Method for rescuing and producing a virus in avian cells
The present invention relates to methods of rescue and/or propagation of paramyxovirus species, particularly wherein both rescue and propagation are carried out in the same cell type; i.e., without the use of helper cells for viral rescue. The paramyxoviruses produced by the disclosed methods may encompass wild-type viruses, chimeric viruses, recombinant viruses or engineered viral products such as virus like particles (VLP). Viruses and/or viral products produced in the method according to the current invention are suitable for medical or veterinary use in such applications as treating or preventing infectious diseases, particularly avian paramyxovirus and human respiratory virus infections, and cancer treatment.
1. A method of producing infectious paramyxovirus particles comprising the steps of
a) transfecting a duck cell line with
(i) a vector comprising a paramyxovirus nucleic acid sequence under T7 control, wherein said vector comprising a paramyxovirus nucleic acid sequence under T7 control additionally comprises a T7 promotor sequence as defined by SEQ ID NO: 6 and a T7 terminator sequence as defined by SEQ ID NO: 7;
(ii) a T7 RNA polymerase expression vector; and
(iii) three further expression vectors comprising, respectively, paramyxovirus phosphoprotein (P), nucleoprotein (N), and polymerase (L) coding sequences; and
b) culturing said transfected duck cell line under conditions for production of infectious paramyxovirus particles.
2. The method of claim 1 , wherein said T7 RNA polymerase expression vector and three further expression vectors are under control of a constitutive promoter.
3. The method of claim 1 , wherein the infectious paramyxovirus is a wild-type paramyxovirus, a chimeric paramyxovirus, or a recombinant paramyxovirus.
4. The method of claim 1 , wherein the paramyxovirus nucleic acid sequence is modified to contain one or more restriction enzyme sites for insertion of a heterologous coding sequence located between the paramyxovirus protein coding sequences.
5. The method of claim 1 , wherein the paramyxovirus nucleic acid sequence contains coding sequences for one or more foreign antigens.
6. The method of claim 1 , wherein said paramyxovirus nucleic acid sequence contains coding sequences for one or more foreign antigens selected from the group consisting of antigens from viral pathogens, oncolytic proteins, and immunomodulating proteins.
7. The method of claim 1 , wherein said paramyxovirus nucleic acid sequence contains coding sequences for one or more human metapneumovirus (hMPV) antigen and/or respiratory syncytial virus (RSV) antigen.
8. The method of claim 1 , wherein said paramyxovirus nucleic acid sequence contains coding sequences for fusion (F) protein and/or matrix (M) protein from human metapneumovirus (hMPV) and/or respiratory syncytial virus (RSV).
9. The method of claim 8 , wherein said F and M proteins assemble to produce hMPV or RSV virus-like particles.
10. The method according to claim 8 , wherein said F protein from hMPV or RSV is a wild-type F protein, a modified F protein, an F protein in pre-fusion conformation, or an F protein in a post-fusion conformation.
11. The method according to claim 8 , wherein said hMPV F protein is selected from the group consisting of SEQ ID NOs: 17-20 and immunogenic variants having at least 95% sequence identity to any one of SEQ. ID NOs: 17-20.
12. The method of claim 1 , wherein the paramyxovirus nucleic acid is a Newcastle Disease Virus (NDV) nucleic acid, an NDV LaSota strain nucleic acid, an NDV Hitchner 131 strain nucleic acid, or an NDV AE2240 strain nucleic acid.
13. The method of claim 1 , wherein the paramyxovirus nucleic acid encodes a wild-type Newcastle Disease Virus (NDV) genome, a chimeric NDV genome, a recombinant NDV genome, or a virus-like particle comprising NDV elements.
14. The method of claim 13 , wherein the recombinant NDV genome is defined by SEQ ID NO: 16.
15. The method of claim 2 , wherein the constitutive promoter is a cytomegalovirus (CMV) promoter.