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  • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

• This invention relates generally to recombinant hepatitis delta viral vectors that can be used, for example, in the treatment or prevention of hepatitis virus infections.
• the invention further relates to compositions and methods that employ these vectors.
• Hepatitis delta virus is a subviral satellite as it can only propagate in the presence of Hepatitis B virus (HBV). Indeed, HDV can only productively infect individuals who have HBV, and currently 15 million are co-infected worldwide. Of significance, superinfection of HBV carriers with HDV causes severe liver disease and results in a high rate of chronicity.
• HDV is a small, spherical virus with a 36 nm diameter. It has an outer coat containing three HBV envelope proteins (designated large, medium, and small hepatitis B surface antigens), and host lipids surrounding an inner nucleocapsid.
• the nucleocapsid contains a negative sense, single-stranded, closed circular RNA of approximately 1,700 nucleotides and about 200 molecules of hepatitis D antigen (HDAg) for each genome.
• HDAg hepatitis D antigen
• the HDV circular genome is unique to animal viruses because it is the smallest known viral genome that infects mammals and has a high G/C nucleotide content (about 60%). Additionally, its nucleotide sequence is about 70% self- complementary, allowing the HDV genome to form a partially double-stranded RNA structure that is described as "rod-like.”
• RNA polymerases are made using host cell RNA polymerases; circular genomic RNA, circular complementary antigenomic
• RNA and a linear polyadenylated antigenomic RNA, which is the mRNA containing the open reading frame for the HDAg.
• RNA polymerase II RNA polymerase II
• the RNA polymerases treat the HDV RNA genome as double-stranded DNA due to the folded rod-like structure it is in and possibly due to the action of the HDAg.
• Figure 1 A schematic representation of wild-type HDV genomic RNA, antigenomic RNA and mRNA is shown in Figure 1.
• HDV RNA template occurs via a rolling circle mechanism that is unique to animal RNA viruses but analogous to that of plant viroids.
• HDV RNA is synthesized first as linear (concatemeric) RNA that contains many copies of the genome.
• the genomic and antigenomic RNA contain a sequence of about 85 nucleotides that acts as a ribozyme, which self-cleaves the linear RNA into monomers. These monomers are then ligated to form circular RNA.
• HDV uses ADAR1 editing of the viral antigenome RNA to switch from viral RNA replication to packaging.
• the virus produces a short form of HDAg, termed HDAg-S, which is required for RNA synthesis.
• HDAg-L the virus produces a long form of HDAg, termed HDAg-L, which is required for packaging and inhibits further RNA synthesis as levels increase.
• HDV superinfection in HBV-infected individuals often results in chronic HDV, which further increases the mortality rate associated with chronic hepatitis B.
• present therapies often fail to resolve chronic hepatitis, are expensive, and have serious side-effects.
• the present inventors have developed a novel strategy for stably inserting heterologous nucleotide sequences into the HDV genome.
• they have discovered that it is possible to stably insert an heterologous nucleotide sequence into a site of a HDV genome provided that a substantially complementary heterologous nucleotide sequence is inserted into another site of the HDV genome whereby the heterologous nucleotide sequences are in juxtaposition to permit annealing to each other and to thereby maintain the rod-like secondary structure of the HDV genome and confer stability, thereto.
• the present invention provides
• HDV hepatitis delta virus
• These genomes generally comprise or consist essentially of in operable connection: a promoter; an open reading frame (ORF) for a hepatitis delta antigen (HDAg); a polyadenylation signal; and a HDV ribozyme, wherein the genomes comprise substantially complementary portions conferring a rod-like secondary structure, wherein the genomes are characterized in that they comprise at a first site a first heterologous nucleotide sequence and at a second site a second heterologous nucleotide sequence that is substantially complementary to the first heterologous nucleotide sequence wherein the first and second sites are spaced from each other to permit annealing between the first and second heterologous nucleotide sequences.
• the present invention provides recombinant single- stranded, circular hepatitis delta virus (HDV) RNA genomes, comprising a first portion and a second portion, wherein the first portion comprises in operable connection: (1) a promoter; (2) an open reading frame (ORF) for a hepatitis delta antigen (HDAg); (3) a polyadenylation signal; (4) a HDV ribozyme; and (5) a first heterologous nucleotide sequence, and wherein the second portion is substantially complementary to the first portion so as o permit annealing between the portions.
• the annealing between the portion confers a rod-like secondary structure on the genome.
• the first heterologous nucleotide sequence comprises a non-coding nucleotide sequence (e.g. ,. a functional RNA molecule such as rRNA, tRNA, RNAi, shRNA, siRNA, miRNA, ribozymes and antisense RNA).
• a non-coding nucleotide sequence e.g. ,. a functional RNA molecule such as rRNA, tRNA, RNAi, shRNA, siRNA, miRNA, ribozymes and antisense RNA.
• the non-coding sequence is only transcribed into RNA and is suitably operably connected to a promoter.
• the first heterologous nucleotide sequence comprises a nucleotide sequence that is both transcribed into mR A and translated into a polypeptide.
• the present invention encompasses embodiments in which the first heterologous nucleotide sequence comprises a coding sequence for an exogenous polypeptide.
• the exogenous polypeptide is selected from a polypeptide of a pathogenic organism (e.g. , other than the HDAg), an alloantigen, an autoantigen, a cancer or tumor antigen or any other polypeptide that has therapeutic activity.
• the exogenous polypeptide is or comprises a cytokine (e.g., a cytokine that attenuates HDV, illustrative example of which include interferons (IFNs) including type I IFNs such as IFN- ⁇ .
• IFNs interferons
• the present invention provides recombinant HDV genomes engineered to stably express heterologous nucleotide sequences including cytokine-encoding sequences, which can provide a means of attenuating virulence (i.e., addressing safety concerns) and/or augmenting immunity against or resistance to HDV and optionally HBV or Hepatitis C virus (HCV) in a subject (e.g., a human) to which they are administered.
• the coding sequence 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 comprising the exogenous polypeptide and the HDAg.
• the coding sequence 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 (e.g., hepatocyte) 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 histocompatability antigen, secreted from the cell, or remains in the cytoplasm of the cell.
• the first heterologous nucleotide sequence is located downstream of the promoter and upstream of the ORF.
• the first heterologous nucleotide sequence comprises a coding sequence for an exogenous polypeptide, which is operably connected to the promoter, and an internal ribosome entry site (IRES) that is operably connected to the ORF.
• the first heterologous nucleotide sequence is located downstream of the ORF and suitably upstream of the polyadenylation site.
• the first heterologous nucleotide sequence comprises an internal ribosome entry site (IRES) operably connected to a coding sequence for an exogenous polypeptide.
• the first heterologous nucleotide sequence comprises a first coding sequence for an exogenous polypeptide and a second coding sequence for a proteolytic cleavage site, wherein the first and second coding sequences are in frame with each other and with the ORF to thereby encode a precursor polypeptide, wherein the proteolytic cleavage site is positioned between the exogenous polypeptide and the HDAg in the precursor polypeptide to facilitate release of the exogenous polypeptide upon proteolytic cleavage of the proteolytic cleavage site.
• the first heterologous nucleotide sequence comprises a first coding sequence for an exogenous polypeptide and a second coding sequence for a so-called "self-cleaving" peptide (e.g., a so-called 2 A or 2A-like self-cleaving peptide), wherein the first and second coding sequences are in frame with each other and with the ORF.
• the first heterologous nucleotide sequence is located downstream of the promoter and upstream of the ORF, and the second coding sequence is downstream of the first coding sequence and upstream of the ORF.
• the first heterologous nucleotide sequence is located downstream of the ORF, wherein the second coding sequence is upstream of the first coding sequence and downstream of the ORF.
• the first heterologous nucleotide sequence is located downstream of the HDV ribozyme.
• the first heterologous nucleotide sequence is operably connected to another promoter (e.g., a promoter other than the promoter that is operably connected to the ORF).
• an operably connected promoter in the recombinant genome is a DNA dependent RNA polymerase (e.g., RNA polymerase I, II or III) promoter, illustrative examples of which include native or wild-type HDV promoters.
• RNA dependent RNA polymerase e.g., RNA polymerase I, II or III
• the first heterologous sequence is not operably connected to a promoter.
• the first heterologous nucleotide sequence has a G/C nucleotide content that substantially accords with the G/C content of the HDV geriome, usually between about 55% and about 65% (e.g., about 60%).
• the first heterologous nucleotide sequence has at least about 50% (and at least 51% to at least 99% and all integer percentages in between) sequence identity to the second heterologous nucleotide sequence.
• the present invention provides methods for producing a recombinant single-stranded, circular hepatitis delta virus (HDV) RNA genome.
• HDV hepatitis delta virus
• These methods generally comprise, consist or consist essentially of: providing a parent single-stranded, circular HDV RNA genome, which comprises in operable connection: a promoter; an open reading frame (ORF) for a hepatitis delta antigen (HDAg); a polyadenylation signal; and a HDV ribozyme, and which has substantially complementary portions that anneal to one another and confer a rod-like secondary structure on the parent genome, and inserting into the parent genome at a first site a first heterologous nucleotide sequence and at a second site a second heterologous nucleotide sequence that is substantially complementary to the first heterologous nucleotide sequence to form the recombinant HDV genome, wherein the first and second sites are spaced from each other in the recombinant genome to permit
• the methods comprise inserting the first and second heterologous nucleotide sequences such that they do not interfere or impair annealing of the complementary portions of the parent genome.
• the methods comprise inserting the first heterologous nucleotide sequence downstream of the promoter and upstream of the ORF and inserting the second heterologous nucleotide sequence downstream of the nucleotide sequence of the parent genome that is substantially complementary and anneals to the ORF and upstream of the nucleotide sequence of the parent genome that is substantially complementary and anneals to the promoter.
• the methods comprise inserting the first heterologous nucleotide sequence downstream of the ORF and upstream of the polyadenylation signal and inserting the second heterologous nucleotide sequence downstream of the nucleotide sequence of the parent genome that is substantially complementary and anneals to the polyadenylation signal and upstream of the nucleotide sequence of the parent genome that is substantially complementary and anneals to the ORF.
• the methods comprise inserting the first heterologous nucleotide sequence downstream of the polyadenylation signal and upstream of the ribozyme and inserting the second heterologous nucleotide sequence downstream of the nucleotide sequence of the parent genome that is substantially complementary and anneals to the ribozyme and upstream of the nucleotide sequence of the parent genome that is substantially complementary and anneals to the
• the methods comprise inserting the first heterologous nucleotide sequence downstream of the ribozyme and upstream of portions of the parent genome that are substantially complementary and anneal to each other and inserting the second heterologous nucleotide sequence downstream of those portions and upstream of the nucleotide sequence of the parent genome that is substantially complementary and anneals to the ribozyme.
• the first heterologous nucleotide sequence has at least 50% (and at least 51% to at least 99% and all integer percentages in between) sequence identity to the second heterologous nucleotide sequence.
• the methods further comprise modifying the
• the G/C content of the first and second heterologous nucleotide sequences is modified so that it is between about 55% to about 65% (e.g. , about 60%).
• nucleic acid molecules e.g., a DNA molecule such as a cDNA molecule
• nucleic acid molecules comprising, consisting or consisting essentially of a sequence corresponding to a recombinant HDV genome as broadly described above and elsewhere herein or to an antigenome thereof.
• the nucleic acid molecules are in isolated form.
• vectors comprising, consisting or consisting essentially of a nucleic acid molecule as broadly described above and elsewhere herein.
• a recombinant hepatitis delta virus comprising, consisting or consisting essentially of a recombinant genome as broadly described above and elsewhere herein.
• the HDV is in isolated form.
• compositions comprising, consisting or consisting essentially of a recombinant HDV as broadly described above and elsewhere herein, and a pharmaceutically acceptable excipient, diluent or carrier.
• Still another aspect of the present invention provides
• immunomodulating compositions comprising, consisting or consisting essentially of a recombinant HDV as broadly described above and elsewhere herein, and optionally an adjuvant or immunostimulant.
• the present invention provides methods for eliciting an immune response to a hepatitis delta virus (HDV) in a subject (e.g., a human). These methods generally comprise, consist or consist essentially of administering to the subject an effective amount of a recombinant HDV as broadly described above and elsewhere herein so as to elicit an immune response to the HDV.
• a hepatitis delta virus e.g., a human
• the present invention provides methods for treating or preventing a hepatitis delta virus (HDV) infection in a subject (e.g., a human). These methods generally comprise, consist or consist essentially of administering an effective amount of a recombinant HDV as broadly described above and elsewhere herein to the subject.
• a hepatitis delta virus HDV
• the first heterologous nucleotide sequence comprises a cytokine-encoding sequence, including an interferon-encoding sequence, which is useful, for example, in the treatment of hepatitis virus infections, including HDV, HBV and/or HCV infections.
• a cytokine-encoding sequence including an interferon-encoding sequence
• the present invention provides methods for treating or preventing a hepatitis infection in a subject (e.g. , a human).
• These methods generally comprise, consist or consist essentially of administering an effective amount of a recombinant HDV as broadly described above and elsewhere herein to the subject, wherein the first heterologous nucleotide sequence comprises a coding sequence for a cytokine (e.g., one that codes for a type I IFN such as IFN- ⁇ or IFN-a, a type II IFN such as IFN- ⁇ or a type III IFN such as IFN- ⁇ ).
• a cytokine e.g., one that codes for a type I IFN such as IFN- ⁇ or IFN-a, a type II IFN such as IFN- ⁇ or a type III IFN such as IFN- ⁇ .
• the present invention provides a recombinant hepatitis delta virus (HDV) as broadly described above and elsewhere herein, or a composition as broadly described above and elsewhere herein, for use in eliciting an immune response to a HDV in a subject (e.g. , a human).
• a subject e.g. , a human
• methods for eliciting an immune response to an exogenous polypeptide in a subject (e.g., a human). These methods generally comprise, consist or consist essentially of
• the exogenous polypeptide is an antigen of the subject or an antigen of a microorganism (e.g., bacteria, protozoa, viruses for example other than the HDV such as hepatitis B virus (HBV) and hepatitis C virus (HCV) and used to generate the recombinant HDV of the invention, yeast, fungi, and the like).
• a microorganism e.g., bacteria, protozoa, viruses for example other than the HDV such as hepatitis B virus (HBV) and hepatitis C virus (HCV) and used to generate the recombinant HDV of the invention, yeast, fungi, and the like.
• the present invention provides a recombinant hepatitis delta virus (HDV) as broadly described above and elsewhere herein, or a composition as broadly described above and elsewhere herein, for use in preventing or treating an infection by a pathogen (e.g. , other than the HDV used to generate the recombinant HDV of the invention) in a subject (e.g., a human).
• a pathogen e.g. , other than the HDV used to generate the recombinant HDV of the invention
• a subject e.g., a human
• Another aspect of the present invention provides methods for delivering an exogenous polypeptide having therapeutic activity to a subject (e.g. , a human). These methods generally comprise, consist or consist essentially of
• a hepatitis delta virus as broadly described above and elsewhere herein to the subject, whereby the exogenous polypeptide is produced in a host cell of the subject.
• the host cell is a hepatocyte.
• the therapeutic polypeptide may remain inside the cell, become associated with a cell membrane, or may be 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).
• a host cell e.g., a vertebrate host cell.
• these methods generally comprise, consist or consist of contacting a susceptible host cell with a recombinant hepatitis delta virus (HDV) composition as broadly described above and elsewhere herein, wherein the first heterologous nucleotide sequence comprises a coding sequence for 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.
• Still another aspect of the present invention provides methods for delivering an exogenous polynucleotide to a subject ⁇ e.g. , a human), wherein the exogenous polynucleotide comprises, consists or consists essentially of a non-coding nucleotide sequence as broadly described above and elsewhere herein.
• these methods generally comprise, consist or consist essentially of administering a hepatitis delta virus (HDV) comprising a non-coding sequence as broadly described above and elsewhere herein to the subject, whereby the exogenous polynucleotide is produced in a host cell of the subject.
• the host cell is a hepatocyte.
• the present invention provides a recombinant single-stranded, circular HDV RNA genome, which comprises or consists essentially of a first site and a second site that are spaced from each other, wherein the first site is distinguished from a corresponding site in a parent HDV genome by the addition, deletion or substitution of at least one nucleotide and the second site is distinguished from a corresponding site in the parent HDV genome by the addition, deletion or substitution of at least one nucleotide, wherein the first and second sites are
• Figure 1 is a schematic representation showing a wild-type hepatitis delta virus (HDV) genome, anti-genome and mRNA.
• HDV hepatitis delta virus
• the single-stranded RNA genome/ anti-genome is highly self-complementary and forms a 'rod-like' secondary structure.
• Characteristic features of the anti-genomic RNA include a putative promoter (pro) sequence upstream of the open reading frame for the short (S) and long (L) form of the hepatitis delta antigen (HDAg), a polyadenylation signal (poly A) and the anti-genomic ribozyme (ribo) sequence; features of the genomic RNA (middle panel) include a promoter sequence (pro) and the genomic ribozyme (ribo) sequence.
• HDV mRNAs bottom panel
• FIG. 2 is a schematic representation showing the recombinant HDV genome rHDV-huIFNbeta-IRES-HDAg.
• the heterologous nucleotide sequence of 1 interest which comprises a human interferon-beta coding sequence (huIFN- ⁇ ) and an internal ribosome entry site (IRES), is inserted between the HDV promoter (pro) sequence and the hepatitis delta antigen (HDSAg) open reading frame.
• pro human interferon-beta coding sequence
• HDSAg hepatitis delta antigen
• SS signal sequence
• asterisk editing site.
• FIG. 3 is a schematic representation showing an illustration of the RNA secondary structures of the HDV genome.
• RNA sequences were analyzed using RNAfold (http://rna. tbi.univie.ac.at). Predicted minimum free energy structures and base pair probabilities are shown for (a) the parental, wild-type HDV genome, (b) a modified genome into which the human IFN-beta coding sequence has been inserted upstream of the HDSAg open reading frame, a manipulation that destroys the typical > 'rod-like' structure of the genome (c) a modified genome that contains the IFN sequence described above and partially complementary, 'stabilizing' sequences which restore the 'rod-like' RNA structure; and (d) a modified genome that contains the IFN sequence, the EMCV IRES sequence and partially complementary, 'stabilizing' sequences.
• the RNA secondary structure in (d) corresponds to the schematic representation rHDV-huIFNbeta-IRES-HDAg shown in Figure 2.
• Figure 4 is a photographic representation showing that the recombinant HDV genome rHDV-huIFNbeta-IRES-HDAg construct is replication competent.
• COS-7 cells were transfected with plasmids encoding recombinant HDV genomes and/ or helper plasmids providing HDAg in trans.
• Total RNA was extracted 6 days after transfection, cDNA synthesis was performed using a genomic-specific primer, and PCR was performed using primers binding to anti-genomic-specific primers flanking the insertion site for the IFN gene.
• Lane 1 co-transfection of helper plasmids pJC126.S/B and pJC126.S/N; lane 2, pJC126 (encoding wild-type rHDV.JC126); lane 3, pJC126S/B and pJC126.IFN.IRES (encoding rHDV-huIFNbeta-IRES-HDAg, also referred to herein as rHDV.IFN-IRES-HDAg); lane 4, pJC126S/N and
• FIG. 5 is a schematic representation showing another embodiment of a recombinant HDV genome, rHDV-HDAg-IRES-huIFNbeta.
• An internal ribosome entry site (IRES) and the sequence of interest - here exemplified by the human interferon-beta coding sequence (huIFN- ⁇ ) - have been inserted between the hepatitis delta antigen (HDSAg) open reading frame and the polyadenylation signal (poly A).
• HDSAg hepatitis delta antigen
• poly A polyadenylation signal
• FIG. 6 is a schematic representation showing another embodiment of a recombinant HDV genome, rHDV-HDAg-2A-huIFNbeta.
• the sequence of interest - here exemplified by the human interferon-beta coding sequence (huIFN- ⁇ ) - has been fused to a '2A-like' motif (2A) and inserted (in-frame) immediately after the hepatitis delta antigen (HDSAg) coding sequence.
• HDSAg hepatitis delta antigen
• an additional, 'stabilizing', partially complementary sequence has been inserted opposite the first insertion.
• SS signal sequence; asterisk, editing site.
• FIG. 7 is a schematic representation showing a further embodiment of a recombinant HDV genome, rHDV-HDAg-ribo-insertion.
• the region between the anti-genomic ribozyme and the tip of the 'rod' is another potential site for inserting sequences/ genes of interest.
• an additional, 'stabilizing', partially complementary sequence has been inserted opposite the first insertion.
• SS signal sequence; asterisk, editing site.
• Figure 8 is a schematic representation showing an embodiment of a recombinant FIDV genome, designated recombinant virus rHDV.JC126.R. (A)
• RNA secondary structure Predicted RNA secondary structure (genomic RNA) at the insertion site of heterologous nucleotide sequences that were left behind after the removal of a much larger insert containing an IRES and the coding sequence for human IFN-beta. Dotted lines indicate relative positions of 'homologous' loops in genuine HDV RNA with an intact secondary structure. Nts, nucleotides.
• Figure 9 is a schematic representation showing recombinant HDV genomes with insertions immediately upstream of the HDAg coding sequence.
• A Schematic representation of recombinant virus rHDV.HA(HA)-HDAg depicting the relative positions of the HA-tag sequence and a partially complementary, 'stabilizing' sequence.
• B Inserted nucleotide sequences with the edited (to increase G/C content) HA-tag sequence on top and the partially complementary, 'stabilizing' sequence underneath (blue, non-functional start codon; black, HA-tag and complementary nucleotides; red/green, non-complementary nucleotides/ deletions).
• FIG. 10 Schematic representation of rHDV.HA(HA)-HDAg, a recombinant virus that contains the HA-tag sequence but lacks a partially complementary, 'stabilizing' sequence.
• D Predicted RNA secondary structures (genomic RNA; at the insertion sites) for rHDV.HA(HA)- HDAg, rHD V .HA(HA)-HD Ag, and wild-type virus rHDVJC126. Dotted lines indicate relative positions of 'homologous' loops in genuine HDV RNA with an intact secondary structure. Nts, nucleotides.
• Figure 10 is a schematic representation showing recombinant HDV genomes with insertions immediately downstream of the HDAg coding sequence.
• FIG. 1 Schematic representation of recombinant virus rHDV.R.HDAg-HA(AH) depicting the relative position of the HA-tag sequence and a partially complementary, 'stabilizing' sequence.
• B Inserted nucleotide sequences with an edited (to increase G/C content) HA-tag sequence on top and a partially complementary sequence underneath (blue, heterologous nucleotides introduced earlier - for details, see construction of pJC126R; black, HA-tag and complementary nucleotides; red/green, non-complementary nucleotides/deletions).
• C Schematic representation of rHDV.R.HDAg-HA, a recombinant virus that contains the HA-tag sequence but lacks a partially
• RNA secondary structures (genomic RNA; at the insertion sites). Dotted lines indicate relative positions of 'homologous' loops in genuine HDV RNA with an intact secondary structure. Nts, nucleotides.
• Figure 11 is a schematic representation showing recombinant HDV genomes with insertions immediately downstream of the ribozyme sequence, close to the end of the rod.
• rHDV.XbaHA(AH) depicting the relative position of the HA-tag sequence and a partially complementary, 'stabilizing' sequence.
• B Inserted nucleotide sequences with an edited (to increase G/C content) HA-tag sequence on top and a partially
• rHDV.XbaHA a recombinant virus that contains the HA-tag sequence but lacks a partially complementary, 'stabilizing' sequence.
• D Predicted RNA secondary structures (genomic RNA, at the insertion sites). Dotted lines indicate relative positions of ⁇ homologous' loops in genuine HDV RNA with an intact secondary structure. Nts, nucleotides.
• Figure 12 is a photographic representation showing HDV RNA replication of a virus that carries an insert of 6 nucleotides plus additional 'stabilizing'
• COS-7 cells were transfected with plasmids encoding rHDV.JC126 (wild-type virus; lane 1) or rHDV.JC126R ('restored' virus with a small insertion downstream of the HDAg ORF; lane 2).
• Total RNA was prepared 4 days post transfection and RNA samples of approx. 2 ⁇ g were analyzed by Northern blotting using an HDV-specific riboprobe. Position of 1.7-kb HDV RNA is indicated. MWS, Roche RNA marker 0.24 to 9.5 kb.
• FIG. 13 is a photographic representation showing HDV RNA replication of viruses that carry inserts of 6, 27 or more nucleotides plus additional 'stabilizing' (partially complementary) sequences.
• COS-7 cells were transfected with plasmids encoding rHDV.JC126 (parental, wild-type virus; lane 4), rHDV.JC126R ('restored' virus with a small insertion downstream of the HDAg ORF; lane 5), rHDV.HA(AH)-HDAg (virus with an insertion upstream of the HDAg ORF; lane 6), rHDV.R.HDAg-HA(AH) (virus with an insertion downstream of the HDAg ORF, lane 7) or rHDV.XbaHA(AH) (virus with an insertion at the end of the rod; lane 8).
• RNA samples were prepared 4 days post transfection and RNA samples of approx. 2 ⁇ g (lanes 4 to 8) or dilutions thereof (lanes 1 and 2) were analyzed by Northern blotting using an HDV-specific riboprobe (upper panel). To demonstrate RNA quality and equal loading, an ethidium bromide stain of the corresponding agarose gel is also shown (lower panel). Positions of 1.7-kb HDV RNA, 18S ribosomal RNA, and 28S ribosomal RNA are indicated. MWS, Roche RNA marker 0.24 to 9.5 kb.
• Figure 14 is a photographic representation showing HDV RNA replication kinetics of viruses that carry inserts of 27 or more nucleotides plus additional 'stabilizing' (partially complementary) sequences.
• COS-7 cells were transfected with plasmids encoding a range of recombinant genomes, including rHDV.JC126.del 10 (replication-deficient control virus; lane 1), rHDV.JC126 (wild-type virus; lane 2), rHDV.HA(AH)-HDAg (virus with an insertion upstream of the HDAg ORF; lane 3), rHDV.R.HDAg-HA(AH) (virus with an insertion downstream of the HDAg ORF, lane 4), rHDV.XbaHA(AH) (virus with an insertion at the end of the rod; lane 5), and rHDV.XbaHA (virus with an insertion at the end of the rod but no 'stabilizing', partially complementary sequence; lane 6
• RNA samples of approx. 2 ⁇ g were analyzed by Northern blotting using an HDV-specific riboprobe (upper and lower panels shows HDV RNA accumulation at 4 and 8 days post transfection, respectively). Position of 1.7-kb HDV RNA is indicated; d.p.t, days post transfection.
• Figure 15 is a map of plasmid pJC126d. This plasmid is largely identical to parental plasmid pJC126. A small DNA fragment was removed from pJC126 using the restriction enzymes Sbfi and EcoRV. The remaining plasmid was flush-ended and relegated, generating plasmid pJC126d. Key features of the vector backbone (in grey) and the inserted 1.2-fold cDNA copy of the HDV genome (in color) are indicated.
• Figure 16 is a map of the HDV genome rHDVJC126 (circularized). Key virus features such as the HDAg coding sequence, polyadenylation site, and ribozyme sequences are shown. Furthermore, the position of important restriction sites (Nhel, Pstl, Banl, etc) and oligonucleotide primer binding sites (HDV661G, HDV768A, HDV1517A, etc) are shown. Numbering follows the established convention,(Wang et al. 1986, Nature 323:508-14; Wang et al. 1987, Nature 328:456; Kuo et al. 1988, J. Virol. 62:1855-61) but is based on the corrected JC126 sequence (i.e. the depicted genome contains 1,677 rather than 1,679 nucleotides).
• an element means one element or more than one element.
• virus also includes a plurality of viruses.
• 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 histocompatability 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.
• antigenome means a positive sense viral RNA molecule or DNA molecule complementary to the entire negative sense single stranded viral RNA genome.
• 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 sequence of 10 nucleotides in length and a nucleotide sequence of 20 residues in length.
• Attenuation or "attenuated” as used herein means a reduction of viral virulence. Virulence is defined as the ability of a virus to cause disease in a particular host. Thus the term “attenuated” is synonymous with “less pathogenic” or sometimes with “apathogenic”.
• 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.
• cis-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 cr ' s-regulatory region may be capable of activating, silencing, enhancing, repressing or otherwise altering the level of expression and/or cell-type- specificity and/or developmental specificity of any structural gene sequence.
• cistron refers to a section ofDNA or RNA that contains the genetic codes for a single polypeptide or a protein, and may function as a hereditary unit.
• the term “bicistronic” refers to the existence in the recombinant viruses of the invention of two unrelated cistrons which are expressed from a single viral transcriptional unit.
• One cistron may comprise an open reading frame of the virus and the other cistron may comprise a coding sequence for an exogenous polypeptide.
• 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.
• control sequences that are suitable for prokaryotic cells for example, include a promoter, and optionally a m-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., at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,
• 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 a ⁇ iministration 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.
• eliciting or “inducing” an immune response as contemplated herein includes stimulating an immune response and/or enhancing a previously existing immune response.
• encode refers to the capacity of a nucleic acid to provide for another nucleic acid or a polypeptide.
• a nucleic acid sequence is said to "encode” a polypeptide if it can be transcribed and/or translated to produce the polypeptide or if it can be processed into a form that can be transcribed and/or translated to produce the polypeptide.
• Such a nucleic acid sequence may include a coding sequence or both a coding sequence and a non- coding sequence.
• the terms "encode,” "encoding” and the like include an RNA product resulting from transcription of a DNA molecule, a protein resulting from , translation of an RNA molecule, a protein resulting from transcription of a DNA molecule to form an RNA product and the subsequent translation of the RNA product, or a protein resulting from transcription of a DNA molecule to provide an RNA product, processing of the RNA product to provide a processed RNA product (e. g. , mRNA) and the subsequent translation of the processed RNA product.
• a processed RNA product e. g. , mRNA
• 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 results from the transcription of the non-coding sequence.
• HDV hepatitis delta virus
• 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.
• the term “gene” includes and encompasses a nucleic acid molecule capable of being used to produce mRNA, antisense RNA, siRNA, shRNA, miRNA and the like. Genes may or may not be capable of being used to produce a functional protein. Genes can include both coding and non-coding regions.
• heterologous refers to molecules (e.g., nucleic acid molecules, polypeptides etc.) that are in a cell or a virus where they are not normally found in nature; or, may comprise two or more subsequences that are not found in the same relationship to each other as are normally found in nature, or are recombinantly engineered so that their level of expression, or physical relationship to other molecules in a cell, or structure, is not normally found in nature.
• heterologous nucleotide sequence 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 heterologous nucleotide sequence 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 recombinant HDV (e.g., attenuation) into which it is introduced, or otherwise leads to a desired therapeutic or diagnostic outcome.
• the heterologous nucleotide sequence comprises a non-coding nucleotide sequence that is not transcribed. In some embodiments, the heterologous nucleotide sequence comprises a non-coding nucleotide sequence that is transcribed. Non-limiting non-coding sequences include functional RNA molecule such as rRNA, tRNA, RNAi, shRNA, siRNA, miRNA, ribozymes and antisense RNA. In some embodiments, the heterologous nucleotide sequence interferes with transcription or translation (e.g. , antisense molecule) or mediates RNA interference (e.g., RNAi, siRNA, shRNA, miRNA, etc.). In some embodiments, the heterologous nucleotide sequence comprises a plurality of coding sequences, which in illustrative examples encode the same exogenous polypeptide or different exogenous polypeptides.
• heterologous polypeptide refers to any peptide or polypeptide which is encoded by a heterologous nucleotide sequence
• foreign nucleotide sequence refers to any peptide or polypeptide which is encoded by a heterologous nucleotide sequence
• foreign nucleotide sequence refers to any peptide or polypeptide which is encoded by a heterologous nucleotide sequence
• foreign nucleotide sequence refers to a cell into which a vector including a recombinant HDV 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, an illustrative example host cell of which includes a hepatocyte ) 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 nucleotide 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 histocompatability antigen molecule), can elicit an immune response in a subject.
• an accessory molecule e.g., a class I or class II major histocompatability antigen molecule
• inducing includes eliciting or 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 (i.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 cis- acting 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.
• live virus refers to a virus that retains the ability of infecting and replicating in an appropriate subject or host cell.
• 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 ⁇ e.g. , a promoter
• operably linked 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" an heterologous nucleotide sequence to the ORF of a HDV encompasses positioning and/or orientation of the heterologous nucleotide sequence relative to the HDV ORF so that (1) the heterologous nucleotide sequence and the ORF are transcribed together to form a single cliimeric transcript and optionally (2) if the heterologous nucleotide sequence itself comprises a coding sequence, the coding sequence of the heterologous nucleotide sequence is 'in-frame' with the HDV ORF to produce a chimeric open reading frame comprising the coding sequence of the heterologous nucleotide sequence and the HDV ORF.
• an IRES operably connected to the coding sequence of an heterologous nucleotide sequence refers to positioning and/or orientation of the IRES relative to the coding sequence to permit cap- independent translation of the 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 (mR A translation).
• the term "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.
• the terms "polynucleotide,” “polynucleotide sequence,” “nucleotide sequence,” “nucleic acid” or “nucleic acid sequence as used herein designate mR A, 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.
• This term also includes within its scope two or more 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.
• nucleic acid molecules As used herein the term "recombinant” as applied to "nucleic acid molecules," “polynucleotides” and the like is understood to mean artificial nucleic acid structures (i.e., non-replicating cDNA or RNA; or replicons, self-replicating cDNA or RNA) which can be transcribed and/or translated in host cells or cell-free systems described herein.
• Recombinant nucleic acid molecules or polynucleotides may be ⁇ inserted into a vector.
• Non-viral vectors such as plasmid expression vectors or viral vectors may be used. The kind of vectors and the technique of insertion of the nucleic acid construct according to this invention is known to the artisan.
• a nucleic acid molecule or polynucleotide according to the invention does not occur in nature in the arrangement described by the present invention.
• an heterologous nucleotide sequence is not naturally combined with elements of a parent virus genome (e.g. , promoter, ORF, polyadenylation signal, ribozyme).
• a parent virus genome e.g. , promoter, ORF, polyadenylation signal, ribozyme.
• 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, Arg, His
• sequence identity will be understood to mean the "match percentage” calculated by the DNASIS computer program (V ersion 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.
• signal sequence or “signal peptide” refers to a short
• peptide that directs co- or post-translational transport of a protein from the cytosol to certain organelles such as the nucleus, mitochondrial matrix, and endoplasmic reticulum, for example.
• organelles such as the nucleus, mitochondrial matrix, and endoplasmic reticulum, for example.
• ER targeting signal peptide the signal peptides are typically cleaved from the precursor form by signal peptidase after the proteins are transported to the ER, and the resulting proteins move along the secretory pathway to their intracellular (e.g., the Golgi apparatus, cell membrane, or cell wall) or extracellular locations.
• ER targeting signal peptides include amino-terminal hydrophobic sequences which are usually enzymatically removed following the insertion of part or all of the protein through the ER membrane into the lumen of the ER.
• a signal precursor form of a sequence can be present as part of a precursor form of a protein, but will generally be absent from the mature form of the protein.
• ER targeting signal peptides or sequences that are functional in mammalian cells include the following: the signal sequence for interleukin-7 (IL-7) described in U.S. Patent No. 4,965,195; the signal sequence for interleukin-2 receptor described in Cosman et al. ((1984), Nature 312:768); the interleukin-4 receptor signal peptide described in EP Patent No. 0 367 566; the type I interleukin-1 receptor signal sequence described in U.S. Patent No. 4,968,607; the type II interleukin-1 receptor signal peptide described in EP Patent No. 0 460 846; the signal sequence of human IgG
• ER-targeting signal sequences include ones from prokaryotes ⁇ e.g., viruses), insects (copepods, ostracods, etc.), reptilians and avians as well as artificial ER targeting signal sequences such as: LLLVGILFWA and MLLLLLLLLPQAQA.
• 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 Research!: 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.
• references to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence,” “comparison window”, J “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. Because two polynucleotides may each comprise (1) a sequence (z. e.
• 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 ahgning 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.
• subject refers to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired.
• Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chord ' ata including primates (e.g.
• monkeys 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., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs),
• 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 and/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 refers to a sequence located upstream (i.e., 5') of a coding region.
• a 5' UTR is located downstream (i.e. , 3') to a promoter region and 5' of a coding region downstream of the promoter region.
• sequence while transcribed, is upstream 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 sequence downstream (i.e., 3') of a coding sequence. It generally 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.
• wild-type refers to a genotype of a virus found in nature.
• the rod-like secondary structure of the HDV genome can be retained, maintained or restored by inserting a "stabilizing" heterologous nucleotide sequence generally at a different site in the HDV genome to the one used for inserting the heterologous nucleotide sequence of interest, in which the stabilizing heterologous nucleotide sequence is substantially complementary to the heterologous nucleotide sequence of interest, whereby the heterologous nucleotide sequences are in juxtaposition to permit annealing to each other and to thereby retain, maintain or restore the rod-like secondary structure of the HDV genome.
• Recombinant HDV genomes constructed according to this strategy are remarkably stable over multiple replication cycles.
• the present invention provides a recombinant single- stranded, circular HDV RNA genome, which comprises or consists essentially of in operable connection: a promoter; an ORF for a HDAg; a polyadenylation signal; and a HDV ribozyme, wherein the genome comprises substantially complementary portions conferring a rod-like secondary structure, and wherein the genome comprises at a first site a first heterologous nucleotide sequence and at a second site a second heterologous nucleotide sequence that is substantially complementary to the first heterologous nucleotide sequence wherein the first and second sites are spaced from each other to permit annealing between the first and second heterologous nucleotide sequences.
• the HDV genome will comprise in order from 5' to 3', the promoter, the ORF, the polyadenylation signal and the ribozyme.
• the first site which defines where the first heterologous nucleotide sequence is inserted into the HDV genome, can be between adjacent genome elements (e.g., between the promoter and the ORF, or between the ORF and the polyadenylation signal or between the polyadenylation signal and the ribozyme or downstream of the ribozyme).
• the second site which defines where the second 'stabilizing' heterologous nucleotide sequence is inserted into the HDV genome, will generally be located between portions of the HDV genome that are substantially complementary and anneal to the adjacent elements between which the first heterologous nucleotide sequence is inserted.
• the first and second sites are chosen so that insertion of the first and second heterologous nucleotide sequences into those sites does not interfere or impair annealing of the complementary portions of the parent genome (e.g. , so that the rod-like secondary structure of the HDV genome is retained, maintained or restored).
• the first and second heterologous nucleotide sequences will generally display at least 50% (and at least 51% to at least 99% and all integer percentages in between) and up to 100% sequence identity to each other to permit annealing therebetween.
• the codon composition of the coding sequence is modified/optimized using the degeneracy of the genetic code so that the G/C content of the coding sequence is substantially in accord with the G/C content of the parent HDV genome.
• RNA structure prediction software/algorithm includes RNAfold available on the RNAfold Webserver (which is currently operated by the Institute for Theoretical Chemistry, University of Vienna, Austria
• Illustrative sources for these software/algorithms can be found for example at: http://en.wikipedia.org/wiki
• the first heterologous nucleotide sequence is inserted downstream of the promoter and upstream of the ORF and the second heterologous nucleotide sequence is inserted downstream of the nucleotide sequence of the parent genome that is substantially complementary and anneals to the ORF and upstream of the nucleotide sequence of the parent genome that is substantially complementary and anneals to the promoter.
• the first heterologous nucleotide sequence comprises a coding sequence for an exogenous polypeptide, which is operably connected to the promoter, and an internal ribosome entry site (IRES) that is operably connected to the ORF, to define a bicistronic recombinant HDV genome.
• the first heterologous nucleotide sequence comprises a first coding sequence for an exogenous polypeptide and a second coding sequence for a proteolytic cleavage site, wherein the first and second coding sequences are in frame with each other and with the ORF to thereby encode a precursor polypeptide, wherein the second coding sequence is downstream of the first coding sequence and upstream of the ORF, wherein the proteolytic cleavage site is positioned between the exogenous polypeptide and the HDAg in the precursor polypeptide to facilitate release of the exogenous polypeptide upon proteolytic cleavage of the proteolytic cleavage site.
• the first heterologous nucleotide sequence is inserted downstream of the ORF and upstream of the polyadehylation signal and the second heterologous nucleotide sequence is inserted downstream of the nucleotide sequence of the parent genome that is substantially complementary and anneals to the polyadenylation signal and upstream of the nucleotide sequence of the parent genome that is substantially complementary and anneals to the ORF.
• the first heterologous nucleotide sequence comprises an internal ribosome entry site (IRES) operably connected to a coding sequence for an exogenous polypeptide, to define a bicistronic recombinant HDV genome.
• IRS internal ribosome entry site
• the first heterologous nucleotide sequence comprises a first coding sequence for an exogenous polypeptide and a second coding sequence for a proteolytic cleavage site, wherein the first and second coding sequences are in frame with each other and with the ORF to thereby encode a precursor polypeptide, wherein the second coding sequence is downstream of the ORF and upstream of the first coding sequence, wherein the proteolytic cleavage site is positioned between the exogenous polypeptide and the HDAg in the precursor polypeptide to facilitate release of the exogenous polypeptide upon proteolytic cleavage of the proteolytic cleavage site.
• the methods comprise inserting the first heterologous nucleotide sequence downstream of the ribozyme and upstream of portions of the parent genome that are substantially complementary and anneal to each other and inserting the second heterologous nucleotide sequence downstream of those portions and upstream of the nucleotide sequence of the parent genome that is substantially complementary and anneals to the ribozyme.
• the first heterologous nucleotide sequence is operably connected to another promoter (e.g., a promoter other than the promoter that is operably connected to the ORF) and suitably to another, polyadenylation site (e.g., a polyadenylation site other than the polyadenylation site that is operably connected to the ORF).
• another promoter e.g., a promoter other than the promoter that is operably connected to the ORF
• an operably connected promoter in the recombinant genome is a DNA dependent RNA polymerase (e.g., RNA polymerase I and/or RNA polymerase II) promoter, illustrative * examples of which include native or wild-type HDV promoters.
• deletion of a nucleotide sequence in an HDV genome should be suitably accompanied by a corresponding deletion of another nucleotide sequence that is substantially complementary and anneals to the first nucleotide sequence so that the resulting recombinant HDV genome retains or maintains a substantially rod-like secondary structure.
• the present invention in another aspect may be more broadly defined as a recombinant single-stranded, circular HDV RNA genome, which comprises or consists essentially of a first site and a second site that are spaced from each other, wherein the first site is distinguished from a corresponding site in a parent HDV genome by the addition, deletion or substitution of at least one nucleotide (e.g., at least about 2 nucleotides to at least about 3000 nucleotides and all integer nucleotides in between) and the second site is distinguished from a corresponding site in the parent HDV genome by the addition, deletion or substitution of at least one nucleotide (e.g., at least about 2 nucleotides to at least about 3000 nucleotides and all integer nucleotides in between), wherein the first and second sites are substantially complementary to permit annealing to each other so that the recombinant HDV genome retains or maintains a substantially rod-like secondary structure.
• 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 suitably in the presence of HBV that has also infected the host cell.
• 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 heterologous nucleotide sequence to produce the recombinant virus.
• a specific type of recombinant virus e.g. , a "recombinant HDV” denotes a parent virus of the indicated type that has been modified to incorporate foreign or exogenous genetic material.
• the present invention encompasses any recombinant viruses classified within the Deltavirus genus under the International Committee on Taxonomy of Viruses (ICTV).
• Such "delta viruses” will include any genotype of HDV including, but not limited to, HDV genotypes I, II, III, IV, V, VI and VII.
• Non-limiting parent HDV genomes for insertion of heterologous nucleotide sequence according to the present invention are listed in Table B:
• HDAg Hepatitis D virus delta antigen
• HDV Hepatitis delta virus
• AM779594.1 Hepatitis delta virus dTk27 LHD gene for large HD antigen, genomic
• HM046802.1 Hepatitis delta virus isolate JN, complete genome
• AM779591.1 Hepatitis delta virus dTk5 LHD gene for large HD antigen, genomic
• RNA strain dTkl 0 U81989.1 Hepatitis delta virus from Ethiopia genotype IC, complete genome
• HDAg Hepatitis delta virus large and small antigens
• genomic RNA genomic RNA
• Hepatitis delta virus dFr2411 LHD gene for large HD antigen genomic RNA
• HDV hepatitis D virus
• the recombinant HDV genome is prepared using the parent HDV RNA genome (-) JC126 (also referred to herein as
• rHDV.JC126 which comprises, consists or consists essentially of the nucleotide sequence:
• cDNA is generally used to make the recombinant HDV genome and thus the present invention encompasses the use ofa cDNA sequence corresponding to the HDV JC126 strainparent R A genome, which comprises, consists or consists essentially ofthe nucleotide sequence:
• nucleotide 45 A to T
• nucleotide 221 T deleted
• nucleotide 619 C deleted
• nucleotide 840 C to G
• nucleotide 1343 G to C
• nucleotide 1389 G to C
• nucleotide 1633 A to T
• the corrected sequence contains only 1,677 nucleotides.
• Recombinant viruses of the present invention comprise a first heterologous nucleotide sequence or interest (HSI), which encompasses any nucleotide sequence inserted into the genome of the parent virus, which does not normally exist or naturally occur in that genome.
• the HSI 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 HSI comprises a nucleotide sequence from a HDV that is different to the parent virus including, but not limited to, different viral strains; and different viral serotypes.
• the HSI may be a non-coding nucleotide sequence, which may be transcribed or not transcribed. Alternatively, or in addition, the HSI may encode an exogenous
• the non-coding sequence may interfere with transcription or translation (e.g. , antisense molecule) or mediate RNA interference.
• the non-coding sequence is used to mediate RNA interference, via interaction with RNA transcripts or alternately by interaction with particular gene sequences, wherein such interaction results in modulation of gene silencing either at the transcriptional level or post- transcriptional level such as, for example, but not limited to, RNAi or through cellular processes that modulate the chromatin structure or methylation patterns of the target and prevent transcription of the target gene, with the nucleotide sequence of the target thereby mediating silencing.
• Non-limiting examples of such non-coding sequences include functional RNA molecule such as rRNA, tRNA, RNAi, shRNA, siRNA, miRNA, ribozymes and antisense RNA.
• the exogenous polypeptide is suitably selected from 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 HDV 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 HDV 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-l (also referred to as HTLV-III, LAV or HTLV-III/LAV, or fflV- ⁇ ); and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses); Caliciviridae (e.g., strains that cause gastroenteritis, including
• caliciviruses such as Norwalk virus); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses, hepatitis C virus); Coronaviridae ⁇ e.g., coronaviruses); 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); Bunyaviridae ⁇ e.g., Hantaan viruses, orthobunyaviruses, phlebo viruses and nairo viruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae ⁇ e.g., reoviruses, orbiviruses and rotaviruses); Birnaviridae; 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)); Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae ⁇ e.g., African swine fever virus); and unclassified viruses; and astroviruses.
• HSV herpes simplex virus
• CMV varicella zoster virus
• Poxviridae variola viruses, vaccinia viruses, pox viruses
• Iridoviridae ⁇ e.g., African swine fever virus
• unclassified viruses and astroviruses.
• Pathogenic bacteria include, but are not limited to, Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophila, Mycobacteria sps ⁇ e.g., M. tuberculosis, M. avium, M. intracellular, M. kansaii, M.
• Streptococcus pyrogenes Group A Streptococcus
• Streptococcus agalactiae Group B Streptococcus
• Streptococcus viridans group
• Streptococcus faecalis Streptococcus bovis
• Streptococcus anaerobic spp.
• 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). Examples of natural, animal and plant allergens include proteins specific to the following genera: Canine (Canis familiaris); Dermatophagoides (e.g. ,.
• 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).
• 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 hal
• any of a variety of known cancer- or tumor-associated antigens can be inserted into a HDV of the invention.
• the entire antigens may be, but need not be, inserted.
• 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 HDV 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, 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 p21ras.
• MAGE-2 MAGE-3
• MUC- 1 MUC-2
• HER-2 high molecular weight melanoma-associated antigen MAA
• CEA carcinoembryonic antigen
• 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. (1995) Human Reprod. Update 1:1-18; Kerretal. (1998) J Reprod. Immunol. 40:103- 118; and U.S. Pat. No. 6,197,940.
• 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.
• a-neurotoxins e.g., snake venoms
• a-neurotoxins e.g., kappa toxins, ⁇ -neurotoxins, dendrotoxins, cardiotoxins, myotoxins, and hemorrhaging.
• modified venoms that elicit specific antibodies, but are not themselves toxic.
• Such 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, mterleukins, 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, mterleukins, 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 interleukins, interferons, tumor necrosis factor-alpha (TNF-a), alpha defensins, RANTES (CCL5), CXCL10 (IP 10) and the like.
• the HSI 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 I interferon.
• the interferon may be a mammalian type I interferon (e.g. , interferon-alpha (IFN- a), interferon-beta (IFN- ⁇ ), interferon-kappa (IFN-K), interferon-delta (IFN- ⁇ ), interferon- epsilon (IFN- ⁇ ), interferon-tau (IFN- ⁇ ), interferon-omega (IFN- ⁇ ), or interferon-zeta (IFN- ⁇ )).
• IFN-alpha IFN-alpha
• IFN- ⁇ interferon-beta
• IFN-K interferon-kappa
• IFN-delta IFN- ⁇
• interferon- ⁇ interferon-epsilon
• IFN- ⁇ interferon-tau
• IFN- ⁇ interferon-omega
• the interferon may be a type II interferon (e.g., interferon- gamma (IFN- ⁇ )) or a type III interferon (e.g., an IFN- ⁇ such IFN- ⁇ , IFN-A2 and IFN- ⁇ A3).
• IFN- ⁇ interferon-gamma
• type III interferon e.g., an IFN- ⁇ such IFN- ⁇ , IFN-A2 and IFN- ⁇ A3
• interferons with the recombinant HDVs of the present invention is particularly advantageous as interferons interfere with viral replication within host cells, activate immune cells, such as natural killer cells and macrophages; increase recognition of infection or tumor cells by up-regulating antigen presentation to T lymphocytes; and increase the ability of uninfected host cells to resist new infection by virus.
• interferon-expressing HDVs are useful in a range of applications including the treatment of viral infections (e.g., HBV infections, HBV/HDV co-in
• the cytokine is interferon-beta (IFN- ⁇ ).
• the cytokine is mammalian interferon-beta (IFN- ⁇ ), and more suitably human interferon-beta (IFN- ⁇ ).
• IFN- ⁇ mammalian interferon-beta
• human interferon-beta IFN- ⁇
• the human interferon-beta may be defined by the amino acid sequence set forth in
• the HSI encoding the exogenous protein to be produced by a host cell following infection of the host cell by a recombinant HDV of 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 RNA of an organism, or by standard recombinant methods (Sambrook et al., (1989) "Molecular Cloning: A Laboratory Manual , (2nd ed., Cold Spring.HarbOr Laboratory Press, Plainview, New York; and Ausubel et al.
• 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 polypeptide can further include sequences that direct secretion of the protein from the cell, sequences that alter RNA and/or protein stability, and the like.
• a coding sequence of the HSI 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 the HDAg.
• the proteolytic cleavage site is a so-called "self- cleaving" peptide sequence.
• Illustrative so-called self-cleaving peptides are encoded by some picornaviruses as well as a number of other single- and double-stranded RNA viruses (Doronina et al, 2008, Biochem Soc Trans. 36:712-716; de Felipe 2004, Genet Vaccines Ther.
• self-cleaving peptides are selected from so-called "2A" and "2A-like" self-cleaving sequences, which suitably comprise the consensus motif D-V7I-E-X-N-P- G-P. These sequences are understood to act co-translationally by preventing the formation of a normal peptide bond between the glycine and last proline in the motif, resulting in the ribosome skipping to the next codon, and the production of separate peptides.
• the short 2 A or 2A-like peptide remains fused to the C-terminus of the upstream protein, while the proline is added to the N-terminus of the downstream protein.
• Other sequences encoding proteolytic cleavage sites and methods for their incorporation into polypeptides of interest are well known in the art and described in standard texts.
• the proteolytic cleavage site-encoding sequence is placed upstream (i.e., 5') of the HSI coding sequence.
• the nucleotide sequence encoding the proteolytic cleavage site is located upstream (i.e., 5') and downstream (i.e., 3') of the HIS coding sequence.
• the proteolytic cleavage site-encoding sequence is placed upstream (i.e., 3') of the HSI coding sequence.
• the proteolytic cleavage sites is positioned to facilitate release of the encoded exogenous polypeptide upon proteolytic processing of a recombinant viral polyprotein precursor comprising the encoded exogenous
• a coding sequence of the HSI 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.
• a coding sequence of the HSI is operably linked to an internal ribosome binding site (IRES).
• IRES internal ribosome binding site
• IRES sequences are known in the art and include, but are not limited to, IRES sequences derived from 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), GTX, Cyr61 a, Cyr61b, poliovirus, the immunoglobulin heavy-chain-binding protein (BiP), immunoglobulin heavy chain, a picomavirus, murine encephalomyocarditis virus, poliovirus, and foot and mouth disease virus (e.g., nucleotide numbers 600-1058 of the nucleotide sequence provided under GenBank Accession No.
• BVDV bovine viral diarrhea virus
• EMCV encephalomyocarditis virus
• HCV hepatitis C virus
• SiP immunoglobulin heavy-chain-binding
• 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 HDVs 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.
• an heterologous nucleotide sequence is from about 12 to about 3000 nucleotides in length, for example from 12 to about 18, from about 15 to about 24, from about 21 to about 30, from about 30 to about 60, from about 60 to about 90, from about 90 to about 120, from about 120 to about 150, from about 150 to about 180, from about 180 to about 240, from about 240 to about 300, from about 300 to about 600, from about 600 to about 1200, from about 1200 to about 1500, from about 1500 to about 2100, from about 2100 to about 2400, or from about 2400 to about 3000 nucleotides in length.
• an heterologous nucleotide sequence is no more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 nucleotides in length.
• the first and second heterologous nucleotides sequences combined are no more than 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 nucleotides in length.
• the exogenous polypeptide is from about 4 to about 1000 amino acids in length, for example from about 4 to about 6, from about 5 to about 8, from about 7 to about 10, from about 10 to about 20, from about 20 to about 30, from about 30 to about 40, from about 40 to about 50, from about 50 to about 60, from about 60 to about 80, from about 80 to about 100, from about 100 to about 200, from about 200 to about 400, from about 400 to about 500, from about 500 to about 700, from about 700 to about 800, or from about 800 to about 1000 amino acids in length.
• an exogenous polypeptide is no more than 3, 4, 5, 6, 7, 8 radical 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, 150, 300, 400, 500, 600, 700, 800, 900, 1000 ⁇ amino acids in length.
• the present invention also encompasses processes for preparing a recombinant HDV RNA genome of the invention.
• Illustrative process include constructing antigenomic cDNA corresponding to the recombinant HDV RNA antigenome and transcribing the cDNA to form a mixture containing an antigenomic RNA; and thereafter isolating the antigenomic RNA.
• These processes are well known to persons of skill in the art and include standard recombinant methods as described for example by Sambrook et al, (1989) supra; and Ausubel et al. (Eds), (2000-2010), supra.
• the present invention is also directed toward a recombinant HDV comprising a recombinant antigenomic RNA that comprises heterologous nucleotide sequences as described herein.
• the recombinant HDV can be produced by:
• a cell e.g. , a hepatocyte such as a differentiated hepatocyte
• cell line e.g., HepG2.2.15
• HBV envelope proteins or envelope proteins from a related virus (e.g. , woodchuck hepatitis virus (WHV));
• WBV woodchuck hepatitis virus
• a cDNA comprising a nucleotide sequence corresponding to a recombinant antigenomic HDV RNA, a cell containing the cDNA, a vector comprising the cDNA, a cell containing the cDNA, a cell containing the recombinant antigenomic RNA, and a recombinant HDV containing the recombinant RNA genome of the invention or antigenome thereof.
• the recombinant HDV containing the recombinant RNA genome of the invention or antigenome thereof is in isolated form or is substantially purified.
• compositions including pharmaceutical compositions, comprising a recombinant HDV of the invention.
• compositions may include a buffer, which is selected according to the desired use of the recombinant HDV, and may also include other substances appropriate to the intended use. Where the intended use is to induce an immune response, the composition is referred to as an "immunogenic" or “immunomodulating" composition.
• compositions include preventative compositions (i.e., compositions administered for the purpose of preventing a condition such as an infection) and therapeutic agents that are administered for the purpose of preventing a condition such as an infection.
• 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 (i.e., different) recombinant HDV of the invention (e.g. , recombinant HDV comprising different heterologous nucleic acid sequences including different HSIs).
• 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 aluminum-based adjuvants that may be utilized 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
• 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 lipopolys
• 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 HDV composition is administered in an "effective amount" that is, an amount effective to achieve production of the exogenous
• 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).
• the appropriate dosage of 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).
• the appropriate dosage of 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).
• the appropriate dosage of 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
• 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
• the dose of recombinant HDV administered to an individual will generally be in a range of from about 10 2 to about 10 s , from about 10 3 to about 10 6 , or from about 10 4 to about 10 5 genome equivalents (GE).
• an "effective amount" of a subject recombinant HDV is an amount sufficient to achieve a desired therapeutic effect.
• an "effective amount" of a subject recombinant HDV is an amount of recombinant HDV 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 HDV, or treated with the parent HDV.
• 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 HDV is an amount of recombinant HDV 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 HDV or treated with the parent HDV.
• Methods of measuring tumor growth and numbers of tumor cells are known in the art.
• the amount of recombinant HDV in each dose is selected as an amount that induces an immune response to the encoded exogenous polypeptide antigen, and/or that 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 HDV nucleic acid-based composition will generally involve administration of from about 1-1000 ⁇ g of nucleic acid.
• an effective dose of recombinant HDV will generally be in a range of from about 10 2 to about 10 8 , from about 10 3 to about 10 6 , or from about 10 4 to about 10 5 genome equivalents (GE).
• 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. Following an assessment of antibody titers in the serum, optional booster immunizations may be desired.
• the immune response to the protein of this invention is enhanced by the use of adjuvant and/or an immunostimulant.
• 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).
• parenteral e.g., intravenous
• a pharmaceutical composition of the present invention may be administered to a recipient in isolation or in conjunction with 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 admimstered 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.
• two or more entities are administered to a subject "in conjunction” or “concurrently” they may be admimstered in a single composition at the same time, or in separate compositions at the same time, or in separate compositions separated in time.
• 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., enzyme-linked immunosorbent assay), solution phase assays (e.g., enzyme-linked immunosorbent assay), solution phase assays (e.g., enzyme-linked immunosorbent assay), solution phase assays (e.g., enzyme-linked immunosorbent assay), solution phase assays (e.g., enzyme-linked immunosorbent assay), solution phase assays (e.g., enzyme-linked immunosorbent assay), solution phase assays (e.g., enzyme-linked immunosorbent assay), solution phase assays (e.g., enzyme-linked immunosorbent assay), solution phase assays (e.g., enzyme-linked immunosorbent assay), solution phase assays (e.g., enzyme-linked immunosorbent assay), solution phase assays
• 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 NK cell responses, IFN- ⁇ production by immune cells, quantification of virus RNA/DNA in tissues or biological fluids (e.g., quantification of HBV RNA (GE) in serum, or quantification of HBV cccDNA in the liver), oxidative burst assays, cytotoxic-specific cell lysis assays, pentamer binding assays, and phagocytosis and apoptosis evaluation.
• GE HBV RNA
• oxidative burst assays oxidative burst assays
• cytotoxic-specific cell lysis assays cytotoxic-specific cell lysis assays
• Recombinant HDVs of the invention are useful to deliver an exogenous polynucleotide (e.g., an RNA molecule such as but not limited to a functional RNA molecule) or an exogenous polypeptide to a vertebrate host (e.g. , in the liver of the host) to produce a desired outcome (e.g., to modulate the expression of a gene of interest, to produce a therapeutic polypeptide, to elicit or increase a immune response to an antigen encoded by the recombinant virus, etc.).
• an exogenous polynucleotide e.g., an RNA molecule such as but not limited to a functional RNA molecule
• an exogenous polypeptide e.g., an exogenous polypeptide
• a vertebrate host e.g., in the liver of the host
• a desired outcome e.g., to modulate the expression of a gene of interest, to produce a therapeutic polypeptide, to
• Recombinant HDVs of the present invention are also useful for producing the exogenous polynucleotides or polypeptide in host cells, such as mammalian, particularly human, cells or other cell types.
• 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 functional RNA molecule to a vertebrate host (e.g., a mammal).
• the methods generally involve administering a recombinant HDV of the invention to a vertebrate host, wherein the recombinant virus enters a host cell and produces a functional RNA molecule.
• the functional RNA molecule effects gene silencing and is useful for gene silencing therapy.
• Illustrative functional RNA molecules of this type include molecules that mediate RNA interference such as RNAi, shRNA, siRNA, miRNA and the like.
• gene silencing therapy refers to administration to a vertebrate host (e.g., a mammal) of a recombinant HDV of the invention from which a functional RNA molecule that mediates RNA interference is produced in the host (e.g. , cells of the host).
• the vertebrate host suitably has a condition that is amenable to treatment with gene silencing therapy, including conditions such as genetic diseases (i.e.
• RNA molecule targets a gene associated with the condition to be treated, which includes a gene that is either the cause, or is part of the cause, of the condition to be treated.
• genes associated with a neurodegenerative disease e.g., a trinucleotide- repeat disease such as a disease associated with polyglutamine repeats, Huntington's disease, and several spinocerebellar ataxias
• genes encoding ligands for chemokines involved in the migration of a cancer cells, or chemokine receptor e.g., a trinucleotide- repeat disease such as a disease associated with polyglutamine repeats, Huntington's disease, and several spinocerebellar ataxias
• RNA molecules that mediate RNA interferences can be used for treating infections of the host by other viruses.
• the present invention provides methods for delivering a polypeptide to a vertebrate host (e.g., a mammal).
• the methods generally involve administering a recombinant HDV of the invention to a vertebrate host, wherein the virus enters a host cell and the exogenous polypeptide is expressed either by itself or as a polyprotein with HDAg, which is optionally processed in a host cell to provide separate polypeptides.
• 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, the exogenous polypeptide 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 exogenous polypeptide is one that has therapeutic activity, such that when the protein is produced in the mammalian host, a therapeutic effect is achieved. Whether, and at what level, 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 HDV 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 HDVs as described herein are useful for inducing an irnmu e response to an antigen in an individual.
• the exogenous polypeptide When the exogenous polypeptide is produced in a vertebrate host, it induces 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.
• Recombinant HDV of the invention are, therefore, useful as immunomodulating compositions (also referred to herein as "immunogenic compositions”) to elicit and/or enhance an immune response to the antigen.
• the exogenous polypeptide is an antigenic polypeptide of a microbial pathogen.
• Such recombinant HDVs can then be administered to a host to prevent or treat infection by the pathogen, or to prevent or treat symptoms of such pathogenic infection.
• microbial pathogens that, during the course of infection, are present intracellularly, e.g. , viruses (e.g. , HIV), bacteria (e.g.
• antigenic polypeptides of such microbial pathogens are well known in the art, and can be readily selected for use in the present recombinant HDV immunomodulating composition by the ordinarily skilled artisan.
• a recombinant HDV 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 HDV 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.
• epitopes which could be delivered to a host in a multivalent HDV 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 naive 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 is inserted into a recombinant HDV of the invention as described herein.
• a recombinant HDV of the invention can be administered to an individual having, or suspected of having, a cancer or tumor.
• such recombinant HDV can be administered to an individual who does not have a cancer or tumor, but in whom protective immunity is desired.
• 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.
• Non-limiting cancer- or tumor-associated antigens which may be inserted into HDV 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, CA-125, CA-50, CA-19-9, renal tumor-associated antigen G250, EGP-40 (also known as EpCAM), S100 (malignant melanoma-associated antigen), p53, prostate rumor-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 ovarian-associated antigens
• 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).
• r with infection, by a pathogenic organism can be readily determined by those skilled in the art using standard assays, e.g., 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 are known in the art and 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.
• 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 HDV 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 recombinant virus stability infection.
• One improvement may be that an heterologous nucleotide sequence inserted in a HDV genome may remain genetically stable (i. e. resist mutation/deletion) over extended numbers of viral replication cycles, provided that a substantially complementary heterologous nucleotide sequence is also inserted into the HDV genome at a different site to permit annealing between the heterologous nucleotide sequences.
• an exogenous cytokine expressed by a recombinant virus of the present invention e.g. , a type-I IFN such as interferon- beta
• a type-I IFN 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 I IFN 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 e.g., a type I IFN such as interferon- beta
• a type I IFN such as interferon- beta
• Still another improvement may be that 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.
• 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.
• the inserted human IFN- ⁇ coding sequence is based on the GenBank reference sequence NM 002176.2 but the sequence has been artificially edited to (i) increase the G/C content, i.e. 88 nucleotides in 78 codons have been edited to increase the G/C content from 45 to 60%; (ii) optimize codon usage; and (iii) destroy alter-native open reading frames. Furthermore, partially complementary, so-called 'stabilizing' sequences that 'mirror' the IFN coding and IRES sequence have been inserted into the genome to restore the 'rod-like' RNA secondary structure of the genome/ anti-genome that has been destroyed by the first insertion.
• sequences have also been artificially edited to (i) reduce complementarity (85 mismatches, 10 insertions, and 9 deletions); (ii) introduce 'Wobble' base-pairing; and (iii) introduce a unique restriction site. Due to the editing of the IFN gene and any complementary sequence, the present inventors were not able to rely oh 'conventional' cDNAs for the generation of recombinant HDVs; instead they employed in vitro gene synthesis to generate all inserts.
• nucleotide sequence of rHDV-huIFNbeta-IRES-HDAg comprises, consists or consists essentially of the sequence:
• IFN-beta coding sequence is shown in gray italics
• the present inventors have extensively modeled the consequences of large insertions into the wild-type HDV genome in silico by using RNAfold, a minimum free energy RNA structure prediction program (University of Vienna,
• the present inventors also conducted a series of experiments in which they transfected cultured (COS) cells with eukaryotic expression plasmids encoding a 1.2-length cDNA clone of rHDVIFNbeta-IRES-HDAg and helper plasmids that provide small amounts of the HDAg in trans.
• COS transfected cultured
• cDNA was synthesized using an genomic-specific primer and then analyzed for HDV RNA by PCR using a primer pair that amplifies a product encompassing the insertion site for the IFN coding and IRES sequence.
• the results shown in Figure 4 indicate that rHDVIFNbeta-IRES-HDAg indeed replicates.
• the present inventors have repeated the experiments with different primers and analyzed different time points, which has revealed that specific bands can be amplified at day 6 and day 9 but disappear later (data not shown) a finding that is in line with the intracellular replication pattern of the wild-type HDV genome.
• FIG. 5 An alternative strategy for insertion and expression of heterologous nucleotide sequences in HDV is shown in Figure 5, which illustrates a schematic representation of the construct rHDV-HDAg-IRES-huIFNbeta.
• an IRES sequence followed by the human IFN- ⁇ sequence is inserted downstream of the HDAg open reading frame and before the poly A signal.
• the nucleotide sequence of rHDV-HDAg-IRES-huIFNbeta comprises, consists or consists essentially of the sequence:
• IFN-beta coding sequence is shown ingray bold italics
• FIG. 6 An alternative strategy for insertion and expression of heterologous sequences in HDV is shown in Figure 6, which illustrates a schematic representation of the construct rHDV-HDAg-2A-huIFNbeta.
• the recombinant HDV genome is capable of expressing a gene of interest but that does not feature an IRES sequence. Instead, a picornavirus '2A-like' motif is inserted in-frame between the HDAg and the IFN- ⁇ coding sequence, creating rHDV-HDAg-2A-huIFNbeta ( Figure 5).
• a '2A-like' motif allows the expression of two or more proteins from a single open reading frame (de Felipe et al., 2006, Trends Biotechnol. 24(2):68- 75) by a translational recoding event in which a peptide bond is 'skipped' during elongation (Doronina et al., 2008, supra). Since the function of the '2A-like' motif is believed to be mediated by amino acids rather than the coding sequence, the G/C content of rHDV-HDAg-2A-huIFNbeta can be fully controlled, i.e., all artificially inserted sequences can be edited to match G/C levels found in the wild-type HDV backbone.
• a '2A-like' sequence element Another advantage of using a '2A-like' sequence element is that these elements are relatively short. For example a particular '2A-like' sequence disclosed by Osborne et al., (2005, Mol Ther 12: 569-574) comprises only 63 nts. As a consequence, rHDV-HDAg-2A-huIFNbeta is much smaller than the other two recombinant genomes, which may enhance efficient packaging of recombinant HDV genomes.
• FIG. 7 illustrates a schematic representation of the construct rHDV-HDAg-ribo-insertion.
• an heterologous nucleotide sequence of interest which comprises a coding sequence ⁇ e.g., IFN- ⁇ coding sequence
• a coding sequence ⁇ e.g., IFN- ⁇ coding sequence
• the genomic sequence of rHDV.JC126 comprises the sequence:
• HDAg-L coding sequence is shown in bold type face (stop codon is also underlined ;
• HDAg-S coding sequence is shown in italics (start codon is also underlined).
• the genomic sequence of rHDV.JC126R comprises the sequence:
• the modifications comprise:
• rHDV.JC126 CGCCCaggaaTGGCGGGACC (original HDV sequence);
• rHDV.JC126R CGCCCcatcgatcggTGGCGGGACC (catcgatcgg replaces aggaa; +5 nts);
• rHDV.JC126 TTTATtcaCTGGGG (original HDV sequence),
• rHDV.JC126R TTTATtgatcatgaCTGGGG (tgatcatga replaces tea; +6 nts);
• HDAg-L coding sequence is shown in shadow typeface (stop codon is also underlined);
• HDAg-S coding sequence is shown in italics (start codon is also underlined):
• HA-tag sequence To test various sites within the HDV genome for their potential to carry slightly larger insertions of heterologous sequences, the present inventors inserted an edited version of the hemagglutinin (HA)-tag sequence into three different sites: (i) immediately upstream but not in frame with the HDAg coding sequence (to exclude potentially detrimental effects caused by adding amino acids to the N-terminus of the HDAg), (ii) downstream and in frame with the HDAg-L, or (iii) downstream of the genomic ribozyme sequences, shortly before the tip of the rod.
• HA hemagglutinin
• the original HA-tag was derived from amino acids (YPYDVPDYA) of the human influenza A virus HA surface glycoprotein.
• the corresponding HA-tag cDNA sequence (5 ' -TACCC ATACGATGTTCC AGA TTACGCT-3') was edited at 6 positions (5'-TACCCCTACGACGTCCCCGACTACGCC-3') to increase the G/C content from 44% to 66%, a level much closer to levels found elsewhere in the HDV genome.
• rHDV.HA(AH)-HDAg This virus contains the edited HA-tag ) sequence immediately upstream of the HDAg coding sequence (new Figure 8A).
• the HA-tag does not include a proper start codon. Instead it contains an ATA codon which will allow us to compare the phenotype of this virus to that with an ATG in future experiments.
• the virus contains an additional 25 nucleotides, designed and positioned to maintain the rod-like secondary structure of the genomic/anti-genomic RNA (for an alignment of the two sequence elements, see new Figure 9B).
• RNA secondary structure predictions revealed that a virus with an HA-tag sequence upstream of the HDAg and a properly positioned, partially complementary, 'stabilizing' sequence is able to maintain the rod-like appearance of its genome, while a 'control' virus that lacks the 'stabilizing' sequence (e.g. rHDV.HA-HDAg; Figure 9C) most likely has a 'unorderly' RNA secondary structure at the insertion site ( Figure 9D).
• Replication competency testing of rHDV.HA(AH)-HDAg revealed that this virus does not replicate well enough to allow for the accumulation of RNA levels that are easily detectable by Northern blotting ( Figures 13 and 14).
• the genomic sequence of rHDV.HA(AH)-HDAg comprises the sequence:
• HDAg-L coding sequence is shown in shadow typeface (stop codon is also underlined):
• HDAg-S coding sequence is shown in italics (start codon is also underlined):
• the genomic sequence of rHDV.HA-HDAg comprises the sequence:
• HDAg-S coding sequence is shown in italics (start codon is also underlined).
• rHDV.R.HDAg-HA(AH) Compared with the sequence of rHDV JC126, rHDV.R.HDAg-HA(AH) contains an edited HA-tag sequence (27 nucleotides) downstream and in frame with the HDAg-L plus additional coding and non-coding nucleotides. Apart form this insertion, rHDV.R.HDAg-HA(AH) contains a second, slightly shorter insert, designed and positioned to maintain the rod-like secondary structure of the genomic/anti-genomic RNA (see, new Figures 10A and B). The combined length of these heterologous sequences is 80 nucleotides (42 + 38), but .
• RNA secondary structure predictions revealed that rHDV.R.HDAg-HA(AH) has a rod-like genome while a virus that lacks the 'stabilizing' sequence (e.g. rHDV.R.HDAg-HA; Figure IOC) does not ( Figure 10D).
• Northern blot analysis of total RNA isolated from COS-7 cells 4 and 8 days after the transfection of rHDV.HA(AH)-HDAg revealed that this virus can indeed replicate efficiently and that host cells accumulate detectable RNA levels ( Figures 13 and 14).
• the genomic sequence of rHDV.R.HDAg-HA(AH) comprises the sequence:
• the modifications comprise:
• rHDVJC126 CGCCCaggaaTGGCGGGACC (original HDV sequence);
• rHDV.JC126 TTTATtcaCTGGGG (original HDV sequence)
• HDAg-L coding sequence is shown in shadow typeface (stop codon is also underlined); [0287] HDAg-S coding sequence is shown in italics (start codon is also underlined); and
• the genomic sequence of rHDV.R.HDAg-HA comprises the sequence:
• the modifications comprise:
• rHDV.JC126 CGCCCaggaaTGGCGGGACC (original HDV sequence);
• rHDV.JC126R CGCCCcatcgatcggTGGCGGGACC (catcgatcgg replaces aggaa; +5 nts) ;
• rHDV.JC126 TTTATtcaCTGGGG (original HDV sequence);
• HDAg-L coding sequence is shown in shadow typeface (stop codon is also underlined);
• HDAg-S coding sequence is shown in italics (start codon is also underlined):
• rHDV.XbaHA(AH) This virus contains an edited HA-tag sequence (27 nucleotides) inserted downstream of the ribozyme sequence, close to the end of the rod and a partially complementary sequence of 22 nucleotides to stabilize the first insertion ( Figures 11 A and B). Similar to the viruses described above, RNA secondary structure prediction programs suggest that rHDV.XbaHA(AH) has a rod-like, genome while a control virus that lacks the 'stabilizing' sequence (e.g., rHDV.XbaHA; Figure 11C) will have a much altered secondary RNA structure, including a different structure at the tip of the rod ( Figure 1 ID). Northern blotting revealed a strong replication competency for rHDV.XbaHA(AH) ( Figures 13 and 14) but not for rHDV.XbaHA (compare lines 5 and 6 in Figure 14).
• genomic sequence of rHDV.XbaHA(AH) comprises the sequence:
• HDAg-L coding sequence is shown in shadow typeface (stop codon is also underlined):
• HDAg-S coding sequence is shown in italics (start codon is also
• the genomic sequence of rHDV.XbaHA comprises the sequence: [0308] CCTGAGCCAAGTTCCGAGCGAGGAGACGCGGGGGGAGGATCAGCtCCCGAG AGGGGATGTCACGGTAAAGAGCATTGGAACGTCGGAGAAACTACTCCCAAGAAGCAAAGAGAGG TCTTAGGAAGCGGACGAGATCCCCACAACGCCGGAGAATCTCTGGAAGGGGAAAGAGGAAGGTG GAAGAAAAAGGGGCGGGCCTCCCGATCCGAGGGGCCCAATCCCAGATCTGGAGAGCACTCCGGC CCGAAGGGTTGAGTAGCACTCAGAGGGAGGAATCCACTCGGAGATGAGCAGAAATCACCTCC AGAGGACCCCTTCAGCGAACAAGAGGCGCTTCGAGCGGTAGGAGTAAGACCATAGCGATAGGAG GAGATGCTAGGAGTAGGGGGAGACCGAAGCGAGGAGGAAAGCAAAGAAAGCAACGGGGCTAGCC GGTGGGTGTTCCGCCCCGCGAGATCCCC
• HDAg-L coding sequence is shown in shadow typeface (stop codon is also underlined ;
• HDAg-S coding sequence is shown in italics (start codon is also underlined).
• GenBank accession number M21012.1 GenBank accession number M21012.1 at the following seven positions: (i) position 45, A to T; (ii) position 221, supernumerary T; (iii) position 619: supernumerary C; (iv) position 840, C to G; (v) position 1343, G to C; (vi) position 1389, G to C; and (vii) position 1633, A to T. Consequently, the resequenced rJC126 HDV genome contains only 1,677 nucleotides (as compared to the previously published 1,679 nucleotides).
• the pJC126 plasmid has provided the 'sequence backbone' to all 'pHDV plasmids described below (recombinant viruses encoded by these plasmids are named
• plasmid pJC126, pJC126R, and pHDV.huTFN- IRES-HDAg contain a 1.2-fold copy of virus rHDV.JC126, rHDVJC126R and rHDV.huIFN-IRES-HDAg, respectively). All newly generated plasmids were verified by both restriction digest and sequencing (see appendix for detailed sequence information).
• pJC126d A small DNA fragment was removed from pJC126 using the restriction enzymes Sbfi and EcoRV. The remaining plasmid was flush-ended using T4 DNA polymerase (Promega, Fitchburg, WI, USA) and religated, generating plasmid pJC126ASbfl-EcoRV (also referred to as pJC126d; for plasmid map of pJC126d, see new Fig. 7 and for a map of the circularized and corrected genome rHDV.JC126, see new Fig. 8).
• pHDV.HDAg-huIFN-IRES Construction of this plasmid relied on two synthetic DNA fragments.
• the first fragment contained the IRES and edited human IFN-beta coding sequence as described above, flanking pJC126 sequences, and terminal Notl arid Stul sites.
• the second fragment contained a sequence partially complementary to the IFN-beta and IRES sequences, flanking pJC126 sequences and terminal Nhe/ and Bam H sites. Fragments were sequentially inserted into plasmid pJC126d using the aforementioned restriction sites.
• Plasmid pJC126R was generated from plasmid pHDV.HDAg-huIFN-IRES by deleting two fragments. Firstly, a Bcli fragment encompassing the IRES and IFN-beta coding sequence was removed, the remaining plasmid backbone was religated, and then cut a second time with Pvul to remove the sequences partially complementary to the IRES and IFN-beta coding sequence. This resulted in a plasmid identical to pJC126d except for two small inserts containing a total of 11 additional nucleotides. [0320] (e) pHDV.HA(AH)-HDAg.
• a nucleotide sequence that encodes the hemagglutinin (HA) epitope tag was used to test and compare potential insertion sites within the HDV genome.
• the original HA-tag was derived from amino acids 98 to 106 (YPYDVPDYA) of the human influenza A virus HA surface glycoprotein.
• the corresponding cDNA sequence (5'-TACCCATACGATGTTCCAGA TTACGCT-3') - was edited to increase the G/C content (resulting in sequence 5'-TACCCCTACG ACGTCCCCGACTACGCC-3'), fitted at its 5' end with a modified, non-functional (ATA) start codon, and positioned immediately upstream of the initiation (ATG) codon of the HDAg coding sequence.
• a sequence partially complementary to the edited HA-tag sequence was generated and appropriately spaced in relation to the HA-tag sequence by using intermittent HDV sequences (see above).
• This sequence information was used to purchase a synthetic DNA cassette containing the edited, G/C-rich HA-tag sequence, an intermittent HDV sequence, a sequence partially complementary to the HA sequence, flanking HDV sequences, and terminal restriction sites for SacU and Nhel (GeneScript).
• the cassette was imported as described above and cloned into plasmid pJC126d, replacing the HDV sequence between restriction sites Sadl and Nhel.
• pHDV.(AH)-HDAg The sequence partially complementary to the edited HA-tag (see above) was inserted three pHDV126.d by fusion PCR using the following four oligonucleotide primers: two HDV-specific primers that contain information for the insertion (5 ' -CCGCCCGAGCCCGAGAGGTACCCACGACG
• pHD V.R.HDAg-HA(AH) pHD V.R.HDAg-HA(AH).
• a DNA Fragment containing the coding sequence for the HA-tag, flanked by NotI and SacU restriction sites was isolated from plasmid pHDV.R.HDAg-HA (see below) and cloned into plasmid pHDV.R.HDAg(AH) (see below), replacing the sequence between the Notl and Sacll restriction sites.
• rHDV.R.HDAg-(AH) A partially complementary HA-tag sequence (see above) was inserted into pHDV126.R by fusion PCR using the following four oligonucleotide primers: two HDV-specific primers that contain information for the insertion (5 ' -GTAGTCGGGGACGTCGT AGGGGTACCATGACTGGGGTCGA- 3' and 5'-CCTACGACGTCCCCGACTACGCCACAT GATC AATAAAGC-3 inserted sequences are underlined), the HDV-specific primer 1328G upstream of the insertion site, and a T7 promoter-specific primer downstream of the insertion site. The resulting PCR product was digested with Nhel and BamHl and cloned into plasmid pJC126R replacing the sequence between restriction sites Nhel and BamHl.
• sequences/plasmid sequences including the BamHl site in the plasmid' s polylinker region.
• pHDV.XbaHA The edited HA-tag sequence (27 nucleotides) was inserted at the Xbal restriction site in HDV genome of pJC126d at position 783 using a synthetic DNA cassette identical for the one described in the paragraph above but for the partially complementary sequence of 22 nucleotides.
• Plasmids pJC126.S/B and pJC126.S/N were generated from pJC126 by removing a fragment flanked by Seal and Bari (position 1,620 to 711), or Seal and Nhel (position 1,620 to 429), respectively. Due to these large deletions, both plasmids cannot generate replication competent RNAs but are still capable of producing 'HDV-like mRNAs' and providing HDAg in trans to rescue any viral replication that is compromised by insufficient HDAg expression. In fact, we have verified recombinant HDAg expression in COS-7 cells after the transfection with helper plasmids by flow cytometry using human HDAg-specific antisera (kindly provided by Prof. Michael Roggendorf, University Clinic Essen-Duisburg, Essen, Germany).
• First strand HDV-specific cDNA synthesis was performed with the genome- specific primer 1517A (5 '-GGCCGGAAGAAAGAAGTTAG-3 ') and after heat inactivation, PCR was performed using primer 1517A and the antigenome-specific primer 661G (5'-CGCGTTCCATCCTTTCTTAC-3'). Note that resulting amplicons encompass the insertion site for the transgene gene and the circularization site.
• RNA samples were incubated with glyoxal loading buffer (Roche) for 30 min at 50°C, separated on 1.3% agarose MOPS/formaldehyde gels, and transferred to positively charged nylon membranes (Roche, Basel, Switzerland) using standard protocols.
• glyoxal loading buffer (Roche) for 30 min at 50°C, separated on 1.3% agarose MOPS/formaldehyde gels, and transferred to positively charged nylon membranes (Roche, Basel, Switzerland) using standard protocols.
• HDV-specific RNAs were detected by chemiluminescence using CDP-Star (Applied Biosystems, Foster City, CA, USA), a digoxigenin (DIG)-labeled SP6-generated riboprobe corresponding to nucleotides 1620 to 429 (Banl-Nhel fragment inserted into pGEM-3Z using Smal and Xbal site, linearized by Sac ), and alkaline phosphatase (AP)-conjugated anti-DIG antibodies (Roche), essentially as recommended by the manufacturers.
• Hybridization signals were documented using the ImageQuant LAS4000 digital imaging system (GE Healthcare, Little Chalfont, UK) and a 0.24 to 9.5-kb RNA ladder (Invitrogen/Life Technologies) was used as molecular weight standard.
• Thermodynamic RNA secondary structure predictions The mimmum free energy (MFE) structure of genomic and antigenomic RNA copies of the JC126 virus and genetically modified derivates were computed at the RNAfold server (http://rna.tbi.univie.ac.at/), Institute for Theoretical Chemistry, University of Vienna (Austria) using a loop-based energy model and the dynamic programming algorithm introduced by Zuker and Stiegler (Zuker and Stiegler, 1981, Nucleic Acid Res 9: 133- 48).
• MFE mimmum free energy

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