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

Abstract

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 a pharmaceutical composition intended for the prevention and/or the treatment of a Flaviviridae infection in a sensitive species.

1 . 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. 
     
     
         2 . Use according to  claim 1 , characterized in that said recombinant lentiviral vector is of triplex type. 
     
     
         3 . Use according to  claim 1  or  claim 2 , characterized in that said recombinant lentiviral vector comprises a 3′ LTR in which the promoter and the activator have been deleted from the U3 region. 
     
     
         4 . Use according to any one of  claims 1  to  3 , characterized in that said recombinant lentiviral vector is pseudotyped with at least one envelope protein of another virus. 
     
     
         5 . Use according to  claim 4 , characterized in that said envelope protein is the vesicular stomatitis virus glycoprotein G. 
     
     
         6 . Use according to any one of  claims 1  to  5 , characterized in that said polynucleotide fragment encodes at least one structural protein of a Flaviviridae or an immunogenic peptide of at least 8 amino acids of said protein. 
     
     
         7 . Use according to  claim 6 , characterized in that said structural protein is a membrane protein or an envelope protein. 
     
     
         8 . Use according to any one of  claims 1  to  5 , characterized in that said polynucleotide fragment encodes at least one non-structural protein of Flaviviridae or an immunogenic peptide of at least 8 amino acids of said protein. 
     
     
         9 . Use according to any one of  claims 1  to  8 , characterized in that said polynucleotide fragment encodes both at least one structural protein of a Flaviviridae or an immunogenic peptide of at least 8 amino acids of said protein, and at least one non-structural protein or a fragment of at least 8 amino acids of said non-structural protein of the same Flaviviridae. 
     
     
         10 . Use according to any one of  claims 1  to  9 , characterized in that said immunogenic peptide consists of the combination of immunogenic fragments of various Flaviviridae and/or of different serotypes of the same Flaviviridae. 
     
     
         11 . Use according to any one of  claims 1  to  10 , characterized in that said Flaviviridae is selected from the group consisting of West Nile virus, dengue virus, yellow fever virus and hepatitis C virus. 
     
     
         12 . Use according to any one of  claims 1  to  11 , characterized in that said polynucleotide fragment is selected from the group consisting of:
 the 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,   the cDNAs 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/or an NS3 protein of hepatitis C virus, and the cDNAs encoding one or more immunogenic peptides of at least 8 amino acids of the above proteins,   the cDNAs encoding one or more different domains III (positions 295 to 394) of an E protein of dengue virus, each corresponding to one of the four types of dengue virus.   
     
     
         13 . Use according to  claim 12 , characterized in that said cDNA encodes the four domains III, the sequences of which are represented by SEQ ID NOs. 1-4 in the sequence listing attached in the appendix. 
     
     
         14 . Use according to  claim 12 , characterized in that 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. 
     
     
         15 . Use according to any one of  claims 1  to  12 , characterized in that said polynucleotide is a fragment of a coding sequence of a Flaviviridae corresponding to an accession number in the NCBI database selected from the group consisting of: M23027, M19197, M93130, M14931, M12294, AF481864, M18370, X03700, U27495, M73835, M31182, M31768, J04358; M62321, D90208 and M58335. 
     
     
         16 . Recombinant lentiviral vector as defined in any one of  claims 1  to  6  and  8  to  15 , this vector comprising a polynucleotide encoding at least one structural protein or a fragment of said protein and, optionally, a non-structural protein or a fragment of said protein, as defined in  claim 6 . 
     
     
         17 . Recombinant vector according to  claim 16 , characterized in that it is of triplex type. 
     
     
         18 . Recombinant lentiviral vector according to  claim 16  or  claim 17 , characterized in that it comprises a cDNA selected from the group consisting of:
 a 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, and a cDNA encoding one or more immunogenic peptides of at least 8 amino acids of the above proteins,   a 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, and a cDNA encoding one or more immunogenic peptides of at least 8 amino acids of the above proteins, and   a 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.   
     
     
         19 . Recombinant lentiviral vector according to  claim 18 , characterized in that 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. 
     
     
         20 . Recombinant lentiviral vector according to  claim 18 , characterized in that said cDNA encodes the four domains III the sequences of which are represented by SEQ ID NOs. 1-4. 
     
     
         21 . Recombinant lentiviral vector according to  claim 18 , characterized in that it comprises the cDNA encoding an E protein and a prM protein of a dengue virus or of West Nile virus. 
     
     
         22 . Recombinant lentiviral vector according to  claim 18 , characterized in that it comprises 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 Docteur Roux, 757244 Paris Cedex 15. 
     
     
         23 . Recombinant lentiviral vector according to any one of  claims 16  to  22 , characterized in that said polynucleotide fragment is placed under the control of the CMV promoter. 
     
     
         24 . Immunogenic composition, characterized in that it comprises at least one recombinant lentiviral vector as defined in  claims 1  to  23 . 
     
     
         25 . Composition according to  claim 24 , characterized in that it comprises a pharmaceutically acceptable vehicle and, optionally, a carrier substance. 
     
     
         26 . Composition according to  claim 24  or  claim 25 , characterized in that said recombinant lentiviral vector is of triplex type. 
     
     
         27 . Composition according to any one of  claims 24  to  26 , characterized in that it comprises viral particles of said recombinant lentiviral vector, pseudotyped with an envelope protein of another virus. 
     
     
         28 . Composition according to  claim 27 , characterized in that said envelope protein is the vesicular stomatitis virus glycoprotein G. 
     
     
         29 . Composition according to  claim 26 , characterized in that it comprises an isolated nucleic acid molecule corresponding to the recombinant genome of said 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 and termination of lentiviral origin and, optionally, the regulatory sequences for the Rev protein 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 in any one of  claims 1  and  6  to  15 . 
     
     
         30 . Cells, preferably eukaryotic cells, modified with a recombinant lentiviral vector as defined in  claims 1  to  23 . 
     
     
         31 . Method for producing viral proteins of Flaviviridae and/or immunogenic fragments of said proteins or else viral pseudoparticles, characterized in that it comprises at least the following steps:
 a) culturing modified cells according to  claim 30 , under conditions which allow the expression of one or more viral proteins of Flaviviridae and/or of one or more of the immunogenic fragments of said proteins encoded by said recombinant lentiviral vector, and   b) separating said proteins, protein fragments or viral pseudoparticles from the culture supernatant or from the cells in a).   
     
     
         32 . Method for screening antiviral compounds, characterized in that it comprises:
 bringing modified eukaryotic cells according to  claim 30 , in particular eukaryotic cells modified with a vector comprising a cDNA encoding a non-structural protein of Flaviviridae such as NS3 or NS5, into contact with various compounds of a library to be tested,   measuring the activity of said protein, in the presence or in the absence of said compounds, and   selecting the compounds capable of modulating said activity.   
     
     
         33 . Method according to  claim 32 , characterized in that the eukaryotic cells are selected from the group consisting of dendritic cells, neuronal cells and hepatocytes. 
     
     
         34 . 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) bringing said biological sample into contact with modified eukaryotic cells expressing at least one Flaviviridae antigen, as defined in  claim 30 , and   b) revealing the antigen-antibody complexes formed in (a).   
     
     
         35 . Method according to  claim 34 , characterized in that said modified eukaryotic cells in a) are permeabilized. 
     
     
         36 . Method for diagnosing an 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) bringing said biological sample into contact with viral pseudoparticles produced from the culture supernatant of cells modified with a lentiviral vector expressing at least one membrane protein and/or envelope protein as defined in any one of  claims 7 ,  9  to  12 ,  15  to  18  and  21  to  23 , and   b) revealing the antigen-antibody complexes formed in (a).   
     
     
         37 . Kit for carrying out the methods according to  claims 31  to  36 , characterized in that it comprises at least modified cells according to  claim 30 .