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
Bats are reservoir hosts for a spectrum of infectious diseases. Some pathogens (such as Hendra, Nipah and Marburg viruses) appear to use mainly fruit bats as reservoir. We describe designed immortalization of primary fetal cells from the Egyptian fruit bat (Rousettus aegyptiacus) to facilitate isolation and characterization of pathogens associated with these mammals. Three cell lines with different properties were recovered and successful immortalization was confirmed by continuous cultivation for over 18 months. Surprisingly, the cell lines are fully permissive for a highly attenuated poxvirus, modified vaccinia Ankara (MVA). MVA is a safe and well characterized vaccine vector that cannot replicate in most mammalian cells. High permissivity of Rousettus cell lines could justify testing bats for susceptibility to MVA as a replication competent vector with low zoonotic potential to induce herd immunity in bat colonies against viruses causing rabies or haemorrhagic fevers.
Assuntos
Linhagem Celular , Quirópteros , Vaccinia virus/fisiologia , Replicação Viral , Animais , Chlorocebus aethiops , Cricetinae , Patos , Feminino , Feto/citologia , Vetores Genéticos , Proteínas de Fluorescência Verde , Humanos , Vacínia/virologia , Células VeroRESUMO
Post-translational processes are essential for the generation and dynamics of mammalian circadian rhythms. In particular, phosphorylation of the key circadian protein PER2 precisely controls the period and phase of circadian oscillations. However, the mechanisms underlying that control are poorly understood. Here, we identified in a high-throughput RNAi-based genetic screen casein kinase 2 (CK2) as a PER2-phosphorylating kinase and novel component of the mammalian circadian clock. When CK2 subunits are silenced by RNAi or when CK2 activity is inhibited pharmacologically, circadian rhythms are disrupted. CK2 binds to PER2 in vivo, phosphorylates PER2 specifically at N-terminal residues in vitro, and supports normal nuclear PER2 accumulation. Mutation of CK2 phosphorylation sites decreases PER2 stability and copies CK2 inhibition regarding oscillation dynamics. We propose a new concept of how PER2 phosphorylation and stabilization can set the clock speed in opposite directions, dependent on the phase of action.