Evaluation of Reverse Transcriptase Assay for Viral Load Monitoring

Retaining an active reverse transcriptase (RT) enzyme is a fundamental requirement for all retroviruses to replicate. Bearing in mind that HIV has a very high propensity to mutate measuring RT activity to determine the level of retroviral replication has the capacity to eliminate problems associated with divergence, as the virus at all costs must retain an active RT enzyme. We show that two p24 antigen assays utilizing different p24 capture antibodies quantify HIV-1 replication inadequately. We also documented the development (version 1 and 2) and use of a RT assay for the quantification of viral load in HIV infected individuals and SIV/SHIV infected macaques. Finally, we defined RT-fitness as the ratio of HIV-1 RT activity/RNA (fg RT/1000 RNA copies) in an attempt to determine if mutations associated with ARV therapy alter the fitness of the RT enzyme. Our results showed that the RT assay strongly correlated with conventional methods for viral load determination of both HIV in humans and SIV/SHIV in macaques. Trends in RT-load and RNA-load mirrored each other even under ARV therapy, indicating that both assays quantified the same fundamental process of viral replication, even though they measured two different replication markers. Regarding RT-fitness, the NRTI resistance mutation T215Y was associated with reduced RT-fitness, while acquisition of L74V in viruses already containing T215Y increased the RT-fitness. To conclude, the RT assay documented in this thesis should be considered as a viable alternative for viral load monitoring, particularly in regions where expensive and complex gene-based technologies are not a viable option. Furthermore, RT-load may have the potential to add a further virological fitness dimension to viral load measurement and should be further investigated