Replication deficient influenza virus for the expression of heterologous sequences
View Patent ↗The present invention relates to 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 having a heterologous sequence inserted between the splice donor site and the splice acceptor site of the NS gene segment. The virus can be used as vector for expression of various proteins like chemokines, cytokines or antigenic structures and to produce vaccines. 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 also provided.
1. A replication deficient influenza virus, characterized in that the virus 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 a complete lack of functional RNA binding and at least one amino acid modification between position 74 and the carboxy-terminal amino acid residue resulting in a complete lack of effector function, and
b) a heterologous sequence between a functional splice donor site and a functional splice acceptor site inserted in the NS gene segment,
wherein the virus comprises a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:78, and SEQ ID NO:79.
2. A replication deficient influenza virus according to claim 1 , further comprising a signal peptide or part thereof fused to the C-terminus of NS1 protein.
3. A replication deficient influenza virus according to claim 1 , wherein the virus further comprises a lariat consensus sequence upstream of the splice acceptor site.
4. A replication deficient influenza virus according to claim 1 , wherein the virus is formulated as a vaccine.
5. A vector comprising a nucleotide sequence coding for a replication deficient influenza virus according to claim 1 .
6. A replication deficient influenza virus according to claim 1 , wherein the virus comprises SEQ ID NO:1.
7. A replication deficient influenza virus according to claim 1 , wherein the virus comprises SEQ ID NO:2.
8. A replication deficient influenza virus according to claim 1 , wherein the virus comprises SEQ ID NO:3.
9. A replication deficient influenza virus according to claim 1 , wherein the virus comprises SEQ ID NO:4.
10. A replication deficient influenza virus according to claim 1 , wherein the virus comprises SEQ ID NO:5.
11. A replication deficient influenza virus according to claim 1 , wherein the virus comprises SEQ ID NO:6.
12. A replication deficient influenza virus according to claim 1 , wherein the virus comprises SEQ ID NO:7.
13. A replication deficient influenza virus according to claim 1 , wherein the virus comprises SEQ ID NO:8.
14. A replication deficient influenza virus according to claim 1 , wherein the virus comprises SEQ ID NO:9.
15. A replication deficient influenza virus according to claim 1 , wherein the virus comprises SEQ ID NO:10.
16. A replication deficient influenza virus according to claim 1 , wherein the virus comprises SEQ ID NO:67.
17. A replication deficient influenza virus according to claim 1 , wherein the virus comprises SEQ ID NO:68.
18. A replication deficient influenza virus according to claim 1 , wherein the virus comprises SEQ ID NO:78.
19. A replication deficient influenza virus according to claim 1 , wherein the virus comprises SEQ ID NO:79.