Quantitative determination of the infectivity of the proviral DNA of a retrovirus in vitro: Evaluation of methods for DNA inactivation

• Published in: Biologicals 2009-08, Vol.37 (4), p.259-269
• Main Authors: Sheng-Fowler, Li, Lewis, Andrew M., Peden, Keith
• Format: Article
• Language: English
• Subjects: AIDS Vaccines - genetics; Cells, Cultured; Cloning, Molecular; Disinfectants - pharmacology; DNA inactivation; DNA infectivity; DNA, Superhelical - physiology; DNA, Viral - analysis; DNA, Viral - isolation & purification; DNA, Viral - metabolism; DNA, Viral - physiology; Endodeoxyribonucleases - metabolism; Endoribonucleases - metabolism; HIV Infections - genetics; HIV-1 - genetics; HIV-1 - pathogenicity; Human immunodeficiency virus; Humans; Jurkat Cells; Propiolactone - pharmacology; Retroviridae - genetics; Retroviridae - pathogenicity; Retroviridae Infections - prevention & control; Vaccine cell substrates; Virus Inactivation
• ISSN: 1045-1056; 1095-8320
• DOI: 10.1016/j.biologicals.2009.04.002

All viral vaccines contain contaminating residual DNA derived from the production cell substrate. The potential risk of this DNA, particularly when derived from tumorigenic cells, has been debated for over 40 years. While the major risk has been considered to be the oncogenicity of the DNA, another potential risk is that a genome of an infectious virus is present in this DNA. Such a genome might generate an infectious agent that could establish an infection in vaccine recipients. To determine the quantity of a retroviral provirus in cellular DNA that can establish a productive infection in vitro, we developed a transfection/co-culture system capable of recovering infectious virus from 1 pg of cloned HIV DNA and from 2 μg of cellular DNA from HIV-infected cells. We demonstrate that infectivity can be reduced to below detectable levels either by lowering the median size of the DNA to 350 base pairs or by treatment with β-propiolactone. From the amount of reduction of infectivity, we calculate that clearance values in excess of 10 7 are attainable with respect to the infectivity associated with residual cell-substrate DNA. Thus, the potential risk associated with DNA can be substantially reduced by degradation or by chemical inactivation.