WO2013188918A1 - Virus recombinants améliorés - Google Patents

Virus recombinants améliorés Download PDF

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Publication number
WO2013188918A1
WO2013188918A1 PCT/AU2013/000658 AU2013000658W WO2013188918A1 WO 2013188918 A1 WO2013188918 A1 WO 2013188918A1 AU 2013000658 W AU2013000658 W AU 2013000658W WO 2013188918 A1 WO2013188918 A1 WO 2013188918A1
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virus
recombinant
protein
flaviviridae
coding sequence
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PCT/AU2013/000658
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English (en)
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Ian Ramshaw
Mario LOBIGS
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The Australian National University
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Priority claimed from AU2012902593A external-priority patent/AU2012902593A0/en
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Publication of WO2013188918A1 publication Critical patent/WO2013188918A1/fr

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    • 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
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/565—IFN-beta
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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • A61K2039/5254—Virus avirulent or attenuated
    • 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
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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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
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16034—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
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—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
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2770/24143—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
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    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/023—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a poxvirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates generally to treatment and/or prevention of viral diseases. More particularly, the present invention relates to recombinant Flaviviridae viruses for stably expressing heterologous nucleic acid molecules, methods for producing such viruses, and their use in immunomodulating compositions for treating and or preventing conditions including Flaviviridae virus infections.
  • arthropods predominantly mosquitoes and ticks
  • Arbovirus infection in mammals can manifest in potentially life-threatening diseases including encephalitis and hemorrhagic fever.
  • Flaviviridae family of arboviruses have been responsible for numerous epidemics in both human and animal populations.
  • Japanese encephalitis virus accounts for up to 50,000 cases of encephalitis in humans annually (with case fatality rates of about 25%), and significant epidemics have occurred in India and Nepal in recent years.
  • Yellow fever virus causes a wide spectrum of disease ranging from mild symptoms to kidney /liver failure and hemorrhaging of the gastrointestinal tract.
  • Dengue virus is a prevalent mosquito-borne member of the Flavivirus genus causing significant human disease ranging from dengue fever to life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). It is estimated that 2.5 billion people are at risk of dengue virus infection with 50-100 million cases of dengue fever annually causing approximately 25,000 deaths (predominantly among children).
  • DHF/DSS is a plasma leakage syndrome characterized by defects in vascular permeability, marked thrombocytopenia, hepatomegaly and bleeding diathesis, which can lead to life-threatening shock.
  • Each of the four dengue virus serotypes can cause the full spectrum of disease.
  • the rapid spread of Dengue virus to most tropical and subtropical countries has led to its classification as an emerging infectious disease and has intensified efforts to prevent infection.
  • Live recombinant viral vectors i.e., viruses engineered to express foreign gene(s)
  • additional component(s) capable of enhancing and/or broadening host immune responses.
  • live recombinant viral vectors few if any have been developed against members of the genus Flavivirus. This arises at least in part from difficulties in maintaining the genetic stability of foreign gene insert(s) over multiple viral replication cycles in the host.
  • the present invention arises in part from the determination that it is possible to insert an heterologous nucleic acid sequence into the open reading frame (ORF) of a flaviviridae virus genome while maintaining stability of the genome over multiple replication cycles, Specifically, it has been discovered that stability of a flaviviridae virus genome is not substantially compromised when an heterologous nucleic acid sequence comprising a transgene and an internal ribosome entry site (IRES) is inserted between adjacent protein-coding sequences of the flaviviridae virus ORF, whereby the transgene is operably connected (e.g.
  • coding sequences e.g., a coding sequence comprising a nucleotide sequence encoding the capsid (C) protein of the Flaviviridae virus
  • IRES is operably connected upstream of the other coding sequence (e.g., a coding sequence comprising a nucleotide sequence encoding proteins of the Flaviviridae virus other than the C protein).
  • This bicistronic strategy is based on the hypothesis that unless a precise deletion of the heterologous nucleic acid sequence occurs, which restores the native viral genome, deletions in the heterologous nucleic acid sequence would be selected against since they would prevent production of essential viral translation products.
  • the present invention provides a recombinant Flaviviridae virus comprising a bicistronic genome that comprises, consists or consists essentially of a flaviviridae virus open reading frame (ORF) and an heterologous nucleic acid sequence inserted between adjacent first and second coding sequences of the ORF, wherein the coding sequences encode different flaviviridae virus proteins, wherein the heterologous nucleic acid sequence comprises a transgene and an internal ribosome entry site (IRES), wherein the transgene is operably connected downstream of the first coding sequence and the IRES is operably connected upstream of the second coding sequence.
  • ORF flaviviridae virus open reading frame
  • the heterologous nucleic acid sequence comprises a transgene and an internal ribosome entry site (IRES), wherein the transgene is operably connected downstream of the first coding sequence and the IRES is operably connected upstream of the second coding sequence.
  • IRES internal ribosome entry site
  • the Flaviviridae virus is a member of the genus Flavivirus (e.g., a virus selected from the group consisting of Dengue virus, Japanese encephalitis virus. Yellow fever virus, Murray Valley encephalitis virus (MVEV), West Nile virus, and St Louis encephalitis virus).
  • the Flaviviridae virus is selected from Dengue virus serotype I, II, III, or IV.
  • the Flaviviridae virus is a live, replication competent and/or attenuated Flaviviridae virus.
  • the first coding sequence and the second coding sequence together comprise all the subunit proteins of a polyprotein encoded by the flaviviridae virus ORF.
  • the Flaviviridae virus is a member of the genus Flavivirus.
  • representative subunit proteins include, for example, the amino terminal structural proteins capsid (C) protein, pre-membrane (prM) protein and envelope (E) protein and the carboxyl-terminal non-structural proteins nonstructural protein 1 (NSl), non-structural protein 2 A (NS2A), non-structural protein 2B (NS2B), non-structural protein 3 (NS3), non-structural protein 4A (NS4A), nonstructural protein 4B (NS4B) and non-structural protein 5 (NS5).
  • the recombinant flaviviridae virus genome comprises, in order from 5' to 3', a flaviviridae virus 5' untranslated region (UTR), the first coding sequence, the heterologous nucleic acid sequence, the second coding sequence and a flaviviridae virus 3' UTR.
  • the Flaviviridae virus is a member of the genus Flavivirus.
  • the first coding sequence comprises a nucleotide sequence encoding at least one flavivirus virus structural protein ⁇ e.g., any one or more of C, prM and E proteins) and optionally at least one flavivirus virus nonstructural protein (e.g., any one or more of NSl , NS2A, NS2B, NS3, NS4A and NS4B proteins).
  • the first coding sequence comprises a nucleotide sequence encoding a C protein.
  • the first coding sequence comprises a nucleotide sequence encoding a C protein and a second nucleotide sequence encoding a prM protein.
  • the first coding sequence comprises a nucleotide sequence encoding a C protein, a second nucleotide sequence encoding a prM protein and a third nucleotide sequence encoding an E protein.
  • the first coding sequence comprises a nucleotide sequence encoding a C protein, a second nucleotide sequence encoding a prM protein, a third nucleotide sequence encoding an E protein, and a fourth nucleotide sequence encoding a NS 1 protein.
  • the first coding sequence comprises a nucleotide sequence encoding a C protein, a second nucleotide sequence encoding a prM protein, a third nucleotide sequence encoding an E protein, a fourth nucleotide sequence encoding a NSl protein and a fifth nucleotide sequence encoding a NS2A.
  • the first coding sequence comprises a nucleotide sequence encoding a C protein, a second nucleotide sequence encoding a prM protein, a third nucleotide sequence encoding an E protein, a fourth nucleotide sequence encoding a NS l protein, a fifth nucleotide sequence encoding a NS2A protein and a sixth nucleotide sequence encoding a NS2B protein.
  • the first coding sequence comprises a nucleotide sequence encoding a C protein, a second nucleotide sequence encoding a prM protein, a third nucleotide sequence encoding an E protein, a fourth nucleotide sequence encoding a NSl protein, a fifth nucleotide sequence encoding a NS2A protein, a sixth nucleotide sequence encoding a NS2B protein and a seventh nucleotide sequence encoding a NS3 protein.
  • the first coding sequence comprises a nucleotide sequence encoding a C protein, a second nucleotide sequence encoding a prM protein, a third nucleotide sequence encoding an E protein, a fourth nucleotide sequence encoding a NSl protein, a fifth nucleotide sequence encoding a NS2A protein, a sixth nucleotide sequence encoding a NS2B protein, a seventh nucleotide sequence encoding a NS3 protein and a eighth nucleotide sequence encoding a NS4A protein.
  • the first coding sequence comprises a nucleotide sequence encoding a C protein, a second nucleotide sequence encoding a prM protein, a third nucleotide sequence encoding an E protein, a fourth nucleotide sequence encoding a NSl protein, a fifth nucleotide sequence encoding a NS2A protein, a sixth nucleotide sequence encoding a NS2B protein, a seventh nucleotide sequence encoding a NS3 protein, a eighth nucleotide sequence encoding a NS4A protein and a ninth nucleotide sequence encoding a NS4B protein.
  • the second coding sequence comprises a nucleotide sequence encoding at least one flaviviridae virus structural protein (e.g., for members of the genus Flavivirus, any one or more of a prM and/or E proteins) and optionally at least one flaviviridae virus non-structural protein (e.g., for members of the genus Flavivirus, any one or more of NSl, NS2A, NS2B, NS3, NS4A, NS4B and NS5 proteins).
  • flaviviridae virus structural protein e.g., for members of the genus Flavivirus, any one or more of a prM and/or E proteins
  • flaviviridae virus non-structural protein e.g., for members of the genus Flavivirus, any one or more of NSl, NS2A, NS2B, NS3, NS4A, NS4B and NS5 proteins.
  • the second coding sequence comprises a first nucleotide sequence encoding a prM protein, a second nucleotide sequence encoding an E protein, a third nucleotide sequence encoding a NSl protein, a fourth nucleotide sequence encoding a NS2A protein, a fifth nucleotide sequence encoding a NS2B protein, a sixth nucleotide sequence encoding a NS3 protein, a seventh nucleotide sequence encoding a NS4A protein, an eighth nucleotide sequence encoding a NS4B protein and a ninth nucleotide sequence encoding a NS5 protein.
  • the second coding sequence comprises a first nucleotide sequence encoding an E protein, a second nucleotide sequence encoding a NS1 protein, a third nucleotide sequence encoding a NS2A protein, a fourth nucleotide sequence encoding a NS2B protein, a fifth nucleotide sequence encoding a NS3 protein, a sixth nucleotide sequence encoding a NS4A protein, a seventh nucleotide sequence encoding a NS4B protein and a eighth nucleotide sequence encoding a NS5 protein.
  • the second coding sequence comprises a first nucleotide sequence encoding a NS1 protein, a second nucleotide sequence encoding a NS2A protein, a third nucleotide sequence encoding a NS2B protein, a fourth nucleotide sequence encoding a NS3 protein, a fifth nucleotide sequence encoding a NS4A protein, a sixth nucleotide sequence encoding a NS4B protein and a seventh nucleotide sequence encoding a NS5 protein.
  • the second coding sequence comprises a first nucleotide sequence encoding a NS2A protein, a second nucleotide sequence encoding a NS2B protein, a third nucleotide sequence encoding a NS3 protein, a fourth nucleotide sequence encoding a NS4A protein, a fifth nucleotide sequence encoding a NS4B protein and a sixth nucleotide sequence encoding a NS5 protein.
  • the second coding sequence comprises a first nucleotide sequence encoding a NS2B protein, a second nucleotide sequence encoding a NS3 protein, a third nucleotide sequence encoding a NS4A protein, a fourth nucleotide sequence encoding a NS4B protein and a fifth nucleotide sequence encoding a NS5 protein.
  • the second coding sequence comprises a first nucleotide sequence encoding a NS3 protein, a second nucleotide sequence encoding a NS4A protein, a third nucleotide sequence encoding a NS4B protein and a fourth nucleotide sequence encoding a NS5 protein.
  • the second coding sequence comprises a first nucleotide sequence encoding a NS4A protein, a second nucleotide sequence encoding a NS4B protein and a third nucleotide sequence encoding a NS5 protein.
  • the second coding sequence comprises a first nucleotide sequence encoding a NS4B protein and a second nucleotide sequence encoding a NS5 protein. In other illustrative examples, the second coding sequence comprises a first nucleotide sequence encoding a NS5 protein.
  • the first coding sequence comprises a nucleotide sequence encoding a C protein and the second coding sequence comprises a first nucleotide sequence encoding a prM protein, a second nucleotide sequence encoding an E protein, a third nucleotide sequence encoding an NS1 protein, a fourth nucleotide sequence encoding a NS2A protein, a fifth nucleotide sequence encoding a NS2B protein, a sixth nucleotide sequence encoding a NS3 protein, a seventh nucleotide sequence encoding a NS4A protein, an eighth nucleotide sequence encoding a NS4B protein and a ninth nucleotide sequence encoding a NS5 protein.
  • the transgene suitably comprises a nucleotide sequence that is both transcribed into mRNA and translated into a polypeptide or a nucleotide sequence that is only transcribed into RNA (e.g., a non-coding sequence, illustrative examples of which include functional RNA molecules such as rRNA, tRNA, RNAi, ribozymes and antisense RNA).
  • the transgene comprises a nucleotide sequence (i.e. , a coding sequence) that encodes an exogenous polypeptide.
  • the transgene coding sequence is in frame with the first coding sequence.
  • the exogenous polypeptide is selected from a polypeptide of a pathogenic organism other than the Flaviviridae virus, an alloantigen, an autoantigen, a cancer or tumor antigen or any other polypeptide that has therapeutic activity.
  • the transgene comprises a nucleotide sequence encoding a cytokine (e.g., a cytokine that attenuates the Flaviviridae virus, illustrative example of which include interferons (IFNs) including type I IFNs such as IFN- ⁇ .
  • IFNs interferons
  • the present invention provides recombinant flaviviridae virus genomes engineered to stably express transgenes including cytokine-encoding sequences, which can provide a means of attenuating virulence (i.e., addressing safety concerns) and/or augmenting immunity against the viruses, in a subject to which they are administered.
  • the transgene further comprises a nucleotide sequence that encodes a proteolytic cleavage site positioned to facilitate release of the exogenous polypeptide upon proteolytic processing of a precursor polypeptide encoded by the first coding sequence and the coding sequence of the transgene.
  • the transgene further comprises a nucleotide sequence encoding a signal peptide (which is suitably upstream of the coding sequence for the exogenous polypeptide) for transit of the exogenous polypeptide to a particular cellular compartment or into an extracellular environment.
  • the signal peptide directs translocation of the exogenous polypeptide across an endoplasmic reticulum (ER) membrane within a host cell infected by the virus.
  • ER endoplasmic reticulum
  • the exogenous polypeptide is exported to the host cell surface, presented on the cell surface as a peptide with a major histocompatibility antigen, secreted from the cell, or remains in the cytoplasm of the cell.
  • the present invention provides a pharmaceutical composition comprising a recombinant Flaviviridae virus as broadly described above and elsewhere herein, and a pharmaceutically acceptable excipient, diluent or carrier.
  • the present invention provides an
  • immunomodulating composition comprising a recombinant Flaviviridae virus as broadly described above and elsewhere herein, and optionally an adjuvant or immunostimulant.
  • the present invention provides methods of eliciting an immune response to a Flaviviridae virus in a subject (e.g., a human). Such methods comprise administering an effective amount of a recombinant Flaviviridae virus as broadly described above and elsewhere herein, to the subject so as to elicit an immune response to the Flaviviridae virus.
  • the exogenous polypeptide also referred to herein as an heterologous polypeptide
  • is a cytokine e.g., an IFN including a type I IFN such as IFN- ⁇
  • the present invention provides methods of treating or preventing a flaviviridae virus infection in a subject (e.g., a human). Such methods comprise administering an effective amount of a recombinant Flaviviridae virus as broadly described above and elsewhere herein, to the subject.
  • the exogenous polypeptide is a cytokine (e.g. , an IFN including a type I IFN such as IFN- ⁇ ) that attenuates the Flaviviridae virus or that augments the immune response to the Flaviviridae virus.
  • the present invention provides a recombinant Flaviviridae virus, or composition as broadly described above and elsewhere herein, for use in inducing an immune response to a Flaviviridae virus (e.g., the Flaviviridae virus used to generate the recombinant Flaviviridae virus) in a subject (e.g., a human).
  • a Flaviviridae virus e.g., the Flaviviridae virus used to generate the recombinant Flaviviridae virus
  • a subject e.g., a human
  • Another aspect of the present invention provides methods of eliciting an immune response to an exogenous polypeptide in a subject (e.g., a human). Such methods comprise administering a recombinant Flaviviridae virus as broadly described above and elsewhere herein, to the subject so as to elicit an immune response to the exogenous polypeptide.
  • the exogenous polypeptide can be a host antigen or an antigen of a microorganism (e.g., bacteria, protozoa, viruses other than the Flaviviridae virus used to generate the recombinant Flaviviridae virus of the invention), yeast, fungi, and the like).
  • the present invention provides a recombinant Flaviviridae virus, or composition as broadly described above and elsewhere herein, for use in preventing or treating an infection by a pathogen (other than the Flaviviridae virus used to generate the recombinant Flaviviridae virus of the invention) in a subject (e.g., a human).
  • a pathogen other than the Flaviviridae virus used to generate the recombinant Flaviviridae virus of the invention
  • a subject e.g., a human
  • Still another aspect of the present invention provides methods of delivering an exogenous polypeptide having therapeutic activity to a subject ⁇ e.g., a human). Such methods generally involve administering a recombinant Flaviviridae virus as broadly described above and elsewhere herein, to the subject. The exogenous polypeptide is then produced in a host cell of the subject. The therapeutic polypeptide may remain inside the cell, becomes associated with a cell membrane, or is secreted from the cell.
  • Yet another aspect of the present invention provides methods for producing an exogenous polypeptide in a host cell ⁇ e.g., a vertebrate host cell), comprising contacting a susceptible host cell with a recombinant Flaviviridae virus as broadly described above and elsewhere- herein, wherein the transgene comprises a nucleotide sequence that encodes the exogenous polypeptide, and culturing the host cell for a period of time to allow production of the exogenous polypeptide by the host cell.
  • the methods further comprise purifying the exogenous polypeptide.
  • Figure 1 is a graph demonstrating that co-expression of IFN- ⁇ boosts
  • Figure 2 is a graph showing virus titers and CD8 + T cell responses in MyD88-/- mice after vaccinia virus (W)-IFN-p treatment.
  • Figure 3 provides schematic representations of wild type (wt) MVEV and recombinant (r) MVEV constructs.
  • Top - MVEV.wt wild-type (wt) MVEV.
  • Middle - MVEV.C-IRES a termination codon and the encephalomyocarditis virus (ECMV) internal ribosome entry site (IRES) were introduced down-stream from the MVEV C protein.
  • Bottom - MVEV.IFNP a functional mouse IFN- ⁇ gene, termination codon and ECMV IRES were introduced down-stream from the MVEV C protein.
  • Black bar signal peptide.
  • Figure 4 is a time-course graph showing growth kinetics of
  • MVEV.wt MVEV.C-IRES and MVEV.IFNp in mouse embryo fibroblasts.
  • Figure 5 is a time-course graph showing survival of BALB/c mice over 35 days infected with MVEV.wt, MVEV.C-IRES and MVEV.IFNp.
  • Figure 6 is a time-course graph showing mortality in BALB/c mice infected with MVEV.wt (circles), or, MVEV.IFNp with MVEV.wt (triangles).
  • Figure 7 are dotplot graphs showing humoral immunity induced by MVEV.wt, rMVEV.C-IRES and rMVEV.IFNp in BALB/c and/or CBA mice.
  • Figure 8 provides schematic representations of chimeric MVEV encoding Dengue virus prM-E for IFN- ⁇ co-expression.
  • Figures 9 A and 9B provide schematic representations of the
  • antigen and “epitope” are well understood in the art and refer to the portion of a macromolecule which is specifically recognized by a component of the immune system, e.g., an antibody or a T-cell antigen receptor.
  • Epitopes are recognized by antibodies in solution, e.g., free from other molecules. Epitopes are recognized by T-cell antigen receptor when the epitope is associated with a class I or class II major histocompatibility complex molecule.
  • a "CTL epitope” is an epitope recognized by a cytotoxic T lymphocyte (usually a CD8 + cell) when the epitope is presented on a cell surface in association with an MHC Class I molecule.
  • An "allergen” refers to a substance that can induce an allergic or asthmatic response in a susceptible subject.
  • “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, position or length that varies by as much 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, position or length.
  • nucleotide sequence of between 10 and 20 nucleotides in length is inclusive of a nucleotide of 10 residues in length and a nucleotide of 20 residues in length.
  • c/s-acting sequence or “cw-regulatory region” or similar term shall be taken to mean any sequence of nucleotides which is derived from an expressible genetic sequence wherein the expression of the genetic sequence is regulated, at least in part, by the sequence of nucleotides.
  • a m-regulatory region may be capable of activating, silencing, enhancing, repressing or otherwise altering the level of expression and/or cell-type- specificity and/or devel opmental specificity of any structural gene sequence.
  • cistron refers to a section of DNA or RNA that contains the genetic codes for a single polypeptide or a protein, and may function as a hereditary unit.
  • cistron refers to the existence in the recombinant viruses of the invention of two unrelated cistrons which are expressed from a single viral transcriptional unit.
  • the first cistron comprises an open reading frame encoding at least 1, 2, 3, 4, 5, 6, 7, 8, 9 protein(s) of the virus and a transgene, while the second cistron initiates translation from an IRES element located between the two cistrons, and includes an open reading frame encoding at least 1, 2, 3, 4, 5, 6, 7, 8, 9 protein(s) of the virus suitably not included in the first cistron.
  • coding sequence is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene.
  • non-coding sequence refers to any nucleic acid sequence that does not contribute to the code for the polypeptide product of a gene.
  • control element or "control sequence” is meant nucleic acid sequences (e.g., DNA) necessary for expression of an operably linked coding sequence in a particular host cell.
  • the control sequences that are suitable for prokaryotic cells for example, include a promoter, and optionally a cw-acting sequence such as an operator sequence and a ribosome binding site.
  • Control sequences that are suitable for eukaryotic cells include transcriptional control sequences such as promoters, polyadenylation signals, transcriptional enhancers, translational control sequences such as translational enhancers and internal ribosome binding sites (IRES), nucleic acid sequences that modulate mRNA stability, as well as targeting sequences that target a product encoded by a transcribed polynucleotide to an intracellular compartment within a cell or to the extracellular environment.
  • transcriptional control sequences such as promoters, polyadenylation signals, transcriptional enhancers, translational control sequences such as translational enhancers and internal ribosome binding sites (IRES), nucleic acid sequences that modulate mRNA stability, as well as targeting sequences that target a product encoded by a transcribed polynucleotide to an intracellular compartment within a cell or to the extracellular environment.
  • a nucleic acid sequence that displays substantial sequence identity to a reference nucleic acid sequence e.g., at least about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 97, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% sequence identity to all or a portion of the reference nucleic acid sequence) or an amino acid sequence that displays substantial sequence similarity or identity to a reference amino acid sequence (e.g.
  • an effective amount in the context of treating or preventing a condition or for modulating an immune response to a target antigen or organism is meant the administration of an amount of an agent (e.g., a recombinant virus) or composition to an individual in need of such treatment or prophylaxis, either in a single dose or as part of a series, that is effective for the prevention of incurring a symptom, holding in check such symptoms, and/or treating existing symptoms, of that condition or for modulating the immune response to the target antigen or organism.
  • the effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • endogenous refers to a gene or nucleic acid sequence or segment that is normally found in a host organism.
  • expression refers to transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein.
  • expression of a coding sequence results from transcription and translation of the coding sequence.
  • expression of a non-coding sequence resul ts from the transcription of the non-coding sequence.
  • Flaviviridae virus encompasses all viruses within the Flaviviridae family as classified under the Baltimore Classification System (BCS) and/or the International Committee on Taxonomy of Viruses (ICTV).
  • BCS Baltimore Classification System
  • ICTV International Committee on Taxonomy of Viruses
  • the name of an organism or group to which it belongs indicates the use of that name as a noun
  • the name of an organism or group to which it belongs e.g., a virus species, genus or family
  • Flaviviridae ' ' ' ' shall mean the family Flaviviridae
  • "Flaviviridae virus” shall indicate a virus of the family Flaviviridae.
  • “flaviviridae genome (polyprotein, nucleotide sequence)” shall indicate a genome (polyprotein, nucleotide sequence) of a virus belonging to the family Flaviviridae.
  • “Flavivirus” shall mean the genus Flavivirus
  • “flavivirus genome (polyprotein, nucleotide sequence)” shall indicate a genome (polyprotein, nucleotide sequence) of a virus belonging to the genus Flavivirus.
  • the term “gene” as used herein refers to any and all discrete coding regions of a genome, as well as associated non-coding and regulatory regions.
  • the gene is also intended to mean an open reading frame encoding one or more specific polypeptides, and optionally comprising one or more introns, and adjacent 5' and 3' non- coding nucleotide sequences involved in the regulation of expression.
  • the gene may further comprise control signals such as promoters, enhancers, termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals.
  • heterologous polynucleotide foreign polynucleotide
  • exogenous polynucleotide refers to any nucleic acid (e.g. , a gene sequence or regulatory sequence) which is introduced into the genome of an organism by experimental manipulations and may include gene sequences found in that organism so long as the introduced gene contains some modification (e.g., a point mutation, the presence of a endonuclease cleavage site, the presence of a loxP site, etc.) relative to the naturally-occurring gene(s).
  • nucleic acid e.g. , a gene sequence or regulatory sequence
  • some modification e.g., a point mutation, the presence of a endonuclease cleavage site, the presence of a loxP site, etc.
  • heterologous polypeptide foreign polypeptide
  • exogenous polypeptide are used interchangeably to refer to any peptide or
  • polypeptide which is encoded by a heterologous polynucleotide "foreign
  • polynucleotide and "exogenous polynucleotide,” as defined above.
  • host cell refers to a cell into which a vector including a recombinant Flaviviridcte virus of the invention is introduced.
  • Host cells of the invention include, but need not be limited to, bacterial, yeast, animal (including vertebrate animals falling within the scope of the term “subject” as defined herein) insect and plant cells.
  • Host cells can be unicellular, or can be grown in tissue culture as liquid cultures, monolayers or the like.
  • Host cells may also be derived directly or indirectly from tissues or may exist within an organism including animals.
  • the term "immunogenic" when used in the context of a given agent such as, for example, a nucleotide sequence, polypeptide, an heterologous nucleic acid sequence, an heterologous polypeptide, an antigen, or an epitope, means that the agent has a capability to induce an immune response, enhance an existing immune response, or alter an existing immune response, either alone, or acting in combination with other agent(s).
  • the immune response may include a humoral and/or cellular immune response in a subject.
  • antigenic amino acid sequence means an amino acid sequence that, either alone or in association with an accessory molecule ⁇ e.g., a class I or class II major histocompatibility antigen molecule), can elicit an immune response in a subject.
  • accessory molecule e.g., a class I or class II major histocompatibility antigen molecule
  • inducing an immune response includes stimulating an immune response and/or enhancing a previously existing immune response.
  • IRES internal ribosomal entry site
  • a viral, cellular, or synthetic ⁇ e.g., a recombinant nucleotide sequence which allows for initiation of translation of an mRNA at a site internal to a coding region within the same mRNA or at a site 3' of the 5' end of the mRNA, to provide for translation of an operably linked coding region located downstream of (/ ' . e. , 3' of) the internal ribosomal entry site. This makes translation independent of the 5' cap structure, and independent of the 5' end of the mRNA.
  • An IRES sequence provides necessary exacting sequences required for initiation of translation of an operably linked coding region.
  • isolated is meant to describe a compound of interest (e.g., a recombinant virus, a nucleic acid molecule such as a genome, a polypeptide, etc.) that is in an environment different from that in which the compound naturally occurs. "Isolated” is meant to include compounds that are within samples that are substantially enriched for the compound of interest and or in which the compound of interest is partially or substantially purified.
  • a compound of interest e.g., a recombinant virus, a nucleic acid molecule such as a genome, a polypeptide, etc.
  • operably connected or “operably linked” as used herein refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a transcriptional control sequence "operably linked" to a coding sequence refers to positioning and/or orientation of the transcriptional control sequence relative to the coding sequence to permit expression of the coding sequence under conditions compatible with the transcriptional control sequence.
  • "operably connecting" a flaviviridae virus coding sequence to a transgene encompasses positioning and/or orientation of the transgene relative to the flaviviridae virus coding sequence so that (1) the flaviviridae virus coding sequence and the transgene are transcribed together to form a single chimeric transcript and optionally (2) if the transgene itself comprises a coding sequence, the coding sequence of the transgene is 'in-frame' with the flaviviridae virus coding sequence to produce a chimeric open reading frame comprising the transgene coding sequence and the flaviviridae virus coding sequence.
  • an IRES operably connected to a flaviviridae virus coding sequence refers to positioning and/or orientation of the IRES relative to the flaviviridae virus coding sequence to permit cap-independent translation of the fiaviviridae virus coding sequence.
  • open reading frame and “ORF” refer to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence.
  • initiation codon and “termination codon” refer to a unit of three adjacent nucleotides ('codon') in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation).
  • parent virus will be understood to be a reference to a virus that is modified to incorporate heterologous genetic material to form a recombinant virus of the present invention.
  • polynucleotide designate mRNA, RNA, cRNA, cDNA or DNA.
  • the term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide.
  • the term includes single and double stranded forms of RNA or DNA.
  • Polypeptide “peptide,” “protein” and “proteinaceous molecule” are used interchangeably herein to refer to molecules comprising or consisting of a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers.
  • complementing or interactive polypeptides comprising different parts or portions (e.g., polypeptide domains, polypeptide chains etc.) of a luciferase polypeptide of the present invention, wherein the individual complementing polypeptides together reconstitute the activity of the different parts or portions to form a functional luciferase polypeptide.
  • complementing polypeptides are used routinely in protein complementation assays, which are well known to persons skilled in the art.
  • the term "recombinant polynucleotide” as used herein refers to a polynucleotide formed in vitro by the manipulation of nucleic acid into a form not normally found in nature.
  • the recombinant polynucleotide may be in the form of an expression vector.
  • expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleotide sequence.
  • recombinant polypeptide is meant a polypeptide made using recombinant techniques, i.e., through the expression of a recombinant polynucleotide.
  • the term "recombinant virus” will be understood to be a reference to a “parent virus” comprising at least one heterologous nucleic acid sequence.
  • sequence identity refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a "percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base ⁇ e.g., A, T, C, G, I) or the identical amino acid residue ⁇ e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison ⁇ i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base ⁇ e.g., A, T, C, G, I
  • the identical amino acid residue ⁇ e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys
  • sequence identity will be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software.
  • Similarity refers to the percentage number of amino acids that are identical or constitute conservative substitutions as defined in Table A below. Similarity may be determined using sequence comparison programs such as GAP (Deveraux et al. 1984, Nucleic Acids ResearchYl: 387-395). In this way, sequences of a similar or substantially different length to those cited herein might be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP. TABLE A: EXEMPLARY CONSERVATIVE AMINO ACID SUBSTITUTIONS
  • references to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence,” “comparison window”, “sequence identity,” “percentage of sequence identity” and “substantial identity”.
  • a “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length.
  • two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • the comparison window may comprise additions or deletions (i.e. , gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • GAP Garnier et al
  • BESTFIT Pearson FASTA
  • FASTA Pearson's Alignment of sequences
  • TFASTA Pearson's Alignin Altschul et al, Nucl. Acids jRes.25:3389.
  • a detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al., "Current Protocols in Molecular Biology", John Wiley & Sons Inc, 1994-1998, Chapter 15.
  • Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates (e.g., humans, monkeys and apes, and includes species of monkeys such from the genus Macaca (e.g., cynomologus monkeys such as Macaca fascicularis, and/or rhesus monkeys (Macaca mulatto)) and baboon (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes such as chimpanzees (Pan troglodytes)), rodents (e.g., rodents, rodents, rodents (e.g., rodents, rodents, rodents, e.g., rodents (
  • transgene is used herein to describe genetic material that has been or is about to be artificially introduced into a genome of a host organism and that is transmitted to the progeny of that host.
  • the transgene will typically comprise a polynucleotide that is capable of being transcribed into RNA and optionally, translated and/or expressed under appropriate conditions. In some embodiments, it confers a desired property to the Flaviviridae virus (e.g. , attenuation) into which it is introduced, or otherwise leads to a desired therapeutic or diagnostic outcome.
  • Flaviviridae virus e.g. , attenuation
  • the transgene is transcribed into a molecule that interferes with transcription or translation (e.g., antisense molecule) or mediates RNA interference (e.g., siRNA or shRNA).
  • the transgene comprises a plurality of coding sequences, which in illustrative examples encode the same exogenous polypeptide or different exogenous polypeptides.
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof arid/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e. , causing regression of the disease.
  • 5' untranslated region or “5' UTR” refers to a sequence located 3' to promoter region and 5' of the downstream coding region. Thus, such a sequence, while transcribed, is upstream (i.e., 5') of the translation initiation codon and therefore is generally not translated into a portion of the polypeptide product.
  • 3' untranslated region refers to a nucleotide sequences downstream (i.e. , 3') of a coding sequence. It extends from the first nucleotide after the stop codon of a coding sequence to just before the poly(A) tail of the corresponding transcribed mRNA.
  • the 3' UTR may contain sequences that regulate translation efficiency, mRNA stability, mRNA targeting and/or polyadenylation.
  • the present invention provides improved recombinant viruses with enhanced stability.
  • maintaining the genetic stability of foreign or heterologous gene insert(s) in the genomes of those viruses over multiple virus replication cycles in a host is still problematic.
  • inadequate attenuation and potential reversion to pathogenic phenotype also remain a concern.
  • Production of recombinant Flavivmdae viruses in accordance with the present invention can provide a means of enhancing the genetic stability of foreign or heterologous genetic material incorporated into the virus and/or prevent (or substantially prevent) reversion of the virus to a pathogenic state upon replication.
  • the present invention provides recombinant Flaviviridae viruses comprising a bicistronic genome.
  • the bicistronic genome comprises a flaviviridae virus open reading frame (ORF) and an heterologous nucleic acid sequence inserted between adjacent first and second coding sequences, of the ORF, wherein the coding sequences encode different flaviviridae virus proteins.
  • the heterologous nucleic acid sequence comprises a transgene and an internal ribosome entry site (IRES), wherein the transgene is operably connected downstream of the first coding sequence and the IRES is operably connected upstream of the second coding sequence.
  • IRES internal ribosome entry site
  • the first and second coding sequences together suitably comprise all the subunit proteins of a polyprotein encoded by the flaviviridae virus ORF.
  • the subunit proteins include for example, the amino terminal structural proteins C, prM and E proteins and the carboxyl- terminal non-structural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5.
  • the recombinant flaviviridae virus genomes of the invention have improved stability over multiple replication cycles and are thus useful for eliciting stronger immune responses to flaviviridae virus protein and optionally a heterologous protein encoded by the transgene.
  • the transgene encodes a cytokine, which provides a means of attenuating virulence (i.e., addressing safety concerns) and/or augmenting immunity against a Flaviviridae virus.
  • the recombinant flaviviridae virus genome comprises, in order from 5' to 3', a flaviviridae virus 5' untranslated region (UTR), the first coding sequence, the heterologous nucleic acid sequence, the second coding sequence and a flaviviridae virus 3' UTR.
  • the recombinant viruses are live attenuated recombinant viruses.
  • the recombinant viruses are replication-competent meaning that they are capable of reproducing in a host cell that they have infected.
  • Recombinant viruses of the present invention may be produced by genetic modification of a "parent" virus.
  • the parent virus is modified to incorporate foreign or exogenous genetic material in the form of a transgene to produce the recombinant virus.
  • reference herein to a specific type of recombinant virus e.g., a "recombinant Flaviviridae virus” or a “recombinant Flavivirus” denotes a parent virus of the indicated type that has been modified to incorporate foreign or exogenous genetic material.
  • a recombinant virus in accordance with the present invention may be a single-stranded RNA (positive-sense) virus classified under "Group I V" of the Baltimore Classification System (BCS).
  • BCS Baltimore Classification System
  • the recombinant virus may be a recombinant Flaviviridae family virus (also referred to herein as a "recombinant Flaviviridae virus") as classified under the BCS and/or the International Committee on Taxonomy of Viruses (ICTV).
  • the recombinant virus may be a recombinant Flavivirus, Pestivirus, or Hepacivirus.
  • the recombinant virus is a recombinant virus of the Flavivirus genus.
  • suitable recombinant flaviviruses include the following: Alfuy virus, Aroa virus, Bagaza virus, Banzi virus, Bouboui virus, Bussuquara virus, Cacipacore virus, Dengue virus (e.g.. Dengue virus serotype I, Dengue virus serotype II, Dengue virus serotype III, Dengue virus serotype IV), Edge Hill virus, Gadgets Gully virus, Kadam virus, Kunjin virus, Kokobera virus, Kyasanur Forest disease virus, Iguape virus, Ilheus virus, Israel Turkey
  • meningoencephalomyelitis virus Japanese encephalitis virus, Jugra virus, Kedougou virus, Koutango virus, Langat virus, Louping ill virus, Meaban virus, Modoc virus, MVEV, Naranjal virus, Negeishi virus, Ntaya virus, Omsk hemorrhagic fever virus, Potiskum virus, Powassan virus, Rio Bravo virus, Rocio virus, Russian spring summer encephalitis virus, Royal Farm virus, Saboya virus, Saumarez Reef virus, Sepik virus, St.
  • the recombinant Flavivirus is a recombinant Dengue virus.
  • the Dengue virus may be a serotype I, II, III, or IV serotype virus or a recombinant form thereof.
  • the , recombinant Flavivirus is a recombinant MVEV, a recombinant West Nile virus, a recombinant Japanese encephalitis virus, or a recombinant Yellow fever virus.
  • Recombinant viruses of the present invention comprise at least one heterologous nucleic acid sequence, which comprises a transgene and an IRES.
  • a transgene as used herein encompasses any nucleotide sequence inserted into the genome of the parent virus to form a recombinant virus.
  • An transgene may therefore include a sequence that is identical to a sequence in the genome of the parent virus, or, a sequence that differs from sequences in the genome of the parent virus.
  • the transgene comprises a nucleotide sequence from a Flaviviridae virus that is different to the parent virus including, but not limited to, different viral strains; different viral serotypes; and viruses from a different species and/or genus.
  • the transgene comprises a nucleotide sequence corresponding to a nucleotide sequence of an organism other than a Flaviviridae virus or encodes a peptide or polypeptide corresponding to a peptide or, polypeptide of an organism other than a Flaviviridae virus.
  • the transgene may encode an exogenous transcript product that interferes with transcription or translation (e.g., antisense molecule) or mediates RNA interference (e.g., siR A or shRNA).
  • the transgene may encode an exogenous polypeptide product.
  • Exogenous polypeptides include polypeptides from any of a variety of pathogenic organisms, including, but not limited to, viruses, bacteria, yeast, fungi, and protozoa; cancer- or tumor-associated antigens; "self antigens (i. e. , autoantigens); foreign antigens (e.g., alloantigens and allergens) from other than pathogenic organisms; proteins that have a therapeutic activity; and the like.
  • any nucleic acid molecule comprising a nucleotide sequence which encodes a polypeptide which, when produced by a cell infected by a recombinant Flaviviridae virus of the invention, increases an immune response is suitable for use in the present invention.
  • Nucleic acid sequences encoding one or more exogenous polypeptides (e.g., antigens or epitopes) of interest can be included in a recombinant bicistronic Flaviviridae virus as defined herein.
  • exogenous antigen or epitope of interest can be antigens or epitopes of a single pathogen or antigens or epitopes from more than one (different) pathogen.
  • an organism is a pathogenic microorganism.
  • exogenous epitope may be found on bacteria, parasites, viruses, yeast, or fungi that are the causative agents of diseases or disorders.
  • the antigen is an allergen.
  • the antigen is a cancer- or tumor-associated antigen.
  • Retroviridae e.g. , human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV -III); and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, Hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviviridae (e.g. , dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses);
  • Retroviridae e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV,
  • Rhabdoviridae e.g., vesicular stomatitis viruses, rabies viruses
  • Filoviridae e.g., ebola viruses
  • Paramyxoviridae e.g. , parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus
  • Orthomyxoviridae e.g., influenza viruses
  • Arenaviridae hemorrhagic fever viruses
  • Reoviridae e.g. , reoviruses, orbiviurses and rotaviruses
  • Bimaviridae Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses);
  • Papovaviridae papilloma viruses, polyoma viruses
  • Adenoviridae most
  • adenoviruses Herpesviridae (Herpes simplex virus (HSV) 1 and 2, Varicella zoster virus, Cytomegalovirus (CMV), herpes viruses); Poxyiridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swine fever virus); Hepatitis C virus; and unclassified viruses (e.g., the agent of delta hepatitis (thought to be a defective satellite of Hepatitis B virus); Norwalk and related viruses, and astroviruses).
  • HSV Herpesviridae
  • CMV Cytomegalovirus
  • Iridoviridae e.g., African swine fever virus
  • Hepatitis C virus Hepatitis C virus
  • unclassified viruses e.g., the agent of delta hepatitis (thought to be a defective satellite of Hepatitis B virus); Norwalk and related viruses, and
  • Pathogenic bacteria include, but are not limited to, Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophila, Mycobacteria sps (e.g. , M. tuberculosis, M. avium, M. intracellulars M. kansaii, M.
  • Streptococcus pyrogenes Group A Streptococcus
  • Streptococcus agalactiae Group B Streptococcus
  • Streptococcus viridans group
  • Streptococcus faecalis Streptococcus bovis
  • Streptococcus anaerobic sps.
  • Streptococcus pneumoniae pathogenic
  • Campylobacter sp. Enterococcus sp., Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, pathogenic strains of Escherichia coli, Streptobacillus moniliformis, Treponema pallidium, Treponema peramba, Leptospira, and Actinomyces israelii.
  • Infectious fungi include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis and Candida albicans.
  • Infectious protozoa include, but are not limited to, Plasmodium spp., e.g., Plasmodium falciparum; trypanosomes, e.g., Trypanosoma cruzi; and Toxoplasma gondii.
  • Allergens include, but are not limited to, pollens, insect venoms, animal dander dust, fungal spores and drugs (e.g., penicillin).
  • natural, animal and plant allergens include proteins specific to the following genera: Canine (Canis familiaris); Dermatophagoides (e.g., Dermatophagoides farinae); Felis (Felis domesticus); Ambrosia (Ambrosia artemiisfolia; Lolium (e.g., Lolium perenne or Lolium multiflorum); Cryptomeria (Cryptomeria japonica); Altemaria (Alternaria alternata); Alder; Alnus (Alnus gultinosa); Betula (Betula verrucosa); Quercus
  • Juniperus e.g., Juniperus sabinoides, Juniperus virginiana, Juniperus communis and Juniperus ashei
  • Thuya e.g., Thuya orientalis
  • Chamaecyparis e.g., Charnaecyparis obtusa
  • Periplaneta e.g., Periplaneta americand
  • Agropyron e.g, Agropyron repens
  • Secale e.g., Secale cereale
  • Triticum e.g., Triticum aestivum
  • Dactylis e.g., Dactylis glomerata
  • Festuca e.g., Festuca elatior
  • Poa e.g., Poa pratensis or Poa compressa
  • Avena e.g., Avena sativa
  • Avena e.g., Avena sativa
  • Anthoxanthum odoratum Arrhenatherum (e.g., Arrhenatherum elatius); Agrostis(e.g., Agrostis alba); Phleum (e.g., Phleum pratense); Phalaris (e.g., Phalaris arundinacea); Paspalum (e.g. , Paspalum notatum); Sorghum (e.g. , Sorghum halepensis); and Bromus (e.g., Bromus inermis).
  • any of a variety of known cancer- or tumor-associated antigens can be inserted into Flaviviridae virus of the invention.
  • the entire antigens may be, but need not be, inserted. Instead, a portion of a cancer- or tumor-associated antigen, e.g., an epitope, particularly an epitope that is recognized by a CTL, may be inserted.
  • Tumor- associated antigens (or epitope-containing fragments thereof) which may be inserted into Flaviviridae virus include, but are not limited to, MAGE-2, MAGE-3, MUC-1, MUC-2, HER-2, high molecular weight melanoma-associated antigen MAA, GD2, earcinoembryonic antigen (CEA), TAG-72, ovarian-associated antigens OV-TL3 and MOV 18, TUAN, alpha-feto protein (AFP), OFP, CA-125, CA-50, CA-19-9, renal tumor-associated antigen G250, EGP-40 (also known as EpCAM), SI 00 (malignant melanoma-associated antigen), p53, prostate tumor-associated antigens (e.g., PSA and PSMA), and p2 Iras.
  • MAGE-2 MAGE-3
  • MUC-1 high molecular weight melanoma-associated antigen MAA
  • GD2 high molecular weight melanoma-
  • antigens of interest include, but are not limited to, sperm- associated antigens, venoms, hormones, and the like.
  • Sperm-associated proteins are known in the art, and a nucleic acid molecule encoding any such protein is suitable for use herein. See, e.g., Primakoff (1994) Reproductive Immunol. 31 :208-210; Naz et al.
  • Hormones of interest include, but are not limited to, human chorionic gonadotrophin (hCG). Hormones such as hCG are useful to elicit specific antibodies, for use as contraceptive.
  • Venoms of interest include those from any poisonous animal, e.g., snake venoms, including, but not limited to, a-neurotoxins, kappa toxins, ⁇ -neurotoxins, dendrotoxins, cardiotoxins, myotoxins, and hemorrhaging.
  • modified venoms that elicit specific antibodies, but are not themselves toxic.
  • modified venoms are useful to elicit an immune response to a venom, and in many embodiments, elicit a protective immune response such that, upon subsequent exposure to the venom from an animal source, any adverse physiological effects of the venom are mitigated.
  • a "therapeutic protein” includes a protein that the host does not produce but is in need of; a protein that the host does not normally produce, but which has a therapeutic activity; a protein that the host produces, but produces in inadequate amounts; a protein that the host produces but in a form which is inactive, or which has reduced activity compared with an activity normally associated with the protein; or a protein that the host produces in adequate amounts and with normal activity associated with that protein.
  • Therapeutic proteins include naturally-occurring proteins, and recombinant proteins whose amino acid sequences differ from a naturally-occurring counterpart protein, which recombinant proteins have substantially the same, an altered activity, or enhanced activity relative to a naturally-occurring protein.
  • Proteins that have therapeutic activity include, but are not limited to, cytokines, including, but not limited to, interleukins, endothelin, colony stimulating factors, tumor necrosis factor, and interferons; hormones, including, but not limited to, a growth hormone, insulin; growth factors, including, but not limited to human growth factor, insulin-like growth factor; bioactive peptides; trophins; neurotrophins; soluble forms of a membrane protein including, but not limited to, soluble CD4; enzymes; regulatory proteins; structural proteins; clotting factors, including, but not limited to, factor XIII; erythropoietin; tissue plasminogen activator; etc.
  • cytokines including, but not limited to, interleukins, endothelin, colony stimulating factors, tumor necrosis factor, and interferons
  • hormones including, but not limited to, a growth hormone, insulin
  • growth factors including, but not limited to human growth factor, insulin-like growth factor
  • the exogenous polypeptide is a cytokine, which according to the present invention also encompasses a chemokine.
  • the cytokine is identical or substantially identical to a cytokine produced in a subject to which the recombinant virus is administered.
  • the cytokine is suitably one that is associated with antiviral immune responses in the host organism.
  • suitable cytokines include interferons, tumor necrosis factor-alpha (TNF-a), alpha defensins, RANTES (CCL5), CXCL10 (IP10) and the like.
  • the transgene may comprise a plurality of cytokine-encoding nucleic acid sequences. This includes duplicate(s) of a nucleic acid sequence encoding a specific cytokine and/or combinations of different nucleic acid sequences encoding different cytokines.
  • the cytokine expressed by the recombinant virus may be sufficient to reduce the virulence (i.e., degree of pathogenicity) of the virus such that potentially adverse effects are avoided in a subject to which the virus is administered.
  • the virulence of a recombinant virus of the present invention may be assessed using a number of methods known in the art.
  • the virulence of a given recombinant virus may be assessed using cell culture-based assays, animal models (e.g., mouse, rat, hamster, primate) and/or assessing the monitoring subjects(s) to which the virus has been administered.
  • the cytokine is an interferon.
  • the interferon is a type-1 interferon.
  • the interferon may be a mammalian type-1 interferon (e.g. , interferon-alpha (IFN-a), interferon-beta (IFN- ⁇ ), interferon-kappa (IFN- ⁇ ), interferon-delta (IFN-6), interferon- epsilon (IFN- ⁇ ), interferon-tau (IFN- ⁇ ), interferon-omega (IFN- ⁇ ), or interferon-zeta (IFN-O).
  • IFN-a interferon-alpha
  • IFN- ⁇ interferon-beta
  • IFN- ⁇ interferon-kappa
  • IFN-6 interferon-delta
  • IFN-6 interferon- epsilon
  • IFN- ⁇ interferon-tau
  • IFN- ⁇ interferon-omega
  • IFN-O interferon
  • the cytokine is interferon-beta (IFN- ⁇ ).
  • the cytokine is mammalian interferon-beta (IFN- ⁇ ), and more suitably human interferon-beta (IFN- ⁇ ).
  • the human interferon-beta (IFN- ⁇ ) may be defined by the amino acid sequence set forth in GenBank accession number AAC41702.1.
  • a coding sequence of the transgene may comprise at least one nucleotide sequence encoding a proteolytic cleavage site.
  • the proteolytic cleavage site may be advantageous in facilitating cleavage and release of the encoded polypeptide from other viral-encoded polypeptides. Suitable sequences encoding proteolytic cleavage sites and methods for their incorporation into other sequences are well known in the art and described in standard texts.
  • the proteolytic cleavage site is at the 5' end of the transgene coding sequence.
  • the nucleotide sequence comprises a proteolytic cleavage site at its 5' end and its 3' end.
  • proteolytic cleavage sites may be positioned to facilitate release of the encoded exogenous polypeptide upon proteol ytic processing of a recombinant viral polyprotein precursor comprising the encoded exogenous polypeptide.
  • a coding sequence of the transgene comprises a nucleotide sequence encoding a signal peptide for directing transport of an exogenous polypeptide within a host cell (e.g. , to the endoplasmic reticulum) and/or to the cell exterior.
  • nucleic acid molecule encoding the exogenous protein to be produced by a host cell following infection of the host cell by the recombinant
  • Flaviviridae virus to the present invention can be obtained by techniques known in the art, including but not limited to, chemical or enzymatic synthesis, purification from genomic DNA of the microorganism, by purification or isolation from a cDNA encoding the exogenous protein, by cDNA synthesis from RN A of an organism, or by standard recombinant methods (Sambrook et al., (1989), "Molecular Cloning: A Laboratory ManuaT (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York; and Ausubel et al (Eds), (2000-2010), "Current Protocols in Molecular
  • nucleotide sequences encoding many of the above-listed exogenous proteins are publicly available. Variant of such sequences can readily be generated by those skilled in the art using standard recombinant methods, including site-directed and random mutagenesis.
  • the nucleic acid molecule encoding the exogenous polypepti de can further include sequences that direct secretion of the protein from the cell, sequences that alter RNA and/or protein stability, and the like.
  • IRES sequences can be used in the recombinant bicistronic Flaviviridae virus of the invention.
  • the IRES may be an IRES from the parent Flaviviridae virus or from a different organism or, an IRES from the same virus such that the recombinant virus genome comprises at least one extra copy of that IRES compared to the parent virus.
  • IRES sequences are known in the art and include, but are not limited to, IRES sequences derived from viruses such as Mengovirus, Bovine viral diarrhea virus (BVDV), Encephalomyocarditis virus (EMCV), Hepatitis C virus (HCV; e.g., nucleotides 1202-1812 of the nucleotide sequence provided under GenBank Accession number AJ242654), picornaviruses such as Poliovirus,
  • Encephalomyocarditis virus, and Foot and mouth disease virus e.g. , nucleotide numbers 600-1058 of the nucleotide sequence provided under GenBank Accession No. AF308157
  • cellular mRNAs such as those encoding GTX, Cyr61a, Cyr61b, the immunoglobulin heavy-chain-binding protein (BiP), and the immunoglobulin heavy chain.
  • IRES sequences such as those reported in WO 96/01324; WO 98/49334; WO 00/44896; and U.S. Pat. No. 6,171,821 can be used in the recombinant flaviviruses of the invention.
  • Mutants, variants and derivatives of naturally occurring IRES sequences may be employed in the present invention provided they retain the ability to initiate translation of an operably linked coding sequence located 3' of the IRES.
  • An IRES sequence suitable for use in the present invention has at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or more, nucleotide sequence identity with a naturally occurring IRES.
  • An IRES sequence suitable for use in the present invention may also be a fragment of a naturally occurring IRES, provided the fragment functions to allow ribosome attachment and initiate translation of an operably linked 3' coding region.
  • heterologous nucleic acid sequence in some embodiments the sequence may be between about 10 to 15, 15 to 25, 20 to 30, 25 to 35, 30 to 40, 35 to 45, 40 to 50, 50 to 75, 75 to 100, 100 to 150, 150 to 200, 200 to 275, 275 to 350, 350 to 500, 500 to 750, 750 to 1000, 1000 to 1250, 1250 to 1500, 1500 to 1750, 1750 to 2000, 2000 to 2500, 2500 to 3000, or more than 3000 nucleotides in length.
  • the heterologous nucleic acid sequence is inserted into an open reading frame (ORF) of the virus.
  • ORF open reading frame
  • the nucleotide sequence may be inserted at a junction between two viral coding sequence, which code two different viral proteins.
  • the heterologous nucleic acid sequence is inserted between the coding sequence for C protein and the coding sequence for prM protein; or the coding sequence for prM protein and the coding sequence for E protein; or the coding sequence for E protein and the coding sequence for NS1 protein; or the coding sequence for NS1 protein and the coding sequence for NS2a protein; or the coding sequence for NS2a protein and the coding sequence for NS2b protein; or the coding sequence for NS2b protein and the coding sequence for NS3 protein; or the coding sequence for NS3 protein and the coding sequence for NS4a protein; or the coding sequence for NS4a protein and the coding sequence for NS4b protein; or the coding sequence for NS4b protein and the coding sequence for NS5 protein.
  • a Flavivir us comprising a recombinant genome, which comprises an heterologous nucleic acid sequence comprising: a transgene that comprises a coding sequence for an exogenous polypeptide (e.g., a cytokine such as a mammalian interferon-beta (IFN- ⁇ ); and an exogenous IRES (e.g., an EMCV IRES).
  • the parent virus may be, for example, a Dengue virus, Japanese encephalitis virus, Yellow fever virus, MVEV, West Nile virus, or St Louis encephalitis virus.
  • the heterologous nucleic acid sequence is inserted into the parent virus ORF, suitably between the coding sequence for the C protein and the coding sequence for the prM protein.
  • the coding sequence of the transgene is inserted downstream (3') (suitably immediately
  • the exogenous polypeptide will generally include a proteolytic cleavage site for cleavage of the exogenous polypeptide from the C protein by host or viral peptidases. If desired, the exogenous polypeptide may include a signal peptide for directing the exogenous polypeptide to the endoplasmic reticulum for luminal translocation.
  • the IRES is generally positioned downstream (3') of the stop codon of the chimeric coding sequence and upstream (5') of the coding sequence for the prM protein. Further, the sequence may comprise mutations inserted to avoid direct repetition with the natural prM signal peptide sequence, reducing the possibility of deletion due to homologous recombination (while maintaining the correct amino acid sequence required for processing). This may in turn reduce the loss of the heterologous nucleic acid sequence during viral replication.
  • a Flavivirus comprising a recombinant genome, which comprises an heterologous nucleic acid sequence comprising: a transgene that comprises a coding sequence for an exogenous polypeptide (e.g., a cytokine such as a mammalian interferon-beta (IFN- ⁇ ); and an exogenous IRES (e.g. , an EMCV IRES).
  • the parent virus may be, for example, a Dengue virus, Japanese encephalitis virus, Yellow fever virus, MVEV, West Nile virus, or St Louis encephalitis virus.
  • the heterologous nucleic acid sequence is inserted into the parent virus ORF, suitably between the coding sequence for the prM protein and the coding sequence for the E protein.
  • the coding sequence of the transgene is inserted downstream (3') (suitably immediately downstream) and in-frame with the coding sequence for the prM protein of the parent virus to produce a chimeric coding sequence, such that translation of the chimeric coding sequence is 5' cap- dependent with production of a polyprotein comprising the viral C and prM proteins and the exogenous polypeptide.
  • the exogenous polypeptide will generally include a proteolytic cleavage site for cleavage of the exogenous polypeptide from the viral proteins of the polyprotein by host or viral peptidases.
  • the exogenous polypeptide may include a signal peptide for directing the exogenous polypeptide to the endoplasmic reticulum for luminal translocation.
  • the IRES is generally positioned downstream (3') of the stop codon of the chimeric coding sequence and upstream (5') of the coding sequence for the E protein.
  • a Flavivirus comprising a recombinant genome, which comprises an heterologous nucleic acid sequence comprising: a transgene that comprises a coding sequence for an exogenous polypeptide (e.g., a cytokine such as a mammalian interferon-beta (IFN- ⁇ ); and an exogenous IRES (e.g., an EMCV IRES).
  • the parent virus may be, for example, a Dengue virus, Japanese encephalitis virus, Yellow fever virus, MVEV, West Nile virus, or St Louis encephalitis virus.
  • the heterologous nucleic acid sequence is inserted into the parent virus ORF, suitably between the coding sequence for the E protein and the coding sequence for the NS1 protein.
  • the coding sequence of the transgene is inserted downstream (3') (suitably immediately downstream) and in-frame with the coding sequence for the E protein of the parent virus to produce a chimeric coding sequence, such that translation of the chimeric coding sequence is 5' cap- dependent with production of a polyprotein comprising the viral C, prM and E proteins and the exogenous polypeptide.
  • the exogenous polypeptide will generally include a proteolytic cleavage site for cleavage of the exogenous polypeptide from the viral proteins of the polyprotein by host or viral peptidases. If desired, the exogenous polypeptide may include a signal peptide for directing the exogenous polypeptide to the endoplasmic reticulum for luminal translocation.
  • the IRES is generally positioned downstream (3') of the stop codon of the chimeric coding sequence and upstream (5') of the coding sequence for the NS1 protein.
  • a Flavivirus comprising a recombinant genome, which comprises an heterologous nucleic acid sequence comprising: a transgene that comprises a coding sequence for an exogenous polypeptide (e.g. , a cytokine such as a mammalian interferon-beta (IFN- ⁇ ); and an exogenous IRES (e.g., an EMCV IRES).
  • the parent virus may be, for example, a Dengue virus, Japanese encephalitis virus, Yellow fever virus, MVEV, West Nile virus, or St Louis encephalitis virus.
  • the heterologous nucleic acid sequence is inserted into the parent virus ORF, suitably between the coding sequence for the NS 1 protein and the coding sequence for the NS2A protein.
  • the coding sequence of the transgene is inserted downstream (3') (suitably immediately downstream) and in-frame with the coding sequence for the NS1 protein of the parent virus to produce a chimeric coding sequence, such that translation of the chimeric coding sequence is 5' cap- dependent with production of a polyprotein comprising the viral C, prM, E and NS 1 proteins and the exogenous polypeptide.
  • the exogenous polypeptide will generally include a proteolytic cleavage site for cleavage of the exogenous polypeptide from the viral proteins of the polyprotein by host or viral peptidases. If desired, the exogenous polypeptide may include a signal peptide for directing the exogenous polypeptide to the ' endoplasmic reticulum for luminal translocation.
  • the IRES is generally positioned downstream (3') of the stop codon of the chimeric coding sequence and upstream (5') of the coding sequence for the NS2A protein.
  • a Flavivirus comprising a recombinant genome, which comprises an heterologous nucleic acid sequence comprising: a transgene that comprises a coding sequence for an exogenous polypeptide (e.g., a cytokine such as a mammalian interferon-beta (IFN- ⁇ ); and an exogenous IRES (e.g., an EMCV IRES).
  • the parent virus may be, for example, a Dengue virus, Japanese encephalitis virus, Yellow fever virus, MVEV, West Nile virus, or St Louis encephalitis virus.
  • the heterologous nucleic acid sequence is inserted into the parent virus ORF, suitably between the coding sequence for the NS2A protein and the coding sequence for the NS2B protein.
  • the coding sequence of the transgene is inserted downstream (3') (suitably immediately downstream) and in-frame with the coding sequence for the NS2A protein of the parent virus to produce a chimeric coding sequence, such that translation of the chimeric coding sequence is 5' cap- dependent with production of a polyprotein comprising the viral C, prM, E, NS1 and NS2A proteins and the exogenous polypeptide.
  • the exogenous polypeptide will generally include a proteolytic cleavage site for cleavage of the exogenous polypeptide from the viral proteins of the polyprotein by host or viral peptidases. If desired, the exogenous polypeptide may include a signal peptide for directing the exogenous polypeptide to the endoplasmic reticulum for luminal translocation.
  • the IRES is generally positioned downstream (3') of the stop codon of the chimeric coding sequence and upstream (5') of the coding sequence for the NS2B protein.
  • a Flavivirus comprising a recombinant genome, which comprises an heterologous nucleic acid sequence comprising: a transgene that comprises a coding sequence for an exogenous polypeptide (e.g., a cytokine such as a mammalian interferon-beta (IFN- ⁇ ); and an exogenous IRES (e.g. , an EMCV IRES).
  • the parent virus may be, for example, a Dengue virus, Japanese encephalitis virus, Yellow fever virus, MVEV, West Nile virus, or St Louis encephalitis virus.
  • the heterologous nucleic acid sequence is inserted into the parent virus ORP, suitably between the coding sequence for the NS2B protein and the coding sequence for the NS3 protein.
  • the coding sequence of the transgene is inserted downstream (3') (suitably immediately downstream) and in-frame with the coding sequence for the NS2B protein of the parent virus to produce a chimeric coding sequence, such that translation of the chimeric coding sequence is 5' cap- dependent with production of a polyprotein comprising the viral C, prM, E, NS1, NS2A and NS2B proteins and the exogenous polypeptide.
  • the exogenous polypeptide will generally include a proteolytic cleavage site for cleavage of the exogenous polypeptide from the viral proteins of the polyprotein by host or viral peptidases. If desired, the exogenous polypeptide may include a signal peptide for directing the exogenous polypeptide to the endoplasmic reticulum for luminal translocation.
  • the IRES is generally positioned downstream (3') of the stop codon of the chimeric coding sequence and upstream (5') of the coding sequence for the NS3 protein.
  • a Flavivirus comprising a recombinant genome, which comprises an heterologous nucleic acid sequence comprising: a transgene that comprises a coding sequence for an exogenous polypeptide (e.g., a cytokine such as a mammalian interferon-beta (IFN- ⁇ ); and an exogenous IRES (e.g., an EMCV IRES).
  • the parent virus may be, for example, a Dengue virus, Japanese encephalitis virus, Yellow fever virus, MVEV, West Nile virus, or St Louis encephalitis virus.
  • the heterologous nucleic acid sequence is inserted into the parent virus ORF, suitably between the coding sequence for the NS3 protein and the coding sequence for the NS4A protein.
  • the coding sequence of the transgene is inserted downstream (3') (suitably immediately downstream) and in-frame with the coding sequence for the NS3 protein of the parent virus to produce a chimeric coding sequence, such that translation of the chimeric coding sequence is 5' cap- dependent with production of a polyprotein comprising the viral C, prM, E, NS1 , NS2A, NS2B and NS3 proteins and the exogenous polypeptide.
  • the exogenous polypeptide will generally include a proteolytic cleavage site for cleavage of the exogenous polypeptide from the viral protei ns of the polyprotein by host or viral peptidases. If desired, the exogenous polypeptide may include a signal peptide for directing the exogenous polypeptide to the endoplasmic reticulum for luminal translocation.
  • the IRES is generally positioned downstream (3') of the stop codon of the chimeric coding sequence and upstream (5') of the coding sequence for the NS4A protein.
  • a Flavivirus comprising a recombinant genome, which comprises an heterologous nucleic acid sequence comprising: a transgene that comprises a coding sequence for an exogenous polypeptide (e.g., a cytokine such as a mammalian interferon-beta (IFN- ⁇ ); and an exogenous IRES (e.g., an EMCV IRES).
  • the parent virus may be, for example, a Dengue virus, Japanese encephalitis virus, Yellow fever virus, MVEV, West Nile virus, or St Louis encephalitis virus.
  • the heterologous nucleic acid sequence is inserted into the parent virus ORF, suitably between the coding sequence for the NS4A protein and the coding sequence for the NS4B protein.
  • the coding sequence of the transgene is inserted downstream (3') (suitably immediately downstream) and in-frame with the coding sequence for the NS4A protein of the parent virus to produce a chimeric coding sequence, such that translation of the chimeric coding sequence is 5' cap- dependent with production of a polyprotein comprising the viral C, prM, E, NS1 , NS2A, NS2B, NS3 and NS4A proteins and the exogenous polypeptide.
  • the exogenous polypeptide will generally include a proteolytic cleavage site for cleavage of the exogenous polypeptide from the viral proteins of the polyprotein by host pr viral peptidases. If desired, the exogenous polypeptide may include a signal peptide for directing the exogenous polypeptide to the endoplasmic reticulum for luminal translocation.
  • the IRES is generally positioned downstream (3') of the stop codon of the chimeric coding sequence and upstream (5') of the coding sequence for the NS4B protein.
  • a Flavivirus comprising a recombinant genome, which comprises an heterologous nucleic acid sequence comprising: a transgene that comprises a coding sequence for an exogenous polypeptide (e.g., a cytokine such as a mammalian interferon-beta (IFN- ⁇ ); and an exogenous IRES (e.g., an EMCV IRES).
  • the parent virus may be, for example, a Dengue virus, Japanese encephalitis virus, Yellow fever virus, MVEV, West Nile virus, or St Louis encephalitis virus.
  • the heterologous nucleic acid sequence is inserted into the parent virus ORF, suitably between the coding sequence for the NS4B protein and the coding sequence for the NS5 protein.
  • the coding sequence of the transgene is inserted downstream (3') (suitably immediately downstream) and in-frame with the coding sequence for the NS5 protein of the parent virus to produce a chimeric coding sequence, such that translation of the chimeric coding sequence is 5' cap- dependent with production of a polyprotein comprising the viral C, prM, E, NS 1 , NS2A, NS2B, NS3, NS4A and NS4B proteins and the exogenous polypeptide.
  • the exogenous polypeptide will generally include a proteolytic cleavage site for cleavage of the exogenous polypeptide from the viral proteins of the polyprotein by host or viral peptidases. If desired, the exogenous polypeptide may include a signal peptide for directing the exogenous polypeptide to the endoplasmic reticulum for luminal translocation.
  • the IRES is generally positioned downstream (3') of the stop codon of the chimeric coding sequence and upstream (5') of the coding sequence for the NS5 protein.
  • compositions comprising a recombinant Flaviviridae virus of the invention.
  • Representative compositions may include a buffer, which is selected according to the desired use of the recombinant Flaviviridae virus, and may also include other substances appropriate to the intended use. Where the intended use is to elicit or increase an immune response, the composition is referred to as an "immunogenic" or “immunomodulating" composition.
  • Such compositions include preventative
  • compositions i.e., compositions administered for the purpose of preventing a condition such as an infection
  • therapeutic compositions i.e., compositions administered for the purpose of treating conditions such as an infection
  • An immunomodulating composition of the present invention may therefore be administered to a recipient for prophylactic, ameliorative, palliative, or therapeutic purposes.
  • composition can comprise a pharmaceutically acceptable excipient, a variety of which are known in the art and need not be discussed in detail herein.
  • Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C.
  • compositions comprise more than one (/. e. , different) recombinant Flaviviridae virus of the invention (e.g., recombinant
  • compositions of the present invention may be in a form suitable for administration by injection, in a formulation suitable for oral ingestion (such as, for example, capsules, tablets, caplets, elixirs), in the form of an ointment, cream or lotion suitable for topical administration, in a form suitable for delivery as an eye drop, in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation, or in a form suitable for parenteral
  • administration that is, subcutaneous, intramuscular or intravenous injection.
  • Supplementary active ingredients such as adjuvants or biological response modifiers can also be incorporated into pharmaceutical compositions of the present invention.
  • adjuvant(s) may be included in pharmaceutical
  • compositions of the present invention they need not necessarily comprise an adjuvant. In such cases, reactogenicity problems arising from the use of adjuvants may be avoided.
  • adjuvant activity in the context of a pharmaceutical composition of the present invention includes, but is not limited to, an ability to enhance the immune response (quantitatively or qualitatively) induced by immunogenic components in the composition (e.g., a recombinant virus of the present invention). This may reduce the dose or level of the immunogenic components required to produce an immune response and/or reduce the number or the frequency of immunizations required to produce the desired immune response.
  • any suitable adjuvant may be included in a pharmaceutical composition of the present invention.
  • an aluminum-based adjuvant may be utilized.
  • Suitable aluminum-based adjuvants include, but are not limited to, aluminum hydroxide, aluminum phosphate and combinations thereof.
  • Other specific examples of aluminium-based adjuvants that may be utilised are described in European Patent No. 1216053 and United States Patent No. 6,372,223.
  • Other suitable adjuvants include Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2
  • aluminium salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A; oil in water emulsions including those described in European Patent No. 0399843, United States Patent No. 7,029,678 and PCT Publication No. WO
  • cytokines such as GM-CSF or interleukin-2, -7, or -12, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF) monophosphoryl lipid A (MPL), cholera toxin (CT) or its constituent subunit, heat labile enterotoxin (LT) or its constituent subunit, toll-like receptor ligand adjuvants such as lipopolysaccharide (LPS) and derivatives thereof ⁇ e.g., monophosphoryl lipid A and 3-Deacylated monophosphoryl lipid A), muramyl dipeptide (MDP) and F protein of Respiratory Syncytial Virus (RSV).
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • TNF tumor necrosis factor
  • MPL tumor necrosis factor
  • CT cholera toxin
  • LT heat labile enterotoxin
  • LPS lipo
  • compositions of the present invention may be provided in a kit.
  • the kit may comprise additional components to assist in performing the methods of the present invention such as, for example, administration device(s), buffer(s), and/or diluent(s).
  • the kits may include containers for housing the various components and instructions for using the kit components in the methods of the present invention.
  • the recombinant Flaviviridae virus composition is administered in an "effective amount" that is, an amount effective to achieve production of the exogenous polypeptide in the host at a desired level.
  • an effective amount that is, an amount effective to achieve production of the exogenous polypeptide in the host at a desired level.
  • One skilled in the art would be able, by routine experimentation, to determine an effective, non-toxic amount of a recombinant virus described herein to include in a pharmaceutical composition of the present invention for the desired therapeutic outcome.
  • a pharmaceutical composition of the present invention can be administered in a manner compatible with the route of administration and physical characteristics of the recipient (including health status) and in such a way that it elicits the desired effect(s) (i. e. therapeutically effective, immunogenic and/or protective).
  • composition of the present invention may depend on a variety of factors including, but not limited to, a subject's physical characteristics (e.g., age, weight, sex), whether the compound is being used as single agent or adjuvant therapy, the type of MHC restriction of the patient, the progression (i.e., pathological state) of a virus infection, and other factors that may be recognized by one skilled in the art.
  • a subject's physical characteristics e.g., age, weight, sex
  • the type of MHC restriction of the patient e.g., the type of MHC restriction of the patient
  • progression i.e., pathological state
  • Flaviviridae virus will be sufficient to generate, upon infection of host cells, about 1- 1000 ⁇ g of protein, generally from about 1-200 g, normally from about 10-100 ⁇ g.
  • the dose of recombinant bicistronic flavivirus administered to an indi vidual will generally be in a range of from about 10 2 to about 10 7 , from about 10 3 to about 10 6 , or from about 10 4 to about 10 5 plaque forming units (PFU).
  • an "effective amount" of a subject recombinant bicistronic Flaviviridae virus is an amount sufficient to achieve a desired therapeutic effect.
  • an "effective amount" of a subject recombinant bicistronic Flaviviridae virus is an amount of recombinant Flaviviridae virus that is effective in a selected route of administration to elicit an immune response to an exogenous polypeptide.
  • an "effective amount” is an amount that is effective to facilitate protection of the host against infection, or symptoms associated with infection, by a pathogenic organism, e.g., to reduce a symptom associated with infection, and/or to reduce the number of infectious agents in the individual.
  • an effective amount reduces a symptom associated with infection and/or reduces the number of infectious agents in an individual by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, or more, when compared to the symptom or number of infectious agents in an individual not treated with the recombinant bicistronic Flaviviridae virus, or treated with the parent flavivirus.
  • Symptoms of infection by a pathogenic microorganism, as well as methods for measuring such symptoms are known in the art. Methods for measuring the number of • pathogenic microorganisms in an individual are standard in the art.
  • an "effective amount" of a recombinant is e.g., where the exogenous polypeptide is a cancer- or tumor-associated antigen.
  • Flaviviridae virus is an amount of recombinant Flaviviridae virus that is effective in a route of administration to elicit an immune response effective to reduce or inhibit cancer or tumor cell growth, to reduce cancer or tumor cell mass or cancer or tumor cell numbers, or to reduce the likelihood that a cancer or tumor will form.
  • an effective amount reduces tumor growth and/or the number of tumor cells in an individual by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, or more, when compared to the tumor growth and/or number of tumor cells in an individual not treated with the recombinant bicistronic flavivirus, or treated with the parent flavivirus.
  • Methods of measuring tumor growth and numbers of tumor cells are known in the art.
  • the amount of recombinant Flaviviridae virus in each dose is selected as an amount which induces an immune response to the encoded exogenous polypeptide antigen, and/or which induces an immunoprotective or other immunotherapeutic response without significant, adverse side effects generally associated with typical vaccines. Such amount will vary depending upon which specific exogenous polypeptide is employed, whether or not the vaccine formulation comprises an adjuvant, and a variety of host-dependent factors.
  • An effective dose of recombinant Flaviviridae virus nucleic acid- based composition will generally involve administration of from about 1-1000 ⁇ g of nucleic acid.
  • an effective dose of recombinant Flaviviridae virus will generally be in a range of from about 10 2 to about 10 7 , from about 10 3 to about 10 6 , or from about 10 4 to about 10 s plaque forming units (PFU).
  • An optimal amount for a particular immunomodulating composition can be ascertained by standard studies involving observation of antibody titers and other responses in subjects. The levels of immunity provided by the immunomodulating composition can be monitored to determine the need, if any, for boosters.
  • a pharmaceutical composition of the present invention can be administered to a recipient by standard routes, including, but not limited to, parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular), oral, topical, or mucosal routes (e.g., intranasal).
  • parenteral e.g., intravenous, intraspinal, subcutaneous or intramuscular
  • oral topical
  • mucosal routes e.g., intranasal
  • a pharmaceutical composition of the present invention may be administered to a recipient in isolation or in combination with other additional therapeutic agent(s).
  • the administration may be simultaneous or sequential (i.e., pharmaceutical composition administration followed by administration of the agent(s) or vice versa).
  • the treatment may be for the duration of the disease state or condition.
  • the optimal quantity and spacing of individual dosages will be determined by the nature and extent of the disease state or condition being treated, the form, route and site of administration, and the nature of the particular individual being treated. Optimum conditions can be determined using conventional techniques.
  • a pharmaceutical composition of the present invention may be administered 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times.
  • the administrations may be from about one to about twelve week intervals, and in certain embodiments from about one to about four week intervals. Periodic re-administration may be desirable in the case of recurrent exposure to a particular pathogen or allergen targeted by a pharmaceutical composition of the present invention.
  • the methods for the prevention (i.e. vaccination) and treatment of infection described herein encompass the administration of multiple separated doses to a subject, for example, over a defined period of time. Accordingly, the methods for the prevention
  • the composition or vaccine is administered at least once, twice, three times or more.
  • Methods for measuring the immune response are known to persons of ordinary skill in the art.
  • Exemplary methods include solid-phase heterogeneous assays (e.g., enzyme-linked immunosorbent assay), solution phase assays (e.g. ,
  • electrochemiluminescence assay electrochemiluminescence assay
  • amplified luminescent proximity homogeneous assays flow cytometry, intracellular cytokine staining, functional T-cell assays, functional B-cell assays, functional monocyte-macrophage assays, dendritic and reticular endothelial cell assays, measurement of K cell responses, oxidative burst assays, cytotoxic-specific cell lysis assays, pentamer binding assays, and phagocytosis and apoptosis evaluation. 5. Uses of Recombinant Flaviviridae Viruses of the Invention
  • Recombinant Flaviviridae viruses of the invention are useful to deliver a polypeptide to a mammalian host; and to elicit or increase an immune response . to an antigen encoded by the recombinant virus.
  • Recombinant Flaviviridae viruses of the present are also useful for producing the exogenous polypeptide in host cells, such as mammalian, particularly human, cells or other cell types.
  • the exogenous protein can further be isolated or purified using standard methods.
  • the present invention provides methods for delivering a polypeptide to a mammalian host.
  • the methods generally involve administering a recombinant bicistronic Flaviviridae virus of the invention to a vertebrate host, wherein the virus enters a host cell and the exogenous polypeptide is expressed as a polyprotein with at least one virus protein.
  • the exogenous polypeptide remains intracellular.
  • the exogenous polypeptide becomes associated with the plasma membrane of a host cell.
  • the exogenous polypeptide is secreted from the cell.
  • the exogenous polypeptide in those embodiments in which the exogenous polypeptide is secreted from the cell, can be secreted into the extracellular milieu, e.g. , the interstitial fluid; and/or the exogenous polypeptide can enter the blood stream; and/or the exogenous polypeptide can bind to and/or enter a cell other than the cell in which it was produced.
  • the extracellular milieu e.g. , the interstitial fluid
  • the exogenous polypeptide can enter the blood stream; and/or the exogenous polypeptide can bind to and/or enter a cell other than the cell in which it was produced.
  • the exogenous polypeptide is one that has therapeutic activity, such that when the protein is produced in the mammalian host, a therapeutic effect is achieved.
  • a therapeutic protein is produced in an individual is readily determined using any known method, e.g., methods for detecting the presence of and or measuring the amount of a protein, including, but not limited to, an enzyme-linked immunosorbent assay, a radioimmunoassay, and the like, using specific antibody; and methods for detecting the presence of and/or measuring the amount of a biological activity associated with the protein.
  • Whether a therapeutic effect is achieved can be determined using a method appropriate to the particular therapeutic effect. For example, whether a therapeutic effect is achieved when insulin is delivered to a host using the subject method can be determined by measuring glucose levels in the individual.
  • the present invention provides methods for eliciting an immune response to an antigen.
  • the methods generally involve administering a recombinant bicistronic Flaviviridae virus of the invention to a vertebrate host, wherein the virus enters a host cell, the exogenous polypeptide is expressed as a polyprotein with at least one virus protein, and an immune response is elicited to the exogenous polypeptide.
  • recombinant Flaviviridae virus as described herein are useful for inducing or increasing an immune response to an antigen in an individual.
  • the exogenous polypeptide When the exogenous polypeptide is produced in a vertebrate host, it elicits an immune response to the exogenous polypeptide.
  • the immune response protects against a condition or disorder caused by or associated with expression of or the presence in the host of, an antigen comprising the epitope.
  • the antigen is a pathogen-associated antigen, and the immune response provides protection against challenge or infection by the exogenous pathogen (bacterial, viral, fungal, parasitic) in which the antigen occurs.
  • Flaviviridae virus of the invention are, therefore, useful as immunomodulating compositions (also referred to herein as "immunogenic compositions") to elicit and/or increase an immune response to the antigen.
  • the exogenous polypeptide is an antigenic polypeptide of a microbial pathogen.
  • a microbial pathogen e.g., HIV
  • bacteria e.g., Shigella, Listeria, mycobacteria, and the like
  • parasites e.g., malarial parasites, illustrative examples of which include Plasmodium falciparum,
  • Antigenic polypeptides of such microbial pathogens are well known in the art, and can be readily selected for use in the present recombinant Flaviviridae virus immunomodulating composition by the ordinarily skilled artisan.
  • a recombinant Flaviviridae virus of the invention can be used as a delivery vehicle to delivery any antigen to an individual, to provoke an immune response to the antigen.
  • recombinant Flaviviridae virus of the invention are used as bivalent or multivalent immunomodulating composition to treat human or veterinary diseases caused by infectious pathogens, particularly viruses, bacteria, and parasites.
  • Examples of epitopes which could be delivered to a host in a multivalent Flaviviridae virus composition of the invention include multiple epitopes from various serotypes of Group B streptococcus, influenza virus, rotavirus, and other pathogenic organisms known to exist in nature in multiple forms or serotypes; epitopes from two or more different pathogenic organisms; and the like.
  • Suitable subjects include nave subjects (i.e., subjects who were never exposed to the antigen such that the antigen or pathogen entered the body), and subjects who were previously exposed to the antigen, but did not mount a sufficient immune response to the pathogenic organism.
  • a polypeptide antigen expressed on a given cancer or tumor cell e.g., a cancer- or tumor-associated antigen
  • a recombinant Flaviviridae virus of the invention as described herein.
  • Such recombinant Flaviviridae virus can be administered to an individual having, or suspected of having, a cancer or tumor.
  • such recombinant Flaviviridae virus can be
  • the immune system does not mount an immune response effective to inhibit or suppress cancer or tumor growth, or eliminate a cancer or tumor altogether.
  • Cancer- or tumor-associated antigens are often poorly immunogenic; perhaps due to an active and ongoing immunosuppression against them.
  • cancer patients tend to be immunosuppressed, and only respond to certain T-dependent antigens.
  • introduction into the host of a recombinant Flaviviridae virus of the invention which expresses an exogenous peptide polypeptide corresponding to an antigen expressed on the tumor cell surface can elicit an immune response to the tumor in the host.
  • Non-limiting cancer- or tumor-associated antigens which may be inserted into Flaviviridae virus include, but are not limited to, MAGE-2, MAGE-3, MUC-1, MUC-2, HER-2, high molecular weight melanoma-associated antigen MAA, GD2, carcinoembryonic antigen (CEA), TAG- 72, ovarian-associated antigens OV-TL3 and MOV 18, TUAN, alpha-feto protein (AFP), OFP, C A- 125, CA-50, CA- 19-9, renal tumor-associated antigen G250, EGP-40 (also known as EpCAM), SI 00 (malignant melanoma-associated antigen), p53, prostate tumor-associated antigens (e.g., PSA and PSMA) and p21ras.
  • MAGE-2 MAGE-3
  • MUC-1 high molecular weight melanoma-associated antigen MAA
  • GD2 carcinoembryonic antigen
  • TAG- 72 ova
  • Suitable subjects include subjects who do not have cancer, but are considered at risk of developing cancer; and subjects who have cancer, but who have not mounted an immune response sufficient to reduce or eliminate the cancer.
  • Whether an immune response has been elicited to a pathogenic organism, cancer or tumor can be determined (quantitatively, e.g. , by measuring a parameter, or qualitatively, e.g., by assessing the severity of a symptom, or by detecting the presence of a particular parameter) using known methods.
  • Methods of measuring an immune response are well known in the art and include enzyme-linked immunosorbent assay (ELISA) for detecting and/or measuring antibody specific to a given pathogenic organism, cancer or tumor antigen; and in vitro assays to measure a cellular immune response (e.g., a CTL assay using labeled, inactivated cells expressing the epitope on their cell surface with MHC Class I molecules).
  • an immune response is effective to facilitate protection of the host against infection, or symptoms associated with infection, by a pathogenic organism
  • determining the number of pathogenic organisms in a host e.g., measuring viral load, and the like
  • measuring a symptom caused by the presence of the pathogenic organism in the host e.g. , body temperature, CD4 + T cell counts, and the like.
  • Whether an immune response is elicited to a given cancer or tumor can be determined by methods standard in the art, including, but not limited to, assaying for the presence and/or amount of cancer- or tumor-associated antigen-specific antibody in a biological sample derived from the individual, e.g. , by enzyme-linked
  • ELISA immunosorbent assay
  • RIA radioimmunoassay
  • assaying for the presence and/or numbers of CTLs specific for a cancer- or tumor-associated antigen include, but are not limited to, chromium-release assays, tritiated thymidine incorporation assays, and the like.
  • Standard immunological protocols may be used, which can be found in a variety of texts, including, e.g., Current Protocols in Immunology (J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W.
  • Whether an immune response is effective in reducing the number of tumor cells in an individual can be determined by standard assays, including, but not limited to, measuring tumor cell mass, measuring numbers of tumor cells in an individual, and measuring tumor cell metastasis. Such assays are well known in the art and need not be described in detail herein.
  • the invention further provides methods of producing an exogenous polypeptide in a vertebrate host cell.
  • the methods generally involve contacting a susceptible host cell with a recombinant bicistronic Flaviviridae virus of the invention with, culturing the host cell for a period of time to allow production of the exogenous polypeptide by the host cell.
  • the methods further comprise purifying the exogenous polypeptide from the host cell or from the culture medium.
  • the exogenous protein remains intracellular (e.g., in the cytoplasm, in a cell membrane, or in an organelle), in which case the cells are disrupted.
  • a variety of protocols for disrupting cells to release an intracellular protein are known in the art, and can be used to extract an exogenous protein from a cell.
  • the exogenous protein is secreted into the medium in which the cells are grown.
  • any convenient protein purification procedures may be employed, where suitable protein purification methodologies are described in Guide to Protein Purification, (Deuthser ed.) (Academic Press, 1990).
  • a lysate may prepared from the infected host cell, or a cell culture supernatant may be produced, and the exogenous protein purified using HPLC, exclusion chromatography, gel
  • the methods disclosed herein may provide several improvements over existing methods, particularly in the context of virus infection.
  • One improvement may be that an heterologous nucleic acid sequence inserted in a Fiaviviridae virus ORF between adjacent protein-encoding sequences may remain genetically stable (i.e. resist mutation/deletion) over extended numbers of viral replication cycles.
  • an exogenous cytokine expressed by a recombinant virus of the present invention e.g. , a type- 1 cytokine such as interferon-beta
  • a type- 1 cytokine such as interferon-beta
  • Another improvement may be that despite the attenuated virulence, the administration of a recombinant virus of the present invention to a subject may induce a similar level of immunity against the targeted microorganism (e.g., a target virus) to that which may be achieved by administering the targeted microorganism (i.e. wild-type).
  • the targeted microorganism e.g., a target virus
  • a further improvement may be that an exogenous cytokine expressed by a recombinant virus of the present invention (e.g., a type-1 cytokine such as interferon-beta) may act as a molecular adjuvant in the host organism enhancing humoral and/or cell-mediated immunity.
  • a recombinant virus of the present invention administered to a subject may continue to propagate until the immune system is sufficiently activated to halt the infection, thereby providing a means of inducing stronger immune responses.
  • Yet another improvement may be that a recombinant virus of the present invention administered to a subject may not revert to a pathogenic state over extended numbers of viral replication cycles.
  • Another improvement may be that the expression of cytokine by the recombinant virus may prevent the appearance of revertant viruses.
  • IFN- ⁇ The expression of IFN- ⁇ was studied using a V model. Compared to control VV, which grew to high titers (10 7 PFU), co-expression of IFN-p controlled the replication of VV-IFN- ⁇ in vivo and infectious virus was not detected at any stage at the limits of the plaque assay (10 2 PFU).
  • VV-IFN- ⁇ is also highly attenuated in other animal models of immunosuppression (IFN-y-R and MyD88 GKO mice), in which wild-type VV is highly virulent.
  • IFN-y-R and MyD88 GKO mice The significant attenuation of VV-IFN- ⁇ observed in both immunocompetent and immunodeficient mice suggests a local antiviral state induced by viral expression of IFN- ⁇ , which is independent of T lymphocytes.
  • IFN ⁇ -expressing viruses Despite their highly attenuated phenotype, IFN ⁇ -expressing viruses elicited robust humoral and cell-mediated immune responses to both co-expressed and viral antigens.
  • ECMV encephalomyocarditis virus
  • IFN- ⁇ proteins a signal peptide at the C-prM junction
  • prM to NS5 the rest of the viral polyprotein
  • the prM signal peptide must remain down-stream from the C protein; accordingly, the prM signal peptide was used for ER luminal translocation of the IFN.
  • the derivative plasmid (pMVEV-FL-C.IRES) was modified by removing a Nsi I restriction site present in the IRES cDNA.
  • mouse IFN- ⁇ gene was inserted into plasmid, pMVEV-FL- C.IRES, as a 1098 bp Apa I fragment excised from a synthetically synthesized
  • the IFN- ⁇ gene lacking its own signal peptide, was positioned directly down-stream of the MVEV prM signal sequence, allowing translocation of interferon into the lumen of the endoplasmic reticulum and subsequent secretion. Cap-dependent translation of viral capsid and IFN- ⁇ proteins is terminated at the authentic C-terminal amino acid of the latter protein.
  • the down-stream EMCV IRES drives translation of the rest of the viral polyprotein (prM-NS5).
  • the prM signal peptide down-stream of the IRES was modified (SEQ ID NO: 1) in order to prevent homologous recombination between it and the upstream prM signal peptide, which drives translocation of IFN- ⁇ .
  • Recombinant virus encoding IFN- ⁇ was generated was generated by standard procedures involving linearization of plasmid DNA with Nsi I, in vitro synthesis of full-length RNA using T7 RNA polymerase, electroporation of RNA into BHK cells and culture of cells for 3 - 4 days to allow accumulation of recombinant virus in the culture supernatant.
  • the bicistronic viruses were recovered following electroporation of in vitro synthesized, full-length genomic RNA into BFfK cells and amplification of the supernatant on Vero cells, a cell line approved for human vaccine production. Growth of the bicistronic control and IFN-P-encoding viruses in Vero cells (type I IFN response-defective monkey fibroblasts) were similar to that of wt in terms of plaque size and virus yield (>10 7 PFU/ml).
  • Humoral immunity is key to vaccine-mediated protection against flaviviral disease.
  • a single dose of MVEV. IFNp (10 s PFU) was effective in eliciting an antibody response against MVEV, which was only marginally lower than that produced with the same dose of wt virus.
  • Two vaccine doses resulted in high levels of humoral immunity, equivalent to that induced with wt or control MVEV.C-IRES vaccination in two different mouse strains ( Figure 7). The experiment shows that despite complete virulence attenuation of the iFN-expressing virus, its immunogenic potency was not ablated.
  • the first recombinant flavivirus encoding a cytokine/IFN was produced, and shown to have stable, prolonged and high levels of IFN- ⁇ expression.
  • the IFN co-expression strategy enhances the safety profile of a live,, attenuated vaccine, because the effective production of the IFN at the site of infection will also restrict the growth of any virulence revertant that may arise.
  • the advantage of the alternative virus model for validation of the novel vaccine technology in the context of flaviviral disease is the availability of highly sensitive assays for viscero- and neuro virulence of MVEV in mice. Attenuation of viscero- and neurotropism of MVE V co-expressing an IFN would strongly predict even greater safety following replacement of the MVEV structural (prM-E) proteins with those of DENV, due to the additional level of attenuation associated with the chimerization.
  • the inventors have developed an innovative strategy for efficient and stable expression of heterologous nucleic acid sequences including cytokine/IFN- encoding sequences from a bicistronic flavivirus genome (see Examples above).
  • This strategy will be used to construct recombinant MVEV expressing mouse IFN-cc4, and the newly defined IFN- ⁇ and IFN- ⁇ genes.
  • IFN-a4 is chosen as this is the predominant subtype detected after infection, and has high antiviral activity compared to other IFNas.
  • Synthetic DNA corresponding to the IFN genes will be inserted into the full- length infectious clone of MVEV, and virus generated.
  • IFN- ⁇ and IFN- ⁇ will be compared to wt MVEV and control bicistronic virus
  • MVEV.C-IRES type I IFN response-deficient Vero (monkey) and type I IFN response-sufficient mouse embryo fibroblasts and/or L929 cells. This comparison will measure the level of attenuation of virus growth associated with the different IFNs. Levels of secreted IFN will be quantitated by ELISA and correlated to the number of infected cells.
  • the present inventors have pioneered the concept of using viruses for the expression of heterologous nucleic acid sequences including cytokine/IFN-encoding sequences in flaviviruses.
  • the parallel production of otherwise identical viruses that encode IFN-a, - ⁇ , - ⁇ or - ⁇ will result in a unique resource for direct comparison of their antiviral and immunological activities. It is envisaged that these experiments may demonstrate that IFN-a, - ⁇ or - ⁇ can also be effectively used for the purposes outlined in the Examples above.
  • Virulent (wt) MVEV gives low (0.1 - 5 PFU) LD 50 values by both injection routes in mice of this age; attenuation of neuroinvasiveness and neurovirulence are reflected in markedly increased i.p. and i.e. LD50 values, respectively.
  • LD50 measurement in 3-wk-old and suckling mice is a standard assay for assessing flavivirus virulence and will allow comparison of the antiviral property of the different IFNs. It will also allow direct virulence comparisons with licensed and experimental live flavivirus vaccines. It is envisaged these experiments may demonstrate that virulence of bicistronic flaviviruses expressing IFN-a, - ⁇ , or - ⁇ is also effectively attenuated.
  • Neutralizing antibody titers will be measured by a plaque-reduction neutralization test (PRNT50).
  • PRNT50 plaque-reduction neutralization test
  • a PRNT5 0 titer of >10 is considered a surrogate measure of protective immunity.
  • IgG isotypes will be monitored to determine if class-switching to IgG2a is promoted which would be indicative of a Thl-type antiviral immune response.
  • MVEV-specific CD8+ T cell immune responses elicited with wt and recombinant viruses will be evaluated in CBA mice by intracellular cytokine staining for IFN- ⁇ and TNF- of ex vivo splenocytes stimulated with the
  • a newly developed assay will be used to assess the protective capacity of the IFN-encoding viruses against lethal challenge with MVEV. It involves the challenge with a low dose (10 3 PFU, i.p.) of wt MVEV of 3-w-old pups from vaccinated (during the 1 st week of gestation) or control-treated mothers. This allows the assessment of protective antibody responses, through placental transfer, in a highly sensitive model system (CIB, unpublished).
  • CIB highly sensitive model system
  • the research is expected to reveal advantageous effects of co- expression of IFN-a, - ⁇ , - ⁇ or - ⁇ on the magnitude and quality of the anti-MVEV adapti ve immune responses in a set-up that allows direct comparison of the IFNs. It is also envisaged the experiments may demonstrate that levels of immunity considered to be protective can be elicited with a live vaccine candidate in the absence of detectable viral toad. Accordingly, the data to be generated is expected to support the notion that the IFN co-expression strategy is beneficial in vaccination against flaviviruses.
  • chimeric viruses will be constructed by substituting the prM and E protein genes in the MVEV genome with those of one of each of the 4 DENV serotypes in the presence or absence of co-expression of the selected mouse type I IFN ( Figure 7).
  • the E protein is the dominant antigen for induction of virus neutralizing and protective antibodies, and DENV prM-E expression from chimeric flaviviruses is considered among the most promising strategies for the development of a dengue vaccine.
  • IFN- ⁇ has all the properties required for virulence attenuation of MVEV, whilst still inducing high levels of immunity, DENV-2 chimeras with or without the IFN are currently being constructed for evaluation.
  • Sera will be collected at 4 wk after priming, animals will be boosted with the priming dose and second serum samples taken 4 wk later.
  • Neutralizing antibody titers of mouse sera against each of the four DENV serotypes will be determined by PRNT50 assay, the "gold-standard" for monitoring immunological protection in flavivirus vaccine recipients.
  • MVEV-DENV(prM E) chimera lacking IFN expression
  • IFN co-expression markedly enhanced vaccine immunogenicity and/or results in more balanced immunity against the 4 DENV following immunization with tetravalent vaccines
  • experiments to measure the durability of the induced neutralizing antibody responses for a period of up to 6 months post-immunization will be conducted.
  • MVEV.IFN /ORF ECMV IRES for translation of essential viral proteins in construct
  • MVEV.IFN /ORF will prohibit loss-of-function mutations from occurring in this sequence element, in contrast to the genome that encodes the IRES-IFN- ⁇ cassette in the 3'-UTR, since in the latter case the presence of the IRES sequence is not required for virus growth.
  • novel strategy has wide application and can, for instance, be expanded to investigate the benefits of co-expression of other immune-modulating molecules for enhancement of flavivirus vaccine immunogenicity or to (non-infectious) flavivirus replicon vaccine technology, where optimal induction of an antiviral immune response is the primary aim in view of the self-limiting replication of the vaccine vectors.

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Abstract

La présente invention concerne des produits et des méthodes pour le traitement et/ou la prévention de maladies virales. Plus particulièrement, la présente invention concerne des virus Flaviviridae recombinants pour l'expression stable de molécules d'acide nucléique hétérologues, des procédés de production de tels virus, et leur utilisation dans des compositions d'immunomodulation pour le traitement et/ou la prévention d'états, dont des infections par des virus Flaviviridae.
PCT/AU2013/000658 2012-06-20 2013-06-20 Virus recombinants améliorés WO2013188918A1 (fr)

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CN107201370A (zh) * 2016-03-17 2017-09-26 中国人民解放军军事医学科学院微生物流行病研究所 一种dna分子与重组病毒及它们的制备方法和用途
CN107201370B (zh) * 2016-03-17 2020-04-24 中国人民解放军军事医学科学院微生物流行病研究所 一种dna分子与重组病毒及它们的制备方法和用途
CN108929877A (zh) * 2017-05-23 2018-12-04 中国人民解放军军事医学科学院微生物流行病研究所 一种编码嵌合寨卡病毒的dna分子及其制备方法和用途
WO2022074453A3 (fr) * 2020-10-05 2022-06-09 Versameb Ag Compositions et méthodes pour la modulation de l'expression de gènes de manière simultanée
CN114015723A (zh) * 2021-11-05 2022-02-08 四川农业大学 一种鸭坦布苏病毒质粒载体、弱毒株及其制备方法和应用

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