WO2014170493A2 - Vecteur d'alphavirus - Google Patents

Vecteur d'alphavirus Download PDF

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WO2014170493A2
WO2014170493A2 PCT/EP2014/058028 EP2014058028W WO2014170493A2 WO 2014170493 A2 WO2014170493 A2 WO 2014170493A2 EP 2014058028 W EP2014058028 W EP 2014058028W WO 2014170493 A2 WO2014170493 A2 WO 2014170493A2
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promoter
alphavirus vector
recombinant alphavirus
virus
utr
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PCT/EP2014/058028
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WO2014170493A3 (fr
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Clayton BEARD
Giulietta MARUGGI
Peter W. Mason
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Novartis Ag
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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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18534—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36141—Use of virus, viral particle or viral elements as a vector

Definitions

  • Alphaviruses are positive sense single-stranded 11.5 kb RNA viruses in the
  • Togaviridae family (Strauss, J.H., et al, Microbiol. Rev. 58:491-562 (1994))
  • the genome has a cap at the 5 'terminus and poly(A)-tail at the 3' terminus and encodes only 4 nonstructural proteins, nsPl-4, which are translated directly from the genomic RNA and interact with host factors to form replicative enzyme complexes.
  • nsPl-4 nonstructural proteins
  • the resulting RNA is capable of directing its own replication and heterologous gene expression when introduced into the cytoplasm of host cells, but is incapable of forming virions or spreading to adjacent cells because it does not encode the alphavirus capsid or glycoprotein genes.
  • the replicon may be packaged into virus replicon particles (VRP) by providing the structural protein in trans.
  • VRP virus replicon particles
  • alphavirus replicons make them attractive candidates for use as vaccine vectors, including high levels expression of the heterologous gene, vector amplification through double-stranded RNA intermediates, which stimulates aspects of innate immunity, such as activation of the IFN cascade, the ability to deliver the replicon and encoded heterologous proteins to a variety of cell types, including antigen-presenting cells, and the overall lack of preexisting immunity to alphavirus in the human population.
  • the immune response induced by the alphavirus replicon has been attributed both to the production of the heterologous protein and to the activation of innate responses.
  • the viral double-strand RNA (dsRNA) replication intermediates are recognized by host pattern recognition receptors, which can activate IRF-3/7-dependent signaling pathways to induce IFN-a and IFN- ⁇ mediated antiviral response.
  • the type I IFN signaling is important for the activation of both the innate and adaptive immune responses.
  • the immune response induced by the VRP is mainly due to the levels and the length of the expression of the heterologous protein, to the levels of activation of IFN- induced antiviral response or to a combination of these components.
  • Alphavirus vectors are a promising technology for vaccination against a variety of diseases.
  • Alpha viruses that have been used for construction of replicon-based RNA vaccines include Sindbis virus (SINV), Semliki Forest virus, and Venezuelan equine encephalitis virus (VEEV).
  • SINV replicon particles avoid the safety concerns of VEEV, but expression levels are lower.
  • the invention relates to alphavirus vectors engineered to produce altered levels of antigen expression and/or interferon (IFN) induction.
  • IFN interferon
  • the invention relates to a recombinant alphavirus vector comprising a nucleotide sequence encoding an exogenous protein with adenine at position 3 and cytosine at position 30 of the 5' UTR.
  • the recombinant alphavirus vector may further encode an amino acid at P3 of the nsPl cleavage site selected from the group consisting of isoleucine at position 533, valine at position 533, and threonine at position 533.
  • the recombinant vector may also encode a glutamic acid at position 536 or serine at position 773 in nsP2.
  • the recombinant vector may further encode an inactivated 26S promoter.
  • the inactivated 26S promoter can comprise at least one point mutation in the 3' region of nsP4, deletion of the last five nucleotides of a promoter between nsP4 and the exogenous nucleotide sequence, and insertion of an internal ribosome entry site (IRES) downstream of the inactivated 26S promoter.
  • IRS internal ribosome entry site
  • the invention relates to a recombinant alphavirus vector
  • a recombinant alphavirus vector comprising a nucleotide sequence encoding an exogenous protein with adenine at position 3 of the 5 ' UTR and encoding an amino acid at P3 of the nsPl cleavage site selected from the group consisting of isoleucine at position 533, valine at position 533 and threonine at position 533.
  • the recombinant alphavirus vector can also have a uracil at position 24 of the 5' UTR or cytosine at position 30 of the 5' UTR.
  • the recombinant alphavirus vector can also encode a glutamic acid at position 536 or serine at position 773 in nsP2.
  • the recombinant vector may further encode a 26S promoter that has a mutation that causes inactivation of the 26S promoter.
  • the mutation may comprise at least one point mutation in the 3' region of nsP4, deletion of the last five nucleotides of a promoter between nsP4 and the exogenous nucleotide sequence, and insertion of an IRES downstream of the inactivated 26S promoter.
  • the invention relates to a recombinant alphavirus vector comprising a nucleotide sequence encoding an exogenous protein with adenine at position 3 of the 5 ' UTR and a glutamic acid at position 536 in nsP2.
  • the recombinant alphavirus vector can further encode an amino acid at P3 of the nsPl cleavage site selected from the group consisting of isoleucine at position 533, valine at position 533 and threonine at position 533.
  • the recombinant alphavirus vector can further encode a 26S promoter comprising a mutation that causes inactivation of the 26S promoter.
  • the mutation can comprise at least one point mutation in the 3 ' of nsP4 region, deletion of the last five nucleotides of a promoter between nsP4 and the exogenous nucleotide sequence, and insertion of an IRES downstream of the inactivated 26S promoter.
  • the invention also relates to plasmids encoding and viral particles comprising the recombinant alphaviruses described herein.
  • the invention also relates to a method for inducing expression of the exogenous nucleotide sequences described herein, comprising administering the recombinant alphavirus vector to a cell.
  • the invention also relates to pharmaceutical compositions comprising the viral particle.
  • the invention further relates to methods of inducing an immune response in an individual.
  • a pharmaceutical composition comprising a viral particle is administered to the individual.
  • a viral particle comprising a recombinant alphavirus vector is administered to the individual.
  • FIGS. 1A-1F show the analysis of the effects of 5' UTR site specific mutations.
  • FIGS. 1A and IB show schematics of computer-predicted (M-fold) secondary structures of the 5' UTR, both on the positive and negative strand, in the genomes of VEE/SIN variants having mutations at nucleotides 3, 24, 30 and of the wild-type sequence (TRD) (SEQ ID NOS 16-25, respectively, in order of appearance).
  • TRD wild-type sequence
  • FIG. 1C is a graph showing antigen expression levels in BHK-V cells infected with the TC-83CR, the G3A-A30C or G3A-C24U VRP and analyzed 24 hours post-infection. The antigen levels were measured by ELISA of the supernatants from BHK-V cells infected with an MOI of 1 and normalized for the number of positive cells.
  • FIG. ID is a graph showing
  • FIGS. 1E-1F are graphs showing infection efficiency and antigen expression levels in BHK-V cells transfected with the TC-83CR, the G3A-A30C or G3A-A30G RNA replicons (10 ⁇ 11 replicon copies/sample) and analyzed 24 hours post-infection.
  • FIGS. 2A and 2B are graphs showing characterization of the TC83CR A533I mutant.
  • FIG. 2 A shows antigen expression levels in BHK-V cells infected with an MOI of 1 and analyzed 24 hours post-infection.
  • FIG. 2B shows the results of Type I IFN ELISA that was performed on the supernatants harvested 24 hours post-infection from L929 cells infected with an MOI of 1.
  • FIG. 3A are schematic representations of control (TC-83CR and VCR) and of the nsP2 mutated (VCR nsP2 G536E, VCR nsP2 P773S and TC-83CR nsP2 G536E) replicons. Arrows indicate the position of the subgenomic promoter. The positions of the mutations in the VEE 5' UTR and nsP2 coding gene are indicated. A3G indicates a nucleotide mutation; G536E and P773S indicate protein mutations; GOI: gene of interest; nsPs: non structural proteins; *: mutations where the TC-83CR sequence differs from the VCR sequence.
  • FIG. 3B is a graph showing antigen expression levels in BHK-V infected cells.
  • BHK-V cells infected with an MOI of 1 and analyzed 24 hours post-infection. The antigen levels were measured by ELISA and normalized for the number of positive cells.
  • FIG. 3C is a graph showing interferon induction in L929 cells by the replicons from (A). Type 1 interferon was measured by ELISA post-infection and normalized for the number of positive cells.
  • FIG. 4A are schematic representations of the TC-83CR and of the designed viral genomes to test the effect of the 26S promoter inactivation.
  • A26S inactivated 26S promoter.
  • FIG. 4B shows an alignment of the subgenomic promoter-containing fragment of the VEE TC- 83 genome and the corresponding fragment of the TC-83CR-A26S-GFP-IRES-GOI genome. The position of the promoter is indicated by the open box. The start of the subgenomic RNA in the VEEV TC-83 genome is indicated by an arrow, and the start of the GFP is labeled. The mutations, introduced into the TC-83CR-A26S-GFP-IRES-GOI genome are shown in lower case letters.
  • FIG. 4B discloses SEQ ID NOS 27, 26, 29, and 28, respectively, in order of appearance.
  • FIG. 4C is a graph showing antigen expression levels in BHK-V infected cells with the VRPs obtained from the genomes from (A) with an MOI of 1. The antigen production was measured by ELISA 24 hours post-infection and normalized for the number of positive cells.
  • FIG. 4D is a graph showing Interferon induction in L929 cells by the replicons from (A). Type I Interferon was measured by ELISA 24 hours post-infection and normalized for the number of positive cells.
  • FIGS. 5A-5B show that modified replicons produce different levels of antigen expression and different levels of the cellular type I IFN response.
  • SEAP placental alkaline phosphatase
  • FIG. 5B is a table summarizes the in vitro characterization of the replicons from (A), the expression levels of the two antigens (SEAP, truncated RSV-F fusion peptide deleted (TFPD)) was measured by ELISA on the supernatants from infected BHK-V cells and the IFN modulation in L929 cells. The SEAP expression levels measured in the sera of the treated mice are indicated.
  • the replicons were tested in vivo at 10 6 IU (VRP) or 0.1 ⁇ g (RNA).
  • FIGS. 6A-6B shows in vivo characterization of the modified replicons after delivery as VRP (A) or replicon formulated with a cationic oil-in- water emulsion (B).
  • VRP vascular endothelial protein
  • B cationic oil-in- water emulsion
  • the VEEV/SINV chimera replicon was tested at two different doses: 10 6 and 10 7 IU (VRP) or 0.1-1 ⁇ (RNA). All the other replicons were tested 10 6 IU (VRP) or O. ⁇ g (RNA).
  • the empty symbols refer to the F-specific IgG titers measured 3 weeks after the first immunization (3wpl, open diamond or open circle), while the shaded symbols indicate the 2 weeks post second immunization titers (2wp2, closed diamond or closed circle).
  • the bar in each group represents the geometric mean (log 10) of the titers of individual mice (5mice/group).
  • FIGS. 7A-7D are graphs showing the replicon carrying the G3A-C24U mutation in the 5' UTR demonstrates the highest immune response both in terms of F-specific IgG (see FIG. 6) and T cells.
  • IFN- ⁇ CD8 and IL2 CD4 responses are represented, as these were the dominant cytokine made by each subset of T cells.
  • Alphavirus vectors are a promising technology for vaccination against a variety of diseases. These vectors comprise alphavirus replicons that contain the genetic elements required for RNA replication, but lack those encoding gene products necessary for particle assembly because the structural genes of the alphavirus genome are replaced by sequences encoding a (one or more) heterologous proteins (immunogens). Upon delivery of the replicons to eukaryotic cells, the positive-stranded RNA is translated to produce four non-structural proteins, which together replicate the genomic RNA and transcribe abundant subgenomic mRNAs encoding the antigenic protein. Alphavirus replicons are able to induce strong innate immune responses that potentiate adaptive immune responses to the encoded immunogen.
  • alphavirus has its conventional meaning in the art and includes various species such as Venezuelan equine encephalitis virus (VEE; e.g., Trinidad donkey, TC83CR, etc.), Semliki Forest virus (SFV), Sindbis virus, Ross River virus, Western equine encephalitis virus, Eastern equine encephalitis virus, Chikungunya virus, S.A.
  • VEE Venezuelan equine encephalitis virus
  • SFV Semliki Forest virus
  • Sindbis virus Sindbis virus
  • Ross River virus Western equine encephalitis virus
  • Western equine encephalitis virus Eastern equine encephalitis virus
  • Chikungunya virus S.A.
  • alphavirus may also include chimeric alphaviruses (e.g., as described by Perri et al, (2003) J. Virol. 77(19): 10394-403) that contain genome sequences from more than one alphavirus.
  • VRP alphavirus replicon particle
  • replicon particle is an alphavirus replicon packaged with alphavirus structural proteins.
  • an "alphavirus replicon” (or “replicon”) is an RNA molecule which can direct its own amplification in vivo in a target cell.
  • the replicon encodes the polymerase(s) which catalyze RNA amplification (nsPl, nsP2, nsP3, nsP4) and contains cis RNA sequences required for replication which are recognized and utilized by the encoded polymerase(s).
  • An alphavirus replicon typically contains the following ordered elements: 5' viral sequences required in cis for replication, sequences which encode biologically active alphavirus nonstructural proteins (nsPl, nsP2, nsP3, nsP4), 3' viral sequences required in cis for replication, and a polyadenylate tract.
  • An alphavirus replicon also may contain one or more viral subgenomic "junction region" promoters directing the expression of heterologous nucleotide sequences, which may, in certain embodiments, be modified in order to increase or reduce viral transcription of the subgenomic fragment and heterologous sequence(s) to be expressed.
  • Other control elements can be used, as described below. It may additionally contain a promoter and/or an IRES.
  • the alphavirus replicon is designed to express a heterologous nucleic acid, e.g., a gene of interest (GOI), also referred to herein as a heterologous RNA or heterologous sequence, which can be chosen from a wide variety of sequences derived from any desired source, e.g., viruses, prokaryotes, eukaryotes, archae.
  • a heterologous RNA or heterologous sequence which can be chosen from a wide variety of sequences derived from any desired source, e.g., viruses, prokaryotes, eukaryotes, archae.
  • categories of heterologous sequences include, for example, immunogens, including antigenic proteins, cytokines, toxins, therapeutic proteins, enzymes, antisense sequences, and immune response modulators.
  • the recombinant alphavirus replicons described herein can produce desired levels of expression of a heterologous protein and/or interferon (IFN) induction.
  • the alphavirus vectors can induce an improved immune response by changing the levels of antigen expression and/or of the type I IFN induction.
  • Suitable replicons include, for example, 5' UTR mutations, changes to the nsPl/nsP2 cleavage domain, modification to the nsP2 gene, inactivation of the 26S subgenomic promoter, insertion of an internal ribosome entry site (IRES), and combinations thereof.
  • recombinant alphavirus vectors have 5' UTR mutations that can result in alterations in interferon response, rate of RNA self-replication, and/or subgenomic RNA transcription.
  • the 5' UTR may be mutated at position 3 to contain an adenine and/or at position 30 to contain a cytosine and/or a uracil at position 24.
  • Recombinant alphavirus vectors of the invention may comprise mutations that interrupt the nsPl/nsP2 cleavage domain. Such mutations can alter regulation of the type I IFN induction. (Cruz, C. et al, Virology, 377(1): 160-169 (2008)) For example, P3 of the nsPl cleavage site may be mutated at position 533 to contain an isoleucine, valine, or threonine.
  • Recombinant alphavirus vectors may comprise modifications to the nsP2 gene that can result in persistent non cytopathic replication.
  • the nsP2 gene may be modified at position 536 to contain a glutamic acid and/or at position 773 to contain a serine.
  • the 26S subgenomic promoter may be attenuated or inactivated as a result of a mutation (including deletion in whole or in part).
  • the mutation may be at least one point mutation in the 3' region of nsP4 and/or deletion of the 3' of the promoter and deletion of the region between the promoter and the following gene.
  • the vectors can include a different expression control sequence (e.g., IRES downstream of the position of the 26S subgenomic promoter) that is operably linked to the gene(s) of interest, or any combination thereof.
  • the 26S promoter may be inactivated by introducing four silent point mutations in the 3 ' region of nsP4 and by deleting the last 5 nucleotides of the promoter and of the region between the promoter and the following gene.
  • Insertion of an IRES in a recombinant alphavirus vector may be used to express the heterologous protein can result in changes in the heterologous protein expression level. For example, insertion of an IRES before the heterologous protein in the recombinant alphavirus vector may increase expression of the heterologous protein in comparison to a 26S subgenomic promoter.
  • An IRES allows multiple proteins to be made from a single mRNA transcript as ribosomes bind to each IRES and initiate translation in the absence of a 5 ' -cap, which is normally required to initiate translation of protein in eukaryotic cells.
  • the IRES can be EV71 or EMCV.
  • Suitable IRES elements include, but are not limited to, viral IRES elements from picornaviruses, e.g., poliovirus (PV) or the human enterovirus 71 , e.g. strains 7423/MS/87 and BrCr thereof; from encephalomyocarditis virus (EMCV); from foot-and-mouth disease virus (FMDV); from flaviviruses, e.g., hepatitis C virus (HCV); from pestiviruses, e.g., classical swine fever virus (CSFV); from retroviruses, e.g., murine leukemia virus (MLV); from lentiviruses, e.g., simian immunodeficiency virus (SIV); from cellular mRNA IRES elements such as those from translation initiation factors, e.g., elF4G or DAP5; from transcription factors, e.g., c-Myc (Yang and Sarnow
  • the IRES element of this invention can be derived from, for example, encephalomyocarditis virus (EMCV, GenBank accession #NC001479), cricket paralysis virus (GenBank accession # AF218039), Drosophila C virus (GenBank accession # AF014388), Plautia stali intestine virus (GenBank accession # AB006531), Rhopalosiphum padi virus (GenBank accession #AF022937), Himetobi P virus (GenBank accession # ABO 17037), acute bee paralysis virus (GenBank accession # AF 150629), Black queen cell virus (GenBank accession # AF 183905), Triatoma virus (GenBank accession # AF 178440), Acyrthosiphon pisum virus (GenBank accession # AF024514), infectious flacherie virus (GenBank accession # AB000906), and/or Sacbrood virus (Genbank accession # AF092924).
  • EMCV encephalomy
  • IRES elements have been described, which can be designed, according to methods know in the art to mimic the function of naturally occurring IRES elements (see Chappell, SA et al. Proc. Natl Acad. Sci. USA (2000) 97(4): 1536-41.
  • the IRES is functional in mammalian cells.
  • the recombinant alphavirus vector may also be modified by 2'-0 methylation of the viral mRNA cap.
  • the alphavirus mRNA cap is methylated at the N-7 positions of the guanosine cap. Additional methylation in position 2'-0 may be useful to evade host restriction by IFN- induced proteins with tetratricopeptide repeats (IFIT family members), which are interferon- stimulated genes.
  • IFIT family members IFN-induced proteins with tetratricopeptide repeats
  • the 2'-0 methylation of the 5' cap of viral RNA could function to subvert innate host antiviral responses through escape of IFIT-mediated suppression (Daffis, S. et al. Nature (2010) Nov 18; 468(7322):452-6).
  • Heterologous proteins suitable for inclusion in the recombinant alphavirus vectors described herein may be derived from any pathogen (e.g., a bacterial pathogen, a viral pathogen, a fungal pathogen, a protozoan pathogen, or a multi-cellular parasitic pathogen), allergen or tumor.
  • pathogen e.g., a bacterial pathogen, a viral pathogen, a fungal pathogen, a protozoan pathogen, or a multi-cellular parasitic pathogen
  • the heterologous protein is derived from a viral pathogen.
  • viral pathogens include, e.g., respiratory syncytial virus (RSV), hepatitis B virus (HBV), hepatitis C virus (HCV), Dengue virus, herpes simplex virus (HSV; e.g., HSV-I, HSV- II), molluscum contagiosum virus, vaccinia virus, variola virus, lentivirus, human
  • HIV immunodeficiency virus
  • HPV human papilloma virus
  • CMV cytomegalovirus
  • VZV varicella zoster virus
  • rhinovirus enterovirus
  • adenovirus coronavirus
  • coronavirus e.g., SARS
  • influenza virus flu
  • para-influenza virus mumps virus
  • measles virus papovavirus
  • the heterologous protein can be CMV glycoprotein gH, or gL; Parvovirus; HIV glycoprotein gpl20 or gpl40, HIV p55 gag, pol; or RSV-F heterologous protein, etc.
  • the heterologous protein is derived from a virus which infects fish, such as: infectious salmon anemia virus (ISAV), salmon pancreatic disease virus (SPDV), infectious pancreatic necrosis virus (IPNV), channel catfish virus (CCV), fish lymphocystis disease virus (FLDV), infectious hematopoietic necrosis virus (IHNV), koi herpesvirus, salmon picorna-like virus (also known as picorna-like virus of atlantic salmon), landlocked salmon virus (LSV), atlantic salmon rotavirus (ASR), trout strawberry disease virus (TSD), coho salmon tumor virus (CSTV), or viral hemorrhagic septicemia virus (VHSV).
  • infectious salmon anemia virus ISAV
  • SPDV salmon pancreatic disease virus
  • IPNV infectious pancreatic necrosis virus
  • CCV channel catfish virus
  • FLDV fish lymphocystis disease virus
  • IHNV infectious hematopoietic necrosis virus
  • the heterologous protein is derived from a parasite from the Plasmodium genus, such as P .falciparum, P.vivax, P.malariae or P. ovale.
  • the invention may be used for immunizing against malaria.
  • the heterologous protein is derived from a parasite from the Caligidae family, particularly those from the Lepeophtheirus and Caligus genera e.g. sea lice such as Lepeophtheirus salmonis or Caligus rogercresseyi.
  • the heterologous protein is derived from a bacterial pathogen.
  • bacterial pathogens include, e.g., Neisseria spp, including N. gonorrhea and N. meningitides; Streptococcus spp, including S. pneumoniae, S. pyogenes, S. agalactiae, S. mutans; Haemophilus spp, including H. influenzae type B, non typeable H. influenzae, H.
  • Moraxella spp including M. catarrhalis, also known as Branhamella catarrhalis;
  • Bordetella spp including B. pertussis, B. parapertussis and B. bronchiseptica; Mycobacterium spp., including M. tuberculosis, M. bovis, M. leprae, M. avium, M. paratuberculosis, M.
  • smegmatis Legionella spp, including L. pneumophila
  • Escherichia spp including enterotoxic E. coli, enterohemorragic E. coli, enteropathogenic E. coli
  • Vibrio spp including V. cholera, Shigella spp, including S. sonnei, S. dysenteriae, S. flexnerii; Yersinia spp, including Y.
  • enterocolitica Y. pestis, Y. pseudotuberculosis, Campylobacter spp, including C. jejuni and C. coli
  • Salmonella spp including S. typhi, S. paratyphi, S. choleraesuis, S. enteritidis
  • Listeria spp. including L. monocytogenes
  • Helicobacter spp including H pylori
  • Pseudomonas spp including P. aeruginosa, Staphylococcus spp., including S. aureus, S. epidermidis
  • Enterococcus spp. including E. faecalis, E. faecium
  • Clostridium spp. including C. tetani, C. botulinum, C.
  • Bacillus spp. including B. anthracis; Corynebacterium spp., including C. diphtheriae; Borrelia spp., including B. burgdorferi, B. garinii, B. afzelii, B. andersonii, B. hermsii; Ehrlichia spp., including E. equi and the agent of the Human Granulocytic Ehrlichiosis; Rickettsia spp, including R. rickettsii; Chlamydia spp., including C. trachomatis, C. neumoniae, C. psittaci; Leptsira spp., including L. interrogans; Treponema spp., including T. pallidum, T. denticola, T. hyodysenteriae.
  • the heterologous protein is derived from a fungal pathogen (e.g., a yeast or mold pathogen).
  • a fungal pathogen e.g., a yeast or mold pathogen.
  • Exemplary fungal pathogens include, e.g., Aspergillus fumigatus, A. flavus, A. niger, A. terreus, A. nidulans, Coccidioides immitis, Coccidioides posadasii, Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, and
  • the heterologous protein is derived from a protozoan pathogen.
  • protozoan pathogens include, e.g., Toxoplasma gondii, Strongyloides stercoralis, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae.
  • the heterologous protein is derived from a multi-cellular parasitic pathogen.
  • exemplary multicellular parasitic pathogens include, e.g. , trematodes (flukes), cestodes (tapeworms), nematodes (roundworms), and arthropods.
  • the heterologous protein is derived from an allergen, such as pollen allergens (tree-, herb, weed-, and grass pollen allergens); insect or arachnid allergens (inhalant, saliva and venom allergens, e.g. mite allergens, cockroach and midges allergens, hymenopthera venom allergens); animal hair and dandruff allergens (from e.g. dog, cat, horse, rat, mouse, etc.); and food allergens ⁇ e.g. a gliadin).
  • pollen allergens tree-, herb, weed-, and grass pollen allergens
  • insect or arachnid allergens inhalant, saliva and venom allergens, e.g. mite allergens, cockroach and midges allergens, hymenopthera venom allergens
  • animal hair and dandruff allergens from e.g. dog
  • Important pollen allergens from trees, grasses and herbs are such originating from the taxonomic orders of Fagales, Oleales, Pinales and platanaceae including, but not limited to, birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus) and olive (Olea), cedar (Cryptomeria and Juniperus), plane tree (Platanus), the order of Poales including grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale, and Sorghum, the orders of Asterales and Urticales including herbs of the genera Ambrosia, Artemisia, and Parietaria.
  • Other important inhalation allergens are those from house dust mites of the genus Dermatophagoides and Euroglyphus, storage mite e.g.
  • Lepidoglyphys, Glycyphagus and Tyrophagus those from cockroaches, midges and fleas e.g. Blatella, Periplaneta, Chironomus and Ctenocepphalides, and those from mammals such as cat, dog and horse, venom allergens including such originating from stinging or biting insects such as those from the taxonomic order of Hymenoptera including bees (Apidae), wasps (Vespidea), and ants (Formicoidae).
  • the heterologous protein is derived from a tumor antigen selected from: (a) cancer-testis antigens such as NY-ESO-1, SSX2, SCP1 as well as RAGE, BAGE, GAGE and MAGE family polypeptides, for example, GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE- 12 (which can be used, for example, to address melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumors; (b) mutated antigens, for example, p53 (associated with various solid tumors, e.g., colorectal, lung, head and neck cancer), p21/Ras (associated with, e.g., melanoma, pancreatic cancer and colorectal cancer), CDK4 (associated with, e.g., melanoma), MUM1 (associated with, e.g.
  • KSA associated with, e.g., colorectal cancer
  • gastrin associated with, e.g., pancreatic and gastric cancer
  • telomerase catalytic protein MUC-1 (associated with, e.g., breast and ovarian cancer)
  • G-250 associated with, e.g., renal cell carcinoma
  • p53 associated with, e.g., breast, colon cancer
  • carcinoembryonic antigen associated with, e.g., breast cancer, lung cancer, and cancers of the gastrointestinal tract such as colorectal cancer
  • shared antigens for example, melanoma-melanocyte differentiation antigens such as MART-l/Melan A, gplOO, MC1R, melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase related protein- 1/TRPl and tyrosinase related protein-2/TRP2 (associated with,
  • tumor immunogens include, but are not limited to, pl5, Hom/Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens, including E6 and E7, hepatitis B and C virus antigens, human T-cell lymphotropic virus antigens, TSP-180, pl85erbB2, pl80erbB-3, c-met, mn-23Hl, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, pl6, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29 ⁇ BCAA), CA 195, CA 242, CA-50, CAM43, CD68 ⁇ KP1, CO-029,
  • a recombinant alphavirus vector comprises a nucleotide sequence with adenine at position 3 and cytosine at position 30 of the 5' UTR.
  • the recombinant alphavirus vector may further encode an isoleucine, valine or threonine amino acid at position 533 P3 of the nsPl cleavage site.
  • the recombinant vector may also encode a glutamic acid at position 536 or serine at position 773 in nsP2.
  • the recombinant vector may further encode an inactivated 26S promoter.
  • the inactivated 26S promoter may comprise at least one point mutation in the 3' region of nsP4 and/or deletion of the 3' of the promoter and of the region between the promoter and the following gene.
  • an internal ribosome entry site IVS
  • the recombinant alphavirus vector contains a nucleotide sequence that encodes one or more heterologous proteins and is operably linked to an expression control sequence.
  • the heterologous protein is located 3' of the nsP4 26S subgenomic promoter.
  • a recombinant alphavirus vector comprises a nucleotide sequence with adenine at position 3 of the 5 ' UTR and encodes an isoleucine, valine or threonine amino acid at position 533 P3 of the nsPl cleavage site.
  • the recombinant alphavirus vector may also have a uracil at position 24 of the 5' UTR or cytosine at position 30 of the 5' UTR.
  • the recombinant alphavirus vector may also encode a glutamic acid at position 536 or serine at position 773 in nsP2.
  • the recombinant alphavirus vector may further encode a 26S promoter that has a mutation that causes inactivation of the 26S promoter.
  • the inactivated 26S promoter may comprise at least one point mutation in the 3 ' region of nsP4 and/or deletion of the 3' of the promoter and of the region between the promoter and the following gene.
  • an internal ribosome entry site IVS
  • the recombinant alphavirus vector contains a nucleotide sequence that encodes one or more heterologous proteins and is operably linked to an expression control sequence.
  • a recombinant alphavirus vector comprises a nucleotide sequence with adenine at position 3 of the 5' UTR and a glutamic acid at position 536 in nsP2.
  • the recombinant alphavirus vector may further encode an isoleucine, valine or threonine amino acid at position 533 P3 of the nsPl cleavage site.
  • the recombinant alphavirus vector may further encode a 26S promoter comprising a mutation that causes inactivation of the 26S promoter.
  • the inactivated 26S promoter may comprise at least one point mutation in the 3' region of nsP4 and/or deletion of the 3' of the promoter and between the promoter and the following gene.
  • an internal ribosome entry site IVS
  • the recombinant alphavirus vector contains a nucleotide sequence that encodes one or more heterologous proteins and is operably linked to an expression control sequence.
  • the heterologous protein is located 3' of the nsP4 26S subgenomic promoter.
  • a recombinant alphavirus vector comprises a nucleotide sequence with adenine at position 3 of the 5' UTR and an asparagine at position 551 in nsP2.
  • the recombinant alphavirus vector may further encode an isoleucine, valine or threonine amino acid at position 533 P3 of the nsPl cleavage site.
  • the recombinant alphavirus vector may further encode a 26S promoter comprising a mutation that causes inactivation of the 26S promoter.
  • the inactivated 26S promoter may comprise at least one point mutation in the 3' region of nsP4 and/or deletion of the 3' of the promoter between the promoter and the following gene.
  • an internal ribosome entry site is inserted downstream of the 26S promoter (e.g., attenuated, inactivated).
  • the recombinant alphavirus vector contains a nucleotide sequence that encodes one or more heterologous proteins and is operably linked to an expression control sequence.
  • the heterologous protein is located 3' of the nsP4 26S subgenomic promoter.
  • self-replicating RNA molecules or VRPs are administered to an individual to stimulate an immune response.
  • self-replicating RNA molecules or VRPs typically are present in a composition which may comprise a pharmaceutically acceptable carrier and, optionally, an adjuvant. See, e.g., U.S. 6,299,884; U.S. 7,641,911; U.S. 7,306,805; and US 2007/0207090.
  • the self-replicating RNA described herein are suitable for delivery in a variety of modalities, such as naked RNA delivery or in combination with lipids, polymers or other compounds that facilitate entry into the cells.
  • Self-replicating RNA molecules can be introduced into target cells or subjects using any suitable technique, e.g., by direct injection, microinjection, electroporation, lipofection, biolystics, and the like.
  • the self-replicating RNA molecule may also be introduced into cells by way of receptor-mediated endocytosis. See e.g., U.S. Pat. No. 6,090,619; Wu and Wu, J. Biol. Chem., 263: 14621 (1988); and Curiel et al, Proc.
  • U.S. Pat. No. 6,083,741 discloses introducing an exogenous nucleic acid into mammalian cells by associating the nucleic acid to a polycation moiety ⁇ e.g., poly-L-lysine having 3-100 lysine residues), which is itself coupled to an integrin receptor-binding moiety ⁇ e.g., a cyclic peptide having the sequence Arg-Gly-Asp).
  • a polycation moiety e.g., poly-L-lysine having 3-100 lysine residues
  • an integrin receptor-binding moiety e.g., a cyclic peptide having the sequence Arg-Gly-Asp
  • the self-replicating RNA molecules can be delivered into cells via amphiphiles. See e.g., U.S. Pat. No. 6,071,890.
  • a nucleic acid molecule may form a complex with the cationic amphiphile. Mammalian cells contacted with the complex can readily take it up.
  • the self-replicating RNA can be delivered as naked RNA ⁇ e.g. merely as an aqueous solution of RNA) but, to enhance entry into cells and also subsequent intercellular effects, the self-replicating RNA is preferably administered in combination with a delivery system, such as a particulate or emulsion delivery system.
  • a delivery system such as a particulate or emulsion delivery system.
  • delivery systems include, for example lipo some-based delivery (Debs and Zhu (1993) WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques 6(7): 682-691; Rose U.S. Pat. No.
  • Three particularly useful delivery systems are (i) liposomes, (ii) non-toxic and biodegradable polymer microparticles, and (iii) cationic submicron oil-in-water emulsions.
  • RNA molecules of the invention may be used to deliver the self-replicating RNA molecules of the invention, as naked RNA or in combination with a delivery system, into a target organ or tissue.
  • Suitable catheters are disclosed in, e.g., U.S. Pat. Nos. 4,186,745; 5,397,307; 5,547,472; 5,674,192; and 6,129,705, all of which are incorporated herein by reference.
  • the present invention includes the use of suitable delivery systems, such as liposomes, polymer microparticles or submicron emulsion microparticles with encapsulated or adsorbed self-replicating RNA, to deliver a self-replicating RNA molecule that encodes alphavirus replicons, for example, to elicit an immune response alone, or in combination with another macromolecule.
  • the invention includes liposomes, microparticles and submicron emulsions with adsorbed and/or encapsulated self-replicating RNA molecules, and combinations thereof.
  • the self-replicating RNA molecules associated with liposomes and submicron emulsion microparticles can be effectively delivered to a host cell, and can induce an immune response to the protein encoded by the self-replicating RNA.
  • the immune response can comprise a humoral immune response, a cell-mediated immune response, or both.
  • an immune response is induced against each delivered CMV protein.
  • a cell-mediated immune response can comprise a Helper T-cell (Th) response, a CD8+ cytotoxic T-cell (CTL) response, or both.
  • the immune response comprises a humoral immune response, and the antibodies are neutralizing antibodies.
  • Neutralizing antibodies block viral infection of cells.
  • Neutralizing antibody responses can be complement-dependent or complement-independent. In some embodiments the neutralizing antibody response is complement-independent.
  • a useful measure of antibody potency in the art is "50% neutralization titer.”
  • serum from immunized animals is diluted to assess how dilute serum can be yet retain the ability to block entry of 50% of viruses into cells.
  • a titer of 700 means that serum retained the ability to neutralize 50% of virus after being diluted 700-fold.
  • higher titers indicate more potent neutralizing antibody responses.
  • this titer is in a range having a lower limit of about 200, about 400, about 600, about 800, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, or about 7000.
  • the 50% neutralization titer range can have an upper limit of about 400, about 600, about 800, about 1000, about 1500, about 200, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, about 7000, about 8000, about 9000, about 10000, about 11000, about 12000, about 13000, about 14000, about 15000, about 16000, about 17000, about 18000, about 19000, about 20000, about 21000, about 22000, about 23000, about 24000, about 25000, about 26000, about 27000, about 28000, about 29000, or about 30000.
  • the 50% neutralization titer can be about 3000 to about 6500.
  • An immune response can be stimulated by administering VRPs or self-replicating RNA to a subject, typically a mammal, including a human. Suitable animal subjects include, for example, fish, birds, cattle, pigs, horses, deer, sheep, goats, bison, rabbits, cats, dogs, chickens, ducks, turkeys, and the like.
  • the immune response induced is a protective immune response, i.e., the response reduces the risk or severity of infection.
  • compositions can be administered intra-muscularly, intra-peritoneally, sub-cutaneously, or trans-dermally. Some embodiments will be administered through an intra-mucosal route such as intra-orally, intra- nasally, intra-vaginally, and intra-rectally. Compositions can be administered according to any suitable schedule.
  • Replicons characterized by the ability to produce different levels of heterologous protein or to modulate the cellular type I IFN response were obtained. These replicons have unique genome structures and the same specific infectivity of TC-83.
  • the replicon is a VEEV/SINV chimera derived from a VEEV sequence and contains the packaging signal and 3' UTR from the SINV.
  • the phenotype of all the mutated replicons was characterized in vitro in BHK-V and L929 cells (IFN-competent cells), by measuring the heterologous protein expression levels and IFN induction after infection with the VRP with a multiplicity of infection (MOI) of 1.
  • MOI multiplicity of infection
  • SEAP secreted placental alkaline phosphatase
  • TFPD truncated fusion peptide deleted form of the F protein
  • RSV Respiratory Syncytial Virus
  • the main result of this first analysis was that the major determinant of specific infectivity is the nucleotide sequence at the 5' end of the alphavirus genome. All the mutants having a G3A mutation had the same phenotype of the TC-83CR replicon. Attention was focused on two mutants: the G3A-C24U mutant, and the G3A-A30C mutant, having the G3A sequence and a folding on the negative strand that differs from the TC-83 one only by the absence of the single strand RNA at the terminus (FIGS. 1 A and IB). The absence of the single strand RNA tail will help to define if it is important in the viral replication cycle.
  • VEEV encodes an alanine at the P3 cleavage position and we examined whether substitution with an Isoleucine (A533I) or Valine (A533V) or Threonine (A533T) would result in a replicon with similar effects on IFN induction as seen with the SINV T538I and the RRV A532V. Substitution of the Alanine 533 resulted in viable VRP that grew at slightly lower titers. BHK-V cells were infected with either the TC-83CR or the 533 mutants expressing the SEAP protein and the heterologous protein expression levels were evaluated at different times post infection by ELISA of the supernatants from infected cells.
  • the heterologous protein expression was reduced with all the nsPl mutants with respect to the TC-83CR.
  • L929 cells were infected with either the mutants or the TC-83CR and type I IFN induction in the supernatant was measured by ELISA. All of the mutants exhibited enhanced IFN induction in comparison to the TC-83CR replicon at 24 hours post infection. The highest induction was observed in the cells infected with the A533I mutant. The Isoleucine mutant was selected for further analysis and for the in vivo study.
  • the TFPD heterologous protein was cloned to study the immunogenicity in the A533I replicon. The in vitro
  • a mutation at position 536 (A536E-lst amino acid of nsP2) in SINV was tested that is responsible of non-cytopathic persistence and packaging efficiency comparable to the wild- type virus mutations in our replicons both in the TC-83CR and in the VCR (TRD sequence) backbone (FIG. 3A) and a mutation in VEE in position 773 of the polyprotein (P773S).
  • the vectors were packaged and tested in BHK and L929 cells with a multiplicity of infection of 1.
  • the G536E (wt VEEV has a glycine in position 536 instead of alanine as in SINV) mutants expressed lower levels of heterologous protein, both in the VCR and in TC-83CR context, and they did not change the IFN production in L929 cells following the infection.
  • the TC-83CR G536E was selected as the replicon of interest in the TC-83CR.
  • the substitution of a serine in position 773, in combination with the A3G mutation replicon, was characterized by the same expression levels of SEAP or TFPD per positive cell of the wt VCR, but induced higher activation of the IFN pathway in L929 cells (FIGS. 3B-3C).
  • 26S subgenomic promoter The 26S subgenomic transcript was modified to change the levels of heterologous protein expression.
  • the transcription of the subgenomic R A is driven by a 24-nt promoter located in the junction region between the coding sequence for the nsP4 and the structural proteins.
  • the inactivation of the 26S promoter has been associated with a change in the host response and in particular with an increase in the IFN induction.
  • the presence of the IRES is an additional factor that can change the kinetic of the heterologous protein expression and the host response.
  • the 26S promoter was inactivated by simultaneous point mutations in the 3 ' of the nsP4 region and deletion of the 3 ' of the promoter and of the region between the promoter and the following gene (FIG. 4B).
  • a reporter gene (GFP, green fluorescent protein) was cloned downstream of the inactivated promoter, to evaluate the inactivation, followed by the Encephalomyocarditis virus (EMCV) IRES and the
  • the delta 26S replicon produced lower levels of heterologous protein and a slight increase in the IFN response (FIGS. 4C-4D).
  • Mutants were selected that are able to induce very low levels of heterologous protein but high IFN induction, as the nspl mutants.
  • the nsP2 mutant in position 536 that produced low levels of heterologous protein without changing the IFN response and the P773S mutant that produced normal levels of heterologous protein but high IFN induction.
  • the inactivation of the 26S promoter slightly increased the IFN induction, while it reduced the heterologous protein expression levels.
  • the mutated replicons were tested in vivo to better characterize their phenotype. Two different in vivo studies were performed, one with SEAP as reporter gene, in order to measure the differences in the protein expression levels among the different vectors, and one with RSV F antigen to look at the immunogenicity of the different mutants. Balb/c 6-8wks old female mice were obtained from Charles River Laboratories.
  • RSV-F antigen immunogenicity screening (low dose screening) immunogenicity of the modified replicons was evaluated in vivo by using the replicons expressing the RSV-F antigen.
  • 6-8 week old BALB/c mice were immunized with replicons as shown in FIG. 5A, either delivered as VRP at l .OE+06 IU or CNE-56 formulated RNA at O. ⁇ g dose on days 0 and 22, and blood was collected on day 0, 21, and 36.
  • the control replicon (TC83CR) was delivered also at 1.0E+07 IU VRP dose or CNE-56 formulated RNA at ⁇ g dose.
  • Immunizations were performed intramuscularly in 100 ⁇ total volume (administered 50 ⁇ in each hind leg for mice). Serum was collected 3 weeks after the 1st vaccination (3wpl, day 21) and 2 weeks after the 2nd vaccination (2wp2, day 36) for IgG analysis. Mouse spleens were collected at the time of sacrifice for T cell analysis (2wp2, day 36). Endpoint immunogenicity assays were ELISA to determine mouse F-specific serum IgG titers and intracellular cytokine staining and flow cytometry to determine F-specific splenic T cell responses.
  • F-specific serum IgG titers showed that each mutation confers to the replicon the ability to elicit different levels of immunogenicity, with a correlation between antigen expression and immune response (FIGS. 6A-6B).
  • FIGS. 7A-7D An example of F-specific splenic T cell response elicited after immunization is shown in FIGS. 7A-7D.
  • the phenotype of all the mutated replicons was characterized in vitro in BHK-V and L929 cells (IFN-competent cells), by measuring the heterologous protein expression levels and IFN induction after infection with the VRP with a multiplicity of infection (MOI) of 1.
  • MOI multiplicity of infection
  • SEAP secreted placental alkaline phosphatase
  • TFPD truncated fusion peptide deleted form of the F protein
  • RSV Respiratory Syncytial Virus
  • Example 1 In the in vivo screening described in Example 1 , we analyzed the phenotype of a replicon having the 5'UTR entirely derived from Sindbis virus in the context of the VEE/SIN chimera.
  • the SINDBIS 5' UTR sequence (Table 1) was introduced in the replicon and replaced the VEE 5'UTR.
  • the SINDBIS 5'UTR replicon had the same specific infectivity, in vitro and in vivo heterologous protein expression levels, and elicited the same F-specific immune response with respect to the VEE/SIN chimera.
  • Table 1 shows an alignment of the sequences modified in the modified replicons described in this study. For each region that has been modified, the sequences are shown together with the sequence of the wild type (wt) replicon in the same region. Lower case letters indicate the mutated nucleotides.
  • the first category shows sequences at the 5'UTR region (nt 1- 44) for wild type (wt) and modified replicon, with the mutated nucleotide positions, or the 5'UTR origin (SINDBIS 5'UTR) indicated.
  • the second category shows sequences at the cleavage site between nsPl and nsP2 (nt 1638-1649). Amino acid changes are indicated for each replicon.
  • the third category shows sequences in part of nsP2, and amino acid changes are indicated for each replicon.
  • the fourth category shows sequences at the 26 promoter (wt-nt 7567-7590), and inactivation by point mutations and nucleotides deletions (dashes).

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Abstract

L'invention concerne d'une manière générale des vecteurs d'alphavirus recombinants comprenant un 5'-UTR, des séquences codant des gènes non de structure nsP1, nsP2, nsP3 et nsP4, et une séquence qui est reliée de manière fonctionnelle à une séquence de contrôle d'expression et codant pour une protéine hétérologue; mis au point pour produire des niveaux souhaités d'expression de protéine hétérologue et/ou d'induction d'interféron.
PCT/EP2014/058028 2013-04-19 2014-04-19 Vecteur d'alphavirus WO2014170493A2 (fr)

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018075235A1 (fr) * 2016-10-17 2018-04-26 Synthetic Genomics, Inc. Systèmes réplicons de virus recombinants et leurs utilisations
CN109328233A (zh) * 2016-03-21 2019-02-12 生物技术Rna制药有限公司 反式复制rna
US10538786B2 (en) 2016-04-13 2020-01-21 Janssen Pharmaceuticals, Inc. Recombinant arterivirus replicon systems and uses thereof
WO2020264138A1 (fr) * 2019-06-27 2020-12-30 Emd Millipore Corporation Expansion cellulaire avec des vecteurs d'arn auto-réplicatifs exprimant des protéines d'immortalisation
US11021692B2 (en) 2017-12-19 2021-06-01 Janssen Sciences Ireland Unlimited Company Hepatitis B virus (HBV) vaccines and uses thereof
US11020476B2 (en) 2017-12-19 2021-06-01 Janssen Sciences Ireland Unlimited Company Methods and compositions for inducing an immune response against Hepatitis B Virus (HBV)
US11083786B2 (en) 2018-01-19 2021-08-10 Janssen Pharmaceuticals, Inc. Induce and enhance immune responses using recombinant replicon systems
WO2021183564A1 (fr) * 2020-03-09 2021-09-16 Arcturus Therapeutics, Inc. Compositions et méthodes pour l'induction de réponses immunitaires
US20210340567A1 (en) * 2020-04-30 2021-11-04 Vlp Therapeutics, Inc. Cytokine immunotherapy
US11389531B2 (en) 2017-12-19 2022-07-19 Janssen Sciences Ireland Unlimited Company Methods and apparatus for the delivery of hepatitis B virus (HBV) vaccines
US11613561B2 (en) 2021-03-19 2023-03-28 Tiba Biotech, Llc Artificial alphavirus-derived RNA replicon expression systems
US11845939B2 (en) 2016-12-05 2023-12-19 Janssen Pharmaceuticals, Inc. Compositions and methods for enhancing gene expression

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005113782A1 (fr) * 2004-05-18 2005-12-01 Alphavax, Inc. Vecteurs alpha viraux dérivés du tc-83, particules et méthodes
US20060198854A1 (en) * 2004-12-28 2006-09-07 Peter Pushko Vector platforms derived from the alphavirus vaccines
WO2011139717A1 (fr) * 2010-04-26 2011-11-10 Novartis Ag Production améliorée de particules de réplicon de virus dans des cellules d'encapsidation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005113782A1 (fr) * 2004-05-18 2005-12-01 Alphavax, Inc. Vecteurs alpha viraux dérivés du tc-83, particules et méthodes
US20060198854A1 (en) * 2004-12-28 2006-09-07 Peter Pushko Vector platforms derived from the alphavirus vaccines
WO2011139717A1 (fr) * 2010-04-26 2011-11-10 Novartis Ag Production améliorée de particules de réplicon de virus dans des cellules d'encapsidation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SEREGIN A V ET AL: "TC83 replicon vectored vaccine provides protection against Junin virus in guinea pigs", VACCINE, ELSEVIER LTD, GB, vol. 28, no. 30, 5 July 2010 (2010-07-05), pages 4713-4718, XP027118985, ISSN: 0264-410X [retrieved on 2010-07-02] *

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109328233A (zh) * 2016-03-21 2019-02-12 生物技术Rna制药有限公司 反式复制rna
US10538786B2 (en) 2016-04-13 2020-01-21 Janssen Pharmaceuticals, Inc. Recombinant arterivirus replicon systems and uses thereof
US11555205B2 (en) 2016-04-13 2023-01-17 Janssen Pharmaceuticals, Inc. Recombinant arterivirus replicon systems and uses thereof
US11364310B2 (en) 2016-10-17 2022-06-21 Janssen Pharmaceuticals, Inc. Recombinant virus replicon systems and uses thereof
CN110073002A (zh) * 2016-10-17 2019-07-30 合成基因组公司 重组病毒复制子体系及其用途
CN110073002B (zh) * 2016-10-17 2024-06-11 杨森制药公司 重组病毒复制子体系及其用途
AU2017347725B2 (en) * 2016-10-17 2024-01-04 Janssen Pharmaceuticals, Inc. Recombinant virus replicon systems and uses thereof
WO2018075235A1 (fr) * 2016-10-17 2018-04-26 Synthetic Genomics, Inc. Systèmes réplicons de virus recombinants et leurs utilisations
US11845939B2 (en) 2016-12-05 2023-12-19 Janssen Pharmaceuticals, Inc. Compositions and methods for enhancing gene expression
US11725194B2 (en) 2017-12-19 2023-08-15 Janssen Sciences Ireland Unlimited Company Hepatitis B virus (HBV) vaccines and uses thereof
US11389531B2 (en) 2017-12-19 2022-07-19 Janssen Sciences Ireland Unlimited Company Methods and apparatus for the delivery of hepatitis B virus (HBV) vaccines
US11020476B2 (en) 2017-12-19 2021-06-01 Janssen Sciences Ireland Unlimited Company Methods and compositions for inducing an immune response against Hepatitis B Virus (HBV)
US11021692B2 (en) 2017-12-19 2021-06-01 Janssen Sciences Ireland Unlimited Company Hepatitis B virus (HBV) vaccines and uses thereof
US11083786B2 (en) 2018-01-19 2021-08-10 Janssen Pharmaceuticals, Inc. Induce and enhance immune responses using recombinant replicon systems
US11826416B2 (en) 2018-01-19 2023-11-28 Janssen Pharmaceuticals, Inc. Induce and enhance immune responses using recombinant replicon systems
CN114072513A (zh) * 2019-06-27 2022-02-18 Emd密理博公司 用表达永生化蛋白的自我复制型rna载体的细胞扩增
WO2020264138A1 (fr) * 2019-06-27 2020-12-30 Emd Millipore Corporation Expansion cellulaire avec des vecteurs d'arn auto-réplicatifs exprimant des protéines d'immortalisation
WO2021183564A1 (fr) * 2020-03-09 2021-09-16 Arcturus Therapeutics, Inc. Compositions et méthodes pour l'induction de réponses immunitaires
US11744887B2 (en) 2020-03-09 2023-09-05 Arcturus Therapeutics, Inc. Coronavirus vaccine compositions and methods
US11759515B2 (en) 2020-03-09 2023-09-19 Arcturus Therapeutics, Inc. Compositions and methods for inducing immune responses
US20210340567A1 (en) * 2020-04-30 2021-11-04 Vlp Therapeutics, Inc. Cytokine immunotherapy
US11613561B2 (en) 2021-03-19 2023-03-28 Tiba Biotech, Llc Artificial alphavirus-derived RNA replicon expression systems

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