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.

List of scientific papers

I. Corrigan GE, Al-Khalili L, Malmsten A, Thorstensson R, Fenyo EM, Kallander CF, Gronowitz JS. (1998). Differences in reverse transcriptase activity versus p24 antigen detection in cell culture, when comparing a homogeneous group of HIV type 1 subtype B viruses with a heterogeneous group of divergent strains. AIDS Res Hum Retroviruses. 14(4): 347-52
https://pubmed.ncbi.nlm.nih.gov/9519896

II. Greengrass VL, Turnbull SP, Hocking J, Dunne AL, Tachedjian G, Corrigan GE, Crowe SM. (2005). Evaluation of a low cost reverse transcriptase assay for plasma HIV-1 viral load monitoring. Curr HIV Res. 3(2): 183-90
https://pubmed.ncbi.nlm.nih.gov/15853722

III. Jennings C, Fiscus SA, Crowe SM, Danilovic AD, Morack RJ, Scianna S, Cachafeiro A, Brambilla DJ, Schupbach J, Stevens W, Respess R, Varnier OE, Corrigan GE, Gronowitz JS, Ussery MA, Bremer JW. (2005). Comparison of two human immunodeficiency virus (HIV) RNA surrogate assays to the standard HIV RNA assay. J Clin Microbiol. 43(12): 5950-6
https://pubmed.ncbi.nlm.nih.gov/16333081

IV. Corrigan GE, Hansson EO, Morner A, Berry N, Kallander CF, Thorstensson R. (2006). Reverse transcriptase viral load correlates with RNA in SIV/SHIV-infected macaques. AIDS Res Hum Retroviruses. 22(9): 917-23
https://pubmed.ncbi.nlm.nih.gov/16989619

V. Corrigan GE, Grutzmeier S, Albert J, Källander CFR and Thorstensson R. (1970). Accumulation of NRTI resistance mutations in the HIV-1 genome in vivo results in an initial but transient eduction in reverse transcriptase fitness. [Submitted]