414. Vitamin B2: A Key Component for Controlled Inactivation of Viruses Suitable for Biological Use
Authors: Shellie M. Callahan, Piyanuch Wonganan, Linda J. Obenauer-Kutner, Suganto Sutjipto, Hong T. Le, Maria A. Croyle
Journal: Molecular Therapy (2005)

Inactivated recombinant adenoviruses (Ads) are important controls for both immunological and analytical studies. The process of inactivation however needs to be considered as it may contribute to alterations in a biological system. Previous studies have shown that Ad inactivation can be achieved with the combined treatment of 8-methoxypsoralen (0.33 |[mu]|g/|[mu]|l) and long-wave length ultraviolet irradiation (PUVA). Although PUVA effectively inactivates Ad, an alternative method of inactivation was needed in order to identify vector components that contribute to changes in hepatic drug metabolism. Cytochromes P450 (CYP), an enzyme system responsible for hepatic drug metabolism, convert psoralens to reactive metabolites, which in turn alter various CYP enzymes. This effect is so striking that subjects in clinical trials must refrain from eating certain foods containing psoralens to prevent extraneous changes in drug levels. In addition, the efficiency of psoralen removal was hindered by the fact that it absorbs light within the same range as Ad. In this report, riboflavin (vitamin B2), a molecule with over 70 years of research detailing its chemistry, toxicology and in vitro ability to sensitize the photochemistry of nucleic acids, was selected as an appropriate compound for inactivation of Ad. First generation Ad expressing E. coli beta-galactosidase (AdlacZ) was mixed with riboflavin (final conc. 50 |[mu]|M) and exposed to long-wave length ultraviolet irradiation (365 nm) for 1.5 hours. AdlacZ was also treated with PUVA and UV alone to compare the effectiveness of the Ribo-UV treatment in inactivating the virus. Samples were taken and titered by limiting dilution at timepoints ranging from 30 seconds to 1.5 hours after UV exposure. DMSO (100%) was used as a diluent for both riboflavin and psoralen, the final volume with which was 3% of the viral preparation. A MTS assay confirmed that this concentration of DMSO was not cytotoxic. Capsid integrity was confirmed by electron microscopy. The kinetics of inactivation were slower with Ribo-UV treatment with respect to PUVA. Ten minutes after UV exposure there was a 2-log drop in Ribo-UV AdlacZ and a 4-log decline in PUVA AdlacZ, as compared to pre-UV titers. At 30 minutes the Ribo-UV treated AdlacZ titer had decreased by 6-logs whereas the PUVA AdlacZ titer had decreased by 9-logs. Forty-five minutes of UV exposure resulted in complete inactivation of both treatment groups. The Ribo-UV preparation was well tolerated in rats given a dose of 5.7|[times]|1012 viral particles/kg as AST and ALT levels were not altered over 14 days after administration. Transgene expression was absent in liver sections from animals treated this preparation. The data presented here show that adenoviral vectors can be inactivated by treatment with riboflavin and UV exposure. The use of vitamin B2 for viral inactivation may be useful in physiological systems sensitive to other photosensitizers and analytical applications where intact capsids are required. Inactivation of lentiviral and adeno-associated viral vectors will also be discussed.