Semple, Malcolm Gracie; (1995) A culture-independent method for the measurement of cell-free HIV1. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Investigations of the pathogenesis of the human immunodeficiency viruses and examination of the efficacy of putative therapies have been hampered by the lack of efficient methods for the quantification and analysis of circulating virus. This thesis details the development of novel molecular techniques for the rescue and quantification of cell-free human immunodeficiency virus type 1 (HIV-1) which are suitable for the investigation of large numbers of patient samples. The new techniques include: for the extraction of cell-free HIV-1; precipitation using polyethylene glycol and capture by immunoadhesion to latex microparticles, synthesis of specific HIV-1 cDNA by site-directed reverse transcription prior to amplification and quantification by end-point dilution in nested PCR and a standardised quantitative PCR. Cell-free HIV-1 was detected in all patients infected with HIV-1 as determined by the presence of HIV-1 specific antibody. In cross-sectional studies, quantification of cell-free HIV-1 demonstrated that viraemia increased with progression from asymptomatic to symptomatic disease and the relationships between HIV-1 viraemia and indirect markers of viraemia were investigated. In longitudinal studies of patients treated with zidovudine, quantification of HIV-1 viraemia demonstrated rapid 10 to 1000 fold decreases in viraemia. Suppression of viraemia was not maintained by uninterrupted zidovudine therapy and individual viraemia levels returned towards pretreatment levels within three months. HIV-1 genome rescued by these techniques is suitable for further genetic analysis. When used in conjunction with an assay which determines the prevalence of point mutations associated with zidovudine resistance, an increase in the prevalence of these mutations was associated with the return of viraemia to pretreatment levels. These methods have proved reliable and precise when applied to the study of antiretroviral therapy. The use of viraemia as a laboratory marker in drug studies to determine therapeutic efficacy is proposed.

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