US20090214589A1 - Recombinant lentiviral vector for expression of a flaviviridae protein and applications thereof as a vaccine - Google Patents

Recombinant lentiviral vector for expression of a flaviviridae protein and applications thereof as a vaccine Download PDF

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US20090214589A1
US20090214589A1 US11/596,675 US59667508A US2009214589A1 US 20090214589 A1 US20090214589 A1 US 20090214589A1 US 59667508 A US59667508 A US 59667508A US 2009214589 A1 US2009214589 A1 US 2009214589A1
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protein
virus
flaviviridae
vector
lentiviral vector
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Philippe Despres
Pierre Charneau
Frederic Tangy
Marie-Pascale Frenkiel
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Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Institut Pasteur
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Priority to US12/929,215 priority patent/US8716013B2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • 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
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a recombinant lentiviral vector for expression of a protein of a Flaviviridae and to its applications as a vaccine intended for the prevention and/or treatment of an infection with a virus of the family Flaviviridae, in a sensitive species (host or reservoir)
  • Flaviviridae The family Flaviviridae is divided up into three genera: Flavivirus, Pestivirus and Hepacivirus or hepatitis C virus; Flaviviridae represent a major human and veterinary health problem due to the large number of both human and veterinary diseases induced by Flaviviridae. Specifically, there are, for example, more than 70 species of Flavivirus, at least 50% of which are the cause of human or veterinary diseases.
  • Flaviviridae are small enveloped viruses. Their genome is a single-stranded RNA molecule of positive polarity, of 9.5 kb to 12.5 kb, depending on the Flaviviridae, and contains a single open reading frame flanked by two short non-coding regions at its 5′ and 3′ ends. This open reading frame is translated into a polyprotein, which is the precursor of the structural proteins, in its N-terminal portion, and of. the non-structural (NS) proteins, in its C-terminal portion.
  • NS non-structural
  • viruses of this family Many serious human and animal pathologies are induced by the viruses of this family; according to the infecting virus, the various symptoms observed are generally fever (cyclic or non-cyclic), haemorrhagic fever, diarrhoea, encephalitis, hepatitis or septic shock. More precisely, the various viruses in question are the following:
  • Migratory birds can be the reservoir of some of these viruses, in particular the West Nile virus, which has also been noted to cross the species barrier, in horses and humans.
  • a recombinant lentiviral vector for expression of at least one immunogenic protein of a virus of the family Flaviviridae effectively makes it possible to induce a strong immune response in the individual (human or animal) immunized, capable in particular of protecting said individual against infection with this virus.
  • the recombinant lentiviral vector was able to induce a very early, long-lasting, fully protective immune response against a high dose West Nile virus challenge.
  • lentiviral vectors are efficient tools for eliciting a humoral protective response against a pathogen. This broadens the applicability of lentiviral vectors as vaccination tools against pathogens like viruses of the Flaviviridae family, in which a neutralizing humoral response is one active arm of the immune system.
  • a subject of the present invention is the use of a recombinant lentiviral vector comprising a polynucleotide fragment encoding at least one protein of a virus of the family Flaviviridae or an immunogenic peptide of at least 8 amino acids of said protein, for preparing an immunogenic composition intended for the prevention and/or the treatment of a Flaviviridae infection in a sensitive species.
  • said lenti-viral vector is selected from the group consisting of those derived from: HIV (human immunodeficiency virus), for example HIV-1 or HIV-2, CAEV (caprine arthritis encephalitis virus), EIAV (equine infectious anaemia virus), VMV (visna/maedi virus), SIV (simian immuno-deficiency virus) or FIV (feline immunodeficiency virus).
  • HIV human immunodeficiency virus
  • CAEV caprine arthritis encephalitis virus
  • EIAV equine infectious anaemia virus
  • VMV visna/maedi virus
  • SIV simian immuno-deficiency virus
  • FIV feline immunodeficiency virus
  • the invention also encompasses the chimeric lentiviruses derived from at least two different lentiviruses.
  • the choice of the lentiviral vector depends in particular on the sensitive species; for example, vectors derived from HIV are advantageously used for human immunization.
  • the lentiviral vectors are known to those skilled in the art; they consist of a recombinant nucleotide sequence (recombinant lentiviral genome) comprising: (i) a sequence of interest (coding sequence of Flaviviridae, in the case of the present invention) placed under the control of regulatory signals for transcription and for expression, and (ii) the regulatory sequences of lentiviral origin necessary and sufficient for encapsidation, reverse transcription and viral integration, and, optionally, regulatory sequences for the Rev protein (RRE or rev responsive element). Mention may in particular be made of lentiviral vectors derived from HIV, described by Poznansky et al. (J.
  • said lenti-viral vectors are vectors capable of expressing the coding sequence(s) as defined above, in a suitable cellular system; said vector comprises an expression cassette that includes the suitable regulatory elements for transcription (promoter, enhancer, Kozak consensus sequence, polyadenylation signal, etc.) under the control of which are inserted the coding sequences as defined above; said coding sequences of interest comprise the signals required for cell transport, for instance a signal for translocation in the endoplasmic reticulum, derived in particular from the ORF preceding said coding sequence in the polyprotein of said Flaviviridae.
  • said expression cassette comprises a strong ubiquitous promoter such as the cytomegalovirus (CMV) early promoter or an enhancer free promoter such as the elongation factor 1 ⁇ (EF1 ⁇ ) or the phosphoglycerate (PGK) promoters.
  • CMV cytomegalovirus
  • EF1 ⁇ elongation factor 1 ⁇
  • PGK phosphoglycerate
  • said vector may also comprise a suicide gene such as herpes type 1 thymidine kinase (HSV 1-TK), so as to eliminate the transduced cells by treatment with the appropriate drug, for example acyclovir in the case of HSV 1-TK.
  • a suicide gene such as herpes type 1 thymidine kinase (HSV 1-TK)
  • HSV 1-TK herpes type 1 thymidine kinase
  • the invention encompasses simple expression vectors and multiple expression vectors that allow simultaneous expression of several coding sequences from the same promoter or from different promoters, said promoters being located in the same region or else in different regions of said expression vector.
  • said recombinant lentiviral vector is of triplex type.
  • the vectors of triplex type are in particular described in Zennou et al., Cell, 2000, 101, 173-185 and in International Applications WO 99/55892, WO 01/27304 and WO 01/27300.
  • the triplex vectors are characterized in that they comprise a DNA region capable of forming a triplex (or DNA trimer) during viral reverse transcription.
  • This triplex DNA region consists of a cis-active region for central initiation, or polypurine tract (cPPT), and a cis-active region for termination (CTS), said regions making it possible to initiate the transcription of a +strand whose synthesis is initiated by the PTT region present at the centre of the genome of the lentivirus, and to interrupt the transcription of a +strand whose synthesis is initiated at a 3′ PPT site upstream of the retroviral LTR.
  • the presence of this triplex DNA region in the lentiviral vectors notably improves the transduction of genes in mitotic or non-mitotic cells, by stimulating the rate of nuclear import of the vector.
  • said recombinant lentiviral vector comprises a 3′ LTR in which the promoter and the activator have been deleted from the U3 region; this deletion provides additional safety features.
  • said recombinant lentiviral vector is pseudotyped with at least one envelope protein of another virus, preferably the vesicular stomatitis virus (VSV) glycoprotein G;
  • VSV glycoprotein G advantageously makes it possible to obtain high titres of vector particles and to produce vector particles having a broad cellular tropism, capable of transducing in particular antigen-presenting cells such as dendritic cells, in any vertebrate species: humans or animals including horses, fowl, and zoo animals at risk.
  • said Flaviviridae is chosen from a Flavivirus, a Pestivirus or a Hepacivirus, as specified above.
  • said Flaviviridae is selected from the group consisting of the West Nile virus, dengue virus, yellow fever virus and hepatitis C virus.
  • said polynucleotide in particular a cDNA or a cDNA fragment of Flaviviridae encodes: (i) one or more different structural proteins (C, prM, M, E, E1, E2), and/or (ii) one or more different non-structural (NS) proteins, and/or (iii) one or more different immunogenic fragments of said proteins, said proteins or their fragments being derived either from the same Flaviviridae (monovalent vaccine) or from various Flaviviridae and/or from different serotypes or different types of the same Flaviviridae, for preparing polyvalent vaccines.
  • Said cDNA can also derive from a coding sequence of a Flaviviridae by a shift in the open reading frame of one or two nucleotides (ribosomal frameshifting).
  • Such cDNAs are known to those skilled in the art, in particular for the C protein of the hepatitis C virus (Xu et al., EMBO, 2001, 20, 3840-3848; Roussel et al., J. Gen. Virol., 2003, 84, 1751-1759; Vassilaki et al., J. Biol. Chem., 2003, 278, 40503-40513; International Application WO 99/63941).
  • said polynucleotide is a fragment of a coding sequence of Flaviviridae corresponding to the accession number in the NCBI database listed in Table 1:
  • Flavivirus M23027 5′ cDNA sequence of the poly- protein of the dengue virus type 1 Flavivirus M19197 DNA equivalent of the genome of the dengue virus type 2
  • Flavivirus M93130 DNA equivalent of the genome of the dengue virus type 3 Flavivirus M14931 DNA equivalent of the genome of the dengue virus type 4
  • Flavivirus M12294 DNA equivalent of the genome of the West Nile virus
  • Flavivirus M73835 cDNA of the structural proteins of the Langat virus (T)
  • said polynucleotide fragment is selected from:
  • cDNAs encoding an E protein and, optionally, a prM or M protein, and/or a C protein, and/or a non-structural protein of West Nile virus or of dengue virus, and the cDNAs encoding one or more immunogenic peptides of at least 8 amino acids of the above proteins,
  • said cDNA encoding a C protein according to a +1 or +2 reading frame is selected from the group consisting of the sequences SEQ ID NOs. 5 to 14.
  • said membrane proteins (prM or M) and/or envelope proteins (E, E1, E2) are expressed by the recombinant lentiviral vector as defined above, either in membrane form, located in the plasma membrane, at the surface of the cells, or in secreted form, i.e. exported from the cell, to the extracellular medium.
  • Flavivirus prM and E proteins when expressed simultaneously in the cells transduced by the recombinant vector (in vitro or in vivo), they assemble as viral pseudoparticles (or virus-like particles, VLPs) that are secreted into the extracellular medium. Such particles are particularly immunogenic and induce the production of neutralizing antibodies.
  • the cDNA encoding said membrane form comprises the sequence encoding the mature protein, preceded by a sequence encoding a signal peptide for translocation in the endoplasmic reticulum, which sequence includes a translation initiation codon (ATG) at its 5′ end.
  • said signal sequence is advantageously derived from the M protein precursor (prM).
  • the cDNA encoding said secreted form comprises the sequence encoding a truncated mature protein, from which the membrane anchoring region has been deleted and which is preceded by a signal peptide as defined above.
  • the cDNAs encoding the membrane form of the E protein, the secreted form of the E protein and the prM and E proteins of the West Nile virus correspond, respectively, to positions 919 to 2469, 919 to 2292 and 399 to 2469 in the sequence of the genome of said virus as defined above.
  • a subject of the present invention is also a recombinant lentiviral vector comprising a polynucleotide fragment encoding at least one structural protein of a Flaviviridae or an immunogenic peptide of at least 8 amino acids of said protein; in addition, as specified above in the context of the use of such vectors, said vector advantageously also comprises a cDNA encoding one or more non-structural proteins and/or one or more immunogenic fragments of said proteins. Said polynucleotide fragment is in particular selected from the sequences as defined above.
  • said recombinant lentiviral vector is a vector of triplex type.
  • said recombinant lentiviral vector can advantageously comprise a 3′ LTR in which the promoter and the activator has been deleted from the U3 region. It is preferably a vector that is pseudotyped with at least one envelope protein of another virus, preferably the vesicular stomatitis virus (VSV) glycoprotein G.
  • VSV vesicular stomatitis virus
  • said vector comprises the cDNA encoding at least one E protein and, optionally, a prM or M protein, and/or a C protein, and/or a non-structural protein of West Nile virus or of dengue virus, or the cDNA encoding one or more immunogenic peptides of at least 8 amino acids of the above proteins.
  • said vector comprises the cDNA encoding an E1 or E2 protein or an E1/E2 heterodimer, and/or a C protein according to a 0, +1 or +2 reading frame and, optionally, an NS3 protein of hepatitis C virus, or the cDNA encoding one or more immunogenic peptides of at least 8 amino acids of the above proteins.
  • said cDNA encoding a C protein according to a +1 or +2 reading frame is selected from the group consisting of the sequences SEQ ID NOs. 5 to 14.
  • said vector comprises the cDNA encoding a domain III (positions 295 to 394) or several different domains III of an E protein of dengue virus, each corresponding to one of the four types of dengue virus (types 1 to 4 or DEN-1 to DEN-4), preferably it comprises a cDNA encoding the four domains III (DEN-1 to DEN-4) the sequences of which are represented by SEQ ID NOs. 1-4 in the sequence listing attached in the appendix.
  • said vector is a vector plasmid called pTRIP ⁇ U3.CMV-sE (WNV), comprising the cDNA encoding a secreted form of the E protein of the IS-98-ST1 strain of West Nile virus, which vector is included in a microorganism deposited under the No. I-3076, on 27 Aug. 2003, with the Collection Nationale de Cultures de Microorganismes [National Collection of Cultures of Microorganisms], 25 rue du Dondel Roux, 75724 Paris Cedex 15.
  • WNV vector plasmid called pTRIP ⁇ U3.CMV-sE
  • the invention encompasses the vector plasmids as defined above and the vector particles derived from the above vector particles, in particular the vector particles pseudotyped with at least one envelope protein of another virus, such as in particular the vesicular stomatitis virus (VSV) glycoprotein G.
  • VSV vesicular stomatitis virus
  • the recombinant lentiviral vectors as defined above are prepared by conventional methods, that are known in themselves, and according to standard protocols such as those described in Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc., Library of Congress, USA).
  • the polynucleotide fragments can be obtained either by amplification of a matrix consisting of a genomic RNA or an mRNA of a Flaviviridae or else a cDNA or a DNA fragment derived from the above, by PCR or RT-PCR using primers specific for the genome of a virus of the family Flaviviridae, or by digestion of the Flaviviridae cDNA using a restriction enzyme, or alternatively by total or partial chemical synthesis.
  • the polynucleotide fragment thus obtained is cloned into a vector plasmid containing the lentiviral vector genome, so as to produce a recombinant vector plasmid.
  • the particles of the recombinant lentiviral vector are produced by cotransfection of cells with the recombinant vector plasmid as defined above, an encapsidation plasmid that provides, in trans, the structural proteins and the enzymes of the viral particle and, optionally, a plasmid for expression of the envelope glycoprotein of a virus such as VSV, for the production of pseudotyped particles.
  • a subject of the present invention is also an immunogenic composition, characterized in that it comprises at least one recombinant vector as defined above.
  • composition it comprises a pharmaceutically acceptable vehicle and, optionally, a carrier substance.
  • the pharmaceutically acceptable vehicles and the carrier substances are those conventionally used.
  • the carrier substances are advantageously selected from the group consisting of unilamellar liposomes, multilamellar liposomes, saponin micelles or solid microspheres of a saccharide or auriferous nature.
  • composition it comprises particles of said recombinant lentiviral vector (vector particles), preferably pseudotyped with an envelope protein of another virus, preferably with the vesicular stomatitis virus glycoprotein G.
  • composition it comprises a recombinant lentiviral vector of triplex type as defined above.
  • composition comprises an isolated nucleic acid molecule corresponding to the recombinant genome of said recombinant lentiviral vector of triplex type, which nucleic acid molecule comprises: (i) the regulatory sequences for encapsidation, reverse transcription and integration and the cis-active sequences for central initiation (or polypurine tract cPPT) and termination (CTS) of lentiviral origin and, optionally, the regulatory sequences for the Rev protein (RRE or Rev Responsive Element) and (ii) a polynucleotide fragment encoding a Flaviviridae protein or an immunogenic peptide of at least 8 amino acids of said protein as defined above.
  • nucleic acid molecule comprises: (i) the regulatory sequences for encapsidation, reverse transcription and integration and the cis-active sequences for central initiation (or polypurine tract cPPT) and termination (CTS) of lentiviral origin and, optionally, the regulatory sequences for the Rev protein (
  • said vector of triplex type comprises an expression cassette that includes the suitable regulatory elements for transcription (promoter, enhancer, Kozak consensus sequence, polyadenylation signal, etc.) under the control of which are inserted the coding sequences as defined above, and said coding sequences of interest optionally comprise the signals required for cellular transport, as defined above.
  • suitable regulatory elements for transcription promoter, enhancer, Kozak consensus sequence, polyadenylation signal, etc.
  • the immunogenic or vaccine compositions according to the invention can be administered generally (orally, intramuscularly, subcutaneously, intraperitonealy or intravenously), locally (nasally, other mucosal routes) or by a combination of these routes, in a sensitive species as defined above (human or non-human mammalian host, or reservoir (birds, reptiles)).
  • they are administered subcutaneously in order to target antigen-presenting cells such as dendritic cells, so as to obtain prolonged expression of the antigen in these cells.
  • antigen-presenting cells such as dendritic cells
  • the immunogenic or vaccine compositions according to the invention are used to modify autologous cells of a host species, in particular antigen-presenting cells such as dendritic cells.
  • the modified cells are then re-administered to the host; such a use is particularly advantageous for the treatment of an infection with a Flaviviridae in a human or non-human host mammal.
  • the dose of vector varies according to the route of administration, and also according to the nature and the weight of the species to be treated (human or animal).
  • a subject of the present invention is also cells modified with a recombinant vector as defined above.
  • said cells are eukaryotic cells that are stably modified with said recombinant vector; such cells that stably express at least one protein or one antigenic peptide of Flaviviridae are useful:
  • the pseudoparticles are advantageously used as a reagent for diagnosing a Flaviviridae infection by immunocapture of the specific immunoglobulins present in the biological fluids of infected individuals,
  • the purification of viral protein(s) or of fragment(s) can be carried out, from a culture supernatant, or from lysates of the cells modified with a recombinant vector as defined above, by conventional techniques such as:
  • the purification of the particles of the type of those of Flaviviridae is carried out, from a culture supernatant from cells modified with a recombinant vector as defined above, by conventional techniques such as:
  • a subject of the present invention is also a method for screening antiviral compounds, characterized in that it comprises:
  • the screenings are carried out on specific target tissues, and in particular on dendritic cells, neuronal cells or hepatocytes.
  • a subject of the present invention is also a method for diagnosing infection with a Flaviviridae, using a sample of biological fluid from an individual of a sensitive species, characterized in that it comprises at least the following steps:
  • Flaviviridae antigen C, E, E1, E2, prM, M, NS (in particular NS1)
  • a subject of the present invention is also a method for diagnosing infection with a Flaviviridae using a sample of biological fluid from an individual of a sensitive species, characterized in that it comprises at least the following steps:
  • a subject of the present invention is also a kit for carrying out the methods as defined above, characterized in that it comprises at least modified cells as defined above.
  • a subject of the present invention is also a method of immunization against a Flaviviridae, characterized in that it comprises a single administration of a recombinant vector as defined above, preferably subcutaneously.
  • the invention also comprises other provisions, which will emerge from the following description that refers to examples of preparation of the recombinant vector according to the present invention and of use of said vector for immunization, and derived modified cells for the production of proteins, and also to the attached drawings in which:
  • FIG. 1 is a diagrammatic representation of the vector plasmid pTRIP ⁇ U3CMV-sE(WNV) corresponding to the sequence SEQ ID NO. 15, containing the cDNA (SEQ ID NO. 16) encoding the truncated E protein (E1-411) of the West Nile virus (SEQ ID NO. 17).
  • FIG. 2 illustrates the analysis by ELISA and by means of a neutralization assay, of the sera from mice immunized with a single intraperitoneal injection of 1 ⁇ g of TRIP ⁇ U3CMV-sE(WNV) vector particles.
  • FIG. 3 represents the immunoprecipitation of the lysates of VERO cells infected with West Nile virus, with the sera from the mice immunized with 1 ⁇ g of TRIP ⁇ U3CMV-sE(WNV) vector particles, by comparison with control sera.
  • Lanes 1 to 10 lysates of VERO cells infected with West Nile virus were precipitated with the following sera:
  • lane 1 serum at D14 post-immunization with the TRIP ⁇ U3CMV-GFP vector
  • lane 2 serum at D23 post-immunization with the TRIP ⁇ U3CMV-GFP vector
  • lane 7 serum at D22 post-challenge (10 LD 50 of the IS-98-ST1 strain) from the mice immunized for 14 days with the TRIP ⁇ U3CMV-sE(WNV) vector,
  • lane 8 serum at D30 post-challenge (10 LD 50 of the IS-98-ST1 strain) from the mice immunized for 14 days with the TRIP ⁇ U3CMV-sE(WNV) vector,
  • lane 9 serum at D22 post-challenge (100 LD 50 of the IS-98-ST1 strain) from the mice immunized for 30 days with the TRIP ⁇ U3CMV-sE(WNV) vector,
  • lane 10 serum from mice immunized with the lymphocytic choriomeningitis virus.
  • Lanes 11 and 12 lysates of non-infected VERO cells were precipitated with the following sera:
  • lane 12 serum at D22 post-challenge (100 LD 50 of the IS-98-ST1 strain) from the mice immunized for 30 days with the TRIP ⁇ U3CMV-sE(WNV) vector.
  • FIG. 4 represents the survival curve for the mice immunized intraperitoneally and then challenged by the same route, either 2 weeks after immunization, with 10 LD 50 of the IS-98-ST1 strain (A), or 4 weeks after immunization, with 100 LD 50 of the IS-98-ST1 strain (B).
  • A 10 LD 50 of the IS-98-ST1 strain
  • B 100 LD 50 of the IS-98-ST1 strain
  • • control mice inoculated with DPBS.
  • ⁇ control mice immunized with 1 ⁇ g of TRIP ⁇ U3CMV-EGFP vector particles.
  • FIG. 5 illustrates the purification of viral pseudoparticles from the supernatant of eukaryotic cells transduced with a recombinant lentiviral vector expressing the prM and E proteins of the West Nile virus.
  • FIG. 6 illustrates the detection of anti-WNV-sE antibodies in sera from TRIP ⁇ U3.CMV-sE(WNV) vaccinated 129 mice.
  • Radio-labeled cell lysates from WNV infected Vero cells were immunoprecipitated with pooled immune sera from lentiviral vector vaccinated 129 mice.
  • A Pre-WNV challenge sera.
  • B Post-challenge sera.
  • HMAF Hyperimmune Mouse Ascitic Fluid.
  • Control sera non immune sera.
  • Antisera to MV antisera to Measle Virus.
  • TRIP/WNsE TRIP ⁇ U3.CMVsE(WNV).
  • TRIP/GFP TRIP ⁇ U3.CMV-GFP.
  • FIG. 7 illustrates the analysis by flow cytometry of the effect of heat treatment on recombinant lentiviral vector transduction efficiency.
  • 293T cells were incubated with TRIP ⁇ U3.CMV-GFP vector particles which have been heat-inactivated for 10 min at 70° C. (heated TRIP/GFP), or not inactivated (TRIP/GFP).
  • Non-infected 293T cells (Mock) were used as control.
  • the GFP fluorescence intensity was measured; the percentage of GFP positive cells is indicated.
  • sequence SEQ ID. NO. 16 contains, successively from 5′ to 3′: an ATG, the sequence encoding the signal peptide derived from the M protein precursor (prM 151-166) and the sequence encoding a truncated E protein (E 1-441), from which the membrane anchoring region has been deleted.
  • E protein which is secreted into the extracellular medium (sE protein); the signal peptide derived from the prM protein is used for translocation of the E protein in the endoplasmic reticulum and for its transport, in secretion vesicles, to the plasma membrane, where it is released into the extracellular medium.
  • the lentiviral vector plasmid pTRIP ⁇ U3.CMV-EGFP (application WO 01/27302) was digested so as to excise the EGFP gene, and then the linearized plasmid was ligated with a linker containing the BsiW I and BssH II sites, so as to give the plasmid called pTRIP ⁇ U3.CMV-BsiW I-BssH II.
  • WNV pTRIP ⁇ U3.CMV-sE
  • WNV pTRIP ⁇ U3.CMV-sE
  • WNV pTRIP ⁇ U3.CMV-sE
  • sequence of the 1.4 kb BsiW I-BssH II insert corresponds to the nucleotide sequence SEQ ID NO. 16 in the sequence listing attached in the appendix; it encodes a secreted E protein, called sE, corresponding to the amino acid sequence SEQ ID NO. 17 in the sequence listing attached in the appendix.
  • VSV-G Vesicular Stomatitis Virus Envelope Glycoprotein
  • TRIP ⁇ U3.CMV-sE vector pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G), also called TRIP ⁇ U3.CMV-sE (WNV) vector particles, are produced by calcium phosphate cotransfection of the 293T cell line with the pTRIP ⁇ U3.CMV-sE (WNV) vector plasmid as defined above, an encapsidation plasmid that provides, in trans, the structural proteins and the enzymes of the viral particle (pCMV ⁇ R8.2: Naldini et al., Science, 1996, 272, 263-267; pCMV ⁇ R8.91 or p8.7: Zufferey et al., Nat.
  • pCMV ⁇ R8.2 Naldini et al., Science, 1996, 272, 263-267
  • pCMV ⁇ R8.91 or p8.7 Zufferey et al., Nat.
  • VSV virus envelope glycoprotein pHCMV-G: Yee et al., P.N.A.S., 1994, 91, 9564-9568
  • Zennou et al., Cell., 2000, 101, 173-185 pHCMV-G: Yee et al., P.N.A.S., 1994, 91, 9564-9568
  • WNV-sE WNV-sE expression was examined by indirect immunofluorescence. Briefly, human 293T cells cultured on 8-chamber Glass-Labteks (NUNC) were transduced with TRIP ⁇ U3.CMV-sE (WNV) vector. After 48 h, cells were fixed with 3% paraformaldehyde (PFA) in PBS for 20 min and permeabilized with 0.1% Triton X-100 in PBS for 4 min. Cells were incubated with anti-WNV HMAF at a 1:100 dilution in PBS for 1 h.
  • PFA paraformaldehyde
  • Quantification of p24 antigen content of concentrated vector particles was done with a commercial HIV-1 p24 ELISA kit (PERKIN ELMER LIFESCIENCES).
  • LTR-FL 5′-CACAACAGACGGGCACACACTACTTGA-FITC-3′
  • LTR-LC 5′-RED640-CACTCAAGGCAAGCTTTATTGAGGC-P-3′ primers AA55M: 5′-GCTAGAGATTTTCCACACTGACTAA-3′
  • M667 5′-GGCTAACTAGGGAACCCACTG-3′.
  • primers and probes were as follows: probes CD3-P1: 5′-GGCTGAAGGTTAGGGATACCAATATTCCTGTCTC- FITC-3′, CD3-P2: 5′RED705-CTAGTGATGGGCTCTTCCCTTGAGCCCTTC- P-3′ primers CD3-in-F: 5′-GGCTATCATTCTTCTTCAAGGTA-3′ CD3-in-R: 5′-CCTCTCTTCAGCCATTTAAGTA-3′.
  • Genomic DNA from approximately 3.10 6 lentiviral vector transduced 293T cells was isolated 48 h after transduction using QIAamp® DNA Blood Mini Kit (QIAGEN).
  • QIAamp® DNA Blood Mini Kit QIAGEN
  • 5 ⁇ L of DNA were mixed with 15 ⁇ L of a PCR master mix consisting of 1 ⁇ JumpstartTM Taq ReadyMixTM (SIGMA), 1.9 mM MgCl 2 , 1.5 ⁇ M of forward and reverse primers (AA55M/M667 or CD3-in-F/CD3-in-R), 200 nM of the probes (LTR-FL/LTR-LC or CD3-P1/CD3-P2) and, 1.5 units of Taq DNA Polymerase (Invitrogen).
  • Amplifications were performed using one cycle of 95° C. for 3 min, and 40 cycles of 95° C. for 5 s, 55° C. for 15 s and 72° C. for 10 s.
  • DNA from 293T cells transduced with heat-inactivated (10 min at 70° C.) vector was always tested in parallel.
  • DNA from untransduced cells was used for negative controls.
  • Each DNA sample was tested in duplicate and the mean values are reported.
  • the total number of vector copies per cell was calculated by normalizing the number of U5-R copies to the number of 293T cells, as quantified by the copy number of CD3 molecules on the same genomic DNA sample, and then subtracting the number of copies obtained for the heat-inactivated vector-transduced cells.
  • the number of physical particles of the vector stock used in this study was first evaluated using a commercially available ELISA assay against the p24 HIV-1 capsid protein. The determined concentration was 58 ng of p24 per microliter.
  • the vector stock actual titer was calculated on the basis of the transfer of vector DNA to the target cell, using a quantitative PCR assay.
  • the quantification of both a vector specific sequence (U5) and a cellular locus (CD3) gives the average DNA vector copy number per cell. This allows the calculation, after transduction with a defined concentration of vector particles, of the titer of the vector preparation.
  • the TRIP ⁇ U3.CMV-sE vector stock used in this study was titrated in human 293T cells at 5.2 ⁇ 10 7 transduction units (TU) per ml. In other words, 1 ng of p24 antigen from this TRIP ⁇ U3.CMV-sE vector preparation can transduce 900 human 293T cells.
  • the quantity of vector particles used will be expressed as ng of p24 antigen.
  • mice Six-week-old BALB/c mice (2 groups of 6 mice; Janvier breeding colony) were inoculated intraperitoneally with 0.1 ml of Dulbecco's PBS (DPBS) containing 1 ⁇ g of TRIP ⁇ U3.CMV-sE vector particles prepared as described in Example 1. The animals were given a single vaccine injection.
  • DPBS Dulbecco's PBS
  • control groups were inoculated, under the same conditions, either with 1 ⁇ g of TRIP ⁇ U3.CMV-GFP vector particles prepared in a similar manner to the TRIP ⁇ U3.CMV-sE (WNV) vector particles (2 groups of 3 mice), or DPBS buffer alone (2 groups of 3 mice).
  • WNV TRIP ⁇ U3.CMV-sE
  • mice sera were taken 14 days (D 14 ) and 23 days (D 23 ) after the vaccine injection and heat-inactivated for 30 min at 56° C. before measurement of the antibody response.
  • the West Nile virus strain used is the IS-98-ST1 strain, described in application FR 01 04599; it is produced on Aedes mosquito cells (AP61 line) and purified according to the protocol described by Dessten et al., Virol., 1993, 196, 209-219. More precisely, AP61 cells are infected, at a multiplicity of infection of 0.4, with the IS-98-ST1 strain of the West Nile virus. Three days after infection, the viral particles present in the culture supernatant are precipitated with PEG 6000 (7%), and then purified on a discontinuous 30-60% sucrose gradient and on a linear 10-50% sucrose gradient. The virions thus obtained are conserved at ⁇ 80° C. in sucrose (30%).
  • the West Nile virus is titered by means of a Focus ImmunoAssay (FIA) on AP61 cells, and the infectious titre is expressed as focus-forming units (FFU AP61 /ml), according to the protocol described by Des briefly et al., mentioned above.
  • FIA Focus ImmunoAssay
  • infectious titres of the purified viral preparations are approximately 10 10 FFU AP61 /ml.
  • Anti-WNV hyperimmune mouse ascitic fluid was obtained by repeated immunization of adult mice with WNV strain IS-98-ST1, followed by the inoculation of sarcoma 180.
  • Mouse polyclonal anti-WNV antibodies were obtained by immunization of adult WNV-resistant BALB/c-MBT congenic mice with 10 3 FFU of IS-98-ST1 as described previously (Mashimo et al., PNAS, 2002, 99, 11311-11316). The WNV-immune serum was collected one month after priming.
  • the anti-E total antibodies titres are measured by ELISA according to the protocol described in Mashimo et al., PNAS, 2002, 99, 11311-11316, using, as antigen, WN IS-98-ST1 virions purified on a sucrose gradient as described in paragraph 1.2 (10 6 FFU AP61 per 96-well microplate).
  • Peroxidase-conjugated anti-mouse immunoglobulin (H+ L) JACKSON IMMUNO RESEARCH
  • peroxidase-conjugated anti-mouse IgM ⁇ -chain specific
  • SIGMA peroxidase-conjugated anti-mouse IgG
  • Sigma peroxidase-conjugated anti-mouse IgG
  • the titres are determined by means of the final dilution of serum that corresponds to the optical density (OD) value which is at least twice that of the serum from the control animals, as defined above.
  • the anti-E IgG and IgM antibodies are also measured using an already described isotype specific ELISA (Despres P et. al., J. Infect. Dis., 2005, 191, 207-214).
  • VERO cells are infected with the IS-98-ST1 strain of the West Nile virus, at the multiplicity of infection of 5 FFU AP61 /cell. Twenty hours after infection, the cell proteins are labelled with Tran 35 Slabel (ICN; 100 ⁇ Ci/ml) for 3 hours.
  • cells are lysed in RIPA buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 10 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, supplemented with 25 ⁇ g/ml aprotinin (SIGMA) for 10 min at +4° C.
  • RIPA buffer 50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 10 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, supplemented with 25 ⁇ g/ml aprotinin (SIGMA) for 10 min at +4° C.
  • SIGMA aprotinin
  • the cell lysates are then clarified by centrifugation at 10,000 rpm for 5 min at +4° C.
  • the lysates are incubated with the sera to be tested at the final dilution of 1:100, in
  • the neutralizing antibody titre of the sera is determined by virtue of the final dilution of the serum that neutralizes at least 90 of the 100 FFUs of viruses inoculated in each well.
  • the results given in FIG. 2 show that the specific antibody titre of the sera from the mice immunized with the TRIP ⁇ U3.CMV-sE (WNV) vector particles is 1/10 000 and 1/20 000, respectively, 14 days and 23 days after the vaccine injection.
  • WNV TRIP ⁇ U3.CMV-sE
  • the specificity of the sera from the animals immunized with the TRIP ⁇ U3.CMV-sE vector was verified by immunoprecipitation.
  • the sera from the mice immunized with the TRIP ⁇ U3.CMV-sE vector react with the envelope protein E of West Nile virus; the reactivity is stronger at D 23 than at D 14 after the vaccine injection ( FIG. 3 ).
  • the titres at D 14 and D 23 after vaccine injection are, respectively, 10 and 20 ( FIG. 2 ).
  • mice were challenged by intraperitoneal inoculation of 10 LD 50 (dose that is lethal in 50% of the mice) or 100 LD 50 of the highly neuroinvasive and neurovirulent IS-98-ST1 strain of West Nile virus.
  • the first group of 6 mice immunized as described in Example 2 received 10 LD 50 of the IS-98-ST1 strain, 15 days after the vaccine injection (D15);
  • the second group of 6 mice immunized as described in Example 2 received 100 LD 50 of the IS-98-ST1 strain, 30 days after the vaccine injection (D30).
  • the challenge virus is diluted in DPBS (pH 7.5), supplemented with 0.2% of bovine serum albumin (Sigma); 1 LD 50 corresponds to 10 FFU AP61 /ml.
  • the survival curve for the first group of mice shows that all the control mice, inoculated with DPBS or with the TRIP ⁇ U3.CMV-EGFP vector, die 13 days after the inoculation of the viral challenge dose.
  • mice immunized with the TRIP ⁇ U3.CMV-sE (WNV) vector are resistant to the lethal dose and showed no morbidity.
  • the resistant mice have anti-West Nile virus antibody titres (1.7 ⁇ 0.1, dilution 1:10 4 ), by ELISA, that are greater than those obtained before the challenge.
  • the sera from the challenged mice react strongly with the E protein of West Nile virus ( FIG. 3 ) and the neutralizing antibodies have a titre of 100, 1 month after the challenge.
  • the survival curve for the second group of mice shows that all the control mice, inoculated with DPBS or with the TRIP ⁇ U3.CMV-EGFPs vector (DPBS), die within 9 days following the inoculation of the viral challenge dose.
  • the 6 mice immunized with the TRIP ⁇ U3.CMV-sE (WNV) vector are resistant to the lethal dose and show no morbidity.
  • the sera from the challenged mice react strongly with the E protein of West Nile virus ( FIG. 3 ) and the neutralizing antibodies have a titre of 100, 1 month after the challenge.
  • a recombinant HIV vector of triplex type comprising a cDNA encoding the prM and E proteins of the IS-98-ST1 strain of West Nile virus, corresponding to positions 399 to 2469 of the sequence of the genome (application FR 01 04599 and Genbank AF481864), was constructed as described in Example 1.
  • Stable lines transduced with the TRIP ⁇ U3.CMV-prM-E (WNV) recombinant vector were obtained as described in Example 1.
  • the culture supernatant of the cells transduced with the TRIP ⁇ U3.CMV-prM-E (WNV) vector is harvested, and precipitated with PEG 6000 (Fluka, 0.7% W/V) 4 to 5 hours at 4° C., with gentle agitation.
  • the precipitate obtained is centrifuged for 30 minutes at 9000 rpm at 4° C., and the pellet containing the VLPs is taken up in 4 ml of TNE (20 mM Tris-HCl, pH 8.0; 150 mM NaCl; 2 mM EDTA) and deposited onto a discontinuous sucrose gradient (20%-60% sucrose in 1 ⁇ TNE).
  • the gradient is centrifuged at 39 000 rpm for 2 h, and the opalescent band at the 20-60% interface is harvested, deposited on a linear gradient (11-55% sucrose in 1 ⁇ TNE) and centrifuged at 35 000 rpm for 16 h.
  • the gradient fractions are collected (11 fractions of 0.5 ml) and then analyzed by ELISA using an anti-WNV immune serum (1:20), by SDS-PAGE gel electrophoresis and Coomassie blue staining, and by Western blotting using an anti-WNV immune serum.
  • the results of the ELISA given in FIG. 5 , indicate the presence of purified VLPs in fractions 6 to 10 of the gradient.
  • mice Six to eight week old 129 mice (six groups of six mice) were intraperitoneally (i.p.) inoculated with varying doses of TRIP ⁇ U3.CMV-sE (WNV) vector particles prepared as described in Example 1, diluted in 0.1 ml Dulbecco's PBS (DPBS; pH 7.5) supplemented with 0.2% bovine serum albumin (BSA).
  • DPBS Dulbecco's PBS
  • BSA bovine serum albumin
  • the animals were given a single vaccine injection.
  • control groups were inoculated, under the same conditions, with 500 ng p24 antigen equivalent of TRIP ⁇ U3.CMV-GFP vector particles prepared in a similar manner to the TRIP ⁇ U3.CMV-sE (WNV) vector particles (one group of six mice), or DPBS buffer alone (one group of six mice).
  • WNV TRIP ⁇ U3.CMV-sE
  • mice were bled periorbitally at 6, 13, 20 or 27 days post-immunization (D 6 , D 13 , D 20 , D 27 ) and pooled sera were heat-inactivated for 30 min at 56° C. before measurement of anti-WNV total antibodies, IgG and IgM, and in vitro neutralizing activity, as described in example 2.
  • 293T cells cultured on 25 cm2 flasks were transduced with TRIP ⁇ U3.CMV-GFP vector particles which have been, either heat-inactivated for 0.10 min at 70° C., or untreated (positive control).
  • TRIP ⁇ U3.CMV-GFP vector particles which have been, either heat-inactivated for 0.10 min at 70° C., or untreated (positive control).
  • cells were detached, washed and fixed with 2% PFA.
  • the GFP fluorescence intensity was measured by FACSscan and analyzed with CellQuest software.
  • mice which are less congenic than BALB/c were selected for assessing the humoral immune response induced by the lentiviral vector expressing WNV-sE.
  • FRNT Focus Reduction Neutralization Test: the highest serum dilution that reduced the number of FFU of WNV by least 90%.
  • mice immunized with a single dose of TRIP ⁇ U3.CMV-sE (WNV) vector particles equivalent to 500 ng of p24 antigen were fully protected against a high viral challenge as early as 7 days post-immunization, since no morbidity or mortality were observed in this group (Table 3).
  • WNV TRIP ⁇ U3.CMV-sE
  • mice were inoculated i.p. with 1,000 i.p. LD50 of WNV strain IS-98-ST1. Survivals were recorded for 21 days. c Determined by ELISA on pooled heat-inactivated sera. ND: not determined
  • the infectious virus dose used in the viral challenge was selected to correspond to the maximal viral inoculum that can be transmitted by a mosquito bite. This dose is estimated to correspond to 10,000 in vitro FFU (Despres et al., J. Infect. Dis., 2005, 191, 207-214; Mashimo et al., 2002, precited), itself corresponding to 1000 in vivo-LD 50 by the intraperitoneal route.
  • RIP assays were performed on pooled sera from immunized mice, collected before and at 21 days after WNV challenge. Sera obtained at day 13, 20 and 27 post-immunization with a single dose of TRIP ⁇ U3.CMV-sE (WNV) vector particles equivalent to 500 ng of p24 antigen, reacted with the E protein of WNV. However, sera obtained from day 6 post-immunization did not react with this protein ( FIG. 6A ).
  • c FRNT Focus Reduction Neutralization Test:: the highest serum dilution that reduced the number of FFU of WNV by least 90%.
  • d Mice were inoculated i.p. with 1000 LD 50 of WNV strain IS-98-ST1, three months post immunization. Survival was recorded for 21 days.
  • mice were immunized i.p. with decreasing doses of TRIP ⁇ U3.CMV-sE (WNV) or a 500 ng dose of TRIP ⁇ U3.CMV-GFP vector particles as a control. Seven days later, all mice were challenged with 1000 LD 50 IS-98-ST1. As expected, all mice that received the control vector died within 11-13 days of challenge. Results showed that the minimal dose of TRIP ⁇ U3.CMV-sE (WNV) required for full protection of mice was a vector particle quantity equivalent to 50 ng of p24 antigen (Table 5).
  • mice cells have a lower permissivity to lentiviral vector transduction than other mammal cells, including human cells (Giannini et al., Hepatology, 2003, 38, 114-122; Nguyen et al., Mol. Ther., 2002, 6, 199-209).
  • Avian cells show a better permissivity to transduction than murine cells allowing to predict that minute lentiviral vector vaccine doses would be effective in fowl.
  • mice were immunized with heat-inactivated (10 min at 70° C.) TRIP ⁇ U3.CMV-sE (WNV) vector particles, a treatment that abrogates transduction ( FIG. 7 ). After WNV challenge, all mice injected with the heat-inactivated TRIP ⁇ U3.CMV-sE (WNV) died (Table 5). It is therefore unlikely that free naked DNA plays a role in protection.
  • the lentiviral vector vaccine can theoretically be used, with no modification, in any vertebrate species, including humans and animals like horses, fowl, and zoo mammals at risk.

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BRPI0510016B1 (pt) 2020-03-10
CN1981043A (zh) 2007-06-13
BRPI0510016A (pt) 2007-09-18
MXPA06013388A (es) 2007-06-19
CN1981043B (zh) 2010-06-09
WO2005111221A1 (en) 2005-11-24
CA2564934A1 (en) 2005-11-24
FR2870126A1 (fr) 2005-11-18
BRPI0510016B8 (pt) 2021-05-25
EP1751291A1 (en) 2007-02-14
EP2371966B1 (en) 2015-11-25
US8716013B2 (en) 2014-05-06
EP2371966A1 (en) 2011-10-05
JP2008508863A (ja) 2008-03-27
US20110206710A1 (en) 2011-08-25
CA2564934C (en) 2014-07-08
FR2870126B1 (fr) 2009-07-17

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