WO2012038832A2 - Génération de particules recombinantes chimères virus de la rougeole - rétrovirus - Google Patents

Génération de particules recombinantes chimères virus de la rougeole - rétrovirus Download PDF

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WO2012038832A2
WO2012038832A2 PCT/IB2011/002586 IB2011002586W WO2012038832A2 WO 2012038832 A2 WO2012038832 A2 WO 2012038832A2 IB 2011002586 W IB2011002586 W IB 2011002586W WO 2012038832 A2 WO2012038832 A2 WO 2012038832A2
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chimeric
protein
nucleic acid
replicating
seq
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PCT/IB2011/002586
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WO2012038832A3 (fr
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Thomas Jacques Gilles Mourez
Frédéric Tangy
Chantal Combredet
Valérie NAJBURG
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Institut Pasteur
Centre National De La Recherche Scientifique
Assistance Publique - Hopitaux De Paris
Universite Paris Diderot- Paris 7
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Publication of WO2012038832A3 publication Critical patent/WO2012038832A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
    • C12N2760/18441—Use of virus, viral particle or viral elements as a vector
    • C12N2760/18443—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
    • C12N2810/6045—RNA rev transcr viruses
    • C12N2810/6054—Retroviridae

Definitions

  • the invention is directed to chimeric measles virus - retrovirus particles, i.e., measles virus (MV) particles expressing the envelope proteins of a retrovirus, or immunogenic regions thereof, at its surface.
  • MV measles virus
  • the present invention provides means, in particular nucleic acids, vectors, cells and rescue systems to produce and to culture these chimeric particles.
  • the invention also relates to the use of these chimeric particles, in particular under the form of a composition or of a vaccine, for the treatment or prevention of retrovirus infection, following induction of cellular and/or humoral immune response(s).
  • MV vaccine is a live- attenuated negative-stranded RNA virus which proved to be one of the safest and most effective human vaccines.
  • this vaccine induces life-long immunity after a single or two injections.
  • this vector reduced up to 4 logs the acute viremia of SHIV89.6p when vaccinated macaques were challenged and that CD4 central memory T-cells from the gut were preserved in protected animals (unpublished results).
  • a clinical development was initiated to evaluate the safety and immunogenicity in humans of a recombinant MV encoding an HIV Gag-Pol-Nef antigen.
  • This first generation of MV-HIV vector was designed to induce cellular immunity in adults with preexisting immunity to measles, to evaluate for the first time a replicating vector as an HIV vaccine, and to build the GMP and regulatory logistics for such a vaccine.
  • This vaccine candidate has been recently introduced into phase I clinical trial.
  • a prophylactic or a therapeutic vaccine to prevent or treat retroviral infection, in particular HIV infection, that elicits efficient long-lasting responses possibly after one or two administrations and be produced at million doses and low cost.
  • Figure 1 Construction of MV-SIVenv-(Cherry) chimeric virus.
  • A Schematic representation and genomic organization of MV (N, P, M, F, H, L are MV proteins);
  • B and C Schematic representation and genomic organization of a chimeric MV in which the fusion and hemagglutininglycoproteins were replaced by a ENV protein of a retrovirus. Intracytoplasmic tail of the fusion protein (FIC) was conserved and placed in fusion with said retroviral ENV protein in order to anchor the glycoprotein with the M matrix protein of the MV.
  • FEC Intracytoplasmic tail of the fusion protein
  • E and F Schematic representation and genomic organization of the chimeric MV-SIVenv; fusion and hemagglutininglycoproteins of the MV- Schw vaccine strain were replaced by the extracellular and transmembrane (TM) part of the gp160 of SIVmac239. Intracytoplasmic tail of the fusion protein (FIC) was conserved and placed in fusion with the gp41 in order to anchor the glycoprotein with the M matrix protein of the measles virus.
  • TM extracellular and transmembrane
  • Figure 2 Rescue of MV-SIVenv chimera.
  • FIG. 3 Infection of CEMX174 T-cells by MV-SIVenv chimera.
  • FIG. 4 Replication of MV-Schw and MV-SIVenv chimera in CEMX174 T-cell line.
  • FIG. 5 Comparison of production titer (TCID50) of MV-SIVenv chimeric virus in MT4 and CEMX174 T-cell lines. The same suspension of virus titrated at 4.105 TCID50/ml_ was used in a titration assay using CEMX174 or MT4 cell lines. Both cell lines are able to replicate the chimera. Spearman and Karber method was used to calculate the TCID50.
  • Figure 7 Expression of MV-Nucleoprotein in MV-SIVenv infected cells.
  • Figure 8 Expression of SIV gp160 glycoprotein on the surface of cells infected with MV-SIVenv chimeric virus.
  • SIV gp160 expression was detected by Immunofluorescence using a mouse anti-SIV gp41 monoclonal antibody and a FITC anti-mouse IgG as secondary antibody (green).
  • Figure 9 Expression of MV-N and SIV gp130 in MV-SIVenv infected cells.
  • FIG. 10 MV-SIVenv chimeric virus has lost the capacity to infect Vero cells.
  • Standard MV expressing the cherry protein induces typical syncytia on Vero cells 3 days post-infection (left), while MV-SIVenvchimeric virus is no more able to infect Vero cells, demonstrating a loss of MV tropism.
  • Figure 11 Infectivity of MV-SIVenv chimera in different cell lines.
  • Figure 12 Electron microscopy analyis of the chimera env expression Ultrathin-section electron microscopic analysis of Hela P4C5 infected with MV-SIVenv chimera. Anti-SIVenv gold immunostaining with 10-nanometer colloidal gold particles was performed (A-C). Cells infected with Measles parental virus was used as a negative control (D). Magnifications are variable. Bars indicate the scale. DESCRIPTION
  • the present invention relates to a nucleic acid construct, suitable for the expression of a retroviral envelope protein, comprising the intergenic M/F region of a Measles virus ⁇ MV), a polynucleotide encoding a retroviral envelope protein, or immunogenic regions thereof, and the intergenic H/L region of a MV, wherein said polynucleotide encoding the retroviral envelope protein is operably linked with said intergenic regions, and wherein optionally the intracytoplasmic tail of said retroviral envelope protein is substituted by the intracytoplasmic tail of the F protein of a MV.
  • the invention also concern a nucleic acid construct consisting in said fragment, especially for use as an insert in further nucleotide constructs. In a particular embodiment, this nucleic acid construct has the sequence as defined in SEQ ID NO:2, or comprises this sequence.
  • the invention also relates to a chimeric nucleic acid, derived from the non- segmented negative-strand MV genome, said chimeric nucleic acid comprising from 5' to 3':
  • chimeric nucleic acid is devoid of polynucleotide(s) encoding functional F and H proteins of said MV, and
  • MV multiple myelogenous virus
  • the expression "Measles virus” is abbreviated MV and refers to any strain of the MV species and in particular an attenuated strain, more particularly a non immunosuppressive strain and/or an approved strain for a vaccine.
  • Particular strains that can be used to implement the present invention are the Schwarz MV strain, the Edmonston strain (Ed), the Zagreb strain, the AIK-C strain and the Moraten strain.
  • An approved strain for a vaccine is defined by the FDA (US Food and drug administration) as having the following provisions: safety, efficacy, quality and reproducibility, after rigorous reviews of laboratory and clinical data (www.fda.gov/cber/vaccine/vacappr.htm). Any embodiment of the invention may be applied more generally to strains of the Morbillivirus genus, that share the essential properties of the MV with respect to the present invention.
  • N protein refers respectively to the nucleoprotein (N), the phosphoprotein (P), the matrix protein (M) and the RNA polymerase large protein (L) of a Measles virus.
  • F protein and ⁇ protein refer to the fusion protein (F) and the hemagglutinin protein (H) of a Measles virus.
  • a polynucleotide encoding a protein when used (in reference to proteins N, P, M, L, F or H of a MV), it means a polynucleotide whose sequence consists of the codons of said protein.
  • this polynucleotide has the same nucleotide sequence as the one found in a viral genome of a MV.
  • this polynucleotide has a sequence which differs, from the nucleotide sequence found in the viral genome of a MV, by neutral nucleotide substitutions, i.e., a sequence that encodes a N, P, M or L protein having the same sequence as the one found in the MV particles.
  • polynucleotide encoding the N protein, the M protein and the P protein of a MV is as defined in SEQ ID NO:7.
  • An example of a polynucleotide encoding the L protein of a MV is as defined in SEQ ID NO:8.
  • intergenic M/F region and “intergenic H/L region” are used interchangeably with the expressions “intergenic region between the M and F proteins” and “intergenic region between the H and L proteins” respectively. These regions refer to the nucleotide sequence found between the coding sequence of the M protein and the coding sequence of the F protein in the viral MV genome, and to the nucleotide sequence found between the coding sequence of the H protein and the coding sequence of the L protein in the viral MV genome.
  • An example of intergenic M/F region of a MV is as defined in SEQ ID NO:3.
  • An example of intergenic H/L region of a MV is as defined in SEQ ID NO:6.
  • nucleic acid' refers to any type of nucleotide concatenation, in particular to DNA, cDNA or RNA, as a single strand or double strands.
  • nucleic acid' refers to a nucleotide sequence derived from or obtained from the non- segmented negative-strand genome of a MV, i.e., the genomic RNA (-) strand of the MV genome, the antigenomic RNA (+) strand of the virus genome or the cDNA encoded by of a non-segmented negative strand RNA genome.
  • cDNA used for the description of the chimeric nucleic acid of the invention merely relates to the fact that originally said molecule is obtained by reverse transcription of the genomic (-) RNA genome of viral particles of the measles virus, including most preferably from the full-length genomic (-) RNA genome of viral particles of a MV. This should not be regarded as a limitation for the methods used for the preparation of this cDNA clone.
  • the invention thus encompasses, within the expression "cDNA”, every DNA provided it has the above defined nucleotide sequence. Purified nucleic acids, including DNA, or plasmids are thus encompassed within the meaning of cDNA according to the invention, provided said nucleic acid, especially DNA fulfils the above-given definitions.
  • the expression "derived from” or “obtained from” means that the chimeric nucleic acid of the invention may have a sequence that is different from the sequence of non-segmented negative-strand genome of MV, and in particular is modified by deletion and/or insertion and/or substitution of one or more nucleotides from said non-segmented negative-strand MV genome.
  • Said expression also includes chimeric nucleic acids wherein the sequence having a counterpart in the non-segmented negative-strand genome of MV is identical to the original MV sequence.
  • chimeric refers to a nucleic acid of the invention (either in the form of cDNA or RNA) comprising the coding sequences for the M, N, P and L proteins of MV and a polynucleotide encoding a retroviral envelope protein or a chimeric retroviral envelope protein.
  • this chimeric nucleic acid is suitable to be used for producing replicating MV-retroviruses chimeric particles in a rescue system (replication competent).
  • the chimeric nucleic acid of the invention is devoid of polynucleotides encoding functional F and H proteins, i.e., either the chimeric nucleic acid is not able to express F and H proteins, or the produced F and H proteins are not able to anchor in the membrane of the MV chimeric particles.
  • the chimeric nucleic acid is devoid of the polynucleotides encoding the F and/or the H proteins.
  • the chimeric nucleic acid is devoid of the polynucleotides encoding the F and the H proteins, with the exception of the nucleotide sequence encoding the intracytoplasmic tail of the F protein which is present in the chimeric construct.
  • the chimeric nucleic acid of the invention is devoid of all or part of the nucleotide sequences encompassing the F and H proteins of the MV genome, which are replaced by a polynucleotide encoding a retroviral envelope protein, provided that the resulting nucleic acid is suitable for producing replicating MV- chimeric particles in a rescue system.
  • the sequence starting with the intergenic M/F region and ending with the intergenic H/L region of the MV genome in the chimeric nucleic acid construct is replaced by a nucleic acid construct as defined in the present invention.
  • operably linked refers to the functional link existing between the different polynucleotides and nucleic acid construct of the chimeric nucleic acid of the invention such that said different polynucleotides and nucleic acid construct are efficiently transcribed and/or translated, in particular in helper cell line used in a rescue system, and/or such that the full-length chimeric nucleic acid is replicated and transcribed in particular in helper cell line used in a rescue system.
  • operably linked in reference to the nucleic acid construct, refers to the functional link existing between the polynucleotide encoding the retroviral envelope protein and the intergenic M/F region and intergenic H/L region of a MV, such that said intergenic regions of a MV drives and controls the transcription and translation of the polynucleotide encoding the retroviral envelope protein, in particular in helper cell line used in a rescue system.
  • the chimeric nucleic acid of the invention is replication competent.
  • replication competent it is meant a nucleic acid, which when transduced into a helper cell line expressing the N, P and L proteins of a MV, is able to be transcribed and expressed in order to produce new particles.
  • the chimeric nucleic acid of the invention comprises the cis-active sequences necessary for its replication and transcription, i.e., the Leader and Trailer sequences of MV.
  • the Leader sequence of MV comprises a viral promoter of said virus.
  • the Trailer sequence comprises a terminator sequence of the transcription.
  • the chimeric nucleic acid of the invention meets the so-called rule of 6. Therefore, the chimeric nucleic is a polyhexameric length, i.e., is a multiple of six.
  • the nucleic acid construct designed herein meets the so-called rule of 6.
  • the chimeric nucleic acid of the invention is derived from a full-length non-segmented negative-strand genome of MV.
  • the invention also relates to a chimeric nucleic acid which has a nucleotidic sequence complementary to the sequence of the chimeric nucleic acid defined above and wherein the elements (a) to (e) are included in the sequence from 3' to 5'.
  • complementary it means a sequence which is 100% complementary, over the entire length, with the sequence of the chimeric nucleic acid defined above.
  • the complementary chimeric nucleic acid and the chimeric nucleic acid defined above have the same size.
  • a complementary chimeric nucleic acid is single stranded and is under the form of RNA or DNA.
  • the expression “from 5' to 3” refers to the sense of transcription of the coding sequences contained in the chimeric nucleic acid of the invention (from the initiation codon to the termination codon).
  • a sequence complementary to a 5' to 3' nucleic acid is defined as a 3' to 5' nucleic acid.
  • the chimeric nucleic acid of the invention is derived from the non-segmented negative-strand genome of a MV.
  • the sequence of the non-segmented negative-strand MV genome is from a Schwarz MV strain, and is especially obtained using nucleic acid extracted from Schwarz particles.
  • non-segmented negative-strand Schwarz strain genome from which the chimeric nucleic acid of the invention may be derived, are illustrated by sequences which comprise or consist of the sequence selected in the group consisting of the nucleotide sequence extending from nucleotides 83 to 15977 of SEQ ID NO:1 , the nucleotide sequence extending from nucleotides 83 to 16202 of SEQ ID NO:1 , the nucleotide sequence extending from nucleotides 29 to 15977 of SEQ ID NO:1 , the nucleotide sequence extending from nucleotides 29 to 16202 of SEQ ID NO:1 , the nucleotide sequence extending from nucleotides 26 to 15977 of SEQ ID NO:1 , the nucleotide sequence extending from nucleotides 26 to 16202 of SEQ ID NO:1 , the nucleotide sequence extending from nucleotides 9 to 15977 of SEQ ID NO:1 , and
  • Both the nucleic acid construct and the chimeric nucleic acid of the invention comprise all or part of an envelope protein of a retrovirus (ENV) or immunogenic regions thereof.
  • said retroviral envelope protein is from a lentivirus especially from a HIV.
  • envelope protein it is meant the protein or polyprotein, including glycoproteins(s) which is expressed at the surface of a retrovirus or lentivirus and possibly anchored in its membrane.
  • lentivirus envelope protein examples include HIV- gp160, gp140 or gp120, HIV-2 gp160, gp140 or gp120, or SIVmac gp160, gp140 or gp120, or an immunogenic fragment thereof, especially an immunogenic fragment targeting CD4 cells in a host.
  • the envelope protein is a combination of a part of an envelope protein of a retrovirus fused in frame with a part of the F protein of the MV, to give a chimeric retroviral-MV envelope protein able to anchor in the membrane of the chimeric particles.
  • a chimeric retroviral-MV envelope protein consists of the gp120 combined with the surface region and transmembrane region of the gp41 of a retrovirus, in particular HIV, fused with the intracytoplasmic tail of the F protein of MV.
  • a chimeric retroviral-MV envelope protein may be obtained by deleting the intracytoplasmic tail of the gp160 retroviral protein and inserting the intracytoplasmic tail of the F protein of MV, especially by replacing the intracytoplasmic tail of gp160 by the intracytoplasmic tail of the F protein of MV.
  • sequence of the polynucleotide encoding the gp120 and the surface region and transmembrane region of gp41 of SIV is as defined in SEQ ID NO:4.
  • the invention also concerns the counterpart of this sequence that may be obtained from a HIV-1 gp160 or a HIV-2 gp120, especially the sequence that may be identified from the available sequences of HIV.
  • sequence of the polynucleotide encoding the intracytoplasmic tail of the F protein of a MV is as defined in SEQ ID NO:5.
  • nucleic acid construct as defined herein and claimed as such or the nucleic acid construct as defined herein and contained in the chimeric nucleic acid of the invention is as defined in SEQ ID NO:2.
  • This nucleic acid construct results from the fusion of the following sequences: the intergenic M/F region of a MV as defined in SEQ ID NO:3, a polynucleotide encoding a SIV envelope protein the intracytoplasmic tail of which is replaced by the intracytoplasmic tail of the F protein of MV resulting in a chimeric envelope protein as defined in SEQ ID NO:4 in combination with SEQ ID NO:5, and the intergenic H/L region of a MV as defined in SEQ ID NO:6.
  • the nucleic acid construct results from the fusion of the following sequences: the intergenic M/F region of a MV as defined in SEQ ID NO:3, a polynucleotide encoding a HIV envelope protein the intracytoplasmic tail of which is replaced by the intracytoplasmic tail of the F protein of MV resulting in a chimeric envelope protein, and the intergenic H/L region of a MV as defined in SEQ ID NO:6.
  • the chimeric nucleic acid of the invention further comprises a polynucleotide encoding at least one further heterologous polypeptide(s).
  • heterologous is used to define a polynucleotide the origin of which is different from measles virus.
  • This polynucleotide encoding at least one further heterologous polypeptide(s) is in particular inserted between said polynucleotide encoding the P protein and said polynucleotide encoding the M protein of MV, or is inserted between the polynucleotide encoding the retroviral envelope protein (chimeric or not) and the polynucleotide encoding the L protein of MV.
  • an Additional Transcription Unit ATU
  • An ATU may comprise a transcription stop sequence, a poyadenylation sequence and a transcription start sequence as contained in an intergenic region of MV, such as for example in the N-P intergenic region, P-M intergenic region or H-L intergenic region of MV.
  • the heterologous polypeptide is any peptide, polypeptide or protein of any origin, whose expression in the target cells of the chimeric MV-retrovirus particles or whose recognition by said target cells is desired.
  • said heterologous polypeptide is a polypeptidic marker such as a fluorescent protein, preferably a protein selected from eGFP, RFP and YFP.
  • said heterologous polypeptide is a biologically active polypeptide such as an immunogenic polypeptide or a therapeutic polypeptide.
  • said heterologous protein is a polypeptide derived from a protein of a retrovirus, in particular from the retrovirus from which said envelope (ENV) protein of the chimeric particles is derived.
  • heterologous polypeptides derived from a retrovirus are selected from GAG, POL, VIF, NEF and TAT proteins of a retrovirus, in particular the p55 GAG of HIV able to form Viral-Like Particles (VLP) or immunogenic fragments thereof.
  • VLP Viral-Like Particles
  • the chimeric nucleic acid is particularly preferred under the form of a genomic RNA (-) strand or under the form of a cDNA.
  • the invention also relates to a nucleic acid comprising or consisting of a polynucleotide encoding the gp120 and the surface region and transmembrane region of gp41 of a retrovirus fused with a polynucleotide encoding the intracytoplasmic tail of the F protein of a MV.
  • the gp120 and the surface region and transmembrane region of gp41 are from a HIV or SIV virus.
  • the polynucleotide encoding the gp120 and the surface region and transmembrane region of gp41 is as defined in SEQ ID NO:4.
  • the polynucleotide encoding the intracytoplasmic tail of the F protein of a MV is as defined in SEQ ID NO:5.
  • the invention is directed to a polynucleotide consisting of SEQ ID NO:4 fused with SEQ ID NO:5.
  • the expression "fused”, used in reference to polynucleotides, means that the sequence of the polynucleotides is genetically linked, in order to express a unique protein consisting of the different polypeptides, encoded by said polynucleotides, in frame.
  • the invention is also directed to a polypeptide consisting of the gp120 and the surface region and transmembrane region of gp41 of a retrovirus fused in frame with the intracytoplasmic tail of the F protein of a MV.
  • the gp120 and the surface region and transmembrane region of gp41 are from a HIV or SIV virus.
  • the gp120 and the surface region and transmembrane region of gp41 is encoded by a polynucleotide as defined in SEQ ID NO:4.
  • the intracytoplasmic tail of the F protein of a MV is encoded by a polynucleotide as defined in SEQ ID NO:5.
  • the polypeptide is encoded by a polynucleotide consisting of SEQ ID NO:4 fused with SEQ ID NO:5, or has the protein sequence as defined in SEQ ID NO:9 or has the sequence in HIV functionally corresponding to SEQ ID NO:4 fused with the sequence of SEQ ID NO:5.
  • An example of a chimeric retroviral-MV envelope protein consists of the gp120 combined with the surface region and transmembrane region of the gp41 of a retrovirus, in particular HIV, fused with the intracytoplasmic tail of the F protein of MV.
  • Such a chimeric retroviral-MV envelope protein may be obtained by deleting the intracytoplasmic tail of the gp160 retroviral protein and inserting the intracytoplasmic tail of the F protein of MV, especially by replacing the intracytoplasmic tail of gp160 by the intracytoplasmic tail of the F protein of MV.
  • the sequence of the polynucleotide encoding the gp120 and the surface region and transmembrane region of gp41 of SIV is as defined in SEQ ID NO:4.
  • the invention also concerns the counterpart of this sequence that may be obtained from a HIV-1 gp160 or a HIV-2 gp120, especially the sequence that may be identified from the available sequences of HIV.
  • the sequence of the polynucleotide encoding the intracytoplasmic tail of the F protein of a MV is as defined in SEQ ID NO:5.
  • the invention also relates to means to produce and to culture the chimeric replicating MV-retrovirus particles.
  • a vector especially an expression vector, in particular a plasmid comprising a chimeric nucleic acid as defined herein is part of the invention.
  • An example of a plasmid of the invention is the plasmid pTM- MVSchw-Chim-SIVenv(Cherry) having the sequence as defined in SEQ ID NO:10.
  • the invention relates to plasmid pTM-MVSchw-
  • the invention is also directed to a cell, in particular an isolated cell, transformed with a vector or a plasmid of the invention.
  • Cells of the invention can be prokaryotic or eukaryotic cells, particularly animal or yeast cells, and more particularly mammalian cells such as human cells or non-human mammalian cells.
  • cells are isolated from either a primary culture or a cell line.
  • Cells of the invention may be dividing or non-dividing cells.
  • HEK 293 human embryonic kidney cells, which cell line 293 is deposited with the ATCC under No. CRL-1573 is preferred.
  • human cells are not germ cells and/or embryonic stem cells.
  • the invention also relates to a RNP complex (or nucleocapsid) consisting of a chimeric nucleic acid as defined herein encapsidated in the N, P and L proteins of a MV.
  • RNP complex or nucleocapsid
  • the invention also concerns a rescue system for the assembly of chimeric replicating MV-retrovirus particles, comprising (a) a vector or a plasmid of the invention as defined herein; and (b) a helper cell expressing an RNA polymerase such as a T7 NRA polymerase and the N, P and L proteins of a MV.
  • a rescue system for the assembly of chimeric replicating MV-retrovirus particles comprising (a) a vector or a plasmid of the invention as defined herein; and (b) a helper cell expressing an RNA polymerase such as a T7 NRA polymerase and the N, P and L proteins of a MV.
  • Helper cell lines used to rescue MV particles are well known in the art and are of two types:
  • a cell line recombined with at least one vector(s) suitable for the expression of a RNA polymerase such as a T7 RNA polymerase and for the expression of the N, P and L proteins of a MV.
  • a RNA polymerase such as a T7 RNA polymerase
  • N, P and L proteins of a MV This first type of helper cell line and its use in a rescue system is disclosed in the literature and illustrated in patent applications WO 2004/000876 and in WO 2009/095791 , incorporated herein by reference.
  • the invention also relates to a method to produce chimeric replicating MV-retrovirus particles, comprising (a) transfecting a vector of the invention as defined herein, in a helper cell line which expresses a RNA polymerase such as a T7 RNA polymerase and the N, P and L proteins of a MV; (b) transfering said helper cell line onto cells competent to sustain the replication and production of a MV, preferably CD4 + cells, such as CEMx174 MT4, HeLa -P4C5 cells; primary monkey or human monocytes; and (c) recovering the chimeric MV-retrovirus particles from said coculture; and
  • helper cells line is the 293-T7-NP cell line deposited with the CNCM on June 14, 2006, under number 1-3618 or the 293-nlsT7-NP MV cell line deposited with the CNCM on August 04, 2006, under number I-3662.
  • a vector comprising a nucleic acid encoding a RNA polymerase large protein (L) of a MV is the pEMC-LSchw plasmid deposited with the CNCM on December 18, 2007, under number 1-3881 .
  • This second type of helper cell line and its use in a rescue system are disclosed in application WO2008/078198, incorporated herein by reference.
  • the invention is also directed to a method to produce chimeric replicating MV-retrovirus particles, comprising: (a) transfecting a vector as defined herein, in a helper cell line which stably expresses a RNA polymerase such as a T7 RNA polymerase and the N and P proteins of a MV and which expresses a L protein of a MV from an expression vector; and(b) recovering the chimeric MV-retrovirus particles from said helper cell line.
  • the invention encompasses chimeric replicating MV particles expressing a retroviral envelope protein or immunogenic regions thereof, wherein said particles comprise (a) a RNP complex consisting of a chimeric nucleic acid of the invention, under the form of a genomic RNA (-) strand, encaspidated in the N, P and L proteins of a MV, (b) the M protein of a MV and (c) said retroviral envelope protein or immunogenic regions thereof anchored at the surface of the particles.
  • chimeric refers to a MV particle comprising the M, N, P and L proteins of MV and an envelope protein (or immunogenic regions thereof) that is not the H and F proteins of MV, and in particular that does not express at its surface functional H and F proteins of MV.
  • functionaf it is meant H and F proteins that are able to ensure their activity of fusion and hemagglutinin respectively.
  • the sole envelope protein (or immunogenic regions) anchored at the surface of the chimeric particle is a retroviral envelope protein as defined herein or a chimeric retroviral-MV envelope protein as defined herein.
  • a chimeric MV particle, devoid of functional H and F proteins and expressing at its surface a retroviral envelope protein (or immunogenic regions thereof) or a chimeric retroviral-MV envelope protein is also called a chimeric MV-retrovirus particle.
  • a chimeric retroviral-MV envelope protein consists of the gp120 and the surface region and transmembrane region of gp41 of a retrovirus especially a H IV fused with the intracytoplasmic tail of the F protein of MV.
  • Such a chimeric envelope protein may be obtained by replacing the intracytoplasmic tail of the gp160 retroviral protein by the intracytoplasmic tail of the F protein of MV.
  • sequence of the polynucleotide encoding the gp120 and the surface region and transmembrane region of gp41 of SIV is as defined in SEQ ID NO:4.
  • the counterpart in the protein sequence of HIV may be defined starting from the sequences available in the art.
  • sequence of the polynucleotide encoding the intracytoplasmic tail of the F protein of a MV as defined in SEQ ID NO:5.
  • An example of chimeric retroviral-MV envelope protein is as defined in SEQ ID NO:9.
  • replicating means a particle that is able to enter a target cell, to express the proteins encoded by its genome and to form new particles.
  • the chimeric nucleic acid of the invention within the particle is under a form appropriate for its encapsidation by N, P and L proteins of MV, i.e., under the form of a genomic RNA (-) strand.
  • N, P, L, M proteins, the sequence of the retroviral envelope protein and of the intracytoplasmic tail of the F protein of MV by the person skilled in the art may be easily determined by the person skilled in the art by translation of the corresponding polynucleotide sequences.
  • the chimeric MV-retrovirus particles are characterized in that they are the product of expression, in a cell, of a chimeric nucleic acid of the invention.
  • the chimeric MV-retrovirus particles are characterized in that they are rescued from a helper cell line, expressing or stably expressing a RNA polymerase such as a RNA polymerase such as a T7 RNA polymerase and the N, P and L proteins of a MV, transfected with a chimeric nucleic acid of the invention, in particular using a rescue system as defined above.
  • the chimeric MV-retrovirus particles are capable of eliciting a humoral and/or a cellular immune response(s) against the retrovirus from which the envelope protein originates or against both MV and said retrovirus, in a host upon administration.
  • the application also discloses means to prevent or to treat retrovirus infection, in particular compositions and vaccines.
  • the response may be initially elicited as a mucosal response.
  • the invention also relates to chimeric measles virus particles expressing a retrovirus envelope polypeptide(s) or immunogenic regions thereof, said chimeric measles virus particles being capable of eliciting a humoral and/or a cellular immune response against said retrovirus or against both measles virus and said retrovirus, said chimeric measles virus comprising a genome which is transcribed from a chimeric construct originating from the Schwarz strain of measles virus (MV) defined by the nucleotide sequence comprising at least nucleotide 83 to nucleotide 15977 of Figure 1 (SEQ ID No 1 ), wherein said chimeric construct comprises from 5' to 3':
  • MV Schwarz strain of measles virus
  • compositions in particular therapeutic compositions or prophylactic compositions, comprising the chimeric replicating MV- retrovirus particles of the invention and a pharmaceutically acceptable vehicle, carrier, excipient and/or diluent.
  • Pharmaceutically acceptable vehicles or carriers encompass any substance that enables the formulation of the particles of the invention within a composition.
  • a vehicle is any substance or combination of substance physiologically acceptable i.e., appropriate for its use in a composition in contact with a mammal, and thus non-toxic. Examples of such vehicles are phosphate buffered saline solutions, distilled water, emulsions such as an oil/water emulsions, various types of wetting agents sterile solutions and the like.
  • the physiologically acceptable diluent can be buffering agents, saline, dextrose, glycerol, water, ethanol and the like and combinations thereof.
  • the composition of the invention may also further comprise at least one adjuvant(s).
  • adjuvants are known in the art and include Complete Freund's Adjuvant (CFA), Incomplete Freund's Adjuvant (IFA), montanide ISA (incompletre seppic adjuvant), muramyl peptides such as muramyl dipeptide (MDP) MDP-Lys (L18) (N a -acetylemuramyl-L- alanyl-D-isoglutaminyl-N e -steoroyl-L-lysine), aluminum hydroxide (alum), CpG oligodeoxynucleotides(CPG ODN) such as CPG ODN 1826 and CPG ODN 2007, and MF59, which is a detergent stabilized oil-in water emul
  • the invention in particular relates to a prophylactic or therapeutic vaccine composition
  • a prophylactic or therapeutic vaccine composition comprising the chimeric replicating MV-retrovirus of the invention, and a pharmaceutically acceptable vehicle as defined above and/or at least one adjuvant(s) as defined above.
  • Said vaccine composition may further comprise further distinct immunogenic ingredient(s).
  • a distinct immunogenic ingredient is defined as a molecule, in particular a peptide, antigen or epitope, able to elicit a cellular and/or an immune response independently of or in addition to the one(s) elicited by the immunogenic regions of the particles of the present invention.
  • the cellular and/or immune response are induced against a molecule of the same retrovirus from which the envelope protein of the particle of the invention is derived.
  • the cellular and/or immune response is directed against epitope(s) of HIV virus, in particular epitope(s) of HIV-1 or HIV-2 virus.
  • the invention also concerns the chimeric replicating MV-retrovirus particles of the invention, the composition of the invention or the vaccine composition of the invention, for use in the prevention or the treatment of retrovirus infection, in particular HIV infection.
  • the chimeric replicating MV-retrovirus particles of the invention, the composition of the invention or the vaccine composition of the invention may be used for eliciting a humoral response and in particular for inducing antibodies directed against epitope(s) of the retroviral envelope protein or of the chimeric retroviral-MV envelope protein, and more particular neutralizing antibodies.
  • the chimeric replicating MV-retrovirus particles of the invention, the composition of the invention or the vaccine composition of the invention may also be used for eliciting a T cell immune response.
  • the T cell immune response may be a CD4 T cell immune response and/or a memory immune response.
  • said cellular and/or humoral immune response is a mucosal immune response.
  • the replicating MV-retroviral particles, composition or vaccine composition of the invention is also used to further elicit an immune response against one or many heterologous biologically active polypeptides.
  • the chimeric MV-retrovirus particles of the invention may also express, in addition to the retroviral envelope protein or retroviral-MV chimeric protein, one or more hetrerologous biologically active polypeptides, such as an immunogenic polypeptide.
  • the replicating MV-retroviral particles, composition or vaccine composition are used in conventional regimen of administration well known from the person skilled in the art, and in particular are used in a prime-boost administration regimen.
  • the invention also relates to a method to prevent retrovirus infection, in particular HIV infection in a host or to treat retrovirus infection, in particular HIV infection in a host, comprising administering, into said host, chimeric replicating MV-retrovirus particles, composition or a vaccine composition of the invention.
  • the invention also discloses an in vitro process for culturing and amplifying the chimeric replicating MV-retrovirus particles of the invention comprising rescuing said chimeric replicating MV-retrovirus particles on CEMx174, MT4, Hel_a-P4C5 cells, in particular in a rescue system as discussed above; and culturing rescued chimeric replicating MV-retrovirus particles obtained in step (a) on CD4 + cells, such as CEMx174 cells.
  • step (b) comprises centrifugating chimeric replicating MV-retrovirus particles together with CD4 + cells before inoculation in a culture media or plating.
  • step (b) also comprises adding fresh media comprising CD4 + cells to the culture.
  • the inventors first generated a MV-SIV chimeric virus based on Simian Immunodeficiency Virus SIVmac239, which is the most widely recognized model for AIDS in macaques.
  • the chimera was constructed obtained from the rescue of a construct based on MV CNDA which was deleted for the fusion (F) and hemagglutinin (H) glycoproteins genes from MV antigenomic cDNA, and substitution with SIVMac239 gp160 coding sequence to provide a MV transcript.
  • the intra cytoplasmic domain of gp160 was substituted (exchanged) with that of MV fusion protein (allowing the anchoring in cell membrane through interactions with the MV matrix protein (Shhofer, Bachi et al. 1998)).
  • the Df-red(Cherry) protein sequence was inserted upstream of the MV-M matrix protein, in order to easily visualize the chimera in infected cells. This additional transcription unit may be used to insert other genes in replacement of RFP.
  • the chimeric genomic sequence was generated as a synthetic cDNA (Genecust) ( Figure 1 E and 1 F, Annex 1 ).
  • the synthetic sequence starts at the Narl site upstream to the MV-F gene and was generated in order to maintain the complete promoter and intergenic sequence of MV-F fusion protein until the initiation codon.
  • the whole sequence between the F initiation codon and the first amino acid of its intracytoplasmic domain was removed and replaced by the sequence of the SIVgp160 comprised between the initiation codon and transmembrane domain included.
  • the sequence of the SIVgp160 was optimized to the Macacca codon usage, and modified to remove a Spel restriction site.
  • optimization of the env sequence was operated avoiding cis-active : internal TATA-boxes, chi-sites and ribosomal entry sites ; AT-rich or GC-rich sequence stretches ; ARE, INS, CRS sequence elements ; repeat sequences and RNA secondary structures ; cryptic splice donor and acceptor sites, branch points. Optimization also avoided measles editing-like sequences. The intergenic sequence between F and H, and the complete sequence of MV-H were removed. After the termination codon, an adenosin was added to respect the « rule of 6 » of measles genome (Calain and Roux, 1993). A BamHI restriction site was finally added to the 3'end of the synthetic sequence to enable the introduction of the cassette into MV cDNA. II. Plasmid construction
  • the plasmid pTM-MVSchw-ATU2 which contains an infectious MV cDNA corresponding to the anti-genome of the Schwarz MV vaccine strain, and expresses the eGFP protein in an additional transcription unit has been described elsewhere (Combredet et al., 2003).
  • the eGFP sequence was replaced by the sequence of RFP-STREPtag protein (red fluorescent protein), more easily detectable than eGFP.
  • the fragment of this plasmid comprised between the two Spel sites flanking the F and H genes was removed and sub-cloned for subsequent re introduction into pTM-MVSchw-RFP-STREPtag vector and designated as pBS-Schw-Spe5803 (Annex 2).
  • the chimeric synthetic sequence (Annex 1 ) was recombined into pBS-Schw-Spe5803 plasmid after Narl/BamHI digestion, introducing the modified SIVenv gene and removing the H and F MV glycoproteins.
  • This intermediate plasmid was designated as pBS-Schw chim/siv/env.
  • the new modified sequence was then introduced into pTM-MVSchw vector after Spel digestion.
  • the resulting vector was designated pTM-MVSchw- Chim-SIVenv (Cherry) (Annex 3).
  • Chimeric MV/SIV virus was recovered from pTM-MVSchw-Chim-SIVenv (Cherry) plasmid using an adapted helper-cell-based rescue system (Tangy, Charneau, and Jacob, 2006).
  • HEK-293-T7-MV cells were transfected as previously described with pTM-MVSchw-Chim-SIVenv (Cherry) plasmid and were co-cultivated with CEMx174 T-cells 3 days after transfection (ATCC®; number CRL-1992). Infection was detected in the coculture by observation of cytopathic effect (CPE) and red fluorescence from the Cherry protein in infected cells ( Figure 2).
  • T-cells present characteristic features of lentiviral infection, with large aggregates of infected cells evolving to form syncytia ( Figure 3).
  • CEMx174 cells were infected 2 hours under slow centrifugation (1000 rpm) in sterile Eppendorff tubes by serial 1 :10 dilutions of virus sample in RPMI growth medium, supplemented with 10% Fetal Calf Serum (FCS). Infected cells were seeded into 96-well plates (25 000 cells in 100ml_ of RPMI/well) and incubated for 24 hours at 37°C. After 24h, 100ml_ of RPMI, 10% FCS, were added in each well. After an additional incubation for 4 days, cells were observed for CytoPathic Effect (CPE) and red fluorescence. TCID50 values were calculated by use of the Spearman and Karber method. V. Virus growth curves
  • Intracytoplasmic immunostaining was operated after incubation of the cells for 1 hour in blocking/permeabilizing solution (PBS, 2% Goat Serum, 0,1 % Saponin), with a mouse anti-MV N mAb (MAB8906F; Chemicon International).
  • a FITC-conjugated sheep anti-mouse IgG antibody (74641 , BioRad) was used as a secondary antibody.
  • the results show that both MV-N and SIV gp160 proteins are expressed simultaneously with Df-red (cherry) protein in infected cells ( Figure 7-8).
  • Df-red (cherry) protein in infected cells.
  • Figure 8 The expression of SIV glycoprotein on the surface of infected cells is demonstrated by immunofluorescence staining in non permeabilized cells ( Figure 8).
  • CEMx174 cells were infected with Chim MV/SIVenv chimeric virus at a multiplicity of infection (MOI) of 0.1 .
  • MOI multiplicity of infection
  • Proteins extracted from cell lysate were separated (50ng per well) by SDS-PAGE gel electrophoresis on 4-12% NUPAGE® Bis-Tris gels with MOPS running buffer (Invitrogen) and transferred to cellulose membranes with the XCell SureLockTM system (Invitrogen).
  • the membranes were blotted with either a mouse anti-SIVgp160/gp120 V2 monoclonal antibody (KK13; obtained from the Programme EVA Centre for AIDS Reagents, NIBSC, UK, supported by the EC FP6/7 Europrise Network of Excellence, AVIP and NGIN consortia and the Bill and Melinda Gates GHRC-CAVD Project and donated by Dr Karen Kent , NIBSC centralized Facility for AIDS Reagents), or a mouse anti-MV N mAb (MAB8906F; Chemicon International).
  • KK13 mouse anti-SIVgp160/gp120 V2 monoclonal antibody
  • MAB8906F mouse anti-MV N mAb
  • the chimeric virus was able to replicate in all CD4 T cells tested: CEMx174, MT4, Jurkat, Hel_a-P4C5, primary macaque splenocytes. On the contrary, the chimeric virus did not grow on Vero, MRC5, HEK293 or U936 cells, all cell lines that do not express CD4 ( Figure 4, 10, 11 ). This result demonstrates that the chimeric virus has acquired the SIV/HIV tropism and the capacity to infect CD4 T-cells.
  • HeLa P4C5 cells (Marechal, V., Clavel, F., Heard, J.M., Schwartz, O., 1998. Cytosolic Gag p24 as an index of productive entry of human immunodeficiency virus type 1 . J. Virol. 72 (3), 2208-2212) were seeded on cover glass strips and infected at a MOI of 0.1 with the MV-SIVenv chimera. Cells were fixed 22 h postinfection with 2% PFA in PBS for 5 min and with 4% PFA for 2 h at room temperature.
  • Non-specific sites were saturated for 10 min with 1 % bovine serum albumin and cells were probed for 40 min with a mouse anti- SIVMac251 gp160/gp120 monoclonal antibody (KK17; NIH-AIDS Research and Reference Reagent Program ; courtesy of NIBSC centralized Facility for AIDS Reagents) in PBS-1 % BSA. After washing, cells were incubated for 20 min with 10-nanometer colloidal gold particles coated with protein A (University Medical Center, Utrecht). Stained cells were then fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer overnight at 4 °C, postfixed with 1 % osmic acid, dehydrated in ethanol, and embedded in Epon.
  • Ultrathin sections of 70-80 nm were cut with a diamond knife, stained with uranyl acetate and Reynolds lead citrate, and observed at 80 kV accelerating voltage using JEOL JEM 1010 Electron Microscope and an Elo ' fse Mega View III camera. HeLa P4C5 cells infected with MV-Schw, and non-infected cells were included in this analysis as controls. Ultrastructure analysis shows that both MV and the chimera have the same structure and size. Immunogold staining with anti SIV gp160/120 shows the presence of SIV glycoprotein at the surface of the chimeric particles ( Figure 12). This result shows that the chimeric virus keeps the MV parental structure with the expression of SIV surface proteins anchored in the virus membrane.
  • the invention relates to an unconventional approach based on a chimera rather than a recombinant vector to elicit both humoral and cellular immune response, and to induce neutralizing antibodies.
  • the present invention describes the generation and characterization of a replicating measles-HIV chimeric virus wherein measles virus surface glycoproteins (hemagglutinin and fusion protein) are substituted with HIV gp160 surface glycoprotein.
  • This chimeric virus was generated from a previously cloned cDNA of live attenuated Schwarz vaccine strain of measles virus (Combredet, Labrousse-Najburg et al. 2003).
  • the insertion of an additional transcription unit (ATU) in the chimeric virus genome allows the expression of additional genes.
  • This chimera will benefit of the capacity of MV to induce very potent mucosal protection through induction of neutralizing antibodies to surface glycoproteins.
  • This new chimeric virus has the capacity of MV to express native SIV gp160 spikes on its surface instead of MV glycoproteins. It thus benefits from both the HIV tropism and the capacity of MV to replicate in dendritic cells, macrophages and lymphocytes, thus mimicking a transient HIV infection. It will also benefit from the genetic stability and safety of MV vaccine strains (absence of recombination in non segmented negative stranded RNA viruses, cytoplasmic cycle, no integration of genetic material, no persistence, no shedding). Non-persisting, this chimera will be cleared from host by the induced immunity.
  • the chimera was designed to induce an efficient processing of native lentiviral gp160 trimeric spikes on particle surface in the context of a budding virus. Based on the safest human live attenuated vaccine, this strategy will also induce cell- mediated immunity and long-lasting memory in the mucosa, as live vaccines do. Such a live non-persisting chimeric virus has never been evaluated as an HIV vaccine. Mimicking a transient non-persisting HIV or SIV infection might result in a similar level of protection to that provided by live-attenuated SIV vaccines, an approach that cannot be transposed to humans.
  • SEQ ID NO :1 corresponds to the pTM-MVSchw-eGFP (ATU2)
  • SEQ ID NO: 2 corresponds to the Narl - Spel sequence of the MV- SlVenv(Cherry) chimeric clone (disclosed in Figure 1 E)
  • SEQ ID NO: 3 corresponds to the intergenic region M/F, including the F promoter (nucleotides 7 to 527 of SEQ ID NO:2)
  • SEQ ID NO: 4 corresponds to the SIVmac 239 gp120 gene, and the surface and transmembrane region of the SIVmac239 gp41 (nucleotides 530-2672 of SEQ ID NO:2)
  • SEQ ID NO: 5 corresponds to the intracytoplasmic region of the F protein from the MV Schwartz strain plus a TGA codon (nucleotides 2673 to 2774 of SEQ ID NO:2)
  • SEQ ID NO: 6 corresponds to the intergenic region H/L of MV (nucleotides 2776-2825 of SEQ ID NO:2)
  • SEQ ID NO: 7 corresponds to the sequence from the first nucleotide to the Narl site of the MV-SIVenv-(Cherry) chimeric clone (disclosed in Figure 1 E)
  • SEQ ID No 8 corresponds to the sequence from the Spel site to the last nucleotide of the MV-SIVenv-(Cherry) chimeric clone (disclosed in Figure 1 E) actagtGTGAAATAGACATCAGAATTAAGAAAAACGTAGGGTCCAAGTGG
  • SEQ ID NO: 10 corresponds to the complete sequence of the plasmid pTM- MVSchw-Chim-SIVenv (Cherry) [SEQ ID NO:7 recombined to SEQ ID NO:2 recombined to SEQ ID NO:8]
  • SEQ ID NO: 11 corresponds to pBS-Schw-Spe5803 plasmid containing the spel/spel region of the pTM-MVSchw-ATU2
  • SEQ ID NO: 12 corresponds to the plasmid comprising SEQ ID NO:2 as defined above.

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Abstract

La présente invention concerne des particules chimères virus de la rougeole - rétrovirus, en d'autres termes, des particules du virus de la rougeole (VR) exprimant les protéines d'enveloppe d'un rétrovirus, ou des régions immunogènes de celui-ci, sur sa surface. La présente invention fournit des moyens, en particulier des acides nucléiques, des vecteurs, des cellules et des systèmes de sauvetage pour cultiver ces particules chimères. L'invention concerne également l'utilisation de ces particules chimères, en particulier sous la forme d'une composition ou d'un vaccin, pour le traitement ou la prévention d'une infection par un rétrovirus, après l'induction d'une (de) réponse(s) immunitaire(s) cellulaire(s) et/ou humorale(s).
PCT/IB2011/002586 2010-09-24 2011-09-23 Génération de particules recombinantes chimères virus de la rougeole - rétrovirus WO2012038832A2 (fr)

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WO2022152818A1 (fr) * 2021-01-13 2022-07-21 Viroxis Vaccin contre le virus de la rougeole-vih ou le virus de la rougeole-htlv

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CN106164258A (zh) * 2013-11-18 2016-11-23 巴斯德研究院 基于作为异源多肽载体的包含非节段负链rna病毒的核蛋白的多聚核糖核蛋白的亚单位疫苗平台
CN106164258B (zh) * 2013-11-18 2021-01-19 巴斯德研究院 基于作为异源多肽载体的包含非节段负链rna病毒的核蛋白的多聚核糖核蛋白的亚单位疫苗平台
EP3253874B1 (fr) * 2015-02-06 2022-03-23 Association Institut de Myologie Traitement de la dystrophie facioscapulohumérale
JP2016202113A (ja) * 2015-04-27 2016-12-08 学校法人北里研究所 組換え麻疹ウイルス
WO2022152818A1 (fr) * 2021-01-13 2022-07-21 Viroxis Vaccin contre le virus de la rougeole-vih ou le virus de la rougeole-htlv

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