EP1324770A2 - Systeme d'apport de vaccin particulaire non replicatif et techniques de preparation et d'utilisation de ce systeme - Google Patents

Systeme d'apport de vaccin particulaire non replicatif et techniques de preparation et d'utilisation de ce systeme

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Publication number
EP1324770A2
EP1324770A2 EP01975564A EP01975564A EP1324770A2 EP 1324770 A2 EP1324770 A2 EP 1324770A2 EP 01975564 A EP01975564 A EP 01975564A EP 01975564 A EP01975564 A EP 01975564A EP 1324770 A2 EP1324770 A2 EP 1324770A2
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EP
European Patent Office
Prior art keywords
tubule
protein
vaccine
virus
fusion protein
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.)
Withdrawn
Application number
EP01975564A
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German (de)
English (en)
Inventor
Polly Roy
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UAB Research Foundation
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UAB Research Foundation
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Publication date
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Publication of EP1324770A2 publication Critical patent/EP1324770A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N2720/12311—Rotavirus, e.g. rotavirus A
    • C12N2720/12322—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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—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
    • C12N2770/32122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates generally to the field of immunizations.
  • Vaccination provides the most effective preventive measures for controlling infectious diseases. As new infections keep emerging and old infections that are thought to be controlled reemerge, the list of vaccines required for children and adults grows. New ways of vaccination including vaccine combinations and simplified immunization schedules are therefore of great value to combat infectious diseases.
  • Some of the well appreciated examples are those of hepatitis B virus core and surface proteins genetically fused to foot- and-mouth disease virus (FMDV) (Clarke et al., 1987) and HIV (Michel et al., 1988; Schlienger et al., 1992) antigens; the development of Ty virus like particles (VLPs) as antigen carriers (Adams et al., 1987) where antigens are genetically fused to the C-terminus of the TYA gene encoded protein of the yeast retro-transposon Ty to form hybrid Ty-VLPs, parvovirus like particles (Miyamura et al., 1993). These technologies ensure that the antigen in question is presented in multiple copies in relatively large particles.
  • FMDV foot- and-mouth disease virus
  • HIV Michel et al., 1988; Schlienger et al., 1992
  • VLPs Ty virus like particles
  • antigens are genetically fused to the C-terminus of the TYA gene encoded protein of the yeast retro-
  • the prior art is deficient because there remains a lack of effective means of delivering multiple peptide components representing viral/tumor epitopes in order to elicit protective immunity.
  • the present invention fulfills this long-standing need and desire in the art.
  • the invention relates to a novel, non-replicating vaccine delivery vehicle and vaccines comprising that delivery vehicle.
  • the invention relates to a vaccine comprising a recombinant viral tubule (e.g., a tubule of bluetongue virus (BTV; Orbivirus: Reoviridae family) carrying one or more immunogens.
  • the vaccine is a multivalent vaccine that contains multiple foreign immunogens or model epitopes/genes.
  • the invention also relates to a fusion protein, comprising the amino acid sequence of a viral tubule protein and the amino acid sequence for one or more immunogens. More than one fusion protein is preferably assembled with, or capable of assembling with, other fusion proteins to form a viral tubule carrying one or more different types of immunogens. Thus, the invention further provides a composition comprising more than one fusion protein, optionally with different immunogenic domains, different tubule protein domain, or both.
  • the invention also relates to a vector (e.g., a baculovirus vector such as an Autographa californica vector) comprising a heterologous DNA encoding a viral tubule protein and one or more immunogens.
  • a vector e.g., a baculovirus vector such as an Autographa californica vector
  • a cell e.g., an insect cell
  • the invention further relates to a method of inducing an immune response in a subject, comprising administering a therapeutic amount of one or more fusion proteins, compositions, or vaccines of the invention to the subject.
  • a method can further comprise administering one or more virus-like particles to the subject, wherein each particle carries an immunogen.
  • the invention also provides methods of detection, diagnosis, and manufacture related to the vaccines, fusion proteins, and compositions of the invention.
  • Figure 1 shows a schematic of BTV-10 NSl protein in which carboxyl terminus is manipulated to carry FMDV peptide.
  • FIG. 2 shows a schematic of BTV-10 NSl protein in which carboxyl terminus is manipulated to carry an influenza A HA (aal 86-205) peptide.
  • BTV 10 NSl gene in a recombinant baculovirus transfer vector pAcYMl containing Spel and Sm ⁇ l sites between the last amino acid and the stop codon were digested by both Spel and Sm ⁇ l, dephosphorylated and ligated to phosphorylated, annealed synthetic oligonucleotides.
  • pAcYMl containing Spel and Sm ⁇ l sites between the last amino acid and the stop codon
  • Figure 3 shows the proliferative response of CD4+ T cell clones obtained from B10(H-2 b ) mouse immunized either with synthetic HA( 186-205) peptide or chimeric NS1.HA( 186-205) tubules.
  • T cells were incubated with irradiated feeder cells and indicated doses of peptide or chimeric tubular proteins and proliferation was determined by the incorporation of [ 3 H] thymidine. Results represent the arithmetic mean for incorporation by triplicate cell cultures.
  • Figure 4 shows gamma interferon (IFN- ⁇ ) production of the CD4+ T cell clones shown in Figure 3 and was measured in ng/ml. Results represent the arithmetic mean for IFN- ⁇ production by triplicate cell cultures.
  • IFN- ⁇ gamma interferon
  • Figure 5 shows the alignment of VP2 sequences of 7 different BTV serotypes (identified as SEQ ID Nos: 75-81).
  • the selected epitope sites are outlined and are indicted by the designation Peptide A (amino acid residues 208-268), Peptide B (amino acid residues 321-346), Peptide C (amino acid residues 398-418), Peptide D (amino acid residues 481-492), Peptide E (amino acid residues 527-539), Peptide F (amino acid residues 638-660).
  • Figure 6 shows a schematic of fusion proteins comprising the NSl protein and various epitopes A-C in Figure 6 A and epitopes D-F in Figure 6B.
  • Figure 6C shows a schematic of the formation of the fusion proteins into a tubule having multiple immatoriums.
  • Figure 7 shows an evolutionary tree of neutralizing protein VP2 and the closely related serotypes.
  • Ranges may be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
  • immunogenic capable of causing a measurable immune response in a subject.
  • An “immunogen” is an antigen capable of causing an immune response.
  • the immune response is a protective immune response, which is either temporary or permanent resistance to a pathogen.
  • isolated as used herein means the vaccine, protein, polypeptide, or peptide of this invention is sufficiently free of naturally occurring contaminants or cell components and is present in such concentration as to be the only significant vaccine, protein, peptide or polypeptide present in the sample. "Isolated” does not mean that the preparation is technically pure (homogeneous), but it is sufficiently pure to provide the peptide or polypeptide in a form in which it can be used therapeutically or diagnostically.
  • Epitope as used herein means a specific amino acid sequence of limited length which, when present in the proper conformation, provides a reactive site for an antibody or T cell receptor.
  • the identification of epitopes on antigens can be carried out by immunology protocols that are standard in the art. (Berzofsky, J.A. & Berkower, 1993.)
  • protein protein
  • peptide polypeptide
  • polypeptide polypeptide
  • a peptide usually describes a chain of amino acids of from two to about 30 amino acids
  • polypeptide usually describes a chain of amino acids having more than about 30 amino acids
  • protein usually describes one or more chains of at least about 100 amino acids.
  • polypeptide or protein can refer to a linear chain of amino acids or to a chain of amino acids which have been processed and folded into a functional protein.
  • peptide and polypeptide may be used interchangeably for a chain of amino acids around 30.
  • polypeptide and protein are used interchangeable for chains of around 100 and larger.
  • the peptides and polypeptides of the present invention are obtained by isolation and purification of the peptides and polypeptides from cells where they are produced naturally or by expression of exogenous nucleic acid encoding the peptide or polypeptide.
  • the peptides and polypeptides of this invention can be obtained by chemical synthesis, by proteolytic cleavage of a polypeptide and/or by synthesis from nucleic acid encoding the peptide or polypeptide.
  • Nucleic acid refers to single- or double-stranded molecules which may be DNA, comprised of the nucleotide bases A, T, C and G, or RNA, comprised of the bases A, U (substitutes for T), C, and G.
  • the nucleic acid may represent a coding strand or its complement.
  • Nucleic acids may be identical in sequence to the sequence which is naturally occurring or may include alternative codons which encode the same amino acid as that which is found in the naturally occurring sequence. Furthermore, nucleic acids may include codons which represent conservative substitutions of amino acids as are well known in the art.
  • the nucleic acid can be part of a recombinant nucleic acid construct comprising any combination of restriction sites and/or functional elements as are well known in the art which facilitate molecular cloning and other recombinant DNA manipulations.
  • the present invention further provides a recombinant nucleic acid construct comprising a nucleic acid encoding a fusion protein of this invention.
  • the invention relates to a means of delivering peptides, polypeptides, or small proteins using viral tubules. More specifically, the invention relates to a novel non-replicating vaccine delivery vehicle and vaccines comprising that delivery system. The invention relates to a vaccine comprising a recombinant, non- infectious viral tubule carrying one or more immunogens. Unlike cellular microtubules, the various functions of which have been analyzed extensively, virus- derived tubules (TUBs) are a rarity with unknown function (Fagerland et al., 1986; Roy et al., 1990; Van Lent et al., 1991).
  • the viral tubule is an orbivirus tubule, preferably, a Bluetongue virus tubule, and even more preferably, a Bluetongue virus NSl tubule.
  • the sequence for NSl is selected from the group consisting of SEQ ID NO:82, which provides the amino acide sequence of BTV10.
  • NSl can also include fragments and variants of the provided sequence. Whenever the specific NSl tubule is referenced herein, other types of tubules and tubule proteins can be substituted. Other types of viral tubules that can be used include, for example, viral tubules of other orbiviruses such as African horse sickness (AHS; see Maree and Huismanns (1997) J Gen Virol 98: 1077-1082), Changuinola, Chenuda, Chobar Gorge, Corriparta, Epizootic hemorrhagic disease (EHD), Equine encephalosis (EE), Eubenangee, Ieri virus, Great Island, Kemerovo, Lebombo, Orungo, Palyam, Umatilla, Wad Medani, Wallall, Warrego, Wongorr virus, or Coltivirus Group. Examples of amino acid sequences for tubule proteins from EHD and AHS include SEQ ID NO:83 and 84 respectively. Given these teachings, one skilled in
  • An object of the present invention is to construct chimeric polypeptides (e.g., NSl-TUBs) containing multiple foreign immunogens or model epitopes/genes for the development of novel multivalent vaccines that would be efficacious against targeted diseases.
  • the tubules constitute a versatile delivery system to develop efficient prophylactic and therapeutic vaccines against various diseases.
  • the present invention provides virus tubules (e.g., Bluetongue virus- based NSl-TUBs) to present multiple, chemically defined antigenic sequences to a host's immune system.
  • virus tubules e.g., Bluetongue virus- based NSl-TUBs
  • the ultimate objective is to produce safe, clinically acceptable, multivalent vaccines that are cost effective, i.e., produced in high yields, physically stable, efficient and capable of eliciting long lasting immunities.
  • such vaccines will be engineered to elicit humoral and mucosal antibodies and possess the MHC association requirements for inducing T-cell immunity.
  • the present invention is equally applicable to veterinary medicine as well as to human medicine.
  • non-infectious NSl-TUBs also have inherent properties that trigger protective immunities against Bluetongue virus and related orbivirus diseases, and further, due to the conservative nature of sequence, it can be used as a group specific diagnostic reagent.
  • the tubules have such characteristic features that allow further manipulation for delivery of foreign genes into the appropriate host cells.
  • tubules of the present invention also can be used as an adjuvant to promote immunogenic responses to other immuns.
  • NSl-TUBs offer several particular advantages over other systems (Table 1).
  • Third, tubules are devoid of any detectable amount of contaminating proteins, RNAs, or DNAs.
  • NSl-TUBs can tolerate a wide range of additional protein sequences without disruption, allowing multiple epitopes to be accommodated.
  • BTV NSl-TUBs have inherent properties of inducing CTL responses in vertebrate hosts (Jones et al., 1996). Like non-replicating parvovirus- like particles have been shown to induce antiviral CTLs (Sedlik et al., 1997), BTV NSl-TUBs are effective to deliver single/multiple CTL epitopes to the host immune system.
  • NSl-TUBs can be developed to deliver multiple peptide components representing epitopes of any immunogens (e.g., pathogenic and tumogenic) in order to elicit protective immunity.
  • Information gained in the present invention would be of great importance for designing vaccine delivery as well as appropriate immunization routes necessary to induce protection.
  • the recombinant non- infectious viral tubule comprises dimerized or multimerized fusion proteins that comprise NSl and one or more immunogens. Because each fusion protein can comprise one or more immunogens and because each tubule can comprise one or more fusion proteins with different immunogens, the vaccine delivery system can elicit a heterotypic antibody response.
  • the immunogens of the vaccine comprise one or more epitopes of a pathogen (e.g., a viral, bacterial, or parasitic pathogen) or a combination thereof.
  • a pathogen e.g., a viral, bacterial, or parasitic pathogen
  • the pathogen epitopes comprise, in one embodiment, one or more epitopes of HIV or bluetongue virus.
  • the HIV epitopes can be selected from the group consisting of SEQ ID NOs: 1-24 and the Bluetongue virus epitopes can be selected from the group consisting of SEQ ID NOs: 34-74.
  • Other embodiments of the vaccine with epitopes of viral pathogens include those with such viral pathogens as foot and mouth disease virus and influenza virus.
  • an immunogen can include various forms of the pathogens that are capable for eliciting an immunogenic response.
  • subunits of a pathogen can be used as an immunogen.
  • the subunit is an epitope or antigenic determinant.
  • immunogenicity for various serotypes of a pathogen can be provided.
  • epitopes of various pathogens are used, immunogenicity for various pathogens can be provided.
  • certain embodiments of the invention provide heterotypic antibody responses.
  • the invention also provides a vaccine, wherein the immunogens comprise one or more tumor immunogens.
  • any immunogen that functions to protect or vaccinate against any disease ca n be used.
  • Amino acid substitutions, deletions, or insertions in the immunogens may enhance immunogenicity.
  • substitutions, deletions, or insertions can be made in the immunogens, delivery vehicles, or in the fusion proteins of this invention by methods standard in the art and as set forth herein and enhanced immunogenicity can be determined according to the methods provided in the Examples herein.
  • the peptides and polypeptides may also contain conservative substitutions where a naturally occurring amino acid is replaced by one having similar properties and which does not alter the function of the polypeptide. Such conservative substitutions are well known in the art.
  • modifications and changes which are distinct from the substitutions which enhance immunogenicity, may be made in the nucleic acid and/or amino acid sequence of the peptides and polypeptides and still obtain a peptide or polypeptide having like or otherwise desirable characteristics.
  • Such changes may occur in natural isolates or may be synthetically introduced using site-specific mutagenesis, the procedures for which, such as mis-match polymerase chain reaction (PCR), are well known in the art.
  • PCR polymerase chain reaction
  • the invention also relates to a vaccine for delivering an immunogen expressed by a nucleic acid.
  • the vaccine delivery system carries a nucleic acid that expresses the immunogen.
  • the nucleic acid is linked to the vaccine delivery system using techniques known in the art.
  • the nucleic acid is a DNA.
  • the vaccine of the invention induces or is capable of inducing in a subject a protective humoral immune response.
  • the vaccine preferably activates CD4+ T cells reactive against the immunogen or immunogens.
  • the invention also relates to a fusion protein, comprising the amino acid sequence of a non-infectious viral tubule protein and the amino acid sequence for one or more immunogens, joined by a peptide bond. More than one fusion protein is preferably assembled with, or capable of assembling with, other fusion proteins to form a viral tubule.
  • the number of amino acid residues in the immurgedic domain is restricted to allow tubule formation. Specifically, up to about 270 amino acid residues can be accomodated without disruption of tubule formation.
  • One skilled in the art would readily be able to test tubule formation using the techniques described in the examples.
  • the tubule protein portion of the fusion protein comprises the amino acid sequence for any non-infectious viral tubule protein, including for example, a viral tubule protein selected from the group consisting of an orbi virus such as bluetongue virus, African horse sickness (AHS), Changuinola, Chenuda, Chobar Gorge, Corriparta, Epizootic hemorrhagic disease (EDH), Equine encephalosis (EE), Eubenangee, Ieri virus, Great Island, Kemerovo, Lebombo, Orungo, Palyam, Umatilla, Wad Medani, Wallall, Warrego, Wongorr virus, and Coltivirus Group.
  • an orbi virus such as bluetongue virus, African horse sickness (AHS), Changuinola, Chenuda, Chobar Gorge, Corriparta, Epizootic hemorrhagic disease (EDH), Equine encephalosis (EE), Eubenangee, Ieri virus, Great Island, Kemero
  • the tubule domain of a preferred embodiment of the fusion protein is an orbivirus tubule protein, preferably, a Bluetongue virus tubule protein, and even more preferably, NSl tubule protein. More specifically, the amino acid sequence for NSl is SEQ ID NO:82 or any comparable amino acid sequence of other serotypes.
  • the tubule protein domain optionally comprises a variant of the naturally occurring tubule protein.
  • the immunogen domain of the fusion protein can include epitopes of pathogens (bacteria, parasitic, viral, fungal, etc.) or tumor immunogens as described above for vaccines.
  • the immunogen domain of the fusion protein can comprise one or more epitopes of a pathogen or tumor immunogen or epitopes for more than one pathogen or tumor immunogen.
  • the fusion protein itself can induce a protective humoral immune response and can activate CD4+ T cells reactive against the immunogen or immunogens.
  • the tubules formed by the fusion proteins When assembled with other tubules which contain the same or different immunogens, the tubules formed by the fusion proteins contain various immunogens and are capable of eliciting a heterotypic antibody response.
  • the invention provides a composition comprising more than one fusion protein.
  • the fusion proteins of the composition comprise fusion proteins with different immunogenic domains, different tubule protein domain, or both.
  • the fusion proteins of the composition assemble or are capable of assembling into
  • compositions of the present invention optionally further comprise a pharmaceutical carrier and/or a suitable adjuvant.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected peptide, polypeptide, nucleic acid, vector or cell without causing substantial deleterious biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained.
  • suitable adjuvant describes an adjuvant capable of being combined with the peptide or polypeptide of this invention to further enhance an immune response without deleterious effect on the subject or the cell of the subject.
  • a suitable adjuvant can be, but is not limited to, viral tubules themselves, MONTANIDE ISA51 (Seppic, Inc., Fairfield, NJ), SYNTEX adjuvant formulation 1 (SAF-1), composed of 5 percent (wt/vol) squalene (DASF, Parsippany, N.J.), 2.5 percent Pluronic, L121 polymer (Aldrich Chemical, Milwaukee), and 0.2 percent polysorbate (Tween 80, Sigma) in phosphate-buffered saline.
  • SAF-1 SYNTEX adjuvant formulation 1
  • Suitable adjuvants include QS-21, Freund's adjuvant (complete and incomplete), alum, aluminum phosphate, aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor- muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N- acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( 1 '-2'-dipalmitoyl-sn-glycero- 3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE) and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trealose dimycolate and cell wall skeleton (MPL+TDM+CWS) in 2% s
  • compositions of the present invention can also include other medicinal agents, pharmaceutical agents, carriers, diluents, immunostimulatory cytokines, etc. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art. See Remington's Pharmaceutical Sciences (Martin, E.W., ed., latest edition), Mack Publishing Co., Easton, PA.
  • the invention also relates to a vector comprising a heterologous DNA encoding a non-infectious viral tubule protein and one or more immunogens.
  • the vector is a baculovirus vector, and, more specifically, an Autographa californica vector.
  • the invention also relates to a cell (e.g., an insect cell) comprising the vector.
  • the vector of the invention can be an expression vector which contains all of the genetic components required for expression of the nucleic acid in cells into which the vector has been introduced, as are well known in the art.
  • the expression vector can be a commercial expression vector or it can be constructed in the laboratory according to standard molecular biology protocols.
  • the expression vector can comprise viral nucleic acid including, but not limited to, vaccinia virus, baculovirus, adenovirus, retrovirus and/or adeno-associated virus nucleic acid.
  • the nucleic acid of this invention can include, for example, expression control sequences, such as an origin of replication, a promoter, an enhancer and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites and transcriptional terminator sequences.
  • expression control sequences are promoters derived from metallothionine genes, actin genes, immunoglobulin genes, CMV, SV40, adenovirus, bovine papilloma virus, etc.
  • a nucleic acid encoding a selected peptide or polypeptide can readily be determined based upon the genetic code for the amino acid sequence of the selected peptide or polypeptide and many nucleic acids will encode any selected peptide or polypeptide. Modifications in the nucleic acid sequence encoding the peptide or polypeptide are also contemplated. Modifications that can be useful are modifications to the sequences controlling expression of the peptide or polypeptide to make production of the peptide or polypeptide inducible or repressible as controlled by the appropriate inducer or repressor. Such methods are standard in the art. See Sambrook et al., 1989.
  • the nucleic acid of this invention can be generated by means standard in the art, such as by recombinant nucleic acid techniques and by synthetic nucleic acid synthesis or in vitro enzymatic synthesis.
  • the nucleic acids and/or vectors of this invention can be transferred into the host cell by well-known methods, which vary depending on the type of cell host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cell hosts.
  • Methods for producing the vaccines, peptides, polypeptides, and proteins fusion proteins of this invention comprise producing the cells that contain the nucleic acids or vectors of this invention as exogenous nucleic acid; culturing the cells under conditions whereby the exogenous nucleic acid in the cell is expressed and the encoded vaccine, peptides, polypeptides, protein, and/or fusion protein is/are produced; and isolating the vaccine, peptides, polypeptides, protein, and/or fusion protein from the cell.
  • vaccine, peptides, polypeptides, protein, and fusion protein of this invention can be produced in quantity in vitro in either prokaryotic or eukaryotic expression systems as are well known in the art.
  • prokaryotic expression systems there are numerous expression vectors that can be used and that are known to one of ordinary skill in the art.
  • Microbial hosts suitable for use include E. coli, bacilli, such as Bacillus subtilis, and other enterobacteria, such as Salmonella, Serratia, as well as various Pseudomonas species.
  • These prokaryotic hosts can support expression vectors which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication).
  • any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (Tip) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
  • the promoters will typically control expression, optionally with an operator sequence and have ribosome binding site sequences for example, for initiating and completing transcription and translation.
  • an amino terminal methionine can be provided by insertion of a Met codon 5' and in-frame with the polypeptide.
  • the carboxy-terminal extension of the polypeptide can be removed using standard oligonucleotide mutagenesis procedures.
  • the nucleic acid sequences can be expressed in hosts after the sequences have been positioned to ensure the functioning of an expression control sequence.
  • These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA.
  • expression vectors can contain selection markers, e.g., tetracycline resistance or hygromycin resistance, to permit detection and/or selection of those cells transformed with the desired nucleic acid sequences. See, e.g., U.S. Patent 4,704,362.
  • Eukaryotic expression systems can also be used.
  • a mammalian or a yeast expression system can be used to achieve post-translational modification.
  • Efficient post-translational glycosylation and expression of recombinant polypeptides can also be achieved in Baculovirus systems in insect cells, as are well known in the art.
  • the vaccine, peptides, polypeptides, protein, and/or fusion protein of the invention are expressed in Baculovirus systems in insect cells (e.g., Spodoptera frugiperda (Sf9) insect cells).
  • insect cells e.g., Spodoptera frugiperda (Sf9) insect cells.
  • the underpinning technology that was utilized in generation of TUBs is based on novel baculovirus expression.
  • the productivity and flexibility of insect baculovirus expression vectors and the ability of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) genome to incorporate (and express) large amount of foreign DNA have permitted this system to be used for the expression of not only a single gene, but also for the simultaneous expression of multiple genes (reviewed by Roy et al., 1997).
  • Autographa californica nuclear polyhedrosis virus vectors synthesize polypeptides and proteins that represent the authentic products in terms of size, post-translational modification (apart from details of glycosylation), antigenicity and cell sorting/trafficking capabilities.
  • Baculovirus multigene expression vectors can concomitantly synthesize several peptides, polypeptides, and/or proteins, including polypeptides and proteins that interact in defined stoichiometries to produce complex multiprotein structures.
  • the Autographa californica nuclear polyhedrosis virus is an ideal expression vector for the production of foreign gene products.
  • the BTV-10 M6 gene product was expressed in an insect baculovirus expression vector derived from the Autographa californica nuclear polyhedrosis virus.
  • the BTV-10 NSl polypeptide expressed by recombinant baculoviruses reacted with Bluetongue virus antibody and induced numerous tubular structures in the cytoplasm of Sf9 cells (Urakawa and Roy, 1988).
  • the BTV NSl polypeptide was expressed to a high level in insect cells infected with recombinant baculovirus. From stained preparation of cell extracts it was estimated that the amount of NSl present in cells infected at high multiplicity with the recombinant virus was ca. 50% of the total stainable protein in the cell extract prepared at the end of the infection course.
  • the TUBs were purified and were demonstrated to be composed of NSl protein and can be used as a group- specific diagnostic reagent (Ritter and Roy, 1988).
  • the BTV-10 NSl polypeptide has a low content of charged amino acids but is rich in cysteines (Lee and Roy, 1987; Monastyrskaya et al., 1994; Roy et al., 1990), thereby suggesting that it has a highly ordered structure. Also, there are several clearly defined hydrophobic regions, particularly in the carboxyl-terminal half of the polypeptide.
  • the baculovirus expression technology has allowed a variety of novel studies to be undertaken, including the formation of protein chimeras (also referred to herein as fusion proteins) by insertion into the Bluetongue virus genes foreign protein sequences representing antigens that are human immunogens.
  • protein chimeras also referred to herein as fusion proteins
  • fusion proteins foreign protein sequences representing antigens that are human immunogens.
  • Preferred vaccine delivery vehicles include a non-infectious viral protein of BTV carrying an immunogenic epitope of a pathogen.
  • the invention further relates to a method of inducing an immune response (e.g., an anti-viral, anti-bacterial, anti-fungal, anti-parasitic, anti-tumorogenic response), preferably a long acting immune response, and even more preferably a protective immune response, in a subject, comprising administering one or more fusion proteins, compositions, or vaccines described above to the subject, wherein the fusion proteins, compositions, or vaccines are administered in a therapeutic amount sufficient to induce the immune response.
  • an immune response e.g., an anti-viral, anti-bacterial, anti-fungal, anti-parasitic, anti-tumorogenic response
  • a long acting immune response preferably a long acting immune response
  • a protective immune response e.g., a protective immune response
  • a "therapeutic amount" of vaccine prepared as disclosed herein can be administered to a subject (human or animal) alone or in conjunction with an adjuvant (e.g. as described in U.S.
  • a therapeutic amount is an amount sufficient to provoke an immune response in the subject.
  • an immune response is recognized by one skilled in the art by any number of ways, including by detecting activation of T-cells and B- cells, the presence of specific antibodies, etc.
  • the immune response is sufficient to confer immunity, preferably long-lasting, which can also be detected by one of skill in the art using no more than routine experimentation. Partial or full protective immunity is detected when protection is afforded against challenge with the pathogen or tumor immunogen by evaluating symptoms of the disease, viremias, and clinical reactions.
  • Determination of a therapeutic amount takes into account such factors as the weight and/or age of the subject and the selected route for administration.
  • the exact amount of the fusion protein, vaccine, or composition required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the particular fusion protein, vaccine, or composition used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every peptide or polypeptide. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. See, e.g., Remington's Pharmaceutical Sciences (Martin, E.W., ed., latest edition), Mack Publishing Co., Easton, PA.
  • a cocktail of vaccines, fusion proteins expressing various pathogen protective epitopes can also be prepared as a vaccine composition.
  • Vaccines can be administered by a variety of methods known in the art. Exemplary modes include transmucosal, oral (e.g. via aerosol), intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, parental, transdermal and intranasal routes. If necessitated by a particular mode, the vaccine may be encapsulated.
  • a virus-like particle is a virus structure lacking genetic material.
  • the virus-like particles are double shelled virus-like particles, and even more preferably they comprise structural proteins of an orbivirus, such as bluetongue virus.
  • Structural proteins of the bluetongue virus present in the viruslike particles are preferably VP2, VP5, VP-3 and VP7.
  • Each virus-like particle optionally carries one immunogen, as compared to the fusion proteins, compositions, or vaccines of this invention, which optionally each contain more than one immunogen.
  • virus-like particles and the fusion proteins, vaccine, or compositions of the invention promotes a more substantial and longer lasting immune response in the subject.
  • Such a combined use provides protection against both homologous and heterologous serotypes of a given pathogen.
  • the virus-like particles and the fusion proteins are administered sequentially in a prime and boost paradigm.
  • the virus-like particles are administered first and in another embodiment the fusion proteins, vaccine, or compositions of the invention are administered first.
  • the invention also relates to a method of generating in a subject one or more antibodies specific for one or more immunogens, comprising administering to the subject one or more fusion proteins, vaccines, or compositions of the invention wherein the fusion protein, vaccine, or composition is administered in a therapeutic dose sufficient to generate antibodies specific for the immunogen or immunogens.
  • a suitable adjuvant can be in the composition of this invention or the adjuvant can be in a separate composition comprising the suitable adjuvant and a pharmaceutically acceptable carrier.
  • the adjuvant can be administered prior to, simultaneous with or after administration of the composition containing any of the peptides, polypeptides, nucleic acids and/or vectors of this invention.
  • QS-21 similar to alum, complete Freund's adjuvant, SAF, etc.
  • SAF complete Freund's adjuvant
  • the effectiveness of an adjuvant can be determined by measuring the immune response directed against the peptide or polypeptide of this invention with and without the adjuvant, using standard procedures, as described in the Examples herein.
  • the invention also provides a single chain antibody fragment (scFv) that binds to an antigen comprising an NSl. tubule protein of Bluetongue virus.
  • scFv single chain antibody fragment
  • the invention further provides methods of making the fusion proteins, vaccines, and compositions of the invention.
  • the invention relates to a method of making the fusion protein, comprising expressing a vector comprising a heterologous DNA encoding the viral tubule protein and one or more immunogens.
  • the invention provides a method of making the vaccine or composition, comprising expressing a vector that comprises a heterologous DNA encoding a viral tubule protein and one or more immunogens, under conditions that allow the expressed viral tubule proteins to assemble into tubules carrying one or more immunogens.
  • the invention also relates to a method of detecting orbivirus antibody (e.g., a Bluetongue virus antibody) in a sample, comprising the steps of contacting the sample with NSl tubule protein of the orbivirus; and detecting the binding of the sample to the NSl tubule, wherein the binding of the sample to the NSl tubule protein indicates the presence of the orbivirus antibody in the sample.
  • orbivirus antibody e.g., a Bluetongue virus antibody
  • the invention relates to a method of detecting orbivirus (e.g., bluetongue virus) in a sample, comprising the steps of contacting the sample with an antibody that binds to NS 1 tubule protein of the orbivirus; and detecting the binding of the antibody with the sample, wherein the binding of the antibody with the sample indicates the presence of orbivirus in the sample.
  • orbivirus e.g., bluetongue virus
  • NSl-TUBs to generate vaccine grade material (e.g., for human use).
  • the present invention is further drawn to methods of group specific diagnostic assays using NSl antigen to detect the presence of anti-Bluetongue virus antibodies or using anti-NSl antibodies to detect the presence of Bluetongue virus antigen.
  • New recombinant transfer vectors were prepared based on the dual foreign gene pAcUW51 plasmid transfer vector. In one region of this vector there is a copy of the AcNPV plO gene promoter and SV40 transcription termination signal inserted in tandem but with a unique Bgl ⁇ restriction site in between. This plO cassette is located upstream, but in the opposite orientation to a cassette involving the polyhedrin promoter, a BamHI restriction site, and the resident polyhedrin transcription termination signal. The two cassettes are flanked with AcNPV sequences.
  • the AcNPV vector facilitates the insertion of the coding region of one foreign gene into the Bani ⁇ site (i.e., under the control of the polyhedrin gene promoter) and a second into the Bgl ⁇ site (i.e., under the control of the plO promoter).
  • the vector has single strand DNA capability to facilitate site directed mutagenesis.
  • the vector is used in conjunction with linearized AcPAK6 DNA to obtain recombinants at frequencies of ca. 100%.
  • AcPAK6 is a polyhedrin-negative AcNPV containing the lacL coding region in place of the polyhedrin gene coding region. It gives blue plaques when stained with XGAL.
  • the lipofection technique is used to co-transfect monolayer of Sf9 cells with the recombinant transfer vector and Bsu361 triple-cut AcNPV DNA (Feigner et al., 1987; Kitts and Possee,1993).
  • Recombinant baculoviruses are selected on the basis of their lacZ-negative phenotypes, plaque purified and propagated as described elsewhere (King and Possee, 1992).
  • recombinant baculoviruses are isolated.
  • Baculovirus-expressed chimeric mutant BTV-10 NSl-TUBs were purified as described previously (Urakawa and Roy, 1988; Monastyrskaya et al., 1994). Sf9 cells were infected in suspension culture with recombinant AcNPV viruses using a m.o.i. of 5. After incubation at 28°C for 72 h, cells were harvested, washed with PBS, resuspended in STE buffer (150 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 7.5) containing 0.5% v/v Triton X-100 and lysed by homogenization.
  • STE buffer 150 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 7.5
  • the lysate is clarified by centrifugation (5 min at 3500 rpm) and the supernatant loaded onto a cushion of 3 ml of 40% (w/v) sucrose in STE and centrifuged in a SW41 rotor (2 h at 35,000 rpm).
  • the resulting pellet is resuspended in a small volume of STE and loaded onto a 10-40% gradient of sucrose in STE and centrifuged in a SW28 rotor (1 h at 19,000 rpm).
  • the gradient is fractionated (1.5 ml) and the pellet and each fraction is analyzed by SDS-PAGE for the presence of chimeric NSl.
  • chimeric NSl-TUBs or wild-type TUBs were resuspended in a sample buffer (50 mM Tris pH 7.5, 1 mM PMSF, 4 M urea, 1%SDS, 2 mM DTT and 2% 2-ME), boiled and electrophoresed in 10% SDS-PAGE and blotted onto an Immobilon P (Millipore) membrane. The membrane is blocked overnight with PBST containing 5% skimmed milk and incubated either with anti-NSl mouse serum to confirm the origin and authenticity of the TUBs or appropriate anti-peptide mouse antiserum whenever possible for 2 h at room temperature.
  • the membrane is then washed and incubated with an alkaline phosphatase labeled anti-mouse IgG rabbit antiserum for 1 h at room temperature, washed and the reaction developed by Amersham enhanced chemiluminescence detection system.
  • Sucrose gradient purified wild-type and chimeric NSl-TUBs in STE buffer are adsorbed onto carbon coated copper 400-mesh electron microscopy grids for 2-3 min, washed with water, and negatively stained with 2% (w/v) phosphotungstic acid. Grids are examined with a Hitachi H 7000 electron microscope at 75 kV.
  • EXAMPLE 3 Simultaneous assembly of different chimeric NSl proteins into a tubule Analysis of a panel of monoclonal antibodies has established that a NS 1 antigenic site is located near to the carboxyl terminus of the protein. It appears to be exposed on the surface of tubules (Monastyrskaya et al., 1995). In order to demonstrate that the foreign sequences are exposed on the surface of the tubules, immunogold labeling experiments were performed. A coinfection experiment was performed to test whether three different chimeric NSl proteins with C-terminal extensions would simultaneously assemble into one tubule.
  • Sf cells were infected at a multiplicity of infection (m.o.i.) of 5 with three different recombinant baculoviruses.
  • Purified NSl-TUBs were then first incubated in electron microscopic grids in the mixture of three anti-epitope sera (mouse anti-C. difficile toxin-A, rabbit anti-bovine leukemia virus [BLV], and human anti-Hepatitis B virus [HBV]). Secondary antibodies were then used. Rabbit anti-BLV antiserum was detected with goat anti-rabbit IgG conjugated with 5 nm gold particles; human anti-HBV antiserum was detected with goat anti-human IgG conjugated with 10 nm gold; and mouse anti-C.
  • NSl- TUBs can be used as carriers for the delivery of multiple epitopes/sequences from the same or different viral pathogens.
  • the use of multiple baculovirus gene expression vectors (Belyaev and Roy, 1993) increases the efficacy of formation of multiple epitope tubules.
  • Sf cells were grown in suspension or monolayer cultures at 28°C in SF900 II serum-free medium (GIBCO BRL).
  • Derivatives of AcNPV containing the wild-type BTV-10 NSl gene (AclOBTV ⁇ ) and the chimeric NSl mutant were plaque purified and propagated as described elsewhere (Brown and Faulkner, 1977).
  • Purified Foot and Mouth Disease Virus (FMDV) particles used as antigens were prepared as described elsewhere (Berinstein et al., 1993).
  • Sucrose gradient purified wild-type and chimeric NSl-TUBs in STE buffer were adsorbed onto carbon coated copper 400-mesh electron microscopy grids for 2-3 min, washed with water, and negatively stained with 2% (w/v) phosphotungstic acid. Grids were examined with a Hitachi H 7000 electron microscope at 75 kV. For in situ localization of tubules, cells infected with recombinant baculoviruses expressing either wild type or chimeric mutantNSl were fixed in 1% glutaraldehyde and embedded in Epon for sectioning. Sections on grids were then stained with lead citrate and uranyl acetate.
  • mice Sixty to ninety day old BALB/c mice were immunized intraperitoneally (i.p.) on days 0, 15, 30 and 45 with either NSl-TUBs carrying VP1 peptide of FMDV or wild-type NSl-TUBs (50 ⁇ g of protein in Incomplete Freund's adjuvant per animal per injection).
  • the antibody response was measured by a direct ELISA using a synthetic peptide pi 35- 160 (which represents the amino acid residues of FMDV VP1 O1C between positions 135-160) as an antigen (Zamorano et al., 1998) or purified virus capsids.
  • mice sera to be tested were added and incubated for 1 h at 37°C. Plates were washed three times with PBST and incubated for 1 h at 37°C with a peroxidase-labeled rabbit anti-mouse IgG antibodies (Dakkopats). After 3 washes, the reactions were developed by addition of O-phenylenediamine-H 2 ⁇ 2 in citrate buffer pH 5 and optical density (OD) read after 10 min at 490 nm in an MR 500 Microplate Reader (Dynatech). Sera were individually tested in a 4 fold dilution series in blocking buffer. Serum titers are expressed as the log of the reciprocal of the highest serum dilution representing OD readings above the mean OD readings plus 3 standard deviation (3SD) of sera from 5 animals immunized with wild- type NSl-TUBs.
  • SD standard deviation
  • mice were challenged with 10 4 lethal doses (SM 50 Lb) of FMDV Ol Campos intraperitoneally (Zamorano et al., 1998). Protection was determined by the absence of viremia in the blood samples of the challenged mice at 36 h post infection. Viremia was assessed by intramuscular (i.m.) inoculation of 50 ⁇ l/mice of a 1/10 dilution of peripheral blood samples of the challenged animals in 5-6 days old new born BALB/c mice. The presence of viremia was indicated by death of the animal.
  • SM 50 Lb lethal doses
  • FMDV Ol Campos intraperitoneally
  • the capsid protein VP1 of the Foot and Mouth Disease Virus (FMDV) carries critical epitopes responsible for the induction of neutralizing antibodies. It has already been demonstrated that a lOaa sequence of the VP1 protein (residues 135-144), harboring both B- and T-cell epitope, when used as a synthetic dimer could elicit protective neutralizing antibodies (Zamorano et al., 1994, 1998; Carrillo et al., 1998).
  • Oligonucleotide 5' - CT AGT AGC TAC AGC AGA AAT GCT GTG CCC AAC GTG 3' (SEQ ID NO: 25) and its complementary were designed to encode FMDV-1 Campos B-T-cell epitope (residues 135-144 of the G-H loop of VP1 protein; Zamorano et al., 1998).
  • a 5' overhang CT AGT (SEQ ID NO. 26) was designed for forward sense and a 3' A for the reverse sense oligonucleotide respectively for insertion into Spel and Smal cut pAcYMlNSlSpSm transfer vector.
  • the oligonucleotides were obtained from Amit of Biotech (Boston, MA), phosphorylated with T4 polynucleotide kinase and annealed at 55°C for 3 min. The annealed oligonucleotides were then ligated into Spel and Smal digested pAcYMlNSlSpSm, which contains the BTV-10 NSl gene manipulated at the carboxyl terminus to incorporate Spel and Smal sites between the last aa Y (aa 552) and the stop codon.
  • the sequence of the inserted oligonucleotides was then confirmed by the dideoxynucleotide sequencing technique (Sanger et al., 1977), within corporation of [ 35 S] dATP, using a sequenase version 2.0 DNA sequencing kit (United States Biochemical).
  • the forward primer (HL3) was some 96 nucleotides upstream of NSl stop codon while the reverse primer (Bac2) corresponds to a region of the AcNPV genome that is some 60 nucleotides downstream of the NSl insertion site and represents the sequence in the opposing DNA strand.
  • Chimeric NSl protein containing FMDV VPl sequence (aa 135-144) was synthesized by the recombinant baculovirus in insect cells and were detected SDS- PAGE analysis. Specifically, extracts obtained from AcNSl.FMDV VPl (pl35-144) infected insect cells along with mock infected cells were separated by SDS- 12.5% PAGE and staiend with Coomassie Blue. Purified NSl tubules werew used as a marker. The level of expression of the chimeric protein appeared to be as high as to the wild-type NSl expression, indicating that the chimeric construct did not interfere in protein folding and overall structure. The origin and authenticity of the two protein bands were confirmed by Western analysis using anti-FMDV VPl (aa 135-160) mouse antiserum.
  • chimeric NSl, not wild-type NSl, from infected insect cell lysates were detected by anti- FMDV VPl (aa 135-160) mouse antiserum.
  • anti- FMDV VPl aa 135-160
  • the SDS-PAGE gels were blotted onto PVDS membrane and probed with an anti-FMDV VPl (pl35-160) mouse serum. Only the chimeric protein, and not the wild-type NSl protein was recognized.
  • the aim of this study was to generate NSl-TUBs that could display the FMDV-VPl peptide.
  • the recombinant chimeric protein synthesized in insect cells was therefore examined, for its capability to form the tubules.
  • sections of AcNSl.FMDV VPl (pl35-144) recombinant baculovirus infected cells were examined the accumulation of tubules were evident in the cytoplasm of infected Sf cells similar to the wild-type tubules generated in AclOBTV ⁇ infected cells (Fig. 4).
  • the data demonstrate that the insertion of FMDV VPl peptide does not perturb the tubular morphology.
  • tubule formation capability of the fusion protein was assessed by biochemical methods using purified chimeric protein.
  • the NSl protein of BTV When the NSl protein of BTV was expressed in insect cells, it formed tubules that could be isolated by sucrose gradient centrifugation (Urakawa and Roy, 1988). Similar procedures were, therefore, used to identify the tubules made by chimeric NSl proteins. Insect cells infected with the recombinant baculovirus were lysed, and the cytoplasmic extracts were centrifuged on a sucrose gradient. To provide a positive control, NSl-TUBs representing the wild type protein were purified similarly.
  • mice A group of seven BALB/c mice was immunized and boosted twice with 50 ⁇ g/dose of either chimeric or wild type TUBs intraperitoneally. Ten days after the last booster, animals were bled and the sera analyzed for the presence of anti-FMDV antibodies. The experiment was independently performed four times. Antibodies raised in immunized mice with chimeric TUBs showed significant antibody response in ELISA against VPl as demonstrated by the reactivity to aa 135-160 peptide (Table 2). Equally strong antibody responses were obtained when intact FMDV particles were used as antigen in ELISA.
  • each immunized mouse was challenged by intraperitoneal inoculation of virulent virus.
  • peripheral blood samples of the challenged mice were tested for viremia in a group of 5-6 day old new born mice as described previously (Zamorano et al., 1995), at least 60% of the mice survived, indicating that the inoculated blood samples lacked the presence of FMDV. Therefore, more than 50% of animals that received the chimeric TUBs had generated sufficient immune response that protected the animals against the FMDV challenges.
  • the VPl sequences that was used for chimeric tubules has previously been shown to constitute the shortest aa sequence of VPl that had the ability to induce a protective anti-FMDV immune response (Zamorano et al., 1998, 1994; Carrillo et al., 1998).
  • the aa 135-144 epitope used as a synthetic peptide, elicited a protective neutralizing antibody response only when presented as a dimer.
  • the aa 135-144 epitope was expressed as a single copy and induced an anti-FMDV antibody response which protected more than 50% of the immunized mice.
  • an improved design of an NSl construct(s) that includes the adjacent flanking aa residues of the aal35-144 region of VPl may enhance the immune response. Nevertheless, this is the first evidence that demonstrated that a single copy FMDV VPl (pl35-144) epitope expressed as a fusion protein could protect immunized mice against a virulent FMDV challenge. It is quite probable that the immunogenicity conferred by the chimeric tubules may be due to the polyvalent nature of the antigen that may be important in cross-activation of immune cells.
  • mice BALB/c(H-2d) and B10.GD(H-2b) strains of mice were bred under specific pathogen-free conditions and used at 2-3 months of age. VIRUSES AND CELLS
  • Influenza A H3 subtype viruses were grown in the allantoic fluid of embryonated eggs and viral titers determined by hemagglutination assay expressed as HAU/ml. Viruses were stored at -70°C. X31 is a recombinant virus between A/Aichi/2/68 and A PR/8/34 with Hong Kong glycoproteins (H3N2) and PR8 internal components (Kilbourn, 1969). Spodoptera frugiperda cells were grown in suspension or monolayer cultures at 28°C in SF-900II serum-free medium (GIBCO BRL).
  • T-cell clones are established by limiting dilution from lines from the spleens of individual BALB/c donors primed by intranasal infection with X31 virus.
  • T-cell clones were maintained by restimulation with X31 virus (100 HAU/ml) and irradiated (3000 rads) syngeneic spleen cells (2 x 10 6 /ml) as feeder cells every 10-12 days, with the addition of interleukin-2 (IL-2)-containing supernatant (prepared from concanavalin A-stimulated rat spleen cells) and feeder cells 3 days after antigen.
  • IL-2 interleukin-2
  • T cells focus on limited regions of foreign proteins which can be defined with synthetic peptides for each mouse la haplotype (Benjamin et al., 1984).
  • Babbit et al (1985) demonstrated that binding of MHC class II antigens with synthetic peptides in vitro correlates with in vivo defined T-cell recognition specificity.
  • Oligonucleotide 5' - CT AGT AGC ACG AAC CAA GAA CAA ACC AGC CTG TAT GTT CAA GCA TCA GGG AGA GTC ACA GTC TCT 3' (SEQ ID NO: 29) and it complementary were designed to encode the Flu HA T helper cell epitope of residues 186-205 (Barnett et al., 1989).
  • a 5' overhang of CT AGT (SEQ ID NO: 26) was designed for forward sense and a 3' A for the reverse sense oligonucleotide respectively for insertion in Spel and Sm ⁇ l cut pAcYMlNS ISpSm transfer vector.
  • the oligonucleotides were obtained from Amitof Biotech (Boston, MA, USA), phosphorylated with T4 polynucleotide kinase and annealed at 55 °C for 3 min. The annealed oligonucleotides were then ligated into Spel and Sm ⁇ l digested pAcYMlNSlSpSm, which contains the BTV-10 NSl gene manipulated at the carboxyl terminus to incorporate Spel and Sm ⁇ l sites between the last amino acid Y (552) and the stop codon, to generate the recombinant transfer vector pNSlSpSm.Flu HAQ86-205).
  • the sequence of the inserted oligonucleotide was then confirmed by the dideoxynucleotide technique (Sanger et al., 1977) using a sequenase version 2.0 DNA sequencing kit (Amersham Life Science, Inc., USA).
  • the forward primer was then confirmed by the dideoxynucleotide technique (Sanger et al., 1977) using a sequenase version 2.0 DNA sequencing kit (Amersham Life Science, Inc., USA).
  • HL3 is some 96 nucleotides upstream of NSl stop codon
  • reverse primer (Bac2) corresponds to a region of the AcNPV genome that is some 60 nucleotides downstream of the NSl insertion site and represents the sequence in the opposing DNA strand.
  • Three additional amino acids, namely a threonine (T) and a serine (S) encoded by the restriction enzyme Spel and one glycine (G) encoded by the residual Sm ⁇ l cut triplet codon had to be accommodated in this protocol ( Figure 2).
  • mutant or wild type BTV-10 NSl gene in insect cells was assessed using SDS-PAGE. Extracts obtained from AcNSl.Flu HA (186-205) virus infected Sf cells along with mock infected cells were separated by SDS-PAGE and staiend with Coomassie Brillant Blue R-250, using purified NSl tubules as a reference marker. A high level of chimeric NSl protein containing Flu HA( 186-205) sequence was synthesized by the recombinant baculovirus in insect cells. The level of expression of the chimeric protein appears to be as high as the wild-type NSl. The origin and authenticity of the two protein bands were confirmed by Western analysis using anti-NSl mouse antiserum, as described above. Both wild-type NSl, and the chimeric NSl from infected insect cell lysates were detected by anti-NSl antiserum, but not the uninfected cell lysates.
  • the aim of this study was to generate NSl tubules that displayed the Flu HA( 186-205) peptide. Therefore, the recombinant chimeric protein synthesized in insect cells was examined for its capability to form the tubules.
  • sections of AcNSl.Flu HA( 186-205) recombinant baculovirus infected cells were examined by electron microscopy, the accumulation of tubules are evident in the cytoplasm of infected S. frugiperda cells similar to wild-type tubules generated in AclOBTV ⁇ infected cells.
  • the data demonstrate that insertion of Flu HA peptide does not perturb the tubular morphology.
  • tubule forming capability was assessed by biochemical methods using purified chimeric protein.
  • the NSl protein of BTV When the NSl protein of BTV is expressed in insect cells, it forms tubules that can be isolated by sucrose gradient centrifugation (Urakawa and Roy, 1988). Similar procedures were therefore used to identify the tubules made by chimeric NSl proteins. Insect cells infected with the recombinant baculovirus were lysed, and the cytoplasmic extracts were centrifuged on a sucrose gradient.
  • NSl tubules representing the wild-type protein were purified similarly. Analysis by SDS-PAGE of sucrose gradient fractions of the resolved wild-type NSl products and the chimeric NSl containing the Flu epitope demonstrated the distribution of tubules in a broad peak. The bands obtained with gradient purified wild-type BTV-10 NSl tubules and chimeric NSl tubules when analyzed by SDS-PAGE, revealed only a single protein band in each case. As expected the chimeric mutant protein was found to be larger in size compared to that of the wild-type NSl protein made by AclOBTV ⁇ , thereby representing the NSl. Flu fusion sequence. To confirm the physical status of the NSl protein, fractions of each gradient containing NSl tubules were examined by electron microscopy. The chimeric mutant NSl protein formed tubules similar to those made by wild-type NSl.
  • Green fluorescent protein (GFP) gene was used as a model to evaluate the capability of the NSl vector to accommodate immunologically relevant full length genes from pathogens of interest.
  • Polymerase chain reaction was used to construct the NSl gene extension mutant containing the GFP gene.
  • the forward and the reverse sense primers were (Green 1: 5' GCG ACT AGT ATG GTG ACG AAG GGC 3', SEQ ID NO: 32) and (Green 2: 5' GCG ACT AGT TTA TCT AGA TCC GGT3', SEQ ID NO: 33) respectively.
  • Plasmid pEGFP-Cl (Clontech Laboratories, Inc., CA), a 4.7kb plasmid encoding a red-shifted variant of wild-type GFP optimized for brighter fluorescence, was used as a template.
  • the forward primer corresponds to the N-terminal end of the GFP gene with a Spel site upstream of the initiation codon.
  • the reverse primer includes sequence in the opposing strand of the C-terminal end of the GFP gene and contains a Spel site downstream of the termination codon.
  • Plasmid DNA for PCR was added to 100 ⁇ l of a solution containing 200 ⁇ M dNTPs, 10 mM KC1, 20 mM Tris-HCl, pH8.8, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% Triton X-100), 5 pmol of the forward and reverse primers (approximately 5 ng each) and 2 units of Vent DNA polymerase (New England Biolabs, Inc., MA).
  • the reaction involved 1 cycle of 94°C for 3 min followed by 30 cycles of 94°C for 1 min, 50°C for 1 min, 72°C for 1 min, then 1 cycle of 72°C for 3 min in a DNA Thermal Cycler (Perkin Elmer, USA).
  • PCR products Five ⁇ l samples were analyzed on an agarose gel to check the presence of PCR amplified product. Prior to restriction enzyme digestion, the PCR products were treated in proteinase K (Boehringer Mannheim, Germany). 25 ⁇ g of the enzyme was added to 92 ⁇ l of PCR mixture in Tris-HCl, pH 8.0 (final concentration 10 mM), EDTA, pH8.0 (final concentration 5 mM) and SDS solution (final concentration 0.1% w/v) and incubated at 37°C for 30 min. Proteinase K was then heat-inactivated by incubation at 70°C for 10 min. Following phenol extraction the PCR products were precipitated with cold ethanol, dissolved in water and digested with Spel and was then purified with GeneClean II Kit (BIO 101, Inc. CA).
  • the purified GFP DNA fragment was then ligated to Spel digested and dephosphorylated pAcYMl.NSlSpSm vector to generate the recombinant transfer vector pNSlSpSm.GFP for baculovirus based expression in Sf9 cells.
  • the sequences at the insertion sites were confirmed by the dideoxynucleotide techniques.
  • the forward primer (HL3) is some 96 nucleotides upstream of NSl stop codon while the reverse primer (Bac2) in the case of baculovirus transfer vector (pAcYMl) corresponds to a region of the AcNPV genome that is some 60 nucleotides downstream of the NSl insertion site and represents the sequence in the opposing DNA strand.
  • the expression and stability of the NSl -GFP fusion protein in mammalian cells were tested in a transient expression system in HeLaT4 + cells.
  • Bright green fluorescence emitted from the GFP of the NSl containing chimeric protein was visualized through fluorescence microscopy and was found to be localized only in the cytoplasm of the transfected cells, but not in their nuclei. This observation is consistent with the distribution pattern of NSl protein either in their native form in BTV infected cells or as an expressed recombinant protein in mammalian cells.
  • HeLaT4+ cells transfected either with the wild-type NSl containing pGEM plasmid or the plasmid DNA alone did not show any sign of fluorescence under similar condition.
  • the NS1-GFP fusion protein was expressed in insect cells through recombinant baculoviruses.
  • Recombinant baculovirus was generated using a baculovirus compatible vector pAcYMl containing the NS1-GFP fusion gene.
  • Infected Sf9 cells exhibited very bright fluorescence indicating the presence of the GFP containing fusion protein as in mammalian cells described above.
  • NSl-TUBs single or multiple CTL, CD4 or other immunogenic epitopes are inserted and delivered by NSl-TUBs. These may represent viral, bacterial, parasitic and other immunogenic fragments (including cancer/tumor). Such chimeric constructs are highly immunogenic.
  • HIV epitopes useful for insertion are HIV epitopes useful for insertion
  • HIV-1 gpl20 H-2Dd help RIQRGPGRAFVTIGK 10
  • HIV-1 gpl20 A2 HIV-1 gpl20 A2, H-2Dd RGPGRAFVTI 11
  • CT Cholera toxin
  • CT Cholera toxin
  • S61F Cholera toxin
  • El 12K in the ADP ribosyltransferase active center of the CT-A subunit, completely lack ADP ribosyltransferase activity and diarrhoeagenicity, but supported antigen specific responses when given parenterally (Yamamoto et al., 1997a).
  • CT may effect a variety of cells including macrophages, epithelial cells, and lymphocytes (Yamamoto et al., 1997b).
  • NS 1-TUBs are internalized by dendritic cells led to the hypothesis that chimeric TUBs carrying mctx-A could have potential application in the enhancement of its adjuvanticity.
  • Chimeric TUBs carrying important immunogenic epitopes delivered in conjunction with the mctx-A-containing TUBs enhance the immune response of the vaccinated hosts. This is tested by comparing the activities of chimeric TUBs containing native CT, mCT, native CT-A or mutant CT-A subunit.
  • Chimeric NSl.ctxB TUBs are generated for in vitro experiments to see whether it is possible to deliver mctx-A into the cytoplasm through chimeric TUBs by incubating the appropriate cell lines with either chimeric ctxB-TUBs or commercially available rCT-B proteins.
  • Immunoaffinity purification is one of the most powerful techniques for the isolation of purified proteins.
  • antibodies are first covalently attached to a solid-phase matrix, e.g., protein A beads.
  • the antigen then is bound to the antibodies, and contaminating macromolecules are removed by serial washing steps.
  • Antibody-antigen interaction is then broken by treating the immune complexes with strong elution conditions thereby releasing the antigens into the elute. Because of the unique properties of the antigen-antibody interaction, no other type of chromatographic technique is likely to yield greater purification in a single step.
  • Fd phage was used to display antibody fragments of choice fused to the fd-gene III coat protein.
  • Hybridoma cells from NSl protein-immunized 10B1 mice were used to engineer a rearranged heavy (V H ) and kappa (Vi ) light chain gene of about 800 base pairs (the scFv fragment). Fd phages displaying the antibody fragments on their surface were generated is shown by ELISA.
  • 96-well plates were coated with 10 ⁇ g/ml NSl in PBS overnight at 4°C followed by blocking with fetal calf serum. Colonies of phage-transduced bacteria were inoculated into 200 ⁇ l medium with antibiotic. 50 ⁇ l phage supernatant was then added to the coated plates and was kept at 4°C for 2 hrs. Since the phage has a c-myc tag epitope fused to fd-gene III, an anti-c-myc monoclonal antibody (1:1000) was then added and incubated for 3 hrs. Anti-mouse IgG.HRP antibody was then added and kept for 1 hr at room temperature and developed with (2,2'-azino bis(3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt.
  • Protein A binds specifically to the Fc domain of antibodies, and after the antibody is bound the interaction is stabilized by cross-linking with a bifunctional coupling reagent.
  • a bifunctional coupling reagent One of the commonly used bifunctional reagents is dimethylpimelimidate (DMP), both the binding groups of which are bound to free amino groups.
  • DMP dimethylpimelimidate
  • Antibodies and protein A beads are mixed in a slurry and incubated at room temperature for 1 hr with gentle rocking. The beads are then washed twice with 10 volumes of 0.2 M sodium borate (pH 9.0) by centrifugation at 3000g for 5 min. After resuspension in 10 volumes of 0.2M sodium borate (pH 9.0), enough DMP (solid) are added to bring the final concentration to 20mM.
  • the mixing will take place for 30 min at room temperature on a rocker.
  • the reaction is stopped by washing the beads once in 0.2M ethanolamine (pH 8.0) and then incubating for 2 hr at room temp in 0.2M ethanolamine with gentle mixing. After final wash, the beads are resuspended in PBS with 0.01% merthiolate.
  • Antibody-beads are then transferred to a suitable column and washed with 20 bed volumes of PBS. Antigens are then allowed to bind to the immunoaffinity columns * Passing the antigen solution through a volume of uncoupled agarose beads equivalent to or larger than the volume of the antibody-beads first should lower the nonspecific background. Antigen solution is applied to the column and passed through it three times by gravity. The column is then washed with 20 bed volume of binding buffer. After washing the column, the buffer is changed to the pre-elution buffer by passing 10 bed volumes through the column. The pre-elution buffer allows a quicker change to the elution conditions and should give a sharper elution profile.
  • NSl tubule as a group-specific diagnostic reagent
  • the NSl antigen in direct or indirect ELISA applications, is a superior group specific diagnostic antigen.
  • Micro ELISA plates were coated overnight with an optimal concentration of NSl antigen (10 ⁇ g/well) in carbonate coating buffer (15 mM Na 2 C0 3 , 36 mM NaHCO 3 , pH 9.6) and subsequently blocked with PBS supplemented with 2% (w/v) skimmed milk.
  • anti-NS 1 antibodies disclosed herein can be used to detect the presence of Bluetongue virus antigen by specific immunofluorescence. Rapid and sensitive diagnosis of BTV is done by simple microscopy and flow cytometry to detect intracellular as well as cell surface BTV antigen.
  • Viral tubules are prepared as described above.
  • Virus-like particles are prepared as described in Roy at al., Vaccine 12:805-811 (1994). Briefly, VLPs containing a VP2 species (e.g., BTV-1, -2, -10, -13, -17, or others), VP3, VP5, or VP7 are made using dual recombinant baculovirus expression vectors. A prime and boost vaccination regime is utilized. Specifically, the animals to be vaccinated are primed with viral tubule vaccine delivery systems as described above and are then boosted with VLPs according to the administration methods of in Roy at al, Vaccine 12:805-811 (1994).
  • a VP2 species e.g., BTV-1, -2, -10, -13, -17, or others
  • VP3, VP5, or VP7 are made using dual recombinant baculovirus expression vectors.
  • a prime and boost vaccination regime is utilized. Specifically, the animals to be vac
  • the animals to be vaccinated are primed with VLPs and boosted with viral tubules.
  • the combination results in a protective antibody response, preferably for more than one serotype of bluetongue virus.
  • Adjuvant e.g., 50% (v/v) ISA-50
  • other portent adjuvants such as MF59 (Chiron, CA) may be used with either or the viral tubules or VLPs or both.
  • Chimeric NSl-TUBs carrying epitopes of various bluevirus serotypes are made according to the methods described above.
  • Epitopes carried by the NSl -TUB are derived, for example, from the VP2 protein sequence according to Figure 5, which shows the sequences for BTV serotypes 10, 17, 11, 1, 13, 2, and 9, with regions designated peptides A-F to indicate epitopes from each serotype.
  • the fusion proteins are then made according to Figure 6A and B.
  • the fusion proteins can comprise three sequences of designated peptide A, for example, from various serotypes to provide protection against various serotypes of BTV, can comprise peptides A-C, for example, from the same serotypes, or a combination thereof.
  • Viral tubules comprising these fusion proteins are represented in Figure 6C.
  • the specific epitopes are indicated in Table 4. Any combination of these epitopes can be combined in a single fusion protein so long as the fusion proteins remain capable of forming viral tubules.
  • PEPTIDE SEROTYPE NOS.
  • PEPTIDE SEROTYPE NOS. PEPTIDE SEQUENCE NO:
  • FIG. 7 shows an evolutionary tree of VP2 proteins and the relatedness of certain strains.
  • One vaccine directed to BTV13 therefore, is likely to have effects that extend to BTV 16 and 3.
  • a vaccine directed to BTV17 is likely to have effects to BTV4, 12, 10, and perhaps to BTV 15.
  • Table 5 shows homologies among nine BTV, EHDV and three AHSV VP2 proteins.
  • a composition of various fusion proteins can be used to provide broad immunity to a wide range of BTV serotypes and various other immunogens.
  • Table 5 Homologies among nine BTV, EHDV and three AHSV VP2 proteins

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Abstract

La présente invention concerne un moyen d'apport d'immunogènes multiples, tels que des épitopes pathogènes/tumorogènes, destinés à induire des réponses immunitaires. Cette invention concerne, en particulier, une protéine de fusion, comprenant la séquence d'acide aminé d'une protéine de tubule viral et la séquence d'acide aminé d'un ou de plusieurs immunogènes. Ces protéines de fusion peuvent s'assembler avec d'autres protéines de fusion de façon à former un tubule viral qui peut servir à apporter des immunogènes multiples lors d'une administration à un sujet sous forme de vaccin. Cette invention concerne aussi des techniques de préparation et d'utilisation des divers vaccins, protéines de fusion et compositions de cette invention.
EP01975564A 2000-09-27 2001-09-27 Systeme d'apport de vaccin particulaire non replicatif et techniques de preparation et d'utilisation de ce systeme Withdrawn EP1324770A2 (fr)

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