RESUMO
Highly attenuated poxviruses are promising vectors for protective and therapeutic vaccines. These vectors do not replicate in human cells and can therefore be safely given even to immunocompromised recipients. They can accommodate very large inserts and provide strong stimulation of the immune system against the vectored antigen. Disadvantages include that very high numbers of infectious units are required per dose for full efficacy. Because they are difficult to produce, improved cellular substrates and processes are urgently needed to facilitate programs intended to reach a large number of vaccinees. We have developed a fully scalable and very efficient chemically-defined production process for modified vaccinia Ankara (MVA), canarypox (CNPV, strain ALVAC) and fowlpox viruses (FPV) based on a continuous cell line.
Assuntos
Vetores Genéticos/genética , Poxviridae/genética , Animais , Reatores Biológicos , Células CHO , Vírus da Varíola dos Canários/genética , Vírus da Varíola dos Canários/imunologia , Linhagem Celular , Proliferação de Células , Cricetinae , Cricetulus , Vírus da Varíola das Aves Domésticas/genética , Vírus da Varíola das Aves Domésticas/imunologia , Vetores Genéticos/imunologia , Humanos , Poxviridae/imunologia , Vacinas Atenuadas/imunologia , Vaccinia virus/genética , Vaccinia virus/imunologia , Vacinas Virais/imunologia , Replicação Viral/genéticaRESUMO
A new method for sampling suspended animal cells by fast filtration is presented that allows rapid quenching of cellular metabolism and efficient separation of the cells from culture medium. Compared to sampling with a microstructure heat exchanger or centrifugation without prior quenching, the adenylate energy charge and the measured concentrations especially of metabolites with a high turnover rate or of metabolites early in metabolic pathways were substantially higher. No leakage of ATP from the cells was observed when using iso-osmotic NaCl solution in the washing step. The combination of fast filtration and cold methanol extraction is therefore suitable for intracellular metabolomic studies of suspended animal cell cultures and superior to other methods currently applied.