Replication deficient influenza virus for the expression of heterologous sequences

Abstract

The present invention covers a novel replication deficient influenza virus comprising a modified NS1 segment coding for a NS1 protein lacking a functional RNA binding domain and functional effector domain and a heterologous sequence inserted between the splice donor site and the splice acceptor site of the NS gene segment. Further the use of the virus as vector for expression of various proteins like chemokines, cytokines or antigenic structures is covered, methods for producing virus particles using said virus vector as well as its use for production of vaccines. Also a fusion peptide comprising part of the N-terminus of an NS1 protein and a signal sequence fused to the C-terminus of said NS1 peptide is covered.

1 . Replication deficient influenza virus characterized in that it comprises
 a) a modified NS segment coding for a NS1 protein comprising at least one amino acid modification within positions 1 to 73 resulting in complete lack of its functional RNA binding and at least one amino acid modification between position 74 and the carboxy-terminal amino acid residue resulting in complete lack of its effector function and   b) a heterologous sequence between a functional splice donor site and functional splice acceptor site inserted in the NS gene segment   
     
     
         2 . Replication deficient influenza virus according to  claim 1  characterized in that it comprises at least 10 amino acids and preferably up to 14, preferably up to 30 amino acids of the N-terminus of the NS1 protein. 
     
     
         3 . Replication deficient influenza virus according to any one of  claim 1  or  2  characterized in that amino acids 134 to 161 are deleted. 
     
     
         4 . Replication deficient influenza virus according to any one of  claim 1  or  2  characterized in that amino acids 117 to 161 are deleted. 
     
     
         5 . Replication deficient influenza virus according to any one of  claims 2  to  4  comprising a signal peptide or part thereof fused to the C-terminus of NS1 protein. 
     
     
         6 . Replication deficient influenza virus according to any one of  claims 1  to  5  characterized in that the heterologous sequence is expressed from the NS1 open reading frame. 
     
     
         7 . Replication deficient influenza virus according to any one of  claims 1  to  5  characterized in that the heterologous sequence is expressed from a separate open reading frame. 
     
     
         8 . Replication deficient influenza virus according to any one of  claims 1  to  7  characterized in that the heterologous sequence is selected from the group consisting of biologically active proteins, antigens or derivatives or fragments thereof. 
     
     
         9 . Replication deficient influenza virus according to any one of  claims 1  to  8  characterized in that the heterologous sequence is a chemokine or cytokine or derivative or fragment thereof. 
     
     
         10 . Replication deficient influenza virus according to any one of  claims 1  to  9  characterized in that the heterologous sequence is derived from mycobacterium tuberculosis. 
     
     
         11 . Replication deficient influenza virus according to any one of  claims 1  to  10  characterized in that the heterologous sequence comprises a signal peptide. 
     
     
         12 . Replication deficient influenza virus according to  claim 11  characterized in that the signal peptide is derived from an antibody light chain, preferably from an Ig kappa chain, more preferably from mouse Ig kappa chain. 
     
     
         13 . Replication deficient influenza virus according to  claim 12  characterized in that the Ig kappa signal peptide comprises at least 10 amino acids, more preferred at least 12 amino acids. 
     
     
         14 . Replication deficient influenza virus according to any one of  claim 12  or  13  characterized in that the Ig kappa signal peptide comprises the sequence METDTLLLWVLLLWVPGSTGD (SEQ ID No. 11) or METDTLLLWVLLLWVPRSHG (SEQ ID No. 82) or part or derivatives thereof. 
     
     
         15 . Replication deficient influenza virus according to any one of  claims 1  to  14  characterized in that the heterologous sequence comprises a fusion protein of a biologically active protein and an antigen. 
     
     
         16 . Replication deficient influenza virus according to any one of  claims 1  to  15  characterized in that the heterologous sequence is selected from the group consisting of IL2, GM-CSF, IL15, MIP 1 alpha and MIP 3 alpha, ESAT-6 or a derivative or fragment thereof. 
     
     
         17 . Replication deficient influenza virus according to any one of  claims 1  to  16  characterized in that the translation of said NS1 protein is terminated by at least one STOP codon and expression of said heterologous sequence is reinitiated by a START codon. 
     
     
         18 . Replication deficient influenza virus according to any one of  claims 1  to  17  characterized in that the heterologous open reading frame is at least partially overlapping with the NS1 open reading frame. 
     
     
         19 . Replication deficient influenza virus according to any one of  claims 1  to  18  characterized in that translation of the heterologous open reading frame is initiated from an overlapping STOP/START codon sequence. 
     
     
         20 . Replication deficient influenza virus according to  claim 19  characterized in that the overlapping START/STOP codon is TAATG (SEQ ID. No. 81) or UAAUG (SEQ ID No. 53). 
     
     
         21 . Replication deficient influenza virus according to any one of  claims 1  to  20  characterized in that translation of the heterologous open reading frame is initiated from an optimized translation initiation sequence, preferably a Kozak consensus sequence. 
     
     
         22 . Replication deficient influenza virus according to any one of  claims 1  to  21  containing an altered sequence downstream of the splice donor site and/or upstream of the splice acceptor site in the NS segment. 
     
     
         23 . Replication deficient influenza virus according to any one of  claims 1  to  22  characterized in that the heterologous sequence is secreted from the infected cell. 
     
     
         24 . Replication deficient influenza virus according to any one of  claims 1  to  23  with a sequence as shown in SEQ ID Nos 1 to 10, 67, 68 or with at least 98% homology therewith. 
     
     
         25 . Combination of at least two replication deficient influenza viruses according to any one of  claims 1  to  24  comprising at least one biologically active molecule or derivative or fragment thereof and at least one antigenic structure. 
     
     
         26 . Vaccine comprising a replication deficient influenza virus according to any one of  claims 1  to  24 . 
     
     
         27 . Use of a replication deficient influenza virus according to any one of  claims 1  to  24  for the preparation of a medicament for therapeutic treatment in patients. 
     
     
         28 . Use of a replication deficient influenza virus according to any one of  claims 1  to  24  for the preparation of a medicament for the tumor treatment in patients. 
     
     
         29 . Vector comprising a nucleotide sequence coding for a replication deficient influenza virus according to any one of  claims 1  to  24 . 
     
     
         30 . Method for producing a replication deficient influenza virus according to any one of  claims 1  to  24 , comprising the steps of:
 transfecting cells, preferably Vero cells, with at least one vector according to  claim 29 , incubating the transfected cells to allow for the development of viral progeny containing the heterologous protein.   
     
     
         31 . Method for producing a replication deficient influenza virus according to any one of  claims 1  to  24 , comprising the steps of:
 transforming a cell, preferably a Vero cell, with a vector according to  claim 29  preferably together with a purified preparation of influenza virus RNP complex, infecting the selected cells with an influenza helper virus, incubating the infected cells to allow for the development of viral progeny and selecting transformed cells that express the modified NS gene and the heterologous sequence,   
     
     
         32 . Method for the creation of a replication deficient influenza virus according to any one of  claims 1  to  24 , comprising the steps of:
 transfecting a cell line, preferably a Vero cell line, with a DNA vector comprising a modified NS gene free of functional RNA binding domain and a heterologous sequence inserted between a functional splice donor site and the splice acceptor site of the NS gene,   selecting transfected cells that express the modified NS gene and the heterologous sequence,   infecting the selected cells with a desired influenza virus,   incubating the infected cells to allow for the development of viral progeny containing the heterologous protein,   selecting and harvesting said viral progeny containing the heterologous protein.   
     
     
         33 . The method according to  claim 29 , characterized in that the DNA vector is a transcription system for minus sense influenza RNA. 
     
     
         34 . The method according to  claim 29  or  30 , characterized in that said viral progeny is further combined with a pharmaceutically acceptable carrier for use as a vaccine. 
     
     
         35 . Fusion protein comprising between 10 and 30 amino acids of the N-terminus of an NS1 protein, a heterologous sequence and a signal peptide fused to the C-terminus of said NS1 peptide. 
     
     
         36 . Fusion protein according to  claim 32  characterized in that the signal peptide consists of 8-50 amino acids. 
     
     
         37 . Fusion protein according to any one of  claim 32  or  33  characterized in that the signal peptide is derived from an antibody light chain, preferably from an Ig kappa chain, more preferably from mouse Ig kappa chain or a derivative thereof. 
     
     
         38 . Fusion protein according to any one of  claims 35  to  37  characterized in that the Ig kappa signal peptide comprises at least 10 amino acids, more preferred at least 12 amino acids. 
     
     
         39 . Fusion protein according to any one of  claims 35  to  38  characterized in that the Ig kappa signal peptide comprises the sequence METDTLLLWVLLLWVPGSTGD (SEQ ID No. 11) or METDTLLLWVLLLWVPRSHG (SEQ ID No. 82) or part or derivatives thereof.