Comparison of egg and high yielding MDCK cell-derived live attenuated influenza virus for commercial production of trivalent influenza vaccine: in vitro cell susceptibility and influenza virus replication kinetics in permissive and semi-permissive cells.

Currently MedImmune manufactures cold-adapted (ca) live, attenuated influenza vaccine (LAIV) from specific-pathogen free (SPF) chicken eggs. Difficulties in production scale-up and potential exposure of chicken flocks to avian influenza viruses especially in the event of a pandemic influenza outbreak have prompted evaluation and development of alternative non-egg based influenza vaccine manufacturing technologies. As part of MedImmune's effort to develop the live attenuated influenza vaccine (LAIV) using cell culture production technologies we have investigated the use of high yielding, cloned MDCK cells as a substrate for vaccine production by assessing host range and virus replication of influenza virus produced from both SPF egg and MDCK cell production technologies. In addition to cloned MDCK cells the indicator cell lines used to evaluate the impact of producing LAIV in cells on host range and replication included two human cell lines: human lung carcinoma (A549) cells and human muco-epidermoid bronchiolar carcinoma (NCI H292) cells. The influenza viruses used to infect the indicators cell lines represented both the egg and cell culture manufacturing processes and included virus strains that composed the 2006-2007 influenza seasonal trivalent vaccine (A/New Caledonia/20/99 (H1N1), A/Wisconsin/67/05 (H3N2) and B/Malaysia/2506/04). Results from this study demonstrate remarkable similarity between influenza viruses representing the current commercial egg produced and developmental MDCK cell produced vaccine production platforms. MedImmune's high yielding cloned MDCK cells used for the cell culture based vaccine production were highly permissive to both egg and cell produced ca attenuated influenza viruses. Both the A549 and NCI H292 cells regardless of production system were less permissive to influenza A and B viruses than the MDCK cells. Irrespective of the indicator cell line used the replication properties were similar between egg and the cell produced influenza viruses. Based on these study results we conclude that the MDCK cell produced and egg produced vaccine strains are highly comparable.

Regulation of mitosis in response to damaged or incompletely replicated DNA require different levels of Grapes (Drosophila Chk1).

Checkpoints monitor the state of DNA and can delay or arrest the cell cycle at multiple points including G1-S transition, progress through S phase and G2-M transition. Regulation of progress through mitosis, specifically at the metaphase-anaphase transition, occurs after exposure to ionizing radiation (IR) in Drosophila and budding yeast, but has not been conclusively demonstrated in mammals. Here we report that regulation of metaphase-anaphase transition in Drosophila depends on the magnitude of radiation dose and time in the cell cycle at which radiation is applied, which may explain the apparent differences among experimental systems and offer an explanation as to why this regulation has not been seen in mammalian cells. We further document that mutants in Drosophila Chk1 (Grapes) that are capable of delaying the progress through mitosis in response to IR are incapable of delaying progress through mitosis when DNA synthesis is blocked by mutations in an essential replication factor encoded by double park (Drosophila Cdt1). We conclude that DNA damage and replication checkpoints operating in the same cell cycle at the same developmental stage in Drosophila can exhibit differential requirements for the Chk1 homolog. The converse situation exists in fission yeast where loss of Chk1 is more detrimental to the DNA damage checkpoint than to the DNA replication checkpoint. It remains to be seen which of these two different uses of Chk1 homologs are conserved in mammals. Finally, our results demonstrate that Drosophila provides a unique opportunity to study the regulation of the entry into, and progress through, mitosis by DNA structure checkpoints in metazoa.