A Fusion Protein Designed with Segments of Different Allergens of Blomia Tropicalis

Abstract

S A T U R D A Y 36 Functional Characterization of A Cis-Acting Temperature Sensitive Mutation From A Respiratory Syncytial Virus Vaccine Candidate Michael Teng, David J. Fitzhugh, MD, Kim Tran, Richard F. Lockey, MD, Peter Collins; University of South Florida Morsani College of Medicine, Tampa, FL, NIAID, Bethesda, MD, James A. Haley Veterans’ Hospital, Tampa, FL. RATIONALE: The current respiratory syncytial virus (RSV) vaccine candidate in clinical trials (MEDI-559) incorporates a number of point mutations that confer temperature sensitivity to the virus. One of these mutations (ts404-M2) is unique in that it is present in a transcription regulatory sequence rather than an open reading frame. Therefore, these studies were designed to elucidate the mechanism by which this mutation causes temperature sensitivity. METHODS: We examined the effect of this mutation on the temperature sensitivity of viral mRNA expression by QRT-PCR and viral replication by bothplaque formationandmultiple step replicationassaysat permissive (32C) andnon-permissive (37C) temperatures.We alsogenerated recombinantRSV that incorporated the ts404-M2 at different positionswithin theviral genome to examine the positional and contextual effects on temperature sensitivity. RESULTS:We found that ts404-M2 caused global temperature sensitivity of viral transcription and replication. The ts404-M2 mutation also conferred temperature sensitivity when imported into other positions in the viral genome. This effect is not sequence specific, but requires wildtype sequence context. CONCLUSIONS: These data indicate that temperature sensitivity due to ts404-M2 is the result of global down-regulation of viral transcription. This phenotype is not position-dependent nor does it require a specific sequence context. Similar mutation of transcription signals may also result in temperature sensitivity in related viruses, resulting in a more universal mechanism of developing attenuated vaccine candidates.