WO1999028487A1 - Systeme d'expression et de diffusion propre au flavivirus - Google Patents

Systeme d'expression et de diffusion propre au flavivirus Download PDF

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Publication number
WO1999028487A1
WO1999028487A1 PCT/AU1998/000993 AU9800993W WO9928487A1 WO 1999028487 A1 WO1999028487 A1 WO 1999028487A1 AU 9800993 W AU9800993 W AU 9800993W WO 9928487 A1 WO9928487 A1 WO 9928487A1
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WIPO (PCT)
Prior art keywords
replicon
flavivirus
vector
c20dx
nucleotide sequence
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PCT/AU1998/000993
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English (en)
Inventor
Edwin G. Westaway
Alexander A. Khromykh
Andrei Varnavski
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The Crown In The Right Of The Queensland Department Of Health
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Publication date
Priority claimed from AUPP0627A external-priority patent/AUPP062797A0/en
Priority claimed from AUPP6096A external-priority patent/AUPP609698A0/en
Application filed by The Crown In The Right Of The Queensland Department Of Health filed Critical The Crown In The Right Of The Queensland Department Of Health
Priority to NZ504725A priority Critical patent/NZ504725A/xx
Priority to CA002311395A priority patent/CA2311395C/fr
Priority to AU15517/99A priority patent/AU733155B2/en
Priority to JP2000523363A priority patent/JP2002500003A/ja
Priority to EP98959672A priority patent/EP1034290A4/fr
Publication of WO1999028487A1 publication Critical patent/WO1999028487A1/fr
Priority to US09/580,476 priority patent/US6893866B1/en
Priority to US11/098,283 priority patent/US20060088937A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24141—Use of virus, viral particle or viral elements as a vector
    • C12N2770/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/60—Vector systems having a special element relevant for transcription from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the present invention generally relates to the field of gene expression and in particular to Flavivirus gene expression and delivery systems and to virus like particles produced from such systems.
  • procaryotic and yeast expression systems are extremely efficient and easy to use, these systems suffer from a number of disadvantages, including an inability to glycosylate proteins, inefficient cleavage of "pre” or “prepro” sequences from proteins (eg., inefficient post translational modification), and a general inability to secrete proteins.
  • Another expression system widely available is the baculovirus expression system.
  • This system is arguably one of the most efficient in protein production, but is limited only to use in insect cell lines.
  • insect cell lines glycosylate proteins differently from mammalian cell lines thus this system has not proven useful for the production of many mammalian proteins.
  • Another disadvantage of this system is that it relies on the use of homologous recombination for the construction of recombinant virus stocks. Thus, this system often proves very laborious when large numbers of genetic variants have to be analysed.
  • eucaryotic host systems typically mammalian host cell systems
  • mammalian host cell systems for mammalian protein production.
  • the protein produced has a structure most like that of the natural protein species and purification often is easier since the protein can be secreted into the culture medium in a biologically active form.
  • One of the most efficient mammalian cell expression systems is based on Vaccinia virus. The main problem with this system, however, is that it uses recombinant viruses that express the heterologous gene upon infection. Thus there is no control over the virus once it has been release.
  • RNA viruses such as Semliki Forest Virus (SFV), Sindbis (SIN) virus, and poliovirus
  • SFV Semliki Forest Virus
  • Sindbis virus Sindbis virus
  • poliovirus a virus that promotes the expression of heterologous genes in vitro and in vivo.
  • the success of these expression systems has been mainly based on each virus' ability to produce high titer stocks of "pseudo" infectious particles containing recombinant replicon RNA packaged by structural proteins.
  • Semliki Forest virus (SFV) and Sindbis virus expression systems this is achieved by co- transfection of replicon RNA with defective helper RNA(s) expressing structural genes, but lacking the packaging signal.
  • Replicon RNA expression provides enzymes for RNA replication and transcription of both RNA's, whereas helper RNA supports the production of structural proteins for packaging of replicon RNA via expression of its subgenomic region.
  • the main problem with these expression systems is that the viruses used in the expression system are cytopathic and often compete out the host protein synthesis.
  • Another major disadvantage of these systems includes possible contamination with infectious particles containing packaged full-length genomic RNA (in other words, infectious virus) due to the high probability of recombination between replicon and helper RNAs.
  • the present invention seeks to provide an improved expression and delivery system that at least ameliorates some of the problems associated with prior art systems.
  • the present invention provides a gene expression system comprising: a) a replicon of flavivirus origin, which is adapted to receive at least a nucleotide sequence without disrupting its replication capabilities; and b) at least a second vector that is capable of expressing flavivirus structural protein(s) and any other proteins required for packaging of the self- replicating expression vector into flavivirus viral particles which vector is engineered to prevent recombination with the self-replicating vector when in its presence.
  • any replicon (self-replicating expression vector) derived from any flavivirus RNA may be used in the present invention.
  • the replicon should however encode a sufficient amount of a flavivirus 5' UTR and at least a portion of the 5' flavivirus coding region for core protein, each of which is required for RNA replication.
  • Both the 5' UTR and the 5' core protein coding region of a flavivirus genome contains regulatory elements that are required for flavivirus RNA replication. It will be appreciated that the flavivirus 5' UTR and the 5' core protein coding region may contain mutations or deletions in these regions and still be able to replicate.
  • the replicon should contain 5' UTR and at least about between 60 and 80 nucleotides from the 5' coding region for flavivirus core protein.
  • the relative number of nucleotides from the 5' core protein coding region that will be required in the replicon for RNA replication will largely depend on the type of flavivirus used in the vector. For example when the replicon is derived from Kunjin virus it must contain at least 60 nucleotides of the 5' core protein coding region.
  • a gene expression system comprising: a) a replicon of flavivirus origin which includes the nucleotide sequence for a flavivirus 5' untranslated region (UTR), at least a portion of the 5' coding region for flavivirus core protein, the nucleotide sequence coding for the flavivirus non-structural proteins, and part or all of the 3'-terminal sequence of a flavivirus 3'UTR, required for self-replication of flavivirus genomic material, which vector is adapted to receive at least a nucleotide sequence without disrupting its replication capabilities; and b) at least a second vector that is capable of expressing flavivirus structural protein(s) and any other proteins required for packaging of the self- replicating expression vector into flavivirus viral particles which vector is engineered to prevent recombination with the self-replicating vector when in its presence.
  • UTR 5' untranslated region
  • the replicon of flavivirus origin is adapted to receive at least a nucleotide sequence. Insertion of such a nucleotide sequence, into the replicon may be achieved at any point in the replicon that does not effect processing of flavivirus proteins.
  • heterologous genes may be inserted into the 3' UTR of the flavivirus replicon, within a structural gene or within the locality of deleted structural genes.
  • heterologous genes are inserted into structural genes or in place of deleted structural genes since such insertions generally produce higher levels of expression and generally do not affect replication efficiency of the replicon.
  • the nucleotide sequence(s) are inserted into the 3'UTR they may be preceded by an internal ribosomal entry site (IRES) sequence.
  • IRES internal ribosomal entry site
  • the 3' UTR is used only for insertion of IRES-Neo (neomycin transferase) or IRES-pac (puromycin N-acetyl transferase) sequences. Such insertions allow the generation of stable cell lines persistently expressing foreign genes via antibiotic (eg Geneticin or puromycin) selection.
  • a gene expression system comprising: a) a replicon of flavivirus origin which includes a nucleotide sequence for a flavivirus 5'UTR, at least a portion of a 5' coding region for flavivirus core protein, a nucleotide sequence coding for a flavivirus non-structural proteins, the complete or most of the 3'-terminal region of a flavivirus 3'UTR required for self-replication of the genomic material and the nucleotide coding sequence for flavivirus structural proteins, wherein (i) the vector is adapted to receive at least a nucleotide sequence without disrupting the replication capabilities of the vector, (ii) the nucleotide sequence is inserted into the vector in a manner which deactivates expression of at least a gene that would otherwise code for a flavivirus structural protein and (iii) the inserted nucleotide sequence does not encode for the structural protein sequence that it deactivates; and b) at least
  • nucleotide sequence When the nucleotide sequence is inserted into the replicon it should be introduced into the vector in a manner which avoids a frame shift in the open reading frame of the vector coding sequence. This may be achieved by either adapting the foreign nucleotide sequence or the vector to ensure the reading frame of the vector coding sequence is maintained. In an alternative arrangement foreign nucleotide sequence can be inserted without preserving open reading frame of the vector if it is followed by a termination codon and an internal ribosomal entry site (IRES) sequence to ensure initiation of translation of the vector's nonstructural proteins.
  • IVS internal ribosomal entry site
  • a replicon which encodes flavivirus structural and non-structural proteins may be either RNA or DNA based provided it is capable of self-replication and encodes flavivirus structural and non-structural protein coding information.
  • the replicon is an RNA sequence the flavivirus genome is first reverse transcribed into complementary DNA sequence. The nucleotide sequence is then inserted into the complementary DNA sequence and the genomic sequence is then transcribed back into RNA prior to delivery to a host cell.
  • the vector is DNA based the flavivirus genome is first transcribed into complementary DNA sequence, a nucleotide sequence can then be inserted into the transcribed complementary sequence and the complementary sequence is then introduced into a host cell.
  • the replicon will in most circumstances be prepared from a single strain of flavivirus it should be appreciated that in some circumstances nucleotide sequences from more than one flavivirus strain may be brought together in a single vector.
  • the replicon is derived from the genomic sequence of a single flavivirus species.
  • the replicon is derived from a single flavivirus species (such as Kunjin virus (KUN)) and includes the entire or a substantial portion of the genome of that strain, the genome being modified in at least one of its structural proteins to accept a nucleotide sequence such that the insertion of the nucleotide sequence into the structural protein nucleotide sequence disrupts coding for part or all of the structural protein.
  • KUN Kunjin virus
  • Nucleotide sequences that may be inserted into the replicon include, for example, parts of flavivirus or non-flavivirus cDNA gene sequences. Nucleotide sequence(s) that are inserted into the replicon must, however, disrupt the expression of at least a structural protein thus preventing viral genome packaging. Desirably the inserted nucleotide sequence is a non-flavivirus nucleotide sequence (hereinafter referred to as a "heterologous nucleotide sequence").
  • the heterologous nucleotide sequence is not limited only to a sequence that encodes an amino acid sequence, but may also include sequences appropriate for promoting replication and or expression of a sequence that encodes an amino acid sequence.
  • Insertion of a heterologous nucleotide sequence into the replicon may occur at any point in a flavivirus structural protein(s) or in any region of the nucleotide sequence where such a protein would normally be expressed in the native flavivirus sequence had the protein not been deleted.
  • the heterologous nucleotide sequence is inserted into at least one of the structural genes deactivating that gene.
  • at least a structural gene is deleted from the vector and the deletion site is adapted to serve as the insertion site for heterologous genetic sequences.
  • the nucleotide sequence is inserted into the locality from where at least a structural gene was deleted.
  • the replicon By positioning heterologous nucleotide sequences within the locality of one or more sites in the replicon that might otherwise code for structural genes in a native flavivirus, the replicon is unable to produce structural proteins for viral packaging.
  • the invention employs a second vector that is engineered to prevent recombination with the replicon.
  • the second vector is heterologous in origin to the origin of the replicon. Any non-flavivirus vector that is engineered to prevent recombination with the replicon may be employed in the expression system to deliver the flavivirus structural protein that is deactivated in the replicon.
  • the second vector may be derived from a virus other than a flavivirus.
  • the second vector could be derived from an alphavirus such as SFV or SIN, or from DNA virus such as adenovirus, fowlpox virus, or vaccinia virus.
  • SFV alphavirus
  • DNA virus such as adenovirus, fowlpox virus, or vaccinia virus.
  • the replicon is derived from KUN while the second vector is derived from SFV to take account of the impossible recombination between KUN RNA and SFV RNA.
  • the second vector may be a plasmid DNA expression vector.
  • highly efficient packaging may be achieved by inserting structural genes into CMV based DNA expression cassettes which are inserted into baculovirus expression vectors which provide very efficient delivery of the cassettes into mammalian cells (see for example Shoji et al, (1997) J.Gen.Virol., 78: 2657-2664 and pBacMam-1 vector described on the Novagen homepage).
  • the second vector may be an inducible plasmid DNA expression vector (for example tetracycline inducible vector (Clontech)) allowing selection of packaging cell lines expressing KUN structural proteins in response to addition or removal of tetracycline in the incubation medium.
  • the present invention also provides a method for producing a stable cell line capable of persistently producing replicon RNA's, comprising the steps of:
  • the described vectors are preferably constructed in selectable form by inserting an IRES-Neo or IRES-pac cassette into the 3'UTR.
  • the invention provides a method for producing a flavivirus like particles containing a replicon as herein described comprising the steps of:
  • the replicon containing virus like particles prepared by this method are purified from cellular and viral proteins and nucleic acids that may cause an adverse immunological or physiological reaction when introduced into an animal.
  • Methods for purifying such viral particles are known in the art.
  • the replicon containing virus like particles are 50%, 60%, 70%, 80%, 90%, 95% or 99% free of all contaminating material including cellular and viral proteins, lipids and nucleic acids.
  • the invention provides a flavivirus like particles containing a flavivirus replicon that is adapted to receive at least a nucleotide sequence without disrupting its replication capabilities.
  • virus like particles are purified from cellular and viral nucleic acids and amino acid sequences that may cause an adverse immunological or physiological reaction when introduced into an animal. Such particles may be used as a therapeutic agent.
  • the described virus particles can be used to deliver to a subject any nucleotide sequence that is inserted into the replicon.
  • the replicon within the virus like particles may be employed to deliver to a cell a nucleotide sequence encoding one or more amino acid sequences which are capable of inducing, for example, a protective immune response to a subject.
  • the invention provides a DNA replicon of flavivirus origin that is adapted to receive at least a nucleotide sequence without disrupting its replication capabilities.
  • the DNA replicon may be introduced into a cell as a naked vector (i.e. flavivirus structural proteins do not surround it) or alternatively in virus like particles prepared in accordance with the described method. Whether the DNA replicon is prepared as a naked vector or in virus like particles it should be purified from cellular and viral nucleic acids and amino acid sequences that may cause an adverse immunological or physiological reaction in an animal prior to introduction into that animal. Such particles may be used as a therapeutic agent.
  • virus particles can be used to deliver to a subject any nucleotide sequence that is inserted into the replicon.
  • the replicon is prepared in DNA form and is administered to a cell in a virus like particle.
  • protein should be understood to include within its scope parts of proteins such as peptide and polypeptide sequences.
  • the replicon is introduced into a host cell where gene expression and hence protein production take place. Because the vector is capable of self- replication, multiple copies of the replicon will also be generated. This leads to an exponential increase in the number of replicons in the host cell as well as an exponential increase in the amount of protein that is produced.
  • the flavivirus expression system has relatively high level of protein expression in eukaryotic cell lines.
  • the flavivirus expression system is capable of expressing proteins in a wide variety of mammalian cell lines and cell types.
  • the replicons used in the flavivirus expression system produce a long- term non-cytopathic replication in host cells. There are no observable effects on the host's translation process. This feature of flavivirus replicons also allows selection of stable cell lines continuously expressing other genes using a replicon vector expressing a gene confirming resistance to an antibiotic (e.g. neomycin transferase (Neo), puromycin N-acetyltransferase (pac), etc.)
  • the flavivirus expression system is an RNA system that does not permit integration of viral genomic material into a host's genomic sequence.
  • flaviviruses differ from alphaviruses (such as SFV and SIN) by their genome structure (structural genes situated at the 5' end of the genome) and by the absence of synthesis of subgenomic RNA. Furthermore, there are no data to date on packaging of flavivirus RNA.
  • the replicon used in the invention should be adapted to include part or all of the following: at least, about the first 150 nucleotides of a flavivirus genome; at least about the last 60 nucleotides of E protein; substantially all of the nonstructural region; and part or all of the 3'UTR.
  • Replication of a flavivirus genome is dependent on the genes in the nonstructural region of the genome being present during transcription and translation.
  • any modification made to the nonstructural region should not interfere with the functional activity of the genes within the nonstructural region of the genome.
  • the replicon is derived from KUN and includes the first 157 nucleotides of the KUN genome, the last 66 nucleotides of E protein, the entire nonstructural region, and all of the 3'UTR.
  • Optimal flavivirus replicon design for transfection into eukaryotic cells might also include such sequences as: sequences to promote expression of the heterologous gene of interest, including appropriate transcription initiation, termination, and enhancer sequences; as well as sequences that enhance translation efficiency, such as the Kozak consensus sequence; internal ribosomal entry site (IRES) of picornaviruses; an alphavirus subgenomic 26S promoter to enhance expression of inserted genes if cotransfection with alphavirus replicon RNA is used.
  • sequences to promote expression of the heterologous gene of interest including appropriate transcription initiation, termination, and enhancer sequences; as well as sequences that enhance translation efficiency, such as the Kozak consensus sequence; internal ribosomal entry site (IRES) of picornaviruses; an alphavirus subgenomic 26S promoter to enhance expression of inserted genes if cotransfection with alphavirus replicon RNA is used.
  • sequences to promote expression of the heterologous gene of interest including
  • the nucleotide sequence may be placed under the control of flavivirus regulatory machinery in the replicon, it may alternatively be controlled by one or more alternate regulatory elements capable of promoting expression.
  • the replicon is derived from KUN virus and contains a eucaryotic promoter sequence (such as CMV or hybrid CMV enhancer-chicken ⁇ - actin promoter [CAG]) upstream of the KUN 5'UTR and a delta virus ribozyme sequence followed by an SV40, bovine growth hormone, or rabbit ⁇ -globin transcription terminator sequences downstream of the KUN 3'UTR.
  • a eucaryotic promoter sequence such as CMV or hybrid CMV enhancer-chicken ⁇ - actin promoter [CAG]
  • Transfection of the resulting plasmid DNA in cells will ensure production of a KUN replicon RNA transcript with the authentic 5'-end by cellular RNA polymerase II and with the authentic 3'-end cleaved by delta virus ribozyme, which is preferred for its efficient replication.
  • nucleotide sequence inserted into the replicon may encode part or all of any natural or recombinant protein except for the structural protein sequence into which or in place of which the nucleotide sequence is inserted.
  • the nucleotide sequence may encode a single polypeptide sequence or a plurality of sequences linked together in such a way that each of the sequences retains their identity when expressed as an amino acid sequence.
  • the nucleotide sequence encodes a plurality of peptides
  • the peptides should be linked together in such a way that each retains its identity when expressed.
  • Such polypeptides may be produced as a fusion protein or engineered in such a manner to result in separate polypeptide or peptide sequences.
  • the nucleotide sequence may encode one or more immunogenic polypeptides in association with a range of epitopes which contribute to T-cell activity.
  • the heterologous nucleotide sequence preferably encodes epitopes capable of eliciting either a T helper cell response or a cytotoxic T-cell (CTL) response or both.
  • the replicon described herein may also be engineered to express multiple nucleotide sequences allowing co-expression of several proteins such as a plurality of antigens together with cytokines or other immunomodulators to enhance the generation of an immune response. Such a replicon might be particularly useful for example in the production of various proteins at the same time or in gene therapy applications.
  • the nucleotide sequence may encode the cDNA sequence of one or more of the following: malarial surface antigens; beta- galactosidase; any major antigenic viral antigen eg Haemagglutinin from influenza virus or a human immunodeficiency virus (HIV) protein such as HIV gp 120 and HIV gag protein or part thereof; any eukaryotic polypeptide such as, for example, a mammalian polypeptide such as an enzyme, e.g. chymosin or gastric lipase; an enzyme inhibitor, e.g. tissue inhibitor of metalloproteinase (TIMP); a hormone, e.g. growth hormone; a lymphokine, e.g.
  • malarial surface antigens beta- galactosidase
  • any major antigenic viral antigen eg Haemagglutinin from influenza virus or a human immunodeficiency virus (HIV) protein
  • HIV gp 120 and HIV gag protein or part thereof any eukary
  • an interferon e.g an interleukin (eg IL-2, IL-4, IL-6 etc); a chemokine eg macrophage inflammatory protein-2; a plasminogen activator, e.g. tissue plasminogen activator (tPA) or prourokinase; or a natural, modified or chimeric immunoglobulin or a fragment thereof including chimeric immunoglobulins having dual activity such as antibody- enzyme or antibody-toxin chimeras.
  • a cytokine e.g an interleukin (eg IL-2, IL-4, IL-6 etc); a chemokine eg macrophage inflammatory protein-2; a plasminogen activator, e.g. tissue plasminogen activator (tPA) or prourokinase; or a natural, modified or chimeric immunoglobulin or a fragment thereof including chimeric immunoglobulins having dual activity such as antibody- enzyme or antibody-toxin
  • the nucleotide sequence may also code for one or more amino acid sequences that serve to enhance the effect of the protein being expressed.
  • ubiquitination of viral proteins expressed from DNA vectors results in enhancement of cytotoxic T-lymphocyte induction and antiviral protection after immunization.
  • the replicon may encode ubiquitin in association with the protein to be expressed thus targeting the resulting fusion protein to proteosomes for efficient processing and uptake by the MHC class I complexes.
  • ubiquitin sequence is inserted into the replicon vector.
  • the ubiquitin sequence is preferably inserted either prior to the 5' end of the heterologous genetic sequence or at the 3' end of the heterologous genetic sequence.
  • the second vector that contains the flavivirus structural gene(s) should be engineered to prevent recombination with the self-replicating expression vector.
  • One means for achieving this end is to prepare the second vector from genetic material that is heterologous in origin to the origin of the self-replicating expression vector.
  • the second vector might be prepared from SFV when the replicon is prepared from KUN virus.
  • the second vector might include such sequences as: sequences to promote expression of the genes of interest, including appropriate transcription initiation, termination, and enhancer sequences; as well as sequences that enhance translation efficiency, such as the Kozak consensus sequence.
  • the second vector contains separate regulatory elements associated with each of the different structural genes expressed by the vector.
  • the flavivirus C gene and the prME genes are placed under the control of separate regulatory elements in the vector.
  • flavivirus structural proteins during virus replication in cells is complex and requires a number of post-translational cleavages by host and viral proteases.
  • Numerous in vitro and in vivo studies on processing of the C-prM region have established two cleavage events: cleavage at a dibasic cleavage site by viral NS2B-NS3 protease generating the carboxy terminus of mature virion C protein, which appears to be a prerequisite for the efficient cleavage at the NH 2 terminus of prM by cellular signalase.
  • the second vector may also be adapted to include genes encoding viral NS2B-NS3 protease.
  • C-prM-E genes can be expressed as a single cassette only if C and prM genes separated by a self-cleaved peptide like for example 2A autoprotease of foot-and-mouth disease virus in order to ensure proper processing of C-pM region in the absence of KUN virus encoded NS2B-NS3 protease.
  • the present invention also provides stable cell lines capable of persistently producing replicon RNAs.
  • the described vectors are preferably constructed in selectable form by inserting an IRES-Neo or IRES-pac cassette into the 3'UTR.
  • Host cell lines contemplated to be useful in the method of the invention include any eukaryotic cell lines that can be immortalised, ie., are viable for multiple passages, (eg., greater than 50 generations), without significant reduction in growth rate or protein production.
  • Useful cell line should also be easy to transfect, be capable of stably maintaining foreign RNA with an unarranged sequence, and have the necessary cellular components for efficient transcription, translation, post-translation modification, and secretion of the protein.
  • Currently preferred cells are those having simple media component requirements, and which can be adapted for suspension culturing.
  • Most preferred are mammalian cell lines that can be adapted to growth in low serum or serum-free medium.
  • Representative host cell lines include BHK (baby hamster kidney), VERO, C6-36, COS, CHO (Chinese hamster ovary), myeloma, HeLa, fibroblast, embryonic and various tissue cells, eg., kidney, liver, lung and the like and the like.
  • a cell line is selected from one of the following: BHK21 (hamster), SK6 (swine), VERO (monkey), L292 (mouse), HeLa (human), HEK (human), 2fTGH cells, HepG2 (human).
  • Useful cells can be obtained from the American Type Culture Collection (ATCC), Rockville, Md. or from the European Collection of Animal Cell Cultures, Porton Down, Salisbury SP40JG, U.K.
  • all means for introducing nucleic acids into a cell are contemplated including, without limitation, CaPO.sub.4 co-precipitation, electroporation, DEAE-dextran mediated uptake, protoplast fusion, microinjection and lipofusion.
  • the invention contemplates either simultaneous or sequential transfection of the host cell with vectors containing the RNA sequences.
  • host cells are sequentially transfected with at least two unlinked vectors, one of which contains flavivirus replicon expressing heterologous gene, and the other of which contains the structural genes.
  • the present invention also provides virus like particles containing flavivirus replicons and a method for producing such particles.
  • virus like particles that contain flavivirus derived replicons can be used to deliver any nucleotide sequence to a cell.
  • the replicons may be of either DNA or RNA in structure.
  • One particular use for such particles is to deliver nucleotide sequences coding for polypeptides that stimulate an immune response.
  • Such particles may be employed as a therapeutic or in circumstances where the nucleotide sequence encodes peptides that are capable of eliciting a protective immune response they may be used as a vaccine.
  • Another use for such particles is to introduce into a subject a nucleotide sequence coding for a protein that is either deficient or is being produced in insufficient amounts in a cell.
  • the replicon containing flavivirus like particles that contain nucleotide coding sequence for immunogenic polypeptide(s) as active ingredients may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the flavivirus replicon therapeutic(s) may also be mixed with excipients that are pharmaceutically acceptable and compatible with the replicon encapsulated viral particle. Suitable excipients are, for example, water, saline, dextrose glycerol, ethanol, or the like and combinations thereof.
  • the therapeutic may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvant which enhance the effectiveness of the therapeutic.
  • the replicon containing flavivirus like particles may be conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly.
  • Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • suppositories traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1 %-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like, These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of virus like particles, preferably 25-70%.
  • the flavivirus like particles may be formulated into the vaccine as neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such an, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from in- organic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamins, trimethylamine, 2-ethylamino ethanol, histidino, procaine, and the like.
  • the flavivirus like particles may be administered in a manner compatible with the dosage formulation and in such amount as will be prophylactically and/or therapeutically effective.
  • the dose of viral particles to be administered depends on the subject to be treated, the type of nucleotide sequence that is being administered and the type of expression efficiency of that sequence and in the case where the nucleotide sequence encodes immunogenic peptide/polypeptides the degree of protection desired. Precise amounts of active ingredient required to be administered may depend on the judgment of the practitioner and may be peculiar to each subject.
  • the flavivirus like particles may be given to a subject in a single delivery schedule, or preferably in a multiple delivery schedule.
  • a multiple delivery schedule is one in which a primary course of delivery may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or re-enforce the effect sought and if needed, a subsequent dose(s) after several months.
  • the delivery regimen will also, at least in part, be determined by the need of the individual and be dependent upon the judgment of the practitioner.
  • Figure 1 illustrates the construction and specific infectivity of the full- length KUN cDNA clones, and the structure of KUN replicon RNAs.
  • Schematic representations of the full-length and deleted (replicon) constructs show consecutive substitutions of the cDNA fragments in AKUN clone (textured boxes) with analogous fragments obtained by RT-PCR from KUN virion RNA (shaded boxes).
  • PFU titers on the right hand side of the figure represent an average (from three experiments) obtained after electroporation of the transcribed RNAs into BHK21 cells and determined by plaque assay; the titer of purified wild type KUN RNA was ⁇ 10 5 -10 6 PFU/ ⁇ g RNA.
  • Bgl(89), Sac(1481), Sph(2467), Dra(8376), Xho(11021) show restriction enzyme sites used in replacement cloning with the numbers in brackets representing nucleotide numbers in the KUN sequence.
  • An Expand High Fidelity PCR kit (Boehringer Mannheim) was used to obtain the indicated cDNA fragment of 6895 nucleotides in the FLSD and FLSDX constructs, and "Pfu PCR" in FLSDX indicates that this cDNA fragment of 2645 nucleotides was obtained using Pfu DNA polymerase (Stratagene).
  • C20DXrep and C20DXrepNeo constructs were prepared as described below in Example 1 (C20DXrep) and in Example 4 (C20DXrepNeo). Open boxes represent the deleted part of the genome; Ires - internal ribosomal entry site of encephalomyelitis virus RNA; Neo - neomycin transferase gene.
  • Figure 2 illustrates a schematic representation of the recombinant SFV constructs.
  • the solid line in all constructs represents the segment of the SFV replicon genome flanking the multiple cloning site, open boxes show the inserted KUN structural genes C, prM, and E as indicated, 26S shows the position of the subgenomic SFV promoter, the filled and partially filled boxes in the KUN prM and E genes represent hydrophobic signal and anchor sequences, respectively.
  • Capital letters in the nucleotide sequences show authentic KUN nucleotides, small letters show nucleotides derived from the pSFV1 vector or encoded in the primers used for PCR amplification of KUN genes.
  • Bold and italicised letters show initiation (ATG) and termination (taa, tag) codons. Numbers with arrows represent amino acid positions in the KUN polyprotein. Msc, Sma, Spe, Bam, and Bgl represent specific restriction sites. Asterisks indicate that these restriction sites were destroyed during the cloning procedure.
  • Figure 3 illustrates expression of KUN C protein by recombinant SFV-C replicon.
  • SFV1 panels 2, 4, and 6) represents IF of cells transfected with the control SFV1 RNA prepared from pSFV1 vector. Cells in panels 1 and 2 were photographed at lower magnification then in panels 3 to 6.
  • Ace is an abbreviation for acetone fixation
  • F+Me is an abbreviation for formaldehyde-methanol fixation.
  • BHK21 cells in 60mm culture dishes at 18h after transfection were continuously labelled with 50 ⁇ Ci/ml of 35 S-methionine/cysteine for 4h. Labelled cell lysates and radioimmunoprecipitates were prepared and samples were electrophoresed in a 15% polyacrylamide gel.
  • Sample volumes were 1 ⁇ l of 500 ⁇ l in SFV-C, 0.5 ⁇ l of 300 ⁇ l in SFV1 , 10 ⁇ l of 30 ⁇ l radioimmunoprecipitate from 160 ⁇ l of both SFV-C and SFV1 (+anti-C) cell lysates.
  • Dots indicate the location of KUN proteins NS5, NS3, E, NS4B, prM, NS2A, C, and NS4A/NS2B (from top to bottom) in the radiolabeled KUN infected cell lysate.
  • the arrow shows position of KUN C protein. Numbers represent molecular weights of low range pre-stained Bio-Rad protein standards. This and following figures were prepared by scanning all the original data (slides, autoradiograms, etc.) on the Arcus II scanner
  • Figure 4 illustrates expression of KUN prME genes by recombinant SFV replicon.
  • A) IF analysis of SFV-prME and SFV1 transfected BHK21 cells at 18h after transfection using KUN monoclonal anti-E antibodies.
  • B) and (C) show the results of pulse-chase labelling and radioimmunoprecipitation analysis with KUN monoclonal anti-E antibodies, respectively, of SFV- prME transfected BHK21 cells, where CF (culture fluid) and C (cells) represent samples collected during chase periods.
  • Lanes 1 to 9 in (B) and (C) represent the same samples either directly electrophoresed in 12.5% SDS-polyacrylamide gel (B), or radioimmunoprecipitated with anti-E antibodies followed by electrophoresis in a 12.5% SDS-polyacrylamide gel (C).
  • Lanes 2 and 9 show samples collected after a 4h-chase period from culture fluid and cells, respectively, after transfection with the control SFV1 RNA.
  • Lanes 3, 4, and 5 show culture fluid samples collected at 1 h, 4h, and
  • Figure 5 illustrates expression of all three KUN structural proteins by the recombinant SFV-prME-C replicon.
  • B) and (C) cells at
  • Figure 6 illustrates packaging of KUN replicon RNA by KUN structural proteins expressed from the recombinant SFV replicons.
  • A IF analysis with KUN anti-NS3 antibodies of BHK21 cells infected with the culture fluid collected from BHK21 cells at 26h after transfection first with C20DXrep RNA and 26h later with SFV-prME-C RNA (panel 1), or with SFV-prME and SFV-C RNAs (panel 2), or with SFV-prME RNA (panel 3).
  • (B) and (C) show Northern blot analysis of RNAs isolated from BHK21 cells infected as described in (A), using labelled KUN-specific (B) and SFV-specific (C) cDNA probes.
  • Lane 1 in (B) and lane 2 in (C) correspond to the cells in panel 1 in (A).
  • Lane 2 in (B) and lane 3 in (C) correspond to the cells in panel 2 in (A).
  • Lane 1 in (C) represents in vitro synthesized SFV-prME-C RNA.
  • Figure 7 illustrates optimisation of conditions for packaging of KUN replicon RNA.
  • samples were collected at a fixed time (24h) after second transfection (with SFV-prME-C RNA) and using different time intervals as shown between transfections of C20DXrep and SFV-prME-C RNAs.
  • samples were collected at different times as shown after the second transfection (with SFV-prME-C RNA) which occurs at a fixed time (30h) after the first transfection (with C20DXrep
  • RNA The probe in both (A) and (B) was a radiolabeled cDNA fragment representing the last 761 nucleotides of the KUN genome.
  • Titers in (A) shown under the lanes in the Northern blot represent the amounts of infectious units (IU) contained in the corresponding samples of culture fluids used for infections and determined by IF analysis with anti-NS3 antibodies and counting of IF positive cells.
  • Figure 8 illustrates characterisation of infectious particles.
  • Panel 1 represents IF with anti-NS3 antibodies of cells infected with culture fluid collected after the transfections and incubated with anti-E monoclonal antibodies for 1 h at 37°C.
  • Panel 2 represents IF with anti-NS3 antibodies of cells infected with the same sample of culture fluid incubated under similar conditions in the absence of anti-E antibodies.
  • FIG. B shows IF analysis with anti-N3 antibodies of ceils infected with equal proportions of resuspended pellet (panel 1 ; 2 ⁇ l from 50 ⁇ l of total volume) or supernatant fluid (panel 2; 200 ⁇ l from 5ml of total volume) from the culture fluid collected from cells transfected with C20DXrep and SFV-prME-C RNAs and subjected to ultracentrifugation.
  • FIG. 9 Sedimentation and electron microscopy analyses of KUN replicon and virion particles.
  • A Sedimentation profiles of virions and replicon particles in parallel sucrose density gradients. Particles were collected from culture fluids of BHK cells either at 35h after sequential transfections with C20DXrep and SFV-prME-C107 RNAs, or at 24h after infection with KUN virus, and were concentrated by ultracentrifugation as described in Materials and Methods. The pelleted particles were resuspended in 300 ⁇ l of PBS-0.1%BSA overnight at 4°C, and clarified by centrifugation at 16,000g in the microcentrifuge for 10 min.
  • the supernatant was overlaid on the top of a 12 ml 5-25% sucrose density gradient which was centrifuged at 38,000 rpm for 70 min at 20°C in an SW41 rotor.
  • 0.5 ml fractions were collected from the bottom of the gradient and diluted 1 :2 (replicon particles) or 1 :100 (KUN virions) for infectivity assays by IF on cover slip cultures of BHK cells at 24h (replicon particles) or at 18h (KUN virions) after infection, using anti-E antibodies; titers of infectious particles were determined as described earlier (see .
  • Resuspended particles were then sonicated in the Transsonic 700/h sonicating water bath (CAMLAB, Germany) for 1 min and pelleted onto a carbon coated formvar grid by centrifugation in an 18° fixed angle A-100 rotor in a Beckman Airfuge for 1 h at 80,000 rpm. Grids were stained with 4% uranyl acetate and particles were visualized by electron microscopy. The bar represents 200nm.
  • FIG. 10 Schematic representation of the Kunjin replicon expression vectors and recombinant constructs.
  • A shows C20DX2Arep(Neo) vector(s) and its derivatives.
  • SP6 shows the position of the SP6 promoter. 5'UTR and 3'UTR represent 5' and 3' untranslated regions, respectively.
  • C20 corresponds to the first twenty amino acids of KUN Core protein.
  • 22E corresponds to the last twenty two amino acids of KUN E protein.
  • NS1- NS5 correspond to the sequence coding for KUN nonstructural proteins.
  • 2A indicates sequence coding for 2A autoprotease of foot-and-mouth disease virus (FMDV) with its cleavage site indicated.
  • FMDV foot-and-mouth disease virus
  • IRESNeo represents a sequence of an internal ribosomal entry site (IRES) of encephalomyocarditis virus (EMCV) RNA followed by a sequence coding for the neomycin transferase gene (Neo).
  • This cassette was inserted at the indicated position in the 3'UTR to obtain C20DX2ArepNeo vector for stable selection of replicon expressing cells (similar to ⁇ ME/76Neo, Khromykh and Westaway, J. Virol. ,1997, 71 :1497-1505).
  • Spel shows a unique restriction site for cloning of heterologous genes.
  • B shows a list of KUN replicons with heterologous genes inserted into the Spel site of
  • C20DX2Arep vector C20DX2Arep vector.
  • hcv-trCore and hcv-flCore sequences coding for the first 160 and 191 amino acids of hepatitis C virus Core protein, respectively; CAT - chloramphenicol acetyltransferase; GFP - green fluorescent protein, hcv-NS3 - sequence coding for amino acids 183 to 617 of hepatitis C virus NS3 protein; VSV-G - glycoprotein G of vesicular stomatitis virus; ⁇ -GAL - Escherichia coli ⁇ -galactosidase.
  • ⁇ IRESNeo signs opposite to CAT and GFP indicate that these genes were also cloned into C20DX2ArepNeo vector.
  • C Dicistronic C20DXIRESrep vector and its derivative construct C20DX/CAT/IRESrep. Ascl-Stop shows the position of a unique site for cloning of heterologous genes followed by the translation termination codon (Stop). The other abbreviations are as in (A).
  • FIG. 11 Expression of heterologous genes in BHK21 cells eiectroporated with recombinant RNAs.
  • A) and (C) show IF analysis of BHK21 cells at 24 to 40 hours after transfection with the recombinant KUN replicon RNAs expressing different heterologous genes (indicated under each panel) using corresponding antibodies. Dilutions of antibodies were as follows: 1/100 for rabbit anti-CAT polyclonal antibodies (panels 1 and 2 in A); 1/150 for rabbit anti-VSV-G polyclonal antibodies (panels 3 and 4 in A); 1/40 for human anti-HCV polyclonal serum (panels 1-4 in C). Mock show parallel IF analyses of untransfected BHK21 cells.
  • (B) GFP panel shows fluorescence of live unfixed BHK21 cells at 24 h after transfection with C20DX GFP/2Arep RNA.
  • ⁇ -Gal panel represents X-gal staining of BHK21 cells at 46 h after transfection with C20DX/ ⁇ -GAL/2Arep RNA performed as described in the examples.
  • Figure. 12. Time course analyses of the CAT and ⁇ -GAL expression in cells transfected with corresponding recombinant KUN replicon RNAs.
  • FIG. 13 Processing of polyproteins translated from the eiectroporated recombinant KUN replicon RNAs.
  • A Radioimmunoprecipitation (RIP) analysis of radiolabelled BHK21 cells transfected with C20DX/CAT/2Arep (lane 1), C20DXCAT/IRESrep (lane 2), and C20DX2Arep (lane 3) RNAs using anti-CAT antibodies.
  • C20DX/VSV-G/2Arep RIP sample was treated with endoglycosidase F (endo F) as described elsewhere and both endo F-treated and untreated samples were electrophoresed on SDS-10% polyacrylamide gel. Arrows show the positions of glycosylated (gVSV-G) and nonglycosylated (VSV-G) proteins.
  • FIG. 14 Packaging of the recombinant KUN replicon RNAs.
  • A GFP fluorescence and IF analysis of BHK21 and Vero cells at 35h after infection with culture fluid collected from BHK21 cells sequentially transfected with recombinant KUN replicon RNAs and SFV-prME-C105 RNA using corresponding antibodies as indicated. Time intervals between transfections were 30 h for C20DX GFP/2Arep, 34 h for C20DX ⁇ SV-
  • FIG. 15 Stable BHK cell lines expressing GFP (repGFP-BHK) and CAT (repCAT-BHK). Cell lines were established by selection of BHK21 cells transfected with C20DX/GFP/2Arep and C20DX/CAT/2Arep RNAs, respectively, in the medium containing 1mg per ml of G418 (Geneticin).
  • A GFP fluorescence of passage 5 of repGFP-BHK cells.
  • B Autoradiogram of the CAT assay of the lysates from repCAT-BHK cells at passages 6 and 17.
  • FIG. 16 (A) Schematic representation of KUN replicon expression vector containing ubiquitin gene (C20DXUb2Arep). Ub shows ubiquitin gene, all the other abbreviations as in Fig. 10. (B) IF analysis of BHK cells at 24h after transfection with C20DXrep and C20DXUb2Arep RNAs using anti-NS3 antibodies. Figure. 17. (A), illustrates the construction of full-length
  • C20DXUb2A_HDVrep vector (Fig. 17A).
  • B) illustrates efficient replication of C20DXUb2A_HDVrep RNA in -100% BHK21 cells compared to -60% positive cells obtained after transfection with the same amounts of parental C20DXUb2Arep RNA (Fig. 17B).
  • FIG. 18 illustrates the construction of DNA-based pKUNRepl vector (Fig. 18A).
  • (B) shows successful detection of expression of the KUN NS3 protein (indicator of the replicating KUN replicon RNA) at 42 h post transfection with pKUNRepl plasmid DNA (Fig. 18B).
  • Figure. 19 illustrates Expression of GFP in mouse lung epithelium after intranasal immunization with recombinant KUN VLPs containing encapsidated C20DX/GFP/2Arep RNA.
  • BHK21 cells were grown in Dulbecco's modification of minimal essential medium (Gibco BRL) supplemented with 10% foetal bovine serum at 37°C in a CO 2 incubator.
  • minimal essential medium Gibco BRL
  • C20rep All deletion constructs were prepared from the cDNA clones used in the construction of the plasmid pAKUN for generation of the infectious KUN RNA (Khromykh and Westaway, J .Virol., 1994, 68:4580-4588) by PCR-directed mutagenesis using appropriate primers and conventional cloning.
  • dME cDNA and its derivatives were deleted from nucleotides 417 to 2404, which represent loss of the signal sequence at the carboxy terminus of C now reduced to 107 amino acids, deletion of prM and E, with the open reading frame resumed at codon 479 in E, preceding the signal sequence for NS1.
  • C20 rep and C2rep cDNAs represent progressive in frame deletions in coding sequence of C leaving only 20 or 2 amino acids of C, respectively, with the open reading frame continued at codon 479 in E, as in dME.
  • FLSDX All RT reactions were performed with Superscript II RNase H- reverse transcriptase (Gibco BRL) essentially as described by the manufacturer using100-200ng of purified KUN virion RNA, or 1 ⁇ g of total cell RNA and appropriate primers. PCR amplification after RT of a 6895bp DNA fragment was performed with the Expand High Fidelity PCR kit (Boehringer Mannheim) using 1/25 volume of RT reaction and appropriate primers as follows.
  • the PCR reaction mixture (50 ⁇ l) containing all necessary components except the enzyme mixture (3 parts of Taq polymerasse and 1 part of Pwo polymerase) was preheated at 95°C for 5 min, then the enzyme mixture was added and the following cycles were performed: 10 cycles of 95°C for 15sec and 72°C for 6min, followed by 6 cycles of 95°C for 15sec and 72°C for 6min with an automatic increase of extension time at 72°C for 20sec in each following cycle. All PCR reactions with Pfu DNA polymerasse (Stratagene) were performed essentially as described by the manufacturer using 1/25-1/10 volumes of RT reactions and appropriate primers.
  • Fig. 1 All plasmids shown in Fig. 1 were obtained from the previously described stable KUN full-length cDNA clone pAKUN (Khromykh and Westaway, J.Virol., 1994, 68:4580-4588) by substitution of the original cDNA fragments with those obtained by RT and PCR amplification of purified KUN RNA using existing unique restriction sites which were also incorporated into the primers for PCR amplification.
  • Sacll 1 81 -Dralll 8376 (6895 bp) fragment in pAKUN clone (Fig. 1) was replaced with the fragment amplified using Expand High Fidelity PCR kit from the cDNA obtained by reverse transcription of purified KUN virion RNA using appropriate primers.
  • RNA transcribed from the resulting cDNA clone (FLSD) had a specific infectivity of ⁇ 2 ⁇ 10 3 PFU per 1 ⁇ g, compared to only 1-5 PFU per 10 ⁇ g for AKUN RNA (Fig.1 ).
  • FLSD RNA transcribed from the resulting cDNA clone
  • Fig.1 RNA transcribed from the resulting cDNA clone
  • KUN replicon cDNA construct C20DXrep was constructed from described above C20rep by replacing an Spfrl 2467 - X/70I 11021 fragment representing the sequence coding for the entire nonstructural region and the 3'UTR with the corresponding fragment from a stable full-length KUN cDNA clone FLSDX.
  • Transfection of BHK cells with 5-10 ⁇ g of C20DXrep RNA resulted in detection of -80% replicon-positive cells compare to only -10% positive after transfection with the same amount of C20rep RNA.
  • SFV-C (iv) SFV-C.
  • An SFV replicon construct expressing KUN core (C) gene was obtained by cloning of the Bg/ll-BamHI fragment, representing the sequence of the last 7 nucleotides of the KUN 5'UTR and the sequence coding for the first 107 of the 123 amino acids of KUN C protein, from the plasmid pCINeoC107 (Khromykh, A. A. and E. G. Westaway. Arch. Virol., 1996, 141 :685-699) into the BamHI site of the SFV replicon expression vector pSFV1 (Gibco BRL; Fig. 2).
  • KUN prME sequence was PCR amplified from another highly efficient full-length KUN cDNA clone FLBSDX modified from FLSDX (which will be described elsewhere), using appropriate primers with incorporated BglU sites. The amplified fragment was digested with BglU and cloned into the BamHI site of the SFV replicon expression vector pSFV1 to obtain the SFV-prME construct (Fig. 2).
  • SFV-prME-C SFV replicon construct expressing both KUN prME and KUN C genes was obtained by cloning a Mscl-Spel fragment from the SFV-C plasmid containing the SFV 26S subgenomic promoter, KUN C sequence and SFV 3'UTR into the SFV-prME vector digested with S al and Spel (Fig. 2).
  • SFV-prME-C should produce SFV replicon RNA which upon transfection into BHK cells will direct synthesis of two subgenomic RNAs expressing KUN prME and KUN C genes.
  • RNA transcripts were prepared from C20DXrep plasmid DNA linearized with Xho ⁇ , and from SFV plasmids linearised with Spel using SP6 RNA polymerase. Electroporation of RNAs into BHK21 cells was performed. Briefly, 10-20 ⁇ g of in vitro transcribed RNAs were eiectroporated into 2 ⁇ 10 6 BHK21 cells in 400 ⁇ l in a 0.2-cm cuvette (Bio-Rad) using the Bio-Rad Gene Pulser apparatus.
  • KUN replicon RNA C20DXrep Replication of KUN replicon RNA C20DXrep after initial electroporation, and after infection of BHK cells in packaging experiments, was monitored by immunofluorescence (IF) analysis with antibodies to KUN NS3 protein.
  • IF immunofluorescence
  • Metabolic labeling with 35 S-methionine/cysteine of eiectroporated BHK cells was performed essentially as described in the SFV Gene Expression System Manual with some minor modifications. Briefly, cells at 18 h after the electroporation with SFV RNAs (with or without prior electroporation with KUN replicon RNA), were pulse labeled with 35 S-methionine/cysteine for 4h, or for 1-2h followed by different periods of incubation (chase) in medium with an excess of unlabeled methionine/cysteine. Cell culture fluid was collected for analysis of secreted proteins by electrophoresis and radioimmunoprecipitation (RIP).
  • RIP radioimmunoprecipitation
  • Labeled cells were lysed in buffer containing 1% Nonidet P40 (NP40), 50 mM Tris-HCI (pH 7.6), 150 mM NaCI, and 2mM EDTA, the nuclei removed by low speed centrifugation and the lysate supernatant was used for parallel analysis with the culture fluid.
  • NP40 Nonidet P40
  • Tris-HCI pH 7.6
  • 150 mM NaCI 1
  • 2mM EDTA 2mM EDTA
  • labeled cell culture fluids were first filtered through 0.45 ⁇ m filter (Sartorius AG, Gottingen, Germany) and digested with RNase A (20 ⁇ g per ml) for 30 min at 37°C to ensure the removal of membrane particulate material and naked RNA. Filtered and RNase treated culture fluids, or untreated cell lysates, were then mixed with 1/20 volume of the pooled anti-E monoclonal antibodies (see above) or with rabbit anti-C antibodies, and incubated overnight at 4°C with constant rotation in microcentrifuge tubes. Protein A-Sepharose beads were then added to a final concentration of about 1%, and incubation was continued for another 1 h at 4°C.
  • RNA Five ⁇ g total RNA, isolated using Trizol reagent (Gibco BRL) from BHK21 cells infected with culture fluid collected from cells doubly transfected with C20DXrep RNA and SFV RNAs expressing KUN structural proteins, was electrophoresed for Northern blotting.
  • the hybridisation probes were [ 32 P]-labelled cDNA fragments representing the 3'-terminal 761 nucleotides of the KUN genome including the 3'UTR region (see Fig. 6B and Fig. 7), and 446 nucleotides of the SFV NSP2 region (see Fig. 6C). Expression of KUN C gene by the recombinant SFV-C replicon.
  • KUN C gene in the pSFV1 vector the Bg/ll-BamHI fragment from plasmid pCINeoC107 was subcloned into the BamHI site of pSFV1 (Fig. 2).
  • This fragment represents the sequence coding for the first 107 amino acids of KUN C protein, equivalent to the mature form of C, from which the carboxy terminal hydrophobic sequence has been removed.
  • the SFV-C construct also contains a native KUN initiation codon with an extra 7 nucleotides of the KUN 5'UTR derived from the pCINeoC107 plasmid and four extra codons at the carboxy-terminus derived from the SFV vector sequence (Fig. 2).
  • Electroporation of SFV-C RNA into BHK21 cells resulted in expression of KUN C protein in almost 100% of cells as judged by IF with antibodies to KUN C protein (Fig.3A, panel 1).
  • KUN C protein expressed in SFV-C RNA transfected cells was localised in the cytoplasm (Fig. 3A, panel 3; acetone fixation) and also in the nuclei (Fig. 3A, panel 5; formaldehyde-methanol fixation). Because of difficulties in identification of KUN C protein in radiolabeled lysates of SFV-C transfected cells (Fig 3B), immunoprecipitation of the radiolabelled lysates with anti-C antibodies was carried out. A labelled band coincident in migration with KUN C protein was apparent in the lysates of SFV-C but not in those of SFV1 transfected cells (compare SFV-C and SFV1 in Fig. 3B).
  • the full-length prME sequence plus the preceding signal sequence in our SFV- prME construct was included in the replicon.
  • As a source of cDNA for prME genes full-length KUN cDNA clone FLBSDX were used. An initiation and a termination codon, as well as BglU sites for conventional cloning, were incorporated into the primers for PCR amplification (see Fig. 2).
  • Pfu DNA polymerase (Stratagene) was used in all our PCR reactions.
  • a labelled band corresponding to KUN prM protein was detected only in cell lysates (cells in Fig. 4B).
  • a labelled band corresponding in migration to the predicted molecular weight of KUN pr protein was detected in the culture fluid only of transfected cells (culture fluid in Fig. 4B).
  • results of the direct pulse-chase labelling and RIP analyses confirmed both the correct processing of prME polyprotein in cells and the secretion of E, and possibly pr and M proteins, into the culture fluid after transfection of SFV-prME RNA into BHK21 cells.
  • cell culture fluid was filtered through a 0.45 ⁇ m filter (Sartorius AG, Gottingen, Germany) and treated with RNase A (20 ⁇ g per ml) for 0.5h at room temperature (followed by 1.5h incubation at 37°C during infection of cells).
  • RNase A 20 ⁇ g per ml
  • the pellets were resuspended in 50 ⁇ l PBS supplemented with RNAse A (20 ⁇ g per ml), left to dissolve overnight at 4°C, and then used for infection of BHK21 cells or for RT- PCR analysis.
  • BHK21 cells on 1.3 cm 2 coverslips were infected with 100-200 ⁇ l of serial 10-fold dilutions of cell culture fluid or of pelleted material for 1.5h at 37°C. The fluid was then replaced with 1 ml of DMEM medium supplemented with 2% FBS; cells were incubated for 24h at 37°C in the CO 2 incubator and then subjected to IF analysis with anti-NS3 antibodies as described above.
  • the infectious titer of packaged particles was calculated using the following formula:
  • N is the average number of anti-NS3 positive cells in the image area, calculated from 5 image areas in different parts of the coverslip;
  • SW is the surface of the well in a 24-well plate (200 mm 2 );
  • SIA is the surface of the image area (1.25 mm 2 using defined magnification parameters, calculated according to the manual for the Wild MPS46/52 photoautomat [Wild Leitz, Heerburg, Germany]);
  • V is the total volume of the culture fluid (usually 3-5 ml per 60 mm dish) collected from the population of 2x10 6 initially eiectroporated BHK21 cells;
  • VI is the volume used for infection of the cover slips (usually 100-200 ⁇ l); and 10 n is the dilution factor.
  • KUN replicon construct C20rep was able to successfully transfect only 10-20% of cells a KUN replicon of greater transfection efficiency was used for attempted packaging in doubly transfected cells (i.e. KUN replicon, and recombinant SFV replicons expressing KUN structural proteins). This significantly improved the efficiency of transfection in BHK21 cells to about 80% using the replicon construct C20DXrep, which was used in all packaging experiments. As noted above, all cell culture fluids from packaging experiments were filtered to remove large membrane fragments and treated with RNase A to remove naked RNA.
  • variable time points between electroporations (Fig. 7A), and between the second electroporation and harvesting of the infectious particles (Fig. 7B), were examined.
  • Initially optimisation of the time between the two electroporations was studied with a fixed time for collection of the infectious particles.
  • Equal amounts of cells were seeded onto cell culture dishes after the first electroporation with C20DXrep RNA, and cells were subsequently eiectroporated with SFV-prME-C RNA at 12h, 18h, 24h, or 30h incubation intervals.
  • KUN replicon RNA was immunoprecipitated in the absence of detergents from the culture fluid of cotransfected and radiolabeled cells using anti-E antibodies.
  • Half of the immunoprecipitated sample was used for separation in the SDS-polyacrylamide gel, and the other half was used to extract RNA by proteinase K digestion.
  • Radioautography of the polyacrylamide gel showed the presence of E, prM, and C proteins in the immunoprecipitates of culture fluid collected from cells either sequentially transfected with C20DXrep and SFV-prME-C RNAs or infected with KUN virus (Fig. 8C, lanes 1 and 3, respectively).
  • RNA extracted from the immunoprecipitates was reverse transcribed and PCR amplified using KUN-specific primers.
  • a DNA fragment of predicted size (-700 bp, NS2A region) was observed in the RT-PCR reactions of RNAs extracted from the immunoprecipitates of the culture fluid collected from cells either transfected sequentially as in Fig. 6 with both C20DXrep and SFV-prME-C RNAs (Fig. 8D, lane 2) or infected with KUN virus (Fig. 8D, lane 4).
  • No RT-PCR product was obtained from RNA extracted from the immunoprecipitate of the culture fluid collected from cells transfected with SFV-prME-C RNA alone (Fig. 8D, lane 3).
  • EXAMPLE 3 Construction of modified KUN replicon vectors and expression of heterologous genes.
  • BHK21 cells were grown in Dulbecco's modification of minimal essential medium (DMEM, Gibco BRL) supplemented with 10% of fetal bovine serum (FBS). Vero cells were grown in M 199 medium (Gibco BRL) supplemented with 5% FBS. Construction of the plasmids.
  • DMEM minimal essential medium
  • FBS fetal bovine serum
  • C20DXrepNeo The dicistronic replicon construct C20DXrepNeo used for generation of replicon-expressing BHK cells (repBHK) was prepared from C20DXrep by cloning an Ires-Neo cassette into the 3'UTR 25 nucleotides downstream of the polyprotein termination codon.
  • Xma ⁇ -Xho ⁇ fragment from ⁇ ME/76Neo plasmid (Khromykh and Westaway, J.Virol.1997, 71 :1497-1505) representing nucleotides 10260 -10422 of KUN sequence, followed by the IRES- Neo cassette and the last 522 nucleotides of KUN sequence was used to substitute Xmal 10260 -X/7ol 11021 fragment in C20DXrep construct.
  • IRES- Neo cassette was initially derived from the mammalian expression vector plresNeol (a derivative of pCIN1 , provided by S. Rees (Rees et al., BioTechniques, 1996, 20:102-110)).
  • the nucleotide sequence at the C-terminus of IRES element in this IRES-Neo cassette was modified by authors in order to decrease the level of Neo expression thus forcing selection of cells expressing only high levels of inserted genes when using this (plresNeol) vector and high concentrations of antibiotic G418.
  • C20DX2Arep and C20DX2ArepNeo To ensure cytosolic cleavage of heterologous genes expressed from the KUN replicon vectors, the C20Dxrep, C20DXrepNeo constructs were modified by inserting sequence coding for 2A autoprotease of the food-and-mouth disease virus (FMDV-2A) between the first twenty amino acids of KUN C and the last twenty two amino acids of KUN E proteins in each plasmid preserving the KUN polyprotein open reading frame. (C20DX2Arep, Fig. 10A).
  • FMDV-2A food-and-mouth disease virus
  • FMDV-2A peptide represents a specific sequence of 19 amino acids which cleaves itself at the C-terminus between the glycine-proline dipeptide and has been used to mediate cleavage of artificial polyproteins.
  • Two unique sites for cloning of foreign genes were also incorporated into these vectors: (1.) a Spel site between the first 20 amino acids of C protein and the 2A sequence, and (2.) a Eagl site between the 2A sequence and the rest of the KUN replicon sequence. Cloning into Spel site ensures the correct cleavage of C20- FG-2A fusion protein from the rest of the KUN polyprotein sequence. Cloning into the Eagl site permits correct N-terminus cleavage, but it will have its C-terminus fused to the 22aa of E protein.
  • C20DXIRESrep and C20DX/CAT/IRESrep were constructed by cloning EMCV IRES sequence PCR amplified from ⁇ ME/76Neo plasmid (Khromykh and Westaway, J.Virol., 1997, 71 :1497-1505) using the appropriate primers with incorporated Ascl (forward primer) and Mlu ⁇ (reverse primer) restriction sites into the Ascl site of the C20DXrep plasmid.
  • C20DX/CAT/IRESrep construct was prepared by cloning CAT gene PCR amplified from the plasmid pT3CAT2A/NAmodll (Percy er a/., J.Virol. 1994, 68:4486-4492) using primers with incorporated Mlu ⁇ restriction sites into the Ascl site of C20DXIRESrep plasmid (Fig. 10C).
  • heterologous genes were PCR amplified from corresponding plasmids using primers with incorporated Spel and/or Xba ⁇ restriction sites (sequences of the primers may be obtained from the corresponding author), and cloned into the Spel site of the C20DX2Arep or C20DX2ArepNeo (Fig. 10A).
  • Plasmids for PCR amplifications of the above genes were: GFP - pEGFP (Clontech), hcv Core - pcDNA3/HCV-Core (obtained from Eric Gowans, Sir Albert Sakzewski Virus Research Center, Brisbane), hcvNS3 - p3B-271 (obtained from Eric Gowans), VSV-G - pHCMV19 (obtained from Michael Bruns, Heinrich-Pette- Institute, University of Hamburg), ⁇ -GAL - pSFV3/LacZ (Gibco BRL).
  • Recombinant KUN replicon RNA transcripts were prepared using SP6 RNA polymerase from the corresponding recombinant KUN replicon plasmid DNAs linearized with Xho ⁇ or from the SFV-prME-C105 plasmid linearized with Spel. Electroporation of RNAs into BHK21 cells was performed according to the method described in Example 1.
  • IF Immunofluorescence analysis of eiectroporated or infected cells was performed as described using antibodies specific to expressed proteins or KUN anti-NS3 antibodies.
  • Rabbit polyclonal anti-CAT antibodies were purchased from 5 Prime ⁇ 3 Prime (Boulder, CO, USA)
  • rabbit polyclonal anti-VSV-G antibodies were obtained from Michael Bruns (Heinrich-Pette-lnstitut, Hamburg, Germany)
  • human anti-HCV polyclonal serum was provided by Eric Gowans (Sir Albert Sakzewski Virus Research Centre, Brisbane, Australia).
  • Preparation and characterization of KUN anti-NS3 antibodies were described previously (Westaway et al., J.Virol., 1997, 71 :6650-6661).
  • CAT activity in lysates of BHK21 cells either eiectroporated with TRCAT and C20DX/CAT/2Arep RNAs, or after infection with VLPs containing encapsidated C20DX/CAT/2Arep RNA, or in stable cell line expressing C20DX/CAT/2ArepNeo RNA was determined using TLS or LSC assays as described previously (Khromykh and Westaway, J.Virol., 1997, 71 :1497-1505).
  • Optimal time of expression of heterologous products In order to estimate the level and the optimal time of expression of heterologous products using this system, as well as to evaluate possible effects of the size of inserted sequences on the replication and packaging efficiency of resulting recombinant KUN replicon RNAs, KUN replicons expressing CAT (218 amino acids), GFP (237 amino acids), and ⁇ -Gal (1017 aa) genes were prepared in C20DX2Arep vector (Fig. 10B). In addition, CAT gene was also inserted into C20DXIRESrep vector producing C20DX/CAT/IRESrep RNA (Fig. 10C).
  • a C20DX/VSV-G/2Arep construct expressing VSV G glycoprotein (Fig. 10B) was prepared.
  • the expression of these genes in eiectroporated BHK21 cells was initially demonstrated by IF analysis with specific antibodies for CAT and VSV-G proteins (Fig. 11 A), by fluorescence analysis of live unfixed cells for GFP protein (panel 1 in Fig. 11 B), and by X-gal staining for ⁇ -Gal protein (panel 2 in Fig. 11 B).
  • the percentage of expressing cells in these experiments varied amongst the constructs from -10% for C20DX/CAT/IRESrep RNA, -20% for C20DX ⁇ - Gal/2Arep, C20DX/VSV-G/2Arep, and C20DX/CAT/2Arep RNAs to -50% for C20DX/GFP/2A RNA at 24-48 after electroporation (data not shown).
  • HCV proteins using the KUN replicon system
  • Core and NS3 genes of an Australian isolate of HCV (Trowbridge and Gowans, Arch.Virol., 1998, 143:501-511) were expressed using the replicon vector C20DX2Arep.
  • a truncated form of HCV NS3 gene (coding for amino acids 183 to 617), containing most of the HCV NS3 cytotoxic T cell epitopes was cloned into C20DX2Arep vector.
  • HCV Core gene was expressed in two forms: as a full length gene (coding for 191 amino acids, C20DX/hcv-flCore/2Arep RNA, Fig. 10B), and as a truncated gene (coding for the first 160 amino acids, C20DX/hcv- trCore/2Arep RNA, Fig. 10B).
  • Electroporation of both RNAs into BHK21 cells resulted in expression of HCV Core protein in -60-70% of transfected cells (data not shown), and at a similar levels, as judged by the intensity of IF with human anti-HCV antiserum (Fig. 11C, panels 3 and 4).
  • truncated HCV Core expressed from KUN replicon vector was localized in the nuclei, while full- length Core was not (data not shown).
  • C20DX/CAT/2Arep RNA were examined using radioimmunoprecipitation (RIP) analysis with anti-CAT antibodies. Strong radiolabelled band of -30 kDa, corresponding to a predicted size of C20/CAT/2A fusion protein (257 amino acids) was observed (lane 1 , Fig. 13A), suggesting that FMDV-2A cleavage indeed occurred. The presence of a very weak band of -33 kDa, corresponding to the predicted size of C20/CAT/2A/22E fusion protein (286 amino acids) was also observed (lane 1 , Fig. 13A), indicating that the cleavage by FMDV-2A protease was not complete.
  • RIP radioimmunoprecipitation
  • KUN replicon vector C20DXIRESrep was demonstrated by detection of -27.5 kDa radiolabelled band corresponding to a predicted size of C20CAT protein (240 amino acids) in the anti-CAT immunoprecipitate from the lysate of BHK21 cells transfected with C20DX/CAT/IRESrep RNA (lane 2, Fig. 13A).
  • Glycosylation of the VSV-G glycoprotein expressed from KUN replicon was demonstrated by the observed reduction in size of the endoglycosidase F treated VSV-G protein immunoprecipitated from the radiolabbeled lysates of BHK21 ceils transfected with C20DX/VSV-G/2Arep RNA (compare lanes 1 and 2 in Fig. 13B).
  • VLP virus-like particles
  • a heterologous packaging system has been developed allowing production of VLPs containing KUN replicon RNA encapsidated by the KUN structural proteins using consecutive transfections with KUN replicon RNA and SFV replicon RNA SFV-prME-C105 expressing KUN structural genes.
  • the highest titer of VLPs was achieved when the second electroporation with SFV- prME-C105 RNA was performed at the time of the maximum replication of KUN replicon RNA (delay of ⁇ 24-27h).
  • C20DX/CAT/2ArepNeo and C20DX/GFP/2ArepNeo were prepared by cloning CAT and GFP sequences into the Spel site of the C20DX2ArepNeo vector (Fig. 10A and B).
  • Transfection of the resulting RNAs into BHK21 cells and subsequent incubation of these cells in the medium supplemented with 1mg/ml G418 (Geneticin) resulted in a rapid enrichment of cells expressing CAT and GFP genes (repCAT-BHK and repGFP-BHK, respectively; Fig.15).
  • noncytopathic flavivirus KUN replicon vectors can be used for transient or stable expression of heterologous genes in mammalian cells. They also show that recombinant KUN replicon RNAs expressing heterologous genes can be encapsidated into pseudoinfectious virus-like particles by subsequent transfection with SFV replicon RNA expressing KUN structural genes. These virus-like particles can be used for delivery of the recombinant self- replicating RNAs into a wide range of cells or animals leading to a long-term production of heterologous proteins. Importantly, because of the heterologous nature of the developed packaging system, no recombination between KUN and SFV RNAs producing an infectious virus can occur.
  • Mouse ubiquitin gene was PCR amplified from the plasmid pRB269 (Baker et al., J Biol Chem 269:25381-25386) using appropriate primers with incorporated unique cloning sites (see Fig. 16A). Resulting PCR fragment containing also Xbal site at the 5'end and Spel site at the 3'end was then cloned into the Spel site of C20DX2Arep plasmid (see Fig. 10A), producing C20DXUb2Arep vector (Fig. 16). Thus the gene of interest can be cloned either between C20 and ubiquitin or between ubiquitin and FMDV 2A protease sequences (Fig. 16A).
  • HDV hepatitis delta virus
  • SV40 simian virus 40
  • HDVribo/SV40polyA simian virus 40 polyadenylation signal
  • the fragment containing the last 1331 nucleotides of the KUN replicon sequence followed by HDVribo/SV40polyA cassette was produced by fusion PCR reaction (Karreman, 1998, BioTechniques 24:736-742) using Pfu DNA polymerase (Stratagene), appropriate primers and two plasmid DNAs pTMSV5A (obtained from Tom Macnaughton, Sir Albert Sakzewski Virus Research Center, Brisbane, Australia) and C20DXrep, as templates.
  • NS5dGDD_F KUN NS5 sequence, forward
  • 3'UTRHDV junction of KUN 3'end and HDV ribozyme
  • SV40pA_R SV40 polyadenylation signal, reverse
  • Resulting PCR product was digested with Xmal (5'end) and Xho ⁇ (3'end) and cloned into Xmal / Xho ⁇ digested C20DXUb2Arep DNA, producing C20DXUb2A_HDVrep vector (Fig. 17A).
  • Electroporation of -5-10 ⁇ g RNA transcribed from this construct resulted in its efficient replication in -100% BHK21 cells compared to -60% positive cells obtained after transfection with the same amounts of parental C20DXUb2Arep RNA (Fig. 17B).
  • KUN replicon vector C20DXUb2A_HDVrep by inserting cytomegalovirus immediate-early (CMV-IE) enhancer/promoter region immediately upstream of the KUN replicon sequence.
  • CMV-IE cytomegalovirus immediate-early
  • the fragment containing CMV-IE promoter sequence followed by 5'end of the KUN replicon sequence was produced in fusion PCR reaction (Karreman, 1998, BioTechniques 24:736-742) using Pfu DNA polymerase, appropriate primers and pCI (Promega) and C20DXUb2Arep plasmid DNAs as templates.
  • CMV_F CMV IE promoter, forward
  • CMV ⁇ 'UTR junction of CMV promoter and 5'UTR of the KUN sequence
  • FMDV2AR end of FMDV-2A autoprotease sequence, reverse
  • Resulting PCR product was digested with Eagl (3'end) and cloned into Nrul (blunt) / Eagl digested C20DXUb2A_HDVrep plasmid, producing pKUNRepl vector (Fig. 18A).
  • SV40polyA sequence was previously incorporated downstream of HDV antigenomic ribozyme sequence (see Fig. 17A) to ensure termination of transcription by RNA polymerase II.
  • mice Two female BALB/c mice were immunized intra-nasally with -10 6 IU per mouse of the recombinant KUN VLPs expressing GFP (for details of the VLP preparation and determination of their titre see Example 3). Mice were anaesthetized with ketamine/xylazine (100ul per 20g of mouse weight) via intra-peritoneal route prior to immunization. At days 2, and 4 after immunization mice were euthanased with
  • mice were immunized intra-dermally (in the base of a tail) with -5x10 5 IU of VLPs containing packaged C20DX/GFP/2Arep RNA (see Example 3). Two weeks after immunization their serum was analyzed on the presence of anti-GFP antibodies by ELISA with purified GFP protein. The results of 50% end point titrations (ELISA t 50 ) for each mouse were: mouse #1 - 1/200, mouse #2 - 1/130, mouse #3 - 1/100.

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Abstract

L'invention concerne un système d'expression génique comprenant les éléments suivants: a) vecteur d'expression d'origine de flavivirus, à autoréplication, incluant la région non convertie 5' du flavivirus (UTR5'), au moins une partie de la région de codage 5' pour la protéine noyau du flavivirus, le codage de séquence nucléotidique pour les protéines non structurelles du flavivirus, et la totalité ou la majeure partie de la séquence terminale 3'- de la région UTR3' du flavivirus, indispensable pour l'autoréplication du matériau génomique du flavivirus; ce vecteur est conçu pour recevoir au moins une séquence nucléotidique sans perturbation de ses capacités de réplication; et b) au moins un second vecteur capable d'exprimer la ou les protéines structurales du flavivirus et toute autre protéine requise pour le conditionnement du vecteur d'expression à autoréplication dans les particules virales du flavivirus; ce vecteur est conçu pour éviter la recombinaison avec le vecteur à autoréplication lorsqu'il est en sa présence.
PCT/AU1998/000993 1997-11-28 1998-11-30 Systeme d'expression et de diffusion propre au flavivirus WO1999028487A1 (fr)

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NZ504725A NZ504725A (en) 1997-11-28 1998-11-30 Flavivirus expression and delivery system
CA002311395A CA2311395C (fr) 1997-11-28 1998-11-30 Systeme d'expression et de diffusion propre au flavivirus
AU15517/99A AU733155B2 (en) 1997-11-28 1998-11-30 Flavivirus expression and delivery system
JP2000523363A JP2002500003A (ja) 1997-11-28 1998-11-30 フラビウイルスの発現および送達のシステム
EP98959672A EP1034290A4 (fr) 1997-11-28 1998-11-30 Systeme d'expression et de diffusion propre au flavivirus
US09/580,476 US6893866B1 (en) 1997-11-28 2000-05-26 Flavivirus expression and delivery system
US11/098,283 US20060088937A1 (en) 1997-11-28 2005-04-04 Flavivirus expression and delivery system

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Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1195438A1 (fr) * 2000-10-06 2002-04-10 Rijksuniversiteit te Groningen L'immunisation génétique contre le carcinome du col de l'utérus
GB2372991A (en) * 2001-03-09 2002-09-11 Fiocruz Fundacao Oswaldo Cruz Flavivirus expression vector
WO2002072803A2 (fr) * 2001-03-09 2002-09-19 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Replicons sous-genomiques du la dengue a flavivirus
FR2829149A1 (fr) * 2001-09-06 2003-03-07 Pasteur Institut Souche neurovirulente du virus west nile et ses applications
WO2003046189A1 (fr) * 2001-11-26 2003-06-05 The University Of Queensland Systeme d'administration d'un vaccin a base de flavivirus
US6589531B1 (en) 2000-01-21 2003-07-08 The Regents Of The University Of California Recombinant yellow fever virus and method of use thereof
WO2004072274A1 (fr) * 2003-01-30 2004-08-26 Shanghai Tengen Biomedical Co., Ltd. Vaccin a base de pseudoparticules virales ayant comme support un replicon de recombinaison du virus de la dengue
US6923969B2 (en) * 1999-03-08 2005-08-02 Id-Lelystad, Instituut Voor Dierhouderij En Diergezondheid B.V. Non-spreading pestivirus
US20070249032A1 (en) * 2004-07-12 2007-10-25 Tengen Biomedical Company Flavivirus vaccine
EP1989316A2 (fr) * 2005-10-31 2008-11-12 Fundacao Oswaldo Cruz - Fiocruz Méthode de production de virus recombinant, de constructions d'adn et de compositions de vaccin
US7517683B2 (en) * 2001-01-08 2009-04-14 The Industry and Academic Cooperation in Chungnam National University HIV-like particles and the use thereof
WO2009114207A2 (fr) 2008-03-14 2009-09-17 Sanofi Pasteur Biologics Co. Vaccins de flavivirus défaillant sur le plan de la réplication et vecteurs de vaccin
US7871814B2 (en) * 2001-05-10 2011-01-18 The Regents Of The University Of California Recombinant bicistronic flaviviruses and methods of use thereof
US9273288B2 (en) 2006-02-27 2016-03-01 The Board Of Regents Of The University Of Texas System Pseudoinfectious flavivirus and uses thereof
EP3061826A1 (fr) * 2015-02-27 2016-08-31 Novartis AG Réplicons du flavivirus
US20200048636A1 (en) * 2010-07-06 2020-02-13 Glaxosmithkline Biologicals Sa Immunisation of large mammals with low doses of rna
US20220125723A1 (en) 2010-07-06 2022-04-28 Glaxosmithkline Biologicals Sa Lipid formulations with viral immunogens
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Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015118146A1 (fr) 2014-02-10 2015-08-13 Univercells Nv Système, appareil et procédé de production de biomolécules

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
GALLER R. et al., BRAZILIAN J. MED AND BIOL. RES., February 1997, 30, pages 157-168. *
KHROMYCH R. and WESTAWAY E., J. VIROL., February 1997, 71(2), pages 1497-1505. *
KHROMYKH A. et al., J. VIROL., July 1998, 72(7), pages 5967-5977. *
LINDENBACH B. and RICE C., J. VIROL., Dec. 1997, 71(12), pages 9608-9617. *
See also references of EP1034290A4 *

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US11690861B2 (en) 2010-07-06 2023-07-04 Glaxosmithkline Biologicals Sa Delivery of RNA to trigger multiple immune pathways
US11690864B2 (en) 2010-07-06 2023-07-04 Glaxosmithkline Biologicals Sa Delivery of RNA to trigger multiple immune pathways
US11696923B2 (en) 2010-07-06 2023-07-11 Glaxosmithkline Biologicals, Sa Delivery of RNA to trigger multiple immune pathways
US11707482B2 (en) 2010-07-06 2023-07-25 Glaxosmithkline Biologicals Sa Delivery of RNA to trigger multiple immune pathways
US11883534B2 (en) 2010-07-06 2024-01-30 Glaxosmithkline Biologicals Sa Immunisation with lipid formulations with RNA encoding immunogens
US11730754B2 (en) 2010-07-06 2023-08-22 Glaxosmithkline Biologicals Sa Delivery of RNA to trigger multiple immune pathways
US11739334B2 (en) 2010-07-06 2023-08-29 Glaxosmithkline Biologicals Sa Immunisation of large mammals with low doses of RNA
US11865080B2 (en) 2010-07-06 2024-01-09 Glaxosmithkline Biologicals Sa Delivery of RNA to trigger multiple immune pathways
US11759475B2 (en) 2010-07-06 2023-09-19 Glaxosmithkline Biologicals Sa Delivery of RNA to trigger multiple immune pathways
US11766401B2 (en) 2010-07-06 2023-09-26 Glaxosmithkline Biologicals Sa Methods of administering lipid formulations with immunogens
US11773395B1 (en) 2010-07-06 2023-10-03 Glaxosmithkline Biologicals Sa Immunization of large mammals with low doses of RNA
US11786467B2 (en) 2010-07-06 2023-10-17 Glaxosmithkline Biologicals Sa Lipid formulations with immunogens
US11839686B2 (en) 2010-07-06 2023-12-12 Glaxosmithkline Biologicals Sa Lipid formulations with viral immunogens
US11845925B2 (en) 2010-07-06 2023-12-19 Glaxosmithkline Biologicals Sa Immunisation of large mammals with low doses of RNA
US11851660B2 (en) 2010-07-06 2023-12-26 Glaxosmithkline Biologicals Sa Immunisation of large mammals with low doses of RNA
US11850305B2 (en) 2010-07-06 2023-12-26 Glaxosmithkline Biologicals Sa Method of making lipid formulations with RNA encoding immunogens
US11857681B2 (en) 2010-07-06 2024-01-02 Glaxosmithkline Biologicals Sa Lipid formulations with RNA encoding immunogens
US11857562B2 (en) 2010-07-06 2024-01-02 Glaxosmithkline Biologicals Sa Delivery of RNA to trigger multiple immune pathways
US11759422B2 (en) 2010-08-31 2023-09-19 Glaxosmithkline Biologicals Sa Pegylated liposomes for delivery of immunogen-encoding RNA
US11639370B2 (en) 2010-10-11 2023-05-02 Glaxosmithkline Biologicals Sa Antigen delivery platforms
WO2016135675A1 (fr) 2015-02-27 2016-09-01 Novartis Ag Réplicons de flavivirus
EP3061826A1 (fr) * 2015-02-27 2016-08-31 Novartis AG Réplicons du flavivirus
US10973899B2 (en) 2015-02-27 2021-04-13 Novartis Ag Flavivirus replicons

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EP1034290A1 (fr) 2000-09-13
CA2311395C (fr) 2006-11-28
CA2311395A1 (fr) 1999-06-10
NZ504725A (en) 2003-02-28
JP2008113666A (ja) 2008-05-22
JP2002500003A (ja) 2002-01-08

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