inventors have found that a leucine-345 substitution to arginine can result in a large increase in AHSV VP7 solubility.
  • Baculoviruses were propagated in Spodoptera frugiperda (Sf9) cells, maintained in suspension or monolayer cultures at 27°C using Grace's or Sf900 medium supplemented with 10% (v/v) foetal calf serum (FCS) and antibiotics.
  • Recombinant baculoviruses were generated by means of the BAC-to-BACTM baculovirus expression system (Life Technologies). Bacmid DNA was transfected into Sf9 cells using Cellfectin® reagent (Life Technologies). Cells were incubated at 27°C until evidence of baculovirus infection was obtained (about 3 days), after which virus-containing supernatants were collected and stored at 4°C until further use.
  • insertion mutant refers to an AHSV VP7 protein into which the multiple cloning sites encoded by the nucleic acid sequences P1 , P2, and/or P3 have been inserted.
  • chimeric protein refers to an AHSV VP7 protein or insertion mutant protein into which a foreign epitope has been inserted in accordance with the invention.
  • Plasmid pBR-VP7 with the complete coding sequence of the AHSV-9 VP7 gene was used to clone the VP7 gene into pFastBac to obtain pFastBac-VP7-WT. This was used for PCR modifications to generate five different pFastBac (pFB)-VP7 vector constructs with different small multiple cloning sites at codon positions corresponding to amino acids 144, 177 and 200 of the encoded VP7 protein.
  • pFB pFastBac
  • the five different vector constructs that were prepared are listed in Table 1 and included three single insertion site vectors (pFB-VP7-144, pFB-VP7-177 and pFB-VP7- 200), a double insertion vector (pFB-VP7-144-200) and a triple vector (pFB-VP7-144- 177-200).
  • the number refers to the VP7 gene codon immediately downstream of which an 18 nucleotide DNA fragment containing the restriction enzyme (RE) sites listed in Table 1 were inserted.
  • These DNA fragments encode six amino acid peptides P1 , P2 or P3, as listed in Table 1.
  • the corresponding five pFB-VP7 vectors including pFB- VP7-WT were then used to prepare bacmid DNA which was used to generate the recombinant baculoviruses by transfection into Sf9 cells. All plasmid constructs were sequenced at several stages of the construction process to ensure that no additional mutations were introduced.
  • the leucine 345 codon was substituted with a codon that encodes arginine in the nucleotide sequences that encode VP7-WT and VP7-177.
  • a PCR strategy was designed to create a Sma ⁇ site at nucleotide position 1047 to 1052, which resulted in the required codon change. It also resulted, however, in a substitution of codon 344 by changing it from a valine to an alanine codon. Since both of these are nonpolar hydrophobic amino acids, this mutation should not affect the hydrophilic character of that site.
  • the modified genes were sequenced and used to generate the respective baculovirus recombinants producing a modified AHSV VP7 L345R protein.
  • VP7 constructs with peptides P4 (SEQ. ID. NO. 17), P5 (SEQ. ID. NO. 18) and P6 (SEQ. ID. NO. 19) inserted into sites 144 and 177:
  • P4 (SEQ. ID. NO. 17) insert The sequence encoding a proposed neutralisation- specific domain of AHSV located within amino acids 377-401 of VP2 of AHSV-9 was amplified from the original cDNA clone adding suitable restriction enzyme sites at the flanking region. This fragment was inserted into site 144 of pFB-VP7-144 and then used to generate the recombinant baculovirus.
  • P5 SEQ. ID. NO.
  • a sequence encoding the ELLELDKWASLW peptide was obtained from annealed complementary oligonucleotide sequences (Life Technologies), inserted into the multiple cloning sites of pFB-VP7-144 and pFB-VP7-177 and expressed as baculovirus recombinants.
  • This peptide contains the so-called ELDKWA epitope located at amino acids 671-677 of HIV gp41.
  • P6 SEQ. ID. NO.
  • Chimeric VP7 proteins were expressed in Sf9 cells seeded in a single 75cm 2 flask and harvested 48 or 72 hours post-infection by dislodging the cells from the surface of the flask and centhfuging them at 1500rpm in a Beckman SW55Ti rotor.
  • the collected cell pellet from each monolayer was resuspended in 800 ⁇ l lysis buffer (0.01 M STE with 0.5% Nonidet P40) and cells were incubated on ice for 30 minutes and dounced before being layered on a discontinuous 50-70% (w/v) sucrose gradient in 0.01 M STE.
  • the gradient was centrifuged in the Beckman SW55Ti rotor at 30 OOOrpm for 18 hours.
  • Fractions of 500 ⁇ l were collected from the bottom volumes, resulting in a total of 10-11 fractions.
  • the pellet from the gradient was resuspended in the same volume 0.01 M STE.
  • Fractions were either stored at -20 ° C or immediately analysed by SDS-PAGE.
  • Ten microlitres of each fraction, including the pellet, was analyzed on a 12% denaturing polyacrylamide gel. The gels were stained with Coomassie blue and the protein content of specific bands quantified using the Sigma GelTM software program (Jandel Scientific).
  • Fractions containing the particulate VP7 proteins were, where necessary, diluted 6-fold with 5OmM Tris-HCI pH 8.0, 5OmM NaCI, and recovered by centrifugation for 45min at 5000 rpm for electron microscopic analysis.
  • Crystallographic analysis of the top domain of AHSV VP7 protein indicated the presence of four hydrophilic, surface exposed ⁇ -loops that connect the ⁇ -sheets in the top domain. These loops include an "RGD" motif located on a highly flexible amino acid loop, spanning amino acids 175-180. These hydrophilic loops were targeted as peptide insertion sites. To identify the most suitable sites within these loops, a model of the three-dimensional structure of the top domain of VP7 was obtained with the PyMOL computer program (Fig 1A). From this model, amino acids 144-145, 177-178 and 200- 201 were identified as putatively being the most suitable of these possible insertion sites. A linear presentation of the insertion sites is shown in Fig 1 B.
  • the insertion mutant VP7 proteins containing the amino acids encoded by the multiple cloning site nucleotides were expressed in Sf9 insect cells and analysed by SDS-PAGE. The results are shown in Fig. 2. A unique protein band, corresponding to the expected size of about 39 kDa, was synthesised for each of the insertion mutant VP7 proteins.
  • the expressed insertion mutant VP7 proteins with double and triple peptide inserts showed a distinct size difference from that of the VP7 control (results not shown). The insertions did not affect the level of VP7 expression and were normally expressed as the most abundant protein with levels of approximately 1 -2 mg VP7 for each batch of 2 x 10 7 Sf9 cells.
  • a typical gradient fractionation pattern of the insertion mutant VP7 proteins indicated that some VP7 proteins sedimented in a position at the top of the gradient together with the bulk of the soluble cellular and baculovirus proteins (fractions 8-9 in Figure 3). These fractions are referred to as the soluble protein fractions and were postulated to contain the soluble VP7 trimers.
  • the particulate proteins are either in the pellet, or in fractions in the lower part of the gradient. In a few exceptional cases the proteins were distributed over several different fractions.
  • the sedimentation profiles of the particulate and soluble VP7 proteins reflected a combination of isopycnic and zonal centrifugation conditions. The soluble proteins sedimented under rate-zonal centrifugation conditions.
  • the particles from the VP7- 177 and VP7-200 gradients were largely indistinguishable from the VP7-WT particles, except that in the case of VP7-177 the larger particles often exhibited a distinct rosette type of morphology indicating some reduced stability of the particles.
  • the particulate fractions from the VP7-177 gradient were also associated with large numbers of smaller particles of an unspecific size which were assumed to represent small VP7-177 particles. There was no indication of any recognisable large particulate structures in either the soluble or the particulate fractions of VP7-144 and VP7-144-200.
  • the particles from the VP7-144-177-200 gradients were clearly distinguishable from those of VP7-200 and VP7-177, as may be seen in Fig. 4F.
  • the particles had a rough surface with a diameter of between 6 and 10 ⁇ m.
  • the particles were invariably distorted and often had a thick "cookie” type appearance with different layers piled on top of each other.
  • Fig. 4E shows a number of such "cookie-like" particles packed next to one another.
  • insertion mutant protein VP7-144 is predominantly soluble with only a relatively small particulate fraction. This is in contrast to insertion mutant protein VP7-200 that only has a very small soluble fraction and insertion mutant protein VP7-144-177-200 which appeared to be even less soluble than VP7-WT.
  • the Coomassie-stained gel profiles were scanned using the Sigma GelTM software package. The amount of soluble VP7 was expressed as a percentage of the total insertion mutant VP7 in a cell lysate at 72 hours after infection. At this late stage of infection there appears to be no further increase in VP7 synthesis and this provided the most consistent comparative results. The results are summarized in Table 2.
  • the L345R mutation was found to have a very large effect on AHSV VP7 solubility.
  • the largest proportional effect was on VP7-WT solubility, in which total solubility of the protein increased from about 8% to more than 40%.
  • the solubility of fusion protein VP7-177 was also increased from about 25% to more than 45%.
  • the different VP7 proteins were analysed under nondenaturing conditions (Fig. 6).
  • the Inventors also investigated the VP7-144-177-200 fusion protein for protein trimehzation. Although the lack of a suitable soluble fraction complicated the assay, the results confirmed that the insertion mutant proteins that aggregated into the distorted VP7 particles, took the form of a trimer (results not shown).
  • Peptide P4 (SEQ. ID. NO. 17) overlaps a region associated with the induction of neutralising antibodies against AHSV.
  • Peptide P5 (SEQ. ID. NO. 18) contains the ELDKWA epitope flanked on each side by 3 additional amino acids that correspond to those in the transmembrane protein gp41 of HIV-1 subtype B.
  • Peptide P6 (SEQ. ID. NO.
  • the effect can most likely be ascribed to a reduction in the hydrophobicity of the trimers, resulting in a reduction in the tendency of hydrophobic trimers to aggregate.
  • This modification is affected by the hydrophilicity of the inserted peptide and the site of insertion in the VP7 top domain.
  • the solubility of the insertion mutant VP7 trimers suggests that the protein is correctly folded with the inserted peptide optimally exposed to the aqueous environment.
  • the constructs of the invention may make it possible to display foreign peptides on the top domain of trimers that could not be displayed on chimeric CLPs. The success of such a strategy is, however, entirely dependent on the solubility of the chimeric VP7 trimers.
  • the simultaneous insertion of three different peptides at three different insertion sites in the VP7 top domain appears to have overly distorted the structure, resulting in a large increase in thmer-trimer aggregation, and the formation of relatively low density protein particles with a distorted and irregular shape.
  • the insertion of P1 into site 144 resulted in an increase in the soluble trimer fraction to close to 70% of the total VP7 at 72 hours post-infection.
  • the simultaneous insertion of P1 in site 144 and P2 into site 200 kept the solubility close to 40%.
  • the site of insertion is important, as shown by the result that when P2 was inserted into site 177, the solubility of the insertion mutant VP7 is at least twice that shown when P2 was inserted into site 200.
  • the particles that formed in the case of the VP7-200, VP7-177 and VP7-144- 177-200 insertion mutant proteins were all analyzed by electronmicroscopy. Examination of the gradient purified particulate structures revealed that, in the case of the VP7-200 and VP7-177 constructs, the insertion mutant proteins aggregated into the typical flat, disc-shaped, usually hexagonal crystals of up to 6 ⁇ m in diameter and 200 nm thick that have been previously described. These crystals are formed of flat sheets of VP7 trimers with each sheet presumed to represent a double layer of VP7 trimers with their hydrophobic bottom domains located on an operatively internal face, away from the aqueous surroundings. The hydrophobic interactions between these lower or bottom domains appear to keep the layers together. The space between these layers most likely account for the relatively low density of these particles that have been observed by the Inventors.
  • the most likely variables that affect the solubility of VP7 chimers are, firstly, the change in hydrophilicity in the VP7 top domain associated with the insertion of each of the different peptides and, secondly, constraints-related differences determined by the location of the insertion sites and the length of the peptide. These site-specific constraints can affect the folding of the inserted peptides. It appears that such differences in constraints can be predicted by viewing the location of the insertion sites in Fig 7.
  • Fig. 1A it is evident that when the three VP7 monomers interact to form a trimer that the location of the 200 top domain insertion sites in each of the VP7 monomers are grouped very closely together in the resultant trimer. This suggests the possibility of some steric hindrance when peptides are inserted into each of the 200 sites of each insertion mutant VP7 monomer, as the peptides will have to compete for the limited available space in the immediate vicinity of these sites, once in trimehc form. This could reduce the exposure of hydrophilic amino acids inserted into this site and reduce the hydrophilic effect of these insertions. It is also clear from Fig.
  • Sites 144 and 177 are very close together and the simultaneous insertion of P1 and P3 may have caused distortions that affect the display of charged amino acids in the VP7 loops in the VP7 top domain, causing the insolubility and distortions that were observed.
  • the hydrophilicity of the inserted peptide is important, its effect is not immediately predictable from hydrophilicity predictions because the insertion site will play a large role in determining how the overall hydrophilicity of the VP7 top domain is affected by the insertion. Therefore, even though P1 is less hydrophilic than P2 and P3 respectively, its insertion into site 144 improves the overall VP7 solubility to a larger extent than when the more hydrophilic P2 is inserted into site 200. It is even more difficult to predict the effect of inserting larger peptides into the different sites because it is not known how these peptides are normally folded and what space constraints are involved.
  • the best characterized of the peptide inserts is the 36 amino acid P6 (SEQ. ID. NO.
  • trimer-trimer interaction is relatively weak.
  • the trimer-trimer interaction may thus be affected by relatively specific changes in hydrophobicity.
  • the trimer concentration threshold at which the trimers will aggregate is increased and more trimers are present in solution.
  • the Inventors have found that it is possible to manipulate the equilibrium between AHSV VP7 soluble trimers, on the one hand, and particle formation, on the other hand, in a cell. This has important applications in the development of recombinant vaccines based on the presentation of small peptides as chimerical VP7 trimers to the immune system, as discussed in the further examples below.
  • the neutralization-specific outer capsid protein VP2 has been the target of most recombinant vaccine strategies.
  • the insolubility of insect cell-produced VP2 proteins remains the main stumbling block to using VP2 peptides and epitopes as particulate vaccines by themselves.
  • the possibility of using epitopic domains of AHSV VP2, rather than the full-length VP2, has also been explored and a number of putative neutralization-specific domains have been located between amino acid residues 253 and 413 on VP2 of AHSV.
  • these peptides are expressed in either bacterial or insect cells they are again largely insoluble and non-immunogenic.
  • VP7 pFB insertion vectors AHSV VP7 with L345R mutations
  • VP7 chimerae with immunologically important peptides.
  • Numbers 144, 177 and 200 in the VP7 refer to the VP7 gene codon immediately downstream of which a multiple cloning site was inserted.
  • the DNA inserts encode one of peptides P1 , P2 or P3.
  • P1 aa "PGQFLQ” encoded by DNA fragment with RE sites Sma ⁇ , EcoR ⁇ and Xho ⁇
  • P2 aa"KLSRVD” encoded by DNA fragment with RE sites Hind ⁇ , Xba ⁇ and Sa/I
  • P3 aa"LQRPAR” encoded by DNA fragment with RE sites BssHW, Stu ⁇ and PsM
  • P4 (SEQ. ID. NO. 17): aa'OPNHDTWKNHVKDIRERMQKEQSAN" (aa377-401 ) of VP2 of AHSV-9
  • P5 (SEQ. ID. NO.
  • FMDV vaccine strain SAT2/ZIM7/83 FMDV vaccine strain SAT2/ZIM7/83.
  • VP7-177-eGFP A full-length eGFP protein (220 amino acids) was inserted into site
  • VP7-C-eGFP The eGFP protein was attached to the C-terminal of VP7.
  • VP7-177-eGFP-truncC The eGFP protein was inserted into site 177 of the VP7 top domain, but with the C-terminal half deleted immediately after insertion of the eGFP.
  • Two separate suspension cultures of 5 x 10 7 Sf9 cells were infected 12 hours apart with recombinant baculovirus expressing VP7-eGFP. About 1 x 10 6 cells were collected at 18 hours post infection and every representative time 6 hours thereafter, up to, and including, 90 h.p.i. Cells were collected by centhfugation at 20Og for 5 minutes, after which the TC100 medium in which the cells were growing was separated and kept, and the cells were resuspended in 1 ml STE (0.01 M NaCI, 0.01 M Tris, 0.05M EDTA) containing protease inhibitors (Pepstatin and Pefabloc). The medium and cells were frozen and kept at -80 0 C until all representative samples were collected.
  • each sample of cells was treated with NP40 for 30 minutes, the cells lysed mechanically by douncing, and collected by centrifugation for 10 minutes at 16,20Og (benchtop centrifuge) to separate soluble and insoluble components.
  • the insoluble component pellet was re- suspended in 1 ml STE (0.01 M NaCI, 0.01 M Tris, 0.05M EDTA). Thereafter, fluorescent measurements of each component (soluble, insoluble, medium) were taken for each representative sample. To account for background fluorescence, samples were also taken pre-infection, as well as at 0 h post infection (i.e. immediately after infection).
  • a discontinuous sucrose gradient was prepared from bottom to top containing equal volumes (4.8 ml total) of 70%, 65%, 60%, 55% and 50% sucrose, upon which all of the cell lysate was loaded.
  • the sucrose gradients were then centrifuged using a Beckman SW55 rotor at 40 OOOrpm (151 00Og) for 18 hours. About 19-21 fractions of approximately 250 ⁇ l (descending density) each were collected, which were then analyzed by fluorometery as well as by SDS-PAGE.
  • a discontinuous sucrose gradient was prepared from bottom to top containing equal volumes (20 ml total) of 70%, 65%, 60%, 55% and 50% sucrose, upon which all of the cell lysate was loaded.
  • the sucrose gradients were then centrifuged using a Beckman SW28 rotor at 20 OOOrpm (53 00Og) for 18 hours. Approximately 30- 33 fractions of approximately 750 ⁇ l each (descending density) were collected, which were then analysed by fluorometery as well as by SDS-PAGE.
  • Cells were harvested at either 48 h.p.i. or at any time after 60 h.p.i. by low speed centhfugation at 5000 rpm for 5 minutes. Cells were resuspended in 0.01 M NaCI STE buffer containing 0.5% detergent (either Triton X100 or NP40), left on ice for 30 minutes, and then dounced 20 times on ice.
  • 0.01 M NaCI STE buffer containing 0.5% detergent either Triton X100 or NP40
  • eGFP as an insert to characterise some of the properties of chimeric VP7 proteins was postulated to hold several advantages. Fluorescence of chimeric VP7-eGFP could provide evidence that the inserted protein is correctly folded and exposed to its aqueous environment. It is also a non-invasive method of quantifying chimeric VP7 expression levels at different times after infection and makes it possible to track the assembly of chimeric VP7 proteins into particles, thereby allowing one to quantify the ratio of soluble versus particulate protein throughout the infection cycle.
  • Chimeric protein VP7-177-eGFP was expressed by means of a baculovirus recombinant.
  • eGFP is positioned on the highly flexible amino acid loop 175-180 in the protein top domain. Expression of the protein was confirmed by SDS-PAGE and by means of Western blot (not shown) using an anti-eGFP antibody (N- terminal, SIGMA).
  • SIGMA anti-eGFP antibody
  • the chimeric protein VP7-eGFP was fluorescent, indicating that the eGFP had retained its conformational and functional integrity, even after having been inserted medially into AHSV VP7.
  • the cells infected with the baculovirus recombinants were harvested at 72 hours after infection and analysed by sucrose gradient density analysis as indicated under the Materials and Methods section for this example above. The relative amount of fluorescence of each fraction was calculated, together with the relative amount of VP7-177-eGFP protein calculated from Coomassie stained gels. The result is shown in Figure 8.
  • the stability of this soluble fraction which constituted about 54% of the total VP7 chimeric protein expressed at 72 h.p.i., was tested under a range of different conditions that included different salt concentrations, as well as freeze drying. Surprisingly, the Inventors found that in all cases tested, the trimers remained soluble and did not aggregate. Apart from the soluble fraction, there were, however, also distinct, but small, particulate peaks at fractions 6-13 and 18-22 respectively.
  • the chimeric VP7 protein values (blue) match these positions in the gradient but not in the same relative amount. The soluble fraction appeared to be proportionally much smaller and the soluble fraction was not much more than about 20% of the total VP7 expressed.
  • the fluorescence per unit protein value may well represent a proportional indicator of exposure on the surface of either soluble trimers or trimer aggregates which is informative about immune display of such an insert.
  • Fig. 9 indicate the following:
  • the percentage of soluble VP7 (pink) reaches a maximum by about 48 h.p.i. Most of the VP7 synthesized beyond that point aggregates to form particles.
  • the relative amount of particles (blue) increases from about 30 h.p.i. and reaches a maximum at about 48 h.p.i. with only a small relative further increase.
  • the apparent decline in the percentage soluble protein after 48 hours is due to cell lysis. As the cellular membrane is disrupted, the soluble protein is released into the cell medium. This is apparent from the increase in fluorescence in the medium after 48 h.p.i (black).
  • the particles on the other hand, remain cell- and cell debris-associated and no such decline is observed (blue).
  • the combined soluble VP7- eGFP chimeric protein (green) reaches a maximum by about 48 h.p.i.
  • the Inventors have found that, in order to ensure maximum solubility, soluble proteins should therefore not be harvested after 48 h.p.i. Particles on the other hand, are best harvested after 48 h.p.i. and until as late as 72 h.p.i.
  • soluble chimeric VP7 The aggregation of soluble chimeric VP7 into protein aggregates is concentration dependant. Over and above a maximum concentration, the tendency of trimers to aggregate will increase proportionally. This threshold concentration is probably linked to the hydrophobicity of the chimeric trimers and may therefore be different for every different chimeric construct that is made. In chimeric VP7-eGFP this threshold level was reached at 48 hours after infection and also just before cellular lysis starts depleting the amount of soluble protein that can be recovered.
  • the fluorescent eGFP in the medium does not reflect an active process of protein trafficking out of the cell but rather a collapse of the cellular membrane and cell death that will release the soluble protein (and very small particles) into the medium.
  • the particulate fraction fluorescence values shown in Figure 8 have not been corrected and will therefore underestimate the amount of particulate protein by a factor of about 7.
  • the data at 36 and 48 h.p.i is, on the other hand, directly comparable to the fluorescence data at 72 h.p.i (green) in Figure 1.
  • the results of the solubility experiments indicate that at 36 h.p.i most of the VP7-eGFP appears to be soluble.
  • the proportion of particulate VP7-eGFP shows an increase leading up to the results shown in Fig. 1 at 72 h.p.i.
  • the 20% soluble VP7-eGFP at 72 h.p.i is therefore a significant underestimation of the total amount of soluble chimeric VP7 that could be recovered.
  • VP7-177-eGFP is a trimer and to determine how trimehzation affects VP7 solubility
  • a chimeric protein was constructed that lacked the C-terminal half of the VP7 protein. This was postulated to eliminate the ability of chimeric VP7 to form trimers.
  • This construct, VP7-177-eGFP-truncC has eGFP inserted into site 177 of the VP7 top domain, but has amino acids 178-349 deleted immediately downstream of the inserted eGFP. This elimination was predicted to prevent the formation of trimers, since this would eliminate two of the major ⁇ -helices involved in trimer formation.
  • the protein was expressed by means of a recombinant baculovirus and sucrose gradient density analysis indicated that the protein was almost completely soluble with almost all fluorescence recovered from the top of the gradient. There was, however, a difference in migration pattern of soluble VP7-177-eGFP and VP7-177-eGFP-truncC. The truncated chimeric protein migrated significantly slower, more or less in agreement with it being a monomer and not a trimer, as is the case for full-length VP7-177-eGFP.
  • trimerization assay is based on analysing the soluble fractions of VP7-177-eGFP and VP7-177-eGFP-truncC under denaturing (boiled) and non- denaturing (not boiled) electrophoresis conditions and assaying the size of the different proteins by means of a Western blot with eGFP serum.
  • the Inventors also prepared a chimeric VP7 fusion protein in which the eGFP was attached to the C- terminal site of VP7.
  • This protein, VP7-C-eGFP was expressed as a recombinant baculovirus. Good expression and fluorescence was obtained, but on sucrose gradient density analysis, the chimeric protein was found to be completely insoluble. This result indicates that the site of insertion is key to the solubility of the VP7-eGFP fusions and that different sites may reflect very different outcomes.
  • the Inventors isolated crude, soluble and particulate fractions of VP7-eGFP from cell lysates.
  • the crude fraction contained a mixture of soluble and particulate VP7- eGFP and was expected to provide the best set of options for eliciting an immune response when introduced into a test animal.
  • the Inventors injected these constructs into guinea pigs and analysed the sera.
  • the Inventors found that the serum against the crude chimeric protein isolate gives a good immune response against eGFP - the antibodies generated recognize a fusion product of NS1 with eGFP (not shown). The protein is therefore displayed correctly by the VP7 trimer.
  • P6 (SEQ. ID. NO. 19): aa 'RYNGECKYTQQSTAIRGDRAVLAAKYANTKHKLPST" (aa129-164) of 1 D of FMDV vaccine strain SAT2/ZIM7/83.
  • This epitope contains a cluster of immunodominant epitopes on the 1 D protein of FMDV.
  • the soluble trimer fraction was generally more than 60%.
  • P7 The full-length 1 D protein of FMDV: This protein includes the P6 (SEQ. ID. NO. 19) epitope. When this protein was expressed in Sf9 cells it was expressed to high levels (results not shown), but it did not appear to have a soluble fraction and all of the protein appeared to be particulate.
  • the VP7-177-P7 fusion protein (VPI inserted into VP7 at site 177) is, however, recognized by FMDV antiserum, indicating that the protein has domains that are recognized by antibodies in the serum.
  • Sf9 insect cells were infected at an m.o.i. of 5 pfu/cell with recombinant baculoviruses expressing vector protein without any insert (VP7-177), the chimeric protein VP7-177-P6, or the chimeric protein VP7-177-P7.
  • a crude extract comprising a combination of both soluble and particulate proteins
  • a soluble fraction only a particulate fraction only.
  • the crude extracts were prepared by resuspending 2 x 10 8 infected cells in 0.01 M STE, incubation on ice for 30 min and mechanical disruption by douncing 20 times.
  • the nuclear fraction was removed by low speed centrifugation at 1500 rpm for 3 min and rinsed once. Expression of the chimeric protein and its concentration in the lysate was estimated from the band intensity after SDS-PAGE analysis and Coomassie Blue staining.
  • lysates were prepared in 0.01 M STE with 0.5% Nonidet P40, incubated on ice, mechanically dounced and the nuclear fraction removed as before. Lysates were then loaded onto 40-70% discontinuous sucrose gradients in 0.01 M STE. Gradients were centrifuged in an SW28 rotor at 20 000 rpm for 16 h. Fractions of 1 ml each were collected using a dialysis pump and analyzed directly on a 12% SDS polyacrylamide gel (not shown). The gels were stained and the protein content of the specific band quantified by the Sigma GelTM software program (Jandel Scientific).
  • the top fractions of the gradient (fractions 21 -27) representing soluble chimeric protein, and the lower fractions (fractions 5-16) representing protein in a particulate form, were pooled respectively.
  • the size of the pools are as indicated in Fig. 11.
  • the fractions were dialyzed individually overnight against 0.01 M STE with three buffer changes, freeze-dhed and resuspended in 1 x PBS. All proteins were stored at -7O 0 C prior to immunizations.
  • Groups of female guinea pigs were injected intra-muscularly with either a crude-, soluble- or particulate sample of VP7-177, VP7-177-P6 or VP7-177-P7.
  • Two to three animals were immunized with 40 ⁇ g of the respective protein samples in 250 ⁇ l 1 x PBS emulsified in an equal volume of Freund's complete adjuvant.
  • Two boosts with identical samples were administered at two weekly intervals. Serum samples were obtained from each animal pre-vaccination, 24 hr before each booster injection and two weeks after the final boost.
  • a sandwich enzyme-linked immunoabsorbent assay was used to detect antibodies in serum to the FMDV. Briefly, 96-well plates coated with rabbit antibodies against FMDV vaccine strain SAT2/ZIM7/83 were used to trap tissue- cultured virus, diluted 1/50. Guinea pig test serums collected at different times pre-and post-vaccination were serially diluted and antibodies detected with peroxidase-labelled anti-guinea pig conjugate. Titres were expressed as the serum dilution that yielded absorption values three times above pre-immunization serum.
  • the presence of antibodies mediating FMDV neutralizing activity was determined in a virus-neutralization assay.
  • a virus-neutralization assay In a 96-well plate, two-fold dilutions of the test sera were added to 100 TCID 50 units of FMDV vaccine strain SAT2/ZIM7/83. The mixture of serum dilutions and virus was incubated at 37°C for 1 hr and then adsorbed to IBRS2 cells. Cells were incubated for 3 days until a cytopathic effect (CPE) was observed.
  • CPE cytopathic effect
  • the serum neutralization titre was defined as the dilution that resulted in a 50% reduction in CPE.
  • Sf9 cells were infected with recombinant baculoviruses expressing chimeric proteins VP7-177-P6 and VP7-177-P7.
  • Cells were harvested at 72 h.p.i. and the crude, soluble and particulate protein fractions prepared as described.
  • the particulate and soluble fractions were obtained from sucrose gradients as described and the specific fractions collected were as indicated in Fig. 11 from the VP7-177-P6 and the VP7-177- P7 gradients.
  • the relative amount of chimeric protein in the different fractions shown in Fig. 11 was estimated from the band intensity after SDS-PAGE analysis and Coomassie Blue staining.
  • the small to very small particles or trimer aggregates are adequate in inducing a weak to satisfactory immune response, as shown by the results obtained with particulate VP7-177-P6.
  • the Inventors therefore do not exclude the possibility that the trimer aggregates or particles are able to induce a good immune response.
  • the results as presented herein show that a reduction in the size of the particles or trimer aggregates is accompanied by an improved display of the antigen, as well as by an enhanced immune response.
  • the soluble nano-sized trimer particles or antigen display units are therefore considered to be the best display system for foreign epitopes or antigens whereas the large hexagonal crystals or large trimer aggregates are probably less suitable for immune display than the smaller trimer aggregates.
  • Guinea pigs were immunized to evaluate the immune response against the following VP7 fusion proteins:
  • the VP7 fusion protein (VP7-177-P6) with the FMDV P6 peptide (SEQ. ID. NO. 19) was inserted into site 177. This is the same fusion protein used in the experiments described in Example 3.
  • a VP7 fusion protein (VP7-144-P4) with a short AHSV P4 peptide (SEQ. ID. NO. 17) was inserted into site 144.
  • P6 (SEQ. ID No. 19): aa 'RYNGECKYTQQSTAIRGDRAVLAAKYANTKHKLPST" (aa129-164) of 1 D of FMDV vaccine strain SAT2/ZIM7/83.
  • This epitope contains a cluster of immunodominant epitopes on the 1 D protein of FMDV.
  • P4 (SEQ. ID No. 17): aa'OPNHDTWKNHVKDIRERMQKEQSAN" (aa377-401 ) of the VP2 protein of AHSV-9.
  • This sequence is part of a domain that has previously been shown to be serotype-specific in an immune blot with AHSV-9 serum and contains a possible neutralization domain of VP2.
  • Sf9 insect cells were infected at an m.o.i. of 5 pfu/cell with recombinant baculoviruses expressing the chimeric protein VP7-177-P6, or the chimeric protein VP7- 144-P4.
  • VP7-177-P6 For immunization with VP7-177-P6, two protein samples were prepared namely (i) a crude extract comprising a combination of soluble and particulate proteins and (ii) a fraction containing soluble protein. For immunization with VP7-144-P4, only a soluble fraction was prepared.
  • the crude extract was prepared by resuspending 2 x 10 8 infected cells in 0.01 M STE with 0.5% Nonidet P40, incubation on ice for 30 min and mechanical disruption by douncing 20 times.
  • the nuclear fraction was removed by low speed centrifugation at 1500 rpm for 3 min and rinsed once.
  • the lysate was then dialised overnight against 0.01 M STE with three buffer changes, freeze-dried and resuspended in 1 x PBS. Expression of the chimeric protein was confirmed and its concentration in the lysate was estimated from the band intensity after SDS-PAGE analysis and Coomassie Blue staining.
  • lysates were prepared in 0.01 M STE with 0.5% Nonidet P40, incubated on ice, mechanically dounced and the nuclear fraction removed as before. Lysates were then loaded onto 50-70% discontinuous sucrose gradients in 0.01 M STE. Gradients were centrifuged in an SW28 rotor at 22 000 rpm for 20 h. Seventeen fractions of 2 ml each were collected using a dialysis pump and analyzed directly on a 12% SDS polyacrylamide gel (not shown). The gels were stained with Coomassie Blue to determine the protein content ofeach fraction.
  • Groups of female guinea pigs were injected intra-muscularly with either crude- or soluble samples of VP7-177-P6 or the soluble sample of VP7-144-P4.
  • Four animals were immunized with 40 ⁇ g of the respective protein samples in 250 ⁇ l 1 x PBS emulsified in an equal volume of either Freund's complete or incomplete adjuvant as detailed in the Table 3 below.
  • a single boost using either incomplete Freunds or no adjuvant was administered after 21 days. Serum samples were obtained from each animal pre-vaccination, hours before each booster injection and three weeks after the boost.
  • a sandwich enzyme-linked immunoabsorbent assay was used to detect antibodies in serum to the VP7-FMDV fusion protein. Briefly, 96-well plates coated with rabbit antibodies against FMDV vaccine strain SAT2/ZIM7/83 were used to trap tissue-cultured virus, diluted 1/50. Guinea pig test serums collected at different times pre-and post-vaccination were serially diluted, added to the ELISA plates. Antibodies detected with peroxidase-labelled anti-guinea pig conjugate. Titres were expressed as the serum dilution that yielded absorption values three times above pre- immunization serum.
  • the presence of antibodies mediating FMDV neutralizing activity was determined in a virus-neutralization assay.
  • a virus-neutralization assay In a 96-well plate, two-fold dilutions of the test sera were added to 100 TCID 50 units of FMDV vaccine strain SAT2/ZIM7/83. The mixture of serum dilutions and virus was incubated at 37°C for 1 hr and then adsorbed to IBRS2 cells. Cells were incubated for 3 days until a cytopathic effect (CPE) was observed.
  • CPE cytopathic effect
  • the serum neutralization titre was defined as the dilution that resulted in a 50% reduction in CPE.
  • AHSV-9 VP2-specific antibodies in serum was detected by Western blot analyses. Immune reactions were tested against a baculovirus-expressed NS1 fusion protein containing an AHSV-9 VP2 domain that overlaps the P4 insert, as well as against VP7-144-P4. The serum was also analysed in a virus neutralization assay for the presence of antibodies that could mediate AHSV-9 neutralization in a plaque-neutralization assay. Two-fold dilutions of the test sera were added to 100 PFU units of AHSV-9 virus, incubated at 37°C for 1 hr and then adsorbed to CER cells in 6- well plates. Cells were incubated for 5 days until plaques were visible, stained and monitored for a decrease in the number of plaques compared to non-neutralised virus- infected controls. RESULTS
  • the average ELISA titre of the 42 days serum was in the order of more than 1/7600 (injection with CF and boost with IF) with one animal having an ELISA titer of 1/12800 even before the booster injection.
  • the boosters did, however, have a significant effect, resulting in titres of more than 1/12800 in the case of at least two animals.
  • These very high ELISA titers correlated very well with high virus-neutralization titers.
  • the VP7-144-P4 fusion protein with amino acids 377-401 of VP2 from AHSV contained a large and significant soluble component which was used to induce an immune response in guinea pigs as outlined in group 4 in Table 3.
  • the immune sera obtained were tested by a AHSV neutralization assay and found to contain no neutralizing antibodies. It was then tested in an immune blot assay against an NS1 fusion protein which contained an AHSV VP2 sequence that overlapped the P4 sequence. The result is shown in Figure 15. The result clearly shows a strong VP2 insert specific immune response. This result confirms that the VP7 soluble trimer, with P4 inserted into site 144, induced antibodies against the 35 amino acid VP2 insert.
  • Results also confirmed the importance of using an appropriate adjuvant. On average, a better immune response was elicited where complete adjuvant was used as an adjuvant for the primary injection and incomplete Freunds adjuvant for the boost. Nonetheless, even when Incomplete Freunds adjuvant was used for the primary immunization and no adjuvant for the boost, positive virus neutralization titers were obtained for all three animals.
  • the VP2 insert also induced a good antibody immune response. This result is quite significant because it has previously been attempted to induce antibodies against this peptide without the VP7 immune display strategy. However, due to the fact that the peptide was insoluble, no peptide-specific antibodies were raised. However, in this experiment the normally insoluble P4 peptide was presented to the immune system as part of a soluble VP7 fusion protein with peptide P4 inserted into site 144 of the VP7 top domain. Under these conditions we were for the first time able to raise peptide P4 specific antibodies. We were also able to test if such antibodies were able to neutralize the virus. The results confirmed that, if there are any neutralizing epiptopes located on the P4 peptide, these epitopes are likely to be conformational rather than linear epitopes.

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Abstract

La présente invention concerne un antigène chimérique comprenant un polypeptide d'orbivirus VP7 avec un peptide étranger inséré dans une région de domaine supérieure dudit polypeptide d'orbivirus VP7. L'antigène chimérique susmentionné peut être utilisé pour traiter ou prévenir une maladie telle que la fièvre aphteuse, la peste équine, ou la fièvre catarrhale du mouton. Cette invention concerne également un procédé permettant d'induire une réponse immunitaire chez un sujet, par administration d'une quantité efficace d'une composition immunogène contenant l'antigène chimérique et un excipient ou diluant pharmaceutiquement acceptable au sujet, de manière à déclencher ou à induire la réponse immunitaire.
EP06821354A 2005-11-07 2006-11-07 Antigenes chimeriques et vaccins Withdrawn EP1951876A2 (fr)

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CHANG-KWANG LIMN ET AL: "Functional Dissection of the Major Structural Protein of Bluetongue Virus: Identification of Key Residues within VP7 Essential for Capsid Assembly", JOURNAL OF VIROLOGY, 1 June 2000 (2000-06-01), pages 8658 - 8669, XP055017800, Retrieved from the Internet <URL:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC116377/pdf/jv008658.pdf> [retrieved on 20120127] *
MONASTYRSKAYA K ET AL: "Effects of Domain-Switching and Site-Directed Mutagenesis on the Properties and Functions of the VP7 Proteins of Two Orbiviruses", VIROLOGY, ACADEMIC PRESS,ORLANDO, US, vol. 237, no. 2, 27 October 1997 (1997-10-27), pages 217 - 227, XP004452094, ISSN: 0042-6822, DOI: 10.1006/VIRO.1997.8776 *
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