Attenuated oncolytic paramyxoviruses encoding avian cytokines
A cytokine and virus technology, applied in the direction of cytokines/lymphokines/interferons, viruses, sexual diseases, etc., to achieve the effect of reducing pathogenicity
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Embodiment 1
[0231] Generation of recombinant NDVs with avian-derived transgenes that cause chicken cytokine expression
[0232] Viral RNA was obtained using the oncolytic strain MTH68 of NDV. Using RT-PCR, several cDNA fragments were obtained and assembled in a multi-step cloning method into full-length genomic cDNA, which was cloned into the vector pX8δT (Schnell et al., 1994), resulting in plasmid pflMTH68. This vector can be used for transfection to rescue recombinant virus from cell lines expressing T7-polymerase.
[0233] An additional transcription cassette was cloned into a single Sfil restriction site between the genes encoding the F and HN proteins in the NDV MTH68 full-length genomic plasmid (pflMTH68). Two DNA oligonucleotides, Sfi forward (5'-aggccttaattaaccgacaacttaagaaaaaatacgggtagaacggcctgag-3', SEQ ID NO: 1) and Sfi reverse (5'-aggccgttctacccgtattttttcttaagttgtcggttaattaaggcctctc-3', SEQ ID NO: 2) were renatured and subsequently ligated to into the SfiI site of pflMTH68....
Embodiment 2
[0237] ChIFN-α and ChIFN-β expressed from recombinant NDV are biologically active
[0238] Materials and Methods:
[0239] CEC-32 cells (avian quail cell line) or Hela cells (cervical cancer cell line) were plated at 1x10 5 cells / well were plated in 6-well plates. After cell attachment, cells were infected with GFP-expressing control viruses NDV-GFP, NDV-ChIFN-alpha, NDV-ChIFN-beta or mock virus using an MOI of 0.01. After 64 hours, the supernatant was harvested and infectious viral particles were inactivated by UV irradiation.
[0240] To show the biological activity of ChIFN-α or ChIFN-β in supernatants of virus-infected cells, a chicken interferon-specific bioassay was used (Schwarz et al., JICR, 2005). The assay is based on the stably transfected quail cell line CEC-511, which carries the luciferase gene under the control of the IFN-responsive chicken Mx promoter. Luciferase activity was induced when CEC-511 indicated cells were incubated with ChIFN-α or ChIFN-β. To c...
Embodiment 3
[0248] Avian cells but not tumor cells infected with recombinant NDV expressing ChIFN-α or ChIFN-β are protected from viral lysis
[0249] Materials and Methods:
[0250] 3a.) CEC-32 cells (avian quail cell line) or Hela cells (cervical cancer cell line) were treated with 1×10 5 cells / well were plated in 6-well plates. After becoming adherent, cells were infected with the control virus NDV-GFP, NDV-ChIFN-alpha, NDV-ChIFN-beta or mock virus at an MOI of 0.01. After 48 hours, cells were fixed with 4% formaldehyde solution and stained with Giemsa solution. Subsequent photodocumention was then performed with a Zeiss Axiophot imaging system.
[0251] 3b.) CEC-32 cells (avian quail cell line) or Hela cells (cervical cancer cell line) were plated at 15,000 cells / well in a 96-well plate. After becoming adherent, cells were infected with control virus NDV-GFP, NDV-ChIFN-α, NDV-ChIFN-β or mock virus (=none) at MOI of 0.01. After a 48-hour incubation time, proceed to CellTiter- Lu...
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