Generating Recombinant Live-Attenuated Influenza Viruses for Vaccines
Transcrição
Begin with bi-directional plasmids carrying cDNA encoding certain viral essential proteins of a cold-adapted influenza strain.
Introduce a new set of bi-directional plasmids containing hemagglutinin and modified neuraminidase cDNA from a different influenza strain.
Introduce a transfection reagent to this mix and incubate to form a complex with the plasmids.
Transfer these complexes onto cultured human epithelial-like cells to enable plasmid entry into the cell.
The bi-directional plasmids with two promoters in opposite orientations enable the production of viral proteins and their corresponding RNA.
Incubate the cells at a lower temperature.
The viral proteins and the viral RNAs self-assemble to form cold-adapted recombinant influenza viruses with reduced replication in warmer conditions.
Overlay with sialic acid-overexpressing MDCK cells and modified trypsin enzymes that allow viral attachment and entry inside cells.
Incubate at the same temperature to induce replication and production of new viruses.
Centrifuge and harvest the recombinant live-attenuated influenza viruses ready for a vaccine preparation.
